Download What Is Behind Antibiotic Resistance?

CE U P D A T E ANTIBIOTIC RESISTANCE
I
David A. Watson, PhD
Debby Bogaert
What Is Behind Antibiotic
Resistance?
What then, accounts for the seemingly rapid
ABSTRACT Antibiotic resistance develops among bacterialincrease in antibiotic resistance during the past
few years? In this series, we will attempt to answer
species just as Darwin might have predicted—that is,
this
through natural selection. Because bacterial species can share question, however speculatively, using the
gram-positive cocci Streptococcus pneumoniae and
DNA by a variety of mechanisms, and because some of these enterococcal species as examples.
microbes are inherently resistant to one or more major
antibiotics, we should not be surprised that clinically
How and Why Bacteria Resist
To understand why antibiotic resistance is
important species of bacteria can become resistant to
increasing among pathogenic bacteria, one must
commonly used antibiotics by chance alone. The rapid
understand the background of the microbes
increase in the percentage of strains resistant to one or more themselves.
antibiotics, especially broad-spectrum agents, may be the
result of increased use of such compounds.
Evolution of Bacteria and the Role of Mutation
This is the first article in a two-part continuing education series on antibiotic
resistance. After completion of the series, the reader will be able to explain, at the
level of the genetic material itself, basic mechanisms by which bacteria become
resistant to the actions of antimicrobial compounds. The reader also will be able to
enumerate reasons for the rapid increase in resistance of clinically important bacteria to commonly used antibiotics and articulate a strategy for maintaining the
effectiveness of currently available broad-spectrum antimicrobials.
From the
Department of
Microbiology and
Immunology,
University of Texas
Medical Branch,
Galveston, Tex (Dr
Watson) and
Eijkman-Winkler
Institute for Medical
and Clinical
Microbiology,
Utrecht University,
Utrecht, The
Netherlands (Ms
Bogaert).
Reprint requests to
Dr Watson, 301
University Blvd,
Galveston, TX 775551019; and e-mail:
david.watson@
utmb.edu
324
More than 50 years ago, British researchers
coaxed from the common mold Penicillium a
compound that later would prove to be, quite literally, a miracle drug. This monumental achievement changed medicine forever. As recently as
the 1960s, it was believed that the availability of a
wide variety of antibiotics would end the threat
to public health from infectious diseases.1
Following the development of penicillin, other
antibacterial compounds were identified and
produced in quantity, first from natural sources,
later by chemical synthesis. In many cases,
researchers were able to induce resistance to the
actions of these miracle drugs, even penicillin
itself.2 Even so, many of these drugs were widely
used for treating bacterial infections, and treatment failures were rare.
LABORATORY MEDICINE
VOLUME 28, NUMBER 5
MAY 1997
Living organisms evolve when changing environmental conditions prompt the selection of genetic
variants within a species over time. Charles
Darwin termed this process natural selection. In
natural selection, an organism does not specifically target its response to a given selection event
(any obstacle to survival) in order to increase the
likelihood of its survival; rather, changes occur
randomly within the organism's genome. This
change may or may not confer increased fitness
on the microbe. These alterations are collectively
known as mutations. They generally occur at a
fixed rate of 1 in 109 to 1 in 1010 replicated base
pairs. Mutations can be of the following two basic
types, which are further described in Fig 1.
1. One nucleotide may be substituted for another
in the organism's DNA. (This process is known as
a base substitution or point mutation.) This substitution results in either early termination of
protein translation (known as a nonsense mutation), or a change in the amino acid specified by
a given codon (a missense mutation).
2. Frameshift mutations may occur, resulting in
either the insertion or deletion of a nucleotide.
This alteration leads to a shift to the left or to the
right in the reading frame used by the ribosome
for protein translation from messenger RNA.
messenger RNA, on
the protein's amino
acid sequence resulting from translation.
Top, the effect of
changes in a single
DNA base. Bottom,
the effect of
frameshift mutations.
Yellow indicates
codons specifying
methionine (always
the first amino acid
in a growing protein);
gray, termination or
stop codons; arrows,
nucleotide (DNA
base) changes, additions, and
(The messenger RNA itself is transcribed from
the coding strand of DNA, hence the paradigm,
DNA ^ RNA * protein.) Mutations in the first
or second position of a triplet, or codon, of the
DNA bases that encode a growing protein's
amino acid are more likely to result in a change
in that amino acid than are such changes at the
third position of the codon; this phenomenon is
known as wobble. If a mutation results in the
addition of a different amino acid to a growing
protein, then the question becomes whether this
altered protein confers greater survivability on
the organism in the event of environmental
changes.
Compared with higher organisms, certain factors are important in the evolution of microbes:
• Small genome. Genomes of bacterial species are
much smaller than the more complex DNA
assemblages of higher plants and animals.
Genome size of most pathogenic bacteria is
between 1 million and 10 million nucleotide
bases, or three orders of magnitude (1,000-fold)
less than that of higher organisms. Replication of
the entire genome for bacteria requires much less
time because far fewer nucleotide bases must be
added to make an entirely new copy of a bacterium's DNA.
• Short doubling time. Bacteria generally are much
smaller than the cells of higher organisms. They
therefore require less time to undergo binary fission. If a random mutation occurring within the
DNA of a given microbe or acquisition of exogenous DNA from another bacterium confers
increased fitness relative to a particular selective
pressure, the mutant organism can expand
quickly into a previously unfilled niche.
Given these two factors, proliferation in an
uncolonized niche can be rapid compared with
that of higher organisms. (A bacterium with a
doubling time of 30 minutes can undergo a sufficient number of divisions to equal the entire
human population of the earth in approximately
16 hours given optimal growth conditions.) This
occurs through rapid proliferation of a clonal line
descended from a single mutated, or otherwise
altered, progenitor bacterial cell. This is precisely
the case with bacteria that become resistant to
antibiotics, as will be discussed in the second article of this series.
deletions. The amino
acid encoded by
each codon is
indicated by its threeletter abbreviation in
the lower right
corner of each box.
Arg indicates
arginine; Cys,
cysteine; Gin,
glutamine; His,
histidine; Leu,
leucine; Met,
methionine; Pro,
proline.
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Other Ways Bacteria Stay "Fit"
Mutation represents only one of a number of ways
in which bacterial strains may acquire new and
potentially fitness-increasing traits. Entirely new
genes can be incorporated from other bacteria
through a number of processes, as depicted in Fig
2. These include:
• Transformation, during which naked DNA from
the ambient environment binds specifically to a
receptor on the surface of the bacterium; the
DNA is internalized and incorporated into the
recipient genome via a process called homologous recombination.
MAY 1997
VOLUME 28, NUMBER 5
in
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Test Your
Knowledge
Look for the CE
Update exam on
Antibiotic Resistance
(707) in the June
issue of Laboratory
Medicine.
Participants will earn
2 CMLE credit hours.
LABORATORY MEDICINE
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1. Transformation
Fig 2. Three routes
by which a bacterial
pathogen acquires
DNA from another
bacterium. Green
indicates exogenous
transforming DNA;
blue, the surface
protein specifically
involved in binding
and internalizing the
DNA. Genetic material incorporated into
the recipient
genome following
transformation is
represented by a
green rectangle
superimposed onto
the dark blue oval
representing the
genomic DNA of the
recipient. Similarly,
DNA incorporated
following conjugation or transduction
is represented by
red and yellow rectangles, respectively.
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• Conjugation, which is the direct transfer of one
or more genes from a donor to a recipient, either
by way of a specialized "conjugation" tube or
through direct cell-to-cell contact
• Transduction, whereby a bacteriophage (a virus
capable of infecting a bacterium) incorporates
fragments of bacterial DNA into its own genome,
which then are transferred to new, previously
uninfected, bacterial strains by newly synthesized
infectious bacteriophage particles
in office visits for both (predominantly bacterial)
otitis media in children and sinusitis in adults.3
These two observations, when considered
together with the well-described increasing rates
of antibiotic resistance by a variety of clinically
important bacterial species, led the authors of the
study to state that: "Given sufficient time and
appropriate circumstances, there is a strong association between the magnitude of use and the
emergence and spread of antimicrobial-resistant
strains."
Even so, data directly bearing on the question
of whether increased use of antimicrobials is
responsible in whole or in part for increased rates
of resistance are lacking. Indirect evidence supporting a link between increasing antibiotic usage
and increasing rates of resistance, however, can
be found in a recent Spanish study demonstrating a correlation between greater use of ampicillin in a hospital setting and higher levels of
resistance to this antibiotic among isolates from
their hospital of the major bacterial pathogens of
childhood infections4 (Fig 3). In the case of the
pneumococcus, among the most statistically significant factors for acquisition of a penicillinresistant strain of this bacterial species is previous
or current 3-lactam antibiotic therapy.5-7 It also
has been suggested that empiric therapy using
vancomycin for infections suspected to be due to
methicillin-resistant staphylococcal species may
be a major factor in the spread of strains resistant
to this antibiotic.8
Each of these mechanisms has been shown to
operate in the movement of clinically relevant
DNA (antibiotic resistance genes and virulence
genes) from one bacterium to another. For
example, it is widely acknowledged that the
pneumococci have acquired genes encoding
altered penicillin-binding proteins via natural Nonantimicrobial Use of Antibiotics
transformation. Under laboratory conditions, Another mechanism thought to contribute to
Enterococcus faecalis has been shown to transfer antibiotic resistance has been the use of such
genes required for expression of the vancomycin- compounds to stimulate growth in agriculturally
resistance phenotype to Staphylococcus aureus by important livestock species. Although this comway of DNase-resistant conjugation. Last, the gene- pound is not used in livestock species as a growth
encoding pyrogenic exotoxin A in Streptococcus promoter, another antibiotic (bacitracin)
pyogenes (group A Streptococcus) is thought to be employed in this fashion leads to overgrowth of
transmitted among isolates of this pathogen by Enterococcus faecium in the gut. Livestock species
transduction.
therefore may serve as an environmental reservoir for vancomycin-resistant E faecium. The use
of antibiotics in livestock feed also has been
Empiric Antibiotic Usage
A recently published comprehensive survey of the linked with the development of antibiotic resis9
prescribing practices of office-based physicians in tance in Salmonella. An example of this is the
the United States indicates that use of third- identification of vancomycin resistance among
generation cephalosporin antibiotics (a category porcine isolates of enterococcal species followed
of antibiotic exhibiting broad-spectrum antimi- by the isolation of genetically identical strains
10
crobial activity) has increased dramatically from humans.
within the past 15 years. 3 This increase has
occurred concomitantly with a notable increase
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Conclusion
Clinical antibiotic usage presents a man-made
selective pressure to bacterial pathogens. Microbes
respond to this challenge by evolving toward resistance to antimicrobial compounds in accordance
with the principles of natural selection by way of
mutation or through acquisition of resistance
from other bacteria. In recent years, increasing use
of empiric therapy and nonantimicrobial administration of antibiotics have contributed to enhancing this selection pressure.©
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References
1. Burnet M. Natural History of Infectious Disease. Oxford,
United Kingdom: Cambridge University Press; 1962:2.
2. Schmidt LH, Sesler CL. Development of resistance to
penicillin by pneumococci. Proc Soc Exp Biol Med. 1943;
52:352-357.
3. McCaig LF, Hughes JM. Trends in antimicrobial drug prescribing among office-based physicians in the United States.
JAMA. 1995;273:214-219.
4. Gomez J, Ruiz-Gomez J, Hernandez-Cardona JL, Nunez
ML, Cameras M, Valdes M. Antibiotic resistance patterns of
Streptococcus pneumoniae, Haemophilus influenzae, and
Moraxella catarrhalis: A prospective study in Murcia, Spain,
1983-1992. Chemotherapy. 1994;40:299-303.
5. Nava JM, Bella F, Garau J, et al. Predictive factors for
invasive disease due to penicillin-resistant Streptococcus pneumoniae: a population-based study. Clin Infect Dis.
1994;19:884-890.
6. Bedos J-P, Chevret S, Chastang C, Geslin P, Regnier B,
and the French Pneumococcus Study Group. Epidemiological
features of and risk factors for infection by Streptococcus pneumoniae strains with diminished susceptibility to penicillin:
findings of a French study. Clin Infect Dis. 1996;22:63-72.
300
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1986
1989
1992
Year
7. Hofmann J, Cetron MS, Farley MM, et al. The prevalence
of drug-resistant Streptococcus pneumoniae in Atlanta. N Engl
JMed. 1995;333:481-486.
8. Centers for Disease Control. Recommendations for preventing the spread of vancomycin resistance: recommendations of the hospital infection control practices advisory
committee. MMWR. 1995;44:1-13.
9. Cohen ML, Tauxe RV. Drug-resistant Salmonella in the
United States: an epidemiologic perspective. Science. 1986;
234:964-969.
10. Bates J, Jordens JZ, Griffiths DT. Farm animals as a
putative reservoir for vancomycin-resistant enterococcal
infection in man. J Antimicrob Chemother. 1994;34:507-514.
Glossary
Amino acid—one of a group of 20 related compounds that
are the component subunits of proteins
(3-lactam antibiotic—one of a group of antibiotics possessing a (3-lactam ring as part of the primary structure of the
molecule
Binary fission—division into two equal daughter cells
Codon—group of three nucleotide bases specifying the
proper amino acid to add to a growing protein molecule
Conjugation—direct passage of DNA from one bacterial cell
to another with which it is in contact
DNase-resistant conjugation—movement of DNA between
cells in a fashion that is resistant to D N A - d e g r a d i n g
enzymes (DNases)
Empiric antibiotic therapy—initiated on the basis of signs
and symptoms, but when definitive identification by bacterial culture or other accepted diagnostic methodology is
lacking
Evolution—a process resulting in genetic changes that can
be passed on to successive generations in a population
Fitness—a relative measure of the ability of an organism to
survive under a given set of selective constraints
Fig 3. Results of a
prospective study
conducted in a
Spanish hospital.
Resistance of
Streptococcus
pneumoniae,
Haemophilus
influenzae, and
Moraxella catarrhalis
to ampicillin were
followed over time
relative to overall
hospital usage of
this same antibiotic.
As usage of
ampicillin increased,
resistance to this
antibiotic increased.
Frameshift mutation—addition or removal of one or a few
nucleotide bases resulting in a shift to the left or right in the
reading frame for a given protein
Genome—the DNA comprising all of the genetic information for a given species
Mutations—changes in the composition of c o m p o n e n t
bases encoding genes within the genome of a given organism
Reading frame—the proper placement of nucleotide bases
into groups of three to allow for the transcription and
translation of a gene product
Substitution mutation—exchange of one nucleotide base for
another at a specific position within the genome of a given
organism
Transcription—the synthesis of a messenger RNA molecule
encoding a protein by an RNA polymerase using DNA as
the template
Transduction—transfer of a gene from one bacterium to
another by way of a bacteriophage (a virus that infects a
bacterium)
Transformation—uptake and expression of exogenous DNA
by a bacterium
Translation—synthesis of a protein by a ribosome from a
messenger RNA molecule used as a template
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