Antimicrobial resistance surveillance of Bacteroides fragilis isolated from blood cultures, Europe, 2022 (ReSuBacfrag).

OBJECTIVES: Bacteroides fragilis is the most frequent cause of anaerobic bacteraemia. Although recent data suggest a rise in antimicrobial resistance (AMR) of this and other anaerobic bacteria, surveillance remains limited due to a lack of both data availability and comparability. However, a newly introduced standardised method for antimicrobial susceptibility testing (AST) of anaerobic bacteria has made larger scale surveillance possible for the first time. The aim of this study was to investigate phenotypic AMR of Bacteroides fragilis isolates from bacteraemia across Europe in 2022. METHODS: In a multicentre approach, clinical microbiology laboratories in Europe were invited to contribute results of AST for Bacteroides fragilis blood culture isolates (including only the first isolate per patient and year). AST of a selection of four antibiotics was performed locally by participating laboratories in a prospective or retrospective manner, using the new EUCAST disc diffusion method on fastidious anaerobe agar (FAA-HB). RESULTS: A total of 16 European countries reported antimicrobial susceptibilities in 449 unique isolates of Bacteroides fragilis from blood cultures in 2022. Clindamycin demonstrated the highest resistance rates (20.9%, range 0 - 63.6%), followed by piperacillin-tazobactam (11.1%, 0-54.5%), meropenem (13.4%, 0-45.5%), and metronidazole (1.8%, 0-20.0%), all with wide variation between countries. CONCLUSION: Considering that the mean resistance rates across Europe were higher than expected for three of the four anti-anaerobic antibiotics under surveillance, both local AST of clinically relevant isolates of Bacteroides fragilis and continued surveillance on an international level is warranted.

Association between Bacteroides fragilis and Fusobacterium nucleatum infection and colorectal cancer in Vietnamese patients.

BACKGROUND: Colorectal cancer (CRC) is a significant global health concern, and understanding the role of specific bacterial infections in its development and progression is of increasing interest. This cross-sectional study investigated the associations between Bacteroides fragilis (B. fragilis) and Fusobacterium nucleatum (F. nucleatum) infections and Vietnamese CRC patients. METHODS: 192 patients with either polyps or CRC at varying stages were recruited from May 2017 to December 2020. Real-time PCR assessed infection rates and bacterial loads in CRC tissues. RESULTS: B. fragilis infection was notably higher in CRC tissues (51.6 %) than polyps (9.4 %), with a fivefold higher relative load. Positive associations were found in stages II and III, indicating a fivefold increase in CRC progression risk. F. nucleatum infection rates were significantly higher in CRC tissues (55.2 %) than in polyps (10.5 %). In stage II, the infection rate exceeded that in adjacent tissues. The relative load of F. nucleatum was higher in stage III than in stages I and II. Positive F. nucleatum patients had a 3.2 times higher risk of CRC progression. CONCLUSION: These findings suggest associations between loading of F. nucleatum or/and B. fragilis with the advanced stages of CRC.

Pangenome comparison of Bacteroides fragilis genomospecies unveils genetic diversity and ecological insights.

Bacteroides fragilis is a Gram-negative commensal bacterium commonly found in the human colon, which differentiates into two genomospecies termed divisions I and II. Through a comprehensive collection of 694 B. fragilis whole genome sequences, we identify novel features distinguishing these divisions. Our study reveals a distinct geographic distribution with division I strains predominantly found in North America and division II strains in Asia. Additionally, division II strains are more frequently associated with bloodstream infections, suggesting a distinct pathogenic potential. We report differences between the two divisions in gene abundance related to metabolism, virulence, stress response, and colonization strategies. Notably, division II strains harbor more antimicrobial resistance (AMR) genes than division I strains. These findings offer new insights into the functional roles of division I and II strains, indicating specialized niches within the intestine and potential pathogenic roles in extraintestinal sites. IMPORTANCE: Understanding the distinct functions of microbial species in the gut microbiome is crucial for deciphering their impact on human health. Classifying division II strains as Bacteroides fragilis can lead to erroneous associations, as researchers may mistakenly attribute characteristics observed in division II strains to the more extensively studied division I B. fragilis. Our findings underscore the necessity of recognizing these divisions as separate species with distinct functions. We unveil new findings of differential gene prevalence between division I and II strains in genes associated with intestinal colonization and survival strategies, potentially influencing their role as gut commensals and their pathogenicity in extraintestinal sites. Despite the significant niche overlap and colonization patterns between these groups, our study highlights the complex dynamics that govern strain distribution and behavior, emphasizing the need for a nuanced understanding of these microorganisms.

Nontoxigenic Bacteroides fragilis: A double-edged sword.

The contribution of commensal microbes to human health and disease is unknown. Bacteroides fragilis (B. fragilis) is an opportunistic pathogen and a common colonizer of the human gut. Nontoxigenic B. fragilis (NTBF) and enterotoxigenic B. fragilis (ETBF) are two kinds of B. fragilis. NTBF has been shown to affect the host immune system and interact with gut microbes and pathogenic microbes. Previous studies indicated that certain strains of B. fragilis have the potential to serve as probiotics, based on their observed relationship with the immune system. However, several recent studies have shown detrimental effects on the host when beneficial gut bacteria are found in the digestive system or elsewhere. In some pathological conditions, NTBF may have adverse reactions. This paper presents a comprehensive analysis of NTBF ecology from the host-microbe perspective, encompassing molecular disease mechanisms analysis, bacteria-bacteria interaction, bacteria-host interaction, and the intricate ecological context of the gut. Our review provides much-needed insights into the precise application of NTBF.

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.

Bacteroides fragilis spondylitis after suspected oral ulcer: a clinical case and comprehensive literature review.

This paper reports a case of Bacteroides fragilis induced spondylitis. Diagnosis was confirmed through blood culture and metagenomic sequencing of pus for pathogen detection. Due to persistent lumbar pain, surgical intervention became imperative, resulting in favorable postoperative outcomes. A detailed patient history revealed a severe episode of oral ulceration two weeks before symptom onset, although a direct link to the infection remained elusive. Leveraging insights from this case, we conducted a comprehensive literature review on B. fragilis spondylitis, elucidating clinical manifestations, diagnostic methodologies, and therapeutic strategies.

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.

Entero-toxigenic Bacteroides fragilis contributes to intestinal barrier injury and colorectal cancer progression by mediating the BFT/STAT3/ZEB2 pathway.

Our previous findings confirmed the high enrichment of Bacteroides fragilis (BF) in fecal samples from patients with colorectal cancer (CRC). The intestinal mucosal barrier is the first defense of the organism against commensal flora and intestinal pathogens and is closely associated with the occurrence and development of CRC. Therefore, this study aimed to investigate the molecular mechanisms through which BF mediates intestinal barrier injury and CRC progression. SW480 cells and a Caco2 intestinal barrier model were treated with entero-toxigenic BF (ETBF), its enterotoxin (B. fragilis toxin, BFT), and non-toxigenic BF (NTBF). Cell counting kit-8, flow cytometry, wound healing and transwell assays were performed to analyze the proliferation, apoptosis, migration, and invasion of SW480 cells. Transmission electron microscopy, FITC-dextran, and transepithelial electrical resistance (TEER) were used to analyze damage in the Caco2 intestinal barrier model. The Azoxymethane/Dextran Sulfate Sodium (AOM/DSS) animal model was established to evaluate the effect of ETBF on intestinal barrier injury and CRC progression in vivo. ETBF and BFT enhanced the viability, wound healing ratio, invasion, and EMT of SW480 cells. In addition, ETBF and BFT disrupted the tight junctions and villus structure in the intestinal barrier model, resulting in increased permeability and reduced TEER. Similarly, the expression of intestinal barrier-related proteins (MUC2, Occludin and Zo-1) was restricted by ETBF and BFT. Interestingly, the STAT3/ZEB2 axis was activated by ETBF and BFT, and treatment with Brevilin A (a STAT3 inhibitor) or knockdown of ZEB2 limited the promotional effect of ETBF and BFT on the SW480 malignant phenotype. In vivo experiments also confirmed that ETBF colonization accelerated tumor load, carcinogenesis, and intestinal mucosal barrier damage in the colorectum of the AOM/DSS animal model, and that treatment with Brevilin A alleviated these processes. ETBF-secreted BFT accelerated intestinal barrier damage and CRC by activating the STAT3/ZEB2 axis. Our findings provide new insights and perspectives for the application of ETBF in CRC treatment.

Clinical significance of Bacteroides fragilis as a potential prognostic factor in colorectal cancer.

INTRODUCTION: Bacteroides fragilis (B. fragilis) is considered to act in an anti-inflammatory manner on the intestinal tract. On the contrary, enterotoxigenic B. fragilis (ETBF), a subtype of B. fragilis, produces an enterotoxin (BFT; B. fragilis toxin), leading to asymptomatic chronic infections and colonic tumor formation. However, the impact of B. fragilis and ETBF on the clinical outcome of colorectal cancer (CRC) remains unclear. We aim to assess whether their presence affects the outcome in patients with CRC after curative resection. METHODS: We obtained 197 pairs of matched formalin-fixed paraffin-embedded samples from cancerous and adjacent non-cancerous tissues of patients with pathological stage (pstage) II and III CRC after curative resection. The presence of B. fragilis and ETBF were estimated using real-time polymerase chain reaction, and recurrence-free survival (RFS) and overall survival (OS) of the patients were analyzed. RESULTS: 16S rRNA for B. fragilis and bft DNA were detected in 120 (60.9%) and 12 (6.1%) of the 197 patients, respectively. B. fragilis-positive patients had better RFS than B. fragilis-negative patients, although that was not statistically significant. In subgroup analysis, better outcomes on RFS were observed in the presence of B. fragilis in pstage II and left-sided CRC. The association of B. fragilis positivity on OS was accentuated in the depth of T4 subgroup. No significant differences were observed in RFS and OS between ETBF and non-toxigenic B. fragilis. CONCLUSIONS: Our findings suggest that the presence of B. fragilis is associated with better outcomes in patients with pstage II and III CRC after curative resection.

Upregulation of the cfiA carbapenemase gene in a Bacteroides fragilis strain by the novel integrative and conjugative element Tn7563.

We describe Tn7563, a 31,844-bp integrative and conjugative element (ICE) carrying promoters upregulating the cfiA carbapenemase gene in Bacteroides fragilis strain Tbg-22. Excision and circularization of Tn7563 was demonstrated by PCR. Previously, only insertion sequences (IS) have been shown to carry mobile promoters for cfiA.

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.

The effects of identical nim gene-insertion sequence combinations on the expression of the nim genes and metronidazole resistance in Bacteroides fragilis strains.

In this study we examined whether the same nim gene-insertion sequence (IS) element combinations give rise to the same expression levels as they harbor shared IS element-borne promoters. From our quantitative analysis, we found that the expressions of the nimB and nimE genes with their cognate IS elements were similar, but the metronidazole resistance of these strains were more diverse.

Geographic patterns of antimicrobial susceptibilities for Bacteroides spp. worldwide: Results from the Antimicrobial Testing Leadership and Surveillance (ATLAS) programme, 2007-2020.

Antimicrobial susceptibilities of 4973 Bacteroides spp. isolates recovered from various sources of patients from 12 countries (99.6% from European countries) in the Antimicrobial Testing Leadership and Surveillance (ATLAS) programme, 2007-2020, were investigated. The minimum inhibitory concentrations (MICs) of the isolates with six commonly used agents were determined using the agar dilution method. Among the isolates, 10 Bacteroides spp. were included: B. fragilis (n=3180, 64.0%) was encountered most frequently, followed by B. thetaiotaomicron (n=675) and B. ovatus (n=409). During the 14 years, the proportion of B. fragilis declined, but the proportion of non-fragilis Bacteroides spp. increased. More than 90% of the isolates tested were susceptible to piperacillin-tazobactam, meropenem and tigecycline. Significantly lower susceptibility rates to cefoxitin (P<0.001), clindamycin (P<0.001), piperacillin/tazobactam (P<0.001) and tigecycline (P=0.006) were observed among non-fragilis Bacteroides spp. isolates than among B. fragilis isolates. Moreover, the susceptibility rates to clindamycin (P=0.003) and tigecycline (P=0.044) decreased significantly among non-fragilis Bacteroides spp. over time. Clindamycin susceptibility rates >80% were found in Greece (100%), Sweden (86.3%) and the UK (80.7%), and the lowest susceptibility rates were found in the USA (42.9%) and Japan (53.9%). In conclusion, the susceptibility of Bacteroides spp. to commonly used antibiotics varied geographically. Empirical antibiotic therapy for suspected anaerobic infections with clindamycin and cefoxitin should be avoided due to high resistance rates. Piperacillin-tazobactam, meropenem, metronidazole and tigecycline could be considered favourable options for the treatment of infections caused by Bacteroides spp.

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.

Screening and epitope characterization of diagnostic nanobody against total and activated Bacteroides fragilis toxin.

Enterotoxigenic Bacteroides fragilis (ETBF) can rapidly secrete an enterotoxin termed B. fragilis toxin (BFT), which is thought to be the only recognized virulence factor in ETBF. ETBF can cause acute diarrhea, inflammatory bowel disease (IBD), colorectal cancer, and breast cancer. BFT is divided into three subtypes, BFT1, BFT2, and BFT3. BFT1 is the most widely distributed in human B. fragilis isolates. BFT can be used as a biomarker for predicting the inflammation-cancer transformation of intestine and breast. Nanobodies have the advantages of small structure, complete antigen recognition capacity, rapid selection via phage display technology, and can be massively produced in microbial expression systems. Nanobodies have become a powerful tool for medical diagnosis and treatment. This study focuses on screening and structural characterization of nanobodies targeting full length and active BFT. By constructing prokaryotic expression systems to obtain recombinant BFT1 protein, high purity BFT1 protein was used to immunize alpacas. Phage display technology was used to construct a phage display library. The positive clones were selected by bio-panning, and the isothermal titration calorimetry was used to select high-affinity nanobodies. Then the three-dimensional structures of BFT1:Nb2.82 and BFT1:Nb3.27 were solved by crystal X-ray diffraction. We got two kinds of nanobodies, Nb2.82 targeting the BFT1 prodomain and Nb3.27 recognizing the BFT1 catalytic domain. This study provides a new strategy for the early diagnosis of ETBF and the possibility for BFT as a biomarker for diagnosing diseases.

High prevalence of cfiA positive Bacteroides fragilis isolates collected at a teaching hospital in Hohhot, China.

OBJECTIVES: Carbapenem-resistant Bacteroides fragilis has emerged globally and cfiA is the key underlying factor. However, the prevalence of cfiA-positive carbapenem-resistant B. fragilis varies among countries. Therefore, we investigated the prevalence of cfiA-positive B. fragilis clinical isolates in a tertiary hospital in China. METHODS: Carbapenem-resistant cfiA-positive B. fragilis isolates were identified using polymerase chain reaction. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used to identify the characteristic mass spectra of cfiA-positive B. fragilis. RESULTS: The prevalence of cfiA among 153 B. fragilis isolates was 22.2% (34/153), when 20.6% (7/34) cfiA-positive B. fragilis strains were isolated from pediatric patients. Twenty-one carbapenem-resistant B. fragilis isolates were identified and were all positive with cfiA gene. Two characteristic peaks (4825 and 9642 Da) were identified using MALDI-TOF MS, and the sensitivity, specificity, and both the positive and negative predictive values of these two peaks were 100%. A new peak shift from 9627 Da for cfiA-negative isolates to 9642 Da for cfiA-positive isolates was observed. CONCLUSIONS: A high prevalence of cfiA was observed among B.fragilis isolates in this study, especially those isolated from pediatric patients. Characteristic MS spectra can accurately discriminate cfiA-positive and -negative B. fragilis isolates and can contribute to the rapid screening of cfiA-positive B. fragilis isolates in clinical laboratories.

Molecular study on metronidazole resistance in Bacteroides fragilis group isolates from a South Indian tertiary care center.

OBJECTIVE: Bacteroides species are an important part of human intestinal microbiota. They can cause infections of significant mortality and morbidity when moved out of their niche in the gut. The cornerstone drug for prophylaxis and therapy, metronidazole, is exhibiting signs of resistance, which are frequently attributed to nitroimidazole (nim) resistance genes. The aim of this study was to use Epsilometer test (E-test) to assess the metronidazole susceptibility and conventional PCR methodology to map the distribution of nim genes in Bacteroides fragilis group (BFG) isolates. METHODS: MALDI-TOF MS was used to identify BFG isolates. Using the E-test methodology, metronidazole minimum inhibitory concentrations (MICs) were determined. The presence of nim genes in these isolates were checked by conventional PCR methodology. Sequencing was done on selected amplicons for determining the nim gene types. RESULTS: Bacteroides fragilis accounted for 55.3% of the total 273 BFG members identified. Of these, 196 (71.8%) were susceptible, 43 (15.8%) intermediate and 34 (12.5%) resistant to metronidazole as determined by the E-test. nim gene was present in 101 (37%) of the total 273 isolates. Out of the 34 phenotypically resistant isolates (MIC ≥32 µg/ml), 29 harboured nim gene (Chi-square test, p < 0.0000001) but nim gene was absent in 5 (14.7%) isolates. Also, nim gene was detected in 72 (30.1%) of the 239 isolates with susceptible and intermediate metronidazole MIC. Sequencing of 20 amplicons gave a nimE gene type. CONCLUSIONS: In view of the rising metronidazole resistance among BFG and its close association with nim genes, there is a need for implementing routine metronidazole susceptibility testing and more researches are needed to find the molecular basis of these nim genes.