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Bacteroides spp. are opportunist pathogens that cause blood and soft tissue infections and are often resistant to antimicrobial agents. We have developed a combined PCR-restriction fragment length polymorphism (RFLP) technique to characterize the 16S rRNA gene for identification purposes and the nitroimidazole resistance (nim) gene for detection of resistance to the major antimicrobial agent used to treat Bacteroides infections: metronidazole (MTZ). PCR-RFLP analysis of 16S ribosomal (rDNA) with HpaII and TaqI produced profiles that enabled discrimination of type strains and identification of 70 test strains to the species level. The 16S rDNA PCR-RFLP identification results agreed with routine phenotypic testing for 62 of the strains. The discrepancies between phenotypic and PCR-RFLP methods for eight strains were resolved by 16S rDNA sequencing in three cases, but five strains remain unidentified. The presence of nim genes was indicated by PCR in 25 of 28 strains that exhibited reduced sensitivity to MTZ. PCR-RFLP of the nim gene products identified the four reported genes (nimA, -B, -C, and -D) and indicated the presence of a previously unreported nim gene in 5 strains. This novel nim gene exhibited 75% DNA sequence similarity with nimB. These rapid, accurate, and inexpensive methods should enable improved identification of Bacteroides spp. and the detection of MTZ resistance determinants.

We used an anaerobic infection model capable of simulating human serum pharmacokinetic parameters (C(max), C(min), half-life, and AUC) to compare the activity of oral immediate-release (IR) and extended-release (ER) MTZ formulations. Four oral regimens of MTZ plus a control regimen were simulated in the model: [i] (IR) 500 mg q8h, [ii] ER 750 mg q12h, [iii] ER 750 mg q24h, and [iv] ER 1500 mg q24h. Two clinical Bacteroides fragilis isolates (MICs 0.5, 4.0 microg/mL) and two non-fragilis Bacteroides isolates (MICs 0.5, 3 microg/mL) were studied. All four oral MTZ regimens exhibited rapid, bactericidal activity (> or =3-log(10) decrease from the starting inoculum) within 12 h against both fragilis and non-fragilis Bacteroides isolates. Overall, no appreciable difference in the rate of bacteria killing was noted among the four MTZ formulations against either B. fragilis isolates (P = 0.907, 0.737) or non-fragilis isolates (P = 0.809, 0.768). We conclude that ER MTZ dosed at 12 or 24-h intervals possesses equivalent bactericidal activity to standard IR MTZ dosed every eight hours against susceptible Bacteroides spp.

The high neuroactive potential of metabolites produced by gut microbes has gained traction over the last few years, with metagenomic-based studies suggesting an important role of microbiota-derived γ-aminobutyric acid (GABA) in modulating mental health. Emerging evidence has revealed the presence of the glutamate decarboxylase (GAD)-encoding gene, a key enzyme to produce GABA, in the prominent human intestinal genus Bacteroides. Here, we investigated GABA production by Bacteroides in culture and metabolic assays combined with comparative genomics and phylogenetics. A total of 961 Bacteroides genomes were analyzed in silico and 17 metabolically and genetically diverse human intestinal isolates representing 11 species were screened in vitro. Using the model organism Bacteroides thetaiotaomicron DSM 2079, we determined GABA production kinetics, its impact on milieu pH, and we assessed its role in mitigating acid-induced cellular damage. We showed that the GAD-system consists of at least four highly conserved genes encoding a GAD, a glutaminase, a glutamate/GABA antiporter, and a potassium channel. We demonstrated a high prevalence of the GAD-system among Bacteroides with 90% of all Bacteroides genomes (96% in human gut isolates only) harboring all genes of the GAD-system and 16 intestinal Bacteroides strains producing GABA in vitro (ranging from 0.09 to 60.84 mM). We identified glutamate and glutamine as precursors of GABA production, showed that the production is regulated by pH, and that the GAD-system acts as a protective mechanism against acid stress in Bacteroides , mitigating cell death and preserving metabolic activity. Our data also indicate that the GAD-system might represent the only amino acid-dependent acid tolerance system in Bacteroides. Altogether, our results suggest an important contribution of Bacteroides in the regulation of the GABAergic system in the human gut. [ABSTRACT FROM AUTHOR]

The binding of fibronectin an vitronectin to 207 Bacteroides strains and the binding of collagen and sialoproteins to 55 Bacteroides strains were investigated by means of latex agglutination tests. The binding of fibronectin, collagen and lactoferrin to the same 55 strains was also tested by using 125I-labelled proteins. The 207 strains, belonging to ten Bacteroides species, were isolated from different infections (51%) and from faeces of healthy subjects (49%). Most of the strains displaying fibronectin binding belonged in the species B. fragilis or B. vulgatus. The binding could be inhibited by preincubation of the cells with an excess amount of fibronectin. No inhibition of the binding was observed with carbohydrates. The vitronectin binding of the strains was less common, but was always observed to accompany fibronectin binding. None of the examined 55 strains exhibited any binding to fetuin or asialofetuin. The radiolabelling method indicated a low binding to 125I-fibronectin. The binding of 125I-collagen-I and 125I-lactoferrin in the Bacteroides strains tested was higher than that of 125I-fibronectin.

The nucleotide sequence of the DNA replication origin region of a Bacteroides vulgatus plasmid, pIP417, encoding 5-nitroimidazole resistance has been determined. This region of 1934 bp presents some characteristics similar to those of other replication protein-dependent origins. It contains a large open reading frame which could encode a basic Rep protein (RepA) of 36.8 kDa. Upstream of this ORF exist an AT-rich region, three direct repeats (iterons) of 21 bp, multiple DnaA binding sites, and sites, and sites for the integration host factor (IHF). Moreover, the amino acid sequence of the pIP417 RepA protein shows similarities with those of other Rep proteins encoded by plasmids of gram-negative bacteria: pRO1600 from Pseudomonas aeruginosa; pPS10 from Pseudomonas syringae; pFA3 from Neisseria gonorrhoeae; and two cryptic plasmids from Campylobacter hyointestinalis and Butyrivibrio fibrisolvens. Although RepA can be expressed in an Escherichia coli in vitro transcription-translation assay, vectors containing the pIP417 replication origin did not replicate in E. coli. The homology of the pIP417 replication region with the corresponding regions of other Bacteroides spp, plasmids was also studied by Southern blot hybridization. The results indicated that the repA gene of plasmid pIP417 is homologous to that of plasmid pIP421, but not of plasmid pIP419. The replication region of plasmid pIP421 was sequenced and showed about 80% identity at the nucleotide level with that of pIP417. A small (3634-bp) cloning vector (pFK12) of entirely defined nucleotide sequence was constructed for Bacteroides spp.

Metronidazole is metabolized to two major oxidative products: an acid metabolite and a hydroxy metabolite. While the activity of the acid metabolite is negligible, the activity of the hydroxy metabolite is approximately 65% of the activity of the parent drug. Pharmacokinetic studies of metronidazole and its hydroxy metabolite have shown that the MICs of both compounds remain above the MICs for most anaerobic organisms over an 8-h dosing interval. By a checkerboard assay, the combined activities of metronidazole and the hydroxy metabolite were examined against 4 quality control strains of Bacteroides species. Macrobroth tube dilutions were set up with Wilkins-Chalgren broth. Serial twofold dilutions of each agent were performed to achieve final concentrations ranging from 0.06 to 4.0 micrograms/ml. The MICs for Bacteroides fragilis and B. distasonis were 1.0 microgram/ml for both parent drug and metabolite. For B. thetaiotamicron and B. ovatus, the MICs of metronidazole and the hydroxy metabolite were 1.0 and 2.0 micrograms/ml, respectively. Synergy was determined by calculating the fractional inhibitory concentration (FIC) index. The interpretative criteria for the FIC index were as follows: synergy, FIC < or = 0.5; partial synergy, 0.51 to 0.75; indifference, FIC 0.76 to 4.0; and antagonism, FIC > 4.0. Partial synergy was observed for the four anaerobes tested, with FIC indices ranging from 0.63 to 0.75. On the basis of this data, in vitro susceptibilities to agents such as metronidazole may ultimately require reevaluation to account for active metabolites.

Lipopolysaccharide (LPS) extracts of reference strains and isolates of Bacteroides spp. prepared by the proteinase K method were resolved by tricine-sodium-dodecyl-sulphate-polyacrylamide gel electrophoresis and located by silver staining. A considerable diversity of LPS profiles was evident within the Bacteroides genus although profiles were essentially species-specific, with some minor interstrain variations apparent among isolates of B. uniformis, B. ovatus, B. eggerthii and B. thetaiotaomicron. The LPS of most species consisted of a major rough LPS component of 2-5 kDa and a series of higher molecular weight bands which varied with species. B. vulgatus LPS was distinctive in showing an extensive ladder of multiple repeating oligosaccharide units with molecular weights ranging from 4 to > 17 kDa B. stercoris LPS included a high molecular weight (> 17 kDa) ladder of repeating oligosaccharide units. B. fragilis and B. thetaiotaomicron differed from most other species in producing a short ladder of repeating oligosaccharide units interspersing the rough LPS and a 5.6 kDa (B. fragilis) or 9 kDa (B. thetaiotaomicron) yellow-staining component. The heterogeneity of LPS profiles within the Bacteroides genus may reflect the differences in pathogenicity among the species and prove useful for typing.

As part of a sustainable water resources management, the Lisbon municipality identified groundwater and treated wastewater use increase as two opportunities for better and sustainable water use, with natural safeguard for public health as a priority. In this context, the aim of our research was to assess the suitability of the human-associated marker gene Bacteroides HF183 and the cattle feces-associated CowM2, in routine water quality monitoring as indicators for water use and reuse, providing a tool to more accurately assess public health risks. To this intent, Real-Time quantitative PCR was used for detection of human-associated marker gene Bacteroides HF183 and the bovine-associated CowM2, in a total of 67 samples - groundwater and wastewater at three different treatment stages of a Waste Water Treatment Plant, in Lisbon. HF183 marker gene was detected in treated and untreated wastewater samples, with significant concentration reductions from untreated (6,07 E+07 copies/mL) to secondary treated effluent (1,86 E+05 copies/mL) and a further decrease in tertiary treatment (5,74 E+04 copies/mL). In groundwater samples, this marker was also detected in concentrations ranging from 2,63 E+02 copies/mL to 2,24 E+03 copies/mL. CowM2 marker gene on the other hand was only detected in wastewater samples, with concentrations ranging from 2,47 E+02 copies/mL to 1,17 E+04 copies/mL. Our research indicates that the use of Bacteroides spp. in association with traditional fecal indicator bacteria (FIB) is advantageous for water managing entities in urban settings, such as Lisbon, were drainage system failures may occur. An integrated approach thus provides crucial and more adequate information towards mitigation and correction measures when fecal contamination is detected in environmental waters. • Bacteroides were evaluated in an integrated hydric resources management approach. • Mutual detection of Bacteroides and FIB is advantageous for water managing entities. • HF183 marker was detected in groundwater, untreated and treated wastewater samples. • Identifying the origin of groundwater contamination allows correction measures. • HF183 marker may have a future role in evaluation of wastewater treatment. [ABSTRACT FROM AUTHOR]

Transmissible cefoxitin (FX) resistance in Bacteroides vulgatus CLA341 was associated with the 12.5-kb, mobilizable transposon, Tn4555, which encoded the beta-lactamase gene cfxA. Transfer occurred by a conjugation-like mechanism, was stimulated by growth of donor cells with tetracycline (TC), and required the presence of a Bacteroides chromosomal Tcr element. Transconjugants resistant to either FX, TC, or both drugs were obtained, but only Fxr Tcr isolates could act as donors of Fxr in subsequent matings. Transfer of Fxr could be restored in Fxr Tcs strains by the introduction of a conjugal Tcr element from Bacteroides fragilis V479-1. A covalently closed circular DNA form of Tn4555 was observed in donor cells by Southern hybridization, and the levels of this circular transposon increased significantly in cells grown with TC. Both the cfxA gene and the Tn4555 mobilization region hybridized to the circular DNA, suggesting that this was a structurally intact transposon unit. Circular transposon DNA purified by CsCl-ethidium bromide density gradient centrifugation was used to transform Tcs B. fragilis 638, and Fxr transformants were obtained. Both the circular form and the integrated Tn4555 were observed in transformants, but the circular form was present at less than one copy per chromosomal equivalent. Examination of genomic DNA from Fxr transformants and transconjugants revealed that Tn4555 could insert at a wide variety of chromosomal sites. Multiple transposon insertions were present in many of the transconjugants, indicating that there was no specific barrier to the introduction of a second transposon copy.

Gut microbiota are significantly associated with the occurrence and development of inflammatory bowel disease (IBD). Panax notoginseng saponins (PNS) could be used for colitis and to modulate gut microbiota. However, the mechanism behind the effects of PNS on anti-colitis that are pertinent to gut microbiota is largely unknown. This study aimed to evaluate the anti-colitis effects of PNS and explore the involved mechanism as it is related to gut microbiota. Results showed that PNS significantly alleviated dextran sulfate sodium (DSS)-induced colitis. Meanwhile, after PNS treatment, the tight junction proteins were enhanced and proinflammatory cytokines, such as TNF- α , IL-6, IL-1 β , and IL-17, were decreased. Furthermore, Bacteroides spp. were significantly increased after modeling, while PNS reduced their abundance and significantly increased the amount of Akkermansia spp. in vivo. Importantly, Akkermansia spp. and Bacteroides spp. were correlated with the IBD disease indicators. Moreover, fecal microbiota transplantation (FMT) experiments confirmed that PNS-reshaped gut microbiota significantly alleviated DSS-induced colitis, while A. muciniphila significantly reduced the levels of the LPS-induced cellular inflammatory factors IL-1 β and TNF- α. In conclusion, PNS alleviated colitis pertinent to the upregulation of Akkermania spp. and downregulation of Bacteroides spp. in the gut. [ABSTRACT FROM AUTHOR]

During the years 1984 through 1987 2,574 isolates of obligately anaerobic bacteria were isolated from samples submitted for analysis. The most common anaerobic isolates were members of the genus Bacteroides, representing 44.6% of the isolates. Of these, the most commonly isolated identifiable microorganisms were bile-resistant and nonpigmented, belonging to the B fragilis group of Bacteroides. Importantly, obvious predilections for any one species or group of Bacteroides were not apparent for animal or site (condition), except that the proportion of isolates belonging to the nonpigmented, bile-resistant group obtained from the respiratory tract was significantly (P less than 0.005) higher than that not belonging to this group. On the other hand, the proportion of isolates of the non-pigmented, bile-resistant group obtained from abscesses was significantly lower than that not belonging to this group.

The quantitative analysis of the incidence of black-pigmented Bacteroides (B.P.B.) spp. in 80 human dental root canal infections (56 with acute symptoms and 24 clinically asymptomatic) in 79 adults were studied. Altogether 101 strains including 7 species of B. P. B. were identified. It was found that the infection rate of B. P. B. in symptomatic group (192.86%) was higher than that in asymptomatic group (41.67%). The means of quantity of cultivable B. P. B. (CFU/ml) and percentage of B.P.B. (CFU/ml) in total CFU/ml of bacteria were not significant. But the percentage of asacchrolytic B.P.B. species in B.P.B. positive samples in symptomatic group (73.08%) was higher than that in B.P.B. positive samples in asymptomatic group (40%). These results suggest that there is a close correlation between symptoms and the asacchrolytic species of B. endodontalis and B. gingivalis.

Transformation of Bacteroides spp. with a variety of plasmid DNAs was accomplished using electroporation. The standard transformation assay system used to deduce the optimal electroporation parameters employed a 50-to 100-fold concentrated cell suspension of mid-logarithmic phase Bacteroides fragilis strain 638 and the 5.4-kb clindamycin resistance (Ccr) vector, pBI191. A variety of electroporation buffers were used successfully in transformation experiments but of these, 1 mM MgCl2 in 10% glycerol was superior. The incorporation of MgCl2 was essential for optimum viability prior to electroporation and for optimum transformation. Transformants were routinely obtained using 5-ms pulses over a range of field strengths from 5 to 12.5 kV/cm, with a maximum of greater than 10(6) micrograms-1 DNA at 12.5 kV/cm. The number of transformants increased linearly with respect to DNA concentration over the range 0.01-2 micrograms tested. Recovery of transformants required an expression period of up to 2.5 h following exposure to the electric field. This period, however, was dependent on the antibiotic resistance marker used for selection of transformants, with a significantly shorter incubation required when chloramphenicol rather than clindamycin was used in the selective medium. The effect of the DNA source on transformation was tested using the shuttle vector pFD288. Plasmid DNA isolated from Bacteroides uniformis, Bacteroides ovatus, or Bacteroides thetaiotaomicron transformed B. fragilis 638 at frequencies 7.5- to 12.5-fold less than those observed for controls with homologous DNA. Further reductions were seen with Escherichia coli purified pFD288, which transformed at 1000-fold lower frequencies. Finally, using homologous pFD288 or pBI191 isolated from strain 638, several strains of B. fragilis, B. uniformis, and B. ovatus were transformed successfully without modification of the standard assay system. Two strains each of B. thetaiotaomicron and Bacteroides ruminicola were not transformed using the methods described here.

The influences of growth conditions and cold storage on the specific activity of beta-lactamases of four strains of Bacteroides spp. was studied. Interbatch variation was observed in extracts prepared in an identical way on separate occasions but less variation was observed in extracts prepared from bacteria grown on Brain Heart Infusion agar supplemented with yeast extract, haemin and menadione, than in similar extracts of bacteria grown in broth or on other solid media. The loss of enzyme activity seen during the stationary phase of growth of some strains in broth was minimal during incubation for 48 h on agar. Storage of enzyme extracts at 4 degrees C was associated with loss of enzyme activity, but activity was retained during storage at -70 degrees C for up to 32 days. Freezing and thawing had little effect on enzyme activity.

Oral, black-pigmented Bacteroides spp. are important pathogens in oral anaerobic infections and dental disease. We detected conjugation systems in isolates of Bacteroides denticola and Bacteroides intermedius that transferred tetracycline resistance (Tetr) and penicillin resistance to Bacteroides buccae and to Bacteroides fragilis, an intestinal Bacteroides species. A cloned Tetr gene from B. fragilis hybridized to the transferable Tetr locus in the oral strains, indicating that genetic exchange occurs between these two groups of anaerobes.

Multivariate step-wise regression analyses demonstrated that in a population of 146 elderly dental patients (mean age 69.8 yr) the salivary level of mutans streptococci was unrelated to the root DFS score, unlike the levels of lactobacilli and yeasts. Mutans streptococci were related to the wearing of partial dentures and the number of snacks eaten per day, while salivary lactobacillus levels were also related to the number of snacks eaten per day and to denture wearing, and negatively related to saliva buffering. Salivary yeast levels were also related to the wearing of partial dentures and the number of snacks eaten per day, and negatively related to salivary neuraminidase activity. The R2 values ranged from 0.21 to 0.28. Salivary level of black-pigmented Bacteroides spp. were related to maximum CPITN score, mean bleeding index and salivary trypsin-like activity (R2 was 0.08).