A new subtype of the metalloprotease toxin gene and the incidence of the three bft subtypes among Bacteroides fragilis isolates in Japan

The bft gene encoding Bacteroides fragilis toxin (BFT) has been devided into two subtypes, bft-1 and bft-2. We found a novel subtype by sequencing a segment of the bft gene from 64 enterotoxigenic B. fragilis (ETBF) strains isolated in Japan. The 1548-bp nucleotide sequences of the new bft, the bft-1, and bft-2 genes were determined for five, four, and four ETBF strains, respectively; the nucleotide sequence was identical among each bft subtype and the degree of identity between each subtype was between 89 and 94%. Most of the variations between the three subtypes were detected in the region encoding mature toxin. A multiplex PCR was developed with a four-primer mix to subtype the bft sequences. The subtyping of 143 ETBF isolates from extraintestinal and stool specimens of humans and cows showed that the bft-1 was the most prevalent subtype, followed by bft-2 and a new bft subtype. No other subtype was found among the strains tested.     

Diarrhea caused by enterotoxigenic Bacteroides fragilis in children less than 5 years of age in Hanoi, Vietnam

Enterotoxigenic Bacteroides fragilis (ETBF) are considered as an emerging enteropathogen causing diarrhea in children. Eight hundred and thirty-six (836) children less than 5 years of age including 587 children with diarrhea and 249 age-matched controls were involved in the study. Within the group of children with diarrhea, 7.3% (43/587) ETBF was detected by immunoseparation in combination with polymerase chain reaction. The corresponding figure for the controls was 2.4% (6/249) (P<0.01). Within the diarrhea group, the prevalence was significantly higher in children older than 1 year of age. Three subtypes of ETBF isolates have been identified with the prevalence of 67.4%, 18.6%, and 16% for bft-1, bft-2, and a new bft, respectively. In the controls, two of the subtypes were identified, 5 bft-1 and 1 bft-2. More than half (55.8%) of the samples harboring ETBF also had other identified pathogens. The clinical symptoms of the single ETBF infection were not different from those of co-infections. This is the first study of the role of ETBF in children's diarrhea in Vietnam and it is concluded that this pathogen is an important causative agent of diarrhea in children in Hanoi, Vietnam.     

The distribution of the bft alleles among enterotoxigenic Bacteroides fragilis strains from stool specimens and extraintestinal sites

Enterotoxigenic Bacteroides fragilis (ETBF) has been implicated in diarrhoeal illness in animals and humans. Recent data suggest that ETBF is associated with flares of inflammatory bowel disease. Toxigenicity is attributed to expression of a toxin referred to as fragilysin, which stimulates fluid accumulation in ligated intestinal segments and alter the morphology of human intestinal cells. Three different isoforms or variants of the enterotoxin gene, designated bft-1, bft-2, and bft-3, have been identified. In this study we investigated the distribution of bft alleles among ETBF strains in stool specimens from patients with colon cancer (n: 31), the control patients (n: 8) and extraintestinal sources (n: 15). We used restriction fragment length polymorphism analysis of the PCR-amplified enterotoxin gene and sequencing the PCR-product to detect the isoforms of bft gene. Among the stool strains, bft-1 was found to be more common than bft-2; as it was detected 27 of 31 strains from colon cancer patients and 7 of 8 control strains. The bft-1 isoform was also found in almost all isolates from extraintestinal sites. No bft-3 subtype was detected among all tested strains.     

Evaluation of the prevalence of enterotoxigenic Bacteroides fragilis and the distribution bft gene subtypes in patients with diarrhea

The aim of this study was to determine the prevalence of enterotoxigenic Bacteroides fragilis (ETBF) in the patients with diarrhea in our region and to assess the association between diarrhea and bft gene subtypes. The presence of ETBF and bft gene subtypes were investigated in 200 stool samples from patients with diarrhea, diagnosed as gastroenteritis, which were sent to Clinical Microbiology Laboratory at Zonguldak Karaelmas University, Training and Research Hospital and in 200 stool samples from age-matched healthy subjects between April 14, 2009 and October 28, 2009. Nested – polymerase chain reaction was used to detect the presence of bft gene directly from stool samples. The bft gene subtypes were determined by PCR in case of ETBF detection. The presence of bft gene was detected in 29 (15%) of patients and 27 (14%) of control group. bft-1 and bft-2 were found in 24 and five stool samples from 29 diarrheic patients with ETBF, respectively. Among 27 control patients with ETBF, bft-1 and bft-2 were found in 24 and three samples, respectively. No bft-3 subtypes were identified in our study. ETBF was found as a single pathogen in 9% of the patients with diarrhea, while there was an accompanying pathogen in 6% of the patients. The proportion of coinfection with another pathogen among ETBF positive patients was 38%. Cooccurance with ETBF was present in nine of 18 patients with Rotavirus and two of five patients with Entamoeba histolytica. In conclusion; there was no statistically significant difference between the prevalence of ETBF in diarrheal patients and that of the control group. When the patients and controls were compared for each age group, no statistically significant difference in ETBF rates was found. There was no significant difference between groups with respect to bft subtypes; bft-1 was identified as the most common subtype. The rate of coinfection of ETBF and Rotavirus was high.     

Case-Control Study on the Role of Enterotoxigenic Bacteroides fragilis as a Cause of Diarrhea among Children in Kolkata,India

A total of 874 fecal specimens (446 diarrheal cases and 428 controls) from diarrheal children admitted in the Infectious Diseases Hospital, Kolkata and age and sex matched asymptomatic subjects from an urban community were assessed for the prevalence of enterotoxigenic Bacteroides fragilis (ETBF). Isolates of B. fragilis were tested for the presence of enterotoxin gene (bft) by PCR. The detection rate of ETBF was 7.2% (63 of 874 specimens) that prevailed equally in diarrheal cases and controls (7.2% each; 32 of 446 cases and 31 of 428 controls). Male children up to one year age group was significantly (p<0.05) associated with ETBF infection as compared to children > 2 years of age in cases and controls. In 25 ETBF isolates, the bft gene was genotyped using PCR-RFLP and only two alleles were identified with prevalence rate of 40% and 60% for bft-1 and bft-3, respectively. All the ETBF isolates were susceptible for chloramphenicol and imipenem but resistant to clindamycin (48%), moxifloxacin (44%) and metronidazole (32%). Resistance of ETBF to moxifloxacin (44%) and metronidazole is an emerging trend. Pulsed-field gel electrophoresis (PFGE) revealed that majority of the ETBF isolates are genetically diverse. In the dendrogram analysis, two clusters were identified, one with ETBF resistant to 5–8 antimicrobials and the other cluster with metronidazole and moxifloxacin susceptible isolates from diarrheal cases. To our knowledge, this is the first detailed report on ETBF from India indicating its clinical importance and molecular characteristics.     

Occurrence of enterotoxigenic and nonenterotoxigenic Bacteroides fragilis in calves and evaluation of their antimicrobial susceptibility

Bacteroides fragilis is considered an important clinical pathogen and the most common anaerobe isolated from human and animal clinical specimens; enterotoxigenic strains produce diarrhea. The presence of enterotoxigenic (ETBF) and nonenterotoxigenic B. fragilis in stool samples from calves with or without acute diarrhea and the antimicrobial susceptibility of the strains were evaluated. The stool samples were plated onto a selective B. fragilis-bile-esculin agar, and incubated anaerobically (10% CO(2)/90% N(2)), at 37 degrees C, for 72 h. Species of the B. fragilis group were identified by using the API 32-A kit. Enterotoxigenic strains were detected by PCR and the cytotoxic assay. From 54 diarrhea and 54 nondiarrhea stools, 124 and 92 members of the B. fragilis group, respectively, were recovered. Only two ETBF strains were isolated from two different diarrhea samples and the bft gene was detected in both. Moreover, the bft gene was detected in DNA from four different diarrheal stools samples but no ETBF strain was recovered. All the bacteria were susceptible to chloramphenicol, imipenem, moxifloxacin, piperacillin/tazobactam, metronidazole and tigecycline. Most of the isolates from both calves with and without diarrhea were resistant to all metals. Our results are of concern, and suggest the need to increase the surveillance of antibiotic and metal resistance of this microbial group isolated from animal production such as calves.     

Clostridium difficile and enterotoxigenic Bacteroides fragilis strains isolated from patients with antibiotic associated diarrhoea

From the fecal samples of 332 patients with a clinical diagnosis of antibiotic associated diarrhoea (AAD), 131 Clostridium difficile strains were isolated. For detection of toxin A in the isolated strains the enzymatic immunoassay was used. The cytopathic effect was determined on McCoy cell line. PCR was used for the detection of non-repeating and repeating sequences of toxin A gene and non-repeating sequences of toxin B gene. One hundred and six isolated C. difficile strains were TcdA(+)TcdB(+), 10 strains TcdA(-)TcB(+) and 15 were non-toxigenic TcdA(-)TcdB(-). Out of the same fecal samples 50 Bacteroides fragilis strains were isolated. All B. fragilis strains were tested in PCR reaction for fragilysine gene detection (bft). In 9 strains (18%) this gene was detected and the strains could be assumed as enterotoxigenic Bacteroides fragilis (ETBF). In 4 fecal samples toxigenic C. difficile (TcdA(+)TcdB(+)) was found simultaneously with ETBF. One sample contained C. difficile (TcdA(-)TcdB(+)) and ETBF. Out of 4 fecal samples only ETBF was isolated. The cytotoxicity of ETBF strains was tested on HT29/C1 human colon carcinoma cell line. The cytotoxicity titer in the range of 20 and 80 was observed.     

Prevalence of enterotoxigenic Bacteroides fragilis in patients with diarrhea: a controlled study

In this age matched controlled study performed in Malatya, a city in east region of Turkey, enterotoxigenic Bacteroides fragilis (ETBF) was investigated in stool specimens obtained from children and adults with and without diarrhea. A nested polymerase chain reaction (PCR) method was used to detect the enterotoxin gene of B. fragilis in a total of 418 stool samples, including 221 samples from 117 children (aged 0-16 years) and 104 adults (aged >16 years) with diarrhea, and 197 samples from 102 children and 95 adults as control group that was the same age group with those having diarrhea. ETBF was detected in 13 of 117 diarrheal children (11.1%) and 8 of 102 control children (7.8%) (P>0.05). In children aged 1-5 years, the rate of ETBF was significantly higher in patients than in controls (25% versus 9.5%, respectively; P<0.05). On the other hand ETBF was detected similar rates (2.2% and 2.4%, respectively) in children younger than 1 year in both patients and controls. ETBF positivity was not significantly difference between patient and control groups who were older than 5 years of age and adults. The frequency of ETBF in the controls was slightly higher in older persons than in younger ones; however, it was not significant. The rate of ETBF as the only enteropathogen in the patients with ETBF was significantly higher than in controls with ETBF (88% versus 39%, respectively; P<0.02). We found that in east region of Turkey, the prevalence of ETBF was higher in the childhood diarrhea, particularly in aged 1-5. As the only enteropathogen, ETBF may play an important role in diarrheal diseases. Persons after 6 years old can be carrier for ETBF regardless diarrhea.     

Characterization of the Bacteroides fragilis pathogenicity island in human blood culture isolates

Bacteroides fragilis is an important anaerobic pathogen accounting for up to 10% of bacteremias in adult patients. Enterotoxin producing B. fragilis (ETBF) strains have been identified as enteric pathogens of children and adults. In order to further characterize the B. fragilis pathogenicity island (BfPAI) and using PCR assays for bft- and mpII-metalloprotease genes, we determined the frequency of B. fragilis strains with pattern I (containing the BfPAI and its flanking region), pattern II (lacking both the BfPAI and the flanking region), and pattern III (lacking the BfPAI but containing the flanking region) in 63 blood culture isolates. The results were compared to 197 B. fragilis isolates from different clinical sources. We found 19% of blood culture isolates were pattern I (ETBF), 43% were pattern II (NTBF) and 38% were pattern III (NTBF). Comparatively, B. fragilis isolates from other clinical sources were 10% pattern I, 47% pattern II and 43% pattern III. This suggests that the pathogenicity island and the flanking elements may be general virulence factors of B. fragilis.     

Molecular Evolution of the Pathogenicity Island of Enterotoxigenic Bacteroides fragilis Strains

Enterotoxigenic Bacteroides fragilis (ETBF) strains, which produce a 20-kDa zinc metalloprotease toxin (BFT), have been associated with diarrheal disease in animals and young children. Studying a collection of ETBF and nontoxigenic B. fragilis (NTBF) strains, we found that bft and a second metalloprotease gene (mpII) are contained in an approximately 6-kb pathogenicity island (termed B. fragilis pathogenicity island or BfPAI) which is present exclusively in all 113 ETBF strains tested (pattern I). Of 191 NTBF strains, 100 (52%) lack both the BfPAI and at least a 12-kb region flanking BfPAI (pattern II), and 82 of 191 NTBF strains (43%) lack the BfPAI but contain the flanking region (pattern III). The nucleotide sequence flanking the left end of the BfPAI revealed a region with the same organization as the mobilization region of the 5-nitroimidazole resistance plasmid pIP417 and the clindamycin resistance plasmid pBFTM10, that is, two mobilization genes (bfmA and bfmB) organized in one operon and a putative origin of transfer (oriT) located in a small, compact region. The region flanking the right end of the BfPAI contains a gene (bfmC) whose predicted protein shares significant identity to the TraD mobilization proteins encoded by plasmids F and R100 from Escherichia coli. Nucleotide sequence analysis of one NTBF pattern III strain (strain I-1345) revealed that bfmB and bfmC are adjacent to each other and separated by a 16-bp GC-rich sequence. Comparison of this sequence with the appropriate sequence of ETBF strain 86-5443-2-2 showed that in this ETBF strain the 16-bp sequence is replaced by the BfPAI. This result defined the BfPAI as being 6,036 bp in length and its precise integration site as being between the bfmB and bfmC stop codons. The G+C content of the BfPAI (35%) and the flanking DNA (47 to 50%) differ greatly from that reported for the B. fragilis chromosome (42%), suggesting that the BfPAI and its flanking region are two distinct genetic elements originating from very different organisms. ETBF strains may have evolved by horizontal transfer of these two genetic elements into a pattern II NTBF strain.     

Evaluation of the Pathogenicity of the Bacteroides fragilis Toxin Gene Subtypes in Gnotobiotic Mice

Enterotoxigenic Bacteroides fragilis (ETBF) strains produce a metalloprotease toxin (BFT) related to diarrheal disease in animals, young children, and adults. Three different isoforms of the enterotoxin, designated BFT-1, BFT-2, and BFT-3, have been identified and sequenced. In the present study, the pathogenicity of the ETBF strains carrying bft-1 or bft-2 was evaluated. Each toxin gene subtype of ETBF (bft-1 or bft-2) was intragastrically monoassociated to germ-free mice during 10 days and histopathological data from intestines and liver compared with those from mice monoassociated to a non-enterotoxigenic B. fragilis. Histopathological alterations were observed in all groups of animals related to ETBF. These alterations were characterized mainly by ulceration, edema, and inflammatory infiltration in intestine. However, these lesions were slightly more severe in mice monoassociated with bft-2 subtype. No alteration or lesion was observed in animals associated with the non-enterotoxigenic B. fragilis. In conclusion, strains harboring bft-1 or bft-2 gene subtypes were able to induce histopathological alterations in intestine of a gnotobiotic mice model and it could explain the effect produced for the enterotoxin.     

Prevalence and Antimicrobial Susceptibility of Enterotoxigenic Bacteroides fragilis in Children with Diarrhoea in Nicaragua

Enterotoxigenic Bacteroides fragilis (ETBF) strains have been reported as a cause of diarrhoeal diseases. Diarrhoea is an important cause of morbidity and mortality in children from developing countries. This study was conducted to determine the prevalence and antimicrobial susceptibility of ETBF in children from León, Nicaragua. Faecal specimens from 106 children under ten years of age with diarrhoea and 60 asymptomatic, age-matched controls were examined for presence of ETBF using an assay based on immunomagnetic separation (IMS) in combination with PCR (IMS-PCR) and HT29/C1 cell assay. ETBF was present in nine children with diarrhoea (8.4%) and was more often identified in children ≤1 year (7/63, 11.1%) but all ETBF positive children were under 2 years of age. ETBF was isolated as the only pathogen in five of nine positive children (55.5%). The agar dilution method was used to determine the antimicrobial susceptibility pattern of the ETBF strains. All strains were resistant to ampicillin (range 8–1024 mg/L) and one strain was also resistant to clindamycin MIC 256 mg/L. All the other antimicrobial agents were active against the strains (MIC₅₀and MIC₉₀): 8 and 16 mg/L for cefoxitin, 0.004 and 0.008 mg/L for imipenem, 0.5 and 0.5 mg/L for clindamycin and for metronidazole, 2 and 4 mg/L for chloramphenicol. A majority (77%) of the ETBF strains were β-lactamase producers.     

Prevalence of enterotoxigenic Bacteroides fragilis strains (ETBF) in the gut of children with clinical diagnosis of antibiotic associated diarrhoea (AAD)

Prevalence of enterotoxin producing Bacteroides fragilis (ETBF) strains in faecal samples of children with clinical diagnosis antibiotic associated diarrhoea (AAD) was investigated. Out of faecal samples collected from sixty children, thirty C. difficile strains were isolated. Enterotoxigenic B. fragilis (ETBF) strains were cultured from four children what made 6.7% of investigated faecal samples. Out of two samples toxinogenic C. difficile strains [tox A(+) tox B(+)] were cultured together with enterotoxinogenic B. fragilis. From sample of one child C. difficile A negative/B positive strains [tox A(-) tox B(+)] was found together with B. fragilis (ETBF). From faecal sample of one child enterotoxinogenic B. fragilis only was isolated. It was shown that in the gut of children with clinical diagnosis of (AAD) enterotoxinogenic B. fragilis (ETBF) can be present. B. fragilis (ETBF) can be observed in concomitance with toxinogenic C. difficile.     

The Bacteroides fragilis pathogenicity island is contained in a putative novel conjugative transposon

The genetic element flanking the Bacteroides fragilis pathogenicity island (BfPAI) in enterotoxigenic B. fragilis (ETBF) strain 86-5443-2-2 and a related genetic element in NCTC 9343 were characterized. The results suggested that these genetic elements are members of a new family of conjugative transposons (CTns) not described previously. These putative CTns, designated CTn86 and CTn9343 for ETBF 86-5443-2-2 and NCTC 9343, respectively, differ from previously described Bacteroides species CTns in a number of ways. These new transposons do not carry tetQ, and the excision from the chromosome to form a circular intermediate is not regulated by tetracycline; they are predicted to differ in their mechanism of transposition; and their sequences have very limited similarity with CTnDOT or other described CTns. CTn9343 is 64,229 bp in length, contains 61 potential open reading frames, and both ends contain IS21 transposases. Colony blot hybridization, PCR, and sequence analysis indicated that CTn86 has the same structure as CTn9343 except that CTn86 lacks a approximately 7-kb region containing truncated integrase (int2) and rteA genes and it contains the BfPAI integrated between the mob region and the bfmC gene. If these putative CTns were to be demonstrated to be transmissible, this would suggest that the bft gene can be transferred from ETBF to nontoxigenic B. fragilis strains by a mechanism similar to that for the spread of antibiotic resistance genes.     

Prevalence of the Bacteroides fragilis Group and Enterotoxigenic Bacteroides fragilis in Immunodeficient Children

Members of the Bacteroides fragilis group are indigenous to the human and animal intestinal microbiota and they are responsible for several endogenous infections. Enterotoxigenic B. fragilis (ETBF) has been associated with acute diarrhea in children and farm animals. Immunodeficient patients are more predisposed to different opportunistic infections, including anaerobic infections. In this study, 130 stool samples were analysed from 56 immunodeficient and 74 healthy children. Enterotoxin production was detected by cytotoxicity assay on HT-29 cells and by PCR. B. fragilis sensu strictu was prevalent in both groups and ETBF species was detected from a single stool sample belonged to an immunodeficient child with AIDS.     

Enteroaggregative and cell-detaching<Emphasis Type="Italic">Escherichia coli</Emphasis> strains among polish children with and without diarrhea

To determine the association of enteroaggregative (EAEC) and cell-detaching (CDEC)Escherichia coli with diarrhea of unknown origin among children from Wroc?aw (Poland),E. coli strains isolated from stool specimens of children with diarrhea were examined for mannose-resistant adherence to HEp-2 cells. EAEC were isolated from 10 of 39 (26%) children examined with diarrhea and 4 of 20 (20%) age-matched controls. CDEC were present in 14 (36%) cases of diarrhea and 7 (35%) healthy subjects. Cell-detaching activity was distinctly associated with hemolysin production. Among hemolytic CDEC strains cytotoxic necrotizing factor 1 (CNF1) synthesis prevailed among isolates obtained from cases of diarrhea (57%) in comparison with isolates obtained from healthy controls (14.3%). Although neither EAEC nor CDECE. coli strains were associated with diarrhea of children in this setting, there were differences among EAEC and CDEC strains isolated from children with and without diarrhea.     

Identification and characterization of conjugative transposons CTn86 and CTn9343 in Bacteroides fragilis strains

The related genetic elements flanking the Bacteroides fragilis pathogenicity island (PAI) in enterotoxigenic B. fragilis (ETBF) 86-5443-2-2 and also present in pattern III nontoxigenic B. fragilis (NTBF) NCTC 9343 were defined as putative conjugative transposons (CTns), designated CTn86 and CTn9343, respectively (A. A. Franco, J. Bacteriol. 181:6623-6633, 2004). CTn86 and CTn9343 have the same basic structures except that their encoded transposases have low similarity and CTn9343 lacks the B. fragilis PAI and contains an extra 7-kb region not present in CTn86. In this study, using DNA hybridization and PCR analysis, we characterized the genetic element flanking the PAI in a collection of ETBF strains and the related genetic elements in a collection of NTBF pattern III strains. We found that in all 123 ETBF strains, the PAI is contained in a genetic element similar to CTn86. Of 73 pattern III strains, 26 (36%) present a genetic element similar to CTn9343, 38 (52%) present a genetic element similar to CTn9343 but lack the 7-kb region that is also absent in CTn86 (CTn9343-like element), and 9 (12%) present a genetic element similar to CTn86 but lacking the PAI (CTn86-like element). In addition to containing CTn86, ETBF strains can also contain CTn9343, CTn9343-like, or CTn86-like elements. CTn86, CTn9343, CTn86-like, and CTn9343-like elements were found exclusively in B. fragilis strains and predominantly in division I, cepA-positive strains.     

Identification and Characterization of Conjugative Transposons CTn86 and CTn9343 in Bacteroides fragilis Strains

The related genetic elements flanking the Bacteroides fragilis pathogenicity island (PAI) in enterotoxigenic B. fragilis (ETBF) 86-5443-2-2 and also present in pattern III nontoxigenic B. fragilis (NTBF) NCTC 9343 were defined as putative conjugative transposons (CTns), designated CTn86 and CTn9343, respectively (A. A. Franco, J. Bacteriol. 181:6623-6633, 2004). CTn86 and CTn9343 have the same basic structures except that their encoded transposases have low similarity and CTn9343 lacks the B. fragilis PAI and contains an extra 7-kb region not present in CTn86. In this study, using DNA hybridization and PCR analysis, we characterized the genetic element flanking the PAI in a collection of ETBF strains and the related genetic elements in a collection of NTBF pattern III strains. We found that in all 123 ETBF strains, the PAI is contained in a genetic element similar to CTn86. Of 73 pattern III strains, 26 (36%) present a genetic element similar to CTn9343, 38 (52%) present a genetic element similar to CTn9343 but lack the 7-kb region that is also absent in CTn86 (CTn9343-like element), and 9 (12%) present a genetic element similar to CTn86 but lacking the PAI (CTn86-like element). In addition to containing CTn86, ETBF strains can also contain CTn9343, CTn9343-like, or CTn86-like elements. CTn86, CTn9343, CTn86-like, and CTn9343-like elements were found exclusively in B. fragilis strains and predominantly in division I, cepA-positive strains.     

Serotype Diversity of Astroviruses in Rawalpindi,Pakistan during 2009–2010

Astroviruses are globally known enteropathogens causing gastroenteritis and diarrhea, with eight well defined serotypes. Epidemiological studies have recognized serotype-1 as the most common subtype but no such data is available in Pakistan. During 2009–2010, we found astroviruses in 41 out of 535 (7%) samples collected from hospitalized children. Thirty one strains belonged to serotype-1 and clustered into two distinct lineages. Serotype-3, -4 and -6 were detected with 97–98% genetic homology to Indian and Chinese strains.