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 ... -  《-》

High prevalence of division II (cfiA positive) isolates among blood stream Bacteroides fragilis in Slovenia as determined by MALDI-TOF MS.

Bacteroides fragilis can be classified into division I (cfiA negative) and division II (cfiA positive) isolates. Division II isolates have a silent chromosomal carbapenemase gene (cfiA) that can become overexpressed by an insertion of a mobile genetic element and thus develop a phenotypic resistance to carbapenems. Aims of our study were (i) to determine the prevalence of B. fragilis division II (cfiA positive) isolates among blood stream and non-blood stream isolates from two major Slovenian tertiary-care hospitals and (ii) to assess its influence on phenotypic resistance to imipenem. Consecutive non-duplicate B. fragilis isolates from blood stream and non-blood stream specimens were included in the analysis from 2015 to 2017 period. Data from laboratory information system were matched with mass spectra obtained with Microflex LT instrument and MALDI Biotyper 3.1 software (Bruker Daltonik, Bremen, Germany). All mass spectra were reanalyzed using Bruker taxonomy library. Spectra with a log(score) > 2.0 were further analyzed with cfiA library that separates B. fragilis division I and II isolates based on a log(score) value difference of >0.3. Minimal inhibitory concentrations (MICs) for imipenem were determined with Etest (bioMérieux, Marcy l'Étoile, France), using supplemented Brucella agar and EUCAST breakpoints (S ≤ 2 mg/L, R > 8 mg/L). Altogether 623 consecutive B. fragilis isolates were included in the analysis; 47 (7.5%) were isolated from blood stream and 576 (92.5%) from non-blood stream specimens. Among all study isolates, 51 (8.2%) proved to belong to division II (cfiA positive). The proportions of division II isolates among blood stream and non-blood stream isolates were 14.9% and 7.6%, respectively (p = 0.081, ns). In total, 1.3% (n = 8) were non-susceptible to imipenem (MIC >2 mg/L); 4.3% (n = 2) among blood stream and 1% (n = 6) among non-blood stream isolates. All imipenem resistant isolates belonged to division II. Modal MICs (MIC range) were 0.064 mg/L (0.016 mg/L-2 mg/L) and 0.125 mg/L (0.064 mg/L-≥32 mg/L) for division I and II isolates, respectively.

Jeverica S ，Sóki J ，Premru MM ，Nagy E ，Papst L ... -  《-》

Rapid detection and surveillance of cfiA-positive Bacteroides fragilis using matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

To perform surveillance of cfiA-positive Bacteroides fragilis using new subtyping software module, MALDI Biotyper Subtyping Module (MBT Subtyping Module), on MALDI-TOF MS system, and to evaluate the detection ability of the module. cfiA-positive strains were presumed using the module against B. fragilis isolated between 2006 and 2019. The cfiA gene was confirmed using PCR. In cfiA-positive B. fragilis, the insertion sequence (IS) elements were examined and the MBT STAR-BL assay was performed to examine meropenem hydrolysis activity. Of the 396 B. fragilis strains included, the MBT Subtyping Module detected 33 presumptive cfiA-positive strains (8.3%), of which 32 harbored the cfiA gene. The sensitivity and specificity of the MBT Subtyping Module for detecting cfiA-positive B. fragilis were 100.0% and 99.7%, respectively. Of the 32 strains harboring the cfiA gene, seven strains possessed IS elements, which were thought to induce high cfiA expression. Meropenem hydrolysis was detected in all seven strains that were positive for both cfiA and IS elements, and they exhibited resistance to meropenem and imipenem. The overall non-susceptibility rates to meropenem and imipenem were 84.8% and 36.4%, respectively, in the 33 presumptive cfiA-positive strains. The MBT Subtyping Module can detect cfiA-positive B. fragilis rapidly and accurately, supporting its use for surveillance of cfiA-positive B. fragilis in clinical settings.

Kawamoto Y ，Kosai K ，Ota K ，Uno N ，Sakamoto K ，Hasegawa H ，Izumikawa K ，Mukae H ，Yanagihara K ... -  《-》

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 ... -  《-》

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.

Nagy E ，Becker S ，Sóki J ，Urbán E ，Kostrzewa M ... -  《-》