MALDI-TOF MS versus 16S rRNA sequencing: Minor discrepancy between tools in identification of Bacteroides isolates

Members of the genus Bacteroides are important components of the normal microbiota of gastrointestinal tract; however, as opportunistic pathogens are also associated with severe or even life-threatening infections with significant mortality. Various species within Bacteroides fragilis group are phenotypically very similar; thus, their identifications with traditional-automated biochemical methods are frequently inaccurate. The identification of the newly discovered or reclassified bacteria can be doubtful because of the lack of biochemical profile in the database of these tests. The aim of this study was to determine the accuracy of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) method by testing of 400 Hungarian Bacteroides clinical isolates. Inaccurate identification results with MALDI-TOF MS were confirmed by 16S rRNA gene sequencing and findings were compared with traditional-automated biochemical test rapid ID 32A method as well

A multicentre survey of the antibiotic susceptibility of clinical Bacteroides species from Hungary.

BACKGROUND: The species of the Bacteroides fragilis group are important components of human microbiota, but as opportunistic pathogens they can be the causative agents of severe infections. METHODS: The major aims of our investigation were the evaluation of the susceptibility of 400 different Hungarian B. fragilis group isolates to 10 antibiotics by the agar dilution method, the comparison of our resistance data with previous national and international antibiotic resistance data and the comparison of present data in regional aspect. The MIC-values on 10 antibiotics of all the strains were determined with the agar dilution method by CLSI. The presence of the cfiA gene in Division II B. fragilis strains was confirmed by RT-PCR. RESULTS: We detected a relatively high resistance rate of ampicillin, moxifloxacin, clindamycin and tetracycline, but amoxicillin/clavulanic acid, metronidazole, tigecycline and chloramphenicol showed excellent activity. In this study, we found that 6.75% of the isolates were resistant to cefoxitin and 7% to meropenem, while 8.58% of our B. fragilis strains harboured the cfiA gene. Most of the meropenem resistant strains were isolated in one of the participating centres. In the case of meropenem, cefoxitin, clindamycin and high-level-ampicillin-resistant strains, we found significant regional differences. DISCUSSION: Most of the results of our study were concordant with previous national and international data, with the exception of amoxicillin/clavulanic acid, cefoxitin and meropenem. CONCLUSIONS: Our study highlighted the importance of the periodic monitoring of the antimicrobial susceptibility of Bacteroides species providing important information for the appropriate therapy

Detection of enterotoxin and protease genes among Hungarian clinical Bacteroides fragilis isolates.

Bacteroides fragilis as a commensal bacterium is a member of the human intestinal flora, but as an opportunistic pathogen it can cause serious infections as well. Some of them, harbouring an enterotoxin gene (bft), may cause diarrhoea mainly in young children. Recently it has been shown that a member of C11 proteases called fragipain (fpn) can activate the enterotoxin, while C10 protease (bfp) is suspected of playing an important role in the invasiveness of the B. fragilis isolates. The objective of this study was to investigate the prevalence and distribution of the bft isotypes in 200 Hungarian B. fragilis isolates collected recently; and in a subset of 72 strains, we wanted to determine the prevalence of bfp1-4 and fpn genes in bft-positive and bft-negative strains. Using the MALDI-TOF MS cfiA identification project file, 19 B. fragilis strains belonging to Division II were identified and the presence of the cfiA gene was confirmed by RT-PCR. Twenty two (13.0%) B. fragilis isolates turned out to be bft gene positive by RT-PCR; 20 isolates harboured bft-1 and six bft-2 isotypes, but no bft-3 isotype containing strains were found. A melting curve analysis and the PCR-RFLP were performed to differentiate between the bft-1 and bft-2 isotypes confirmed by sequencing. Thirty eight strains harboured bfp1, 58 isolates contained bfp2 gene, while 17 isolates proved positive for bfp3. Morever, no bfp4 positive isolate was found, and some of the B. fragilis strains tested harboured two or three bfp isotypes simultaneously. Among the 26 bft-positive strains, 24 contained the fpn gene, which confirms the role of fragipain in the activation of B. fragilis enterotoxin. In experiments, a significant negative correlation between fpn and cfiA was demonstrated (p < 0.000), a positive correlation was found between bfp2 and fpn genes (p = 0.0000803), and a negative correlation between bfp2 and cfiA genes (p = 0.011)

Molecular characterization of multidrug-resistant Bacteroides isolates from Hungarian clinical samples

INTRODUCTION: Members of the Bacteroides fragilis group are the most important components of the normal human gut microbiota, but these bacteria can cause severe infections as well. Due to the frequent usage of antibiotics, the spreading of the MDR strains is a real threat worldwide. MATERIALS AND METHODS: In a multicentre study 400 Bacteroides isolates from five Hungarian microbiological laboratories were cultured and identified by MALDI TOF-MS. The MIC values of ten antibiotics were determined by the agar dilution method and evaluated by the breakpoints of EUCAST or CLSI. RESULTS: We found six MDR strains, and their antibiotic resistance genes were investigated by molecular methods The DNA amplicon of B. fragilis SZ38 strain was sequenced to look for a mutation in the gyrA gene. Among the six MDR isolates we found one cfiA-, two cepA, three cfxA-, two ermG-, six tetQ- three tetX- and two bexA-positive strains. None of them harboured cepA, nim, ermB and tetX1 genes. DISCUSSION: In the past 12 years only a few cases of MDR Bacteroides infections have been published. Within a comprehensive multicenter survey we demonstrated the relatively large prevalence of MDR strains isolated in one centre with five isolates and one from another centre during a relatively short period of time. This study focused on the importance of antimicrobial susceptibility testing and surveillance among B. fragilis group isolates