Next-generation probiotics: the upcoming biotherapeutics.

Recent and continuing advances in gut microbiome research have pointed out the role of the gut microbiota as an unexplored source of potentially beneficial probiotic microbes. Along the lines of these advances, both public awareness and acceptance of probiotics are increasing. That's why; academic and industrial research is dedicated to identifying and investigating new microbial strains for the development of next-generation probiotics (NGPs). At this time, there is a growing interest in NGPs as biotherapeutics that alter the gut microbiome and affect various diseases development. In this work, we have focused on some emergent and promising NGPs, specifically Eubacterium hallii, Faecalibacterium prausnitzii, Roseburia spp., Akkermansia muciniphila, and Bacteroides fragilis, as their presence in the gut can have an impact on the development of various diseases. Emerging studies point out the beneficial roles of these NGPs and open up novel promising therapeutic options. Interestingly, these NGPs were found to enhance gastrointestinal immunity, enhance immunotherapy efficacy in cancer patients, retain the intestinal barrier integrity, generate valuable metabolites, especially short-chain fatty acids, and decrease complications of chemotherapy and radiotherapy. Although many of these NGPs are considered promising for the prevention and treatment of several chronic diseases, research on humans is still lacking. Therefore, approval of these microbes from regulatory agencies is rare. Besides, some issues limit their wide use in the market, such as suitable methods for the culture and storage of these oxygen-sensitive microbes. The present review goes over the main points related to NGPs and gives a viewpoint on the key issues that still hinder their wide application. Furthermore, we have focused on the advancement in NGPs and human healthiness investigations by clarifying the limitations of traditional probiotic microorganisms, discussing the characteristics of emerging NGPs and defining their role in the management of certain ailments. Future research should emphasize the isolation, mechanisms of action of these probiotics, safety, and clinical efficacy in humans.

Microbiota Effects on Carcinogenesis: Initiation, Promotion, and Progression.

Carcinogenesis is a multistep process by which normal cells acquire genetic and epigenetic changes that result in cancer. In combination with host genetic susceptibility and environmental exposures, a prominent procarcinogenic role for the microbiota has recently emerged. In colorectal cancer (CRC), three nefarious microbes have been consistently linked to cancer development: (a) Colibactin-producing Escherichia coli initiates carcinogenic DNA damage, (b) enterotoxigenic Bacteroides fragilis promotes tumorigenesis via toxin-induced cell proliferation and tumor-promoting inflammation, and (c) Fusobacterium nucleatum enhances CRC progression through two adhesins, Fap2 and FadA, that promote proliferation and antitumor immune evasion and may contribute to metastases. Herein, we use these three prominent microbes to discuss the experimental evidence linking microbial activities to carcinogenesis and the specific mechanisms driving this stepwise process. Precisely defining mechanisms by which the microbiota impacts carcinogenesis at each stage is essential for developing microbiota-targeted strategies for the diagnosis, prognosis, and treatment of cancer.

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.

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.

Inflammatory modulation and outer membrane vesicles (OMV) production associated to Bacteroides fragilis response to subinhibitory concentrations of metronidazole during experimental infection.

OBJECTIVE: An experimental infection based on a tissue cage model was reproduced to evaluate the interference subinhibitory concentration (SIC) of metronidazole in Bacteroides fragilis OMV production patterns and immunological and histological characteristics of the host facing the experimental challenge. METHODS: A tissue cage model was reproduced for B. fragilis experimental challenge in three Wistar rats groups: negative control group (NC) without bacterial inoculation; positive control group (PC) infected with parental strain; and experimental group (EG) infected with the parental strain and treated with metronidazole SIC. Tissue cage sections and histological preparations were evaluated under optical and transmission electron microscope. Observations included OMV identification and count and cellular envelope evaluation. Transcriptional analyses were performed to evaluate cytokines expression levels. RESULTS: Total counts of leukocytes and neutrophils were higher for EG, and slight increase in PC group. It was observed an exacerbated inflammatory infiltrate after 8 days on infection. The expression of TNF-α was increased during the experiments, along with IL-1α and IL-6. MCP-1 levels were suppressed in almost every evaluated time-point. The IL-10 was exacerbated in EG group. A massive production and release of OMV and cell wall thickening were observed especially the EG group. CONCLUSIONS: Despite literature data suggest positive association between OMV and antimicrobial stress for Gram negatives, no correlations are made for B. fragilis and drug-response during experimental model of infection. Results corroborate observations in which OMV may be involved in bacterial pathogenicity once the phenomenon was observed along histological evidence of exacerbated inflammation and cytokines modulation.

A comparison of the antimicrobial resistance of fecal Bacteroides isolates and assessment of the composition of the intestinal microbiotas of carbapenem-treated and non-treated persons from Belgium and Hungary.

The antimicrobial susceptibilities of Bacteroides strains isolated from the feces of imipenem-treated patients from Belgium and Hungary were compared with those isolated from the normal microbiota from these two and five other European countries and assessed. Of the 10 antibiotics tested, highly significant differences were found with cefoxitin (decrease for Belgium and for this two and the five countries from the previous study), clindamycin (decrease for Belgium and for this two and the five countries from the previous study) and moxifloxacin (increase for Belgium and for this two and the five countries from the previous study) relative to normal microbiota strains reported earlier. Imipenem treatment brought about modest, but notable differences in the compositions of the microbiomes where there was less diversity in the treated group relative to the non-treated group.

Identification of enterotoxigenic Bacteroides fragilis in patients with diarrhea: A study targeting 16S rRNA, gyrB and nanH genes.

OBJECTIVES: We aimed to identify the enterotoxigenic Bacteroides fragilis (ETBF) and bft subtypes among patients with diarrhea. In addition, we assessed whether DNA gyrase subunit B (gyrB) and neuraminidase (nanH) genes are useful determinants for identification of B. fragilis compared to 16S rRNA sequencing as a reference method. METHODS: The 530 fecal specimens were cultured on BBE agar. The colonies which supposed to be a member of B. fragilis group were subjected to 16S rRNA gene sequencing and PCR assays targeting the Bacteroides fragilis group (BFG), gyrB and nanH. The B. fragilis toxin (bft) gene and its subtype was detected by PCR. The specificity of PCR assays was calculated considering the 16S rRNA gene sequencing as the reference method. RESULTS: A total of 111 Gram-negative anaerobic coccobacilli were isolated from 530 fecal specimens using BBE agar. Of the 111 isolates, 100 (90.09%) were assumed to be a member of Bacteroides fragilis group as they yielded an amplicon through PCR using the group-specific primers (Bfra-F/g-Bfra-R). However, only 28 isolates out of 100 were definitively identified as species of Bacteroides using16S rRNA gene sequencing; of which 15 isolates were B. fragilis and the remaining 13 isolates were identified as B. thetaiotaomicron (n = 6), Parabacteroides distasonis (n = 3), B. vulgatus (Phocaeicola vulgatus) (n = 1), B. ovatus (n = 1), B. congonensis (n = 1) and B. nordii (n = 1). Among the 15 isolates of B. fragilis, 4 were found to be ETBF. Compared to the reference method, the specificity and accuracy of the PCR targeting gyrB gene (64.7% and 65%) was higher than of nanH (36.4% and 46%, respectively. CONCLUSIONS: This study demonstrated that more than one-fourth of B. fragilis isolates harbored bft gene and less than 1% of patients with diarrhea harbored ETBF. The slight agreement between the PCR assays -already used for identification of B. fragilis which targeting gyrB or nanH - and 16S rRNA gene sequencing as the reference method was noted.

Analysis of changes in microbiome compositions related to the prognosis of colorectal cancer patients based on tissue-derived 16S rRNA sequences.

BACKGROUND: Comparing the microbiome compositions obtained under different physiological conditions has frequently been attempted in recent years to understand the functional influence of microbiomes in the occurrence of various human diseases. METHODS: In the present work, we analyzed 102 microbiome datasets containing tumor- and normal tissue-derived microbiomes obtained from a total of 51 Korean colorectal cancer (CRC) patients using 16S rRNA amplicon sequencing. Two types of comparisons were used: 'normal versus (vs.) tumor' comparison and 'recurrent vs. nonrecurrent' comparison, for which the prognosis of patients was retrospectively determined. RESULTS: As a result, we observed that in the 'normal vs. tumor' comparison, three phyla, Firmicutes, Actinobacteria, and Bacteroidetes, were more abundant in normal tissues, whereas some pathogenic bacteria, including Fusobacterium nucleatum and Bacteroides fragilis, were more abundant in tumor tissues. We also found that bacteria with metabolic pathways related to the production of bacterial motility proteins or bile acid secretion were more enriched in tumor tissues. In addition, the amount of these two pathogenic bacteria was positively correlated with the expression levels of host genes involved in the cell cycle and cell proliferation, confirming the association of microbiomes with tumorigenic pathway genes in the host. Surprisingly, in the 'recurrent vs. nonrecurrent' comparison, we observed that these two pathogenic bacteria were more abundant in the patients without recurrence than in the patients with recurrence. The same conclusion was drawn in the analysis of both normal and tumor-derived microbiomes. CONCLUSIONS: Taken together, it seems that understanding the composition of tissue microbiomes is useful for predicting the prognosis of CRC patients.

To resist and persist: Important factors in the pathogenesis of Bacteroides fragilis.

Bacteroides fragilis is a most frequent anaerobic pathogen isolated from human infections, particularly found in the abdominal cavity. Different factors contribute to the pathogenesis and persistence of B. fragilis at infection sites. The knowledge of the virulence factors can provide applicable information for finding alternative options for the antibiotic therapy and treatment of B. fragilis caused infections. Herein, a comprehensive review of the important B. fragilis virulence factors was prepared. In addition to B. fragilis toxin (BFT) and its potential role in the diarrhea and cancer development, some other important virulence factors and characteristics of B. fragilis are described including capsular polysaccharides, iron acquisition, resistance to antimicrobial agents, and survival during the prolonged oxidative stress, quorum sensing, and secretion systems.

A Europe-wide assessment of antibiotic resistance rates in Bacteroides and Parabacteroides isolates from intestinal microbiota of healthy subjects.

Here, we sought to assess the levels of antibiotic resistance among intestinal Bacteroides and Parabacteroides strains collected between 2014 and 2016 in Europe and also attempted to compare resistance levels between clinical and commensal isolates. Bacteroides and Parabacteroides isolates were recovered from faecal samples via the novel Bacteroides Chromogenic Agar (BCA) method. Antibiotic susceptibilities were determined by agar dilution for ten antibiotics. The values obtained were then statistically evaluated. Altogether 202 Bacteroides/Parabacteroides isolates (of which 24, 11.9%, were B. fragilis) were isolated from the faecal specimens of individuals taken from five European countries. The percentage values of isolates resistant to ampicillin, amoxicillin/clavulanate, cefoxitin, imipenem, clindamycin, moxifloxacin, metronidazole, tetracycline, tigecycline and chloramphenicol were 96.6, 4.5, 14.9, 2.0, 47.3, 11.4, 0, 66.2, 1.5 and 0%, respectively. These values are close to those reported in the previous European clinical Bacteroides antibiotic susceptibility survey except for amoxicillin/clavulanate and clindamycin, where the former was lower and the latter was higher in normal microbiota isolates. To account for these latter findings and to assess temporal effects we compared the data specific for Hungary for the same period (2014-2016), and we found differences in the resistance rates for cefoxitin, moxifloxacin and tetracycline.

Anti-biofilm and antimicrobial effects of zerumbone against Bacteroides fragilis.

Enterotoxigenic Bacteroides fragilis (ETBF) can form biofilms in the colon mucosal membrane and may promote chronic infection and tumor formation. The objective of this study was to determine the effect of zerumbone extracted from Zingiber zerumbet (L.) Smith, on B. fragilis biofilm formation. Inhibitory effects of zerumbone on planktonic cell growth and biofilm formation were examined by crystal-violet biofilm assays and XTT metabolic assays. Results showed that zerumbone significantly inhibited biofilm formation and eradicated established biofilms. Furthermore, B. fragilis biofilms inhibited by zerumbone were observed by confocal laser scanning microscopy. qRT-PCR was used to support the phenotypic results and to investigate the expression levels of an efflux pump-related gene (bmeB). Specifically, among the efflux pump-related genes, zerumbone significantly suppressed the expression level of bmeB12. These results indicate that zerumbone might be a promising candidate as an anti-biofilm and antimicrobial agent to treat and prevent biofilm-related infections caused by B. fragilis.

Detection of B. fragilis group and diversity of bft enterotoxin and antibiotic resistance markers cepA, cfiA and nim among intestinal Bacteroides fragilis strains in patients with inflammatory bowel disease.

We compared frequency of the members of B. fragilis group in 100 and 20 colon biopsy specimens of inflammatory bowel disease (IBD) and non-IBD patients. Agar dilution and PCR were orderly used to detect minimal inhibitory concentration of ampicillin, imipenem, and metronidazole, and carriage of related resistance genes cepA, cfi, and nim. B. fragilis group was detected in 38% of IBD (UC: 36/89; CD:1/11) and 25% (5/20) of non-IBD patients. While B. vulgatus (UC: 20/36, CD: 1/2, control: 1/6); B. fragilis (UC: 18/36, CD: 1/2, control: 5/6); B. ovatus (UC: 2/36); B. caccae (UC: 1/36); and B. eggerthii (UC: 1/36) were characterized, colonization of B. thetaiotamicron, B. merdae, B. distasonis, B. stercoris and B. dorei species was not detected in these specimens. Co-existence of B. fragilis + B. vulgatus (5 patients) and B. vulgatus + B. caccae (1 patient) was detected just in UC patients. bft was detected among 31.5% (6/19) of B. fragilis strains in the IBD and 40% (2/5) in the non-IBD groups. Nearly, 73.6% of the strains from the patient group and 80% in control group harbored cepA; 31.5% and 20% in the patients and control groups harbored cfiA, and none of them harbored nim determinant. Co-occurrence of the cepA and cfiA was orderly detected in 10.5% (2/19) and 20% (1/5) of the strains in these groups. The resistance rates were detected as 95.8% (23/24 (to ampicillin (MIC range of ≤0.5-≥16 µg/ml), 0% to metronidazole and 29.1% to imipenem (7/24, MIC range ≤4-32 µg/ml). Nearly 25% (6/24) of the strains were resistant to ampicillin and imipenem, simultaneously. No statistically significant difference was detected between the IBD and control groups for drug resistance phenotypes. Statistical analysis showed significant associations between resistance to ampicillin or imipenem and carriage of cepA or cfiA, respectively (p value = 0.0007). PCR results on the extracted plasmids confirmed their roles in carriage of cfiA and cepA. These data provide guide for antibiotic therapy and highlights wide distribution of ß-lactam resistant B. fragilis strains in patients with IBD and non-IBD intestinal disorders.

The underappreciated in vitro activity of tedizolid against Bacteroides fragilis species, including strains resistant to metronidazole and carbapenems.

Because Bacteroides fragilis has the ability to develop mechanisms of resistance to almost all antibiotics, we studied the comparative in vitro activity of tedizolid against 124 Bacteroides group species clinical isolates, including carbapenem, metronidazole and piperacillin-tazobactam resistant strains. Tedizolid had an MIC90 of 2 µg/ml (range, 0.5-4 µg/ml) and was 1-4 times more active than linezolid that had an MIC90 of 8 µg/ml (range, 2-16 µg/ml). It was also active (MICs 0.5-2 µg/ml) against the 27 ertapenem, 2 metronidazole and 12 piperacillin-tazobactam resistant strains tested. This suggests that tedizolid may be useful treating infections, including bacteremias, due to resistant B. fragilis group species, as well as, mixed skin and soft tissue infections such as diabetic foot infections caused by Gram-positive aerobes and B. fragilis group species.

Antimicrobial susceptibility of clinical isolates of anaerobic bacteria in Ontario, 2010-2011.

The local epidemiology of antimicrobial susceptibility patterns in anaerobic bacteria is important in guiding the empiric treatment of infections. However, susceptibility data are very limited on anaerobic organisms, particularly among non-Bacteroides organisms. To determine susceptibility profiles of clinically-significant anaerobic bacteria in Ontario Canada, anaerobic isolates from sterile sites submitted to Public Health Ontario Laboratory (PHOL) for identification and susceptibility testing were included in this study. Using the E-test method, isolates were tested for various antimicrobials including, penicillin, cefoxitin, clindamycin, meropenem, piperacillin-tazobactam and metronidazole. The MIC results were interpreted based on guidelines published by Clinical and Laboratory Standards Institute. Of 2527 anaerobic isolates submitted to PHOL, 1412 were either from sterile sites or bronchial lavage, and underwent susceptibility testing. Among Bacteroides fragilis, 98.2%, 24.7%, 1.6%, and 1.2% were resistant to penicillin, clindamycin, piperacillin-tazobactam, and metronidazole, respectively. Clostridium perfringens was universally susceptible to penicillin, piperacillin-tazobactam, and meropenem, whereas 14.2% of other Clostridium spp. were resistant to penicillin. Among Gram-positive anaerobes, Actinomyces spp., Parvimonas micra and Propionibacterium spp. were universally susceptible to ß-lactams. Eggerthella spp., Collinsella spp., and Eubacterium spp. showed variable resistance to penicillin. Among Gram-negative anaerobes, Fusobacterium spp., Prevotella spp., and Veillonella spp. showed high resistance to penicillin but were universally susceptible to meropenem and piperacillin-tazobactam. The detection of metronidazole resistant B. fragilis is concerning as occurrence of these isolates is extremely rare. These data highlight the importance of ongoing surveillance to provide clinically relevant information to clinicians for empiric management of infections caused by anaerobic organisms.

Therapeutic Potential of Bacteroides fragilis SNBF-1 as a Next-Generation Probiotic: In Vitro Efficacy in Lipid and Carbohydrate Metabolism and Antioxidant Activity.

This study explores the potential of aerotolerant Bacteroides fragilis (B. fragilis) strains as next-generation probiotics (NGPs), focusing on their adaptability in the gastrointestinal environment, safety profile, and probiotic functions. From 23 healthy infant fecal samples, we successfully isolated 56 beneficial B. fragilis strains. Notably, the SNBF-1 strain demonstrated superior cholesterol removal efficiency in HepG2 cells, outshining all other strains by achieving a remarkable reduction in cholesterol by 55.38 ± 2.26%. Comprehensive genotype and phenotype analyses were conducted, including sugar utilization and antibiotic sensitivity tests, leading to the development of an optimized growth medium for SNBF-1. SNBF-1 also demonstrated robust and consistent antioxidant activity, particularly in cell-free extracts, as evidenced by an average oxygen radical absorbance capacity value of 1.061 and a 2,2-diphenyl-1-picrylhydrazyl scavenging ability of 94.53 ± 7.31%. The regulation of carbohydrate metabolism by SNBF-1 was assessed in the insulin-resistant HepG2 cell line. In enzyme inhibition assays, SNBF-1 showed significant α-amylase and α-glucosidase inhibition, with rates of 87.04 ± 2.03% and 37.82 ± 1.36%, respectively. Furthermore, the cell-free supernatant (CFS) of SNBF-1 enhanced glucose consumption and glycogen synthesis in insulin-resistant HepG2 cells, indicating improved cellular energy metabolism. This was consistent with the observation that the CFS of SNBF-1 increased the proliferation of HepG2 cells by 123.77 ± 0.82% compared to that of the control. Overall, this research significantly enhances our understanding of NGPs and their potential therapeutic applications in modulating the gut microbiome.

New functions of pirin proteins and a 2-ketoglutarate: Ferredoxin oxidoreductase ortholog in Bacteroides fragilis metabolism and their impact on antimicrobial susceptibility to metronidazole and amixicile.

The understanding of how central metabolism and fermentation pathways regulate antimicrobial susceptibility in the anaerobic pathogen Bacteroides fragilis is still incomplete. Our study reveals that B. fragilis encodes two iron-dependent, redox-sensitive regulatory pirin protein genes, pir1 and pir2. The mRNA expression of these genes increases when exposed to oxygen and during growth in iron-limiting conditions. These proteins, Pir1 and Pir2, influence the production of short-chain fatty acids and modify the susceptibility to metronidazole and amixicile, a new inhibitor of pyruvate: ferredoxin oxidoreductase in anaerobes. We have demonstrated that Pir1 and Pir2 interact directly with this oxidoreductase, as confirmed by two-hybrid system assays. Furthermore, structural analysis using AlphaFold2 predicts that Pir1 and Pir2 interact stably with several central metabolism enzymes, including the 2-ketoglutarate:ferredoxin oxidoreductases Kor1AB and Kor2CDAEBG. We used a series of metabolic mutants and electron transport chain inhibitors to demonstrate the extensive impact of bacterial metabolism on metronidazole and amixicile susceptibility. We also show that amixicile is an effective antimicrobial against B. fragilis in an experimental model of intra-abdominal infection. Our investigation led to the discovery that the kor2AEBG genes are essential for growth and have dual functions, including the formation of 2-ketoglutarate via the reverse TCA cycle. However, the metabolic activity that bypasses the function of Kor2AEBG following the addition of phospholipids or fatty acids remains undefined. Overall, our study provides new insights into the central metabolism of B. fragilis and its regulation by pirin proteins, which could be exploited for the development of new narrow-spectrum antimicrobials in the future.

Bacteroides fragilis alleviates necrotizing enterocolitis through restoring bile acid metabolism balance using bile salt hydrolase and inhibiting FXR-NLRP3 signaling pathway.

Necrotizing enterocolitis (NEC) is a leading cause of morbidity and mortality in premature infants with no specific treatments available. We aimed to identify the molecular mechanisms underlying NEC and investigate the therapeutic effects of Bacteroides fragilis on NEC. Clinical samples of infant feces, bile acid-targeted metabolomics, pathological staining, bioinformatics analysis, NEC rat model, and co-immunoprecipitation were used to explore the pathogenesis of NEC. Taxonomic characterization of the bile salt hydrolase (bsh) gene, enzyme activity assays, 16S rRNA sequencing, and organoids were used to explore the therapeutic effects of B. fragilis on NEC-related intestinal damage. Clinical samples, NEC rat models, and in vitro experiments revealed that total bile acid increased in the blood but decreased in feces. Moreover, the levels of FXR and other bile acid metabolism-related genes were abnormal, resulting in disordered bile acid metabolism in NEC. Taurochenodeoxycholic acid accelerated NEC pathogenesis and taurodeoxycholate alleviated NEC. B. fragilis displayed bsh genes and enzyme activity and alleviated intestinal damage by restoring gut microbiota dysbiosis and bile acid metabolism abnormalities by inhibiting the FXR-NLRP3 signaling pathway. Our results provide valuable insights into the therapeutic role of B. fragilis in NEC. Administering B. fragilis may substantially alleviate intestinal damage in NEC.

Phenotypic and Genotypic Correlation of Antimicrobial Susceptibility of Bacteroides fragilis: Lessons Learnt.

Background Bacteroides fragilis is an opportunistic pathogen causing severe infections, including bacteremia. There have been increased reports of antimicrobial resistance in B. fragilis. However, phenotypic testing of susceptibility is time consuming and not cost effective for anaerobes. The present study investigates the correlation of phenotypic susceptibility with genotypic markers; to determine if these could be considered for deciding empirical therapy for B. fragilis. Material and methods Bacteroides fragilis isolates from various clinical samples including exudates, tissue, and body fluids were collected between November 2018 and January 2020 in the Department of Clinical Microbiology, Christian Medical College (CMC) Vellore. Species identification was done by Matrix Assisted Laser Desorption Ionization time of flight mass spectrometry (MALDI TOF) according to the manufacturer's instructions. A total number of 51 B. fragilis isolates were tested against metronidazole, clindamycin, piperacillin/tazobactam, and meropenem phenotypically by agar dilution method using Clinical & Laboratory Standards Institute (CLSI) 2019 guidelines and minimum inhibitory concentrations (MIC) were interpretated. The genotypic markers for antimicrobial resistance genes (nim, emrF, and cfiA) were studied by polymerase chain reaction (PCR) assay as per the standard protocol on all isolates to detect resistance genes. Results B. fragilis isolates in this study expressed 45%, 41%, and 16% phenotypic resistance to clindamycin, metronidazole, and meropenem, respectively, with least resistance to piperacillin/tazobactam (6%). Among the metronidazole resistant isolates, 52% harbored nim gene. Nim gene was also present in 76% (23/30) of the metronidazole susceptible isolates. Similarly, cfiA was present in all eight meropenem resistant isolates in addition to 22% (9/41) of the susceptible isolates. All cfiA negative isolates were phenotypically susceptible. Interestingly, 74% (17/23) of the clindamycin resistant isolates were positive for ermF. Conclusions Detection of a limited set of genes does not always correlate with phenotypic resistance to metronidazole and clindamycin due to the reported influence of insertion sequence (IS) elements, efflux, and other genetic determinants. Certainly, the absence of the cfiA gene can be employed to rule out meropenem resistance. However, redundant use of antibiotics such as meropenem along with metronidazole could be avoided for B. fragilis, which might otherwise elevate meropenem resistance. Recommendation of metronidazole requires prior phenotypic testing due to the reported 41% resistance.

Gut colonization with an obesity-associated enteropathogenic microbe modulates the premetastatic niches to promote breast cancer lung and liver metastasis.

Introduction: Obesity, an independent risk factor for breast cancer growth and metastatic progression, is also closely intertwined with gut dysbiosis; and both obese state and dysbiosis promote each other. Enteric abundance of Bacteroides fragilis is strongly linked with obesity, and we recently discovered the presence of B. fragilis in malignant breast cancer. Given that enterotoxigenic B. fragilis or ETBF, which secretes B. fragilis toxin (BFT), has been identified as a procarcinogenic microbe in breast cancer, it is necessary to examine its impact on distant metastasis and underlying systemic and localized alterations promoting metastatic progression of breast cancer. Methods: We used syngeneic mammary intraductal (MIND) model harboring gut colonization with ETBF to query distant metastasis of breast cancer cells. Alterations in the immune network and cytokines/chemokines in the tumor microenvironment and distant metastatic sites were examined using flow cytometry, immunohistochemistry, and multiplex arrays. Results: ETBF infection initiates a systemic inflammation aiding in the establishment of the premetastatic niche formation in vital organs via increased proinflammatory and protumorigenic cytokines like IL17A, IL17E, IL27p28, IL17A/F, IL6, and IL10 in addition to creating a prometastatic immunosuppressive environment in the liver and lungs rich in myeloid cells, macrophages, and T regulatory cells. It induces remodeling of the tumor microenvironment via immune cell and stroma infiltration, increased vasculogenesis, and an EMT-like response, thereby encouraging early metastatic dissemination ready to colonize the conducive environment in liver and lungs of the breast tumor-bearing mice. Discussion: In this study, we show that enteric ETBF infection concomitantly induces systemic inflammation, reshapes the tumor immune microenvironment, and creates conducive metastatic niches to potentiate early dissemination and seeding of metastases to liver and lung tissues in agreement with the "seed and soil hypothesis." Our results also support the ETBF-induced "parallel model" of metastasis that advocates for an early dissemination of tumor cells that form metastatic lesions independent of the primary tumor load.