Susceptibility trends of Bacteroides fragilis group and characterisation of carbapenemase-producing strains by automated REP-PCR and MALDI TOF.

Susceptibility testing of clinical isolates of anaerobic bacteria is not considered, often, mandatory in routine clinical practice and the treatments are empirically established. Thus, periodic monitoring of the susceptibility patterns of anaerobic bacteria is advisable. The aim of this study was to update on resistance of Bacteroides fragilis group in our Institution with special attention to carbapenems reporting metallo-beta-lactamase producing strains for the first time in Spain, and to compare fingerprinting analysis results obtained by using automated rep-PCR (DiversiLab System) and MALDI-TOF MS. A total of 830 non-duplicated clinical isolates of the B. fragilis group recovered from the years 2006 to 2010 were studied. B. fragilis was the most prevalent species (59.5%). The total susceptibility of B. fragilis group isolates were: penicillin, 13.3%; amoxicillin/clavulanic, 89.6%; piperacillin-tazobactam, 91.8%; cefoxitin, 65.8%; ertapenem, 95.9%; imipenem, 98.2%; clindamycin, 53.4% and metronidazole, 96.4%. The percentage of sensitive isolates did not change significantly over time for amoxicillin/clavulanic, cefoxitin, clindamycin and metronidazole. A slight increase in the rate of resistance to ertapenem and imipenem was observed. Imipenem resistance and carbapenemase production were detected for the first time in our laboratory in the year 2007. No other report of carbapenemase-producing B. fragilis in our country has been previously published. Six imipenem-resistant isolates were MBL-producing and PCR positive for cfiA gene. Four of them were PCR positive for IS-like immediately upstream cfiA gene and two of them were negative. Both, automated rep-PCR (DiversiLab) and MALDI-TOF MS, revealed a great genetic diversity among carbapenem-producing strains suggesting the acquisition of novel resistance genes more than clonal dissemination of them. Both methods seem to be useful tools for fast and accurate identification and strain typing of B. fragilis group in the daily laboratory routine. Because of the relevant increase observed in Bacteroides species isolated from blood cultures and the appearance of carbapenemase-producing strains in our Institution, we recommend to test the antimicrobial susceptibility of the isolates, at least in the most severe patients.

Treviño M ，Areses P ，Peñalver MD ，Cortizo S ，Pardo F ，del Molino ML ，García-Riestra C ，Hernández M ，Llovo J ，Regueiro BJ ... -  《-》

Update on resistance of Bacteroides fragilis group and related species with special attention to carbapenems 2006-2009.

The susceptibility trends for the species of the Bacteroides fragilis group against various antibiotics were determined using data from 4 years [2006-2009] on 1957 isolates referred by 8 medical centers participating in a National Survey for the Susceptibility of B. fragilis. The antibiotic test panel included doripenem, ertapenem, imipenem, meropenem, ampicillin:sulbactam, piperacillin:tazobactam, cefoxitin, clindamycin, moxifloxacin, tigecycline, chloramphenicol and metronidazole. MICs were determined using agar dilution methods following CLSI recommendations. Genetic analysis of isolates from 2008 with elevated MICs (>2 μg/mL) to one or more of the carbapenems to detect presence of the cfiA gene was performed using PCR methodology. The results showed an increase in the resistance rates to the β-lactam antibiotics. High resistance rates were seen for clindamycin and moxifloxacin (as high as 60% for clindamycin and >80% for moxifloxacin), with relatively stable low resistance (5.4%) for tigecycline. For carbapenems, resistance in B. fragilis was 1.1%-2.5% in 2008-9. One isolate resistant to metronidazole (MIC 32 μg/mL) was observed as well as isolates with elevated MICs to chloramphenicol (16 μg/mL). Genetic analysis indicated that the cfiA gene was present in some but not all of the isolates with high MICs to the carbapenems. These data indicate that there continue to be changes in susceptibility over time, and that resistance can be seen among the carbapenems. High antibiotic resistance rates tend to be associated with specific species.

Snydman DR ，Jacobus NV ，McDermott LA ，Golan Y ，Goldstein EJ ，Harrell L ，Jenkins S ，Newton D ，Pierson C ，Rosenblatt J ，Venezia R ，Gorbach SL ，Queenan AM ，Hecht DW ... -  《-》

Molecular and epidemiological analysis of nosocomial carbapenem-resistant Klebsiella spp. using repetitive extragenic palindromic-polymerase chain reaction and matrix-assisted laser desorption/ionization-time of flight.

Treviño M ，Navarro D ，Barbeito G ，García-Riestra C ，Crespo C ，Regueiro BJ ... -  《-》

Examination of cfiA-mediated carbapenem resistance in Bacteroides fragilis strains from a European antibiotic susceptibility survey.

Of 1284 Bacteroides strains collected in Europe in 2000 for antibiotic susceptibility surveillance, 65 isolates displayed imipenem minimum inhibitory concentrations (MICs) > or =1 mg/L and were chosen for a thorough analysis of their resistance mechanism. Twenty-five of the isolates were positive for the cfiA carbapenem resistance gene. The resistance rates were 0.8% and 1.3% for imipenem and meropenem, respectively. In six of the strains, insertion sequence (IS) elements (IS613, IS614B, IS1186 and IS1187) activated the cfiA gene. However, other strains displayed at least elevated carbapenem MICs or were carbapenem resistant and produced measurable carbapenemase activities but did not harbour IS elements in the region upstream of the cfiA gene. The major determinant of carbapenem resistance in Bacteroides fragilis is production of CfiA metallo-beta-lactamase via activation of the cfiA gene by IS elements (higher level resistance) or by activation of its putative own promoter.

Sóki J ，Edwards R ，Hedberg M ，Fang H ，Nagy E ，Nord CE ，ESCMID Study Group on Antimicrobial Resistance in Anaerobic Bacteria ... -  《INTERNATIONAL JOURNAL OF ANTIMICROBIAL AGENTS》

Bacteroides fragilis: A whole MALDI-based workflow from identification to confirmation of carbapenemase production for routine laboratories.

Bacteroides fragilis is a frequent anaerobic pathogen and can cause severe infections. Resistance to carbapenems, associated with the cfiA gene encoded carbapenemase, represents an emerging problem. To date, no rapid methods are available to detect and confirm this resistance mechanism in routine laboratories, and the missed recognition of carbapenemase-producing strains can lead to therapeutic failures. In this study we have investigated a whole MALDI-TOF MS-based workflow to detect carbapenemase-producing B. fragilis, using the largest set of B. fragilis clinical isolates ever tested. The presence of the cfiA gene was predicted by MALDI subtyping into Division I (cfiA-negative) or Division II (cfiA-positive). The carbapenemase activity in cfiA-positive strains was confirmed by a MALDI-TOF MS imipenem hydrolysis assay (MBT STAR-Carba, Bruker Daltonik, Germany), that was further used for a characterization of the strains in terms of cfiA expression level. The validity of MALDI subtyping was verified by PCR for the cfiA gene, while results of MALDI hydrolysis assay were compared to conventional methods for susceptibility testing and carbapenemase detection (Carba-NP and disk diffusion synergy test). A genetic analysis of the IS elements upstream cfiA was performed, for the evaluations regarding the expression level of cfiA. A total of 5300 B. fragilis isolates (406 from Bologna, Italy, and 4894 from Dortmund, Germany) were identified and subtyped by MALDI-TOF MS, yielding 41/406 (10.1%) strains from Bologna and 374/4894 (7.6%) from Dortmund to belong to Division II. Molecular verification by PCR for the cfiA gene on a subset of strains confirmed the MALDI typing results in all cases (sensitivity and specificity of 100%). MBT STAR-Carba assay detected the carbapenemase activity in all of the 70 cfiA-carrying strains tested. Moreover, it allowed distinct separation into slow (59) and fast (11) imipenem hydrolyzers corresponding to cfiA expression levels as well as to low or high MICs for carbapenems, respectively. Among the 11 cfiA-positive strains with high carbapenem MIC, only 7 harboured IS elements upstream the carbapenemase gene showing low expression level as well. The MALDI-TOF MS-based workflow was superior to the currently available phenotypic methods for carbapenemase detection as it proved to be more sensitive and accurate than Carba NP and disk diffusion synergy test. The whole MALDI-TOF MS-based workflow allows an accurate identification of B. fragilis clinical strains with reliable classification into Division I/II, and confirmation of the carbapenemase-production, together with estimation of carbapenemase activity, within less than 2 h. This may be of particular interest for early therapeutical decisions in life-threatening infections.

Cordovana M ，Kostrzewa M ，Sóki J ，Witt E ，Ambretti S ，Pranada AB ... -  《-》