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Correspondence |
1 Department of Clinical Microbiology, Faculty of Medicine, University of Szeged, H-6725 Szeged, Hungary
2 Anaerobe Reference Laboratory, NPHS Microbiology Cardiff, University Hospital of Wales, Cardiff, UK
Correspondence
Elisabeth Nagy
(nagye{at}mlab.szote.u-szeged.hu)
Although a common member of the normal human gut flora, Bacteroides fragilis possesses various virulence factors, such as capsule, LPS and special surface proteins, and certain types of strains may produce an extracellular enterotoxin, termed fragilysin (BFT). The fragilysin-encoding gene (bft) has three different isoforms (termed bft-1, bft-2 and bft-3) (Chung et al., 1999; Franco et al., 1997; Kling et al., 1997). Because of the increasing number of resistant B. fragilis isolates, the therapeutic possibilities have been restricted to only some antibiotics, such as piperacillin/tazobactam, imipenem, metronidazole, clindamycin and cefoxitin (Rasmussen et al., 1993). Out of these, metronidazole and imipenem are the drugs of choice in the treatment of infections caused by B. fragilis; however, the emergence of imipenem or metronidazole resistant isolates has been reported (Rasmussen et al., 1993). Carbapenem and metronidazole resistances are frequently associated with the presence of the cfiA gene encoded class B metallo-ß-lactamase (Thompson & Malamy, 1990) or various types of 5-nitroimidazole resistance genes (nimA–G) (Gal & Brazier, 2004). Strains carrying the cfiA gene may show susceptibility to imipenem, because in most isolates this gene is silent, but the presence of insertion (IS) elements in the upstream region of this gene can lead to the development of carbapenem resistance (Podglajen et al., 1992).
By using various molecular typing methods, such as arbitrarily primed-PCR, PFGE and multilocus enzyme electrophoresis, bft-positive, cfiA-negative strains and bft-negative, cfiA-positive strains belonged to two different DNA homology groups, and the coexistence of bft and cfiA genes was not detected (Gutacker et al., 2000; Vallim et al., 2002).
During examination of the prevalence of bft- or cfiA-positive B. fragilis strains isolated from various clinical specimens, we found a multi-resistant B. fragilis strain R19811 isolated from a blood culture and identified by rDNA RFLPs in the Anaerobe Reference Laboratory, Cardiff (Wareham et al., 2005). This strain co-harboured the cfiA and bft genes; therefore, our aim was to characterize its genetic background. B. fragilis strain R19811 and control strains were cultured as previously described (Sóki et al., 2000). The list and the description of the control strains have been described before (Sóki et al., 2006).
MICs to a range of antibiotics were determined by using the E-test method (AB Biodisk) according to the manufacturer's instructions. B. fragilis strain R19811 was multi-resistant to penicillin G (MIC 
256 µg ml–1), amoxicillin/clavulanic acid (MIC 256 µg ml–1), cefoxitin (MIC 256 µg ml–1), imipenem (MIC 256 µg ml–1), clindamycin (MIC 256 µg ml–1) and metronidazole (MIC=64 µg ml–1). Because of the imipenem resistance, the presence of cfiA gene encoding metallo-ß-lactamase was tested. cfiA PCR (Podglajen et al., 1992; Thompson & Malamy, 1990) revealed that the strain carried the cfiA gene, at the same time the presence of bft gene was also detected by PCR according to Pantosti et al. (1997).
We analysed the upstream region of the cfiA gene to detect IS elements, hypothesizing that the occurrence of one of these sequences may contribute to the development of carbapenem resistance mechanisms; in B. fragilis strain R19811 an insertion was detected in the upstream region of cfiA gene by G and Up2 primers, as described by Podglajen et al. (1994). To determine the type of this element, various primers, IS614 (IS614-1i, 5'-GAAATTGTGTATCAATGCCG-3' and IS614-2i, 5'-CTGATACCATCCTCAGAGCC-3'), IS942 (Rasmussen & Kovacs, 1991), IS1169 (Trinh et al., 1995), IS1170/1 and IS1170/2 (Trinh et al., 1995), IS1186 (Podglajen et al., 1994), and IS4351 (Podglajen et al., 1994; Rasmussen et al., 1987) were tested for use in PCR. During these amplification reactions, standard PCR reaction mixture was used, as described previously by Sóki et al. (2006). Of the examined IS elements, only IS614 was detected by PCR. Partial nucleotide sequence data of the upstream region of the cfiA gene by Up2, G, IS614-1i and IS614-2i primers, as described by Sóki et al. (2004), confirmed that it is an IS614B element.
B. fragilis strain R19811 contained plasmids with an estimated molecular mass of 5.6 and 9.9 kb using HindIII restriction enzyme (Pharmacia Biotech) analysis. In spite of the fact that cfiA gene could be on a plasmid (Bandoh et al., 1992; Nakano et al., 2004), in our case, Southern blot hybridization, showed it was not.
In spite of the observed metronidazole resistance, nim genes commonly responsible for metronidazole resistance in the B. fragilis group could not be detected using the nim-3 and nim-5 primer pair (Trinh & Reysset, 1996), which is suitable for the detection for all described nim genes. The annealing temperature of nim PCR was reduced from 62 to 52 °C to show incidental sequence variation in the nim gene, but PCR products could not be detected. Further investigations are necessary to establish the metronidazole resistance mechanism in this organism.
In numerous earlier studies, the presence of the cfiA gene was never observed together with the cepA gene encoding endogenous class A ß-lactamase, because cfiA-positive and cepA-positive strains belong to two genetically distinct groups. The first group was characterized by the presence of cfiA gene, while in the second group, the presence of the cepA gene is typical with or without the presence of the bft gene. In spite of this observation, in 2005, Ayala et al. (2005) described two B. fragilis strains (7160 and 213E), which possess both the cepA and the cfiA gene. In strain R19811, no amplification product was detected with cepA PCR as described by Gutacker et al. (2000). This suggests that the strain originally belonged to the cfiA-positive group.
In the cytotoxicity assay, as described by Pantosti et al. (1994), B. fragilis strain R19811 caused a reversible cytopathic effect on the HT-29 cell line, this result indicated that in the cell culture supernatant, functional toxin, namely fragilysin was present. PCR-RFLP analysis of the bft gene by BTT1 (5'-CATGTTCTAATGAAGCTGATTC-3') and BTT2 (5'-ATCGCCATCTGCTGTTTCCC-3') primers, with minor modification according to Chung et al. (1999), revealed a bft-1 allele.
To clarify the epidemiological background of this strain, enterobacterial repetitive intergenic consensus (ERIC) PCR typing (Versalovic et al., 1991) was applied in the case of bft-positive and cfiA-negative, bft-negative and cfiA-positive, bft- and cfiA-negative strains, and the PCR patterns were compared with the result of ERIC PCR in B. fragilis strain R19811. Two homology groups of B. fragilis can be distinguished by using molecular typing methods as described previously by Gutacker et al. (2000), Moraes et al. (1999) and Ruimy et al. (1996): the first group is characterized by the presence of the cfiA gene, these strains were bft-negative, while the strains in the second group carried only the bft gene (Fig. 1
). ERIC PCR typing suggested that strain R19811, which harboured the bft and cfiA gene simultaneously, may be related to the first group, which contained only cfiA-positive strains (Fig. 1).
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ACKNOWLEDGEMENTS
This work was supported by grants KMA0304 and T037475 from Ministry of Economy and Transport, Hungary and the Hungarian National Research Foundation, respectively.
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