Bft Gene Research Articles

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

ObjectivesData 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). MethodsQuantitative 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. ResultsWe 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. DiscussionThe 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.

Methylome and transcriptome analysis of flowering branches building of Citrus plants induced by drought stress

Citrus plants exhibit positive floral response under water stress conditions, however, the mechanistic understanding of floral induction remains largely unexplored in water deficit. In this study, DNA methylomic and transcriptomic analyses were integrated to explore the flowering bud formation as well as branches building after light drought stress. While comparing with the conventional watering group (CK), the light drought group treated with five months (LD) showed a significant increase in the flowering branches, whereas an apparent decrease in vegetative branches. Global DNA methylation analysis showed that the LD Group acquired DNA methylation in more than 70,090 genomic regions and lost DNA methylation in about 18,421 genomic regions compared with normal watering group, this indicates that water deficiency leads to a global increase in the expression of DNA methylation in citrus. In the same time, we verified that the increase of DNA methylation level in LD group was correlated with the decrease of DNA demethylase related gene expression.Interestingly, in transcription analysis, it was found that the promoting flower genes of the LD group did not increase but decreased similarly with repressing genes, which is contrary to the intended result. Thus, we thought the lower expression of suppressors FLC and BFT were the key influencing factor to stimulate the flowering branches formation after LD treatment. Moreover, there was a strong negative correlation between the genes expression level and methylation level of the flowering induction/flower development genes. In general, we thought high global DNA methylation level induced by water deficit regulate the flowering branches building by reducing FLC and BFT genes expression.

Molecular identification of enterotoxigenic Bacteroides fragilis and the Distribution 16S rRNA gene, bft gene subtypes and gyrb gene in Iraqi patients with diarrhea

Molecular identification of enterotoxigenic Bacteroides fragilis and the Distribution 16S rRNA gene, bft gene subtypes and gyrb gene in Iraqi patients with diarrhea

Experimental study on the effect of bft toxin isolated and purified from clinical isolates of Enterotoxigenic Bacteroides fragilis on the liver, spleen and intestine of mice

This study includes three parts: isolation of Enterotoxigenic Bacteroid fragilis from 94 stool samples collected from different hospitals in Baghdad city from the beginning of March/2020 to the end of April/2021. Stool samples were streaked on BBE media in an anaerobic condition for 24-48h. Identification of Fragilis was done based on morphological characteristics on BBE media: gray convex small rounded colonies surround black zone colonies and molecular method using specific genes 16S rRNA and bft gene. Results showed 34 Fragilis isolates were positive for the 16S rRNA gene and 5 Fragilis positive for the bft gene were classified as Enterotoxigenic Fragilis (ETBF). ETBF isolate which was positive for the bft gene and 16S rRNA was purified by using the Van Tassel method. 30 male mice were divided into three groups with 10 mice for each group the first group as control, the second group is positive control mice administered daily2% dextran sulfate sodium for 30 days, the third group mice administered by stomach tube 2%DSS for 10 days after 10 days mice administered with 20 µg of bft toxin by stomach tube for 30 days. At the end of the experiment, all groups of mice were killed by euthanized ethics. Tissue samples (liver, intestine, and spleen) from mice were removed. The organs were fixed in 10% neutral buffered formalin for histological techniques. Histopathological changes in the third group, in the liver section of a mouse inoculated with DSS+bft toxin, showed: necrotic hepatocytes and dilated sinusoids with hemorrhage. Histopathological changes in the intestine section of a mouse inoculated with DSS+bft toxin showed: sloughing and degenerated villi and shorten villi. Histopathological changes in the spleen section of a mouse inoculated with DSS+bft toxin showed: amyloid infiltration and all lymphoid follicles depleted with necrotic lymphocytes

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

ObjectivesWe 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. MethodsThe 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. ResultsA 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. ConclusionsThis 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.

Antibiotic resistance pattern of Bacteroides fragilis isolated from clinical and colorectal specimens

BackgroundBacteroides fragilis is a part of the normal gastrointestinal flora, but it is also the most common anaerobic bacteria causing the infection. It is highly resistant to antibiotics and contains abundant antibiotic resistance mechanisms.MethodsThe antibiotic resistance pattern of 78 isolates of B. fragilis (22 strains from clinical samples and 56 strains from the colorectal tissue) was investigated using agar dilution method. The gene encoding Bacteroides fargilis toxin bft, and antibiotic resistance genes were targeted by PCR assay.ResultsThe highest rate of resistance was observed for penicillin G (100%) followed by tetracycline (74.4%), clindamycin (41%) and cefoxitin (38.5%). Only a single isolate showed resistance to imipenem which contained cfiA and IS1186 genes. All isolates were susceptible to metronidazole. Accordingly, tetQ (87.2%), cepA (73.1%) and ermF (64.1%) were the most abundant antibiotic-resistant genes identified in this study. MIC values for penicillin, cefoxitin and clindamycin were significantly different among isolates with the cepA, cfxA and ermF in compare with those lacking such genes. In addition, 22.7 and 17.8% of clinical and GIT isolates had the bft gene, respectively.ConclusionsThe finding of this study shows that metronidazole is highly in vitro active agent against all of B. fragilis isolates and remain the first-line antimicrobial for empirical therapy.

Prevalence of Enterotoxigenic Bacteroides Fragilis in Stool Specimens Collected from Children Less Than 5 Years Of Age in Iraq

Although Bacteroides fragilis is a bacterium present within gut microbiota, the toxin producer strain, known as enterotoxigenic B. fragilis (ETBF), is associated with diarrhea in children less than 5 years of age. This study includes 69 diarrheal and 29 non-diarrheal (control) samples collected from children less than 5 years old. DNA was extracted directly from stool specimens and directed to conventional PCR targeting beta-isopropylmalate dehydrogenase (leuB) gene, used for detection of B. fragilis, and Bacteroides fragilis toxin (bft) gene, used for the detection of ETBF. The results showed that the prevalence of leuB gene was 78 (79.6%) including 56 (81.2%) in diarrheal and 22 (75.9%) in non-diarrheal subjects, while that of bft gene was only 3 (3.1%) including 2 (2.9%) in diarrheal and 1 (3.4%) in non-diarrheal subjects. Based on sequencing of bft-positive specimens, both bft-1 and bft-2 isoforms were represented in diarrheal specimens, whereas only bft-1 was found in the control specimens. In conclusion, this study examined for the first time the leuB and bft gene in a specimen of Iraqi children with diarrhea and showed no significant differences between diarrheal and control groups in both genes.

Toxigenic and non-toxigenic patterns I, II and III and biofilm-forming ability in Bacteroides fragilis strains isolated from patients diagnosed with colorectal cancer

BackgroundEnterotoxigenic Bacteroides fragilis (ETBF) associated with the initiation and progression of colorectal cancer (CRC) has been alarmingly reported all over the world. In this study, simultaneous investigation of toxigenic and non-toxigenic patterns I, II and III and biofilm formation ability of Bacteroides fragilis isolated from patients with colorectal cancer was performed.MethodsThirty-one patients diagnosed with CRC and thirty-one control subjects were recruited in this study. Specimens were cultured on BBE and BBA culture media. Classical phenotypic identification tests and PCR was performed to verify Bacteroides fragilis presence. Also, biofilm-forming ability and expression of bft gene were assessed under biofilm and planktonic forms.ResultsA total of 68 B.fragilis was isolated from all colorectal tissue, of which 13 isolates (19.1%) (11 isolates from CRC and 2 from normal tissue) were positive for bft gene. The abundance patterns of I, II and III were as follow in descending order; pattern I > pattern III > pattern II in CRC subjects and pattern II > pattern III > pattern I in normal tissues. Also, pattern I showed higher biofilm formation ability compared to other patterns. Toxin expression was significantly reduced in biofilm form comparing with planktonic form.ConclusionsBased on our findings, there was a difference between the abundance of patterns I, II, and III and biofilm formation in isolates obtained from CRC and normal tissues. Biofilm formation ability and toxin encoding gene (bft) are two main virulence factors in B. fragilis pathogenicity which require more investigation to treat B. fragilis infections effectively.

Enterotoxigenic Bacteroides fragilis: A Possible Etiological Candidate for Bacterially-Induced Colorectal Precancerous and Cancerous Lesions.

Enterotoxigenic Bacteroides fragilis (ETBF) produces Bacteroides fragilis toxin (BFT), which is associated with acute diarrheal, inflammatory bowel disease, and colorectal cancer (CRC). In experimental models, ETBF has been shown to contribute to colon carcinogenesis. The present study was conducted to investigate mucosal colonization of ETBF in the colon to find a possible association between the presence of ETBF and precancerous and cancerous lesions. The mucosal biopsies of involved sites were obtained from 68 patients with precancerous and cancerous lesions and 52 healthy controls (HC). The samples were cultured on Bacteroides Bile Esculin agar. Then, specific primers were designed to detect B. fragilis and bft gene using quantitative real-time PCR, and the possible links of ETBF with clinicopathological characteristics was evaluated. Also real-time PCR was performed to detect the bft gene subtypes. Bacteroides fragilis was detected in 51% of the patients and 48% of HCs cultures. The 16SrRNA gene was found to be present in 63 and 81% of the patients and HCs' samples, respectively. Moreover, the bft gene was detected in 47 and 3.8% of the patients and HCs, respectively. Also, B. fragilis was significantly more abundant in the patients' samples compared to those of HCs. In the patient group, higher odds ratio (OR) of ETBF was significantly associated with serrated lesions and adenoma with low-grade dysplasia. The bft1 gene was the most prevalent subtype of bft gene, followed by the bft2 gene. This was the first study in Iran to demonstrate increased positivity of ETBF in patients with precancerous and cancerous lesions. In this study, the bft gene was found to be associated with CRC, especially in the patients with precancerous lesions and initial carcinogenic lesions. Moreover, the results suggest that mucosal BFT exposure is common and could be a risk factor and a screening marker for developing CRC.

The association between fecal enterotoxigenic B. fragilis with colorectal cancer

BackgroundEnterotoxigenic Bacteroides fragilis (ETBF) is an enterotoxin-producing bacterium that possibily has a role in the occurrence and progression of colorectal cancer (CRC) by modulating the mucosal immune response and inducing epithelial cell changes. The aim of this study was to investigate the frequency of ETBF in stool samples of CRC patients and healthy volunteers.MethodsA total of 60 stool samples from confirmed CRC patients and 60 stool samples from healthy volunteers with no personal or familial history or diagnosis of colorectal disease were collected. Stool samples were screened for direct detection of B. fragilis using PCR targeting the marker genes of neu and bft. Enterotoxin isotypes bft-1, bft-2 and bft-3 were also detected in B. fragilis positive samples.ResultsThe frequency of B. fragilis among CRC and control cases was 58.3 and 26.6%, respectively (P < 0.05). The rate of bft gene in CRC cases was significantly higher than in controls (P < 0.05). Also, the presence of bft gene in CRC patients stage III was significantly higher than stages I and II (P < 0.05). Enterotoxin isotype bft-2 was detected with higher frequency among CRC patients than healthy control (P < 0.05).ConclusionOur results show the association between fecal ETBF and CRC, and we suggest that detection of ETBF may be a potential marker for colorectal cancer diagnosis. However, additional investigations on tumor and paired normal tissue samples are required to substantiate this possible correlation.

Detection of the Bacteroides fragilis toxin gene in sheep with and without small intestinal adenocarcinoma

ABSTRACTAims: To determine if presence of the Bacteroides fragilis toxin (bft) gene, a molecular marker of colonic carriage of entertoxigenic Bacteroides fragilis (ETBF) in humans, was associated with a finding of small intestinal adenocarcinomas (SIA) in sheep in New Zealand.Methods: Samples of jejunal tissue were collected from the site of tumours and from grossly normal adjacent tissue in 20 sheep, in different consignments, diagnosed with SIA based on gross examination of viscera following slaughter. Two jejunal samples were also collected from a control sheep in the same consignment that had no gross evidence of SIA. A PCR assay was used to detect the presence of the bft gene in the samples.Results: Of the sheep with SIA, the bft gene was amplified from one or both samples from 7/20 (35%) sheep, and in sheep that had no gross evidence of SIA the bft gene was amplified from at least one sample in 11/20 (55%) sheep (RR 0.61; 95% CI = 0.30–1.25; p = 0.34). Of 11 positive samples analysed, ETBF subtype bft-1 was detected in one, bft-2 was detected in 10, and none were bft-3.Conclusions and Clinical Relevance: There was a high prevalence of detection of the bft gene in both SIA-affected and non-affected sheep, but there was no apparent association between carriage of ETBF, evidenced by detection of the bft gene, and the presence of SIA. ETBF are increasingly implicated in the aetiology of human colorectal cancer, raising the possibility that sheep may provide a zoonotic reservoir of this potentially carcinogenic bacterium.Abbreviation: Bft: Bacteroides fragilis toxin; ETBF: Enterotoxigenic Bacteroides fragilis; SIA: Small intestinal adenocarcinoma

Antimicrobial resistance, prevalence of resistance genes, and molecular characterization in intestinal Bacteroides fragilis group isolates.

Since the level of antimicrobial resistance in Bacteroides fragilis has increased, monitoring the antimicrobial susceptibility could be necessary. The objectives of this study were to (i) investigate the prevalence of species, the occurrence of reduced antimicrobial susceptibility (E-test method), and antibiotic resistance genes in the B.fragilis group and (ii) evaluate the prevalence of enterotoxigenic B.fragilis and the distribution of bft gene subtypes in hospitalized patients. As many as 475 isolates out of 250 stool samples were detected to be B.fragilis group by using conventional biochemical tests (API-32A system) and multiplex-PCR. In addition, 48.2%, 13.9%, 76.6%, and 1.2% of B.fragilis group isolates were resistant (according to EUCAST breakpoint) to piperacillin-tazobactam, meropenem, clindamycin, and metronidazole, respectively. Six metronidazole-resistant strains were isolated; B.fragilis (n: 3), B.thetaiotaomicron, B.vulgates, and B.ovatus. The presence of the cfiA, cepA, ermF, and nim genes was observed in 3.8%, 15.9%, 34.1%, and 0.7% of the B.fragilis isolates, respectively. One hundred thirty-two B.fragilis isolates (27.8%)and 21 B fragilis isolates (15.9%) turned out to be bft gene positive by multiplex-PCR; eleven isolates (52.4%) harbored bft-1, eight isolates (38%) harbored bft-2 isotypes, and two isolates (9.5%) harbored bft-3 isotype (16.66%). These bacteria harbor antimicrobial resistance genes that could be transferred to other susceptible intestinal strains. Further investigations on lineage analysis are needed for a better understanding of these bacteria in Iran.

The presence of antibiotic resistance genes and bft genes as well as antibiotic susceptibility testing of Bacteroides fragilis strains isolated from inpatients of the Infant Jesus Teaching Hospital, Warsaw during 2007–2012

The purpose of this study was to assess drug susceptibility of clinical B. fragilis strains and to determine any correlation between drug resistance and the presence of specific genes. Antimicrobial susceptibility was assessed using E-tests. All isolates were analyzed with the PCR technique for the presence of antibiotic resistance genes (cepA, cfxA, cfiA, ermF, ermB, ermG, nim), insertion sequences elements (IS1186, IS1187, IS1188, IS942), and enterotoxin-encoding genes (bft). Susceptibility tests yielded the following rates of resistance to the evaluated antibiotics: penicillin G (100%), clindamycin (22.5%), cefoxitin (6.3%), amoxicillin/clavulanic acid (1.8%). All strain were susceptible to imipenem, and metronidazole. The following antibiotic resistance genes were detected in the evaluated isolates: cepA (in 96.4% of isolates), cfxA (in 12.6%), cfiA (in 1.8%), and ermF (in 25.2%). Genes ermB, ermG, and nim were not found. The presence of the cepA gene showed no correlation with the penicillin G MIC. However, we observed a high correlation between cefoxitin MIC values and the presence of gene cfxA as well as a nearly complete correlation between clindamycin MIC values and the presence of gene ermF. The presence of a bft gene was detected in 14.4% of the analyzed B. fragilis isolates; with the bft-1 allele found in 75%, bft-2 in 25%, and bft-3 in none of the isolates. Antibiotic susceptibility profiles of enterotoxin gene-positive isolates in our study did not differ from those of enterotoxin gene-negative isolates.

Detection of enterotoxigenic Bacteroides fragilis in patients with ulcerative colitis

PurposeUlcerative colitis (UC) as a type of inflammatory bowel disease (IBD), presumed to occur as a consequence of increased immune responses to intestinal microbiota in genetically susceptible individuals. Enterotoxigenic Bacteroides fragilis (ETBF) strains are important intestinal bacteria that can be involved in IBD. The aim of this study was to design a quantitative assay for detection of B. fragilis and ETBF and also to find their association with UC.MethodsNinety-five biopsies were collected from patients with UC (n = 35) and with no IBD (nIBD, n = 60). All the specimens were cultured in Bacteroides bile esculin agar medium. Specific primers and probes were designed for quantitative real-time PCR (QRT-PCR) based on 16S rRNA and bft genes sequences of ETBF.ResultsThe bft genes were detected in 51.4% of UC samples and 1.6% of nIBD samples, respectively. In UC patients, 37.1% of samples with diarrhea and 11.4% of samples without diarrhea, harbored the bft gene. Mean value of the number of ETBF with bft gene in UC and nIBD samples were 4.46 ן 102 and 1.96, respectively. Likewise these result for 16S rRNA gene in UC and nIBD samples were 2.0 × 103 and 8.4 × 103, respectively.ConclusionsThere was no significant association between presence and numbers of 16S rRNA gene of B. fragilis and UC. ETBF was detected more in UC specimens and biopsies of UC patients with diarrhea than in the control group. These data demonstrated that ETBF is associated with development of UC and as a causative agent for the development of diarrhea in these patients.

Detection of enterotoxin and protease genes among Hungarian clinical Bacteroides fragilis isolates

Bacteroides fragilis as a commensal bacterium is a member of the human intestinal flora, but as an opportunistic pathogen it can cause serious infections as well. Some of them, harbouring an enterotoxin gene (bft), may cause diarrhoea mainly in young children. Recently it has been shown that a member of C11 proteases called fragipain (fpn) can activate the enterotoxin, while C10 protease (bfp) is suspected of playing an important role in the invasiveness of the B. fragilis isolates. The objective of this study was to investigate the prevalence and distribution of the bft isotypes in 200 Hungarian B. fragilis isolates collected recently; and in a subset of 72 strains, we wanted to determine the prevalence of bfp1-4 and fpn genes in bft-positive and bft-negative strains. Using the MALDI-TOF MS cfiA identification project file, 19 B. fragilis strains belonging to Division II were identified and the presence of the cfiA gene was confirmed by RT-PCR. Twenty six (13.0%) B. fragilis isolates turned out to be bft gene positive by RT-PCR; 20 isolates harboured bft-1 and six bft-2 isotypes, but no bft-3 isotype containing strains were found. A melting curve analysis and the PCR-RFLP were performed to differentiate between the bft-1 and bft-2 isotypes confirmed by sequencing. Thirty eight strains harboured bfp1, 58 isolates contained bfp2 gene, while 17 isolates proved positive for bfp3. Morever, no bfp4 positive isolate was found, and some of the B. fragilis strains tested harboured two or three bfp isotypes simultaneously. Among the 26 bft-positive strains, 24 contained the fpn gene, which confirms the role of fragipain in the activation of B. fragilis enterotoxin. In experiments, a significant negative correlation between fpn and cfiA was demonstrated (p < 0.000), a positive correlation was found between bfp2 and fpn genes (p = 0.0000803), and a negative correlation between bfp2 and cfiA genes (p = 0.011).

Three FT and multiple CEN and BFT genes regulate maturity, flowering, and vegetative phenology in kiwifruit.

Kiwifruit is a woody perennial horticultural crop, characterized by excessive vegetative vigor, prolonged juvenility, and low productivity. To understand the molecular factors controlling flowering and winter dormancy, here we identify and characterize the kiwifruit PEBP (phosphatidylethanolamine-binding protein) gene family. Five CEN-like and three BFT-like genes are differentially expressed and act as functionally conserved floral repressors, while two MFT-like genes have no impact on flowering time. FT-like genes are differentially expressed, with AcFT1 confined to shoot tip and AcFT2 to mature leaves. Both act as potent activators of flowering, but expression of AcFT2 in Arabidopsis resulted in a greater impact on plant morphology than that of AcFT1. Constitutive expression of either construct in kiwifruit promoted in vitro flowering, but AcFT2 displayed a greater flowering activation efficiency than AcFT1, leading to immediate floral transition and restriction of leaf development. Both leaf and flower differentiation were observed in AcFT1 kiwifruit lines. Sequential activation of specific PEBP genes in axillary shoot buds during growth and dormancy cycles indicated specific roles in regulation of kiwifruit vegetative and reproductive phenologies. AcCEN and AcCEN4 marked active growth, AcBFT2 was associated with suppression of latent bud growth during winter, and only AcFT was activated after cold accumulation and dormancy release.

Comparison of standard, quantitative and digital PCR in the detection of enterotoxigenic Bacteroides fragilis.

Gut colonization with enterotoxigenic Bacteroides fragilis (ETBF) appears to be associated with the development of colorectal cancer. However, differences in carriage rates are seen with various testing methods and sampling sites. We compared standard PCR, SYBR green and TaqMan quantitative PCR (qPCR) and digital PCR (dPCR) in detecting the B. fragilis toxin (bft) gene from cultured ETBF, and from matched luminal and faecal stool samples from 19 colorectal cancer patients. Bland-Altman analysis found that all three quantitative methods performed comparably in detecting bft from purified bacterial DNA, with the same limits of detection (<1 copy/μl). However, SYBR qPCR under-performed compared to TaqMan qPCR and dPCR in detecting bft in clinical stool samples; 13/38 samples were reported positive by SYBR, compared to 35 and 36 samples by TaqMan and dPCR, respectively. TaqMan qPCR and dPCR gave bft copy numbers that were 48-fold and 75-fold higher for the same samples than SYBR qPCR, respectively (p < 0.001). For samples that were bft-positive in both fecal and luminal stools, there was no difference in relative abundance between the sites, by any method tested. From our findings, we recommend the use of TaqMan qPCR as the preferred method to detect ETBF from clinical stool samples.

Characterization ofbftGenes among EnterotoxigenicBacteroides fragilisIsolates from Extraintestinal Specimens at a University Hospital in Korea

Enterotoxigenic Bacteroides fragilis (ETBF)는 병독소 인자로 알려진 장독소를 생성하는 균종이다. B. fragilis enterotoxin (BFT)은 bft- 1, bft- 2 및 bft- 3 세 개의 유전자 아형들이 밝혀졌다. 본 연구에서는 임상 검체에서 분리된 B. fragilis에서 bft 유전자의 유무와 BFT 음성 및 양성 균주의 항균제 내성을 조사하였다. 국내의 한 대학병원에서 8년간(2006∼2013년) 장외 검체에서 분리된 B. fragilis는 총 537주이었다. 다중중합연쇄반응으로 시험하여 bft유전자 아형을 확인하였다. BFT 음성 74주와 양성 33주를 포함한 B. fragilis 107주의 항균제 감수성은 CLSI 한천희석법으로 시험하였다. 임상 검체에서 분리된 B. fragilis의 bft 유전자 검출율은 30% 이었고, 이 중 혈액과 혈액 외 장외 검체 분리주에서는 각각 33%, 29% 이었다. ETBF 중에서 가장 흔한 아형은 bft- 1 이었고, 그 다음은 bft- 2, bft- 3 순이었다( bft- 1: 77%, bft- 2: 14%, bft- 3: 9%). BFT-음성과 양성 균주의 내성률은 일부 항균제에 대해서 차이가 있었다(BFT-음성 균주: piperacillin-tazobactam 3%, cefoxitin 5%, imipenem 1%, clindamycin 38%; BFT-양성 균주: piperacillin-tazobactam 3%, cefoxitin 6%, imipenem 3%, clindamycin 42%). BFT-음성 및 BFT-양성 균주 모두는 chloramphenicol과 metronidazole에 대한 내성은 없었다. 결론적으로, 혈액 분리주에서의 ETBF 검출율은 혈액 외 장외 검체 분리주에서와 비슷하였고, 가장 흔한 유전자 아형은 bft- 1 이었다. 항균제 내성은 BFT 양성 균주가 음성 균주보다 대체로 높았으나 통계학적으로 유의한 차이는 없었다.

Screening for enterotoxigenic Bacteroides fragilis in stool samples

Bacteroides fragilis is a commensal bacterium found in the gut of most humans, however enterotoxigenic B. fragilis strains (ETBF) have been associated with diarrhoea and colorectal cancer (CRC). The purpose of this study was to establish a method of screening for the Bacteroides fragilis toxin (bft) gene in stool samples, as a means of determining if carriage of ETBF is detected more often in CRC patients than in age-matched healthy controls. Stool samples from 71 patients recently diagnosed with CRC, and 71 age-matched controls, were screened by standard and quantitative PCR using primers specific for the detection of the bft gene. Bacterial template DNA from stool samples was prepared by two methods: a sweep, where all colonies growing on Bacteroides Bile Esculin agar following stool culture for 48 h at 37 °C in an anaerobic environment were swept into sterile water and heat treated; and a direct DNA extraction from each stool sample. The bft gene was detected more frequently from DNA isolated from bacterial sweeps than from matched direct DNA extractions. qPCR was found to be more sensitive than standard PCR in detecting bft. The cumulative total of positive qPCR assays from both sample types revealed that 19 of the CRC patients had evidence of the toxin gene in their stool sample (27%), compared to seven of the age-matched controls (10%). This difference was significant (P = 0.016). Overall, ETBF carriage was detected more often in CRC patient stool samples compared to controls, but disparate findings from the different DNA preparations and testing methods suggests that poor sensitivity may limit molecular detection of ETBF in stool samples.

Heterogeneity in resistant fecal Bacteroides fragilis group collected from healthy people

Normal nonpathogenic flora would represent a constant lake of resistance genes potentially transferable to human pathogens. To assess the prevalence of resistance genes and genetic variability of Bacteroides fragilis group (BFG) from normal flora, 177 Bacteroides isolates obtained from the fecal samples of healthy individuals. These isolates were subjected to antibiotic susceptibility testing and pulsed field gel electrophoresis (PFGE). The isolates were further tested for the presence of ermF, tetQ and bft genes by PCR. Our results indicated the presence of different clonal strains (1 common type and 57 single types) among the resistant isolates. The resistance rate for the six antibiotics in this study was between 1% and 95%. Most of the isolates (99%) were susceptible to metronidazole. ermF and tetQ were detected in all erythromycin and tetracycline resistant isolates. None of the isolates were carried bft gene. These data suggest dissemination of heterogenic clonal groups in healthy persons and resistance to 5 high commonly used antibiotics.