Detection of the antibiotic resistance genes content of intestinal Bacteroides, Parabacteroides and Phocaeicola isolates from healthy and carbapenem-treated patients from European countries.

BACKGROUND: Bacteroides fragilis group (BFG) species are the most significant anaerobic pathogens and are also the most antibiotic-resistant anaerobic species. Therefore, surveying their antimicrobial resistance levels and investigating their antibiotic resistance mechanisms is recommended. Since their infections are endogenous and they are important constituents of the intestinal microbiota, the properties of the intestinal strains are also important to follow. The aim of this study was to investigate the main antibiotic gene content of microbiota isolates from healthy people and compare them with the gene carriage of strains isolated from infections. RESULTS: We detected 13, mainly antibiotic resistance determinants of 184 intestinal BFG strains that were isolated in 5 European countries (Belgium, Germany, Hungary, Slovenia and Turkey) and compared these with values obtained earlier for European clinical strains. Differences were found between the values of this study and an earlier one for antibiotic resistance genes that are considered to be mobile, with higher degrees for cfxA, erm(F) and tet(Q) and with lower degrees for msrSA, erm(B) and erm(G). In addition, a different gene prevalence was found depending on the taxonomical groups, e.g., B. fragilis and NBFB. Some strains with both the cepA and cfiA ß-lactamase genes were also detected, which is thought to be exceptional since until now, the B. fragilis genetic divisions were defined by the mutual exclusion of these two genes. CONCLUSIONS: Our study detected the prevalences of a series of antibiotic resistance genes in intestinal Bacteroides strains which is a novelty. In addition, based on the current and some previous data we hypothesized that prevalence of some antibiotic resistance genes detected in the clinical and intestinal BFG strains were different, which could be accounted with the differential composition of the Bacteroides microbiota and/or the MGE mobilities at the luminal vs. mucosal sites of the intestine.

Co-occurrence of the cephalosporinase cepA and carbapenemase cfiA genes in a Bacteroides fragilis division II strain, an unexpected finding.

BACKGROUND: Bacteroides fragilis, an anaerobic gut bacterium and opportunistic pathogen, comprises two genetically divergent groups (or divisions) at the species level. Differences exist both in the core and accessory genomes and the beta-lactamase genes, with the cephalosporinase gene cepA represented only in division I and the carbapenemase gene cfiA only in division II. METHODS: Multidrug resistance in a clinical B. fragilis strain was examined by whole-genome sequencing. RESULTS: Strain CNM20200260 carried the antimicrobial resistance genes cepA, cfiA2, ant(6'), erm(F), mef(En2), est(T), tet(Q) and cat(A), along with 82-Phe mutation in gyrA (together with 47 amino acid changes in gyrA/B and parC/parE). bexA/B and other efflux pump genes were also observed. None of the detected insertion sequences was located upstream of cfiA2. The genome-based taxonomy coefficients (average nucleotide identity, DNA-DNA hybridization similarity and difference in genomic G + C%) with respect to genomes of the strains of B. fragilis division II and the novel species Bacteroides hominis (both cfiA-positive) met the criteria for CNM20200260 to belong to either species (>95%, >70% and <1%, respectively). No such similarity was seen with type strain NCTC 9343 or the representative genome FDAARGOS 1225 of B. fragilis (division I, cfiA-negative). Strain CNM20200260 harboured four out of nine Kyoto Encyclopedia of Genes and Genomes orthologues defined for division I and one of two defined for division II. CONCLUSIONS: This is the first description of the co-occurrence of cepA and cfiA in a Bacteroides strain, confirming the complexity of the taxonomy of this species.

Novel and rare ß-lactamase genes of Bacteroides fragilis group species: Detection of the genes and characterization of their genetic backgrounds.

OBJECTIVES: This study screened the prevalence of rare ß-lactamase genes in Bacteroides fragilis group strains from clinical specimens and normal microbiota and examined the genetic properties of the strains carrying these genes. METHODS: blaHGD1, blaOXA347, cblA, crxA, and pbbA were detected by real-time polymerase chain reaction in collections of Bacteroides strains from clinical (n = 406) and fecal (n = 184) samples. To examine the genetic backgrounds of the samples, end-point PCR, FT-IR, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry were used. RESULTS: All B. uniformis isolates were positive for cblA in both collections. Although crxA was B. xylanisolvens-specific and associated with carbapenem resistance, it was only found in six fecal and three clinical B. xylanisolvens strains. Moreover, the crxA-positive strains were not clonal among B. xylanisolvens (contrary to cfiA in B. fragilis), implicating a rate of mobility or emergence by independent evolutionary events. The Phocaeicola (B.) vulgatus/P. dorei-specific gene blaHGD1 was detected among all P. vulgatus/P. dorei isolates from fecal (n = 36) and clinical (n = 26) samples. No blaOXA347-carrying isolate was found from European collections, but all US samples (n = 6) were positive. For three clinical isolates belonging to B. thetaiotaomicron (n = 2) and B. ovatus (n = 1), pbbA was detected on mobile genetic elements, and pbbA-positive strains displayed non-susceptibility to piperacillin or piperacillin/tazobactam phenotypically. CONCLUSIONS: Based on these observations, ß-lactamases produced by rare ß-lactamase genes in B. fragilis group strains should not be overlooked because they could encode important resistance phenotypes.

Enrichment of Bacteroides fragilis and enterotoxigenic Bacteroides fragilis in CpG island methylator phenotype-high colorectal carcinoma.

OBJECTIVES: Data support that enterotoxigenic Bacteroides fragilis (ETBF) harbouring the Bacteroides fragilis toxin (bft) gene may promote colorectal tumourigenesis through the serrated neoplasia pathway. We hypothesized that ETBF may be enriched in colorectal carcinoma subtypes with high-level CpG island methylator phenotype (CIMP-high), BRAF mutation, and high-level microsatellite instability (MSI-high). METHODS: Quantitative PCR assays were designed to quantify DNA amounts of Bacteroides fragilis, ETBF, and each bft gene isotype (bft-1, bft-2, or bft-3) in colorectal carcinomas in the Health Professionals Follow-up Study and Nurses' Health Study. We used multivariable-adjusted logistic regression models with the inverse probability weighting method. RESULTS: We documented 4476 colorectal cancer cases, including 1232 cases with available bacterial data. High DNA amounts of Bacteroides fragilis and ETBF were positively associated with BRAF mutation (p ≤ 0.0003), CIMP-high (p ≤ 0.0002), and MSI-high (p < 0.0001 and p = 0.01, respectively). Multivariable-adjusted odds ratios (with 95% confidence interval) for high Bacteroides fragilis were 1.40 (1.06-1.85) for CIMP-high and 2.14 (1.65-2.77) for MSI-high, but 1.02 (0.78-1.35) for BRAF mutation. Multivariable-adjusted odds ratios for high ETBF were 2.00 (1.16-3.45) for CIMP-high and 2.86 (1.64-5.00) for BRAF mutation, but 1.09 (0.67-1.76) for MSI-high. Neither Bacteroides fragilis nor ETBF was associated with colorectal cancer-specific or overall survival. DISCUSSION: The tissue abundance of Bacteroides fragilis is associated with CIMP-high and MSI-high, whereas ETBF abundance is associated with CIMP-high and BRAF mutation in colorectal carcinoma. Our findings support the aetiological relevance of Bacteroides fragilis and ETBF in the serrated neoplasia pathway.

First Report of Antibiotic Resistance Markers cfiA and nim Among Bacteroides fragilis Group Strains in Ecuadorian Patients.

In recent years, increasing resistance of Bacteroides fragilis to several antibiotics has been reported in different countries. The aim of this study was to evaluate the antibiotic resistance profiles of Bacteroides spp. isolated from clinical samples by phenotypic and molecular methods. A total of 40 nonrepetitive isolates of the B. fragilis group were studied from 2018 to 2019. The species was identified by API 20A system. The minimum inhibitory concentrations (MICs) were determined by Sensititre anaerobe MIC plate. The presence of the nim and cfiA genes was checked by conventional PCR. The association between genes and insertion sequence (IS) was performed by whole genome sequencing. Eleven isolates were categorized as metronidazole-resistant and only 2 isolates harbored the nim gene. Five isolates were imipenem-resistant, but cfiA gene was detected in two isolates. cfiA gene was closely related to the cfiA-4 allele and associated with IS614B. The nim gene was not related to any nim gene type and was considered a new variant named nimL. IS612 was found upstream of nimL gene. In view of the scarcity of data on B. fragilis, there is a need to surveil antibiotic resistance levels and molecular mechanisms to implement better antimicrobial therapies against this important group of bacteria.

Comparison of microbiological profile of enterotoxigenic Bacteroides fragilis (ETBF) isolates from subjects with colorectal cancer (CRC) or intestinal pre-cancerous lesions versus healthy individuals and evaluation of environmental factors involved in intestinal dysbiosis.

OBJECTIVE: The aim of this study was to analyze enterotoxigenic Bacteroides fragilis (ETBF) isolates from colorectal biopsies of subjects with a histological analysis positive for colorectal cancer (CRC), pre-cancerous lesions (pre-CRC) or with a healthy intestinal tissue and to evaluate the environmental factors that may not only concur to CRC development but may also affect gut microbiota composition. METHODS: ETBF isolates were typed using the ERIC-PCR method, while PCR assays were performed to investigate the bft alleles, the B. fragilis pathogenicity island (BFPAI) region and the cepA, cfiA and cfxA genes. Susceptibility to antibiotics was tested using the agar dilution method. Environmental factors that could play a role in promoting intestinal dysbiosis were evaluated throughout a questionnaire administered to the subjects enrolled. RESULTS: Six different ERIC-PCR types were identified. The type denominated C in this study was the most prevalent, in particular among the biopsies of subjects with pre-CRC, while an isolate belonging to a different type, denominated F, was detected in a biopsy from a subject with CRC. All the ETBF isolates from pre-CRC or CRC subjects had a B. fragilis pathogenicity island (BFPAI) region pattern I, while those from healthy individuals showed also different patterns. Furthermore, 71% of isolates from subjects with pre-CRC or CRC were resistant to two or more classes of antibiotics vs 43% of isolates from healthy individuals. The B. fragilis toxin BFT1 was the most frequently detected in this study, confirming the constant circulation of this isoform strains in Italy. Interestingly, BFT1 was found in 86% of the ETBF isolates from patients with CRC or pre-CRC, while the BFT2 was prevalent among the ETBF isolates from healthy subjects. No substantial differences based on sex, age, tobacco and alcohol consumption were observed between healthy and non-healthy individuals included in this study, while most of the subjects with CRC or pre-CRC lesions were subjected to pharmacological therapy (71%) and showed a body mass index (BMI) that falls within the overweight range (86%). CONCLUSIONS: Our data suggest that some types of ETBF seem to better adapt and colonize the human gut and that the selective pressure exerted by factors related to lifestyle, such as pharmacological therapy and weight, could facilitate their persistence in the gut and their possible involvement in CRC development.

Characterization of mobile genetic elements in multidrug-resistant Bacteroides fragilis isolates from different hospitals in the Netherlands.

OBJECTIVES: Five human clinical multidrug-resistant (MDR) Bacteroides fragilis isolates, including resistance to meropenem and metronidazole, were recovered at different hospitals in the Netherlands between 2014 and 2020 and sent to the anaerobic reference laboratory for full characterization. METHODS: Isolates were recovered from a variety of clinical specimens from patients with unrelated backgrounds. Long- and short-read sequencing was performed, followed by a hybrid assembly to study the presence of mobile genetic elements (MGEs) and antimicrobial resistance genes (ARGs). RESULTS: A cfxA gene was present on a transposon (Tn) similar to Tn4555 in two isolates. In two isolates a novel Tn was present with the cfxA gene. Four isolates harbored a nimE gene, located on a pBFS01_2 plasmid. One isolate contained a novel plasmid carrying a nimA gene with IS1168. The tetQ gene was present on novel conjugative transposons (CTns) belonging to the CTnDOT family. Two isolates harbored a novel plasmid with tetQ. Other ARGs in these isolates, but not on an MGE, were: cfiA, ermF, mef(EN2), and sul2. ARGs harboured differed between isolates and corresponded with the observed phenotypic resistance. CONCLUSIONS: Novel CTns, Tns, and plasmids were encountered in the five MDR B. fragilis isolates, complementing our knowledge on MDR and horizontal gene transfer in anaerobic bacteria.

Prevalence of antimicrobial resistant genes in Bacteroides spp. isolated in Oita Prefecture, Japan.

INTRODUCTION: Bacteroides spp. are the most common anaerobic bacteria isolated from the human gastrointestinal tract. Several resistant genes are present in Bacteroides spp. However, most studies have focused on the prevalence of the cfiA gene in Bacteroides fragilis alone. We assessed the susceptibility to antimicrobial agents and the prevalence of cepA, cfiA, cfxA, ermF, nim, and tetQ genes in Bacteroides strains isolated from clinical specimens in our hospital. METHODS: We isolated 86 B. fragilis and 58 non-fragilis Bacteroides strains from human clinical specimens collected from January 2011 to November 2021. Resistance against piperacillin (PIPC), cefotaxime (CTX), cefepime (CFPM), meropenem (MEPM), clindamycin, and minocycline was determined. RESULTS: The resistant rates of penicillins and cephalosporins in non-fragilis isolates were significantly higher than those in B. fragilis isolates. In B. fragilis isolates, the resistant rates of PIPC, CTX, and CFPM in cfxA-positive isolates were significantly higher than those in cfxA-negative isolates (71% vs. 16%, 77% vs. 19%, and 77% vs. 30%, respectively). Thirteen B. fragilis isolates harbored the cfiA gene, two of which were resistant to MEPM. Six of the 13 cfiA-positive B. fragilis isolates were heterogeneously resistant to MEPM. CONCLUSION: It is important to evaluate the use of MEPM as empirical therapy for Bacteroides spp. infections, considering the emergence of carbapenem resistance during treatment, existence of MEPM-resistant strains, and heterogeneous resistance.

Carbapenem resistance in Bacteroides fragilis: A review of molecular mechanisms.

Carbapenems are an applicable subclass of ß-lactam drugs in the antibiotic therapy of anaerobic infections, especially for poly-microbial cases, due to their broad antimicrobial spectrum on aerobic and anaerobic bacteria. Bacteroides fragilis is the most commonly recovered anaerobic bacteria in the clinical laboratories from mono- and poly-microbial infections. B. fragilis is relatively non-susceptible to different antibiotics, including ß-lactams, tetracyclines, fluoroquinolones, and macrolides. Carbapenems are among the most effective drugs against B. fragilis strains with high-level resistance to different antibiotics. Increased antibiotic resistance of B. fragilis strains has been reported following the overuse of an antimicrobial agent. Earlier contact with carbapenems is linked with increased resistance to them that limits the options for treatment of B. fragilis caused infections, especially in cases caused by multidrug-resistant strains. Several molecular mechanisms of resistance to carbapenems have been described for different carbapenem-resistant bacteria. Understanding the mechanisms of resistance to antimicrobial agents is necessary for selecting alternative antimicrobial agents and the application of control strategies. In the present study, we reviewed the mechanisms contributing to resistance to carbapenems in B. fragilis strains.

Comparative Genomics of Bacteroides fragilis Group Isolates Reveals Species-Dependent Resistance Mechanisms and Validates Clinical Tools for Resistance Prediction.

Bacteroides fragilis group (BFG) are the most frequently recovered anaerobic bacteria from human infections, and resistance to frontline antibiotics is emerging. In the absence of routine antimicrobial susceptibility testing (AST) for BFG in most clinical settings, we assessed the utility of clinical and modern genomics tools to determine BFG species-level identification and resistance patterns. A total of 174 BFG clinical isolates supplemented with 20 archived carbapenem-resistant B. fragilis sensu stricto (BFSS) isolates underwent antimicrobial susceptibility testing, MALDI-ToF mass-spectrometry, and whole-genome sequencing (WGS). Bruker BioTyper and VITEK-MS MALDI-ToF systems demonstrated accurate species-level identifications (91% and 90% agreement, respectively) compared to average nucleotide identity (ANI) analysis of WGS data. Distinct ß-lactamase gene profiles were observed between BFSS and non-fragilis Bacteroides species, with significantly higher MICs to piperacillin-tazobactam in B. vulgatus and B. thetaiotaomicron relative to BFSS (P ≤ 0.034). We also uncovered phylogenetic diversity at the genomospecies level between division I and division II BFSS (ANI <0.95) and demonstrate that division II BFSS strains harbor an increased capacity to achieve carbapenem resistance through chromosomal activation of the CfiA carbapenemase. Finally, we report that CfiA detection by the Bruker BioTyper Subtyping Module accurately detected carbapenem resistance in BFSS with positive and negative percent agreement of 94%/90% and 95%/95% compared to ertapenem and meropenem susceptibility, respectively. These comparative analyses indicate that resistance mechanisms are distinct at both the phenotypic and genomic level across species within the BFG and that modern MALDI-ToF identification systems can be used for accurate species-level identification and resistance prediction of the BFG. IMPORTANCE Anaerobic infections present unique challenges in terms of detecting and identifying the etiologic agent and selecting the optimal antimicrobial therapy. Antimicrobial resistance is increasing in anaerobic pathogens, and it is critical to understand the prevalence and mechanisms of resistance to commonly prescribed antimicrobial therapies. This study uses comparative genomics to validate clinical tools for species-level identification and phenotypic resistance prediction in 194 isolates of Bacteroides fragilis group (BFG) bacteria, which represent the most commonly isolated organisms among anaerobic infections. We demonstrate species-specific patterns in antimicrobial resistance and validate new strategies for species-level organism identification and phenotypic resistance prediction in a routine clinical laboratory setting. These findings expand our understanding and management of anaerobic infections and justify further investigations into the molecular basis for species-specific resistance patterns observed within this study.

Haemin deprivation renders Bacteroides fragilis hypersusceptible to metronidazole and cancels high-level metronidazole resistance.

BACKGROUND: Infections with Bacteroides fragilis are routinely treated with metronidazole, a 5-nitroimidazole antibiotic that is active against most anaerobic microorganisms. Metronidazole has remained a reliable treatment option, but resistance does occur, including in B. fragilis. OBJECTIVES: In this study we tested whether haemin, a growth supplement for B. fragilis in vivo and in vitro, had an influence on the susceptibility of resistant B. fragilis strains to metronidazole. We further tested whether haemin-deprived B. fragilis would be more susceptible to oxygen and oxidative stress. Metronidazole has been described to cause oxidative stress, which we argued would be exacerbated in haemin-deprived B. fragilis because the bacteria harness haemin, and the iron released from it, in antioxidant enzymes such as catalase and superoxide dismutase. METHODS: Haemin was omitted from growth media and the effect on metronidazole susceptibility was monitored in susceptible and resistant B. fragilis strains. Further, haemin-deprived B. fragilis were tested for resistance to aeration and hydrogen peroxide and the capacity for the removal of oxygen. RESULTS: Omission of haemin from the growth medium rendered metronidazole-resistant B. fragilis strains, including an MDR isolate from the UK, highly susceptible to metronidazole. Haemin deprivation further rendered B. fragilis highly susceptible to oxygen, which was further exacerbated in resistant strains. B. fragilis was incapable of scavenging oxygen when haemin was omitted. CONCLUSIONS: We propose that haemin deprivation overrules resistance mechanisms by rendering B. fragilis hypersusceptible to metronidazole due to a compromised antioxidant defence. Monitoring of haemin concentrations is imperative when conducting metronidazole susceptibility testing in B. fragilis.

Analysis of Six tonB Gene Homologs in Bacteroides fragilis Revealed That tonB3 is Essential for Survival in Experimental Intestinal Colonization and Intra-Abdominal Infection.

The opportunistic, anaerobic pathogen and commensal of the human large intestinal tract, Bacteroides fragilis strain 638R, contains six predicted TonB proteins, termed TonB1-6, four ExbBs orthologs, ExbB1-4, and five ExbDs orthologs, ExbD1-5. The inner membrane TonB/ExbB/ExbD complex harvests energy from the proton motive force (Δp), and the TonB C-terminal domain interacts with and transduces energy to outer membrane TonB-dependent transporters (TBDTs). However, TonB's role in activating nearly one hundred TBDTs for nutrient acquisition in B. fragilis during intestinal colonization and extraintestinal infection has not been established. In this study, we show that growth was abolished in the ΔtonB3 mutant when heme, vitamin B12, Fe(III)-ferrichrome, starch, mucin-glycans, or N-linked glycans were used as a substrate for growth in vitro. Genetic complementation of the ΔtonB3 mutant with the tonB3 gene restored growth on these substrates. The ΔtonB1, ΔtonB2, ΔtonB4, ΔtonB5, and ΔtonB6 single mutants did not show a growth defect. This indicates that there was no functional compensation for the lack of TonB3, and it demonstrates that TonB3, alone, drives the TBDTs involved in the transport of essential nutrients. The ΔtonB3 mutant had a severe growth defect in a mouse model of intestinal colonization compared to the parent strain. This intestinal growth defect was enhanced in the ΔtonB3 ΔtonB6 double mutant strain, which completely lost its ability to colonize the mouse intestinal tract compared to the parent strain. The ΔtonB1, ΔtonB2, ΔtonB4, and ΔtonB5 mutants did not significantly affect intestinal colonization. Moreover, the survival of the ΔtonB3 mutant strain was completely eradicated in a rat model of intra-abdominal infection. Taken together, these findings show that TonB3 was essential for survival in vivo. The genetic organization of tonB1, tonB2, tonB4, tonB5, and tonB6 gene orthologs indicates that they may interact with periplasmic and nonreceptor outer membrane proteins, but the physiological relevance of this has not been defined. Because anaerobic fermentation metabolism yields a lower Δp than aerobic respiration and B. fragilis has a reduced redox state in its periplasmic space-in contrast to an oxidative environment in aerobes-it remains to be determined if the diverse system of TonB/ExbB/ExbD orthologs encoded by B. fragilis have an increased sensitivity to PMF (relative to aerobic bacteria) to allow for the harvesting of energy under anaerobic conditions.

High frequency of enterotoxigenic Bacteroides fragilis and Enterococcus faecalis in the paraffin-embedded tissues of Iranian colorectal cancer patients.

BACKGROUND: The association between specific bacteria and colorectal cancer (CRC) has been proposed. Only a few studies have, however, investigated this relationship directly in colorectal tissue with conflicting results. So, we aimed to quantitate Streptococcus gallolyticus, Fusobacterium spp, Enterococcus faecalis and enterotoxigenic Bacteroides fragilis (ETBF) in formalin-fixed and paraffin-embedded (FFPE) colorectal tissue samples of Iranian CRC patients and healthy controls. METHODS: A total of 80 FFPE colorectal tissue samples of CRC patients (n = 40) and healthy controls (n = 40) were investigated for the presence and copy number of above bacterial species using quantitative PCR. Relative quantification was determined using ΔΔCT method and expressed as relative fold difference compared to reference gene. RESULTS: Relative abundance and copy number of E. faecalis and ETBF were significantly higher in CRC samples compared to control group. E. faecalis was more prevalent than ETBF in tumor samples. Frequency of ETBF and E. faecalis in late stages (III/IV) of cancer was significantly higher than early stages (I/II). We did not detect a significant difference in abundance of S. gallolyticus and Fusobacterium spp between two groups. CONCLUSION: Our study revealed the higher concentration of E. faecalis and ETBF in FFPE samples of CRC patients than controls. However, additional investigations on fecal and fresh colorectal cancer tissue samples are required to substantiate this correlation.

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

OBJECTIVES: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

UK Bacteroides species surveillance survey: Change in antimicrobial resistance over 16 years (2000-2016).

OBJECTIVES: To assess the differences in antimicrobial susceptibility of UK Bacteroides species across two distinct cohorts from 2000 to 2016. METHODS: Strain identification was performed using matrix-assisted laser-desorption ionisation time of flight mass spectrometry (MALDI-TOF MS) or by partial 16S rRNA sequencing. Minimum inhibitory concentrations (MICs) were determined using agar dilution, following CLSI guidelines (CLSI, 2012; 2017). RESULTS: 224 isolates were included from 2000 to 168 from 2016. Bacteroides fragilis was the most common species, comprising 68% of the 2000 cohort, and 77% in 2016. For all antimicrobials tested, there was an overall increase in the rates of non-susceptible isolates between the cohorts. CONCLUSIONS: The antibiogram of Bacteroides species in the UK is no longer predictable. Multi-drug resistant isolates although rare, are on the rise, and require testing to guide therapy. The monitoring and surveillance of resistance trends is imperative, as is the development of standardised, robust and accessible antimicrobial susceptibility testing methodology for clinical laboratories.

Comparative evaluation of agar dilution and broth microdilution by commercial and in-house plates for Bacteroides fragilis group: An economical and expeditious approach for resource-limited settings.

OBJECTIVE: To compare the performance of agar dilution and broth microdilution by commercial and in-house prepared plates for the Bacteroides fragilis group. The cost analysis was performed to demonstrate that in-house prepared BMD plates were a suitable alternative to agar dilution given the high cost and low feasibility of incorporating commercial BMD plates in routine, particularly in the tertiary care institutes of many low- and middle-income countries. METHODS: Thirty B. fragilis group isolates were tested against six antibiotics, frequently used as empirical therapy for anaerobic infections including metronidazole, clindamycin, imipenem, piperacillin-tazobactam, cefoxitin, and chloramphenicol. The running consumable expenditure for all methodologies was calculated. RESULTS: The results demonstrated essential and categorical agreement of >90% for all antibiotics except cefoxitin, which showed <90% categorical agreement. No major or very major errors were observed. We observed a high agreement and strong concordance for MIC values between both methods and inter-rate reliability of >0.9 by Cohen's kappa analysis, indicating almost perfect agreement between both methods using either of the plates. In contrast to agar dilution, a 20.5 fold cost reduction was seen in BMD using in-house plates and a 5.8 fold reduction using commercial plates to test a single isolate. However, when testing 30 isolates concurrently the cost significantly increased for commercial BMD plates by 8.4 folds, and only 1.03 fold cost reduction was seen with in-house BMD plates. CONCLUSION: BMD gives comparable results to agar dilution and can be considered a method of choice to test a small number of samples. The technique is an economical option when plates are standardized in-house and could be employed for susceptibility testing of the B. fragilis group.

An update on ampicillin resistance and ß-lactamase genes of Bacteroides spp.

Introduction. There are several ß-lactamase genes described for Bacteroides strains, of which cepA and cfiA are specific for Bacteroides fragilis and define two genetic divisions. The expression and phenotypic effects of these genes are usually regulated by insertional activation.Hypotheses/Gap Statement. Information is lacking about how cepA is regulated for most of the B. fragilis strains and whether there could be a genetic element for it.Aim. We aimed to investigate the molecular background of ampicillin (and other ß-lactam) resistance among Bacteroides strains as mediated mainly by cepA and also to find a genetic element for it as known for cfiA.Methodology. Various PCR methods were used for ß-lactamase-resistance gene and insertion sequence (IS) element detection in 42 Bacteroides strains. ß-Lactamase activity measurements and antimicrobial-susceptibility testing using agar dilution were also applied. Further molecular experiments involved sequencing, gene targeting, Southern blotting and bioinformatic analyses.Results. We found that high antibiotic resistance and ß-lactamase levels are brought about by insertional activation of the cepA gene or by similar or dissimilar activation of cfxA or cfiA, or by the newly described pbbA genes. Non-activated cepA genes produced low levels of specific ß-lactamase activities that did not correlate with ampicillin resistance. We found a genetic element for cepA and another region close to it that are characteristic for division I B. fragilis strains, which are replaced by other sequences in division II B. fragilis strains.Conclusion. cepA usually is not activated by IS elements and usually produces low ß-lactamase activities that do not correlate with the ampicillin MICs; therefore, it probably involves some non-ß-lactamase-mediated resistance mechanism(s). pbpA is a newly described, effective ß-lactamase gene that is located on a plasmid, and cepA resides on a well-defined chromosomal segment that is mutually replaced in division II B. fragilis strains. This latter finding demonstrates the genetic dichotomy of cepA-cfiA in B. fragilis and requires further investigation.

Bacteroides uniformis CECT 7771 alleviates inflammation within the gut-adipose tissue axis involving TLR5 signaling in obese mice.

This study investigated the immune mechanisms whereby administration of Bacteroides uniformis CECT 7771 reduces metabolic dysfunction in obesity. C57BL/6 adult male mice were fed a standard diet or a Western diet high in fat and fructose, supplemented or not with B. uniformis CECT 7771 for 14 weeks. B. uniformis CECT 7771 reduced body weight gain, plasma cholesterol, triglyceride, glucose, and leptin levels; and improved oral glucose tolerance in obese mice. Moreover, B. uniformis CECT 7771 modulated the gut microbiota and immune alterations associated with obesity, increasing Tregs and reducing B cells, total macrophages and the M1/M2 ratio in both the gut and epididymal adipose tissue (EAT) of obese mice. B. uniformis CECT 7771 also increased the concentration of the anti-inflammatory cytokine IL-10 in the gut, EAT and peripheral blood, and protective cytokines TSLP and IL-33, involved in Treg induction and type 2 innate lymphoid cells activation, in the EAT. It also restored the obesity-reduced TLR5 expression in the ileum and EAT. The findings indicate that the administration of a human intestinal bacterium with immunoregulatory properties on the intestinal mucosa helps reverse the immuno-metabolic dysfunction caused by a Western diet acting over the gut-adipose tissue axis.

Dietary Fiber Drives IL-1ß-Dependent Peritonitis Induced by Bacteroides fragilis via Activation of the NLRP3 Inflammasome.

Intestinal barrier is essential for dietary products and microbiota compartmentalization and therefore gut homeostasis. When this barrier is broken, cecal content overflows into the peritoneal cavity, leading to local and systemic robust inflammatory response, characterizing peritonitis and sepsis. It has been shown that IL-1ß contributes with inflammatory storm during peritonitis and sepsis and its inhibition has beneficial effects to the host. Therefore, we investigated the mechanisms underlying IL-1ß secretion using a widely adopted murine model of experimental peritonitis. The combined injection of sterile cecal content (SCC) and the gut commensal bacteria Bacteroides fragilis leads to IL-1ß-dependent peritonitis, which was mitigated in mice deficient in NLRP3 (nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3) inflammasome components. Typically acting as a damage signal, SCC, but not B. fragilis, activates canonical pathway of NLRP3 promoting IL-1ß secretion in vitro and in vivo. Strikingly, absence of fiber in the SCC drastically reduces IL-1ß production, whereas high-fiber SCC conversely increases this response in an NLRP3-dependent manner. In addition, NLRP3 was also required for IL-1ß production induced by purified dietary fiber in primed macrophages. Extending to the in vivo context, IL-1ß-dependent peritonitis was worsened in mice injected with B. fragilis and high-fiber SCC, whereas zero-fiber SCC ameliorates the pathology. Corroborating with the proinflammatory role of dietary fiber, IL-1R-deficient mice were protected from peritonitis induced by B. fragilis and particulate bran. Overall, our study highlights a function, previously unknown, for dietary fibers in fueling peritonitis through NLRP3 activation and IL-1ß secretion outside the gut.

What we actually know about the pathogenicity of Bacteroides pyogenes.

The aim of the study was to evaluate the pathogenic potential of Bacteroides pyogenes, rarely identified in clinical laboratories anaerobic bacteria. To increase the knowledge about this poorly understood anaerobic microorganism, the study also includes cases of infections described so far in the literature. Only the use of 16S rRNA sequencing and mass spectrometry technique allowed the identification of B. pyogenes from clinical specimens. We reported 13 severe human infections caused by B. pyogenes. Bacteria were cultured from the wound after biting by animals, chronic infections within the oral cavity, from patients with histologically or radiological proven osteomyelitis, surgical site infection, and from urine sample collected after a urological procedure. Most (9/13) of the patients required hospitalization. Almost 70% of them needed urgent admission via the emergency room. Two inpatients due to a life-threatening condition were admitted to the intensive care unit. Almost 50% of isolates were resistant to penicillin. All resistant to penicillin strains were isolated from skin and mucous membrane infections.