No abstract

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is increasingly used in clinical microbiological laboratories to identify bacteria and fungi at a species level and to subtype them. The cfiA gene encoding the unique carbapenemases found in Bacteroides is restricted to division II Bacteroides fragilis strains. The aim of this study was to evaluate whether MALDI-TOF MS is suitable for differentiating B. fragilis strains which harbour the cfiA gene from those that do not. A well-defined collection of 40 B. fragilis isolates with known imipenem MICs (0.062-.32 mg l "1 ) were selected for this study. Twelve B. fragilis strains with known cfiA status, including NCTC 9343 (division I) and TAL3636 (division II), were measured by means of microflex LT MALDI-TOF MS and well-defined differences in mass spectra between the cfiA-positive and cfiA-negative strains were found in the interval 4000-5500 Da. A further 28 strains were selected for the blind measurements: 9 cfiA-positive clinical isolates with different imipenem MICs ranging between 0.06 and .32 mg l "1 (different expressions of the metallo-b-lactamase gene) were clearly separated from the 19 cfiA-negative isolates. The presence or absence of the selected peaks in all tested strains clearly differentiated the strains belonging to B. fragilis division I (cfiA-negative) or division II (cfiA-positive). These results suggest a realistic method for differentiating division II B. fragilis strains (harbouring the cfiA gene) and to determine them at a species level at the same time. Although not all cfiA-positive B. fragilis strains are resistant to carbapenems, they all have the possibility of becoming resistant to this group of antibiotics by acquisition of an appropriate IS element for full expression of the cfiA gene, leading to possible treatment failure.

Fourteen strains harboured the well-known genetic elements: pIP417- and pIP419-like plasmids, chromosomal nimB genes and a common nimE plasmid. However, a rate of interchangeability was also demonstrated, mostly due to combinations of nim genes and their associated IS elements harboured on different replicons.

The aim of this work was to study the toxin types of Clostridium difficile isolates originating from different parts of Hungary. A PCR method was used for amplification of the two major toxin genes and the binary toxin gene and to detect the deletion or insertion in the 3 end of the toxin A gene. The findings were compared with the results of cytotoxicity assays on the HeLa cell line. One hundred twelve isolates were tested; the toxin A and toxin B genes were detected in 79 strains by the PCR method. All of the isolates that were positive by the PCR method were also positive by the cytotoxicity assay. All of the other strains (n ‫؍‬ 33) were negative for the toxin A and toxin B genes; in these cases, cytopathic effects on the cell line were not observed. No tcdA-negative and tcdB-positive isolates were found by the PCR method. In two cases, the presence of a binary toxin gene was observed by PCR; both isolates that were isolated from diarrheal feces carried the tcdA and tcdB genes. No prior hospitalization had occurred in either case.Clostridium difficile is an anaerobic, gram-positive, sporeforming rod. Toxigenic strains are causative agents of antibiotic-associated diarrhea, antibiotic-associated colitis, and pseudomembranous colitis (1, 2). Such strains produce two major toxins: toxin A (TCDa), a cytotoxic enterotoxin that induces fluid secretion, and toxin B (TCDb), a cytotoxin. These toxins cause tissue damage in the intestinal mucosa (3). Both toxins evoke morphological changes in cell lines such as Vero, HeLa, and Hep2, but toxin B is a more potent cytotoxin than toxin A, and cytotoxicity assays therefore mainly detect toxin B.Toxin A variant strains (tcdA negative, tcdB positive) have been reported earlier; these strains lack the repeating sequences of the tcdA gene. The deletion was detected by PCR because in this case the amplified fragment differs in length from the PCR product of reference strain VPI 10463. These strains are toxin A negative by commercial enzyme immunoassay, which detects only the presence of toxin A, giving a negative result, as the strains are deficient in the portion of toxin A that is immunodominant; this repeating unit is recognized by the antibodies used in the toxin A-specific detection kits. In contrast, the cytotoxicity assays are positive because of the presence of toxin B, which is an effective cytotoxin (6).Some strains are known to produce actin-specific ADP-ribosyltransferase (CDT) (binary toxin), which is organized according to A-B toxin type. CDTa (48 kDa) is an enzymatic component, and CDTb (94 kDa) is a binding domain that recognizes the surface receptors of the host cells and mediates the cell entry of CDTa. Two subunits are encoded by separate genes: cdtA and cdtB. Actin modified by CDT induces damage to the actin cytoskeleton and leads to a cytopathic effect on the cell line (5, 9). Binary toxin-producing strains can be divided into three main groups: both toxin A and toxin B positive, both toxin A and toxin B negative, and toxin A negative and toxin B positive (...

Fifteen Bacteroides fragilis isolates from the USA, Hungary and Kuwait were examined for carbapenem resistance, for carbapenemase activity and, with the use of various PCR-based methods and nucleotide sequencing, for cfiA genes and activating insertion sequence (IS) elements. All the B. fragilis isolates were cfiA-positive, 10 of the cfiA genes being upregulated by IS elements that are already known. Of these 10, one was of a novel type (designated IS943) and two further ones (IS614B and IS614C) were suspected hybrids of IS612, IS614 and IS942. There were five cfiA-positive imipenem-resistant B. fragilis isolates with elevated imipenem MICs (minimal inhibitory concentration) that harboured no IS insertion upstream of the cfiA gene, but produced carbapenemase; these isolates might possess a novel activation mechanism. On the basis of the available phenotypic and genotypic evidence, the present data suggest that there are at least two cfiA activation mechanisms among B. fragilis isolates.