Preliminary Safety Evaluation of a New Bacteroides xylanisolvens Isolate

ABSTRACT Besides conferring some health benefit to the host, a bacterial strain must present an unambiguous safety status to be considered a probiotic. We here present the preliminary safety evaluation of a new Bacteroides xylanisolvens strain (DSM 23964) isolated from human feces. First results suggest that it may be able to provide probiotic health benefits. Its identity was confirmed by biochemical analysis, by sequencing of its 16S rRNA genes, and by DNA-DNA hybridization. Virulence determinants known to occur in the genus Bacteroides, such the bft enterotoxin and other enzymatic activities involved in the degradation of the extracellular matrix and the capsular polysaccharide PS A, were absent in this strain. The investigation of the antibiotic susceptibility indicated that strain DSM 23964 was sensitive to metronidazole, meropenem agents, and clindamycin. Resistance to penicillin and ampicillin was identified to be conferred by the β-lactamase cepA gene and could therefore be restored by adding β-lactamase inhibitors. The localization of the cepA gene in the genome of strain DSM 23964 and the absence of detectable plasmids further suggest that a transfer of β-lactamase activity or the acquisition of other antibiotic resistances are highly improbable. Taken together, the presented data indicate that the strain B. xylanisolvens DSM 23964 has no virulence potential. Since it also resists the action of gastric enzymes and low-pH conditions, this strain is an interesting candidate for further investigation of its safety and potential health-promoting properties.

[1]  H. Birnboim,et al.  A rapid alkaline extraction procedure for screening recombinant plasmid DNA. , 1979, Nucleic acids research.

[2]  M. Sakamoto,et al.  Bacteroides finegoldii sp. nov., isolated from human faeces. , 2006, International journal of systematic and evolutionary microbiology.

[3]  M. Nei,et al.  MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. , 2007, Molecular biology and evolution.

[4]  J. Tagg,et al.  Detection and Specific Enumeration of Multi-Strain Probiotics in the Lumen Contents and Mucus Layers of the Rat Intestine After Oral Administration , 2009, Probiotics and antimicrobial proteins.

[5]  D. Kasper,et al.  Polysaccharide Biosynthesis Locus Required for Virulence of Bacteroides fragilis , 2001, Infection and Immunity.

[6]  A. Schauss,et al.  Safety assessment of a proprietary preparation of a novel Probiotic, Bacillus coagulans, as a food ingredient , 2009, Food and chemical toxicology : an international journal published for the British Industrial Biological Research Association.

[7]  Jeffrey I. Gordon,et al.  Angiogenins: a new class of microbicidal proteins involved in innate immunity , 2003, Nature Immunology.

[8]  Hannah M. Wexler,et al.  Bacteroides: the Good, the Bad, and the Nitty-Gritty , 2007, Clinical Microbiology Reviews.

[9]  S. Akimoto,et al.  Detection of Bacteroides fragilis by PCR assay targeting the neuraminidase‐encoding gene , 1996, Letters in applied microbiology.

[10]  T. Riley Agar dilution susceptibility of Bacteroides spp. to sulfamethoxazole and trimethoprim: correlation with a disk diffusion technique , 1981, Antimicrobial Agents and Chemotherapy.

[11]  M. Jacobs,et al.  Beta-lactamase production and susceptibility of US and European anaerobic gram-negative bacilli to beta-lactams and other agents , 1992, European Journal of Clinical Microbiology and Infectious Diseases.

[12]  R. Porschen,et al.  Extracellular deoxyribonuclease production by anaerobic bacteria. , 1974, Applied microbiology.

[13]  R. Aminov,et al.  16S/18S ribosomal DNA clone library analysis of rumen microbial diversity , 2005 .

[14]  B. Evers,et al.  Caco-2 cell differentiation is associated with a decrease in stat protein levels and binding , 1999, Journal of Gastrointestinal Surgery.

[15]  L. Comstock Importance of glycans to the host-bacteroides mutualism in the mammalian intestine. , 2009, Cell host & microbe.

[16]  R. Tulley,et al.  Dietary resistant starch upregulates total GLP-1 and PYY in a sustained day-long manner through fermentation in rodents. , 2008, American journal of physiology. Endocrinology and metabolism.

[17]  G. Gottschalk,et al.  Pathway of Succinate and Propionate Formation in Bacteroides fragilis , 1978, Journal of bacteriology.

[18]  M. J. Ávila-Campos,et al.  Virulence markers and antimicrobial susceptibility of bacteria of the Bacteroides fragilis group isolated from stool of children with diarrhea in São Paulo, Brazil. , 2004, Memorias do Instituto Oswaldo Cruz.

[19]  Wen-Tso Liu,et al.  Relative Abundance of Bacteroides spp. in Stools and Wastewaters as Determined by Hierarchical Oligonucleotide Primer Extension , 2008, Applied and Environmental Microbiology.

[20]  R. Jones,et al.  The E-Test applied to susceptibility tests of gonococci, multiply-resistant enterococci, and Enterobacteriaceae producing potent beta-lactamases. , 1992, Diagnostic microbiology and infectious disease.

[21]  M. Sakamoto,et al.  Bacteroides dorei sp. nov., isolated from human faeces. , 2006, International journal of systematic and evolutionary microbiology.

[22]  E. A. Ahmed Effect of Encapsulation on some Probiotic Criteria , 2010 .

[23]  W. Rudek,et al.  Extracellular enzymes of the genus Bacteroides , 1976, Journal of clinical microbiology.

[24]  K. Itoh,et al.  Bacteroides Induce Higher IgA Production Than Lactobacillus by Increasing Activation-Induced Cytidine Deaminase Expression in B Cells in Murine Peyer’s Patches , 2009, Bioscience, biotechnology, and biochemistry.

[25]  G. Tsujimoto,et al.  Sucrase-Isomaltase Gene Expression Is Inhibited by Mutant Hepatocyte Nuclear Factor (HNF)-1α and Mutant HNF-1β in Caco-2 Cells , 2006 .

[26]  D. Kasper,et al.  Structural features of polysaccharides that induce intra-abdominal abscesses. , 1993, Science.

[27]  S. Targan,et al.  Molecular cloning of a Bacteroides caccae TonB-linked outer membrane protein identified by an inflammatory bowel disease marker antibody. , 2001, Infection and immunity.

[28]  Thomas L. Madden,et al.  Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. , 1997, Nucleic acids research.

[29]  E. R. Rocha,et al.  Characterization of Bacteroides fragilis Hemolysins and Regulation and Synergistic Interactions of HlyA and HlyB , 2006, Infection and Immunity.

[30]  J. Chun,et al.  EzTaxon: a web-based tool for the identification of prokaryotes based on 16S ribosomal RNA gene sequences. , 2007, International journal of systematic and evolutionary microbiology.

[31]  T. Maier,et al.  Species identification of clinical isolates of Bacteroides by matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry. , 2009, Clinical microbiology and infection : the official publication of the European Society of Clinical Microbiology and Infectious Diseases.

[32]  C. Sears,et al.  Bacteroides spp. and diarrhea , 2010, Current opinion in infectious diseases.

[33]  R. Domingues,et al.  Association Between the cfxA Gene and Transposon Tn4555 in Bacteroides distasonis Strains and Other Bacteroides Species , 2007, Current Microbiology.

[34]  P. Lawson,et al.  Bacteroides xylanisolvens sp. nov., a xylan-degrading bacterium isolated from human faeces. , 2008, International journal of systematic and evolutionary microbiology.

[35]  D. Hicks,et al.  Apoptosis in colorectal tumour cells: Induction by the short chain fatty acids butyrate, propionate and acetate and by the bile salt deoxycholate , 1995, International journal of cancer.

[36]  S. Mazmanian,et al.  Inducible Foxp3+ regulatory T-cell development by a commensal bacterium of the intestinal microbiota , 2010, Proceedings of the National Academy of Sciences.

[37]  M. Leclerc,et al.  Bacteroides sp. Strain D8, the First Cholesterol-Reducing Bacterium Isolated from Human Feces , 2007, Applied and Environmental Microbiology.

[38]  N. Saitou,et al.  The neighbor-joining method: a new method for reconstructing phylogenetic trees. , 1987, Molecular biology and evolution.

[39]  M. Adams,et al.  An in vitro model for investigating intestinal adhesion of potential dairy propionibacteria probiotic strains using cell line C2BBe1 , 2003, Letters in applied microbiology.

[40]  W. Verstraete,et al.  Propionate as a health-promoting microbial metabolite in the human gut. , 2011, Nutrition reviews.

[41]  S. Pignata,et al.  The enterocyte-like differentiation of the Caco-2 tumor cell line strongly correlates with responsiveness to cAMP and activation of kinase A pathway. , 1994, Cell growth & differentiation : the molecular biology journal of the American Association for Cancer Research.

[42]  Lukas Wagner,et al.  A Greedy Algorithm for Aligning DNA Sequences , 2000, J. Comput. Biol..

[43]  S. Finegold,et al.  Susceptibility of Anaerobic Bacteria to 23 Antimicrobial Agents , 1976, Antimicrobial Agents and Chemotherapy.

[44]  -. Steffen,et al.  Hydrolytic enzymes of anaerobic bacteria isolated from human infections , 1981, Journal of clinical microbiology.

[45]  J. Heimbach,et al.  Safety assessment of probiotics for human use , 2010, Gut microbes.

[46]  D. Kasper,et al.  Beneficial effects of Bacteroides fragilis polysaccharides on the immune system. , 2010, Frontiers in bioscience.

[47]  R. F. Smith,et al.  Rapid plate method for screening hyaluronidase and chondroitin sulfatase-producing microorganisms. , 1968, Applied microbiology.

[48]  D. Kasper,et al.  Novel tools for modulating immune responses in the host-polysaccharides from the capsule of commensal bacteria. , 2010, Advances in immunology.

[49]  S. Finegold,et al.  Rapid identification of the species of the Bacteroides fragilis group by multiplex PCR assays using group- and species-specific primers. , 2003, FEMS microbiology letters.

[50]  W. P. Reed,et al.  Adherence of group A streptococci to pharyngeal cells: a role in the pathogenesis of rheumatic fever. , 1978, Science.

[51]  K. T. Holland,et al.  Xylan-regulated delivery of human keratinocyte growth factor-2 to the inflamed colon by the human anaerobic commensal bacterium Bacteroides ovatus , 2009, Gut.

[52]  M. Gutacker,et al.  Identification of two genetic groups in Bacteroides fragilis by multilocus enzyme electrophoresis: distribution of antibiotic resistance (cfiA, cepA) and enterotoxin (bft) encoding genes. , 2000, Microbiology.

[53]  K. Itoh,et al.  Absence of Cecal Secondary Bile Acids in Gnotobiotic Mice Associated with Two Human Intestinal Bacteria with the Ability to Dehydroxylate Bile Acids In Vitro , 1999, Microbiology and immunology.

[54]  A. Quesada,et al.  Genetic determinants for cfxA expression in Bacteroides strains isolated from human infections. , 2008, The Journal of antimicrobial chemotherapy.