Download catarrhali

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, BANGALORE
KARNATAKA
PROFORMA FOR REGISTRATION OF SUBJECT FOR PROJECT.
1.
NAME OF THE CANDIDATE
SAVITHA RAVEENDRAN
ADDRESS
POST GRADUATE (M.Sc. M.L.T)
DEPARTMENT OF MICRIBIOLOGY,
ST.JOHN’S MEDICAL COLLGE AND
HOSPITAL, BANGALORE- 34.
2.
COURSE OF STUDY
1ST YEAR M.Sc. MLT
3.
SUBJECT
MICROBIOLOGY
4.
DATE OF ADMISSION
3 SEPTEMBER 2012
5.
TITLE OF THE TOPIC
IDENTIFICATION AND ANTIBIOTIC
SUSCEPTIBILITY PATTERN OF
Moraxella catarrhalis.
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6. BRIEF RESUME OF THE INTENDED WORK
6.1 NEED FOR THE STUDY
Moraxella catarrhalis is an opportunistic pathogen. It is found as a normal commensal in
respiratory tract of human. It is a common cause of bronchitis and bronchopneumonia
seen in patients.(1)In infants it appears that seasonal nasopharyngeal colonization,
peaking in winter is common. Infections are thought to represent extensions of colonized
areas.(2).Studies have shown that organisms from the nasopharynx can spread easily to
the middle ear and lungs. Since the late 1970s it has been clear that Moraxella catarrhalis
is an important and common human respiratory pathogen. In immune compromised
hosts, the bacterium can cause a variety of severe infections including pneumonia,
endocarditis,septicemia and meningitis (5). Moraxella catarrhalisis frequently present in
the sputum of adults with chronic obstructive pulmonary disease (COPD). COPD is the
fourth most common cause of death in the world and is associated with enormous
morbidity and health care costs (3).Adults with COPD develop variable humoral immune
responses to Moraxella catarrhalis after exacerbations, including new serum IgG and
new mucosal IgA to epitopes on the bacterial surface (4).Doren (1986) suggested that it is
the third ranking pathogen in this particular patient population following streptococcal
pneumoniae and Haemophilus influenzae.
Moraxella catarrhali has also been reported to cause keratitis, suppurative arthritis,
bacteremia, meningitis, endocarditis, urogenital infection, otitis media and sinusitis.
Additionally it has been established as a nosocomial pathogen(2).
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Moraxella catarrhalis is resistant to Penicillin due to the production of beta lactamase(1).
The production of Beta -lactamase by Moraxella catarrhalis, was first reported in1977.
Today, more than 90% of the strains isolated worldwide produce beta-lactamase.In
Moraxella catarrhalis two types of Beta-lactamases can be found that are phenotypically
identical: the BRO-1 and BRO-2 types. The enzymes are encoded by chromosomal
genes, and these genes can be relatively easily transferred from cell to cell by
conjugation. Beta-Lactamase from Moraxella catarrhalis not only protects the bacteria
producing the enzyme but also is thought to inactivate penicillin therapy of concomitant
infections by serious airway pathogens such as S. pneumonia and/or nontypeable H.
influenzae. This phenomenon is referred to as the indirect pathogenicity of Moraxella
catarrhalis. Indeed, in such circumstances, treatment failures have been reported (5).
Therefore, taking in to consideration that Moraxella catarrhalis has evolved to become a
beta lactamase producer it is important to isolate Moraxella catarrhalis and study their
antibiotic susceptibility pattern for the management and prevention of the infections.
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6.2 REVIEW OF LITERATURE
History
Moraxella catarrhalis has an interesting and checkered taxonomic history. (6) Initially
Seifert (1882) described a case of purulent tracheobronchitis and penned the putative
agent as Micrococcus catarrhalis defined as a species as Micrococcus (2).After having
initially been named Micrococcus catarrhalis, the organism's name was subsequently
changed to Neisseria catarrhalis, because of its similarities in phenotype and ecological
niche to commensal Neisseria species. The bacterium was transferred to a new genus,
Branhamella, in 1970, because of limited DNA homology with Neisseria species.
Branhamella catarrhalis was subsequently placed in the genus Moraxella on the basis of
biochemical and genetic relatedness, and Moraxella catarrhalis is now the widely
accepted name. (6)Ghon and Pfeiffer (1902) recognized the species as a pathogen.
Thereafter nvestigators in both Europe and the USA (Benzancon1905, Jordan 1908,
Hissand Zinsser 1910) confirmed its pathogenic potential. In1921 Gordon attempted to
define a relationship between the organism and common cold. Failing to find any
association he submitted that Moraxella catarrhalis should be considered as a
Commensal. Berger(1963) and Knapp etal(1984) realized that part of the problem in
defining the pathogenic nature of Moraxella catarrhalis was the organisms frequent
misidentification withthe respiratory commensal Neisseria cinerea(2).However, the
bacterium was described as an “organism whose pathogenic propensities are
known to be slight or non-existent” and was thus ignored for decades. Point
prevalence studies indicate that Moraxella catarrhalis colonizes the respiratory tract
of 5 to 32% of adults with COPD at any one time. (3)
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More recently, the widespread use of pneumococcal conjugate vaccines has altered
nasopharyngeal colonization patterns and caused an increased prevalence of colonization
and infection by Moraxella catarrhalis. More than 90% of Moraxella catarrhalis produce
a β-lactamase and are, thus, resistant to ampicillin. Two types of β-lactamases have been
identified, BRO-1 and BRO-2. After the rapid acquisition of β-lactamase in the 1970s
and 1980s, the antimicrobial susceptibility of Moraxella catarrhalis has remained
relatively stable in surveys of worldwide collections of strains .Sensitive methods, such
as polymerase chain reaction, to detect Moraxella catarrhalis and other bacterial
pathogens in respiratory secretions are in development. The application of such sensitive
assays is likely to contribute important new observations about the epidemiology and
disease patternsof Moraxella catarrhalis, but they are not yet commercially available. (6)
Taxonomy and Description of the Genus
The Genus Moraxella is classified in family Moraxellaceae. Species belonging to Genus
Moraxella are Gram negative,non-motile,coccobacilli (exception Moraxella catarrhalis
are diplococci) with occasional formation of short chains. Cellular pleomorphisam is
typical as the culture ages or with growth at increased temperature. All species have
fimbriae and with exception of Moraxella bovisare encapsulated. They are asaccharolytic
and negative for urease,indole and hydrogensulfide. Several species are weakly hemolytic
on blood agar. The most important species is Moraxella catarrhalis.
The other species associated are
Moraxella nonliquefaciens- isolated from nose, throat, mouth and eye.
Moraxella lacunate -
isolated from mucous membrane of humans and animals
Moraxella osloensis– common resident of human genital tract and other mucous
membrane.
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MINOR SPECIES – Moraxella atlantae, Moraxella lincolinii
Animal associated Species
Moraxella caviae–isolated from throat of guinea pig
Moraxella bovis – bovine keratoconjuctivitis
Moraxella ovis– from sheep
Moraxella canis– from Dogs
Apart from Moraxella catarrhalis other Moraxella’s have little pathogenic potential (2)
Epidemiology and Transmission
Moraxella catarrhalis is an exclusively human pathogen with an ecological niche in the
human respiratory tract. The prevalenceof colonization of the upper respiratory tract is
highly dependent on age. Whereas the rate of colonization among adults is low (1%-5%),
nasopharyngeal colonization is quite common through infancy. A substantial variation in
rates of colonization is observed in different geographic regions. (6) Higher colonization
rate have also seen in preschool children with asthma than in healthy children. (7)A
change in patterns of nasopharyngeal colonization is occurring in countries where
pneumococcal conjugate vaccines are used widely. Colonization by vaccine serotypes of
pneumococcus is decreasing, and colonization by the nonvaccine pneumococcal
serotypes Hemophilus influenza andMoraxella catarrhalis is increasing, resulting in a
shift in the pathogens that cause otitis media in children who had received the vaccine,
compared with the prevalence among episodes that occurred before introduction of the
pneumococcal conjugate vaccine. Pneumococcal vaccination does not affect the genetic
diversity of nasopharyngeal isolates of Moraxella catarrhalis, suggesting that the
increase in prevalence of colonization will translate to increased rates of otitis media.
Similar shifts are being observed among children and adults with sinusitis (6).
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Serological studies have indicated that the children with otitis media due toMoraxella
catarrhalis produce antibodies against a variety of protein antigen of Moraxella
catarrhalis.(7) Biofilms of Moraxella catarrhalis are present in the middle ears of
children with otitis media and may account, in part, for recurrent and chronic otitis
media. However, the role of biofilms in the pathogenesis of otitis media is not yet fully
elucidated(6).Moraxella catarrhalis behave as a primary or as a secondary pathogen
superimposed on an antecedent viral infection. (e.g. Respiratory syncytial virus).Lower
respiratory tract infection due to Moraxella catarrhalis in adults occur predominantly in
elder and immunocompromised patients particularly with COPD, bronchiectasis,
congestive heart failure and predisposition of aspiration. Immunologic abnormalities
because of underlying disease (e.g. diabetes,alcoholism, HIV,transplantation) are also
important contributing factor. Nosocomially acquired pneumonia due toMoraxella
catarrhalis occur in hospital respiratory units and paediatric intensive care unit. (7)
Virulence Factors
Hellio etal (1988) demonstrated the presence of a polysaccharide capsule. Antigenic
characters has determined that the antigenic nature of outer membrane proteins is
conserved among various strains.(7) Resistance to β-lactam antibiotics, mediated by
either the BRO-1 or BRO-2 β-lactamase, is a characteristic acquired by the vast majority
of strains within the past 20 years . The ability of Moraxella catarrhalis to resist
complement – mediated lysis is considered to be an important virulence factor of this
organism. (8)
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Lipoologosaccharide – Studies using monoclonal antibodies demonstrate its structure
Fimbriae – Functionas adhesins
Hemagglutinins-Believed to contribute to adherence
Outer membrane proteins
UspA protein
UspA 1 – Used as adhesins
UspA 2 –Binds to complement system,mucosal adherence,resistance to non-immune sera
C D Protein – Specifically binding tonasal and middle ear mucins
LpbA and LpbB– lactoferrinbinding protein
TbpA and TbpB– Transferrin binding protein
CopB protein – Function in iron acquisition in growth.
E- protein – It may assist in transport of fatty acid to cells.(7)
Laboratory Diagnosis
Specimens
Clinical samples include sputum, blood, middle ear effusion, tracheal aspirate or BAL.
Direct examination
Grams stain
Moraxella catarrhalis is a gram – negative diplococcus with flattened abutting sides. The
sample should be evaluated by microscopy prior to culture to make sure that the Gram
negative diplococcic are associated with inflammatory cells and there is adequate single
morphotype bacteria present.
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Culture methods
Moraxella catarrhalis are non-fastidious organism produce 1-3 mm whitish grey colonies
on blood agar and chocolate agar within 24 hour incubation. Optimum temperature for
growth is 35 – 370C(7).Moraxella catarrhalis colonies can be solid along the agar surface
without disruption this is termed the “hockey puck sign.” In addition, after 48 h of growth
Moraxella catarrhalis colonies tend to be larger and take on a pink color.(6) A selective
media containing acetazolamide and antibiotics has been described.(7)
Biochemical Reactions
Catalase positive
Oxidase positive
Asaccharolytic
Indolenegative
H2S negative
Nitrate positive
DNAse positive
Identification based on
Butyrate esterase (tributyrin test)
The rapid identification of Moraxella catarrhalis is important. The tributyrin spot test
will differentiate Moraxella catarrhalis from Neisseria species. The enzyme butyrate
esterase produced by Moraxella catarrhalis hydrolyses the tributyrin substrate in buffered
salt medium. The reaction yields butyric acid which lowers the pH of the medium
resulting in a yellow colour change. This test can be tested using commercially available
kits.
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Complement resistance
Moraxella catarrhalis strains can be tested for their sensitivity to serum complement –
mediated lysis in a microtiter bactericidal assay,that used 50% pooled human serum.
Strains were subdivided into 3 categories: resistant (R; >50% survival of the bacteria
during a 3-h incubation in 50% pooled human serum), sensitive (S ;< 3% survival of the
bacteria after incubation for 1 h in 50% pooled human serum), and intermediate. (8)
Adherence of bacteria to HEp-2 cells
An assay developed to measure adherence of group B streptococci to epithelial cells was
adapted for use with Moraxella catarrhalis. This assay is based in the binding of
fluorescein – labeled bacteria to monolayers of larynx (HEp-2) epithelial cells in vitro, as
determined by a fluorescence-based micro titer plate reader(8).
Molecular Techniques
Methods for strain typing
These methods include enzymatic bio typing, polyacrylamide gel electrophoresis of
whole cell protein, immunoblotting, and pulsed – field gel electrophoresis and restriction
fragment length polymorphism endonuclease analysis. Two DNA typing method probe
generated restriction fragment length polymorphism analysis and single adapter modified
fragment polymorphism analysis were recently used study the genetic relationships
among 90 Moraxella catarrhalis strains. (7)
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Treatment, Prevention and Control
Animal models. Studies of the pathogenesis and host response to Moraxella catarrhalis
infection have been limited by the absence of a good animal model of infection. Several
studies suggest that Moraxella catarrhalis are generally susceptibleto amoxicillinclavulnate,2nd and 3rd generation cephalosporins, Macrolids, fluroquinolones etc.
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6.3AIMS AND OBJECTIVES
To identify
Moraxella catarrhalis from
clinical specimen (sputum, tracheal
aspirates, BAL , blood, middle ear specimen).
To determine the Antibiotic susceptibility pattern by disc diffusion method for the
isolates.
7.MATERIALS AND METHODS
7.1 Source of data
All the Moraxella catarrhalis isolated from clinical specimen like blood,middle ear
effusions, sputum, tracheal
aspirates, BAL
in Microbiology department St Johns
Medical college,Bangalore for a period of one year (approximately 30 samples)starting
from December 2012.
7.2 Inclusion criteria
1. Moraxella catarrhalis grown from sputum samples where WBC’s is 10 times
the number of Squamous epithelial cells and there are 3 to 4 + of Gram
negative diplococcic seen will be included .
2. In all other samples ( middle ear effusion,tracheal aspirates,BAL ) if there is
Gram negative diplococci associated with inflammatory cells is included.
3. In blood sample where there is pure growth of Moraxella catarrhalis will be
included.
7.3 Exclusion criteria
Nil
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7.4 Methods
a) Gram’s stain
Gram negative diplococcus with flattened abutting sides. The sample should be
evaluated by microscopy prior to culture to make sure that the Gram negative
diplococci are present . In sputum samples if the number of WBC’s is 10 times the
number of Squamous epithelial cells and there
are 3 to 4 + of a single
morphotype of bacteria, accept the specimen for culture.
b)Culture
These sample will be plated on the following media
Blood agar
Chocolate agar
Mac Conkey agar
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Colonies on Blood agar and Chocolate agar are round, opaque, convex and greyish
white. The colonies remains intact when pushed across the surface of the agar and
no growth will be obtained in MacConkey agar.
Do Grams stain ,if gram negative diplococcous with flattened abutting sides
present
Do Catalase and Oxidase test, if positive
Go to further identification of Moraxella catarrhalis by following method
c)DNAse test
This test detects the degradation of DNA by bacterial species that produce DNase. It
can be tested by using DNAse agar. The agar should be inoculated with the
organisms and incubated at 370C for 18 – 24 hours. The plate should be flooded
with 1 M HCl that precipitates DNA and turns the medium cloudy. The presence of
a zone of clearing around the area of growth indicates DNAse production.
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d)Method of rapid identification of Moraxella catarrhalis
 Butyrate esterase (tributyrin test)
The rapid identification of Moraxella catarrhalis is important. The tributyrin spot test
will differentiate Moraxella catarrhalis from Neisseria species. The enzyme butyrate
esterase produced by Moraxella catarrhalis hydrolyses the tributyrin substrate in buffered
salt medium. The reaction yields butyric acid which lowers the pH of the medium
resulting in a yellow colour change.
Antibiotic Susceptibility
 Disc Diffusion Method
Media – Muller Hinton Agar with (5%) sheep blood
Method
Inoculum is prepared visually to match the turbidity of 0.5 McFarland standard. Do
a lawn culture on MHA with sheep blood.Place the disc on media. Incubate at
350 C for 16 – 18 hours in 5 %co2.
Antimicrobial agents used
Selected according to CLSI guidelines 2011
Amoxicillin/clavulanate
Cefuroxime
Cefpodoxime
Cefixime
Azithromycin
Clarithromycin
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Ciprofloxacin
Levofloxacin
Beta lactamase testing
Nitrocefin has very high affinity for bacterial beta lactamase. A nitrocefin disc is
removed from the container and placed on a slide or on a empty petri dish lid. The disc is
moistened with a loopfull of distilled water. A small amount of organism is applied on
the moistened nitrocefin disc and incubated for 5 minutes in room temperature.
Development of pink red colour if the isolate is beta lactamase positive
7.3 Does the study require any investigation or interventions to be conducted
on patients or other human beings?
No, study will be done on isolates obtained from clinical sample only.
7.4 Has ethical clearance been obtained from your institution in case of 7.3
Not applicable
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8.LIST OF REFERENCES
1)Subhash Chandra parija 2009,page 334, Parijas text book of Microbiology and
Immunology,Elsevier.A division of Reed Elsevier
2)S. Peter Borriello,Pattrick . R . Murray and Guido funke, 2005 Chapter 49 page
1305-1308Acinetobacter and Moraxella,Topley and Wilsons Microbiology and Microbial
Infections,Bacteriology Volume 2,10th edition.Edward Arnold (Publishers) Ltd.
3) . Timothy F Murphy, Aimee L. Brauer, Brydon J. B. Grant and Sanjay Sethi
Moraxella catarrhalis in Chronic Obstructive Pulmonary Disease Burden of Disease and
Immune Response. Journal of Respiratory Critical.Care Med.2005.vol 172.pp 195-199
DOI: 10.1164/rccm.200412-1747oc on April 1,2005
4)FarisBakri,Aimee L. Brauer, Sanjay Sethi and and Timothy F. Murphy Systemic
and Mucosal Antibody Response to Moraxella catarrhalis after Exacerbations of Chronic
Obstructive Pulmonary Disease.Journal of Infectious Diseases.2002 ;185:632-40
5) H. V.Prashanth , R. M. Dominic Saldanha ,ShaliniShenoy Moraxella catarrhalis A Rediscovered Pathogen. International Journal of Biological& Medical Research2011; 2
(4):979-981
6) Timothy F. Murphy G.IyerParameswaran ,Ellie J.C. Goldstein Moraxella catarrhalis,
a Human Respiratory Tract Pathogen . Clinical Infectious Diseases. 2009; 49:124-31
7)Washingtonc.Winnetalchapter 11.Konemans color Atlas and Textbook of Diagnostic
Microbiology,Edition 6 .Lippincot Williams and Wilkins.
8) Hester J. Bootsma1, Han G. J. van der Heide, Simone van de Pas, Leo M. Schoulsand
Frits R. Mooi Analysis of Moraxella catarrhalis by DNA Typing: Evidence for a Distinct
Subpopulation Associated with Virulence Traits Journal of Infectious Diseases.
2000;181:1376-87
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9.
SIGNATURE OF CANDIDATE
DR. MARY DIAS
10.
NAME AND DESIGNATION OF
ASSISTANT PROFESSOR
THE GUIDE
DEPARTMENT OF
MICROBIOLOGY,
ST.JOHN’S MEDICAL
COLLEGE,
BANGALORE – 34
11.
REMARKS OF THE GUIDE
SIGNATURE OF THE GUIDE
HEAD OF THE DEPARTMENT
DR. S. MURALIDHARAN
PROFESSOR AND HEAD
DEPT. OF MICROBILOGY
ST.JOHN’S MEDICAL
COLLEGE BANGALORE –
560034
12.
SIGNATURE OF THE HEAD OF
THE DEPARTMENT
REMARKS OF THE DEAN OF
THE INSTITUTION
SIGNATURE OF THE DEAN
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