József Sóki

Community-Acquired Clostridium difficile Diarrhea Caused by Binary Toxin, Toxin A, and Toxin B Gene-Positive Isolates in Hungary

ABSTRACT 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.

Differentiation of division I (cfiA-negative) and division II (cfiA-positive) Bacteroides fragilis strains by matrix-assisted laser desorption/ionization time- of-flight mass spectrometry

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-β-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.

Comparison of a Rapid Molecular Method, the BD GeneOhm Cdiff Assay, to the Most Frequently Used Laboratory Tests for Detection of Toxin-Producing Clostridium difficile in Diarrheal Feces

ABSTRACT Six hundred diarrheal stool specimens were collected from inpatients and outpatients at local university hospitals for the detection of toxigenic Clostridium difficile using three parallel methods, the BD GeneOhm Cdiff assay, the tissue culture cytotoxicity assay, and a commercially available enzyme-linked fluorescence immunoassay (ELFA) (Vidas C. difficile toxin A and B assay; bioMérieux). Toxigenic C. difficile culture was also performed to further clarify discordant results. During a 3-month study period, 58 (9.7%) of the 600 diarrheal samples examined were positive by the BD GeneOhm Cdiff assay, while the Vidas C. difficile toxin A and B assay and the cytotoxicity assay performed directly on stool samples gave 4.7% and 6.3% positivity rates, respectively. In the case of four samples, BD GeneOhm Cdiff assay results were not evaluable at first because of the presence of PCR inhibitors, but upon repeat testing from the frozen lysates, all of these samples proved to be negative. After resolution with toxigenic culture, the cytotoxicity assay proved to be positive in 55 samples (9.2%), while the ELFA was positive in 37 samples (6.2%). Results of culture and repeated cytotoxicity assays emphasized the importance of the culture method, because the use of ELFA or enzyme immunoassay without a culture method may lead to a substantial portion of toxigenic C. difficile strains being missed.

The prevalence of antibiotic resistance genes in Bacteroides fragilis group strains isolated in different European countries

From the 2008-2009 European Bacteroides antibiotic resistance survey, we selected 161 strains for detection of antibiotic resistance genes (cepA, cfxA, cfiA, nim, ermB, ermF, ermG, linA, mefA, msrSA, tetM, tetQ, tetX, tetX1, tet36 and bexA). To facilitate the throughput, the genes were detected by Real-Time PCR. The presence of the genes was correlated with the known MIC data of the strains for the appropriate antibiotics. For the β-lactams, the cepA gene was found in 70.8% of the tested strains (all resistant to ampicillin), but its presence did not correlate with the ampicillin MIC values. The cepA gene occurred at different frequencies among Bacteroides fragilis and non-fragilis Bacteroides strains. The cfxA gene was not a major factor in determining cefoxitin resistance and it was found with higher prevalence in non-fragilis Bacteroides strains than in B. fragilis. Among the five possible clindamycin resistance genes, ermF was the most common and had the highest effect on clindamycin resistance after linA. The ermG-mefA-msrSA combination was found in a set of strains and their linked occurrence implied that they were harbored by the conjugative transposon CTnGERM1. All strains tested were susceptible to metronidazole and none of them harbored nim genes. TetQ was prevalent among both the B. fragilis and non-fragilis Bacteroides strains (78.9 and 84.8%, respectively) and no gene could be clearly linked to tigecycline resistance other than tetQ. BexA, which codes for the fluoroquinolone efflux pump, was found in 7.5% of strains and occurred at different frequencies among B. fragilis and non-fragilis Bacteroides strains, but was represented only in a minor proportion of moxifloxacin-resistant strains.

Investigation of the prevalence of tetQ, tetX and tetX1 genes in Bacteroides strains with elevated tigecycline minimum inhibitory concentrations

In this study, the antibiotic susceptibilities to tigecycline and tetracycline of 35 selected Bacteroides fragilis group strains were determined by Etest, and the presence of tetQ, tetX, tetX1 and ermF genes was investigated by polymerase chain reaction (PCR). tetQ was detected in all 12 B. fragilis group isolates (100%) exhibiting elevated tigecycline minimum inhibitory concentrations (MICs) (≥ 8 μg/mL) as well as the 8 strains (100%) with a tigecycline MIC of 4 μg/mL, whilst tetX and tetX1 were present in 15% and 75% of these strains, respectively. All of these strains were fully resistant to tetracycline (MIC ≥ 16 μg/mL). On the other hand, amongst the group of strains with tigecycline MICs< 4 μg/mL (15 isolates), tetQ, tetX and tetX1 were found less frequently (73.3%, 13.3% and 46.7%, respectively). All but two strains harbouring the tetQ gene in this group were non-susceptible to tetracycline, with a MIC> 4 μg/mL. These data suggest that in most cases tigecycline overcomes the tetracycline resistance mechanisms frequently observed in Bacteroides strains. However, the presence of tetX and tetX1 genes in some of the strains exhibiting elevated MICs for tigecycline draws attention to the possible development and spread of resistance to this antibiotic agent amongst Bacteroides strains. The common occurrence of ermF, tetX, tetX1 and tetQ genes together predicted the presence of the CTnDOT-like Bacteroides conjugative transposon in this collection of Bacteroides strains.

Multidrug-resistant Bacteroides fragilis group on the rise in Europe?

We report a case of multidrug-resistance (MDR) in a strain of Bacteroides fragilis from a blood culture and abdominal fluid in a Danish patient. The patient had not been travelling for several years and had not received antibiotics prior to the present case. We also summarize the cases that have been reported to date of MDR B. fragilis group in Europe. As far as we know, a case like this with MDR B. fragilis has not been described in Scandinavia before.

Molecular analysis of the carbapenem and metronidazole resistance mechanisms of Bacteroides strains reported in a Europe-wide antibiotic resistance survey.

Here we examine the carbapenem and metronidazole resistance mechanisms of 640 Bacteroides strains reported in the 2008-2009 European antibiotic susceptibility survey. Of the 22 strains with elevated imipenem minimum inhibitory concentrations (≥4 μg/mL), 10 were cfiA-positive and out of these 5 carried activating insertion sequence (IS) elements in the upstream regions of the cfiA genes. However, resistant strains with cfiA genes but with no activating IS elements were found (n=2) as well as a resistant strain with no cfiA gene. In the former the resistance phenotypes by Etest were heterogeneous, whilst in the latter no carbapenemase production was seen; both mechanisms have been rarely observed, examined and characterised. Interestingly, few (n=3) nim-positive strains were found, including one metronidazole-resistant strain harbouring nimE activated by ISBf6, and two susceptible strains harbouring chromosomally located nim genes.

Instant screening and verification of carbapenemase activity in Bacteroides fragilis in positive blood culture, using matrix-assisted laser desorption ionization--time of flight mass spectrometry.

Rapid identification of isolates in positive blood cultures are of great importance to secure correct treatment of septicaemic patients. As antimicrobial resistance is increasing, rapid detection of resistance is crucial. Carbapenem resistance in Bacteroides fragilis associated with cfiA-encoded class B metallo-beta-lactamase is emerging. In our study we spiked blood culture bottles with 26 B. fragilis strains with various cfiA-status and ertapenem MICs. By using main spectra specific for cfiA-positive and cfiA-negative B. fragilis strains, isolates could be screened for resistance. To verify strains that were positive in the screening, a carbapenemase assay was performed where the specific peaks of intact and hydrolysed ertapenem were analysed with matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). We show here that it is possible to correctly identify B. fragilis and to screen for enzymic carbapenem resistance directly from the pellet of positive blood cultures. The carbapenemase assay to verify the presence of the enzyme was successfully performed on the pellet from the direct identification despite the presence of blood components. The result of the procedure was achieved in 3 h. Also the Bruker mass spectrometric β-lactamase assay (MSBL assay) prototype software was proven not only to be based on an algorithm that correlated with the manual inspection of the spectra, but also to improve the interpretation by showing the variation in the dataset.

Susceptibility profiles and resistance genes for carbapenems (cfiA) and metronidazole (nim) among Bacteroides species in a Turkish University Hospital

Sixty-six nonduplicate Bacteroides clinical isolates collected at Marmara University Hospital were tested to investigate carbapenem and metronidazole resistance profiles and to detect the resistance genes (cfiA and nim) and related insertion sequence (IS) elements. The study found that there were no strains resistant to metronidazole and nim genes were not detected in any of the strains. Five Bacteroides fragilis strains were resistant to meropenem, one of which was also resistant to imipenem. The cfiA gene was detected in 27% of strains, 32% of strains had the IS1187 element, and five strains harbored both gene cfiA and IS1187. These results indicate higher rates of carriage of the cfiA gene and IS1187 insertion elements than have been reported in other countries.

Use of MALDI-TOF/MS for routine detection of cfiA gene-positive Bacteroides fragilis strains

Blondeau JM, Zhao X, Hansen G, Drlica K. Mutant prevention concentrations of fluoroquinolones for clinical isolates of Streptococcus pneumoniae. Antimicrob of 1–8 g/mL] is also detected with an appreciable frequency. cfiA-positive strains that are either resistant to carbapenems or Agents Chemother 2001;45:433–8. Zhao XL, Drlica K. Restricting the selection of antibiotic-resistant mutant bacteria: measurement and potential use of the mutant selection window. J Infect Dis 2002;185:561–5. Lee HH, Molla MN, Cantor CR, Collins JJ. Bacterial charity work leads to population-wide resistance. Nature 2010;467:82–5. merely ‘silent’ form a subgroup (Division II) of B. fragilis strains that may be differentiated by various typing methods and also by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF/MS) [2,3]; the latter is currently revolutionising the clinical microbiological identification of bacterial and fun in B cep me TOF