INTEGRONS AND ETA-LACTAMASES – A NOVEL PERSPECTIVE ON RESISTANCE

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CHAPTER 2: AMBLER CLASS A EXTENDED-SPECTRUM ETA-LACTAMASES IN PSEUDOMONAS AERUGINOSA – NOVEL DEVELOPMENTS AND CLINICAL IMPACT.

Introduction and epidemiology.

The so-called clavulanic-acid inhibited extended-spectrum beta-lactamases (ESBLs) belong mostly to class A of the Ambler classification scheme (1) and confer resistance to at least several expanded-spectrum cephalosporins (21, 22, 34) (Table 1-1, 1.1). They have been extensively reported in Enterobacteriaceae from the early 1980`s whereas they have been described in Pseudomonas aeruginosa only more recently (22, 34, 40).
These enzymes described in P. aeruginosa, are either of the TEM- and SHV-types that are also well known in Enterobacteriaceae, of the PER-type mostly originating from Turkish isolates, or of the VEB-type from Southeast Asia and, more recently, of the GES /IBC types reported from France, Greece and South Africa, respectively (14, 33,37-39, 41, 48, 52). These five types of enzymes are remotely related, both from a genetic point of view and similarities in hydrolytic profiles. Recent studies indicated that these enzymes may play an important role in the dissemination of antibiotic resistant isolates and may condition future choices of antibiotic regimens for treating life-threatening infections due to ESBL-producing P. aeruginosa (12, 17, 51).
As summarized in Table 2-1, these enzymes have been found so far in a limited number of geographical areas, suggesting that some of these beta-lactamase genes may at least in several cases represent a specific local selection.The SHV-type ESBLs have been identified in very rare isolates of P. aeruginosa, SHV2a from France, whereas SHV-5 and SHV-12 were from Thailand (6, 37). These isolates were nosocomial strains except the SHV-12 producer, isolated from a clinical sample from an outpatient of a Thai hospital (6). The TEM enzymes described in P. aeruginosa namely, TEM-4, TEM-21, TEM-24 and TEM-42 have been reported in rare isolates from France (3, 13, 31, 36, 42, 49). A French survey indicated that only 10% of ticarcillin-resistant P. aeruginosa produce a TEM-type beta-lactamase, whereas other narrow-spectrum beta-lactamases (OXA,CARB) constitute a higher proportion in that species (3). Conversely, the TEM-type enzymes are widely distributed in Enterobacteriaceae, whereas OXA-type and CARBtype beta-lactamases are rare (27). The few reports of P. aeruginosa strains harbouring TEM and SHV-type genes may have several explanations. Firstly, the rarity of narrowspectrum TEM and SHV-type enzymes may limit antibiotic selection of these enzymes with an expanded-spectrum hydrolysis. Secondly, a higher proportion of acquired oxacillinase and carbenicillinase genes (most of them being chromosome-encoded) may fulfil the function of genes encoding narrow-spectrum enzymes such as the TEM and SHV types. Indeed, several oxacillinases (OXA-2 and OXA-10 derivatives, and OXA18) have been reported in P. aeruginosa that have extended substrate profiles including extended-spectrum cephalosporins (7, 40, 45). Thirdly, expression of the chromosomeencoded cephalosporinase of P. aeruginosa may be up regulated (derepression) and may thereby be a convenient way for acquisition of resistance to expanded-spectrum cephalosporins (23), without the need for expanding its genetic repertoire. It is likely that the origin of TEM- and SHV-type ESBLs in P. aeruginosa may result from gene transfer from Enterobacteriaceae (27). This has been shown for TEM-24 (31) and the downstream-located DNA sequences of the chromosome of P. aeruginosa RP-1 that produces SHV-2a, which were found to be identical to those reported as plasmidencoded in a Klebsiella pneumoniae isolate (37, 46). Differences in replication origins of plasmids from Enterobacteriaceae and P. aeruginosa may however limit such intergeneric transfers. Additionally, difficulty of detection of TEM- and SHV-type ESBLs in the clinical laboratory may underestimate their true prevalence in P. aeruginosa.
Beta-lactamase PER-1 was the first ESBL identified and fully characterized in P. aeruginosa in 1993 (39, 41). It was found from a P. aeruginosa isolate of a Turkish patient hospitalised in the Paris area in 1991 (39). A subsequent study on thedistribution of the blaPER-1 gene revealed that it is widespread in Turkey, with PER-1 being identified in up to 46% of Acinetobacter strains and 11% of P. aeruginosa analysed in a nation-based survey performed over a three-month period in 1999 (61). PER-1 was identified in up to 38% of ceftazidime-resistant P. aeruginosa isolates, with ribotyping results indicating spread of different clones (61). Since screening for the blaPER-1 gene has not been performed in P. aeruginosa isolates originating from countries located to the south and east of Turkey such as Syria, Iran and Iraq, no current data are available on the prevalence of PER-1 in the Middle East .It is possible that the spread of PER-1 in Western Europe may be mostly related to the widespread immigration of Turkish nationals. Interestingly, although reported in several enterobacterial species including community-acquired pathogens such as Salmonella spp. (62), beta-lactamase PER-1 seems mostly expressed from P. aeruginosa and Acinetobacter spp. isolates in Turkey (61, 63). A large nosocomial outbreak of PER-1-producing P. aeruginosa has been documented in Varese, Italy, occurring over a 10-month period in a tertiary hospital (30). During that outbreak, a total of 108 clinical isolates were recovered from 18 patients, reflecting the propensity of P. aeruginosa to widely colonize hospitalised patients. In that case, apart from the beta-lactam resistance phenotype conferred by PER-1, epidemic strains were resistant to several disinfectants, including chlorhexidine, iodide povidone, and toluene-psulphochloramide (30). Control of the outbreak was achieved by implementing strict hygienic measures, carbapenem therapy and disinfection of decubitus ulcers and surgical wounds with mercurochrome or silver nitrate solutions (30). As a result of increased carbapenem consumption, selection of several carbapenem-resistant
organisms occurred in the nosocomial environment including OprD-defective P. aeruginosa, Stenotrophomonas maltophilia and Pseudomonas putida producing the class B carbapenemase VIM-1 (30). The same group had reported a P. aeruginosa strain that produced the plasmid-mediated beta-lactamase VIM-2 together with betalactamase PER-1 (12) thus showing that the same P. aeruginosa strain may produce two unrelated beta-lactamases both with expanded-spectrum hydrolysis. Recently, another P. aeruginosa strain that produced PER-1 has been isolated from a patient hospitalised in Clermont-Ferrand in the central part of France (11). Indeed, this latter patient had been hospitalised previously in Strasbourg, in the eastern part of France where the patient might have been in contact with hospitalised Turkish patients (P.Nordmann, personal communication). A pseudo-outbreak (false positive culture results due to specimen contamination) has also been reported from Belgium (8), revealing the obstacles that face investigators when searching for the source of multiresistant P.aeruginosa isolates. Although no mention is made about the antibiotic regimen used for treating the infected patient, the pseudo-outbreak was successfully terminated by decontamination of a side-room urine densitometer (8).

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SUMMARY 
SAMEVATTING 
ACKNOWLEDGEMENTS 
LIST OF FIGURES 
LIST OF TABLES 
LIST OF ABBREVIATIONS
CHAPTER 1: INTRODUCTION.
1.1 General introduction 
1.2 Objectives
1.3 Hypothesis 
1.4 References 
CHAPTER 2: AMBLER CLASS A EXTENDED-SPECTRUM BETA-LACTAMASES IN PSEUDOMONAS AERUGINOSA – NOVEL DEVELOPMENTS AND CLINICAL IMPACT.
2.1 Introduction and epidemiology 
2.2 Substrate profile 
2.3 Genetic determinants 
2.4 Current detection methods 
2.5 Clinical consequences 
2.6 Conclusion 
2.7 References 
CHAPTER 3: INTEGRONS AND ETA-LACTAMASES – A NOVEL PERSPECTIVE ON RESISTANCE.
3.1 Introduction 
3.2 Epidemiology 
3.3 Genetic determinants 
3.4 Expression of co-resistance 
3.5 Detection 
3.6 Conclusion 
3.7 References 
CHAPTER 4: SEQUENCE-SELECTIVE RECOGNITION OF EXTENDED-SPECTRUM BETALACTAMASE GES-2, BY A COMPETITIVE, PEPTIDE NUCLEIC ACID BASED,
MULTIPLEX-PCR ASSAY.
4.1 Introduction 
4.2 Materials and methods 
4.2.1 Isolate collection and storage.
4.2.2 Susceptibility testing.
4.2.3 DNA extraction.
4.2.4 Standard PCR amplification.
4.2.5 Competitive PNA-based multiplex PCR.
4.2.6 DNA sequencing analysis.
4.3 Results
4.4 Discussion 
4.5 References 
CHAPTER 5: RAPID DETECTION AND SEQUENCE SPECIFIC DIFFERENTIATION OF EXTENDED-SPECTRUM BETA-LACTAMASE GES-2 FROM PSEUDOMONAS AERUGINOSA,
WITH A REAL-TIME PCR ASSAY.
5.1 Introduction 
5.2 Materials and methods
5.2.1 Bacterial strains
5.2.2 Susceptibility testing
5.2.3 DNA extraction
5.2.4 LightCycler mediated mutation assay
5.2.5 Nested-PCR amplification
5.2.6 DNA sequencing analysis
5.3 Results 
5.4 Discussion 
5.5 References
CHAPTER 6: GENETIC STABILITY OF CLASS 1 INTEGRON-BORNE BLAGES–TYPE GENES UNDER SHORT TERM, SELECTIVE, IN-VITRO ANTIBIOTIC PRESSURE.
6.1 Introduction 
6.2 Materials and methods
6.2.1 Bacterial strains
6.2.2 Antibiotic challenge assay
6.2.3 DNA extraction
6.2.4 PCR amplification and detection
6.2.5 Restriction enzyme analysis
6.2.6 DNA sequencing
6.3 Results 
6.4 Discussion 
6.5 References 
CHAPTER 7: GENERAL DISCUSSION AND FINAL CONCLUSIONS
7.1 References 
APPENDIX A: WHOLE-CELL DNA EXTRACTION METHOD. 
APPENDIX B: PUBLICATIONS FROM THIS THESIS. 

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