Enterobacteriaceae

Enterobacteriaceae are one of the most important groups of pathogens causing a wide range of diseases. They form a large potential reservoir for resistance genes and their close relationship offers the potential for exchange of these resistance genes between different family members. This is exactly what has happened, and continues to occur. Many resistance genes are shared by different species of Enterobacteri-aceae, but also a multitude of different resistance genes or variations on existing themes have developed. Examples of the first group are the aminoglycoside-modifying enzymes and the TEM ESBLs are an example of the second group. In addition to gene acquisition, resistance also can be caused by mutations, for example, fluoroqui-nolone resistance. Enterobacteriaceae are fast-growing organisms, and thus a quick resistance determination may be important in treating severe illness. Effective empiric treatment is still available, and several molecular methods have been developed that can offer the required speed for detection.

In Enterobacteriaceae, fluoroquinolone resistance is an increasing problem. Resistance is mainly caused by mutations in the gyrA gene that encodes the A subunit of DNA gyrase, but mutations in gyrB, parC and parE, encoding the B subunit of DNA gyrase and the two subunits of DNA topoisomerase IV, respectively, can also contribute. Mutations in the gyrA gene are clustered in the quinolone resistance determining region (QRDR). Several tests have been described that detect fluoroqui-nolone resistance in Salmonella and Yersinia pestis. Y. pestis has received increased interest because of its potential in biological warfare or bioterrorism.

An rtPCR using FRET technology with three probes (specific for Asp87Asn, Asp87Gly, Ser83Phe) was described for S. enterica serovar Typhimurium DT104 gyrA. A total of 92 isolates were evaluated and 86 showed expected mutations. Six isolates had a lower melting temperature indicating a mutation, and DNA sequencing showed that five isolates had a mutation different from those expected, but a sixth had no mutation in gyrA and its resistance was caused by an undetermined mechanism [147]. The reason for the lower melting temperature of these last isolates remained unexplained. A rtPCR for Y. pestis using FRET technology could detect 5 CFU in crude lysates [148]. In another variation, 5'-exonuclease detection technology was used instead of FRET. This method reached an analytical sensitivity of 1 CFU with partially purified lysates [149]. Therefore, both methods are rather comparable. However, the performance on clinical samples was not reported.

Denaturing HPLC is another popular method to detect mutations associated with fluoroquinolone resistance in S. enterica and Y. pestis. Evaluation of the method for the detection of mutations in gyrA, gyrB, parC, and parE using standard HPLC equipment showed that the method correctly predicted the presence or absence of mutations for 50 Salmonella isolates when compared to conventional DNA sequencing [150]. A second group used a similar approach, but only investigated gyrA mutations.

The method clearly identified the mutations and in addition detected more rare mutations. It was shown that an rtPCR method with mutation-specific probes required additional effort in case no match with one of the probes was found. The authors therefore concluded that denaturing HPLC is easier to perform when rare mutations are present in the population [151]. The same method has also been used to detect mutations in gyrA of Y. pestis. The method was shown to be satisfactory when tested on nearly 100 isolates and compared to conventional DNA sequencing [152].

Although tetracycline is an older antibiotic, there is still an interest in it. Furthermore, some of the genes conferring resistance to tetracycline are also responsible for resistance against newer tetracyclines including tigecycline [153]. The mechanisms of resistance are efflux, ribosomal protection, and modification of the antibiotic. A large number of different resistance genes encode these mechanisms. A number of molecular techniques have been developed to detect tetracycline resistance, but these are usually limited to a single gene thereby limiting their utility for diagnostic purposes. An example of single resistance determinant assays is an rtPCR assay for tetR of Tn10 [154].

P-Lactam antibiotics form an important class of antimicrobials to treat infections with Enterobacteriaceae. However, resistance to the older members of this class is widespread and is increasing against the newer members. The presence of P-lactamases is the most important mechanism of resistance. Sometimes the activity of these P-lactamases can be blocked by an inhibitor like clavulanic acid or tazo-bactam. However, P-lactamases that became inhibitor resistant have been described. It is therefore not surprising that molecular tests, such as SCCP, have been described to detect these P-lactamases [155]. These methods are principally of value as epide-miological tools.

Trimethoprim is also a frequently used antibiotic to treat infections with Entero-bacteriaceae and it is usually prescribed in combination with sulfamethoxazole. Resistance against trimethoprim is common, however, and can be mediated by at least a score of different genes. This makes these genes an important target for epidemiological studies and molecular tests are useful for this purpose. This led, for example, to the development of a PCR-RFLP to detect up to 16 different trimethoprim resistance encoding genes [156]. Probe-based assays have also been developed for epidemiological studies of multiple resistance determinants [157]. For the same purposes DNA microarrays are being developed. An example is a microarray that detects 25 virulence and 23 antibiotic resistance encoding genes in Salmonella and enterovirulent E. coli. The array used probes that were amplified by PCR [158]. The same method to generate probes for the microarray was also used in another study, in which a variety of resistance encoding genes were analyzed [159]. A final example of a microarray for epidemiological and surveillance purposes targeted Vibrio spp. It tested for a number of markers including resistance genes and was composed of long oligonucleotides [160].

In principle, the use of microarrays holds the possibility to check many resistance-encoding genes simultaneously, but much development has to be performed both in terms of number of genes and mutations covered, as well as costs, before they can compete with conventional phenotypic assays. rtPCR and techniques such as denaturing HPLC may have applications in some specialized niches in which resistance levels are high and speed is of importance, for example, in critically ill intensive-care patients.

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