GR Micro Limited, 79 William Road, London NW1 3ER, UK
Received 12 April 2005; returned 19 May 2005; revised 30 June 2005; accepted 14 July 2005
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Abstract |
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Methods: For 14 870 H. influenzae, MIC testing was performed using NCCLS broth microdilution methodology. For 2225 ß-lactamase-positive (BLP) H. influenzae, TEM-1 and ROB-1 genes were detected using a Taqman PCR method.
Results: ß-Lactamase positivity was 15.0% overall but varied greatly by country (<5% in several countries to 67.9% in Taiwan). Prevalences of TEM-1 and ROB-1 BLP H. influenzae were 93.7% and 4.6%, respectively, however almost all ROB-1 isolates were found in Canada, the USA and Mexico. ROB-1 isolates (n = 102) were less susceptible against cefaclor (29.4% versus 87.6%) and cefprozil (42.2% versus 91.9%) than TEM-1 (n = 2085) isolates. Differences in susceptibility rates for chloramphenicol, co-trimoxazole and tetracycline were also found between the two groups.
Conclusions: The ROB-1 ß-lactamase was found almost exclusively in North America and was more active against cefaclor and cefprozil than the TEM-1 ß-lactamase.
Keywords: surveillance , resistance genes , cefaclor , cefprozil
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Introduction |
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Previous reports on the distribution of ß-lactamase genes have only contained data from Canada, Japan, Italy and the USA.25 Hence, global prevalence and distribution are unknown. The aim of this study was to determine the global distribution of ROB-1 and TEM-1 and to investigate any differences in in vitro antibacterial efficacy associated with each enzyme.
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Materials and methods |
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MICs were determined at a central reference laboratory (GR Micro Ltd, London, UK) using the NCCLS broth microdilution method.7 Breakpoints were established using Clinical Laboratory Standards Institute (CLSI) criteria.8 ß-Lactamase production was determined for isolates of H. influenzae using nitrocefin reagent as per the manufacturer's instructions (Oxoid Ltd, Basingstoke, UK).
Primers and probes for the ROB-1 and TEM-1 genes were designed using Primer Express software (Applied Biosystems, Warrington, UK). For ROB-1, oligonucleotides used were: primers MGROB31F 5'GCGCCTGTGCAACAATCA3' and MGROB87R 5'CAAATTCGCCAAAGTCTGTTGA3' and probe MGROB50T 5'VIC-CCACACAAGCCACCTT-MGB3'. For TEM-1, oligonucleotides used were: primers MGTEM35F 5'AAGTTGGCCGCAGTGTTATCA3' and MGTEM101R 5'ATGGCATGACAGTAAGAGAATTATGC3' and probe MGTEM56T 5'FAM-CTCATGGTTATGGCAGCAC-MGB3'. Amplification was performed as a duplex PCR in an ABI 2700 thermocycler (Applied Biosystems) using the following cycling parameters: 50°C for 2 min, 95°C for 10 min, and 30 cycles of 95°C for 15 s and 60°C for 1 min. Allelic discrimination was performed using an ABI PRISM 7000 Sequence Detection System (Applied Biosystems). In our laboratory, this methodology for large-scale gene detection has proven to be the most time efficient and cost-effective approach. In addition, no post-amplification manipulation of amplified material occurs minimizing laboratory and specimen contamination.
Statistical analysis was performed using the 2 test and InStat software (GraphPad Software, Inc., San Diego, CA, USA).
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Results |
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Discussion |
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As seen by the range of MICs in Table 2, many ROB-1 and TEM-1 isolates had high levels of resistance to cefaclor and cefprozil (up to >128 mg/L). It is unclear why such variation in MIC levels exists but a recent study showed that mutations in the controlling region of the TEM-1 ß-lactamase were associated with increased resistance to cefaclor.9 We plan to sequence the fts1 gene (which is the transpeptidase region of penicillin binding protein 3A and/or 3B) to determine whether the same or similar mutations are responsible here.
A total of 27 ß-lactamase-positive H. influenzae isolates were negative for both ROB-1 and TEM-1 genes suggesting either a mutation has occurred in either or both ROB-1 and TEM-1 gene(s) to prevent detection by our methodology, or a previously undescribed enzyme (or enzymes) is (are) responsible. Further research is needed to investigate this. Although the prevalence of BLNAR isolates was low overall, the high rate of BLNAR and particularly BLNAI found in Japan supports the previously reported high rate of BLNAR in this country.4 The observed trend of increasing BLNAI/BLNAR prevalence over the 4 years of the study warrants close monitoring as these isolates have high levels of non-susceptibility to the cephalosporins tested and amoxicillin/clavulanate. Of the non-ß-lactam antibiotics, telithromycin, azithromycin, and the fluoroquinolones tested (levofloxacin and ciprofloxacin), all demonstrated high in vitro activity against H. influenzae regardless of H. influenzae ß-lactamase status.
This is the first study to assess and provide baseline data on the global prevalence and distribution of ß-lactamases in H. influenzae. We have shown that the distribution of these enzymes is variable and have confirmed previous reports that in vitro susceptibility to cefaclor is decreased and demonstrated that susceptibility to cefprozil is also decreased. This study is being continued in current PROTEKT studies and is planned to continue in future years.
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Acknowledgements |
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References |
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