INTRODUCTION

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The determination of antimicrobial susceptibility of a clinical isolate is often crucial for the optimal antimicrobial therapy of infected patients. This need is only increasing with increasing resistance and the emergence of multidrug-resistant microorganisms (88, 89, 91). Testing is required not only for therapy but also to monitor the spread of resistant organisms or resistance genes throughout the hospital and community. Standard procedures and breakpoints have been defined to predict therapeutic outcome both in time and at different geographic locations. In some cases the presence of a resistance gene is highly predictive for clinical outcome of antimicrobial therapy. For example, the presence of a -lactamase in Neisseria gonorrhoeae correlates well with the outcome of penicillin treatment. However, the presence of a resistance gene does not necessarily lead to treatment failure (198), because the level of expression may be to low. For example, -lactamase production among members of the Enterobacteriaceae is common, but the development of resistance is dependent on the mode and level of expression (180, 183).

Resistance can be caused by a variety of mechanisms: (i) the presence of an enzyme that inactivates the antimicrobial agent; (ii) the presence of an alternative enzyme for the enzyme that is inhibited by the antimicrobial agent; (iii) a mutation in the antimicrobial agent's target, which reduces the binding of the antimicrobial agent; (iv) posttranscriptional or posttranslational modification of the antimicrobial agent's target, which reduces binding of the antimicrobial agent; (v) reduced uptake of the antimicrobial agent; (vi) active efflux of the antimicrobial agent; and (vii) overproduction of the target of the antimicrobial agent. In addition, resistance may be caused by a previously unrecognized mechanism. On the other hand, a gene which is not expressed in vitro may be expressed in vivo.

Nucleic acid-based detection systems offer rapid and sensitive methods to detect the presence of resistance genes and play a critical role in the elucidation of resistance mechanisms. During the last decade, nucleic acid-based detection systems have expanded tremendously and are becoming more accessible for clinical microbiology laboratories. This accessibility is not limited to the detection and identification of microorganisms but is extended to the detection of properties of these microorganisms, such as virulence factors and antimicrobial resistance. The application of nucleic acid-based technology is particularly useful for slow-growing or nonculturable microorganisms and for the detection of point mutations or certain genotypes. Nucleic acid-based technology can be divided into hybridization systems and amplification systems, although most amplification technologies are also partly based on hybridization technology.

All these factors complicate the debate regarding the determination of phenotypic versus genotypic resistance. The objective of the present review is to discuss examples where molecular techniques were used to detect antimicrobial resistance as part of diagnostic microbiology. The most commonly used or new molecular methods will be described first, followed by the applications of these techniques to detect resistance.

Hybridization is one of the oldest molecular techniques and is based on the fact that in nucleic acids a cytosine forms base pairs with a guanine and an adenine forms base pairs with either a thymidine (in DNA) or a uracil (in RNA). In hybridization, the DNA in a sample is rendered single stranded and allowed to combine with a single-stranded probe. Early hybridizations were performed with target DNA immobilized on a nitrocellulose membrane, but nowadays a variety of different solid supports, including magnetic beads, are used. Other variations include the binding of a capture probe to a solid support. After binding of the target, the probe can hybridize. Probes can be labeled with a variety of reporters, including radioactive isotopes, antigenic substrates, enzymes or chemiluminescent compounds. For an overview of hybridization see references (344) and (158). Despite the fact that hybridization is a relatively old technology, new developments lead to new applications. One important development is molecular beacons (discussed below).

PCR is well known and will be discussed only briefly. Two years after its first description by Mullis (202), and thus only 15 years ago, the first diagnostic application of PCR was published by Saiki et al. (281). The technique became broadly used after the introduction of a thermostable DNA polymerase from Thermus aquaticus (Taq DNA polymerase) (280) and the development of automated oligonucleotide synthesis and thermocyclers. PCR involves cycles of heating the sample for denaturing, annealing of the primers, and elongation of the primers by a thermostable DNA polymerase. In theory, each round of amplification gives a doubling of the number of DNA target molecules, but the process is seldom 100% efficient because of the presence of inhibitors, and in later rounds of amplification DNA polymerase may become limited.

However, during the last few years, new developments in labeling technology have expanded the applicability of PCR. One such development was the use of 5'-fluorescence-labeled oligonucleotides that were blocked at their 3' ends, thereby preventing elongation by DNA polymerase. Besides this special oligonucleotide, PCR has two traditional oligonucleotides which function as primers and are chosen in regions flanking the special oligonucleotide. The special oligonucleotide hybridizes with the target and is removed by the 5'3' exonuclease activity of Taq DNA polymerase during primer extension, resulting in enhanced fluorescence that can easily be detected (Fig. 1) (121).

View larger version (4K): [in this window] [in a new window]   FIG. 1.   Schematic representation of 5'3' exonuclease cleavage of a 5'-labeled (small black star) probe with a 3'-phosphate (grey circle) extension blocker. After cleavage of the label from the probe by DNA polymerase (large black star), the small labeled fragments generated can be separated from the larger probe.

Another advance is the development of molecular beacons. Basically, molecular beacons are hairpin-shaped oligonucleotide probes with a fluorophore attached and a molecule that quenches this fluorescence when it is next to the fluorophore. On hybridization with the target, the fluorophore and quenching molecule are spatially separated and fluorescence is possible (Fig. 2) (360, 361). In principle, the use of fluorophores with different emission spectra makes it possible to discriminate multiple targets. The addition of molecular beacons to PCR amplifications makes possible real-time monitoring of amplification. Furthermore, it allows a relatively easy quantitative PCR (360, 361). This technology has now been commercially realized with the TaqMan (ABI/Perkin-Elmer Corp., Foster City, Calif.) and Lightcycler (Roche Molecular Biochemicals, Mannheim, Germany) systems. A variation on this theme is the Scorpions primer (Oswell Research Products, Southampton, United Kingdom) (393). In this PCR-based method, the primer, probe, and fluorescent label are integrated into one molecule and form part of a homogeneous (closed-tube) assay (Fig. 3).

View larger version (8K): [in this window] [in a new window]   FIG. 2.   Principle of molecular beacons. The stem of the hairpin is less stable than the hybridization of the specific probe (loop region) with its target (top). Hybridization leads to denaturation of the stem and the physical separation of the fluorophore (white star) and its quencher (black star), allowing fluorecence to occur (bottom).

View larger version (8K): [in this window] [in a new window]   FIG. 3.   The Scorpions primer is an extension of molecular beacons. To a molecular beacon, a blocker (grey circle) and PCR primer are added (top). The blocker prevents the copying of the molecular beacon part of the molecule. After one round of amplification (middle), the molecular beacon extension of the primer is able to hybridize with the newly synthesized DNA strand, allowing fluorescence (bottom).

In PCR-single-strand conformation polymorphism (PCR-SSCP), the PCR amplication product is denatured into two single-stranded molecules and subjected to nondenaturing polyacrylamide gel electrophoresis. Under nondenaturing conditions, the single-stranded DNA (ssDNA) molecule has a secondary structure that is determined by the nucleotide sequence, buffer conditions, and temperature. The mobility of the ssDNA molecule depends on both its size and secondary structure. ssDNAs at different positions in the gel indicate a difference in sequence. The technique was originally described for the detection of mutations in oncogenes and allelic variants in human genes (228, 229). PCR-SSCP is capable of detecting more than 90% of all single-nucleotide changes in a 200-nucleotide fragment (115).

Branched DNA (bDNA) was developed by Chiron Corp. and uses multiple hybridization sites for enzyme-coupled probes (221, 285) (Fig. 4). Target-specific probes bound to a solid surface are allowed to capture target ssDNA. A second probe is allowed to hybridize with the target. This probe has a 5' extension that does not hybridize with the target. This extension can hybridize with a bDNA probe. This probe has a bristle-like structure. At least 15 bristles are attached to each probe, and as many as three alkaline phosphatase reporter molecules can bind to each bristle. Using multiple target-specific probes for each target nucleic acid, up to 1,700 enzyme molecules can be bound to a single target molecule. A signal is generated by the addition of a chemiluminescent substrate. The sensitivity of the system is in the range of 10³ to a 10⁵ thousand target molecules.

View larger version (25K): [in this window] [in a new window]   FIG. 4.   Schematic presentation of bDNA amplification. For details, see the text. Reprinted from M. N. Widjojoatmodjo, Diagnosis of infections based on DNA amplification: obstacles and solutions, Academic thesis, University of Utrecht, Utrecht, The Netherlands, 1995, with permission of the author.

DNA sequencing is almost universally performed by dideoxy sequencing (287) and is a well-known technique. Technological developments over the past few years, largely driven by the genome-sequencing efforts, have led to advances in DNA sequencing. These developments, such as the ability to read longer sequences faster and cheaper, brought DNA sequencing within the capabilities of at least some diagnostic laboratories and is the method of choice for determining the resistance of human immunodeficiency virus to antiviral drugs (301).

DNA arrays and DNA chips are based on the principle of hybridization. DNA arrays and chips are devices which allow the mass screening of sequences. In contrast to conventional hybdrization assays, where target DNA is blotted onto a membrane, a large collection of probes is bound to a solid surface. The target DNA is generally tagged with a fluorescent label, and hybridization is detected by using an epifluorescence microscope. In DNA arrays cDNA fragment probes are usually used, whereas DNA chips employ oligonucleotides. Arrays are larger and generally use either a nylon membrane type of material or glass as the solid surface, whereas DNA chips use either glass or silicon. The fragment probes are applied to the solid surface after they are generated, whereas oligonucleotides are either applied after synthesis or synthesized in situ. Various schemes for applying probes onto solid surfaces have been reported (see, e.g., references 43, 217, 275, and 310). On DNA arrays, cDNAs or PCR products are attached to a solid surface and used for large-scale assessment of gene expression by measuring mRNA levels. On DNA chips, most often oligonucleotides are used which can be used not only for measurement of gene expression but also for sequencing (sequencing by hybridization) (43). The sequencing strategy can be explained as follows. A complete set of 65,536 octamer probes, each in a separate spot on a solid phase, is mixed with a 12-mer target oligonucleotide, AGCCTAGCTGAA. When only perfect hybridization is considered, five of the probes will bind the target oligonucleotide. Alignment of these overlapping octamer probes will reconstruct the complement of the 12-mer target (Fig. 5). In its first application (43), 256 tetranucleotides were generated in situ on a solid-phase support by photolithographic techniques. The DNA chip generated proved to be specific for the detection of complementary octanucleotides which were fluorescently labeled. The production of more than 400,000 different 20-mer oligonucleotides on a 1.6-cm² glass slide should be feasible (101). For reviews, see references 94 and 105.

View larger version (13K): [in this window] [in a new window]   FIG. 5.   Principle of oligonucleotide array sequencing. Alignment of the overlapping probes reconstructs the complement of the original target (see the text for details).

Besides the molecular techniques described above, a number of other amplification techniques are used in the clinical microbiology laboratory, although not to detect antibiotic resistance determinants. These techniques include the DNA amplification techniques of strand displacement amplification (377, 378) and ligase chain reaction (16, 19, 25, 398, 405) and the RNA amplification techniques of Q replication (156) and self-sustained sequence replication or nucleic acid-based sequence amplification (55, 148). This latter method can be modified to amplify DNA (99).

The variety of molecular techniques used for diagnostic applications demonstrate that no universal technique exists which is optimal for detection of nucleic acids. The choice of a particular technique is also dependent on the information required or the targets under consideration, but some techniques are more favored than others. New techniques continue to be developed that involve a new approach to amplification, hybridization, formats, and labels (158).

ANTIBIOTIC RESISTANCE IN MYCOBACTERIUM TUBERCULOSIS

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Introduction

In the wake of the human immunodeficiency virus epidemic and the breakdown of medical services in several Eastern European countries, the incidence of tuberculosis is rising rapidly. Of note, the treatment of tuberculosis is threatened by the emergence of multidrug-resistant strains of Mycobacterium tuberculosis. M. tuberculosis is usually treated with only a limited number of antimicrobial agents, the most important ones being rifampin, isoniazid, streptomycin, and ethambutol. Resistance to rifampin is conferred by mutations resulting in at least eight amino acid substitutions in the RpoB subunit of RNA polymerase (335). Isoniazid acts by inhibiting an oxygen-sensitive pathway in the mycolic acid biosynthesis of the cell wall. At least four genes have been described to be involved in resistance to isoniazid: the katG gene, which encodes a catalase; the inhA gene, which is the target for isoniazid; and the oxyR gene and neighboring aphC gene and their intergenic region (739). Streptomycin resistance has been associated with mutations in the rrs gene encoding 16S rRNA and the rspL gene encoding the S12 ribosomal protein (69, 196, 208). Ethambutol resistance is associated with an altered EmbB protein (2, 322), a protein involved in the synthesis of the cell wall component arabinogalactan.

Because the organism is slow growing, traditional diagnosis is time-consuming. Traditional phenotypic determination of resistance may take up to 10 weeks after referral of a sample to the laboratory, but both commercial and in-house amplification assays can greatly improve the detection time. Therefore, it is not surprising that within the past 8 years a multitude of different resistance assays based on molecular techniques were specifically developed for M. tuberculosis. However, many laboratories have had trouble with the technical rigor imposed by these assays (222). For review of mycobacterial resistance, see reference 128.

One of the first assays for the detection of rifampin resistance using PCR-SSCP was published by Telenti et al. (336). In a second paper the assay was more extensively evaluated both in a manual format with radioactively labeled amplification products and with 5'-fluorescein-labeled primers for detection on an automated DNA sequencer (337). Evaluation of the results showed that all 17 of the then known mutations in the rpoB gene leading to resistance could be detected. Equally important, the assays could be applied to minimally grown cultures in Bactec 12B medium with a growth index of 100 or on sputa with at least 10 organisms per field at a magnification of ×250. This clearly established the potential of PCR-SSCP as a powerful technique for the early detection of antimicrobial drug resistance in M. tuberculosis. The application of rifampin resistance detection by PCR-SSCP to cerebrospinal fluid specimens from patients with tuberculosis of the central nervous system also yielded excellent results (289).

PCR-SSCP requires careful control over electrophoresis conditions, which is difficult to achieve in many laboratories. This recognition led to a comparison of PCR-SSCP and dideoxy fingerprinting (84). Dideoxy fingerprinting is in fact an extension of SSCP. After PCR amplification of the gene fragment of interest, a second PCR is performed with a radioactively labeled primer. A dideoxynucleotide is added, which leads to chain termination similar to that obtained in dideoxy sequencing. The products are then analyzed in a similar manner to that in SSCP. Because more fragments are generated, differences between the susceptible and resistance types are more easily obtained in accordance with conventional susceptibility testing and PCR-SSCP. A drawback of this method is its use of a radioactive label. However, by using fluorescent labels, this assay can probably be adapted for use with an automated sequencer.

However, Kim et al. (150) observed that PCR-SSCP reported some isolates as resistant whereas their phenotype was susceptible, but in these isolates the part of the rpoB gene that was amplified contained a silent mutation and a deletion of two amino acids. Apparently, these mutations do not affect the susceptibility to rifampin. These authors therefore concluded that sequencing probably could rule out false-positive results.

Direct testing of a clinical specimen for resistance to rifampin by PCR without prior species determination is believed to be difficult because of the high levels of homology reported between different mycobacterial species, but Whelen et al. (392) devised a rpoB-based seminested amplification which was specific for M. tuberculosis. The assay correctly identified 21 of 24 culture-positive specimens, 13 of which were acid-fast smear negative in a panel of 51 clinical specimens. Three specimens were false-positive and tested negative after aerosol carryover was eliminated. This assay demonstrates that concurrent resistance determination and species identification are possible. However, it also clearly illustrates the dangers of (hemi)nested PCR assays and the need to carefully eliminate potential contamination of amplification reactions by unintended target DNA. SSCP analysis and automated sequencing of these isolates both showed that one isolate was resistant to rifampin and the others were susceptible, in accordance with phenotypic testing.

This group also analyzed the results of the heminested PCR using sputum samples and heteroduplex analysis (401). A total of 655 sputa were tested. The assay correctly detected 41 of 44 culture-positive sputa. In addition, 19 culture-negative sputa were identified as positive. Three assay-positive isolates belonged to either M. avium or M. tuberculosis. Thirty-five sputa which contained non-M. tuberculosis complex bacteria were negative in the assay as well as all other samples. The heteroduplex assay identified 39 of the 44 culture-positive isolates as rifampin susceptible, whereas Bactec radiometric susceptibility testing reported 38 susceptible isolates. The study showed the feasibility of a PCR-based assay directly with sputum samples for both identification and rifampin resistance determination. This is especially true for patients with either smear-positive untreated tuberculosis or suspected of having multidrug-resistant tuberculosis.

A modification of the heteroduplex method has also been described for the detection of rpoB-associated rifampin resistance (209). The region of interest was amplified by PCR with primers which contained the T7 or SP6 RNA polymerase promoter. The amplified fragments were then transcribed using either T7 or SP6 RNA polymerase. The resulting RNA was then hybridized to a reference RNA molecule (e.g., susceptible type sequence) and treated with RNase. In the case of a mismatch, the heteroduplex was cleaved whereas a homoduplex could not be cleaved. This assay reached 100% sensitivity and 96% specificity and took less then 24 h to perform after receipt of an isolate or a smear-positive sample.

Another modification of the heteroduplex assay is the double-gradient (DG) variant of the original denaturing gradient gel electrophoresis (DGGE). DG-DGGE uses both a temperature and a polyacrylamide gradient to optimize separation. DG-DGGE was able to detect all rifampin-resistant isolates among a set of 117 isolates (290). Direct testing on clinical samples showed that the rpoB-specific PCR was positive for 54 of the 84 IS6110 PCR-positive isolates (IS6110 is specific for M. tuberculosis). Of the rpoB PCR-positive isolates, 30 that were classified as susceptible by DG-DGGE were also susceptible in a conventional assay. The remaining 24 isolates were classified as rifampin resistant, but 1 isolate proved to be susceptible by conventional testing. The single discrepancy was due to the presence of at least two clones, one of which carried a mutation in the rpoB gene. Analysis of 48 cerebrospinal fluid specimens showed complete agreement between genotypic and phenotypic testing. The results obtained by direct testing of clinical specimens showed that the type of specimen can have an important effect on the outcome of a molecular assay. This underscores the need for controls and appropriate sample preparation methods.

Analysis of rpoB gene amplification products is possible not only by SSCP or heteroduplex analysis but also by a line probe assay (LiPA; Innogenetics) (63). In the nested PCR, the inner primers were biotinylated and nine different probes were immobilized on a nitrocellulose membrane in addition to a color control and an M. tuberculosis probe. The DNA was directly isolated from sputum and lymph node biopsy specimens. The results of this assay correctly matched classical resistance testing in 65 of 67 isolates. However, 2 isolates of a total of 23 phenotypically resistant isolates gave a susceptible hybridization pattern. DNA sequencing proved the absence of mutations in the sequence used for the assay, thus suggesting another mechanism of rifampin resistance, although this mechanism was not clarified. This underscores that although PCR assays may be valuable tools, they are not absolute in their outcome and unrecognized mechanisms of resistance may lead to therapeutic failure.

A second study (278) evaluated the ability of LiPA to detect mutations in the rpoB gene of 107 M. tuberculosis and 52 non-M. tuberculosis isolates in pure culture, as well as 61 and 203 unidentified clinical isolates that were rifampin resistant and susceptible, respectively. No discrepancies with sequencing were observed. All susceptible isolates were correctly identified. Only four resistant isolates yielded a resistant phenotype, but no mutation was observed in both sequencing and LiPA. It was not clear whether mutations elsewhere in the rpoB gene or a different mechanism of resistance were involved. In another evaluation of this assay, sputum and bronchoalveolar lavage fluid from only two patients were used. Within 48 h, the assay correctly identified mutations in the rpoB gene associated with resistance as confirmed by sequencing (104). A fourth evaluation of the assay with 30 M. tuberculosis isolates showed a 93.3% concordance between culture and LiPA, whereas sequencing was completely concordant with culture (192).

A South African study (149) into the detection of rifampin resistance compared the results from heteroduplex analysis with sequencing and LiPA. Sequencing revealed that some mutations in the rpoB gene apparently do not lead to resistance to rifampin, and a few isolates did not have mutations in the analyzed region. LiPA correlated with the sequence results. Heteroduplex hybridization results also agreed with DNA sequencing, although the presence or absence of some mutations was difficult to ascertain because of small differences in mobility when the mutant sequence was compared with the susceptible wild-type sequence. Despite these limitations, the authors concluded that the assay was a valuable tool for identifying and managing patients with multidrug-resistant M. tuberculosis.

The LiPA and the RNA-RNA heteroduplex assay (MisMatch Detect II; Ambion) for rifampin resistance were first compared with a phenotypic assay by using 16 M. tuberculosis isolates. The MisMatch assay missed one sample. A further evaluation was performed with 38 sputa and bronchoalveolar lavage fluid specimens and 21 isolates submitted by clinicians. The LiPA and MisMatch assay correlated with 36 and 38 of the primary samples, respectively, whereas all 21 isolates were classified correctly (383).

Another examination of the LiPA involved 75 clinical specimens from 70 patients suspected of having tuberculosis (193). A final diagnosis of tuberculosis was reached for 51 of these patients. In a nested PCR, only 31 specimens yielded a PCR product and 30 of these hybridized with the M. tuberculosis-specific probe of the LiPA. Nevertheless, culture yielded only 18 positive specimens. In the PCR-LiPA, only 13 of these culture-positive isolates yielded a positive result. For these same isolates, LiPA testing correlated with the resistance phenotype for 11 isolates. One isolate was phenotypically resistant but susceptible according to LiPA; however, sequencing did not reveal a mutation in rpoB. The other isolate was phenotypically susceptible but yielded a resistant genotype in LiPA, which was confirmed by sequencing.

PCR-SSCP has also been used for the detection of katG-related resistance to isoniazid. A major problem, however, is that compared to rifampin resistance caused by mutations in the rpoB gene, the mutations causing isoniazid resistance are spread over a much longer sequence. This, combined with the fact that PCR-SSCP is most discriminatory when the amplified fragments are less than 400 bp, necessitates the analysis of multiple amplification fragments. In addition, all or part of the katG gene is lacking, meaning that appropriate controls are needed to distinguish the absence of amplification products from PCR inhibition. Initially, resistance was believed to be caused by complete deletion of the katG gene (6), but with the use of PCR and PCR-SSCP, it was shown that both deletion and mutation of the katG gene could lead to resistance (410). Heym et al. (118), who studied the relationship between mutations in the katG gene and isoniazid resistance, developed a discriminatory PCR-SSCP assay which involved 12 amplification products. Although this approach was successful, it is cumbersome to perform on a routine basis.

An extension of PCR-SSCP used Cleavage I, a structure-specific endonuclease (73). After PCR amplification, the DNA fragments were made single stranded and allowed to assume their secondary structures. After incubation with Cleavage I, the resulting fragments were analyzed by acrylamide gel electrophoresis. The technique allowed for a better discriminatory power than classical SSCP. Results with a 620-bp fragment of the katG gene showed the potential applicability of this technique to the detection of resistance in M. tuberculosis. This alternative technique may use larger fragments. This would alleviate the problem of the large number of PCR-SSCPs required for full coverage of the katG gene.

In a complex study, Nachamkin et al. (207) compared techniques to detect resistance caused by different resistance genes. For the katG gene, restriction fragment length polymorphism (RFLP) analysis of a 620-bp amplified PCR fragment was used. RFLP analysis detected the S315T mutation in 12 of 27 specimens, but other mutations were also responsible for isoniazid resistance. Rifampin resistance caused by mutations in the rpoB gene was analyzed by both heteroduplex analysis and sequencing. Heteroduplex analysis for the rpoB gene reached 76.2% sensitivity and 97.2% specificity compared to conventional testing, but sequencing reached 100% sensitivity and specificity. RFLP, despite its shortcomings, was also used to detect resistance to streptomycin caused by mutations in the rslP gene. Although the specificity reached 100%, the sensitivity was only 28.1%. Direct sequencing reached 99% specificity and 67.7% sensitivity. The authors concluded that the results only partly matched those of conventional testing. This is not entirely unexpected, because not all known mutations involved in resistance lead to a change in potential restriction sites. One mutation, although implicated in resistance, did not result in a resistant phenotype. The low sensitivity of both methods appeared to be related to other mechanisms of resistance to streptomycin, e.g., via the rrs gene. The RFLP results are in agreement with the results obtained by Temesgen et al. (338), who compared PCR-SSCP with RFLP for detection of the R463L mutation. The data obtained by these authors indicate that the appropriate molecular method must be chosen and that all resistance genes known to be involved in a particular resistance within a species should be investigated. Nevertheless, it cannot be excluded that unknown mechanisms play a role in resistance, and these mechanisms will be missed by these techniques.

The development of multidrug resistance in classical treatment options for M. tuberculosis has led to the consideration of alternative antimicrobial agents including fluoroquinolones. However, quinolone resistance was observed among clinical isolates and was easily induced with ciprofloxacin at 2 µg/ml (frequency, 1 in 10⁷ to 10⁸), although no induction of resistance was observed with ciprofloxacin at higher concentrations (333). Therefore, a PCR-SSCP was evaluated to distinguish fluoroquinolone-susceptible from fluoroquinolone-resistant isolates (333). The results demonstrated that discrimination between these isolates was possible.

Isolates often show multidrug resistance, and the number potential mutations involved means that the number of assays needed to cover them all can be quite large. Therefore, a number of PCR assays have been developed which do not directly determine the presence or absence of resistance-causing genes and mutations but either identify multiresistant strains by other properties or monitor the effect of chemotherapy. One such completely different approach to the determination of resistance in M. tuberculosis was taken by Cangelosi et al. (38). This group developed a reverse transcriptase PCR (RT-PCR) probe assay that was specific for M. tuberculosis precursor rRNA. Precursor rRNA carries terminal stems which are removed when mature rRNA subunits are formed. The number of these stems present in the bacterial cell is markedly affected by inhibition of RNA synthesis. Hybridization results showed that the assay was specific for M. tuberculosis, and resistance to rifampin and ciprofloxacin could correctly be predicted. As expected, no influence on the levels of precursor rRNA by isoniazid and ethambutol were seen. Instead of RT-PCR, quantitative PCR was also used to determine resistance to isoniazid. Bacteria were inoculated into medium containing different concentrations of isoniazid. After 1 to 3 weeks, more than a 1-log-unit difference in the amount of DNA was observed between isoniazid-resistant and susceptible isolates (1).

The problems with multiresistant M. tuberculosis isolates belonging to strain W, which was associated with several outbreaks in New York state, led to the development of a PCR assay to identify this strain (250). This assay used an internal control and primers to identify a specific fragment called NTF-1 and the orientation of this fragment, which were considered specific for this strain. The assay successfully identified all 48 W strain isolates among 193 isolates tested.

Molecular diagnostic techniques play an important role not only in detecting resistance but also in monitoring chemotherapy. This was demonstrated as early as 1994 in a small study (171). Sixteen patients with tuberculosis were monitored, who all became smear negative after 2 months of treatment. Treatment continued for 4 months. Although all the patients were smear negative after two months, four, two, and one patients were still positive in M. tuberculosis-specific PCR after 2, 3, and 6 months, respectively. These results demonstrated that in principle the identification of patients at high risk for a relapse is possible.

Piatek et al. (248) investigated the use of molecular beacons for the detection of mutations in rpoB. This group designed five fluorogenic probes which were used in five separate PCRs which were monitored in real time. In addition, a species-specific internal control and another complementary to a species-specific multicopy gene were used. The outcome correctly predicted the susceptibility of the 75 isolates tested. A potential drawback of this method is that new mutations might be missed. However, the big advantage of the technique is that the product formation can be analyzed visually without the need for additional equipment.

DNA chips are logical candidates for the analysis of mutations involved in resistance to antimicrobial agents used for tuberculosis treatment. Although the technique potentially holds great promise, it is still very expensive. Head et al. (116), in a limited study involving only nine rifampin-resistant isolates, investigated the application of DNA chip technology to the detection of rifampin resistance. The chip technology chosen sequenced the rpoB gene. Sequencing by chip technology identified two point mutations in all nine isolates. This group concluded that the technique is suitable for the detection of mutations.

Molecular techniques hold great promise for the detection of susceptibility and resistance to antimicrobial agents in M. tuberculosis. In fact, a number of useful assays already exist. However, the use of molecular techniques for the analysis of resistance is dependent on the prevalence of the resistance-causing mutation. With the current assays, this coverage appears incomplete and may vary between different geographical areas (see, e.g., reference 149). Nevertheless, molecular techniques may be especially helpful in the quick identification of multidrug-resistant isolates and the evaluation of culture-negative specimens. It should be kept in mind that isolates that are susceptible according to molecular assays may contain other mechanisms of resistance.

However, molecular techniques, owing to their extreme sensitivity, are prone to contamination, and their execution requires extra care as demonstrated (289).

RESISTANCE TO -LACTAM ANTIBIOTICS

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Mechanisms of Resistance

-lactam antibiotics are among the most commonly used antimicrobial agents. They act on penicillin binding proteins (PBPs), which are involved in cell wall synthesis. Penicillin, a -lactam antibiotic, was one of the first antibiotics. -Lactam antibiotics are still the most widely used and diverse class of drugs used clinally, and new members are still being developed. It is therefore not surprising that resistance to many -lactam compounds is commonplace and still evolving. Resistance is most often caused by the presence of -lactamases, but mutations in PBPs resulting in reduced affinity for -lactam antibiotics are also commonly observed. Resistance is less frequently caused by reduced uptake due to changes in the cell wall or active efflux. Several classification systems for -lactamases have been published. One classification scheme is based on their nucleotide sequence, classes A through D (36). Class A, C, and D enzymes have a serine at their active site, while class B enzymes have four zinc atoms at their active site. Class A enzymes are highly active against benzylpenicillin. The extended-spectrum -lactamases (ESBLs) also belong to this class. ESBLs also inactivate benzylpenicillins as well as some cephalosporins and/or monobactams. Class B -lactamases are equally active against penicillins and cephalosporins, and at least some of these enzymes are able to inactivate carbapenems. Class C genes are usually inducible, but mutations can lead to overexpression. Class D is composed of the OXA-type enzymes, which are capable of hydrolyzing oxacillin. Genes encoding -lactamases can located either on plasmids or the bacterial chromosome and are found among both gram-negative and gram-positive organisms. For purposes of discussion, we divide the -lactamases into metallo--lactamases, ESBLs, and other -lactamases. The -lactamases in the last group are most common and were the first -lactamases encountered in clinical practice. These -lactamases will be referred to as common -lactamases.

Introduction of methicillin into medical practice in the early 1960s quickly resulted in the isolation of methicillin-resistant staphylococci. Methicillin and its analogues bind and inactivate the PBPs involved in cell wall synthesis. Low-level resistance is generally the result of -lactamase overproduction, increased levels of intrinsic PBPs, or reduction of their binding affinity (17, 351). High-level resistance is always dependent on the expression of an alternative PBP (PBP2a) encoded by the mecA gene, which has low affinity for most -lactam antibiotics (21, 113). The mecA gene is located on the chromosome. Expression of mecA is either constitutive or inducible by some -lactam antibiotics, but not by methicillin or oxacillin, or heterogeneous, with only a few cells in a population expressing the gene (21).

Ten years ago a DNA fragment used as a probe for the detection of mecA in Staphylococcus aureus and coagulase-negative staphylococcus (CNS) isolates was described (11). The probe was tested with both a radioactive label and digoxigenin as a label. The results showed that the nonradioactive label performed as well as the radioactive label, bringing probes a step closer to routine use in a clinical laboratory. The probe assay was in complete agreement with the spread plate screening technique for assessing methicillin resistance in S. aureus isolates. Probe assay results for CNS did not correlate completely with the spread plate technique results, but differences with the broth microdilution and agar dilution methods were also observed. The interpretation of the result for CNS should take into account that the National Committee for Clinical Laboratory Standards (NCCLS) lowered the the breakpoint for oxacillin as determined by broth microdilution from 4 to 0.5 µg/ml (211). However, we were not able to reinterprete the results, because MICs for the isolates were not provided, but bigger differences between the techniques are probable. A year later, a digoxigenin-labeled fragment probe to mecA generated by PCR was described (178). Evaluation with isolates of S. aureus and CNS showed some discrepancies. Borderline-resistant S. aureus isolates (MIC, 8 µg/ml) were probe negative in some cases but produced -lactamase. This -lactamase may slowly hydrolyze oxacillin. The correlation for CNS was good, but when the new NCCLS breakpoint criteria were applied, some discrepancies resulted; isolates reported as resistant lacked the mecA gene.

In the past decade, several PCR assays have been described for the detection of methicillin-resistant S. aureus. Murakami et al. (204) evaluated their PCR against approximately 200 S. aureus isolates. Two isolates were mecA negative in the PCR and resistant to oxacillin but were not resistant to methicillin in a broth microdilution assay. Three isolates were mecA positive but susceptible to both methicillin and oxacillin. Evaluation against 100 CNS isolates showed a larger number of discrepancies, especially oxacillin-susceptible but mecA-positive isolates. However, these results would be different when the new NCCLS interpretative breakpoints for oxacillin and CNS were applied. In the same year Predari et al. compared the results of a mecA PCR with those obtained by dot blot hybridization and phenotypic testing for 74 CNS isolates (256). The PCR results correlated perfectly with phenotypic analysis when high inocula were used (10⁸ CFU), an effect also observed by Hedin and Löfdahl (117). PCR performed better than hybridization did. This difference was believed to be the result of the loss of the mecA gene in a large proportion of the bacteria which belong to certain strains. These isolates also gave a weak amplification signal. Another study (349) investigated a total of 58 clinical isolates of S. aureus. Based on MICs of oxacillin and methicillin, 27 of these isolates were resistant and 1 isolate showed an oxacillin MIC of 32 µg/ml and a methicillin MIC of only 2 µg/ml. Thirty isolates were negative in the PCR, and 28 were positive for the mecA gene. These results were confirmed by a hybridization assay on HincII-restricted DNA probed with a mecA-specific 30-mer oligonucleotide. The PCR-positive isolates included not only the oxacillin-resistant but methicillin-susceptible isolate but also isolates with a methicillin MIC of 2 µg/ml and an oxacillin MIC of either 1 or 2 µg/ml. After preincubation of these isolates with ceftizoxime, the MICs of both methicillin and oxacillin increased for all but one isolate. This demonstrates that these isolates had an inducible phenotype. No oxacillin- or methicillin-resistant isolate was negative in either the PCR or hybridization assay.

An integrated PCR detection assay was published by Ubukata et al. (363). This group used a simple mecA PCR with one of the primers carrying a biotin group and the other primer carrying a dinitrophenol group. After PCR, the product was captured on a 96-well microtiter plate coated with streptavidin. After capture and washing, an anti-dinitrophenol antibody conjugated to alkaline phosphatase was added, followed by substrate. The sensitivity of the assay was >5 × 10² CFU for S. aureus and >5 × 10³ CFU for CNS. A total of 97 S. aureus isolated and 64 CNS isolates were tested. One S. aureus isolate and 22 CNS isolates carried the mecA gene but were phenotypically susceptible using the older NCCLS breakpoints. The assay was also applied to the detection of mecA in staphylococcal isolates (n = 40) directly in blood culture bottles. Thirty-three isolates (18 S. aureus and 15 CNS) in the culture bottles were resistant to oxacillin, 31 of which were also positive in the PCR assay. Two CNS isolates were PCR negative. The authors conclude that the assay can be used with reasonable confidence in the clinical microbiology laboratory for the detection of MRSA in blood culture bottles. A possible cause of the difference may be the inhibition of the PCR by components of the blood or culture medium (119).

Comparison of mecA PCR with API ATB Staph (bioMérieux, Balme-les-Grottes, France), oxacillin disk test, agar dilution MIC test, and the BBL Crystal MRSA system (Becton Dickinson, Cockeysville, Md.) using the PCR as "gold standard" with 57 S. aureus isolates showed agreement with the first two methods, but the last two methods missed one isolate (379). However API ATB and the oxacillin disk test reported three isolates as methicillin resistant. The two other methods reported no methicillin-resistant isolates in mecA-negative isolates. The results were poor (negative predictive value of 68 and 82% for the BBL Crystal MRSA system and API ATB Staph, respectively) for 100 CNS isolates belonging to a number of different species. It was concluded that there was a good correlation with the oxacillin MIC for S. aureus in contrast to CNS, but it should be noted that this conclusion was based on the old breakpoints (211). No MIC data for individual isolates were provided, so no evaluation based on the current breakpoint could be made.

The PCR assays with only a single primer pair as described above are robust and simple to perform. However, these assays are vulnerable to inhibition. The addition of a second primer set designed to amplify a gene which is present in all isolates can solve this problem. The use of a species-specific primer also can confirm the identity of the isolate. However, the addition of extra primer pairs leads to increased complexity of the assay, which may be critical when testing is performed directly on clinical samples, although for optimized assays this risk is reduced. Brakstad et al. (32) developed a multiplex PCR in which both the mecA gene and the nuc gene, encoding the S. aureus-specific thermonuclease gene, were amplified. A total of 135 S. aureus isolates and 84 CNS isolates belonging to different species were evaluated. The amplification of nuc-specific sequences was in complete agreement with species determinations. The MICs of oxacillin for S. aureus agreed with the PCR results with the exception of one isolate, for which the MIC was 4 µg/ml. However, a number of discrepant results were reported for CNS using the older NCCLS breakpoints. Three isolates for which the MIC was 8 µg/ml were mecA negative, whereas two isolates for which the MICs were 2 and 1 µg/ml, respectively, were positive for mecA. However, when the new NCCLS breakpoint (211) for oxacillin resistance in CNS (0.5 µg/ml) is applied, seven isolates would be resistant but lacking the mecA gene. No false-positive results would be reported. This multiplex could be applied to samples from either blood culture bottles or whole blood. In the latter case, a sensitivity of 20 and 80 CFU per 3-µl sample was obtained for nuc and mecA products, respectively.

A variation using the S. aureus-specific coa (coagulase) gene was described recently (145). Complete agreement was observed between the results of the coa-specific PCR and conventional slide coagulase testing with S. aureus and CNS isolates. In contrast to most studies, there was also complete agreement between the mecA PCR and standard disk diffusion testing.

Salisbury et al. (283) developed a PCR for mecA which also included an internal control by amplifying 16S rRNA gene sequences. This study not only involved methicillin-resistant S. aureus (MRSA), methicillin-susceptible S. aureus (MSSA), methicillin-susceptible CNS (MSCNS), and methicillin-resistant CNS (MRCNS), but also involved high--lactamase producing S. aureus isolates. The results were in full agreement for MSSA, MRSA, and MRCNS, but one of the -lactamase-overproducing isolates and three MSCNS isolates were also mecA positive. However, it should be noted that for CNS isolates the old breakpoint of 8 µg/ml for resistance was used. Because no MICs were listed in this study, no comparison with the new breakpoint was possible (211).

Using a multiplex PCR assay with the gyrA gene as an internal control, Zambardi et al. (407) evaluated 468 S. aureus isolates for methicillin resistance. Good results were obtained compared with MIC testing, the only exception being -lactamase producers. When a part of the 16S rRNA gene was amplified as an internal control in conjunction with amplification of mecA, only four discrepant results were obtained when results for 228 S. aureus isolates were compared with those of agar dilution and disk susceptibility tests (93). Three of these discrepancies were due to -lactamase hyperproducers. No discrepancies were observed for MSCNS isolates, but 11 mecA-positive isolates were phenotypically susceptible, again confirming findings with other studies of CNS isolates. This PCR was also compared with a hybridization assay (155). This assay was composed of a capture probe which was immobilized on magnetic beads and a acridinium ester-labeled probe for chemiluminescence detection. A bacterial lysate was mixed with the probes, and, after hybridization, excess material was easily removed after magnetization of the mixture. For S. aureus (n = 147), complete agreement between the methods was observed, while for CNS isolates (n = 253) only one mismatch was observed (one isolate was PCR negative). However, compared to disk diffusion, 14 isolates were CNS mecA negative but phenotypically resistant. Of these 14 isolates, 13 proved to be -lactamase hyperproducers. Thirteen CNS isolates were mecA negative and phenotypically resistant. Only three of these isolates proved to be -lactamase hyperproducers. In contrast to most other studies described, no phenotypically susceptible but mecA-positive isolates were observed.

A limited study by Kitagawa et al. (151) in Japan demonstrated the ability of PCR to directly detect MRSA in clinical specimen. This group developed a nested multiplex PCR for mecA and TSST-1 for use with 1-ml blood samples. The sensitivity of the PCR reached 10³ CFU/ml of blood, and the result was obtained in 4 h, whereas culture took 48 h. A total of 35 patients with high fever or watery diarrhea who had undergone major gastrointestinal surgery and 6 healthy volunteers were examined. The PCR was positive for both products for isolates from 12 patients from whom MRSA was also cultured. None of the other patients or healthy volunteers were positive by culture or PCR. This result clearly showed the advantage of PCR over conventional culture, although the sample size was small.

Ünal et al. (365) developed a mecA PCR with the femA gene involved in peptiglycan synthesis of S. aureus as internal control. The results obtained by testing 79 clinical staphylococcal isolates showed that femA was S. aureus specific, and the results of PCR and phenotypic testing generally agreed. In a second study, results obtained using these primers for amplification and microdilution testing were compared. Among 1,450 S. aureus, 186 isolates were resistant to methicillin but 13 isolates for which the MIC was 4 µg/ml were mecA negative (366). A study in Turkey (153) using the same PCR method for the detection of mecA in both S. aureus and CNS was judged by the authors as usable for the clinical microbiology laboratory. In this study, hyperproducing -lactamase strains demonstrating a methicillin-resistant phenotype but mecA-negative genotype were also reported.

Towner et al. (352) described a multiplex PCR based on the mecA and femB genes. The latter gene is involved in peptidoglycan synthesis and is believed to be absent in CNS. Primers for the former gene were labeled with either dinitrophenol or biotin, whereas the primers for the latter gene were labeled with either dinitrophenol or digoxigenin. Detection was performed using the Clearview (Unipath) immunoassay detection system involving a membrane with anti-biotin and anti-digoxigenin antibodies and blue latex beads coated with anti-dinitrophenol antibodies. A total of 480 S. aureus and CNS isolates were evaluated, and five discrepant results were obtained when femB PCR was compared with conventional identification. Of the 152 S. aureus isolates, 40 were considered MRSA by routine MIC testing whereas only 24 harbored the mecA gene. In addition, two isolates were mecA positive but methicillin- susceptible. A similar result was observed when the assay was used to detect MRSA in routine patient screening swabs.

bDNA signal amplification has also been used to detect the mecA gene (154). The results obtained with 416 clinical staphylocococcal isolates were 100% concordant with the results of a mecA PCR. The results of this assay was subsequently evaluated on BACTEC blood cultures with positive growth indices from 225 patients showing staphylococcus-like organisms on gram stain and compared to PCR (411); conventional identification showed 50 S. aureus and 175 CNS isolates. A total of 122 bottles (16 S. aureus and 106 CNS isolates) were positive in both mecA PCR and bDNA assays, and 102 bottles (including 33 with S. aureus) were negative in both assays. One S. aureus-containing blood culture was positive in the bDNA assay but PCR negative. This erroneous result remained after retesting and was confirmed by phenotypic testing. Despite this discrepancy, the bDNA test has the advantages that it does not require elaborate sample preparation, is not subject to inhibitors known to influence the PCR, and a result can be obtained in 6 h in a 96-well format.

In conclusion, the results obtained with various molecular techniques and isolates compared to phenotypic assays reflect the fact that mecA expression may be regulation dependent (21) but also that other mechanisms, may lead to low-level methicillin resistance (64). In addition, the population of MRSA may be composed of only a few clonal lineages (C. L. C. Wielders, M. Vriens, S. Brisse, E. D. J. Peters, A. T. A. Box, J. Verhoef, F. J. Sehmitz, and A. C. Fluit, submitted for publication), which may bias the results to some extent. Nevertheless, mecA PCR is the most robust and reliable way of detecting oxacillin-resistant staphylococci, and the results of this PCR assay are used as a "gold standard" to compare screening methods to identify MRSA (141, 342). Similarly, this is true for CNS, independent of whether the old or the new NCCLS breakpoint recommendations are followed. Alternative tests may provide a valuable addition, especially for quick initial screening.

Resistance to penicillin in pneumococci is due to the presence of altered PBPs, especially PBP2, which have a reduced affinity for penicillin. -Lactamases have never been detected in pneumococci (108). Streptococcus pneumoniae encodes six PBPs, termed PBP1a, PBP1b, PBP2a, PBP2b, PBP2x, and PBP3. In susceptible strains, these PBPs are highly conserved. The genes for low-affinity PBP1a, PBP2b, and PBP2x found in resistant isolates diverge significantly. These genes are called mosaic genes because part of the sequence is highly homologous to the counterpart in susceptible isolates whereas other parts are highly divergent (71, 162). The divergent regions appear to come from other related species, such as Streptococcus mitis (70, 71). In S. pneumoniae, PBP2b is the main target for penicillin but PBP2x is the target for cefotaxime (157).

Ubukata et al. (362) studied PBP2b-related pencillin resistance in 1,062 pneumococcal isolates. Three primer sets were developed: one set specific for the susceptible (wild-type) PBP2b and the other two sets specific for the forms of low-affinity PBP2b termed types A and B. In addition, a primer set was used for the lytA gene, which encodes autolysin and is S. pneumoniae specific. A total of 614 isolates from 621 phenotypically susceptible isolates yielded a product with the wild-type primers. Only 1.8% of the isolates for which the MIC was 0.125 µg/ml yielded a PCR product with the type A primers, and 70.3% yielded a product with the type B primers. The remaining isolates gave no PCR product. However, when only high-level resistance (MIC 1 µg/ml) was taken into account, 89.8% of 275 isolates showed a type B PCR product. Phenotypic analysis of the highly penicillin-resistant isolates, which yielded no PCR product (n = 123), showed the presence of a low-affinity PBP, indicating the presence of non-type A or B low-affinity PBP2bs. A comparable PCR was developed by a South African group (74), which used four different primers for the four known South African low-affinity PBP2b genes and a primer for the wild-type gene in combination with a primer chosen in a conserved region for all five types. PCR analysis of both cerebrospinal fluid and colonies obtained after culture of the cerebrospinal fluid specimens showed 100% concordance. These investigators also developed a seminested PCR for the detection of two resistance variants of the gene encoding PBP1a which are involved in high-level penicillin resistance. For 180 of 183 isolates, MIC data and PCR results were in agreement. Differences were caused by unknown mutations (75).

A somewhat different approach was taken by Jalal et al. (130). This group developed primers for the genes of the susceptible forms of PBP2b, PBP2x, and PBP1a. The latter gene is supposedly also present in resistant isolates and was used as an amplification control. A total of 230 pneumococcal isolates from different geographical locations were analyzed. PCR correctly identified 93% of 116 penicillin-susceptible isolates, 85% of the intermediate-resistant isolates, and all 49 highly penicillin-resistant isolates. Only two of the intermediate penicillin-resistant isolates were classified as susceptible by PCR analysis. A third approach using PCR followed by restriction enzyme digestion (PCR-RFLP) distinguished between susceptible and resistant genotypes of the gene for PBP2b (227). All susceptible isolates had identical RFLP patterns. This pattern was also observed in 14 of 30 intermediate-resistant isolates. A large number of different patterns were observed for both intermediate and high-level penicillin-resistant isolates.

The combined data from these PCR assays suggest that the presence of unknown mutations or mosaic genes for the PBPs can result in false-negative results for penicillin-resistant isolates. However, the formation of a PCR product using wild-type (susceptible) primers in a properly controlled PCR assay may be used as a screen for pencillin-resistant S. pneumoniae. The wild-type gene was not associated with high-level resistance.

Several groups used a variety of techniques including PCR in combination with restriction enzyme analysis (PCR-RFLP) to perform epidemiological typing studies on penicillin-resistant S. pneumoniae, but in none of the cases was PCR-RFLP sufficient (68, 98, 126, 188, 406). The results of these studies indicate widespread genetic exchange and clonal dissemination of strains.

One of the first probes described was used to investigate the relationship of TEM-1, which is the mostly frequenctly encountered -lactamase among Escherichia coli isolates with other -lactamases. The radiolabeled probe hybridized with TEM-2 and OXA-2 -lactamase-encoding plasmids. No hybridization was observed with OXA-1-, OXA-3-, HMS-1-, SHV-1-, CARB-1 (PSE-4)-, and PSE-3-encoding plasmids (57), indicating that discrimination between genes encoding -lactamases frequently encountered among gram-negative pathogens could be discriminated. This probe was also used in colony hybridization a assay of 328 isolates belonging to 11 gram-negative genera (140). TEM genes were detected in 53.6% of the isolates, and the results were 92.7% concordant with the results of isoelectric focusing. Sixteen isolates were positive in hybridization but yielded no result in isoelectric focusing. Although no explanation was offered for this discrepancy, the method was considered convenient for the rapid screening of isolates. Radiolabeled fragment probes were also used to detect the genes for TEM-1, SHV-1, OXA-1, OXA-2, PSE-1, PSE-2, and PSE-4 (127). Some of the probes cross-hybridized with related -lactamase genes. Despite this drawback, the authors declared the probes a success. At least the probes are useful to indentify -lactamase genes at the gene family level.

Ouellette and Roy (233) sequenced the OXA-1 -lactamase gene, and from the data they developed a specific 15-mer oligonucleotide probe. The advantage of an oligonucleotide probe, if well designed, is that its hybridization is more specific than that of a fragment probe. In an extension of their work, a TEM family fragment probe was developed, together with two oligonucleotide probes which discriminated between the two (then known) members, TEM-1 and TEM-2, which differ by only one nucleotide (232). A danger noted by the authors is that when new TEM mutants arise, a negative hybridization may result due to the specificity of the oligonucleotides. No major difference between colony blotting and dot blotting of partially purified DNA was observed. The TEM-1- and OXA-1-specific oligonucleotides were used to assess the presence of these genes in 114 -lactamase-producing isolates belonging to 16 species of gram-negative bacteria (231). The correlation of the hybridization with isoelectric focusing was 96 and 100% for the TEM-1 and OXA-1 probes, respectively. Five P. aeruginosa isolates with the same serotype originating from the same hospital reacted with the probe, but no silent TEM-1 gene could be detected. The authors suggested increasing the length of the probe to circumvent the problem. The study illustrated that with shorter oligonucleotides, the chance is increased that the same sequence in an unrelated gene will be present in a different organism. On the other hand, longer fragment probes often lack sufficient specificity and therefore also hybridize with (partly) related sequences. In the late 1980s and early 1990s, a number of fragment and oligonucleotide probes were found to detect specific -lactamase genes including genes for TEM-1 (30, 40, 42), OXA-1 (30), SHV-1 (26), and AmpC (400).

The determination of the presence of TEM -lactamases in bacteria directly in urine by filtering the urine over nitrocellulose followed by lysis of the bacteria, denaturation, and hybridization with a nonradioactive probe demonstrated problems with direct testing (40). One of the 81 samples tested was positive due to urine pigmentation. A false-negative result was obtained due to insufficient numbers of cells in the sample. One of the isolates did not express the -lactamase. Whether the gene could be expressed in vivo is not clear, but the result illustrates the problem of phenotypic versus genotypic resistance determinations.

PCR products are generally detected by agarose gel electropheresis. A different approach to the detection of PCR products in a TEM-specific PCR was the use of an immunoassay. In this approach, a 16S rRNA-specific internal control primer and a TEM-specific primer are labeled with dinitrophenol, the second internal control primer is labeled with digoxigenin, and the second TEM-specific primer is labeled with biotin. A membrane is then coated with an anti-biotin detection line and an anti-digoxigenin control line. The PCR product is captured by the antibodies on the membrane and detected by blue latex beads coated with anti-dinitrophenol antibodies. Application of the assay to 477 E. coli isolates from urine samples showed 185 PCR immunoassay-positive isolates among 187 -lactamase-producing isolates; after repeat testing, the two PCR immunoassay-negative isolates also became positive. All ampicillin-susceptible isolates except one were immunoassay negative. No explanation was found for this discrepancy (58). The major advantage of the assay is speed, with detection of the amplifcation products taking only minutes.

A very sensitive (10⁴ to 10⁵ CFU) and fast (4-h) chemiluminescent oligonucleotide hybridization assay for TEM-1 was designed for the detection of TEM-1 -lactamase in N. gonorrhoea (286). The assay used 7 capture probes and 19 detection probes for the detection of TEM-1 and 10 capture probes and 26 labeling probes for the N. gonorrhoea-specific TEM-1 probe. No explanation for the large number of probes was given, and although the assay proved to be very specific, the large number of individual oligonucleotides required will make quality control a problem, especially with the current availability of PCR, which can more easily achieve similar levels of sensitivity. Nevertheless, the study showed the potential of signal amplification assays. In another study, a probe for TEM-1 was biotinylated and after hybridization streptavidin-coupled europium was added as fluorescent marker (59). The results demonstrated that 4 × 10⁴ molecules could be detected.

In N. gonorrhoea, six related plasmids are responsible for -lactamase production and only three of these, the Asia, Africa, and Toronto type plasmids, have been associated with epidemics. To differentiate among these plasmids, a PCR assay was developed that differentiated each plasmid type by its differently sized amplification products. The assay was 100% specific and sensitive and identified 16 isolates with the Asia plasmid, 41 isolates with the Africa plasmid, and 16 isolates with the Toronto plasmid, demonstrating the value of the assay for epidemiological typing of N. gonorrhoea -lactamase plasmids (66). Whole-plasmid DNA probes were also used to investigate the prevalence of the ROB -lactamase in Haemophilus influenzae (60). Before this investigation, only two isolates carrying the ROB -lactamase were known. Hybridization of this probe and a TEM-1 probe with DNA from 161 clinical isolates from the United States showed that 13% of the isolates harbored the gene for the ROB -lactamase and the others carried the TEM-1 gene. This investigation demonstrated that quick screening for the presence of newly discovered resistance genes can be performed easily with the use of probes. A PCR assay with primers specific for the TEM and ROB genes was developed to directly detect these genes in cerebrospinal fluid in combination with a 16S rRNA gene amplification (340). The 16S rRNA amplification product was identified by hybridization with an H. influenzae-specific probe. A few isolates, which were all ampicillin resistant, carried the TEM gene, but no ROB gene was detected. The authors noted that they had to use the native Taq DNA polymerase preparation, because the AmpliTaq preparations were contaminated with vector DNA containing TEM gene sequences. A PCR assay was also used to determine the presence of TEM and ROB-1 -lactamase in 157 ampicillin-resistant H. influenzae isolates from Canada (305). The results for the ROB-1-specific PCR were confirmed by restriction enzyme analysis. Eleven isolates carried the ROB-1 gene, whereas the other isolates harbored a TEM gene. Although digestion with the restriction enzyme MboI allowed discrimination between TEM-1 and TEM-2, no TEM-2 -lactamase genes were found.

Cephalosporinases are common -lactamases among anaerobes. To obtain a better understanding of the distribution of cepA- and cfxA-encoded cephalosporinases among Bacteroides isolates, two specific oligonucleotides were designed (194). All 80 resistant isolates of Bacteroides fragilis carried the cepA gene, as well as 2 of 7 resistant Bacteroides distasonis isolates, 1 of 7 resistant Bacteroides vulgatus isolates, and 1 of 5 Bacteroides thetaiotaomicron isolates. Only one B. fragilis isolate harbored the cfxA gene compared to 20% of the B. fragilis group isolates. These results agreed with the isoelectric points for the corresponding -lactamases.

With few exceptions, ESBLs belong either to the TEM or SHV family of -lactamase genes and are located on plasmids and ESBL are usually found in the Enterobacteriaceae. The -lactamases belonging to one of the families differ only in a few amino acids, but these alterations influence the spectrum of acitivity of these enzymes. The vast majority of ESBLs are inhibited in vitro by clavulanic acid. The first ESBLs studied with probes belong to the TEM family. Point mutations in the TEM sequence which led to amino acid changes often broadened the spectrum of activity of these -lactamases. Biotinylated oligonucleotide probes discriminated between TEM-1, TEM-3, and TEM-6 (348), and 12 radiolabeled primers were described that enabled the discrimination of TEM-1 to TEM-7 (185). The latter assay found 14 variants, including 7 new enzymes, among 265 clinical isolates. One of these enzymes had the same substrate profile and isoelectric point as TEM-2 but differed by a single amino acid change. The other enzymes (TEM-14 to TEM-19) were ESBLs which had novel combinations of known amino acid substitutions. The use of radioactively labeled fragment probes also led to the identification of novel ESBLs (13), although these these probes are less sensitive for the detection of mutations which are responsible for the extended substrate range. In some cases these mutations lead to the appearance or disappearance of restriction sites. Amplification of the relevant part of the gene by PCR followed by restriction enzyme analysis can thus indicate the presence or absence of some specific TEM or SHV derived ESBLs (12, 224).

PCR-SSCP has also been applied to the study of ESBLs. Five SHV-type ESBLs were analyzed by PCR-SSCP with satisfactory results, although only few isolates were tested (205). These results were later extended to include SHV-7 and the ability to detect more than one SHV-type gene in a single isolate (206). Another investigation applied PCR-SSCP to TEM-1, TEM-2, TEM-30, and TEM-32 to TEM-39 with good results (319). The results showed the presence of a novel TEM ESBL (TEM-58), which was confirmed by sequencing.

In Turkey, the distribution of the PER-1 ESBL was investigated (368). This ESBL is not related to either TEM or SHV ESBLs. A total of 72, 92, and 362 isolates of Acinetobacter, Klebsiella, and Pseudomonas, respectively, from eight university hospitals were studied by colony hybridization. The enzyme was not present in Klebsiella isolates, but 46% of the Acinetobacter isolates and 11% of the P. aeruginosa isolates carried the gene for this -lactamase. Southern blot analysis with a PER-1 gene-specific probe showed that the gene is present on a number of different DNA fragments. The PER-1 -lactamase currently appears to be restricted to Turkey, but the high prevalence and the presence in different clones suggest a high potential for spread.

The bla_IMP encoded metallo--lactamase efficiently hydrolyzes both carbapenems and cephalosporins. The enzyme was found in gram-negative rods in Japan (9, 306) and is located on an integron. Integrons are genetic elements which are associated with multidrug resistance (90) (see "Multidrug resistance" below). A total of 54 highly ceftazidime-resistant isolates were therefore tested with bla_IMP-specific PCR; 22 isolates (9 P. aeruginosa, 9 Serratia marcescens, 2 Alcaligenes xylosoxidans, 1 Pseudomonas putida, and 1 Klebsiella pneumoniae) were identified which carried the gene. PCR assays for the integron integrase gene intI3 and the aac(6')-Ib aminoglycoside resistance gene associated with the original isolates showed that these genes were well conserved. Rapid PCR detection of novel genes is therefore helpful in the early recognition of emerging resistance.