Carbapenem resistance in Bacteroides fragilis

Sir, Carbapenems are broad-spectrum antibiotics used in the treatment of infections predominantly caused by Gram-negative bacteria, including Bacteroides fragilis. Such infections are mainly those with intra-abdominal aetiology and polymicrobial wound infections. Carbapenem-resistant B. fragilis isolates were first reported in Japan over two decades ago and subsequently in the USA. Carbapenem resistance in B. fragilis is most commonly due to the presence of Ambler class B metallo-β-lactamase CfiA.1cfiA is normally poorly expressed; however, increased expression of cfiA, caused by the acquisition of an insertion sequence (IS) upstream of the gene, can lead to high-level carbapenem resistance.2 The in vivo selection of high-level carbapenem-resistant mutants from carbapenem-susceptible cfiA-encoding B. fragilis has been shown to occur during carbapenem therapy.2 Thus, it is important to be able to detect cfiA-encoding isolates so that treatment with carbapenems can be avoided. Although PCR can be used for detecting isolates encoding cfiA, its use may not be applicable in a routine diagnostic laboratory. We investigated whether a simple disc susceptibility method could be used to facilitate the detection of cfiA-encoding B. fragilis. We determined the meropenem susceptibility of 77 B. fragilis clinical isolates collected from City Hospital, Birmingham, UK (1995–2005). The MIC of meropenem was determined by an agar dilution method3 using Iso-Sensitest agar (Oxoid Ltd, Basingstoke, UK) supplemented with 5% defibrinated horse blood and 20 mg/L β-nicotinamide adenine dinucleotide (NAD) with an inoculum of 104 cfu/spot. Using the same medium, the disc susceptibility of isolates to meropenem was determined using the BSAC standardized disc susceptibility method and a meropenem 10 µg disc.4 All plates were incubated at 35–37°C in an anaerobic atmosphere (GENbox, bioMérieux, Basingstoke, UK) for 24 h. The detection of cepA and cfiA (including adjacent upstream region) was carried out by PCR using primers and conditions described previously.5,6 Additionally, the MICs of ertapenem, imipenem, co-amoxiclav and metronidazole for cfiA-positive isolates were determined using the BSAC standardized agar dilution method, employing supplemented Iso-Sensitest agar as above. B. fragilis NCTC 9343 was used as a control for all susceptibility testing and as a cfiA-negative control. A cfiA-positive control strain, B. fragilis strain 15, was obtained from the Anaerobic Reference Laboratory, NPHS Microbiology, Cardiff, Wales. Of the 77 clinical isolates tested, 7 were cfiA-positive; all 7 had PCR amplimers of ∼400 bp, which is evidence that they do not have an IS immediately upstream of cfiA. In contrast, the cfiA-positive control (B. fragilis strain 15) produced an amplimer of ∼2000 bp, which is consistent with the presence of an IS just upstream of cfiA. The seven cfiA-positive clinical isolates formed a discrete population showing reduced susceptibility to meropenem (Table 1). All had higher meropenem MICs (range 1–4 mg/L) compared with the MICs of meropenem for the 70 cfiA-negative isolates (MIC range 0.06–0.25 mg/L). By disc susceptibility method, the 7 cfiA-positive isolates all had zones of inhibition for meropenem of <30 mm (22–27 mm), whereas the 70 cfiA-negative isolates had zones of inhibition of >30 mm (range 32–45 mm). The positive control strain with upstream IS had no zone of inhibition to meropenem and a meropenem MIC of >128 mg/L. Susceptibility and PCR results for cfiA-positive B. fragilis isolates MEM, meropenem; IPM, imipenem; EPM, ertapenem; AMC, co-amoxiclav; MET, metronidazole; NZ, no zone; NA, not applicable. aMeropenem 10 µg disc. Susceptibility and PCR results for cfiA-positive B. fragilis isolates MEM, meropenem; IPM, imipenem; EPM, ertapenem; AMC, co-amoxiclav; MET, metronidazole; NZ, no zone; NA, not applicable. aMeropenem 10 µg disc. Our results would suggest that a standardized disc susceptibility method could be used for detecting B. fragilis isolates harbouring cfiA (both high-level and low-level carbapenem resistance). The detection of potential cfiA-encoding isolates would help avoid inappropriate carbapenem usage, which might lead to treatment failure or the selection of high-level carbapenem-resistant mutants. The finding of concomitant metronidazole resistance in two of the cfiA-positive isolates is worrying, as it further reduces the therapeutic options for treating infection caused by these organisms. We would like to thank the Anaerobic Reference Laboratory, HPA Cardiff, Wales, for supplying the high-level carbapenem-resistant control strains. None to declare.

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