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CHAPTER ONE
1.0
INTRODUCTION AND LITERATURE REVIEW
1.1 INTRODUCTION
The genus Klebsiella belongs to the tribe Klebsiellae, a member of the
family Enterobacteriaceae. The organisms are named after Edwin Klebs, a 19th
century German microbiologist. Klebsiellae are nonmotile, rod-shaped, gramnegative bacteria with a prominent polysaccharide capsule. This capsule encases
the entire cell surface, accounts for the large appearance of the organism on gram
stain, and provides resistance against many host defence mechanisms (Orskov,
1984; Ewing, 1986; Holt et al., 1994).
Members of the genus Klebsiella typically express 2 types of antigens on
their cell surface. The first is a lipopolysaccharide (O antigen); the other is a
capsular polysaccharide (K antigen). Both of these antigens contribute to
pathogenicity. About 77 K antigens and 9 O antigens exist. The structural
variability of these antigens forms the basis for classification into various
serotypes. The virulence of all serotypes appears to be similar.
1.2 LITERATURE REVIEW
1.2.1 Pathophysiology of Klebsiella infections
Host defense against bacterial invasion depends on phagocytosis by
polymorphonuclear granulocytes and the bactericidal effect of serum, mediated in
large part by complement proteins. Both classic-pathway and alternate-pathway
complement activation have been described, but the latter, which does not require
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the presence of immunoglobulins directed against bacterial antigens, appears to be
the more active pathway in Klebsiella pneumoniae infections. Recent data from
preclinical studies suggest a role for neutrophil myeloperoxidase and
lipopolysaccharide-binding protein in host defense against Klebsiella pneumoniae
infection. Neutrophil myeloperoxidase is thought to mediate oxidative
inactivation of elastase, an enzyme implicated in the pathogenesis of various
tissue-destroying diseases.
The bacteria overcome innate host immunity through several means. They
possess a polysaccharide capsule, which is the main determinant of their
pathogenicity. The capsule is composed of complex acidic polysaccharides. Its
massive layer protects the bacterium from phagocytosis by polymorphonuclear
granulocytes. In addition, the capsule prevents bacterial death caused by
bactericidal serum factors. This is accomplished mainly by inhibiting the
activation or uptake of complement components, especially C3b. The bacteria
also produce multiple adhesins. These may be fimbrial or nonfimbrial, each with
distinct receptor specificity. These facilitate the microorganism to adhere to host
cells, which is critical to the infectious process (Mitscher, 1995).
Lipopolysaccharides (LPS) are another bacterial pathogenicity factor.
They are able to activate complement, which causes selective deposition of C3b
onto LPS molecules at sites distant from the bacterial cell membrane. This
inhibits the formation of the membrane attack complex (C5b-C9), which prevents
membrane damage and bacterial cell death. Availability of iron increases host
susceptibility to Klebsiella pneumoniae infection. Bacteria are able to compete
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effectively for iron bound to host proteins because of the secretion of highaffinity, low molecular weight iron chelators known as siderophores. This is
necessary because most host iron is bound to intracellular and extracellular
proteins. In order to deprive bacteria of iron, the host also secretes iron-binding
proteins. (Collatz et al., 1984; Ardanuy et al., 1998).
1.2.2 Epidemiology of Klebsiellae
Klebsiellae are ubiquitous in nature. In humans, they may colonize the
skin, pharynx, or gastrointestinal tract. They may also colonize sterile wounds and
urine. Klebsiellae may be regarded as normal flora in many parts of the colon and
intestinal tract and in the biliary tract. Oropharyngeal carriage has been associated
with endotracheal intubation, impaired host defenses and antimicrobial use
(Ashiru and Osoba, 1986; Akindele and Rotilu, 1997; Oral et al., 1998; Hiran and
Vishwanathan, 1999; Khaneja et al., 1999; Bouza and Cercenado, 2002).
Klebsiella pneumoniae and Klebsiella oxytoca are the 2 members of this
genus responsible for most human infections. They are opportunistic pathogens
found in the environment and in mammalian mucosal surfaces. The principal
pathogenic reservoirs of infection are the gastrointestinal tract of patients and the
hands of hospital personnel. Organisms can spread rapidly, often leading to
nosocomial outbreaks (Casewell and Philips, 1981; Traub et al., 2000).
Infection with Klebsiella organisms occurs in the lungs, where they cause
destructive changes. Necrosis, inflammation and hemorrhage occur within lung
tissue, sometimes producing thick, bloody, mucoid sputum described as currant
jelly sputum. The illness typically affects middle-aged and older men with
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debilitating diseases such as alcoholism, diabetes or chronic bronchopulmonary
disease. This patient population is believed to have impaired respiratory host
defenses. The organisms gain access after the host aspirates colonizing
oropharyngeal microbes into the lower respiratory tract.
Klebsiellae have also been incriminated in nosocomial infections.
Common sites include the urinary tract, lower respiratory tract, biliary tract, and
surgical wound sites. The spectrum of clinical syndromes includes pneumonia,
bacteremia, thrombophlebitis, urinary tract infection (UTI), cholecystitis,
diarrhea, upper respiratory tract infection, wound infection, osteomyelitis and
meningitis (Hiran and Vishwanathan, 1999). The presence of invasive devices,
contamination of respiratory support equipment, use of urinary catheters, and use
of antibiotics are factors that increase the likelihood of nosocomial infection with
Klebsiella species. Sepsis and septic shock may follow entry of organisms into the
blood from a focal source (Cryz et al., 1991).
Extensive use of broad-spectrum antibiotics in hospitalized patients has
led to both increased carriage of Klebsiellae and subsequently the development of
multidrug-resistant strains that produce extended-spectrum beta-lactamase
(ESBL). These strains are highly virulent, show capsular type K55, and have an
extraordinary ability to spread. Most outbreaks are due to a single clone or single
gene. The bowel is the major site of colonization with infection of the urinary
tract, respiratory tract and wounds. Bacteraemia and significant increased
mortality have resulted from infection with these species (Cryz et al., 1991).
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1.2.3
Laboratory diagnosis and identification of the genus
Klebsiella
The genus Klebsiella belongs to the family Enterobacteriaceae and
according to Orskov (1984), Ewing (1986) and Holt et al., (1994), Klebsiella are
gram-negative
capsulated,
nonmotile,
facultatively
anaerobic
chemo-
organotrophic rods having both a respiratory chain and a fermentative type of
metabolism, with an optimal growth temperature of 37C. A complete blood cell
count for patients infected with Klebsiella usually reveals leukocytosis with a left
shift, but this is not invariably present. Persistence of leukocytosis may signify
empyema formation.
A sputum sample for Gram stain should be obtained. Klebsiellae appear as
short, plump, gram-negative bacilli. They are usually surrounded by a capsule that
appears as a clear space. Serology results are not useful for detection of infection
with Klebsiella organisms. Cultures should be obtained from possible sites such
as; wounds, peripheral or central intravenous access sites, urinary catheters and
respiratory support equipment (Holt et al., 1994).
Klebsiellae may be isolated from blood, urine, pleural fluid, and wounds.
Klebsiellae are microaerophilic and thus, can grow in the presence of oxygen or in
its absence. They have no special culture requirements. Most species can use
citrate and glucose as sole carbon sources. Thus, they grow well on most ordinary
media. Klebsiellae are lactose-fermenting, urease-positive and indole-negative
organisms, although Klebsiella oxytoca and some strains of Klebsiella
pneumoniae are exceptions. Klebsiellae do not produce hydrogen sulfide and they
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yield positive results on both Voges-Proskauer and methyl red tests. Wounds may
be infected with Klebsiella organisms as the sole pathogens or as a component of
a multipathogenic infection. Swabs for Gram stain and culture taken from
possible sites may aid in establishing the diagnosis (Orskov, 1984; Ewing, 1986;
Holt et al., 1994).
These bacteria lack the cytochromes and the cytochrome oxidase. Catalase
involved in the break down of hydrogen peroxide into water and oxygen is
present, while indole, methyl red and Simmons citrate reactions vary among
species. In addition members of this genus typically do not produce hydrogen
sulphide, arginine dihydrolase, ornithine decarboxylase or phenylalanine
deaminase. When urease reactions occur, they are slower and less intense than
those exhibited by the genus Proteus. Members of the genus can grow on
potassium cyanide, reduce nitrates and most species ferment all commonly tested
carbohydrates except ducitol and erythritol, with the production of acid and gas.
However, anaerogenic strains also occur (Holt et al., 1994).
Some strains of Klebsiella can fix nitrogen, a property that is not related to
the source of the strain (Orskov, 1994; Ewing, 1986). It is reported that some
strains may give atypical biochemical reactions and it has therefore been
recommended that routine identification of clinical isolates should integrate
colonial morphology and biochemical reactions for maximum accuracy (Gross
and Holmes, 1990; John and Twitty, 1986).
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1.2.4 Main species of the genus Klebsiella
The genus has four species with Klebsiella pneumoniae being the type
species. The other species are Klebsiella oxytoca, Klebsiella planticola, Klebsiella
terrigena and Klebsiella trevisanii. Orskov (1984) reported that the greatest
problem in identification is to distinguish Klebsiella pneumoniae strains from
nonmotile Enterobacter aerogenes strains. Ewing (1986) reported that DNA
relatedness studies show that Klebsiella ozaenae and Klebsiella rhinoscleromatis
earlier classified as individual species are actually metabolically less active
biotypes of Klebsiella pneunoniae. He proposed that Klebsiella pneumoniae,
should be recognised to have three subspecies. These are:
i)
Klebsiella pneumoniae subspecies pneumoniae
ii)
Klebsiella pneumoniae subspecies ozaenae
iii)
Klebsiella pneumoniae subspecies rhinoscleromatis
Ewing (1986) also proposed that this classification should be recognized in
formal communications. The original classification could remain acceptable in
reporting diagnostic and epidemiological findings.
Gross and Holmes (1990), while in agreement with Orskov (1984) and
Ewing (1986) with respect to the classiffication of Klebsiella pneumoniae into
subspecies added the subspecies atlantae and edwardsii and suggested that
Klebsiella pneumoniae be designated Klebsiella pneumoniae subspecies
aerogenes.
Thus, they suggested that for the sake of uniformity the genus
Klebsiella should be recognised to comprise of the type species Klebsiella
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pneumoniae with its various biochemical variants and four other species which
would include the following:
i) Klebsiella oxytoca referring to organisms that produce indole and liquefy
gelatin
ii) Klebsiella ornithinolytica to describe the only Klebsiella species that
decarboxylates ornithine
iii) Klebsiella planticola (synonym K. trevisanii) to refer to a species that rarely
occurs in clinical specimens and some of whose members produce indole
iv)Klebsiella terrigena to describe a species that is indistinguishable from
Klebsiella pnemoniae but is found only in soil and water.
Holt et al. (1994) agreed with the classification by Orskov (1984) and Ewing
(1986) but however reported that there is doubt as to whether, based on DNA
relatedness studies Klebsiella ornithinolytica should be retained as a subgroup of
Klebsiella planticola or it should be a separate species.
1.2.5 Infections caused by Klebsiella species
Klebsiella species cause various infections including pneumonia,
septicaemia, bacteraemia, meningitis, osteomyelitis, wound infections, urinary
tract infections, childhood gastroenteritis and other conditions (Ashiru and Osoba,
1986; Akindele and Rotilu, 1997; Oral et al., 1998; Hiran and Vishwanathan,
1999; Khaneja et al., 1999; Bouza and Cercenado, 2002). Klebsiella have been
shown to be important opportunistic pathogens featuring prominently among
nosocomial infections (Casewell and Philips, 1981; Traub et al., 2000). Sepsis
and pneumonia due to Pseudomonas aeruginosa and Klebsiella species have been
8
reported to carry high mortality rates, often in the range of 25% to 50% (Cryz et
al., 1991). While Salmonella was the recognised cause of osteomyelitis in sickle
cell disease, Klebsiella pneumoniae has been shown to be an emerging cause of
osteomyelitis (Hiran and Vishwanathan, 1999). Klebsiella pneumoniae has also
been reported as a major cause of both nosocomial and community acquired
bacteraemia and infections at other sites (McGowan, 1985; Bouza and Cercenado,
2002).
Epidemics caused by antimicrobial resistant Klebsiella species have been
reported to have resulted in septicaemia rates of up to 15% with inevitable
mortality. Such epidemics have led to closures of hospital specialist units or even
whole hospitals (Casewell and Philips, 1981). Klebsiella has also been reported to
be a major genus of bacteria causing urinary tract infections in spinal injury
patients. In these patients, Klebsiella pneumoniae was found to be the most
prevalent species often associated with the colonization of urine bags
(Montgomerie et al., 1993) and causing colonization of the urethra, perineum and
rectum.
Isaack et al., (1992) found Klebsiella species and Escherichia coli to be
the commonest Gram-negative bacteria causing infections among children with
severe protein and energy malnutrition in Tanzania. Septicaemia and urinary tract
infections were found to be prevalent in these children.
Infection caused by drug resistant Klebsiella species is sometimes
associated with resistance encoding plasmids that can be transferred to other
genera. Transfer of multiple antibiotic resistance to Escherichia coli-K12 by
9
Klebsiella aerogenes has been reported (Casewell et al., 1981). The transfer of
cephamycin resistance determinants from Klebsiella pneumoniae into Escherichia
coli by plasmid DNA has also been reported (Papanicolaou et al., 1990). As
Klebsiella and other enteric bacteria share a common environment in the hospital,
the spread of resistance determinants among the bacteria is highly likely.
1.2.6 Antimicrobial resistance
Appropriate use of antibiotics is central to limiting the development and
spread of resistant bacteria in hospitals and communities. Use of broad-spectrum
antibiotics, in particular the third generation cephalosporins in nosocomial
infections have been linked to the emergence of antibiotic resistance and increase
in costs (Mc Gowen and Tenover, 1997). The hospital setting is particularly
conducive to the development of antibiotic resistance as patients who are severely
ill, immuno-compromised or have devices or implants in them are likely to
receive frequent courses of empirical or prophylactic antibiotic therapy (Patterson,
2001).
Liberal use of the Third generation cephalosporins (3GC) antibiotics has
resulted in the ESBLs conferring resistance among Enterobacter and
Enterobacteriacae worldwide compromising their clinical use. Prior antibiotic use
is an important risk factor for colonization and bacterial infection. However,
antibiotic use cannot always be correlated with emergent antibiotic resistance.
Studies have reported the association of resistant Klebsiella pneumoniae and other
10
Enterobacteriaceae and vancomycin-resistant enteroccocci with cephalosporin
use (Lautenbach et al., 2003).
1.2.7 Biochemical basis of resistance
Acquisition of resistance is associated with one or a combination of the
following mechanisms (Cohen andAuxe, 1992; Mitscher, 1995):
i)
Bacterial enzymes that inactivate antibiotics
ii)
Alteration of drug target
iii)
Permeability changes that prevent drug entry
iv)
Efflux of the drug from the bacterial cell
1.2.7.1 Bacterial enzymes that inactivate antibiotics
1.2.7.1.1 Beta-lactamases
The discovery of penicilins was accompanied by the discovery of
penicillin destroying enzymes initially referred to as penicillinases and
subsequently as -lactamases (Ross and O’Callaghan, 1975). The introduction of
cephalosporins and broad spectrum penicillins provided substrates for the
detection of a wide range of -lactamases (Bush and Sykes, 1986). Betalactamases are serine proteases which catalyse hydrolysis of the -lactam bond,
thus inactivating the -lactam antibiotics for penicillins and cephalosporins.
There are many beta-lactamases. Some are efficient at hydrolysing
penicillins, some at hydrolysing cephalosporins and some are indiscriminate
(Mitscher, 1995).
Hedges et al. (1974) described the beta-lactamase TEM
specified by plasmid RGK (formerly RTEM). All enzymes showing biochemical
11
properties similar to those of TEM were collectively termed TEM-like lactamases. Hedges et al. (1974) also described an oxacillin hydrolysing lactamase termed OXA, also with several subtypes. Mathew and Hedges (1976)
subdivided the TEM and OXA enzymes into TEM-1 and TEM-2 and OXA-1,
OXA-2 and OXA-3, respectively. Mathew et al. (1979) described two more lactamases, one specified by plasmid R997 and designated HMS-1 and the other
from several sources as SHV-1 (Sulfhydryl variable, because of its ability to
hydrolyse cephaloridine but not benzyl penicillin in the presence of the Sulfhydryl
inhibitor, p-chloromercuribenzoate). Mathew et al. (1979) further reported that
TEM-1 was the commonest -lactamase followed in order by TEM-2 and then the
OXA group.
A multiplicity of enzymes with a variety of hydrolysing capacities was
also described. For example Kleibe et al. (1985) reported the production of lactamases that attacked broad spectrum cephalosporins in Enterobacteriaceae and
Pseudomonas aeruginosa.
Similar findings were reported by Philipon et al.
(1989) who found the existence of plasmid mediated extended spectrum betalactamases which were derivatives of TEM and SHV enzymes. Brun-Buisson et
al. (1987) had described an extended spectrum -lactamase with properties
similar to those of SHV-2 and CTX-1which caused nosocomial infections in
French hospitals.
Petit et al. (1988) described clinical isolates of Klebsiella
pneumoniae that exhibited resistance to ceftazidime, susceptibility to aztreonam
and cefotaxime and upon which aztreonam and cetofaxime showed synergism
when combined with clavulanic acid. Beta-lactamases that hydrolyze expanded
12
spectrum cephalosporins and monobactams are designated as extended spectrum
-lactamases (ESBLs) and arise from one of three parental enzymes TEM-1,
TEM-2 or SHV-1 (Philippon et al., 1989; Arlet et al., 1993; Sirot, 1995; 1
Randegger et al., 2000; Dipersio et al., 2005).
Three major groups of beta lactamases with wide spectra of substrate specificity
are distinguishable (Gniadkowski, 2001). These are:
i.
Class C cephalosporinases (Amp C),
ii.
Extended spectrum beta-lactamases (ESBL)
iii.
Enzymes with carbapenemase activity, for example, class B metallo-betalactamases.
The genes responsible for extended spectrum beta-lactamases are easily
transferable because they are located on plasmids leading to a situation referred to
as a plague of plasmids (Fierer and Guiney, 1999).
Chromosomal enzymes similar to SHV-2 that confer resistance to
expanded spectrum cephalosporins like cefotaxime have also been reported
(Thomson et al., 1991). Many variants of the parental enzymes are now known
and many more -lactamases are likely to be described in future since point
mutations of the parental enzymes give rise to variants with completely new
substrate profiles (Heritage et al., 1999; Perilli et al., 2000).
1.2.7.1.2 Aminoglycoside modifying enzymes
Aminoglycosides contain a 1,3-diaminoinositol derivative (aminocyclitol)
some of whose hydroxyl groups are substituted through glycosidic linkages with
amino sugars. The substitution gives rise to a class of antibiotics with some
13
differences in their chemical properties. The chemical differences among
aminoglycosides are important in determining antimicrobial spectrum, potency,
toxicity, pharmacokinetics and resistance to degradation by bacterial enzymes
(Mitscher, 1995; Kudo et al., 2005).
Aminoglycosides are inactivated by a large number of aminoglycosidemodifying-enzymes originating from both Gram-positive and Gram-negative
bacteria (Wohlleben et al., 1989; Azucena and Mobashery, 2001; Smith and
Baker, 2002). These enzymes fall into three groups depending on the reaction
they catalyse (Haas and Dowding, 1975; Neu, 1984; Lambert and O’Grady, 1992;
Wright and Thomson, 1999):
i)
N-acetyltransferases (AAC) which acetylate amine groups
ii)
O-phosphotransferases (APH) which phosphorylate hydroxyl groups and
iii)
O-nucleotidyl transferases (ANT) which act upon hydroxyl groups. The
nucleotidyl-transferases were initially called O-adenyl transferases (AAD)
because the adenylated antibiotic is the major product. However, the guanylate or
inosinate may be formed (Lambert and O'Grady, 1992).
A number of enzymes that confer resistance to amikacin and other
aminoglycosides have been reported (Jacoby et al., 1990; Tolmasky, 2000; Poirel
et al., 2001; Sarno et al., 2003).
Aminoglycoside modifying enzymes often have the ability to inactivate a
broad range of substrates. Certain antibiotics can also be modified by multiple
enzymes. Thus, cross resistance exists among many of aminoglycosides (Rice
and Bonomo, 1996). In Enterobacteriaceae, aminoglycoside modifying enzymes
14
are often plasmid encoded and are found in association with extended-spectrum
-lactamases, frequently on multiple antibiotic-resistance-encoding plasmids
(Kagan and Davies, 1980; Rice et al., 1990; Fernandez-Rodriguez et al., 1992;
Mendes et al., 2004). In a survey in some eighteen centres worldwide, O'Brien et
al., (1987) found that resistance to kanamycin in Klebsiella pneumoniae ranged
from 1% to 50% and involved several types of resistance mechanisms which
could be transmitted by a similar plasmid. Resistance to aminoglycosides among
Acinetobacter has been attributed to the production of three classes of enzymes of
which aminoglycoside-3"-phosphotransferase VI was more predominant (Nemec
et al., 2004). The production of aminoglycoside-6-N-acetyltransferase, AAC(6'),
3'-0-phosphotransferase type VI, 4'-aminoglycoside nucleotidyl transferase type
II and 4"-0-nucleotidyltransferase type II [ANT(4')-II] that mediated resistance to
amikacin and other aminoglycosides in Gram negative bacteria have been
reported (Jacoby et al., 1990; Rather et al., 1992; Gallimand et al., 1993).
Aminoglycosides exert their bactericidal effects primarily by binding to
the bacterial 30S ribosomal subunit rendering the ribosome unavailable for
translation (Tolmasky, 2000). An enzymatically modified molecule cannot bind
to the ribosomal target and it is therefore unable to inhibit protein synthesis. The
modified molecule also fails to promote the energy dependent uptake of
unmodified antibiotic into the cell as happens in absence of modification
(Williams, 1990; Llano-Sotelo et al., 2002).
The large number of aminoglycoside modifying enzymes is a threat to the
successful treatment of infections using these agents.
15
In some instances,
resistance has developed during therapy. It is therefore common to combine
aminoglycosides with other agents like the beta-lactams or fluoroquinolones
(Mayer and Nagy, 1999) to avoid such development of resistance. The
combination of aminoglycosides with cell wall active agents like vancomycin and
the beta-lactams has been compromised following the observation that the
synergistic effect disappears in strains of enterococci that show high levels of
resistance to aminoglycosides (Gutierrez et al., 1992; Chow, 2000).
1.2.7.1.3 Enzymes that inactivate chloramphenicol
Since all the functional groups in chloramphenicol contribute to its
effectiveness as an inhibitor of ribosomal peptidyltransferase activity, there are
several possible ways of inactivation (Murray and Shaw, 1997). Enzyme
catalysed reactions in the inactivation include dehalogenation, nitrogroup
reduction and hydrolysis of the amide bond (Shaw, 1975). Chloramphenicol
hydrolase, which hydrolyses the amide bond was first isolated from a
Streptomyces species that produces chloramphenicol and later from a number of
other bacteria (Vila et al., 1975). Inactivation by phosphorylation of the hydroxyl
groups has also been reported (Mosher et al., 1995; Izard and Ellis, 2000).
In addition to these modifications, O-acetylation of the hydroxyl groups
has been found to be the major mechanism of inactivation of chloramphenicol. It
involves
the
production
of
an
intracellular
enzyme,
chloramphenicol
acetyltransferase (Gaffney et al., 1981; Bissonette et al., 1991; Shaw and Leslie,
1991; Schwaz et al., 2004). The enzyme is produced by Gram-positive bacteria
16
through induction by chloramphenicol and by Gram-negative bacteria
constitutively (Franklin, 1992). Three variants of the enzyme produced by Gramnegative bacteria have been designated types I, II and III (Murray et al., 1990).
Chloramphenicol inactivation is a two step acetylation in which the acetyl group
is supplied by acetyl-CoA, to form the 1,3 - diacetoxy chloramphenicol which is
inactive. Chloramphenicol-acetyl-transferase mediated-resistance results from the
failure by the modified antibiotic to bind to the 50S ribosomal subunit, hence
failure to inhibit peptide elongation (Shaw, 1975).
A novel group of enzymes that catalyse the transfer, of an acetyl group
from acety - CoA to chloramphenicol but distinct from classical chloramphenicol
acetyl transferase and designated xenobiotic acetyl transferases have been
reported.
They are however, not primarily associated with chloramphenicol
inactivation (Tennigkeit and Matzura, 1991; Murray and Shaw, 1997). In addition
to the large variety of enzymes capable of inactivating chlorampheniol, other
mechanisms of resistance yet to be elucidated may exist. For example, a novel
type of plasmid borne chloramphenicol-florfenicol resistance gene whose product
confers resistance to chloramphenicol and florfenicol in staphylococci and
Escherichia coli by a mechanism that does not involve enzymatic inactivation or
efflux of the drugs was reported by Schwarz et al., 2000).
1.2.7.2 Alteration of drug target
Enzymes are the major targets whose alteration results in resistance. Many
antibiotics are structural analogues of natural metabolites and therefore inhibit
17
enzymes that recognise the antibiotics or their metabolites as substrates (Bennett
and Howe, 1990). Resistance develops when enzymes lose affinity for antibiotics.
For example, one form of resistance to -lactam antibiotics involves the
production of penicillin binding proteins (PBPs) with reduced affinity for lactams but unaltered affinity for peptidoglycan precursors (Collatz et. al., 1984).
Penicillin binding proteins have transpeptidase activity and control such
fundamental processes as cell growth and division so that their inhibition can
cause cell death, lysis or arrest of growth (Georgopapadakou, 1993). Relative
affinities of -lactamases and PBPs for -lactam antibiotics in -lactamase
producing bacteria could expressed in terms of the and that the target accessibility
index (TAI). A higher affinity of PBPs for the antibiotics than beta-lactamases
may lead to cell death while a higher affinity for -lactamases may mean cell
survival in presence of the antibiotics (Georgopapadakou, 1993). However, betalactamase overproduction may render the sensitivity of PBPs irrelevant since the
beta-lactams get inactivated before they get to the target (Lakaye et al., 1999).
Target alteration is more significant in Gram-positive than in Gram-negative
bacteria (Murray, 1991; Hand, 2000; Tillotson and Watson 2001). Targetmediated-resistance may involve production of proteins that have reduced affinity
for antimicrobial agents, production of novel proteins that assume the functions of
antimicrobial targets, hyperproduction of an antimicrobial target that overwhelms
the agents, development of alternative metabolic pathways that bypass a sensitive
target or modification of ribosomal ribonucleic acid (Cooksey, 1991; Tillotson
and Watson, 2001).
18
There is a form of resistance to sulphonamides that involves production of
dihydropteroate synthetase that has reduced affinity for sulphonamides but
unchaged affinity for para-aminobenzoic acid (Skold, 1976, 2000). Resistance to
trimethoprim sometimes involves the production of some forms of dihydrofolate
reductase that have reduced affinities for the antibiotic (Franklin, 1992).
Resistance to fluoroquinolones may be associated with target changes involving
DNA gyrase and/or topoisomerase IV (Deguchi et al., 1997; Heisig et al., 1993;
Piddock, 1999; Weigel et al., 1998).
Inhibition of DNA gyrase which is
responsible for the coiling and supercoiling of DNA within the cell results in the
failure of DNA transcription hence protein synthesis or DNA replication does not
occur (Kidwai et al., 1998; Gruger et al., 2004).
In Streptococcus pneumoniae resistance to fluoroquinolones appears to
result from single step mutational alterations to type II DNA gyrase and
topisomerase IV, the primary fluoroquinolone targets in Streptococcus
pneumoniae. When two or more mutations are present, higher levels of resistance
are experienced (Gruger et al., 2004). Resistance to fluoroquinolones was for a
long time associated sorely with chromosomal mutations but cases of plasmid
mediated resistance have been reported (Martinez-Martinez et al., 1999; Paterson
et al., 2000; Wang et al., 2004).
Resistance may involve over production of a target. Examples include one
type of resistance to sulphonamides in which resistant strains produce large
amounts of para-amino benzoic acid which competes with and displaces the
sulphonamide from the active site of dihydropteroate synthetase (Franklin, 1992)
19
and a reported case of resistance to aztreonam due over production of a
chromosomal beta-lactamase in Klebsiella oxytoca (Jeong et al., 2001).
In Escherichia coli one type of resistance to streptomycin involves the
replacement of a single amino acid on protein S12 of the 30S ribosomal subunit
resulting in ribsomes that cannot bind streptomycin (Franklin, 1992) while
macrolides, lincosamides and streptogramin B resistance may involve a methylase
which methylates the mRNA group to which these antibiotics bind to inhibit
protein synthesis (Skinner et al., 1983; Ackermann et al., 2003).
Resistance involving utilization of an alternative pathway is found in
mutated bacterial cells that utilize thymidine directly to synthesize thymidylate
through salvage pathways, thus causing resistance to trimethoprim (Maskell et al.,
1978).
A novel penicillin binding protein, PBP2a also designated PBP2' that is
not found in susceptible strains of Staphylocccus aureus is found in methicillin
resistant Staphyloccus aureus. This novel protein appears to help resistant strains
bypass the methicillin- sensitive proteins (Ubukata et al., 1985; Dever and
Dermody, 1991; Georgopapadakou, 1993).
When the genetic loci responsible for alterations are located on plasmids
and transposons, the dissemination of this type of resistance mechanisms can
occur among many species or even genera (Murray, 1991; Rowe-Magnus et al.,
2002).
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1.2.7.3 Permeability changes that prevent drug entry
This change of permeability by the bacteria results to inability of the
antibiotic to gain access into the bacteria and the mechanism is more important in
Gram-negative than in Gram-positive bacteria. Gram-positive bacteria possess a
single cytoplasmic membrane below the peptidoglycan layer, where as, the Gram
negative bacteria have an additional membrane external to the peptidoglycan layer
and also a periplasm between the cytoplasmic membrane and the peptidoglycan
layer (Mitscher, 1995). The outer membrane acts as a selective barrier to the
entry of antibiotics into the Gram-negative bacterial cells.
Hydrophilic antibiotics cross the outer membrane by passive diffusion
through water filled pores called porins or outer membrane proteins (OMPs).
Hydrophobic antibiotics may enter by facilitated diffusion or by self promoted
uptake (Collatz et al., 1984; Ardanuy et al., 1998). Porin loss or other changes to
the porins will result in antimicrobial resistance that transcends antimicrobial
classes since porins are not specific to any group of agents (Sanders et al., 1984).
Porin mediated outer membrane permeability loss is reported to be the most
common mechanism of resistance in multiply resistant Pseudomonas cepacia
(Burns and Clark, 1992).
Porin loss has also been reported as a cause of
resistance in a number of members of the family Enterobacteriaceae including
Klebsiella species (Ardanuy et al., 1998; Bradford et al., 1997; DomenechSanchez et al., 1999). In Klebsiella pneumoniae, one form of resistance to lactam antibiotic is associated with loss of three outer membrane proteins
OmpK35, OmpK36 and OmpK37 (Domenech-Sanchez et al., 1999, 2003).
21
It has been found that a causal relationship between the size of the porin
lost and development of resistance exists. For example the mutational loss of a
large outer membrane protein OmpF was found to result in development of
resistance to moxalactam in Escherichia coli while loss of the smaller porins
ompC and ompR did not alter susceptibility (Nikaido, 1989).
In Klebsiella
pneumoniae it has been found that the expression of the smaller porin ompK37 in
absence of the larger porins ompK 35 or ompK36 resulted in low susceptibility to
some -lactams while expression of the larger porins resulted in higher
susceptibility (Domenech-Sanchez et al., 1999). The outer membrane can also
serve as a barrier that prevents the leakage or secretion of some endogenous
products within the cell. In this respect, it has been linked to the inhibition of
diffusion out of -lactamases leading to their concentration in the periplasmic
space (Livermore, 2002; Lakaye et al., 1999). By this type of concentration, the
permeability barrier which may result in low level resistance is amplified
especially by production of destructive enzymes (Nikaido, 1989; MartinezMatinez, et al., 1999; Chevalier et al., 2000).
Pseudomonas aeruginosa is
particularly adept at combining several mechanisms simultaneously and hence its
ability to resist about all classes of antimicrobial agents (Aires et al., 1999;
Livermore, 2002).
1.2.7.4 Efflux of the drugs from the bacterial cell
One of the most common resistance mechanisms in both prokaryotes and
eukaryotes is the transmembrane-protein-catalysed extrusion of drugs from the
22
cell with transmembrane proteins acting like pumps that reduce the intracellular
drug concentration to subtoxic levels (Kohler et al., 1999; Borges - Walmsley and
Walmslsey, 2001; Pages et al., 2003). Efflux systems occur in many Grampositive and Gram-negative bacteria in which pumps specific for only one
substrate or multiple efflux pumps accommodating a wide range of substrates are
found (Kohler et al., 1999; Poole, 2001).
Bacterial antimicrobial efflux transporters have generally been grouped
into five super families primarily on the basis of amino acid sequence homology.
They include the following: the major facilitator super family (MFS), the ATPbinding cassette family, the resistance nodulation division (RND), the small
multi-drug resistance protein family and the multi-drug and toxic compound
extrusion (MATE) family (Poole, 2000; Van Bambeke et al., 2000; Chen et al.,
2003). The RND and MATE families are found in Gram-negative bacteria while
the MFS type predominates among Gram-positive bacteria (Poole, 2000). Several
efflux systems may exist in a single microorganism giving rise to multiple
resistance or intrinsic resistance as appears in Pseudomonas aeruginosa (Aires et
al., 1999; Piddock, 1999).
The level of efflux mediated resistance depends on whether a single
component efflux pump which exports the drug into the periplasm, a multicomponent pump that accomplishes the efflux of the drug directly into the
external medium, or a combination of these is involved. Simultaneous expression
of different structural types of pumps in the same cell results in a higher level of
resistance than expression of single types of pumps (Lee et al., 2000).
23
Resistance to a number of antibiotics is mediated by efflux mechanisms:
Chloramphenicol (Arcangioli et al., 1999), aminoglycosides (Aires et al., 1999),
fluoroquinolones (Piddock, 1999), tetracycline (Roberts, 1996, 1997; McMurray
et al., 1980).
Antimicrobial efflux systems act synergistically with other resistance
mechanisms, especially loss of outer membrane proteins to promote drug
exclusion (Poole, 2001). For instance, Klebsiella adaptation against drugs present
in the environment has been linked to active efflux of drugs in combination with
decreased membrane permeability and presence of beta-lactamases (Chevalier et
al., 2000). Efflux systems are usually chromosomally encoded but some are
present on plasmids (Marshall and Piddock, 1997). For example, tetracycline
resistance is primarily due to energy dependent efflux pumps frequently
associated with conjugative plasmids in Gram negative bacteria and small
mobilizable plasmids or the chromosome in Gram-positive bacteria (Roberts,
1997). It is expected that the selection of novel antimicrobial agents that are not
subject to extrusion from cells and development of potent specific inhibitors of
pumps may lead to the renaissance of drugs rendered noneffective by efflux
mechanisms (Van Bambeke et al., 2000; Pages et al., 2003).
1.2.8 Antimicrobial Resistance In Klebsiella Species
Antimicrobial chemotherapy plays an important role in the management of
infectious diseases.
In this regard, antibiotics are very significant. It was
estimated that antibiotics account for 15% to 30% of drug expenditures, the
24
largest share of any therapeutic group of drugs worldwide (Col and O'Connor,
1987). The determination of antimicrobial susceptibility of clinical isolates is
often of crucial importance for the optimal antimicrobial therapy of infected
patients. This is the requirement in increasing resistance and emergence of
multidrug resistance organisms. Testing is required not only for therapy but to
monitor the spread of resistant organisms or resistant genes throughout the
community. Standard procedures and breakpoints have been identified to predict
therapeutic outcome both in time and at different geographic locations (Fluit et
al., 2001).
The wide spread use of antimicrobial agents has failed to eradicate
microbial infections despite their benefits. Antibiotic resistant bacteria have been
a source of ever-increasing therapeutic problem. Continued mismanaged selective
pressure has contributed towards the emergence of multiple drug resistant bacteria
and that has been regarded as an inevitable genetic response to antimicrobial
therapy (Cohen and Auxe, 1992). The antibiotic resistant mutants that arise
spontaneously are generally resistant to only one antibiotic. However, Klebsiella
species exhibit simultaneous resistance to multiple drugs (Gutmann et al., 1985).
Successful
antimicrobial
chemotherapy is
however
hindered
by
antimicrobial resistance. It has been observed that while antibiotics revolutionized
the treatment of infectious diseases in the 20th century, resistance threatens to
render many of them ineffective in the 21st century (Burk and Berghuis, 2002;
Powers, 2004). Resistance is defined as the ability of a cell and its progeny to
survive under conditions that would kill or inhibit such a cell (Franklin, 1992).
25
Resistance appears as an inevitable consequence of antimicrobial use. As soon as
a new antimicrobial agent is discovered or synthesized, bacteria evolve
mechanisms to overcome the effects of the new agent (Neu, 1983; Ridley, 1970;
Sanders and Sanders, 1992). For instance, resistance to sulphonamides and
penicillin began to appear soon after their introduction into clinical use (Levy,
1982). Bacterial resistance has been reported against most available antibiotics
(Rao, 1998). Almost all pathogenic bacteria have developed some degree of
resistance to one or more antimicrobial agents (Levy and Marshall, 2004).
Infections caused by resistant micro-organisms may result in more hospital
admissions, longer periods of hospitalization, longer and more expensive or toxic
therapy and higher mortality (Cryz et al., 1991; Cohen, 1992; Kunin, 1993;
Waiyaki, 1993; Peres-Bota et al., 2003; Ang et al., 2004).
Some bacteria are inherently resistant to certain antimicrobial agents and
the pattern of this resistance is well known and stable.
Such resistance is
therefore not a hindrance to the selection of effective antimicrobial agents.
However, acquired resistance whose appearance is unpredictable is a major
problem in the selection of therapeutic agents (Franklin, 1992; Sefton, 2002).
Acquired resistance is reflected in treatment failure of infections by
previously susceptible micro-organisms. Sometimes, multiple resistance is
encountered when micro-organisms are simultaneously resistant to several
antimicrobial agents. Klebsiella and other bacteria like Enterobacter, Serratia,
Salmonella species and Escherichia coli are the major reservoirs of resistance
among the Gram-negative bacteria (Brun-Buisson et al., 1987; Guiney, 1984).
26
Infections caused by resistant Klebsiella are widespread and multiple drug
resistance is common (Traub et al., 2000; Yuan et al., 2000; Livermore and Yuan,
1994). Hable et al. (1972) reported an incidence of Klebsiella pneumoniae type
33 septicaemia in an infant intensive care unit in which isolates were resistant to
ampicillin and kanamycin. Thomas et al. (1977) reported a wave of infections of
multiple antibiotic resistant Serratia marcescens followed in its wake by similarly
resistant Klebsiella pneumoniae.
At the intensive care unit of the Kenyatta
National Hospital, Nairobi, Muthotho et al., (1990) reported an endemic strain of
Klebsiella ozaenae resistant to ampicillin, co-trimoxazole, streptomycin,
sulphamethoxazole, gentamicin, chloramphenicol and amoxycillin+ clavulanic
acid, which were antimicrobial agents used frequently in the hospital.
Scheel and Invarsen (1991) reported an increased prevalence of multiple
drug resistant Klebsiella strains in 1989 than in 1985. They also reported a
decrease in the prevalence of Proteus mirabilis over the same period. Hospital
outbreaks of multidrug-resistant Klebsiella pneumoniae, especially those in
neonatal wards, are often caused by strains producing the extended-spectrumbeta-lactamases (ESBLs).
The gastrointestinal tract is the major reservoir of bacteria that cause
systemic infections in the neonates or immuno-compromised children. Burman et
al., (1992) reported that about 16% of Klebsiella faecal isolates from infants in
intensive care units carried SHV-1 -lactamase. The presence of the -lactamase
producing bacteria in the gut would thus present a treatment dilemma if these
strains caused an infection. Klebsiella species have been reported to be resistant
27
to a number of antimicrobial agents including the aminoglycosides (Akindele and
Rotilu, 1997; Jacoby et al., 1990), the -lactam antibiotics (Papanicolaou et al.,
1990; Siu et al., 1999; Yuan et al., 2000), the quinolones (Paterson et al., 2000),
sulphamethoxazole-trimethoprim, (Ananthan and Alavandi, 1999), tetracycline
(Omari et al., 1997) and others (Chevalier et al., 2000).
1.2.9 Cephalosporins
Cephalosporin compounds were first isolated from
cultures of
Cephalosporium acremonium from a sewer in Sardinia in 1948 by Italian scientist
Giuseppe Brotzu. He noticed that these cultures produced substances that were
effective against Salmonella typhi, the cause of typhoid. Researchers at the Sir
William Dunn School of Pathology at the University of Oxford isolated
cephalosporin C, which had stability to β-lactamases but was not sufficiently
potent for clinical use.
The cephalosporin nucleus, 7-aminocephlosporanic acid (7-ACA), was
derived from cephalosporin C and proved to be analogous to the penicillin
nucleus 6-aminopenicillanic acid. Modification of the 7-ACA side-chains resulted
in the development of useful antibiotic agents, and the first agent cephalothin
(cefalotin) was launched by Eli Lilly in 1964. Consequently, this led to the
creation of multiple classes of cephalosporins that differed in spectrum, potency,
β-lactamase stability and pharmacokinetic properties (Williams and Lemke,
2002).
28
Where R1 and R2 represent the side chains.
1.2.9.1 Third generation cephalosporins
These agents possess the same mechanism of action as the previous two
generations of cephalosporins and β-lactams, in general. The reactive portion of
the molecule is the carbonyl carbon on the lactam ring. Penicillin binding proteins
attack this portion of the molecule and form a very stable intermediate due to
tautomerisation and steric hinderance, which prevents a nucleophile from
attacking and displacing the penicillin binding protein (Williams and Lemke,
2002).
The third generation cephalosporins have two R-group substitutions that
can be placed on them. The R1- group is at the number seven position on the
lactam ring, and the R2-group is at the number three position on the
dihydrothiazine ring. Modifications at the R1 position will affect such parameters
as stability, spectrum and resistance to β-lactamases. Modifications at the R2
position will influence the pharmacokinetic properties of the cephalosporin, such
as the duration of action and potency (Williams and Lemke, 2002).
29
1.2.9.2 Resistance to third generation cephalosporins
The third generation cephalosporins have the reputation for being useful
against a broad range of bacterial infections. However, resistance to these agents
is something that must still be considered and creates obstacles for their clinical
use. As of now, the two main mechanisms of resistance to the third generation
agents are altered bacterial penicillin binding proteins and certain species of βlactamases that are capable of hydrolyzing the lactam ring. Alterations in
penicillin binding proteins, in particular 1A and 2X, result in cephalosporins
binding these proteins less effectively. Consequently, peptidoglycan cross-linking
is not inhibited to such a great extent, and bacterial cell lysis is inhibited. Even
though this type of resistance is known to occur, hydrolysis by β-lactamases is a
much more common (Collatz et. al., 1984; Lakaye et al., 1999).).
The third generation agents are more resistant to Gram-negative βlactamases than both the first and second-generation cephalosporins. However,
they have the distinct ability to induce the production of chromosomally encoded,
type I, β-lactamases in aerobic Gram-negative bacteria. Consequently, using third
generation agents to treat these types of infections can result in the bacterial
infections becoming resistant to all third generation agents (Hardman et al.,
2001). Researchers have recognized resistance to the third generation
cephalosporins and, consequently, have made efforts to combat this problem.
Thus far, the most prevalent effort has been the launch of the fourth generation
cephalosporins (Cosgrove et al., 2002).
30
1.2.10 Extended spectrum β-lactamase enzymes (ESBL)
ESBLs are modified beta-lactamase enzymes mainly derived from the
ubiquitous TEM1/2, SHV-1 and CTX-M plasmid-mediated enzymes, which
hydrolyse expanded spectrum cephalosporins to varying degrees. ESBLs are
widespread all over the world, but the prevalence and phenotypic characteristics
among clinical isolates may vary between geographical areas. Production of
plasmid-mediated extended-spectrum β-lactamases (ESBLs) has emerged as an
important mechanism of resistance to β-lactam antibiotics among Klebsiella
pneumoniae. Plasmid mediated -lactamases among the Enterobacteriaceae are
reportedly encountered most frequently in Escherichia coli and Klebsiella species
whereas chromosomally mediated enzymes predominate in the Proteus and
Enterobacter species (Bellon and Mouton, 1992; Nagy et al., 1998; Araque and
Rivera, 2004)).
Klebsiella pneumoniae is a successful opportunistic pathogen and has
been associated with various ailments such as urinary tract infections,
septicaemia, respiratory tract infections and diarrhoea. Resistance of this species
to third generation cephalosporins such as oxyimino β-lactams was first described
in 1980 and since then a linear increase in resistance has occurred. The resistant
strains gain their resistance by producing Extended-spectrum β-lactamases
(ESBLs) which are class A enzymes. ESBLs are the derivatives of common βlactamases (TEM and SHV β-lactamases) that have undergone one or more amino
acid substitutions near the active site of the enzyme, thus increasing their affinity
31
and the hydrolytic activity against third generation cephalosporins and
monobactams (Sirot et al., 1987; Jacoby and Archer, 1991).
CTX-M-group of extended-spectrum -lactamases (ESBLs) represents a
rapidly emerging problem in many countries. Extensive use of newer generation
cephalosporins has been the strong factor for the evolution of newer β-lactamases
such as ESBLs. ESBLs are encoded by transferable conjugative plasmids, which
often code resistance determinants to other antimicrobial agents such as
aminoglycosides.
These
conjugative
plasmids
are
responsible
for
the
dissemination of resistance to other members of gram negative bacteria in
hospitals and in the community (Knoth et al., 1983; Phillipon et al., 1989).
ESBL are distinguished into more than 30 types based on their physical properties
and all are inhibited by clavulanate, sulbactam and tazobactam, a property which
has been used to detect them in the laboratory (Livermore, 1993).
ESBLs are more prevalent in Klebsiella pneumoniae than in any other
enterobacteria species, and outbreaks of infections caused by ESBL producing
strains have been reported widely. ESBL producing strains are probably more
prevalent than currently recognized because they are often undetected by routine
susceptibility testing methods. Occurrence of ESBL producing Klebsiella species
has also been reported from South India and Central India (Hansoita et al., 1997).
Recent reports have highlighted the emergence of ESBL producing strains
endowed with an extremely wide spectrum of antibiotic resistance, including
resistance to trimethoprim, amikacin, streptomycin and gentamicin (Laura et al.,
2000).
32
Due to the extensive spread of multidrug resistant ESBL producing
strains,
there
has
been
renewed
interest
in
Klebsiella
infections.
ESBL producing Klebsiella pneumoniae were first reported in 1983 from
Germany and since then a steady increase in resistance against cephalosporins has
been seen. ESBLs are encoded by transferable conjugative plasmids which also
quite often code resistant determinants to other antibiotics (Bauernfieind et al.,
1989). An ESBL variant may be selected de novo in a given hospital or it may be
introduced from another centre. Its further spread within the hospital can be
consequence of plasmid transmission. Persistence and outbreaks of ESBL
producers have been convincingly correlated with extensive use of cephalosporins
(Sirot et al., 1991).
The plasmid mediated resistance against cephalosporins can be spread
among related and unrelated gram negative bacteria. Klebsiella pneumoniae is an
important cause of nosocomial infection and infections due to ESBL producing
Klebsiella pneumoniae are of concern as third generation cephalosporins (3GC)
are commonly used for treatment of infections due to gram negative organisms.
These infections are difficult to control as they are usually associated with
resistance to aminoglycosides (Ananthakrishnan et al., 2000).
1.2.10.1 Detection of Extended spectrum β-lactamases production
Detection of ESBL producers’ poses a special challenge for clinical
microbiology laboratories, although ESBL producers are able to hydrolyze
extended-spectrum penicillins, cephalosporins, and aztreonam, the minimum
33
inhibitory concentrations of some and perhaps even all of these agents may be
within the susceptible range.
The production of extended-spectrum β-lactamases (ESBL) has been
documented since the introduction of third-generation cephalosporins (3GCs) into
clinical usage (Bush and Sykes, 1986). Plasmid-mediated or hyperproduction of
AmpC-type and other β-lactamases have been the most common cause of
resistance to 3GCs (Philippon et al., 1989). Currently, the ESBLs associated with
3GC-resistant Enterobacteriaceae can be divided into the ‘big three’ families of
TEM, SHV and CTX-M-type β-lactamases. TEM and SHV variants are reliant on
key amino acid substitutions to increase their substrate profile to include the
3GCs, whereas the CTX-Ms have an intrinsic extended-spectrum profile. There
has been an emergence and global dissemination of the CTX-M-type βlactamases, which have become the predominant ESBL type in a number of Asian
and South American countries. The other mechanism causing resistance to the
extended spectrum β–lactams is the production of high levels of AmpC betalactamases. AmpC β-lactamases are usually chromosomally encoded in organisms
such as Citrobacter and Enterobacter species (Gniadkowski, 2001). However,
there has been an isolation of Escherichia coli and Klebsiella resistant to third
generation cephalosporins with characteristics of ampC β-lactamases. AmpC
ESBL may be distinguished from TEM and SHV-type ESBLs using the Double
Disk test plus cefoxitin. In contrast to the TEM and SHV ESBLs, ampC βlactamases are not inactivated by clavulanic acid, sulbactam or tazobactam. In
addition, organisms with high level ampC production are typically resistant to
34
cefoxitin (Jacoby and Han, 1996; Jacoby and Medeiores, 1991). The study aimed
at detecting the presence of ESBL-producing Klebsiella pneumoniae within the
clinical isolates and to characterize the molecular type of CTX-M, SHV and TEM
ESBLs present in this setting.
A study which was done in Chennai, in the University of Madras showed
that the incidence of ESBL producing strains among clinical Klebsiella isolates
has been steadily increasing over the past years and accounts for 6 to 17% of all
nosocomial urinary tract infections. The same study showed that the emergence of
the multiple resistant Klebsiella strains is unfortunately accompanied by a
relatively high stability of the plasmids encoding ESBLs (Subha and Ananthan,
2002).
The detection rate of ESBL producing Klebsiella isolates in stool samples
ranges from 5% to 38%, while rates in the nasopharynx ranges from 1% to 6%
(Podschun and Ullmann, 1998). Conjugative dissemination of ESBL coding
plasmids might facilitate the spread of antibiotic resistance among different
members of Enterobacteria.
1.2.11
Bacterial conjugation
Bacteria without resistance-encoding genes can acquire them from other
bacteria through the processes of conjugation, transformation, transduction or
cell-cell fusion (Bennet and Howe, 1990). Conjugation involves cell to cell
contact and active passage of DNA directly from one bacterial cell to another. It is
35
far more important in the horizontal transfer of genes in bacteria than
transformation or transduction (Bennet and Howe, 1990).
Bacterial conjugation is the transfer of genetic material, which in this case
could be resistance, between bacteria through cell-to-cell contact (as opposed to
transformation or transfection). Conjugation is mediated by plasmids which are
extrachromosomal DNA elements capable of autonomous or semi-autonomous
replication (Novick, 1980).
Bacterial conjugation is often regarded as the bacterial equivalent of
sexual reproduction or mating. However, it is not actually sexual, as it does not
involve the fusing of gametes and the creation of a zygote. It is merely the transfer
of genetic information from a donor cell to a recipient. Such genetic information
could be the antibiotic resistance which could be transferred from a donor cell to a
recipient. In order to perform conjugation, one of the bacteria, the donor, must
play host to a conjugative or mobilizable genetic element, most often a
conjugative plasmid. Most conjugative plasmids have systems ensuring that the
recipient cell does not already contain a similar element.
These elements are best viewed as genetic parasites on the bacterium and
conjugation as a mechanism evolved by the element to spread itself into new
hosts. The prototype for conjugative plasmids is the F-plasmid, also called the Ffactor. The F-plasmid is an episome (a plasmid that can integrate itself into the
bacterial chromosome by genetic recombination of about 100 kb length).
Plasmids carry only a fraction of bacterial genes that are not essential for the
36
survival of the bacterium in its natural environment but encode a multiplicity of
accessory traits that may provide their host with selective advantage under
unfavourable conditions (Novick, 1980; Timmis et al., 1986).
1.2.12 Rationale of the Study
The wide spread use of antibiotics in hospitals has led to emergence of
multidrug resistant organisms of low virulence like Klebsiella causing serious
opportunistic infections. Over the last 15 years numerous outbreaks of infection
with organisms producing extended spectrum β-lactamases (ESBLs) have been
observed world wide. The advent of ESBL producers has posed a great threat to
the use of many classes of antibiotics particularly cephalosporins. There are
indications that poor outcome occurs when patients with serious infections due to
ESBL producing organisms are treated with antibiotics to which the organism is
resistant.
The real challenge is the ESBL producing organism for which minimum
inhibitory concentrations of third generation cephalosporins is in the susceptible
range and they may not be truly susceptible when serious infections are
considered and these isolates may be reported susceptible. The role of third
generation cephalosporins in the treatment of Klebsiella pneumoniae infection is
limited as ESBL mediated resistance is on the increase. Recent reports have
highlighted the emergence of ESBL producing strains endowed with an extremely
wide spectrum of antibiotic resistance, including resistance to trimethoprim,
amikacin, streptomycin and gentamicin. Due to the extensive spread of multidrug
37
resistant ESBL producing strains, there has been renewed interest in Klebsiella
infections (Laura et al., 2000).
Several studies in Kenya have noted an increase in resistance to third
generation cephalosporins such as cefotaxime, ceftriaxone and ceftazidime in
their Klebsiella isolates. This has partly been attributed to the production of
Extended spectrum β-lactamase enzymes by some of these bacteria. For instance,
the reported existence of an extended-spectrum β-lactamase producing Klebsiella
pneumoniae at the Kenyatta National Hospital (Kariuki et al., 2001) and the
observation of resistance to ceftriaxone. The alarming situation with global
dissemination
of
CTX-M-producing
strains
urges
the
need
for
their
epidemiological monitoring, studying resistance mechanisms and also ensuring
prudent use of third generation cephalosporins for treatment of serious infections.
The emergence of extended spectrum β–lactamase strains as potential pathogens
requires careful screening to ensure accurate identification of these organisms and
appropriate reporting of resistance to the physicians who are prescribing treatment
for these patients.
A recent increase in multidrug resistant gram-negative bacilli, particularly
ESBLs is of great concern. The association between emergent ESBL-mediated
infections and 3GC use emphasizes the importance of better describing 3GC drug
utilization to best optimize their use. However, small amount of data is available
in this regard from developing countries. The extensive use of third generation
cephalosporin antibiotics has caused the emergence of extended spectrum beta-
38
lactamases in Gram-negative bacteria worldwide. More third generation
cephalosporins are being widely used in hospitals for empirical and prophylactic
therapy, and as their use extends across the board, more organisms will develop
resistance to them presenting the threat of antimicrobial ineffectiveness in life
threatening infections (Grave et al., 1999).
The increasing resistance to the third generation cephalosporins
accompanied by an increasing cost burden has raised concerns about the
detection, prevalence, and clinical implications of infections with Klebsiella
species. An important source of this resistance results from the production of
extended-spectrum β-lactamases (ESBLs) by bacteria. Many of these βlactamases result in resistance to 3GCs in Enterobacteriaceae.
1.3 HYPOTHESES
1.3.1 NULL HYPOTHESIS (H0)
Extended spectrum β-lactamase enzymes do not contribute to Klebsiella
pneumoniae resistance to third generation cephalosporins.
1.3.2 ALTERNATIVE HYPOTHESIS (HA)
Extended spectrum β-lactamase enzymes contribute to Klebsiella pneumoniae
resistance to third generation cephalosporins.
39
1.4
OBJECTIVES OF THE STUDY
1.4.1 GENERAL OBJECTIVE
The general objective of the study was to determine the occurance of extended
spectrum β-lactamases (ESBLs) among third generation cephalosporins resistant
Klebsiella pnuemoniae isolates.
1.4.2 SPECIFIC OBJECTIVES
The specific objectives of the study were:
1. To determine antimicrobial susceptibility of the Klebsiella pneumoniae
isolates to commonly available antibiotics and to third generation
cephalosporins (3GC).
2. To detect ESBL production using Double Disc Diffusion Synergy Test
(DDST) and phenotypic confirmatory disc diffusion test (PCDDT).
3. To detect and characterise CTX-M, SHV and TEM β-lactamase genes.
4. To investigate possible conjugal transfer of third generation cephalosporin
resistance from Klebsiella pneumoniae to Escherichia coli.
40
CHAPTER TWO
2.0 MATERIALS AND METHODS
2.1 MATERIALS
2.1.1 Reagents and enzymes
Phenol: Chloroform: Isoamyl alcohol 25:24:1 (Molecular Biology
Reagent), Sodium Dodecyl Sulphate, Tris. HCl, 10xTris EDTA buffer were from
Sigma Chemical Company Ltd, St. Louis MO, USA. Ethanol (96%),
Hydrochloric acid and 18 M Sulphuric acid were analytical grade chemicals from
Fisher Scientific (UK) Ltd, Leicestershire, UK; Glucose and Glycerol were from
Rhone Polenc Ltd, Nairobi, Kenya; E test ESBL strips were from AB Biodisk,
Solna, Sweden; Bio-Stat, Stockport, UK.
2.1.2 Bacteriological media
Tryptone Soya Broth, Luria Bertani broth and Yeast extract were from
Oxoid Ltd, Basingstoke Hampshire, England; Triple Sugar Iron media, Blood
agar, Mac Conkey media and Mueller Hinton agar were from Hi Media
laboratories, India.
2.1.3 Glassware and other consumables
Adatab tablets were from Mast Laboratories Ltd, Merseyside, England;
Agarose for Molecular Biology analysis, Ethidium bromide, and polypropylene
micro-centrifuge tubes (1.5 ml ) were from Sigma Chemical Company Ltd, St.
Louis MO, USA; Sensitivity testing disks were from Hi-Media laboratories
41
Limited, India; The conical flasks which were used, the 13x100 mm tubes, and
universal bottles ( Pyrex), were from Bibby Science Products Ltd., UK; API 20 E
Kit was from La Balme les Grotles, Montalieu, Vercieu, France; Micropipette
tips (Finnpipette) were from Labsystems, Pulttitie, Helsinki, Finland; Disposable
Petri dishes, Disposable pipettes of the following volumes;- 1 ml, 2 ml, 5 ml, 10
ml, 20 ml were from Bibby Sterilin, Ltd, UK; Cryo vials (2 ml) were from
Nagene Products, Nalge Company Rochester, New York, USA; Polaroid Type
667 black and white film was from Polaroid Corp., Cambridge, MA, USA.
2.1.4 Equipment
Refrigerated Micro-centrifuge, Model MP50 was from Tomy Seiko Co
Ltd, Tokyo, Japan; Shaking water bath type 1468 was from Kemoto Chemical
Industries Co Ltd, Tokyo, Japan; The shaking orbital incubator was from
GallenKamp & Company Ltd, England; Ultra violet trans-illuminator model TL
33 was from Ultra violet Products Inc., San Gabriel, California, USA;
Micropipettes, 10 µl, 20 µl, 50 µl, 100 µl, 200 µl, 1000 µl, (Finnpipette) were
from Lab Systems , Pulttitie, Helsinki, Finland; Polaroid MP4 Land Camera was
from Polaroid, Cambridge, Massachusetts, USA; Ultra violet trans-illuminator
model TL 33 was from Ultra violet Products Inc., San Gabriel, California, USA;
2.1.5 Reference bacterial strains
Escherichia coli strains K-12F-, 39R861, ATCC 25922 and V517 were
kindly donated by Dr. Kariuki of the Kenya Medical Research Institute/Wellcome
Trust Research Laboratory, Nairobi.
42
2.1.6 Laboratory safety of the investigators
Generally, patient specimens pose a great risk to the laboratory workers
than microorganisms in culture. This is because the nature of the etiologic agents
in the patient specimen is initially unknown. Therefore, all the investigators who
were involved were advised to wear gloves and laboratory coats while handling
the samples as a precaution to minimise the risks of infections to the investigators.
All specimens of blood and body fluids were only put in containers which had
secure leads to prevent leakage during transportation. The investigators were
reminded to disinfect the laboratory bench surface working areas before and after
working using 70% alcohol.
Sharp objects, including scapels and needles were put in the sharps’ containers as
a safety measure. These sharps’ containers would then be incinerated once they
were full.
2.2 METHODS
2.2.1 Clinical isolates from a previous study
A total of 80 multi drug resistant clinical isolates of Klebsiella
pneumoniae were used in the study. The bacteria isolates were obtained from a
previous study which was carried out on anaerobes associated with pelvic
inflammatory disease (P.I.D), KEMRI S.S.C No.495 and the principal
investigator of that study was Dr.Craig Cohen. The study was cleared by both the
KEMRI Scientific Steering Committee (SSC) and the National Ethical Review
Committee. The current study was also cleared by the two committees above and
43
assigned SSC No.1171. The isolates were identified based on colony morphology
on blood agar, MacConkey agar and by standard biochemical tests. As many of
these isolates as could be revived from storage were identified. However, since
the identified stored isolates were 32, while my minimum sample size was 79,
then additional isolates had to be sought in order to obtain the minimum sample
size number. These 48 additional isolates were obtained from the Kenyatta
National Hospital medical microbiology laboratory samples.
2.2.2 Isolation and Identification of Klebsiella pneumoniae from the hospital
laboratory samples.
Isolations were done by routine laboratory diagnosis of patients with
pneumonia, Urinary Tract Infections, burns and wound infections. Urine, blood,
cerebrospinal fluid, sputum, stool and pus, high vaginal and throat swabs were
examined for the presence of Klebsiella species at the routine Microbiology
Laboratory of the Department of Medical Microbiology, University of Nairobi at
the Kenyatta National Hospital.
An isolate was identified as a member of the genus Klebsiella according to
the guidelines of Orskov (1984) and Ewing (1986) as the described in section
1.2.1 of the literature review and also by using the following tests: ability to
ferment inositol, utilization of citrate as the sole source of carbon, the methyl red
reaction, the Voges-Proskaeur reaction and growth characteristics on triple sugar
iron (TSI) medium. TSI medium was used for testing of hydrogen sulphate
production. TSI also contains Phenol red which acts as an acid-base indicator.
44
These biochemical tests were done alongside the use of API 20 E kit for
identification of Klebsiella pneumoniae subspecies pneumoniae.
2.2.3 Minimum sample size determination
In Asia, the percentage of β-lactam resistance due to ESBL production in
Klebsiella pneumoniae remains very low. For instance, the percentage of ESBL
production in Klebsiellae pneumoniae varies, from 8% in Korea to 8.5% in
Taiwan and up to 12% in Hong Kong (Pai et al., 1999). However, the lowest
ESBL production that has been documented amongst Klebsiella pneumoniae in
similar studies done is 6.67% in Chennai, India. The study was done in the
department of microbiology at the University of Madras. Whereas the highest
ESBL production that has been documented among Klebsiella pneumoniae in
similar studies is 52% in Tel Aviv, Israel in the year 2002 (Subha and Ananthan,
2002).
Therefore after taking the average value of the highest and the lowest
documented ESBL production ranges, that is (6.67%+52%) 1/2 . The value
obtained was the estimated prevalence of this characteristic (p) which in this case
is the ESBL production among Klebsiella pneumoniae. Therefore, p is:
(6.67+52)×½ = 29.335%
Hence p is approximately 29%
The minimum sample size was determined using the following mathematical
formula.
N≥
Z2 α/2 × P (1-P)
δ2
45
Whereby;
N is the minimum sample size
P is the estimated prevalence of the characteristic
δ2 is the degree of precision, which is ± 10%
α is the level of significance
Z is the standard normal deviate
P =0.29
δ2 = (0.1)2
α = 5%
Confidence level = 95%
Therefore, N ≥
(1.96)2 × 0.29(0.71)
= 79.09854
(0.1)2
N≥ 79
Hence, the minimum sample size was 79
2.2.4 Disk diffusion tests
The antibiotic sensitivity testing was performed by disk diffusion
technique with commercially available disks on Mueller Hinton agar plates. The
disks which were used were amikacin (30μg), ampicilin (10 µg), aztreonam (10
µg), cefepime (30μg), cefoxitin (30μg), chloramphenicol (10 µg), ciprofloxacin (5
µg), cotrimoxazole (30μg), gentamicin (10 µg), imipinem (10 µg), kanamycin (10
µg), augmentin®, piperracillin (10 µg), streptomycin (10 µg), sulphamethoxazole
(30μg), tetracycline (10 µg) and amoxycillin (10 µg). For sensitivity to third
46
generation cephalosporins, cefotaxime, ceftriaxone and ceftazidime each 30 µg
disk were used. The diameter of the zone of inhibition for each antibiotic was
measured and interpreted as resistant, intermediate susceptible or susceptible
according to NCCLS criteria (2005).
2.2.5 Preparation of agar plates
The agar medium for disk diffusion tests which was Mueller Hinton was
prepared by suspending 31.4 g of the agar in a litre of distilled water in a conical
flask and the flask plugged with cotton wool. The agar was dissolved by boiling
over a bunsen burner flame with constant swirling of the conical flask.
The agar was then sterilized by autoclaving at 121oC for 15 minutes. The sterile
agar was let to cool to 56oC in a water bath and then using an automatic pipette,
20 ml of this agar was transferred aseptically into a 90 ml disposable Petri dish
placed on the bench top and left to set into a firm gel. The set plates were stored at
4oC. The agar plates were dried by holding them in a slanting position over the
lids at 37oC for 10 minutes in an incubator. The dried plates were then inoculated
with the bacterial isolates to be tested.
2.2.6 Preparation of McFarland nephelometer
A McFarland nephelometer was used to adjust the turbidity of bacteria
suspensions to predetermined levels. For bacterial susceptibility tests the 0.5
McFarland turbidity standard corresponding to approximately 106 colony forming
units per millilitre of bacterial cell suspension was used. The 0.5 McFarland
47
turbidity standard was prepared by adding 0.5 ml of a solution of 0.048 M BaCl 2
(1.175% w/v BaCl2.2H20) to 99.5 ml of 0.18 M H2SO4 (1% v/v H2SO4). A 2
McFarland standard was prepared by adding 2 ml of a solution of 0.048 M BaCl2
(1.175% w/v BaCl2. 2H20) to 98 ml of 0.18M H2SO4 (1% v/v H2SO4). The
standards were kept in tubes wrapped in aluminium foil to protect them from light
and stored in the dark.
2.2.7 Preparation of inoculum
Frozen stocks of the isolates were removed from the freezer at -80oC by
picking the beads using a straight wire loop. The isolates recovered on
MacConkey agar by carefully rolling the beads on the agar plates. The frozen
stocks which were not stored in beads were recovered by picking using an
inoculating wire loop and were spread over the surface of the agar using the
inoculating wire loop. The agar was then incubated at 37oC overnight and the
frozen stock was returned to the freezer.
The next day, a sterile wire loop was used to transfer bacterial cells from
about five colonies into 4 ml of sterile Tryptone Soy Broth in a universal bottle.
Sterile saline was added into the universal bottle to produce turbidity which
looked similar to a 0.5 McFarland turbidity standard. A sterile cotton swab was
then dipped into the adjusted cell suspension and excess fluid removed by
pressing and rotating the swab against the inside wall of the universal bottle. The
swab was then used to uniformly inoculate the entire surface of Mueller Hinton
agar plate. The agar plates were then allowed to dry for 3 minutes and disks of
48
antimicrobial agents applied onto the agar surface using sterile forceps. The disks
were then pressed down slightly to ensure contact with the agar surface. The
plates were incubated at 37oC for 18 hours. The diameters of the zones of
inhibition were measured by use of a ruler. The measurements of the zone sizes in
terms of millimetres were used to group the isolates as susceptible, intermediate
or resistant according to the guidelines of the National Committee for Clinical
Laboratory Standards (2005).
2.2.8 Minimum Inhibitory Concentration (MIC) determination
An inoculum from a 24 hour culture was suspended in 5 ml of tryptic soy
broth and adjusted to match a 0.5 McFarland turbidometric standard.
After proper turbidity was achieved, a new sterile cotton swab was submerged in
the suspension, lifted out of the broth, and the excess fluid was removed by
pressing and rotating the swab against the wall of the tube. The swab was then
used to inoculate the entire surface of the Mueller Hinton agar plate three times,
rotating the plate 60 degrees between each inoculation. The inoculum was then
allowed to dry for a few minutes and then the E-test strips were placed on the
plates.
The E-test strips were placed on the agar with sterile forceps and tapped
gently to ensure the adherence to the agar. The plates containing the strips were
incubated at 37oC for 16 to 20 hours in an inverted position in a 5% CO2
incubator. The MIC was read at the point where the zone of inhibition intersected
the antimicrobial agent concentration scale on the strip. The antimicrobial agents
49
whose MIC values were determined were, augmentin, amikacin, ampicillin,
cefoxitin,
ciprofloxacin,
chloramphenicol,
co-trimoxazole,
gentamicin,
kanamycin, streptomycin, sulphamethoxazole, tetracycline and trimethoprim.
Two independent observers (Mutharia and Alice) interpreted the E-test
MICs. While a specific blinding protocol was not used, neither observer had
knowledge of the other’s measurements prior to making his or her own MIC
determinations. Discrepancies were resolved by consensus.
2.2.9 Phenotypic confirmatory disc diffusion test (PCDDT) for
ESBL production.
This test required use of both cefotaxime (30 µg) and ceftazidime (30 µg)
disk alone and in combination with clavulanic acid (30 µg). Disks of ceftazidime
and cefotaxime with clavulanic acid (30 µg/10 µg) were prepared using a stock
solution of clavulanic acid at 1000 µg/mL. Ten microlitres of clavulanic acid
solution was added to these discs within an hour before the discs were applied to
the plates.
A suspension of the isolates was prepared in tryptic soy broth and was
adjusted to match the 0.5 McFarland standard. A sterile cotton swab was used to
spread the homogenous solution of the organisms on the surface of the Mueller
Hinton agar plates. The plates which had now been swabbed were allowed to dry
for a few minutes and the antibiotic disks were applied using forceps. The plates
were incubated at 37°C for 18 hours.
An increase in zone diameter for either antimicrobial agent tested in
combination with clavulanic acid versus its zone when tested alone was observed.
50
For ceftazidime an increase in zone diameter of more than 5mm and for
cefotaxime, an increase of more than 3 mm was considered as an ESBL producer.
E. coli ATCC 25922 and Klebsiella pneumoniae ATCC 700603 were used as
negative and positive controls for ESBL production, respectively.
2.2.10 Double Disc Synergy test (DDST)
Double Disk Synergy Test was also done to determine synergy between a
disk of augmentin® (20 µg amoxycillin and 10 µg clavulanic acid) and 30 µg disk
of each 3GC antibiotics. Mueller Hinton agar plates were prepared and inoculated
with standardized inoculum (0.5 McFarland tube). Thirty microgram disks of each
3GC antibiotics were placed using forceps on the agar at a distance of 15 mm
centre to centre from augmentin® disc. E. coli ATCC 25922 was used as the
negative control and Klebsiella pneumoniae ATCC 700603 was used as the
positive control for ESBL production. ESBL production was interpreted if the
inhibition zone around the test antibiotic disk increased towards the augmentin®
disk or if neither disks were inhibitory alone but bacterial growth was inhibited
where the two antibiotics diffused together.
2.2.11 Isolation of Plasmid DNA
This was done according to the procedure of Birnboim and Doly (1979)
and Ish-Horowicz and Burke (1981) with a few modifications as detailed below.
Two to three isolated colonies of Klebsiella pneomoniae isolates were picked
using a wire loop and inoculated into 2 ml of Luria Burteni broth. The suspension
51
was incubated on an orbital incubator at 370C for 18 hours. A volume of 1.5 ml of
the culture was transferred into a 2 ml eppendorf tube and centrifuged at 12,000
revolutions per minute (rpm) for 30 seconds at 40C in a high speed refrigerated
microcentrifuge. The supernatant was discarded and a fresh portion of 1.5 ml of
culture added and centrifuged again.
The supernatant was again discarded and the last portion of 1.5 ml of
culture material added and the process repeated, to provide bacterial cells from a
total of 4.5 ml broth culture. The medium was removed by aspiration leaving the
pellet as dry as possible.
The cell pellet was washed by adding 0.5 ml STE [0.1 M Sodium chloride,
100 mM Tris. HCl (pH 8.0), 1 mM EDTA (pH 8.0] and vortexing. The
suspension was again centrifuged at 14,000 rpm for two minutes at 40C in a high
speed refrigerated microcentrifuge.
The bacterial pellet was resuspended by vortexing in 100 µl of solution 1
containing 50 mM Tris HCl (pH 8.0), 10 mM EDTA (pH 8.0) and 50 mM
glucose.
To the suspension was added 200 µl of freshly prepared solution II (lysis
solution) containing 0.2 M NaOH and 1% w/v SDS at pH 12.55. The microfuge
tube was closed tightly and inverted rapidly 5 times until the suspension became
clear. The cells were further incubated in an ice bath for 3 to 5 minutes to ensure
complete lysis.
A volume of 150 µl of ice cold solution III containing 5 M potassium
acetate, glacial acetic acid and sterile distilled water was then added to the tube
52
and the mixture vortexed gently in an inverted position to ensure mixing of the
solution with the sticky cell mass. The mixture was stored for 1 hour on an ice
bath to effect precipitation of cell debris.
The mixture was then centrifuged at 12,000 rpm for 5 minutes at 4oC in a
microcentrifuge and the supernatant rich in plasmid DNA transferred into a fresh
microfuge tube. To the supernatant was added an equal volume of a mixture of
phenol: chloroform: isoamyl alcohol 25:24:1and mixed by inverting about 150
times until an emulsion formed. The emulsion was then centrifuged for 2 minutes
at 12,000 rpm at 4oC to separate the phases. The upper aqueous phase was then
removed into a fresh microfuge tube and two volumes of ice-cold absolute ethanol
added to precipitate out plasmid DNA and mixed by vortexing. The mixture was
then allowed to stand for two minutes at room temperature to separate plasmid
DNA.
DNA was recovered by centrifuging the mixture at 12,000 rpm for 5
minutes at 4oC in a microcentrifuge and the supernatant drained by gentle
aspiration taking care not to disturb the pellet of DNA. The tubes containing the
DNA were inverted over paper towels for one hour to drain off all fluids.
The pellet of double stranded DNA was then rinsed with 1ml of 70%
ethanol at 4°C and then centrifuged at 12,000 rpm for five minutes at 4°C. The
supernatant was carefully removed by gentle aspiration without disturbing the
pellet of DNA and the pellet of nuclei acid was allowed to dry in the air for ten
minutes.
53
The pellet of DNA was redissolved in 50 µl of TE buffer [10 mM
Tris.HCl(pH 8.0), 0.1mM EDTA] containing 20 µg/ml of deoxyribonuclease
(DNase) free ribonuclease (RNase). The mixture was then incubated at 37oC for
ten minutes to allow the RNase to act. The plasmid DNA was separated by
agarose gel electrophoresis. The remainder of the plasmid DNA was stored at 20oC until required for further analysis.
2.2.12 Agarose gel Electrophoresis
Agarose gels (1%w/v) was prepared by suspending 1.0 gram of agarose
powder in 100 ml of Tris borate EDTA (TBE) buffer (0.09 M Tris, 0.09 M boric
acid, 2.5 mM EDTA). The agarose was dissolved by bringing the suspension to
boil in a water bath. The agarose solution was then cooled to 55oC in a water bath
maintained at this temperature. Five microlitres of ethidium bromide was added in
to the agarose solution in order to stain it.
The cooled agarose solution was poured into a plastic electrophoresis tray
whose open end had been covered by placing the tray in a horizontal position such
that the open ends faced the walls of the electrophoresis tank and were thus
blocked. A comb was positioned into the agarose solution and then let to cool for
30 minutes on a flat bench top at room temperature.
The comb was removed and the electrophoresis tray containing the
horizontal slab of gel was placed in the proper position in the electrophoresis tank.
TBE (0.5%) buffer was added into the electrophoresis tank up to a level of about
2 mm above the wells in the gel. Samples of DNA were prepared for
54
electrophoresis by mixing 25 µl of DNA with 15 µl of loading buffer (0.25%
bromophenol blue, 0.25% xylene cyanol FF, 30% w/v glycerol in water) and then
loaded into the wells. Each gel run for the plasmids included a molecular weight
marker consisting of DNA from the Escherichia coli strain R39 (Plate 4). The gel
electrophoresis for the PCR products included the Quick-load™ 2-log DNA
Ladder as the molecular weight marker that had fragments ranging from 0.1-10 kb
(Plate 5).
Electrophoresis was done at 125 volts for 2.5 hours. Separated DNA was
visualised by placing the gel on an ultraviolet trans-illuminator, and photographed
using a Polaroid MP-4 Land Camera. Polaroid type 55 positive/negative instant
sheet film was used.
2.2.13 Conjugation
Escherichia coli K12F- (nalidixic acid resistant) was used as the recipient
of DNA donated by the Klebsiella pneumoniae isolates. Three millilitres of the
Luria-Bertani (LB) broth was dispensed in to bijou bottles and labelled. The
typical colonies of both donor and recipient were inoculated into respective bijou
bottles containing the 3 ml of Luria-Bertani (LB) broth and incubated at 37oC
overnight while shaking.
The donors and the recipient were sub cultured in 3 ml of the LB broth
and incubated while shaking at 37°C for three hours so as to get them in the log
phase of the bacterial growth. Four and a half millilitres of fresh and warm (37°C)
LB broth was dispensed in to universal bottles.
55
After the three hours of incubation, both the donors and the recipient were
diluted in 1:10 ratio. This was done by taking 0.5 ml of the subcultures into the
4.5 ml of the fresh and warm LB broth which had been dispensed in to universal
bottles.
The donor and the recipient were mixed in equal proportions of 2 ml of the
donor and 2 ml the recipient and these mixtures were prepared in duplicates. One
group of the mixtures was incubated at 37°C overnight and the other at room
temperature overnight. The two groups of bacterial cells which had been
separately incubated at 37°C and at room temperature overnight, were centrifuged
at 13,000 rpm for 1 minute in microcentrifuge. The pellet was washed twice with
sterile phosphate buffered saline by resuspending the cells using a vortexer.
Using a sterile loop, each of these mixtures was subcultured onto:
MacConkey agar plate containing 30 μg/ml cefotaxime and 30 μg/ml
i.
nalidixic acid, so as to select transconjugants.
ii.
MacConkey agar containing 30 μg/ml cefotaxime to inhibit growth of the
recipient strain.
iii.
MacConkey agar plate containing 30 μg/ml nalidixic acid to inhibit the
growth of the donor.
The plates were incubated at 37°C overnight and examined for
transconjugants.
Plasmid DNA was extracted from the transconjugants as described earlier in
section 2.2.11 for comparison with those from donor Klebsiella pneumoniae
isolates.
56
2.2.14 Detection of ESBL Genes By Polymerase Chain Reaction.
Bacterial cell lysates were used as templates in the specific PCR
amplifications for detection of the blaTEM, blaSHV and blaCTX-M genes. The
bacterial DNA in the cell lysate was prepared from overnight cultures grown on
solid medium (Luria-Bertani medium [LB]) by inoculating about two colonies
using a wire loop into 100 μl of double-distilled sterile water in sterile eppendorf
tubes. The colonies were mixed until a homogenous solution was obtained. The
solution was boiled at 95°C for 10 minutes, and then centrifuged at 12,000 rpm
for two minutes at 4°C. The supernatant was poured into fresh sterile labelled
eppendorf tubes. These bacterial cell lysates were used as templates in the specific
PCR amplifications for detection of the blaTEM, blaSHV and blaCTX-M genes. PCR
was performed with Taq DNA polymerase, according to the instructions of the
manufacturer, in the presence of 2 μl of the template DNA preparation in a total
volume of 50 μl. The PCR conditions were as follows: 15 min at 95°C and 35
cycles of 1 min at 94°C, 1 min at an annealing temperature designed for each
primer set, and 1 min at 72°C, followed by 10 min at 72°C. The resulting PCR
products were analysed and visualized in a 1.5% agarose gel electrophoresis with
ethidium bromide staining and UV light from the UV transmitter. The primers
described in Table 1 were used to determine whether the cell lysate could be
amplified.
57
TABLE 1
Primers used in PCR for detection of resistance genes.
Target(s)
Primer
Sequencea
Primer
length
CTX-M
CTXM-F
TTT GCG ATG TGC
AGT ACC AGT AA
CGA TAT CGT TGG
TGG TGC CAT A
TCG GCC TTC ACT
CAA GGA TG
ATG CCG CCG CCA
GTC ATA TC
TTA GAC GTC AGG
TGG CAC TT
GGA CCG GAG TTA
CCA ATG CT
23
Nucleotide
positions
(bp)b
205-227
21
727-748
20
44-64
20
991-1011
20
76-96
20
1065-1077
CTXM-R
SHV
SHV-F
SHV-R
TEM
TEM-F
TEM-R
KEY
a
b
is the Sequence of primer as synthesized 5′ to 3′.
is the Nucleotide position in base pairs for the GenBank accession number
sequence.
58
CHAPTER THREE
3.1 RESULTS
3.1.1 Disk Diffusion tests
The isolates had large, elevated and mucoid colonies on solid media such
as MacConkey (plate 1) after twenty four to fourty eight hours of incubation at
37°C. In this study, 80 Klebsiella pneumoniae isolates resistant to at least three of
the antibiotics tested were included. ESBL production was detected in 30 (37.5%)
isolates. Among the ESBL producers 16.7% isolates for cefotaxime, 10% for
ceftazidime and 13.3% for ceftriaxone were found to be sensitive by disc
diffusion test. All the isolates were found susceptible to imipenem (Table 2).
When the isolates were subjected to susceptibility testing, resistance to ampicillin
was the most prevalent among 70 (87.5%) isolates followed by resistance to
piperracillin.
3.1.2 MIC determination
The minimum inhibitory concentration of the antimicrobial agents was
assessed against 80 Klebsiella isolates using the E test strips as described under
materials and methods, section 2.2.8. The number of isolates resistant to each
antimicrobial agent was determined by considering the susceptibility of each of
the 80 isolates to individual antimicrobial agents. Table 3 shows the number of
isolates that were resistant to augmentin, amikacin, ampicillin, cefoxitin,
ciprofloxacin,
chloramphenicol,
co-trimoxazole,
gentamicin,
kanamycin,
streptomycin, sulphamethoxazole, tetracycline and trimethoprim. MIC50 and
MIC90 were considered an indication of the overall response of the 80 isolate to
59
each antimicrobial agent. The MIC50 and MIC90 were defined as the MICs of each
antimicrobial agent against 50% and 90%, respectively of isolates against which
the agents were tested.
TABLE 2:
Susceptibility profiles of all the Klebsiella pneumoniae isolates in the study
by the disk diffusion technique.
Antimicrobial
agent
Amikacin
Sensitive
n (%)
20(25)
Intermediate Resistant
n (%)
n (%)
4(5)
56(70)
Ampicilin
8(10)
2(2.5)
70(87.5)
Aztreonam
23(28.75)
7(8.75)
50(62.5)
Cefepime
49(61.25)
4(5)
27(33.75)
Cefoxitin
48(60)
4(5)
28(35)
Chloramphenicol
46(57.5)
6(7.5)
28(35)
Ciprofloxacin
38(47.5)
12(15)
30(37.5)
Cotrimoxazole
29(36.25)
7(8.75)
44(55)
Gentamicin
50(62.5)
8(10)
22(27.5)
Imipinem
80(100)
0
0
Kanamycin
40(50)
2(2.5)
38(47.5)
Augmentin®
65(81.25)
6(7.5)
9(11.25)
Piperracillin
9(11.25)
7(8.75)
64(80)
Streptomycin
46(57.5)
5(6.25)
29(36.25)
Sulfamethoxazole 50(62.5)
8(10)
22(27.5)
Tetracycline
3(3.75)
20(25)
57(71.25)
60
TABLE 3
Resistance of all the eighty isolates to individual antimicrobial agents using
the MIC data.
Antimicrobial agent
Number resistant
Number
n (%)
Susceptible n (%)
*
10 (12.5)
70 (87.5)
Amikacin
58 (73)
22 (27)
Amoxycillin
68 (85)
12 (15)
Ampicillin
70 (87.5)
10 (12.5)
Cefoxitin
30 (38)
50 (62)
Ciprofloxacin
36 (45)
44 (55)
Chloramphenicol
30 (38)
50 (62)
Co-trimoxazole
48 (60)
32 (40)
Gentamicin
25 (31)
55 (69)
Kanamycin
38 (48)
42 (52)
Streptomycin
32 (40)
48 (60)
Sulphamethoxazole
26 (33)
54 (67)
Tetracycline
21 (26)
59 (74)
Trimethoprim
46 (58)
34 (42)
Augmentin®
*Augmentin® consists of amoxicillin and clavulanic acid in the ratio 2:1.
Breakpoint MIC values were interpreted as recommended by the National
Committee for Clinical Laboratory Standards (2005). According to the MIC
results, resistance to trimethoprim and to co-trimoxazole was very close, that is,
58% and 60% respectively. The isolates were most susceptible to augmentin
(87.5%).
61
PLATE 1
The distinctive mucoid colonies of Klebsiella pneumoniae grown on
MacConkey agar.
3.1.3 Susceptibility to cefoxitin.
Although ESBLs derived from TEM and SHV b-lactamases do not
provide resistance to cefoxitin, Table 5 shows that 22 of the 30 ESBL-producing
isolates of Klebsiella pneumoniae were resistant to cefoxitin by disk diffusion
test. Among the isolates shown not to produce an ESBL, 8 of 50 isolates were
cefoxitin resistant, while none of 58 oxyimino-β-lactam-susceptible isolates was
resistant to cefoxitin.
62
TABLE 4
Sensitivity of ESBL non producer Klebsiella pneumoniae isolates to third
generation cephalosporins by disk diffusion method (n = 50)
Antimicrobial
agent
Cefotaxime
Sensitive
Intermediate
Resistant
11(22%)
15(30%)
24(48%)
Ceftazidime
5(10%)
13(26%)
32(64%)
Ceftriazone
12(24%)
11(22%)
27(54%)
3.1.4 Detection of ESBL production
Of the two methods which were used for detection of ESBL production in
this study, phenotypic confirmatory disc diffusion test was a more sensitive
procedure for detection of ESBL than the DDST. Twenty seven (90%) of the 30
ESBL producing strains were detected by DDST using cefotaxime, ceftriaxone
and ceftazidime antibiotics.
Using standard double disk synergy test (DDST) as screening method for
identifying potential ESBL producers, ceftriaxone was the most efficient
antimicrobial in screening isolates as potential ESBL producers followed by
cefotaxime. In this test, a disk containing amoxicillin-clavulanate (AMC) was
placed in proximity to disks containing ceftazidime, cefotaxime and ceftriaxone
antibiotics. The results showed that the clavulanate in the amoxicillin-clavulanate
disk diffused through the agar and inhibited the β-lactamase surrounding the
cefotaxime and ceftriaxone antibiotics disks (Plate 3). Enhancement of the
63
inhibition zone of any of the third generation cephalosporins tested, on the side
facing the amoxicillin-clavulanate disk was interpreted as a positive test.
Ceftriaxone missed three ESBL harbouring isolates. Cefotaxime and
aztreonam missed seven and nine ESBL harbouring isolates respectively.
Ceftazidime was the least efficient among these four antimicrobials missing 14
isolates that were later identified as harbouring ESBLs.
While using the Phenotypic confirmatory disc diffusion test (PCDDT)
method, each of the isolate was tested for susceptibility by the disk diffusion
method with 30 μg disks of cefotaxime, ceftriazone or ceftazidime, with and
without 10 μg of Clavulinic acid. Clavulinic acid enhancement of the diameter of
the zone of inhibition around any oxyimino-β-lactam disk by at least 5 mm was
taken as presumptive evidence for the presence of an ESBL on the basis of
enhancement observed with strains proven to transfer ceftazidime resistance
(plate 2). Maximal clavulinic acid enhancement was found with the aztreonam
disk in 25 isolates, with the ceftazidime disk in 21 isolates, and with the
cefotaxime disk in 19 isolates.
Thirty isolates had a ≥5-mm enhancement of susceptibility in the presence
of the β-lactamase inhibitor, while no susceptible control strains showed a ≥5-mm
clavulinic acid effect. With a 5-mm breakpoint for clavulinic acid enhancement,
30 of the 80 isolates of Klebsiellae pneumoniae were designated ESBL producers.
When the remaining isolates were tested for the ability to transfer ceftazidime
resistance by conjugation to E. coli K12, one additional ESBL-producing
Klebsiella pneumoniae isolates was detected.
64
PLATE. 2.
Phenotypic confirmatory disc diffusion method for ESBL detection showing
the response of one of the ESBL-producing isolates with disks containing 30
μg of ceftazidime (left) and 30 μg of ceftazidime plus 10 μg of clavulanic acid
(right).
PLATE 3.
Double-disk diffusion test for detection of ESBL production by the isolates
showing 30 μg disks of 3GC antibiotics placed at a distance of 15 mm centre
to centre from augmentin® disk.
Inhibition zone of the
amoxicillin-clavulanate disk.
Inhibition zones of Third
generation cephalosporins’
disks.
65
3.1.5 Susceptibility of ESBL producers to 30 μg third generation
cephalosporin disks.
The interpretation of the susceptibility profiles of 30 μg cefotaxime,
ceftazidime and ceftriaxone disks are shown in Table. 4 for the thirty ESBLproducing isolates of Klebsiella pneumoniae. The distribution of zone diameters
did not differ between strains able or not able to transfer ESBL production.
It is evident that many ESBL producing isolates appeared to be susceptible by
disk diffusion.
TABLE 5
Sensitivity of ESBL producing Klebsiella pneumoniae to third generation
cephalosporins by disc diffusion method (n = 30)
Antimicrobial
Sensitive
Intermediate
Resistant
Cefotaxime
5(16.7%)
7(23.3%)
18(60%)
Ceftazidime
3(10%)
3(10%)
24(80%)
Ceftriazone
4(13.3%)
8(26.7%)
18(60%)
agent
66
TABLE 6
Distribution of ESBL-producing isolates in susceptibility categories by disk
diffusion.
Drug
Sensitive
Intermediate
Resistant
n (%)
n (%)
n (%)
Amikacin
9(30%)
3(10%)
18(60%)
Ampicilin
8(26.7)
6(20%)
16(53.3%)
Augmentin®
0
0
30(100)
Aztreonam
10(33.3%)
9(30%)
11(36.7%)
Cefepime
17(56.7%)
5(16.7%)
8(26.6%)
Cefoxitin
5(16.7%)
3(10%)
22(73.3%)
Chloramphenicol
13(43.3%)
3(10%)
14(46.7%)
Ciprofloxacin
17(56.7%)
3(10%)
10(33%)
Cotrimoxazole
8(26.6%)
4(13.3%)
18(60%)
Gentamicin
9(30%)
2(6.7%)
19(63.3%)
Imipinem
30(100%)
0
0
Kanamycin
2(6.7%)
0
28(93.3%)
Piperracillin
0
0
30 (100%)
Streptomycin
22(73.3%)
2(6.7%)
6(20%)
Sulfamethoxazole
16(53.3%)
5(16.7%)
9(30%)
Tetracycline
19(63.3%)
4(13.3%)
7(23.4%)
67
3.1.6 Characterization of plasmids from multi-drug resistant ESBL
producing Klebsiella isolates.
In order to determine the presence of plasmid DNA and possible
correlation with resistance, plasmid DNA was isolated from all the thirty ESBL
producing isolates for which minimum inhibitory concentrations against various
antimicrobial agents had been determined. The DNA was separated by agarose
gel electrophoresis. Escherichia coli R39 which carries plasmids of 147, 63, 43.5
and 6.3 mega Daltons was used as the plasmid size standard in the study. All the
thirty isolates appeared to have plasmids.
Twenty four isolates (80 %) had large plasmids while the remaining six
isolates (20%) had both large and small plasmids. Plate 4 shows the plasmid
patterns of multi-drug resistant ESBL producing isolates carrying a common large
plasmid.
68
PLATE 4. Plasmid profiles of some of the isolates of Klebsiella pneumoniae.
Lane A shows the plasmid pattern of the E.coli strain R39 marker. Lane 1 to 13
represents the plasmids profiles for various Extended spectrum β-lactamases
producing Klebsiella pneumonie isolates
A
1 2
3
4
5
6
7
8
9
10
11 12 13
63.0 MDa
63.0
43.5 MDa
3.1.7 Amplification of ESBL genes by PCR
Amplification of the ESBL genes was done by PCR using the designed primers
for each of the three ESBL genes (Table 1). Seven (23.3%) of the ESBL
producing isolates had the CTX-M gene. Eight (26.7%) of the ESBL producing
isolates had the SHV gene while six (20%) of the ESBL producing genes had the
TEM gene.
69
PLATE 5: Gel electrophoresis showing bands which represent the presence of
CTX-M genes in the respective isolates after amplification by PCR.
1
2
3 4 5 6 7 8 9 10 11 12 13 14 15
8.0 kb
5.0 kb
Lane 1 shows the Quick-load™ 2-log DNA which is a pre-mixed, ready-to-load
molecular weight marker. Lanes two, three, eleven to fifteen shows the presence
of a band after PCR. This means that, the target gene, CTX-M, was detected in
those isolates, where as in the blank lanes, the gene was not detected.
3.1.8 Conjugation experiment
In order to determine whether the plasmids carried by the isolates were
transmissible and responsible for resistance transfer the resistant isolates were
conjugated with Escherichia coli K-12 that does not carry any plasmids. The
DNA from E. coli K-12 transconjugants was then analysed for plasmids.
Transconjugants were tested for resistance transfer by inoculating about 105 cells
on agar plates containing a series of antibiotic concentrations and by the disk
70
diffusion technique. Resistance to cefotaxime was transferred in all the 18 cases
where resistance was detected. Plasmid DNA analysis of the transconjugants
showed that resistance was mainly effected by the large plasmids.
In this study, resistance to 3GC antibiotics and ESBL mediated resistance to
cefotaxime was transferable to recipient Escherichia coli K 12 from ESBL
positive isolates.
71
CHAPTER FOUR
4.1 DISCUSSION
The principal distinguishing features of the genus Klebsiella from other
genera of the family Enterobacteriacae are that all its members are nonmotile, do
not decarboxylate ornitine and have mucoid colonies (Plate 1). Thus, the
distinction between members of this genus and nonmotile strains of Enterobacter
is difficult to make. It has even been suggested that the species Enterobacter
aerogenes be transferred to the genus Klebsiella under the designation Klebsiella
mobilis (Orskov, 1984), a move that would disarray the characteristics of the
genus Klebsiella as is conventionally known. In this study the identification of the
Klebsiella isolates from the Clinical microbiology laboratory of the Kenyatta
National Hospital and the Centre for Microbiology Research was limited only to
Klebsiella pneumoniae subspecies pneumoniae using API 20 E kit.
In the present study, resistance patterns from both disk diffusion tests and
the determination of minimum inhibitory concentrations showed resistance to
most of the drugs tested. Disk diffusion results showed that all the isolates were
sensitive to imipinem. Among all the eighty isolates for which MICs were
determined, 68 (85%) isolates were resistant to amoxicillin, but tests with
Augmentin® (amoxicillin-clavulanic acid) showed resistance in only 10 (12.5%)
of the isolates. The higher activity of Augmentin® is attributable to inhibition of
β–lactamases by the clavulanic acid in the amoxicillin-clavulanic acid
combination. Amoxycillin is a derivative of ampicillin which differs from
72
ampicillin by only hydroxyl group but has a similar antibacterial spectrum of
activity.
A comparison between the phenotypic confirmatory disk diffusion method
and double-disk synergy test (ceftazidime, ceftriaxone, cefotaxime, and
amoxicillin/clavulanic acid) was made to assign the appropriate method of
detection for ESBLs. The double disk synergy test (DDST) was found to be a
useful, simple and cost effective test for the detection of ESBL producing
strains. The results indicated that thirty out of the eighty klebsiellae pneumoniae
isolates in the present study were producing Extended spectrum β-lactamases.
This is relatively a high prevalence (37.5%) of ESBL production. Ampification
by polymerase chain reaction (PCR) indicated that, seven (23.3%) of the ESBL
producing Klebsiella pneumoniae isolates had the CTX-M gene, eight (26.7%)
isolates had the SHV gene and six (20%) of the ESBL producing isolates had
the TEM gene. Carriage of other β-lactamases apart from the ones that were
detected, could also be responsible for the observed resistance.
Extended spectrum β-lactamases appear to have been a major cause of the
resistance encountered in the Klebsiella isolates studied.
In a similar study,
Muthotho et al., (1990) also showed that Klebsiellae organisms sometimes exhibit
multiple resistance. Brun-Buisson et al. (1987) associated the emergence and
spread of Klebsiella pneumoniae resistant to third generation cephalosporins to
the over use of these agents especially in intensive care units.
73
The “susceptible” category implied that an infection due to the strain may
be appropriately treated with the dosage of antimicrobial agent recommended for
that type of infection and infecting species, unless otherwise contraindicated.
The “intermediate” category includes isolates with antimicrobial agent MICs that
approach usually attainable blood and tissue levels and for which response rates
may be lower than for susceptible isolates. The “intermediate” category implied
clinical applicability in body sites where the drugs are physiologically
concentrated (for example, quinolones and β-lactams in urine) or when a high
dosage of a drug can be used (for example, β-lactams). The “intermediate”
category also includes a “buffer zone” which should prevent small, uncontrolled
technical factors from causing major discrepancies in interpretations, especially
for drugs with narrow pharmacotoxicity margins.
Although enzymatic destruction is the most common mechanism of
resistance to the β-lactam antibiotics, this may be accompanied by reduced
accumulation of drugs in the cell and there could as well be an alteration of
penicillin-binding-proteins which are the targets of these antibiotics. It has been
reported that usually, decreased accumulation and destructive enzyme act
synergistically to cause resistance (Nikaido, 1989, 1998). If the rate of
accumulation is so rapid as to saturate the destructive enzyme, the effect of the
enzyme may be nullified since there would be enough drug to inhibit the bacterial
metabolism (Lakaye et al., 1999). The resistance to amoxycillin observed in this
study may therefore reflect a combination of mechanisms that could not be
inferred directly from the results.
74
Resistant strains are not inhibited by the usually achievable systemic
concentrations of the agent with normal dosage schedules and/or fall in the range
where specific microbial resistance mechanisms are likely (for example, ßlactamases) and clinical efficacy has not been reliable in treatment studies
(NCCLS, 2005).
These results show that most of the Klebsiella isolates dealt with in this
study exhibited multiple resistance to the antimicrobial agents tested. The results
have significant implications. It has been reported that many Klebsiella
pneumoniae strains that produce extended spectrum β-lactamases also show
associated resistance to other antimicrobial agents (Medeiros, 1993; Procop et al.,
2003).
Conjugation studies showed the presence of self transmissible plasmids
that conferred resistance to Escherichia coli K-12. Resistance to cefotaxime was
shown to be transferred by conjugation. This shows that, besides chromosomal
carriage of resistance determinants inherent in Klebsiella, there may be additional
determinants on plasmids. In case of exclusive chromosomal carriage, plasmid
content would be irrelevant but the presence of additional resistance genes on
plasmids may raise the level of resistance by increasing gene dosage (Arakawa et
al., 1989).
Extended spectrum β-lactams are commonly included in the empirical
antibiotic regimens for treatment of gram negative sepsis. The increasing use of
broad spectrum cephalosporins has become one of the major factors responsible
75
for the high rate of selection of extended spectrum beta lactamase producing
microorganisms (Palucha et al., 1999).
NCCLS established break point for expanded spectrum cephalosporin
antibiotics shortly after the clinical release of these antibiotics in the early 1980s.
MIC of <8 μg/mL correlated with >92% clinical success for all relevant species
including Klebsiella pneumoniae and Escherichia coli. These break points were
established prior to the era of the ESBLs. In the late 1980s and early 1990s it was
recognized that MICs of some cephalosporin for isolates producing ESBL may be
<8 μg/mL of which, it is in the susceptible range (Paterson et al., 2001).
Good clinical outcomes were observed when extended spectrum
cephalosporins were used to treat conditions like urinary tract infection due to
ESBL producing organism but the outcome in serious infections was
questionable. However, some authors are of the view that ESBL screening is not
likely to affect patient outcome and hence neither clinically necessary nor cost
effective for the laboratories (Emery and Weymouth, 1997).
There are published reports that patients had good clinical outcome despite
receiving cephalosporins for treatment of infections with ESBL producing
organisms and has been an argument against routine screening for ESBL
production (Kevin et al., 1995).
The role of 3GC in the treatment of Klebsiella pneumoniae infection is
limited as ESBL mediated resistance is on the increase in the last decade.
The real challenge is the ESBL producing organisms for which MICs of 3GC are
in the susceptible range and they may not be truly susceptible when serious
76
infections are considered although these isolates may have been reported
susceptible. Among the ESBL producers in this study, 16.7% of isolates for
cefotaxime, 10% for ceftazidime and 13.3% for ceftriaxone were in the
susceptible range. Resistance to third generation cephalosporins may exist and yet
not be shown by routine disk sensitivity tests (Jacoby and Archer, 1991;
Sturenburg et al., 2004). Imipenem was highly active against all the ESBL
producing strains in the present study.
In view of these facts NCCLS approved the standard methods for dilution
in antimicrobial susceptibility tests published in 2000 and recommended that
MICs of >2 μg/mL for cefpodoxime, ceftazidime, aztreonam, cefotaxime and
ceftriaxone should be regarded as possibly indicating ESBL production. It further
suggested that for all ESBL producing strains the test result be reported as
resistant for all penicillins, cephalosporins and aztreonam (NCCLS, 2000). The
laboratories which do not perform tests for detection of ESBLs and do not report
ESBL producers as resistant to cephalosporins risk poor outcome. Among the
non-β-lactam antibiotics, which showed higher sensitivity against these ESBL
producers, were amikacin and ciprofloxacin.
ESBL production is encoded by genes on conjugation plasmids which are
easily transmitted among different members of the Enterobacteriaceae. Usually
this plasmid also carries genes coding resistance for other antibiotics which result
in strains that are multidrug resistant (Palucha et al., 1999).
It is generally thought that patients having infections caused by an ESBLproducing organism are at an increased risk of treatment failure with an expanded-
77
spectrum β-lactam antibiotic. Therefore, it is recommended that any organism that
is confirmed for ESBL production according to NCCLS criteria be reported as
resistant to all expanded-spectrum β-lactam antibiotics, regardless of the
susceptibility test result (NCCLS, 2000). While some ESBL-producing strains
have overt resistance to expanded-spectrum β-lactam antibiotics, many isolates
will not be phenotypically "resistant" according to guidelines such as those
previously used by the NCCLS. Therefore, it is important for the clinical
microbiology lab to be aware of isolates that may show increased MICs of
oxyimino-cephalosporins even though they may not be reported as resistant, as
this might suggest the presence of an ESBL. It is also important for the clinical
microbiology lab to then implement one or more methods to detect ESBLs.
The results obtained in this study showed that extended spectrum βlactamases enzymes contributed to Klebsiella pneumoniae resistance to third
generation cephalosporins. Therefore, i accepted the alternative hypothesis and
rejected the null hypothesis.
4.2 CONCLUSIONS
According to the results obtained in this study, ESBL production among
the multi drug resistant isolates was prevalent. The Klebsiella pneumoniae
isolates were resistant to most of the antimicrobial agents tested against them.
Resistance to cefotaxime was transferable by conjugation. Therefore, it is possible
that the Klebsiella isolates could easily transmit resistance determinants to
pathogens sharing the same environment.
78
4.3 RECOMMENDATONS AND SCOPE FOR FURTHER WORK
Further studies are needed to establish the optimal technique for detecting
ESBL production. Meanwhile, it should be recognized that the present disk
diffusion criteria underestimates the prevalence of the ESBL producing strains.
My observations suggested that additional testing to detect ESBL production in
the clinical isolates on a routine basis would be necessary to institute appropriate
antibiotic therapy and at the same time, it is necessary that measures be taken to
limit the spread of resistant organisms.
4.3.1 Application of Computers in Antibacterial Susceptibility Testing
Antimicrobial resistance is a global problem. Emergence of multidrug
resistance has limited the therapeutic options, hence monitoring resistance is of
paramount importance. Antimicrobial resistance monitoring will help to review
the current status of antimicrobial resistance locally, nationally and globally and
helpful in minimizing the consequence of drug resistance, limit the emergence
and spread of drug resistant pathogens.
Thousands of laboratories are distributed world-wide and need to be
linked to integrate data on most clinically relevant organism on a daily basis to
obtain an accurate picture of “real resistance”.
4.3.2 Collective efforts to control the spread of resistant organisms.
The pharmaceutical industry, clinicians, microbiologists and all other
people who handle drugs, whether for therapy, prophylaxis or other purpose
79
should be involved in the control of the spread of resistant micro-organisms. The
microbial genetic pool appears to be available for sharing between all
microorganisms. Thus, resistance arising from any setting is likely to complicate
therapy.
4.3.3 Inclusion of β-lactamase inhibitor
It would be important to include clavulanic acid (-lactamase inhibitor)
disks in all tests involving cephalosporins for surveillance of extended spectrum
of β-lactamases. Such inclusion may also serve as a surveillance strategy since
development of resistance is sometimes not accompanied by any changes
detectable by the NCCLS breakpoint values
80
REFERENCES
ACKERMANN, G., DEGNER, A, COHEN, S. H., SILVA, J. Jr., and RODLOFF,
A.
C.
(2003).
Prevalennce
and
association
of
macrolide-lincosamide-
streptogramin B (MLS(B)) resistance with resistance to moxifloxacin in
Clostridium difficile. J.Antimicrob. Chemother. 51: 599 – 603.
AIRES, J. R., KOHLER, T., NIKAIDO, H., and PLESIAT, P. (1999).
Involvement of an active efflux system in the natural resistance of Pseudomonas
aeruginosa to aminoglycosides. Antimicrob. Agents Chemother. 43: 2624 – 2628.
AKINDELE, J. A., and ROTILU, I. O. (1997). Outbreak of neonatal Klebsiella
septicaemia: a review of antimicrobial sensitivities. Afr. J. Med. Sci. 26: 51-53.
ANANTHAKRISHAN A.N., KANUNGO. R, KUMAR A, BADRINATH S.
(2000). Detection of extended spectrum beta-lactamase producers among surgical
wound infections and burn patients in JIPMER. Indian J. Med Microbiol ; 18:160165.
ANANTHAN, R. S., and ALAVANDI, S. (1999). Enterotoxigenicity of
Klebsiella pneumoniae associated withchildhood gastroenteritis in Madras, India.
Jpn. J. Infect. Dis. 52: 16-17.
ANG, J. Y., EZIKE, E., and ASMAR, B. I. (2004). Antibacterial resistance.
Indian J. Pediatr. 71: 229 –239.
81
ARAKAWA, Y., OHTA, M., KIDO, N., MORI, M., ITO, H., KOMATSU, T.,
FUJI, Y., and KATO, N. (1989). Chromosomal β-lactamase of Klebsiella
oxytoca, a new class A enzyme that hydrolyzes broad spectrum β-lactam
antibiotics. Antimicro .Agents Chemother. 33: 63 - 70.
ARAQUE, M., and RIVERA, I. (2004). Simultaneous presence of blaTEM
and blaSHV genes on a large conjugative plasmid carried by extended-spectrum
beta-lactamase-producing Klebsiella pneumoniae. Amer Med J. Sci.
327:118 – 122.
ARCANGIOLI, M. A., LEROY-SETRIN, S., MARTEL, J. L., and CHASLUSDANCLA, E. (1999). A new chloramphenicol and florfenicol resistance gene
flanked by two integron structures in Salmonella typhymurium DT 104. FEMS
Microbiol. Lett. 174: 327 -332.
ARDANUY, C., LINARES, J., DOMINGUEZ, A., HERNANDEZ-ALLES, S.,
BENEDI, V. J., and MARTINEZ-MARTINEZ, L. (1998). Outer membrane profiles
of clonally related Klebsiella pneumoniae isolates from clinical samples and
activities of cephalosporins and carbapenems. Antimicrob. Agents Chemother. 42:
1636 -1640.
ARLET, G., ROUVEAU, M., FOURNIER, G., LAGRANGE, P. H., and
PHILIPPON, A. (1993). Novel plasmid-encoded TEM - derived extended-spectrum
β-lactamase in Klebsiella pneumoniae conferring higher resistance to aztreonam than
82
to extended spectrum cephalosporins. Antimicrob. Agents Chemother. 37: 2020 2023.
ASHIRU, J. O., and OSOBA, A. O. (1986). Gram-negative septicaemia in Ibadan,
Nigeria. E .Afr. Med. J. 63: 471 - 476.
AZUCENA, E. and MOBASHERY, S. (2001). Aminoglycoside-modifying
enzymes: mechanisms and inhibition. Drug Resist. Updat. 4: 106 – 117.
BAUERNFIEIND A., CHANG, Y. and SCHWEIGHART, S. (1989). Extended
Broad Spectrum β-lactamase in Klebsiella pneumoniae including resistance to
Cephamycins. Infection; 17: 316-321.
BELLON, J., and MOUTON, R. P. (1992). Distribution of β-lactamases in
Enterobacteriaceae: indoor versus outdoor strains. Chemotherapy. 38: 77 - 81.
BENNET, P. M., and HOWE, T. G. B. (1990). Bacterial and bacteriophage
genetics. In: PARKER, M. T., COLLIER, L. H. LINTON, A. H., and DICK, H.
M. (eds). TOPLEY and WILSON Principles of Bacteriology, Virology and
Immunity Vol. 1, General Microbiology and Immunity. Edward Arnold, London.
p 153 - 210.
BISSONNETTE, L., CHAMPETIER, S., BUISSON, J., and ROY, P. H. (1991).
Characterization of the nonenzymatic chloramphenicol resistance (cmlA) gene of
83
the In4 integron of Tn 1696: similarity of product to transmembrane transport
proteins. J. Bacteriol. 173: 4493 - 4502.
BORGES-WALMSLEY, M. I., and WALMSLEY, A. R. (2001).The structurre
and function of drug pumps. Trends Microbiol. 9: 71 – 79.
BOUZA, E., and CERCENADO, E. (2002). Klebsiella and Enterobacter:
antibiotic resistance and treatment implications. Semin. Respir. Infect. 17: 215 –
230.
BRADFORD, P. A., URBAN, C., MARIANO, N., PRIJAN, S. J., RAHAL, J. .J.,
and BUSH, K. (1997). Imipenem resistance in Klebsiella pneumoniae is
associated with the combination of
ACT-1, a plasmid-mediated AmpC -
lactamase, and the loss of an outer membrane protein. Antimicrob. Agents
Chemother. 41: 563 - 569.
BRUN-BUISSON, C., LEGRAND, P., PHILIPPON, A., MONTRAVERS, F.,
ANSQUER, M., and DUVAL, J. (1987). Transferable enzymatic resistance to
third generation cephalosporins during nosocomial outbreak of multiresistant
Klesiella pneumoniae. Lancet ii: 302 - 306.
BURK, D. L, and BERGHUIS, A. M. (2002). Protein kinase inhibitors and
antibiotic resistance. Pharmacol. Ther. 93: 283 – 292.
84
BURMAN, L. G., HAEGGMANN, S., KUISTILLA, M., TULLUS, K., and
HUOVINEN, P. (1992). Epidemiology of plasmid mediated β-lactamases in
enterobacteria in Swedish neonatal wards and relation to antimicrobial therapy.
Antimicrob. Agents Chemother. 36: 989 - 992.
BURNS, J. C., and CLARK, D. K. (1992). Salicylate-inducible antibiotic
resistance in Pseudomonas cepacia associated with absence of a pore-forming
outer membrane protein. Antimicrob. Agents Chemother. 36: 2280 – 2285.
BUSH, K., and SYKES, R. B. (1986). Methodology for the study of β-lactamases.
Antimicrob. Agents Chemother. 30: 6 - 10.
CASEWELL M.W, and PHILLIPS I (1981). Aspects of plasmid mediated
antibiotic resistance and epidiemology of Klebsiella spp. Amer. Med. J.; 70 459462.
CASEWELL, M. W., TALSANIA, H. G., and KNIGHT, S. (1981). Gentamicin
resistant Klebsiella aerogenes as a clinically significant source of transferable
antibiotic resistance. J. Antimicrob. Chemother. 8: 153 - 160.
CHEN, J., KURODA, T., HUDA, M. N., MIZUSHIMA, T., and TSUCHIYA, A.
(2003). An RND-type multidrug efflux pump SdeXY from Serratia marcescens.
Journal of Antimicrob. Chemother. 52: 176 – 179.
85
CHEVALIER, J., PAGES, J. M., EYRAUD, A., and MACLEA, M. (2000).
Membrane permeability modifications are involved in antibiotic resistance in
Klebsiella pneumoniae. Biochem. Biophys. Res. Commun. 274: 496-499.
CHOW, J. W. (2000). Aminoglycoside resistance in enterococci. Clin. Infect. Dis.
31: 586 –589.
COHEN, M.L. and R.V. AUXE. (1992). Drug resistant Salmonella in the United
States: an epidemiologic perspective. Science, 234: 964-970.
COL, N. F., and O'CONNOR, R. W. (1987). Estimating worldwide current
antibiotic usage: report of task force 1. Rev. Infect. Dis. 9 (suppl.3): 232 - 243.
COLLATZ, E., GUTMANN, L., WILLIAMSON, R., and ACAR , J. F. (1984).
Development of resistance to beta-lactam antibiotics with special reference to
third-generation cephalosporins. J. Antimicrob. Chemother. 14 (Suppl B):13-21.
Col, N. F., and O'Connor, R. W. (1987). Estimating worldwide current antibiotic
usage: report of task force 1. Rev. Infect. Dis. 9 (suppl.3): 232 - 243.
COOKSEY ,R. C. (1991). Mechanisms of resistance to antimicrobial agents. In:BALOWS A., HERRMANN, K.L., ISENBERG, H.D., and SHADOMY, H.J.
(eds) Manual of Clinical Microbiology. American Society for Microbiology
Washington, D.C. p 1099 – 1104.
86
COSGROVE .S.E., KAYE .K.S., ELIOPOULOUS, G.M., CARMELI, Y.
Emergence of third-generation cephalosporin resistance in Enterobacter species.
Arch Intern Med. 2002; 162:186–190.
CRYZ S.J., FURER, E., SADOFF J. C., FREDEKING, T., QUE, J. U., and
CROSI, A. S. (1991). Production and characterization of human-hyper-immunne
intravenous immunoglobulins against Pseudomonas aeruginosa and Klebsiella
species. J. Infect. Dis. 163: 1055 - 1061.
D.A. WILLIAMS and T.L. LEMKE. (2002). Foyes Principles of Medicinal
Chemistry, 5th edition. 842, 849.
DEGUCHI, T., FUKAOKA, A., YASUDA, M., NAKANO., M., OZEKI, S.,
KANEMATSU, E., NISHINO, Y., ISHIHARA, S.,BAN, Y., and KAWADA, Y.
(1997). Alterations in the gyrA subunit of DNA gyrase and the Par C subunit of
topoisomerase IV in quinolone-resistant clinical isolates of Klebsiella
pneumoniae. Antmicrob. Agents Chemother. 41: 699-701.
DEVER, L. A., and DERMODY, T. S. (1991). Mechanisms of bacterial
resistance to antibiotics. Arch. Intern. Med. 151: 886 – 895.
DIPERSIO, J.R., DESHPANDE, L. M., BIEDENBACH, D. J., TOLEMAN, M.
A., WALSH, T. R., and JONES, R. N. (2005). Evolution and dissemination of
extended-spectrum
beta-lactamase-producing
87
Klebsiella
pneumoniae:
epidemiology and molecular report from the SENTRY Antimicrobial Surveillance
Program (1997-2003). Diagn. Microbiol. Infect. Dis. 51: 1 - 7.
DOMENECH-SANCHEZ,
A.,
HERNANDEZ-ALLES,
S.,
MARTINEZ-
MARTINEZ, L., BENEDI, V. J., and ALBERTI, S. (1999). Identification and
characterization of a new porin gene of Klebsiella pneumoniae: its role in betalactam antibiotic resistance. J. Bacteiol. 181: 2726 - 2732.
DOMENECH-SANCHEZ, A., MARTINEZ-MARTINEZ, L., HERNANDEZALLES, S., DEL CARMEN, C. M., PASCUAL, A., TOMAS, J. M., ALBERTI,
S., and BENEDI, V. J. (2003). Role of Klebsiella pneumoniae OmpK35 porin in
antimicrobial resistance. Antimicrob. Agents Chemother. 47: 3332 – 3335.
EMERY C.L, WEYMOUTH L.A. Detection and clinical significance of extended
spectrum β - lactamases in a tertiary care medicine centre(1997). J. Clin.
Microbiol.;35:2061-2067.
EWING, W. H. (1986). The genus Klebsiella. In: Edward and Ewings
identification of Enterobacteriaceae. Elsevier Science Publishers Inc. New York.
365 - 380.
FIERER, J., and GUINEY,D. (1999). Extended-spectrum -lactamases: a plague
of plasmids. J. Am. Med. Assoc. 281: 563 – 564.
88
FERNANDEZ-RODRGUEZ,
A.,
CANTON,
R.,
PEREZ-DIAZ,
J.
C.,
MARTINEZ-BELTRAN, J., PICAZO, J. J., and BAQUERO, F. (1992).
Aminoglycoside-modifying enzymes in clinical isolates harboring extendedspectrum beta-lactamases. Antimicrob. Agents Chemother. 36: 2536 – 2538.
FRANKLIN, T. J. (1992). Bacterial resistance to antibiotics. In: HUGO, H.B.,
and RUSSEL, A. D. (editors). Pharmaceutical Microbiology. Blackwell Scientific
publications, Oxford. 208 - 230.
GAFFNEY,
D.
Chloramphenicol
F.,
CUNDLIFFE,
resistance
that
E.,
and
does
not
FOSTER,
involve
T.
J.
(1981).
chloramphenicol
acetyltransferase encoded by plasmids from bacteria. J. Gen. Microbiol. 125: 113
– 121.
GALLIMAND, M., LAMBERT, T., GERBAUND, G., and COURVALIN, P.
(1993). Characterization of the aac (6') - 1b gene encoding an aminoglycoside 6N-acetyltranferase in Pseudononas aeruginosa BM 2656. Antimicrob. Agents
Chemother. 37: 1456 - 1462.
GEORGOPAPADAKOU, N. H. (1993). Penicillin-binding proteins and bacterial
resistance to -lactams. Antimicrob. Agents Chemother. 37: 2045-2053.
89
GNIADKOWSKI, M. (2001). Evolution and epidemiology of extended-spectrum
beta-lactamases (ESBLs) and ESBL-producing microorganisms. Clin. Microbiol.
Infect. 7: 597 – 608.
GROSS, R. J., and HOLMES, B. (1990). The Enterobacteriaceae. In: PARKER,
T.M., COLLIER, L.H., and DUERDEN, B.I. (eds). Topley and Wilsons
principles of Bacteriology, Virology and Immunity. Vol. 2, Systematic
Bacteriology Edward Arnold, London. p 401 - 414.
GRUGER, T., NITISS, J. L., MAXWELL, A., ZECHIEDRICH, E. L., HEISIG,
P., SEEBER, S., POMMIER, Y., and STRUMBERG, D. (2004). A mutation in
Escherichia coli DNA gyrase conferring quinolone resistance results in sensitivity
to drugs targeting eukaryotic topoisomerase II. Antimicrob. Agents Chemother.
48: 4495 – 4504.
GUINEY, D. G. Jr. (1984). Promiscuous transfer of drug resistance in Gramnegative bacteria. J. Infect. Dis. 149: 320 - 329.
GUTIERREZ, J., HOYOS, A, and PIEDROLA, G. (1992). Aminoglycoside
resistance in enteroccoci. Ann. Biol. Clinic. Paris. 50: 671 - 674.
GUTMANN, L., R. WILLIAMSON, R. MOREAU, M.D. KITZIS, E.
COLLATZ, J.F. ACAR and F.W. GOLDSTAIN. 1985. Cross resistance to
nalidixic acid, trimethoprim and chloramphenicol associated with alterations in
90
the outer membrane proteins of Klebsiella, Enterobacter and Serratia. J. Infect.
Dis., 151: 501-507.
HABLE, K. A., MATSON, J. M., WHEELER, D. J., HUNT, C. E., and QUIE, P.
G. (1972). Klebsiella type 33 septicaemia in an infant intensive care unit. J. Paed.
80: 920 - 924.
HAND, W. L. (2000). Current challenges in antibiotic resistance. Adolesc. Med.
11: 427 – 438.
HANSOITA, J.B, AGARWAL V, PATHAK A.A, SAOJI A.M. Extended
Spectrum β-lactamase mediated resistance to third generation cephalosporins in
Klebsiella Pneumoniae in Nagpur, Central India. 1997; 105:158-161.
HAAS, M. J., and DOWDING, J. E. (1975). Aminoglycoside modifying
enzymes. Methods Enzymol. 43: 611 - 628.
HEDGES, R. W., DATTA, N., KONTOMICHALOU, P., and SMITH, J. T.
(1974). Molecular specificities of R-factor determined β-lactamases: correlation
with plasmid compatibility. J. Bacteriol. 117: 56 - 62.
HEISIG, P., SCHEDLETZKY, H., and FALKENSTEIN-PAUL, H. (1993).
Mutations in the gyrA gene of a highly fluoroquinolone-resistant clinical isolate
of Escherichia coli. Antimicrob. Agents Chemother. 37: 696 – 701.
91
HERITAGE, J., M’ZALI, F. H., GASCOYNE-BINZI, and HAWKEY, P. M.
(1999).
Evolution and spread of SHV extended-spectrum beta-lactamases in
gram-negative bacteria. J. Antimicrob. Chemother. 44: 309 – 318.
HIRAN, S., and VISWANATHAN, K. A. (1999). Klebsiella pneumoniae – an
emerging cause of osteomyelitis in sickle cell disease. J. Ass. Physic. India. 47:
637 – 638.
HOLT, J. G., KRIEG, N .R., SNEATH, P. H. A., STALEY, J. T., and
WILLIAMS, S. T. (1994). Facultative anaerobic Gram-negative rods: The genus
Klebsiella. In: Bergeys manual of determinative bacteriology. 9th edition,
Williams & Wilkins, Baltimore. p 181.
ISAACK, H., MBISE, R. L., and HIRJI, K. F. (1992). Nosocomial bacterial
infections among children with severe protein energy malnutrition. E. Afr. Med. J.
69: 433 - 436.
IZARD, T., and ELLIS, J. (2000). The crystal structures of chloramphenicol
phospho transferase reveal a novel inactivation mechanism. EMBO J. 19: 2690 –
2700.
92
JACOBY, G. A and Han P. Detection of Extended spectrum-lactamases in
clinical isolates of Klebsiella pneumoniae and Escherichia coli. Clin. Microbiol.
1996; 34: 908-911.
JACOBY, G. A., BLASER, M. J., SANTANAM, P., HÄCHLER, H., KAYSER,
.F. H., HARE, R. S., and MILLER, G. H. (1990). Appearance of amikacin and
tobramycin resistance due to 4'-aminoglycoside nucleotidyl transferase(ANT(4')II) in Gram-negative pathogens. Antimicrob. Agents Chemother. 34: 2381 - 2386.
JACOBY, G. A., and ARCHER, G. L. (1991). New mechanisms of bacterial
resistance to antimicrobial agents. N. Engl. J.Med. 9: 601 - 612.
JACOBY, G.A, MEDEIORES, A.A. More Extended Spectrum lactamases.
Antimicrob. Agents chemother 1991; 35:1697-1704.
JEONG, S. H., KIM, W. M., CHANG, C. L., KIM, J. M., LEE, K., CHONG, Y.,
HWANG, H.Y., BAEK, Y. W., CHUNG, H. K., WOO, I. G., and KU, J. Y.
(2001). Neonatal intensive care unit outbreak caused by a strain of Klebsiella
oxytoca resistant to aztreonam due to overproduction of chromosomal betalactamase. J. Hosp. Infect. 48: 281 -288.
JOHN, J. F.Jr., and TWITTY, J. A. (1986). Plasmids as epidemiologic markers in
nosocomial Gram-negative bacilli: experience at a university and review of the
literature. Rev. Infect. Dis. 8: 693 - 704.
93
J.G. HARDMAN, L.E. LIMBIRD, and A.G. GILMAN
(2001). The
Pharmacological Basis of Therapeutics, 10th edition. pp. 1206, 1209.
KAGAN, S. A., and DAVIES, J. E. (1980). Enzymatic modification of
aminoglycoside antibiotics: mutations affecting the expression of aminoglycoside
acetyltransferase-3. Plasmid 3: 312 – 318.
KARIUKI, S., C. GILKS, G. REVATHI, and C. A. HART. (2000). Genotypic
analysis of multidrug resistant Salmonella enterica Serovar Typhi, Kenya.
Emerging Infectious Diseases.
KARIUKI, S., CORKILL, J. E., REVATHI, G., MUSOKE, R., and HART, C. A.
(2001). Molecular characterization of a novel plasmid-encoded cefotaximase
(CTX-M-12) found in clinical Klebsiella pneumoniae isolates from Kenya.
Antimicrob. Agents Chemother. 45: 2141 – 2143.
KEVIN S, PAUL S, XIAQQIN X, ALAN J, HAMISH B, FATIMA E.B, BARRY
C, GARY F. (1995) Extended spectrum β – lactamase producing Klebsiella
pneumoniae strain causing Nosocomial outbreaks of infection in the United
Kingdom. J. Clin. Microbiol. ; 36:3105-3110.
KHANEJA, M., NAPRAWA, J., KUMAR, A., and PIECUCH, S. (1999).
Successful treatment of late-onset infection due to resistant Klebsiella
94
pneumoniae in an extremely low birth weight infant with ciprofloxacin. J.
Perinatol. 19: 311 – 314.
KIDWAI, M., MISRA, P., and KUMAR, R. (1998). The fluorinated quinolones.
Curr. Pharm. Res. 4: 101 – 118.
KLIEBE, C., NIER, B. A., MEYER, J. F., TOLXDORFF-NEUTZLING, R. M.,
and WIEDEMANN, B. (1985). Evolution of plasmid coded resistance to broad
spectrum cephalosporins. Antimicrob. Agents Chemother. 28: 302 - 307.
KNOTH .H Shah P, KRCMERY V. ANTAL M, MITSUHASI S. Transferable
resistance to cefotaxime, cefoxitin, cefomandole and cefuroxime in clinical
isolates of Serratia marcesens infections 1983;11:316-317.
KOHLER, T., PECHERE, J. C., PLESIAT, P. (1999). Bacterial antibiotic efflux
systems of medical importance. Cell. Mol. Life Sci. 56: 771 – 778.
KUDO, F., KAWABE, K., KURIKI, H., EGUCHI, T., and KAKINUMA, K.
(2005). A new family of glucose-1-phisphate/glucosamine nucleotidyltransferase
in the biosynthetic pathways for antibiotics. J. Am. Chem. Soc, 127: 1711 - 1718.
KUNIN, C. M. (1993). Resistance to antimicrobial drugs: a worldwide calamity.
Ann.Intern.Med. 118: 557 - 561.
95
LAKAYE, B., DUBUS, A., LEPAGE, S., GROSLAMBERT, S., and FRERE, J.
M. (1999). When drug inactivation renders the target irrelevant to antibiotic
resistance: a case story with beta-lactams. Mol. Microbiol. 31: 89 - 101.
LAMBERT, H. P. and O’GRADY, F. W. (1992). Aminoglycosides and
aminocyclitols. In: Antibiotics and chemotherapy. Churchill Livingstone,
Edinburgh. p 2 – 26.
LAURA V, PEZELLA C, TOSINI F, VISCA P, PETRUCCA A, CARRATOLI
A. Multiple antibiotic resistance mediated by structurally related plasmids
carrying an ESBL gene and a class 1 integron. Antimicrobial agents
Chemotherapy2000; 44:2911-2914.
LAUTENBACH E, La ROSA L.A, MARR A.M, NACHAMKIN I, BILKER
W.B and FISHMAN N.O (2003). Changes in the prevalence of vancomycinresistant enterococci in response to antimicrobial formulary interventions: impact
of progressive restrictions on use of vancomycin and third generation
cephalosporins. Clin. Infect. Dis. 36: 440-446.
LEE, A. MAO, W., WARREN, M. S., MISTRY, A., HOSHINO, K.,
OKUMURA, R., ISHIDA, H., and LOMOVSKAYA, O. (2000). Interplay
between efflux pumps may provide either additive or multiplicative effects on
drug resistance. J. Bacteriol. 182: 3142 – 3150.
96
LEVY, S. B. (1982). Microbial resistance to antibiotics: an evolving and
persistent problem. Lancet ii: 83 - 88.
LEVY, S. B., and MARSHALL, B. (2004). Antibacterial resistance worldwide:
causes, challenges and responses. Nat. Med. 10 (12 suppl): S122 – 129.
LIVERMORE, D. M. (2002). Multiple mechanisms of antimicrobial resistance in
Pseudomonas aeruginosa: our worst nightmare? Clin. Infect. Dis. 34: 634 - 640.
LIVERMORE, D. M., and YUAN, M. (1994). Antibiotic resistance and
production of extended spectrum -lactamases amongst Klebsiella species from
intensive care units in Europe. J. Antimicrob. Chemother. 38: 409 – 424.
LLANO-SOTELO, B., AZUCENA, E. F. Jr., KOTRA, L. P., MOBASHERY, S.,
and CHOW, C.S. (2002). Aminoglycosides modified by resistance enzymes
display diminished binding to the bacterial ribosomal aminoacyl-tRNA site.
Chem. Biol. 9: 455 – 463.
MARSHALL, N. J., and PIDDOCK, L. J. (1997). Antibacterial efflux systems.
Microbiologia 13: 285 – 300.
MARTINEZ-MARTINEZ, L., PASCUAL, A., HERNENDEZ- ALLES, S.,
ALVAREZ-DIAZ. D., SUAREZ, A. I., TRAN, J.,
BENEDI, V. J., and
JACOBY, G. A. (1999). Roles of -lactamases and porins in activities of
97
carbapenems and cephalosporins against Klebsiella pneumoniae Antimicrob.
Agents Chemother. 43: 1669 – 1673.
MASKELL, R., OKUBAGEJO, A. O., and PAYNE, R. H. (1978). Human
infections with thymine-requiring bacteria. J. Med. Microbiol. 11: 33 – 45.
MATHEW, M., and HEDGES, R. W. (1976). Analytical isoelectric focusing of
R-factor-determined β-lactamases: correlation with plasmid compatibility. J.
Bacteriol. 125: 713-718.
MATHEW, M., HEDGES, R. W., and SMITH, J. T. (1979). Types of β-lactamase
determined by plasmids in Gram-negative bacteria. J. Bacteriol. 138: 657 - 662.
MAYER, I., and NAGY, E. (2000). Investigation of the synergic effects of
aminoglycoside-fluoroquinolone and third-generation cephalosporins combination
against clinical isolates of Pseudomonas species. J. Antimicrob. Chemother. 43:
651 – 657.
McGOWAN, J. E. Jr. (1985). Changing etiology of nosocomial bacteremia and
fungemia and other hospital acquired infections. Rev. Infect Dis. 7 (Suppl3): 357
- 370.
McGOWEN J.E, TENOVER FC. Control of antimicrobial resistance in the
health- care system. Infect Dis Clin N Am. 1997; 11:297–311.
98
McMURRAY, L., PETRUCCI, E. Jr., and LEVY, S. B. (1980). Active efflux of
tetracycline encoded by four genetically different tetracycline resistance
determinants in Escherichia coli. Proc. Natl. Acad. Sci. USA. 77: 3974 –3977.
MEDEIROS, A. A. (1993). Nosocomial outbreaks of multiresistant bacteria:
extended-spectrum beta-lactamases have arrived in North America. Ann. Intern.
Med. 119: 428 -430.
MENDES, R. E., TOLEMAN, M. A., RIBEIRO, J., SADER, H. S., JONES, R. N,
and WALSH, T. R. (2004). Integron carrying a novel metallo-beta lactamase gene
blaIMP-16, and a fused form of aminoglycoside-resistant gene aac(6’)30/aac(6’)-Ib’: report from the SENTRY antimicrobial surveillance program.
Antimicrob. Agents Chemother. 48: 4693 – 4702.
MITSCHER, L. A. (1995). Antibiotics and antimicrobial agents. In: FOYE, W.
O., LEMKE, T. L., and WILLIAMS, D. A. Principles of medicinal chemistry.
Williams and Wilkins Philadelphia p 764 –802.
MONTGOMERIE, J. Z., JOHN, J. F., ATKINS, L. M., GILMORE, D. S., and
ASHLEY, M. A. (1993). Increased frequency of large R-plasmids in Klebsiella
pneumoniae colonizing patients with spinal cord injury. Diagn. Microbial. Infect.
Dis. 16: 25 - 29.
99
MOSHER, R. H., CAMP, D. J., YANG, K., BRAWN, M. P., SHAW, W.V., and
VINING, L. C. (1995). Inactivation of chloramphenicol by O-phosphorylation. J.
Biol. Chem. 27: 2700 – 2706.
MURRAY, B. E. (1991). New aspects of antimicrobial resistance and the
resulting therapeutic dilemmas. J. Infect. Dis. 163: 1185 - 1194.
MURRAY, B. E., MATHEWSON, J. J., DuPONT, H. L., ERICCSON, C. D., and
REVES, R. R. (1990). Emergence of resistant fecal Escherichia coli in travelers
not taking prophylactic antimicrobial agents. Antimicrob. Agents chemother: 34:
515 - 518.
MURRAY, I. A., and SHAW, W. V. (1997). O-acetyltransferases f or
chloramphenicol and other natural products. Antimicrob. Agents Chemother. 41:
1 – 6.
MUTHOTO, J. N., WAIYAKI, P. G., MONINDA, M., and KARURI, J. (1990).
Outbreak of respiratory infections due to a multiresistant Klebsiella ozaenae strain
at the intensive care unit, Kenyatta National Hospital, Nairobi. Abstract No. 569,
Programme Abstracts, International Congress for Infectious Diseases, Montreal,
Canada.
NAGY, E., PRAGAI, Z., KOCZIAN, Z., HADJU, E., and FODOR, E. (1998).
Investigation of the presence of different broad-spectrum beta-lactamases among
100
clinical isolates of Enterobacteriacae. Acta Microbiol. Immunol. Hung. 45:43346.
NATIONAL COMMITTEE FOR CLINICAL LABORATORY STANDARDS:
Performance Standards for antimicrobial disk Susceptibility test, 5th edition,
(Villanova, PA: NCCLS) 1993: document M2-A5.
NATIONAL COMMITTEE FOR CLINICAL LABORATORY STANDARDS
(2000). Methods for dilution antimicrobial susceptibility test for bacteria that
grow aerobically. Approved standard M7-A5, 5th edition. National Committee
for clinical laboratory standard Wayne, Pa.
NATIONAL COMMITTEE FOR CLINICAL LABORATORY STANDARDS:
Performance Standards for antimicrobial susceptibility testing, 15th informational
supplement (2005). NCCLS antimicrobial susceptibility testing standards M2-A8
and M7-A6.
NEMEC, A., DOLZANI, I., BRISSE, S., VAN DEN BROEK, P., and
DIJKSHOORN, I. (2004). Diversity of aminoglycoside-resistance genes and their
association with class1 integrons among strains of pan-European acinetobacter
baumannii clones. J. Med. Microbiol. 53: 1233 – 1240.
NEU, H. C. (1983). The emergence of bacterial resistance and influence of
empiric therapy. Rev. Infect .Dis. 5 (suppl.): 9 - 20.
101
NEU, H. C. (1984). Current mechanisms of resistance to antimicrobial agents in
microorganisms causing infections in the patients at risk for infection. Am. J.
Med. 76: 11 - 27.
NIKAIDO, H. (1989). Outer membrane barrier as a mechanism of antimicrobial
resistance. Antimicrob. Agents Chemother. 33: 1831 - 1836.
NIKAIDO, H. (1998). Multiple antibiotic resistance and efflux. Curr. Opin.
Microbiol. 1: 516 – 523.
NONICK, R. P. (1980). Plasmids: these accessory genetic elements in bacteria,
best known as carriers of resistance to antibiotics and as vehicles for genetic
engineering are actually sub cellular organisms poised on the threshold of life.
Sci. Amer. 243 (6): 76 -90.
OMARI, M. A., MALONZA, I. M., BWAYO, J. J., MUTERE, A. N., MURAGE,
E. M., MWATHA, A. K., and NDINYA-ACHOLA, J. O. (1997). Patterns of
bacterial infections and antimicrobialsusceptibility at the Kenyatta National
Hospital, Nairobi, Kenya. E. Afr. Med. J. 74:
ORAL, R., AKISU, M., KULTURSAY, N., VARDAR, F., and TANSUG, N.
(1998). Neonatal Klebsiella pneumoniae sepsis and imipenem/ cilastin. Ind. J.
Pediatr. 65: 121 – 129.
102
ORSKOV, I. (1984). Facultatively anaerobic Gram-negative rods: genus V;
Klebsiella. In: Kreig, N. R, and Holt, J. G. (eds). Bergeys Manual of Systematic
Bacteriology. Williams and Wilkins, Baltimore. p 461 - 465.
O'BRIEN, T. F., and Members of Task Force 2. (1987). Resistance of bacteria to
antimicrobial agents: report of task force 2. Rev .Infect. Dis. 18 (suppl3): 244 253.
PAGES, J. M., DIMARCQ, J. L., QUENIN, S., and HETRU, C. (2003). Thanatin
activity on multidrug resistant clinical isolates of Enterobacter aerogenes and
Klebsiella pneumoniae. Int. J. Antimicrob. Agents. 22: 365 – 369.
PAI H, LYU S, LEE J.H, KIM J, KWON Y, KIM J-W, CHOE K.W. Survey of
extended-spectrum β-lactamases in clinical isolates of Escherichia coli and
Klebsiella pneumoniae: prevalence of TEM-52 in Korea. J. Clin Microbiol.
1999;37:1758–1763.
PALUCHA A, MIKIEWIEZ B, HRYNIEWIEZ W, GRIADKOWSKI M.
Concurrent outbreaks of extended spectrum β-lactamase producing organisms of
the family Enterobacteriaceae in a Warsaw Hospital. J.Antimicrob. Chemother.
1999;44:489-499.
103
PAPANICOLAOU, G. A., JACOBY, G. A., and MEDEIROS, A. A. (1990).
Novel plasmid mediated β-lactamase (MIR-1) conferring resistance to oxyimino
and methoxy beta-lactams in clinical isolates of Klebsiella pneumoniae.
Antimicrob. Agents Chemother. 34: 2200- 2209.
PATERSON, D. L., MULAZIMOGLU, L., CASELLAS, J. M., KO, W.,
GOOSSENS, H., Von GOTTBERG, A., MOHAPATRA, S., TRENHOLME, G.
M., KLUGMAN, K. P., McCORMACK, J. G., and YU, V. L. (2000).
Epidemiology of ciprofloxacin resistance and its relationship to extended
spectrum β-lactamase production in Klebsiella pneumoniae isolates causing
bacteremia. Clin. Infect. Dis. 30: 473 – 478.
PATERSON L.D, WEN-CHIEN K.O, GOTTBERG A.V, CASELLAS J.M, RICE
L.B, MACORMACK J.G, VICTOR LYU. (2001). Outcome of cepaholsporin
treatment for serious infections due to apparently susceptible organisms
producing extended spectrum β-lactamases. Implications for the clinical
Microbiology Laboratory. J. Clin. Microbiol.;39:2206-2212.
PATTERSON, J.E. Antibiotic utilization. Is there an effect on antimicrobial
resistance? Chest. 2001; 119:426–430.
PERES-BOTA, D., RODRIGUEZ, H., DIMOPOULOS, G., DAROS, A.,
MELOT, C., STRUELENS, M. J., and VINCENT, J. L. (2003). Are infections
104
due to resistant pathogens associated with a worse outcome in critically ill
patients? J. Infect. 47: 307 – 316.
PERILLI, M., SEGATORE, B., DE MASSIS, M. R., RICCIO, M. L., BIANCHI,
C., ZOLLO, A., ROSSOLINI, G. M., and AMICOSANTE, G. (2000). TEM-72, a
new extended-spectrum -lactamase detected inProteus mirabilis and Morganella
morganii in Italy. Antimcrob. Agents Chemother. 44: 2437 – 2539.
PETIT, A., SIROT, D. L., CHANAL, G. M., SIROT, J. L., LABIA, R.,
GERBAUD, G., and CLUZEL, R. A., (1988). Novel plasmid -mediated βlactamase in clinical isolates of Klebsiella pneumoniae more resistant to
ceftazidime than other broad-spectrum cephalosporins. Antimicrob. Agents
Chemother. 32: 626 - 632.
PHILIPPON, A., LABIA, R., and JACOBY, G. A. (1989). Extended spectrum βlactamases. Antimicrob. Agents Chemother. 33: 1131- 1136.
PIDDOCK, L. J. (1999). Mechanisms of fluoroquinolone resistance: an update
1994 – 1998. Drugs. 58 (Suppl. 2): 11 – 18.
PODSCHUN R. and ULLMANN U. (1998). Klebsiella spp. As Nosocomial
Pathogens: Epidemilogy, Taxonomy, Typing Methods and Pathogenicity Factors.
Clin. Microb. Rev. 11: 589-603.
105
POIREL, L., LAMBERT, T., TURKOGLU, S., RONCO, E., GAILLARD, J. L.,
and NORDMAN, P. (2001). Characterization of class-1 integrons from
Pseudomonas aeruginosa that contain the bla(VIM- 2) carbapenem hydrolyzing
beta- lactamase gene and two novel aminoglycoside resistance gene cassettes.
Antimicrob. Agents Chemother. 45: 546 – 552.
POOLE, K. (2000). Efflux mediated resistance to fluoroquinolones in
Gram-positive bacteria and the Mycobacteria. Antimcrob. Agents Chemother. 44:
2595 – 2599.
POOLE, K. (2001). Multidrug resitance in Gram-negative bacteria. Curr. Opin.
Microbiol. 4: 500 – 508.
POWERS, J. H. (2004). Antimicrobial drug development – the past, the present
and the future. Clin. Microb. Infect. 10 (Suppl 4): 23 – 31.
PROCOP, G. W., TUOHY, M. J., WILSON, D. A., WILLIAMS, D.,
HADZIYANNIS, E., and HALL, G. S. (2003). Cross-class resistance to non-betalactam antimicrobials in extended-spectrum beta-latamase-producing Klebsiella
pneumoniae. Am. J. Pathol. 120: 265 – 267.
RAO, G. G. (1998). Risk factors for the spread of antibiotic-rresistant bacteria.
Drugs. 55: 323 – 330.
106
RANDEGGER, C. C., KELLER, A., IRLA, M., WADA, A., and HÄCHLER, H.
(2000). Contribution of natural amino acid substitutions in SHV extendedspectrum β-lactamases to resistance against various β-lactams. Antimicrob.
Agents Chemother. 44: 2759 – 2763.
RATHER, P. .N., MUNAYYER, H., MANN, P. A., HARE, R. S., and MILLER,
G. H. (1992). Genetic analysis of bacterial acetyltranferases: identification of
aminoacids determining the specificities of the aminoglyside 6'-N-acetyltranferase
Ib and IIa proteins. J. Bacteriol. 174: 3196 - 3023.
RICE, L. B., and BONOMO, R. A. (1996). Genetic and biochemical mechanisms
of bacterial resistance to antimicrobial agents. In: Lorian, V. (editor). Antibiotics
in laboratory medicine. 4th edition. Williams & Wilkins, Baltimore.p 453 – 501.
RICE, L. B., WILLEY, S. H., PAPANICOLAU, G. A., MEDEIROS, A. A.,
ELIOPOULOS, G. M., MOELLERING, R. C. Jr., and JACOBY, G. A. (1990).
Outbreak of ceftazidime resistance caused by extended-spectrum -lactamases at
a Massachusetts chronic-care facility. Antimicrob. Agents Chemother. 34: 2193 –
2199.
107
RIDLEY, M., BARRIE, D., LYNN, R., and STEAD, K. C. (1970). Antibiotic
resistant Staphylococcus aureus and hospital antibiotic policies. Lancet i: 230 233.
ROBERTS, M. C. (1996). Tetracycline resistance determinants: mechanisms of
action, regulation of expression, genetic mobility and distribution. FEMS
Microbiol. Rev. 19: 1 – 24.
ROBERTS, M. C. (1997). Genetic mobility and distribution of tetracycline
resistance determinants. Ciba Found. Symp. 207: 206 – 222.
ROSS, W. G., and O'CALLAGHAN, C. H. (1975). Beta-lactamase assays.
Methods Enzymol. 43: 69 - 85.
ROWE-MAGNUS, D. A., GUEROUT, A. M., and MAZEL, D (2002). Bacterial
resistance evolution by recruitment of super-integron gene cassettes. Mol.
Microbiol. 43: 1657 -1669.
SANDERS C.C., SANDERS W.E. Beta-lactam resistance in Gram negative
bacteria: global trends and clinical impact. Clin Infect Dis. 1992; 15:824–839.
SANDERS, C. C., SANDERS, W. E., Jr., GEORING, R. V., and WERNNER, V.
(1984). Selection of multiple antibiotic resistance by quinolones, beta lactams,
108
and aminoglycosides with special reference to cross-resistance between unrelated
drug classes. Antimicrob. Agents Chemother. 26: 797 – 801.
SARNO, R., HA, H., WEINSETEL, N., and TOLMASKY, M. E. (2003).
Inhibition of aminoglycoside 6’-N-acetyltransferase type Ib-mediated amikacin
resistance by antisense oligodeoxynucleotides. Antimicrob. Agents Chemother.
47: 3296 – 3304.
SCHEEL, O., and IVERSEN, G. (1991). Resistant strains isolated from
bacteremia patients in Northern Norway. Scand. J. Infect. Dis. 23: 599 - 605.
SCHWARTZ, S., KEHRENBERG, C., DOUBLET, B., and CLOECAERT, A.
(2004). Molecular basis of bacterial resistance to chloramphenicol and florfenicol.
FEMS Microbiol. Rev. 28: 519 –142.
SCHWARTZ, S., WERCKENTHIN, C., KEHRENBERG, C. (2000). The
identification of a plasmid borne chloramphenicol-florfenicol resistance gene in
Staphylococcus sciuri. Antimicrob. Agents Chemother. 44: 2530 – 2533.
SEFTON. A. M. (2002). Mechanisms of antimicrobial resistance: their clinical
relevance in the new millennium. Drugs. 62: 557 – 566.
SHAW, W. V. (1975). Chloramphenicol acetyltransferase from chloramphenicol
resistant bacteria. Methods Enzymol. 43: 737 - 755.
109
SHAW, W. V., and LESLIE, A. G. W. (1991). Chloramphenicol acetyl
transferase. Annu. Rev. Biophys. Biophys. Chem. 20: 363 – 386.
SIROT, D., SIROT, J., LABIA, R., MORAND, A., COURVALIN, P.,
DARFENNILLE MICHAND, A., and PERRONX, R. Transferable resistance to
third generation cephalosporins in clinical isolates of Klebsiella spp:Identification
of CTX-1, a novel-Lacatamase. Antimicrob. Agents Chemother.1987; 20:323324.
SIROT, D. (1995). Extended spectrum plasmid-mediated β-lactamases. J.
Antimicrob. Chemother. 36: 19 – 34.
SIROT, D., De CHAMPS, C., CHANAL, C., LABIA, F., DARFEUILLEMICHAUD, A. Translocation of antibiotics resistance determinants including
extended spectrum β-lactamase between conjugative plasmids of Klebsiella
pneumoniae and Escherichia coli. Antimicrobial Agents and Chemotherapy.
1991; 35: 1576-1581.
SIU, L. K., LU, P., HSUAH, P., LIN, F. M., CHANG, S., LUH, K., HO, M., and
LEE. C. (1999). Bacteremia due to extended-spectrum β-lactamase producing
Escherichia coli and Klebsiella pneumoniae in a pediatric oncology ward: clinical
features and identification of different plasmids carrying both SHV-5 and TEM- 1
genes. J. Clin. Microbiol. 37: 4020 – 4027.
110
SKINNER, R., CUNDLIFFE, E., and SCHMIDT, F. J. (1983). Site of action of
ribosomal RNA methylase responsible for resistance to erythromycin and other
antibiotics. J. Biol. Chem. 258: 12702 – 12706.
SKOLD, O. (1976). R-factor-mediated resistance to sulfonamides by a plasmidborne, drug resistant dihydropteroate synhase. Antimicrob. Agents Chemother. 9:
49 – 54.
SKOLD, O. (2000). Sulfonamide resistance: mechanisms and trends. Drug Resist.
Updat. 3: 155 – 160.
SMITH, C. A., and BAKER, E. N. (2002). Aminoglycoside antbiotic resistance
by enzymatic deactivation. Curr. Drug Targets Infect. Disord. 2: 143 – 160.
STURENBURG, E., LANG, M., HORSTKOTTE, M. A., LAUFS, R., and
MACK, D. (2004). Evaluation of the MicroScan ESBL plus confirmation panel
for detection of extended-spectrum {beta}-lactamases in clinical isolates of
oxyimino-cephalosporin-resistant
Gram-negative
bacteria.
J.
Antimicrob.
Chemother. 54: 870 - 875.
SUBHA. A and ANANTHAN. S. 2002. Extended spectrum beta lactamase
(ESBL) mediated resistance to third generation cephalosporins among klebsiella
pneumoniae in Chennai. Volume: 20; 92-95.
111
TENNIGKEIT, J., and MATZURA, H. (1991). Nucleotide sequence analysis of a
chloramphenicol-resistance determinant from Agrobacterium tumefaciens and
identification of its gene product. Gene. 98: 113 – 116.
THOMAS, F. E., JACKSON, R. T., MELLY. M. A., and ALFORD, R. H. (1977).
Sequential hospital wide outbreaks of resistant Serratia and Klebsiella infections.
Arch. Intern. Med. 137: 581 - 584.
THOMSON, K. S., SANDERS, C. C., and WASHINGTON, J. A., II. (1991).
High level resistance to cefotaxime and ceftazidime in Klebsiella pneumoniae
isolates from Cleveland, Ohio. Antimicrob. Agents Chemother. 35: 1001 - 1003.
TILLOTSON, G. S., and WATSON, S. J. (2001). Antimicrobial resistance
mechanisms: what’s hot and what’s not in respiratory pathogens. Semin. Respir
Infect. 16: 155 - 168.
TIMMIS, K. N., GONZALEZ-CARRERO, M. I., SEKIZUKI, T., and ROJO, F.
(1986). Biological activities specified by antibiotic resistance plasmids.
J.Antimicrob.Chemother. 18 (Suppl. C): 1 - 12.
TOLMASKY, M. E. (2000). Bacterial resistance to aminoglycosides and betalactams: the Tn 1331 transposon paradigm. Front. Biosci. 5: 20 – 29.
112
TRAUB, W. H., SCHWARZE, I., and BAUER, D. (2000). Nosocomial outbreak
of cross-infection due to multiple-antibiotic-resistant Klebsiella pneumoniae:
characterization of the strain and antibiotic susceptibility studies. Chemotherapy
46: 1 – 14.
UBUKATA, K., YAMASHITA, N., and KONNO, M. (1985). Occurrence of a βlactam inducible penicillin-binding protein in methicillin-resistant staphylococci.
Antimicrob. Agents Chemother. 27: 851 – 857.
Van BAMBEKE, F., BALZI, E., and TULKENS, P. M. (2000). Antibiotic efflux
pumps. Biochem. Pharmacol. 60: 457 – 470.
VILA, J., MARCOS, A., MARCO, F., ABDALLA, S., VERGARA, Y., REIG,
R., VIRING, L. C. (1975). Chloramphenicol hydrolase. Methods Enzymol. 43:
734 - 737.
WAIYAKI, P. G. (1993). Bacterial drug resistance, diarrhoeal diseases, and
laboratory diagnosis of pulmonary tuberculosis (Editorial). E. Afr .Med. J. 70:
153 - 154.
WANG, M., SAHM, D. F., JACOBY, G. A., and HOOPER, D. C. (2004).
Emerging plasmid-mediated quinolone resistance associated with the qnr gene in
113
Klebsiella pneumoniae in the United States. Antimicrob. Agents Chemother. 48:
1295 – 1299.
WEIGEL, L. M., STEWARD, C. D., and TENOVER, F. C. (1998). GyrA
mutations associated with fluoroquinolone resistance in eight species of
Enterobacteriaceae. Antimicrob. Agents Chemother. 42: 2661 – 2667.
WILLIAMS, J. D. (1990). Antimicrobial substances. In: LINTON, A. H., and
DICK, H. M. (editors). Topley and Wilsons Principles of Bacteriology, Virology
and Immunity, Volume 1, General Microbiology and Immunity. Edward Arnold,
London.105 - 152.
WOHLLEBEN, W., ARNOLD, W., BISSONNETTE, L., PELLETIER, A.,
TANGUAY, A., ROY, P. H., GAMBOA, G. C., BARRY, G. F., AUBERT, E.,
DAVIES, J., and KAGAN, S. A. (1989). On the evolution of Tn21-like
multiresistant transposons: sequence analysis of the gene(aac C1) for gentamicinacetyltransferase-3-I(AAC(3)-I), another member of the Tn21-based expression
cassette. Mol. Gen. Genet. 217: 202 - 208.
WRIGHT,
G.
D.
and
THOMSON,
P.
R.
(1999).
Aminoglycoside
phosphotransferases: proteins, structure and mechanism. Front. Biosci. 4: 9 – 21.
114
YUAN, M., HALL, L. M. C., SAVELKOUL, P. H. M., VANDENBROUCKE, C.
M. J. E., and LIVERMORE, D. M. (2000). SHV-13. A novel extended spectrum
-lactamase, in Klebsiella pneumoniae isolates from patients in an intensive care
unit in Amsterdam. Antimicrob. Agents Chemother. 44: 1081 – 1084.
115