JOURNAL OF CLINICAL MICROBIOLOGY, Aug. 1998, p. 2169–2172 Vol. 36, No.

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MINIREVIEW

Preventing Antibiotic Resistance through Rapid Genotypic

Identification of Bacteria and of Their Antibiotic Resistance
Genes in the Clinical Microbiology Laboratory
MICHEL G. BERGERON* AND MARC OUELLETTE
Centre de Recherche en Infectiologie de l’Université Laval and Division of Microbiology,
Faculty of Medicine, Université Laval, Québec, Canada G1V 4G2

INTRODUCTION sible for the infection and its susceptibility to antibiotics be-
come available. With the actual state-of-the-art technology,
The emergence of drug resistance in microorganisms is a which dates back to the last century, we cannot even tell ac-
serious problem, and several strategies have been proposed to curately before 18 to 24 h whether a clinical sample has bac-
try to tackle it. Prevention should be the ultimate solution, and teria or not. This is of importance because no bacteria can be
vaccines have been suggested as a strategy that can be used to grown out of more than 80% of all normally sterile clinical
slow down the emergence of drug resistance by decreasing the samples sent to clinical microbiology laboratories (4). The lack
infection rate and hence antibiotic usage (15). Unfortunately, of a timely response by the laboratory has consequences on
we are far from this ideal. Broad surveillance programs (22) antibiotic usage and prescription. Patients must be treated
and education of clinicians, pharmacists, veterinarians, drug empirically. When severe or nosocomial infections are sus-
company representatives, and the public about the spread of pected, they are often treated with broad-spectrum antibiotics.
antimicrobial resistance and the consequences of antibiotic The increased use of broad-spectrum antibiotics is not re-
misuse should also have a significant impact (21). Restrictive stricted to hospitalized patients in intensive care units or pa-
use of newer and broad-spectrum antibiotics has also been ap- tients seen in emergency rooms, however. Indeed, a recent
plied and advocated. Strict application of therapeutic guide- American survey has indicated that toxic and expensive broad-
lines might also be useful. spectrum antibiotics are prescribed more frequently for the
Another strategy is to increase our understanding of the treatment of common infections by office-based physicians
biochemical basis of antimicrobial resistance mechanisms (14). Clearly, with 80% of normally sterile specimens received
which should suggest preventive and therapeutic strategies for in the microbiology laboratory not growing any microorganism,
overcoming resistance (17). The understanding of resistance several patients are receiving antibiotics even if they do not
mechanisms is also necessary for the development of the tools have a bacterial infection because there are no accurate ways
necessary to detect resistance by methods other than pheno- of determining before the next day whether the clinical sample
typic testing, which now includes disc diffusion or dilution tests harbors bacteria. In line with this latter argument, a recent
(MIC tests) to evaluate the susceptibilities of microbes to study in Spain has indicated that on any particular day, the
antibiotics. We advocate that rapid (#1 h) identification of number of antibiotic prescriptions exceeded by three times the
microorganisms should literally modify the habits of the pre- number of bacterial infections diagnosed (3). Moreover, mi-
scribers and contribute to a reduction of the dissemination crobiologic results are available so slowly that physicians rarely
of antimicrobial resistance. While most DNA-based tests consult them unless the patient is not responding to the given
are presently used to identify viruses or bacteria whose iden- antibiotic. If physicians could have in hand the identity of the
tification is tedious, like Mycobacterium tuberculosis or chla- microorganism and its resistance profile from the microbiology
mydia, we are suggesting that the time is ripe for the use of laboratory at the same time that they have the biochemistry
these tests to identify bacteria causing common and deadly and hematology results, antibiotic prescription rates could go
bacterial diseases. We believe that the simultaneous rapid ge- down dramatically, and when antibiotics are needed, more
notypic identification of bacteria and their antibiotic resistance targeted and inexpensive antibiotics could be used. On the
genes will have a major impact on the treatment of infectious other hand, whether you are using phenotypic or genotypic
diseases while contributing to a better control of antimicrobial identification systems, the presence of bacteria or even the
resistance (14). absence of bacteria in the clinical specimens does not neces-
Speed is the essence when one deals with bacterial infec- sarily mean the presence or the absence of infection because
tions. Although the Gram stain can sometimes be helpful, clinical judgment should always prevail.
presently, diagnosis in the clinical microbiology laboratory is
only confirmatory because a clinical decision has been made
long before (usually 48 h) the identity of the organism respon- RAPID IDENTIFICATION OF MICROORGANISMS AS A
MEANS OF DECREASING THE EMERGENCE OF
ANTIMICROBIAL RESISTANCE
* Corresponding author. Mailing address: Centre de Recherche
en Infectiologie de l’Université Laval, CHUQ, Pavillon CHUL, 2705 The advances in sample preparation, DNA-based amplifica-
boul. Laurier, Ste-Foy, Québec G1V 4G2, Canada. Phone: (418)-654- tion techniques, and product detection have evolved to the
2705. Fax: (418)-654-2715. E-mail: [email protected] extent that it is now possible to identify microorganisms di-
.ca. rectly from clinical specimens in 1 h (13). Moreover, as these

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DNA-based tests evolve, their sensitivity will allow the detec- nique of susceptibility testing is relatively simple, it requires
tion of a single copy of the genome of a microorganism. If the bacterial isolation, and hence, the result is not available until 2
precise identification of the microbial agents responsible for days after a treatment is started. The phenotypic approach also
infections were available within 1 h when the results of other has some shortcomings; since different bacterial species have
laboratory tests are available to the physician, it would have a different susceptibilities to the same antibiotics, breakpoints of
major impact on the management and treatment of patients. different values must be tested. There is also no international
The use of universal probes based either on the rRNA gene agreement for the interpretation of breakpoints in antibiotic
(11) or on some other conserved region of microorganism susceptibility tests. Finally, several of the presently performed
genomes should indicate whether or not the patient is infected susceptibility tests are highly dependent on experimental con-
with a bacterium. Because more than 80% of normally sterile ditions, and often, more than one method would need to be
clinical specimens (blood, cerebrospinal fluid, joint fluid, etc.) performed to obtain an accurate susceptibility profile. If we
sent to the microbiology laboratory are not “infected” or do take only b-lactam antibiotic testing as an example, special pre-
not harbor bacteria (4), the use of universal primers should cautions must be taken for testing for penicillin resistance in
permit determination in 1 h of whether or not the patient Streptococcus pneumoniae, for methicillin resistance in Staph-
suffers from a bacterial infection. Obviously, universal probes ylococcus aureus, and for the presence of extended-spectrum
would not be useful for sputum or surgical wound specimens or b-lactamases in members of the family Enterobacteriaceae.
specimens from other nonsterile clinical sites. Provided that To increase the rapidity and accuracy of resistance testing,
appropriate controls are included and relevant sensitivity is the use of a genotypic approach has recently been advocated
reached, the absence of amplification products would suggest (6, 23), and numerous DNA-based assays for the detection of
the absence of bacterial infections and the use of antibiotics bacterial resistance have been developed (1, 19). This novel
could be avoided. In contrast, the detection of an amplification approach is a true revolution because it relies on a completely
product with universal primers would indicate that a bacterium different concept; testing for resistance instead of testing for
is present. However, it would not provide information on the susceptibility. Several clinical studies will be required, how-
nature of the bacterium and hence on the antibiotic to be used. ever, to validate the genotypic approach. Indeed, is the pres-
Therefore, universal primers are useful for screening negative ence of a resistance gene always indicative of a resistant bac-
samples but are of limited value for orienting the choice of terium? If a gene coding for a resistance to a drug is not
antibiotics in the case of a positive reaction. detected, does it mean that the bacterium is susceptible to that
There are now specific DNA probes or amplification primers drug? One stumbling block in using DNA-based assays for
for almost every relevant pathogenic organisms (8, 26), and resistance testing is the formidable complexity of resistance
these primers can be used to identify the bacteria present in mechanisms. Drug resistance may arise because drug uptake
clinical specimens. Because multiple bacteria can be isolated may be thwarted by loss of the uptake system or alteration of
from different sites, it would be advisable to carry out reactions the membrane composition; once the drug is inside the cell it
under multiplex conditions, i.e., with more than one pair of may be inactivated or excreted (modified or not), or if drug
primers per reaction. It would be possible to discriminate the activation is required, activation mechanisms may be sup-
amplicon either by size on agarose gel electrophoresis or with pressed. Drug-microbial target interactions may be less effec-
a different fluorochrome if fluorescence was to be used as the tive because the target is modified or alternative pathways may
detection method. It should also be possible to decrease sub- bypass the blocked target. Resistance to the same drug can be
stantially the number of primers by generating genus-specific due to several different mechanisms. For example, resistance
or even group-specific PCR primers. This approach has the to b-lactam antibiotics can be due to decreased uptake, in-
benefit on the one hand of decreasing the complexity of the creased efflux, inactivation enzymes, or modified target. Fur-
amplification reactions and on the other of increasing the pro- thermore, there are several different enzymes that can confer
portion of bacteria detected. With group-, genus-, and species- resistance by the same biochemical pathway. For instance,
specific amplification primers it should be possible to detect several dozen plasmid-mediated b-lactamases confer resis-
most microorganisms responsible for any type of infection. tance by inactivating b-lactam antibiotics. Nevertheless, as our
Nevertheless, there will always be the rare uncommon patho- understanding of drug resistance mechanisms increases, we
gen that is responsible for an infection but that may not be should be able to generate the appropriate tools to detect
detected with the available primers. Because the universal resistance. In addition, new resistance genes will undoubtedly
primers would have detected the presence of an infection but arise in bacteria in the future. To prevent the possibility that a
none of the genus- or species-specific primers would have clinician could unknowingly use an antibiotic to which the
produced an amplification product, it would indicate that the organism is resistant, continuously updated epidemiological
infection is due to an uncommon pathogen. In those rare studies would help in the selection of the right set of primers
instances, culture may be requested if species determination for the detection of relevant new types of resistance in partic-
was thought to be useful, but with time, most microorganisms ular organisms. Finally, technological innovations in DNA-
could be identified by DNA-based tests. Rapid bacterial iden- based diagnostics should also allow the detection of multiple
tification would be of major benefit to the clinician, but be- alleles or genes at once.
cause the antibiotic susceptibility profile is an important pa- Although the multiplicity of resistance mechanisms will
rameter in the management of infections, we believe that a complicate the detection of the resistance genotype for certain
rapid identification system will fully blossom only when both specimens, there are clear cases in which detection of resis-
bacterial identification and the resistance profile are provided tance could easily be implemented and could have an imme-
simultaneously. diate impact on the treatment of infectious diseases. Molecular
diagnostic methods have already found a niche in the clinical
FROM PHENOTYPIC TO GENOTYPIC TESTING microbiology laboratories where they are used.
OF RESISTANCE The detection of antibiotic resistance genes in gram-positive
bacteria should also be relatively easy. Indeed, resistance to
Presently, susceptibility (in contrast to resistance) is the pa- key antibiotics such as methicillin and vancomycin is due to few
rameter provided to clinicians. Although the phenotypic tech- genes. This small heterogeneity of resistance determinants is in
VOL. 36, 1998 MINIREVIEW 2171

contrast to the situation prevailing in several gram-negative achieved by prescribing antibiotics only to the patients who
bacteria. Methicillin resistance in S. aureus and coagulase- require them. Because the bacteria will be identified rapidly,
negative staphylococci is due to the synthesis of a novel peni- targeted antibiotics will be used and broad-spectrum antibiot-
cillin-binding protein encoded by the mecA gene. Resistance to ics will be used only when dealing with resistant organisms.
methicillin has important implications, often necessitating pa- These tests would appropriately and rapidly identify the pa-
tient isolation and the use of vancomycin. However, despite tients who should be isolated to prevent the disastrous spread
numerous guidelines for optimization of the phenotypic de- of multidrug-resistant organisms within institutions. It should
tection of methicillin resistance, it is becoming increasingly thus prevent the unnecessary isolation of patients suspected of
clear that mecA detection is becoming the “gold standard” for carrying VRE, methicillin-resistant S. aureus, or other resistant
the detection of methicillin resistance (25). All the studies pathogens. On occasion, emergency rooms and hospitals had
reported to date indicate an excellent correlation between the to be closed in Canada pending the phenotypic identification
presence of the mecA gene and methicillin resistance (19). In of these pathogens and the results of susceptibility tests. The
mecA-positive staphylococci, the use of vancomycin is war- continuing implementation of molecular tests in the routine
ranted. When mecA is absent, cells could exhibit intermediate microbiology laboratory will contribute to a definitive diagno-
levels of methicillin resistance due to overexpression of a b-lac- sis and should have a major impact on the clinical management
tamase. In these cases, however, b-lactam antibiotics or b-lac- of infectious diseases. It would also reduce global health care
tam–b-lactamase inhibitor combinations would likely be more costs and, it is hoped, save lives while contributing to a major
effective and appropriate than vancomycin. Several multiplex reduction in the spread of antibiotic resistance.
PCR assays that permit both S. aureus identification and mecA
detection have been developed, and some reports have de- ACKNOWLEDGMENTS
scribed the utility of those tests directly with positive blood
culture vials (5, 24). The rapid identification of mecA should Marc Ouellette is a research fellow from the FRSQ and is the
restrict the use of vancomycin and hence the emergence of recipient of a Burroughs Wellcome Fund New Investigator Award in
resistance to this drug of last resort. Molecular Parasitology. Michel G. Bergeron is recipient of a Fonds de
la Recherche en Santé du Québec (FRSQ) grant on antimicrobial
Indeed, resistance to vancomycin is now widespread in en- resistance.
terococci (10), and there are now enterococci that seem to
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