Search

Microbial communities play a significant role in maintaining ecosystems in a healthy homeostasis. Presently, in the human gastrointestinal tract, there are certain taxonomic groups of importance, though there is no single species that plays a keystone role. Bacteroides spp. are known to be major players in the maintenance of eubiosis in the human gastrointestinal tract. Here we review the critical role that Bacteroides play in the human gut, their potential pathogenic role outside of the gut, and their various methods of adapting to the environment, with a focus on data for B. fragilis and B. thetaiotaomicron. Bacteroides are anaerobic non-sporing Gram negative organisms that are also resistant to bile acids, generally thriving in the gut and having a beneficial relationship with the host. While they are generally commensal organisms, some Bacteroides spp. can be opportunistic pathogens in scenarios of GI disease, trauma, cancer, or GI surgery, and cause infection, most commonly intra-abdominal infection. B. fragilis can develop antimicrobial resistance through multiple mechanisms in large part due to its plasticity and fluid genome. Bacteroidota (formerly, Bacteroidetes) have a very broad metabolic potential in the GI microbiota and can rapidly adapt their carbohydrate metabolism to the available nutrients. Gastrointestinal Bacteroidota species produce short-chain fatty acids such as succinate, acetate, butyrate, and occasionally propionate, as the major end-products, which have wide-ranging and many beneficial influences on the host. Bacteroidota, via bile acid metabolism, also play a role in in colonization-resistance of other organisms, including Clostridioides difficile, and maintenance of gut integrity., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Hannah Wexler reports no conflict of interest. Cirle Warren: Medical Advisory Board for Seres Therapeutics and site PI for Ferring Pharmaceuticals Inc. Jae Shin: site co-investigator for Ferring Pharmaceuticals Inc. Tiffany MacKenzie is an employee of Ferring Pharmaceuticals Inc. Glenn Tillotson is a consultant to Ferring Pharmaceuticals Inc., Spero Therapeutics and Dynavax. Ellie Goldstein reports no financial conflict of interest. He is a prior Associate Editor at Anaerobe., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Essential genes are those that are indispensable for the survival of organism under specific growth conditions. Investigating essential genes in pathogenic bacteria not only helps to understand vital biological networks but also provides novel targets for drug development. Availability of genetic engineering tools and high-throughput sequencing methods has enabled essential genes identification in many pathogenic gram-positive and gram-negative bacteria. Bacteroides fragilis is one of the major bacteria specific of human gastrointestinal microbiota. When B. fragilis moves out of its niche, it turns into deadly pathogen. Here, we describe detailed method for the essential gene identification in B. fragilis. Generated transposon mutant pool can be used for other applications such as identification of genes responsible for drug resistance in B. fragilis., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

Bacteroides fragilis is Gram-negative obligatory anaerobe which usually resides in the gut of humans and animals. As an important member of the human gut microbiota it plays a vital role in digestion and absorption of nutrients as well as shaping of host immune system. B. fragilis is also infamous for causing serious infections. Treatment of B. fragilis infections caused emergence of multidrug-resistant strains. Molecular biology tools such as transposon mutagenesis help to decipher and understand commensal and pathogenic faces of B. fragilis. Using two mariner transposon vectors we describe the detailed methodology for the transposon mutagenesis of B. fragilis. We also describe two methods for the identification of transposon integration site (TIS) in transposon mutants. Transposon mutagenesis methods described in this chapter serve as a great tool for studying B. fragilis.

Background: While CRISPR-Cas systems have been identified in bacteria from a wide variety of ecological niches, there are no studies to describe CRISPR-Cas elements in Bacteroides species, the most prevalent anaerobic bacteria in the lower intestinal tract. Microbes of the genus Bacteroides make up ~25% of the total gut microbiome. Bacteroides fragilis comprises only 2% of the total Bacteroides in the gut, yet causes of >70% of Bacteroides infections. The factors causing it to transition from benign resident of the gut microbiome to virulent pathogen are not well understood, but a combination of horizontal gene transfer (HGT) of virulence genes and differential transcription of endogenous genes are clearly involved. The CRISPR-Cas system is a multi-functional system described in prokaryotes that may be involved in control both of HGT and of gene regulation. Results: Clustered regularly interspaced short palindromic repeats (CRISPR) elements in all strains of B. fragilis ( n = 109) with publically available genomes were identified. Three different CRISPR-Cas types, corresponding most closely to Type IB, Type IIIB, and Type IIC, were identified. Thirty-five strains had two CRISPR-Cas types, and three strains included all three CRISPR-Cas types in their respective genomes. The cas1 gene in the Type IIIB system encoded a reverse-transcriptase/Cas1 fusion protein rarely found in prokaryotes. We identified a short CRISPR (3 DR) with no associated cas genes present in most of the isolates; these CRISPRs were found immediately upstream of a hipA/hipB operon and we speculate that this element may be involved in regulation of this operon related to formation of persister cells during antimicrobial exposure. Also, blood isolates of B. fragilis did not have Type IIC CRISPR-Cas systems and had atypical Type IIIB CRISPR-Cas systems that were lacking adjacent cas genes. Conclusions: This is the first systematic report of CRISPR-Cas systems in a wide range of B. fragilis strains from a variety of sources. There are four apparent CRISPR-Cas systems in B. fragilis -three systems have adjacent cas genes. Understanding CRISPR/Cas function in B. fragilis will elucidate their role in gene expression, DNA repair and ability to survive exposure to antibiotics. Also, based on their unique CRISPR-Cas arrays, their phylogenetic clustering and their virulence potential, we are proposing that blood isolates of B. fragilis be viewed a separate subgroup.

Bacteroides fragilis, an important component of the human gastrointestinal microbiota, can cause lethal extra-intestinal infection upon escape from the gastrointestinal tract. We demonstrated transfer and recombination of large chromosomal segments from B. fragilis HMW615, a multidrug resistant clinical isolate, to B. fragilis 638R. In one example, the transfer of a segment of ~435 Kb/356 genes replaced ~413 Kb/326 genes of the B. fragilis 638R chromosome. In addition to transfer of antibiotic resistance genes, these transfers (1) replaced complete divergent polysaccharide biosynthesis loci; (2) replaced DNA inversion-controlled intergenic shufflons (that control expression of genes encoding starch utilization system outer membrane proteins) with more complex, divergent shufflons; and (3) introduced additional intergenic shufflons encoding divergent Type 1 restriction/modification systems. Conjugative transposon-like genes within a transferred segment and within a putative integrative conjugative element (ICE5) ~45 kb downstream from the transferred segment both encode proteins that may be involved in the observed transfer. These data indicate that chromosomal transfer is a driver of antigenic diversity and nutrient adaptation in Bacteroides that (1) contributes to the dissemination of the extensive B. fragilis pan-genome, (2) allows rapid adaptation to a changing environment and (3) can confer pathogenic characteristics to host symbionts.

Background: Metronidazole is the most commonly used antimicrobial for Bacteroides fragilis infections and is recommended for prophylaxis of colorectal surgery. Metronidazole resistance is increasing and the mechanisms of resistance are not clear., Methods: A transposon mutant library was generated in B. fragilis 638R (BF638R) to identify the genetic loci associated with resistance to metronidazole., Results: Thirty-two independently isolated metronidazole-resistant mutants had a transposon insertion in BF638R_1421 that encodes the ferrous transport fusion protein (feoAB). Deletion of feoAB resulted in a 10-fold increased MIC of metronidazole for the strain. The metronidazole MIC for the feoAB mutant was similar to that for the parent strain when grown on media supplemented with excess iron, suggesting that the increase seen in the MIC of metronidazole was due to reduced cellular iron transport in the feoAB mutant. The furA gene repressed feoAB transcription in an iron-dependent manner and disruption of furA resulted in constitutive transcription of feoAB, regardless of whether or not iron was present. However, disruption of feoAB also diminished the capacity of BF638R to grow in a mouse intraperitoneal abscess model, suggesting that inorganic ferrous iron assimilation is essential for B. fragilis survival in vivo., Conclusions: Selection for feoAB mutations as a result of metronidazole treatment will disable the pathogenic potential of B. fragilis and could contribute to the clinical efficacy of metronidazole. While mutations in feoAB are probably not a direct cause of clinical resistance, this study provides a key insight into intracellular metronidazole activity and the link with intracellular iron homeostasis., (Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy 2014. This work is written by US Government employees and is in the public domain in the US.)

Objectives:   Bacteroides fragilis , a Gram-negative anaerobic bacterium, is alternately a gut commensal or virulent pathogen and is an important reservoir for horizontal gene transfer (HGT) of bacterial resistance and virulence genes in the human gastrointestinal tract. We identified a unique conjugative transposon (CTn) in a multidrug resistant clinical isolate of B. fragilis (BF-HMW615); we named this element CTnHyb because it included a hybrid mosaic of foreign elements. This study reports the characterization of CTnHyb and discusses the potential impact on horizontal spread of resistance genes. Results: CTnHyb contains several efflux pump genes and several genes that confer or may confer antibiotic resistance to tetracycline, kanamycin, metronidazole and spectinomycin (truncated gene). CTnHyb also contains a mosaic of mobile elements from Gram-positive organisms. CTnHyb is easily transferred from BF-HMW615 (the original isolate) to BF638R (lab strain) and integrated into the BF638R chromosome. The "foreign" (from Gram-positive bacteria) nucleotide sequences within CTnHyb were > 99% preserved indicating that the gene acquisition from the Gram-positive bacteria was very recent. Conclusion: CTnHyb is a novel CTn residing in a multidrug resistant strain of B. fragilis . The global nature and wide phylogenetic reach of HGT means that any gene in any bacterium can potentially be mobilized. Understanding the mechanisms that drive the formation and transfer of these elements and, potentially, ways to limit the transfer are necessary to prevent a devastating spread of resistance elements.

Background: Bacteroides fragilis is a Gram-negative anaerobe that is normally a human gut commensal; it comprises a small percentage of the gut Bacteroides but is the most frequently isolated Bacteroides from human infections. Identification of the essential genes necessary for the survival of B. fragilis provides novel information which can be exploited for the treatment of bacterial infections., Results: Massive parallel sequencing of saturated transposon mutant libraries (two mutant pools of approximately 50,000 mutants each) was used to determine the essential genes for the growth of B. fragilis 638R on nutrient rich medium. Among the 4326 protein coding genes, 550 genes (12.7%) were found to be essential for the survival of B. fragilis 638R. Of the 550 essential genes, only 367 genes were assigned to a Cluster of Orthologous Genes, and about 290 genes had Kyoto Encyclopedia of Genes and Genomes orthologous members. Interestingly, genes with hypothetical functions accounted for 41.3% of essential genes (227 genes), indicating that the functions of a significant percentage of the genes used by B. fragilis 638R are still unknown. Global transcriptome analysis using RNA-Seq indicated that most of the essential genes (92%) are, in fact, transcribed in B. fragilis 638R including most of those coding for hypothetical proteins. Three hundred fifty of the 550 essential genes of B. fragilis 638R are present in Database of Essential Genes. 10.02 and 31% of those are genes included as essential genes for nine species (including Gram-positive pathogenic bacteria)., Conclusions: The essential gene data described in this investigation provides a valuable resource to study gene function and pathways involved in B. fragilis survival. Thorough examination of the B. fragilis-specific essential genes and genes that are shared between divergent organisms opens new research avenues that will lead to enhanced understanding of survival strategies used by bacteria in different microniches and under different stress situations.

Two multidrug-resistant Bacteroides fragilis clinical isolates contain and express a novel nim gene, nimJ, that is not recognized by the "universal" nim primers and can confer increased resistance to metronidazole when introduced into a susceptible strain on a multicopy plasmid. HMW615, an appendiceal isolate, contains at least two copies of nimJ on its genome, while HMW616, an isolate from a patient with sepsis, contains one genomic copy of nimJ. B. fragilis NimJ is phylogenetically closer to Prevotella baroniae NimI and Clostridium botulinum NimA than to the other known Bacteroides Nim proteins. The predicted protein structure of NimJ, based on fold recognition analysis, is consistent with the crystal structures derived for known Nim proteins, and specific amino acid residues important for substrate binding in the active site are conserved. This study demonstrates that the "universal" nim primers will not detect all nim genes with the ability to confer metronidazole resistance, but nimJ alone cannot account for the very high metronidazole MICs of these resistant clinical isolates.

The mariner transposon vector pYV07 was tested for use in the mutagenesis of Bacteroides fragilis 638R. The transposon vector efficiently generated mutants in B. fragilis 638R. The transposon disrupted genes were scattered throughout the genome of B. fragilis 638R. This method serves as a powerful tool to study B. fragilis., (Published by Elsevier Ltd.)

Susceptibility testing of anaerobic bacteria recovered from selected cases can influence the choice of antimicrobial therapy. The Clinical and Laboratory Standards Institute (CLSI) has standardized many laboratory procedures, including anaerobic susceptibility testing (AST), and has published documents for AST. The standardization of testing methods by the CLSI allows comparisons of resistance trends among various laboratories. Susceptibility testing should be performed on organisms recovered from sterile body sites, those that are isolated in pure culture, or those that are clinically important and have variable or unique susceptibility patterns. Organisms that should be considered for individual isolate testing include highly virulent pathogens for which susceptibility cannot be predicted, such as Bacteroides, Prevotella, Fusobacterium, and Clostridium spp.; Bilophila wadsworthia; and Sutterella wadsworthensis. This review describes the current methods for AST in research and reference laboratories. These methods include the use of agar dilution, broth microdilution, Etest, and the spiral gradient endpoint system. The antimicrobials potentially effective against anaerobic bacteria include beta-lactams, combinations of beta-lactams and beta-lactamase inhibitors, metronidazole, chloramphenicol, clindamycin, macrolides, tetracyclines, and fluoroquinolones. The spectrum of efficacy, antimicrobial resistance mechanisms, and resistance patterns against these agents are described.

Genetic analysis of Bacteroides fragilis (BF) is hindered because of the lack of efficient transposon mutagenesis methods. Here, we describe a simple method for transposon mutagenesis using EZ::TN5, a commercially available system that we optimized for use in BF638R. The modified EZ::TN5 transposon contains an Escherichia coli conditional origin of replication, a kanamycin resistance gene for E. coli, an erythromycin resistance gene for BF , and 19 basepair transposase recognition sequences on either ends. Electroporation of the transposome (transposon-transposase complex) into BF638R yielded 3.2 ± 0.35 × 10(3) CFU μg(-1) of transposon DNA. Modification of the transposon by the BF638R restriction/modification system increased transposition efficiency sixfold. Electroporation of the EZ::TN5 transposome results in a single-copy insertion of the transposon evenly distributed across the genome of BF638R and can be used to construct a BF638R transposon library. The transposon was also effective in mutating a BF clinical isolate and a strain of the related species, Bacteroides thetaiotaomicron. The EZ::TN5-based mutagenesis described here is more efficient than other transposon mutagenesis approaches previously reported for BF., (Published 2012. This article is a U.S. Government work and is in the public domain in the USA.)

Bacteroides fragilis is a gram-negative anaerobic commensal that can be a virulent pathogen when it escapes its normal niche in the human gut. Recent increases in reports of multi-drug resistance strains of this organism have lent urgency to understanding its mechanisms of antimicrobial resistance. We have identified and characterized RND-type multi-drug efflux pumps in B. fragilis which can pump out a variety of substrates and whose transcription levels can be elevated by a wide variety of antimicrobials, antiseptic agents, bile and other stressors. Our research is directed toward understanding how the efflux pump genes are controlled and how we may exploit that understanding to develop more effective, targeted therapy that will cure the infection without disrupting the entire gut microbiome that is so important in many aspects of human health., (Published by Elsevier Ltd.)

Objective: This study examined the antimicrobial resistance profile and the prevalence of resistance genes in Bacteroides spp. and Parabacteroides distasonis strains isolated from children's intestinal microbiota., Methods: The susceptibility of these bacteria to 10 antimicrobials was determined using an agar dilution method. β-lactamase activity was assessed by hydrolysis of the chromogenic cephalosporin of 114 Bacteriodales strains isolated from the fecal samples of 39 children, and the presence of resistance genes was tested using a PCR assay., Results: All strains were susceptible to imipenem and metronidazole. The following resistance rates were observed: amoxicillin (93%), amoxicillin/clavulanic acid (47.3%), ampicillin (96.4%), cephalexin (99%), cefoxitin (23%), penicillin (99%), clindamycin (34.2%) and tetracycline (53.5%). P-lactamase production was verified in 92% of the evaluated strains. The presence of the cfiA, cepA, ermF, tetQ and nim genes was observed in 62.3%, 76.3%, 27%, 79.8% and 7.8% of the strains, respectively., Conclusions: Our results indicate an increase in the resistance to several antibiotics in intestinal Bacteroides spp. and Parabacteroides distasonis and demonstrate that these microorganisms harbor antimicrobial resistance genes that may be transferred to other susceptible intestinal strains.

OmpA1 is the major outer membrane protein of the Gram-negative anaerobic pathogen Bacteroides fragilis. We identified three additional conserved ompA homologues (ompA2-ompA4) and three less homologous ompA-like genes (ompAs 5, 6 and 7) in B. fragilis. We constructed an ompA1 disruption mutant in B. fragilis 638R (WAL6 OmegaompA1) using insertion-mediated mutagenesis. WAL6 OmegaompA1 formed much smaller colonies and had smaller, rounder forms on Gram stain analysis than the parental strain or other unrelated disruption mutants. SDS-PAGE and Western blot analysis (with anti-OmpA1 IgY) of the OMP patterns of WAL6 OmegaompA1 grown in both high- and low-salt media did not reveal any other OmpA proteins even under osmotic stress. An ompA1 deletant (WAL186DeltaompA1) was constructed using a two-step double-crossover technique, and an ompA 'reinsertant', WAL360+ompA1, was constructed by reinserting the ompA gene into WAL186DeltaompA1. WAL186DeltaompA1 was significantly more sensitive to exposure to SDS, high salt and oxygen than the parental (WAL108) or reinsertant (WAL360+ompA1) strain. No significant change was seen in MICs of a variety of antimicrobials for either WAL6 OmegaompA1 or WAL186DeltaompA1 compared to WAL108. RT-PCR revealed that all of the ompA genes are transcribed in the parental strain and in the disruption mutant, but, as expected, ompA1 is not transcribed in WAL186DeltaompA1. Unexpectedly, ompA4 is also not transcribed in WAL186DeltaompA1. A predicted structure indicated that among the four OmpA homologues, the barrel portion is more conserved than the loops, except for specific conserved patches on loop 1 and loop 3. The presence of multiple copies of such similar genes in one organism would suggest a critical role for this protein in B. fragilis.

The outer membrane proteins of Desulfovibrio piger and Bilophila wadsworthia (Omp-DP and Omp-BW, respectively) and the genes encoding them (omp-DP and omp-BW) were isolated and characterized. Native Omp-DP and Omp-BW form a trimeric structure of approximately 120 kDa. These proteins disaggregated into monomers with a molecular weight of approximately 53 kDa after heating at 95 degrees C for 10 min. The pore-forming abilities of these oligomeric proteins demonstrated that they form small nonspecific channels with an exclusion limit of 260-300 Da. The omp-DP and omp-BW genes were cloned and sequenced. Sequence analyses revealed an open reading frame of 1,512 bp for omp-DP and 1,440 bp for omp-BW. The mature Omp-DP protein consisted of 480 amino acids and had a calculated MW of 53,290 Da. The mature Omp-BW protein consisted of 456 amino acids and had a calculated MW of 50.050 Da. Alignment of Omp-DP with Omp-BW revealed 54% homology, whereas alignment with other known porins showed a low level of homology. Analysis of the secondary structures indicated that both proteins span the outer membrane 18 times with amphipathic beta-strands. This research presents porins which were isolated and characterized for the first time from bacteria belonging to the Desulfovibrionaceae family.

Bacteroides fragilis constitutes 1-2% of the natural microbiota of the human digestive tract and is the predominant anaerobic opportunistic pathogen in gastrointestinal infections. Most bacteria use quorum sensing (QS) to monitor cell density in relation to other cells and their environment. In Gram-negative bacteria, the LuxRI system is common. The luxR gene encodes a transcriptional activator inducible by type I acyl-homoserine lactone autoinducers (e.g., N-[3-oxohexanoyl] homoserine lactone and hexanoyl homoserine lactone [C6-HSL]). This study investigated the presence of QS system(s) in B. fragilis. The genome of American-type culture collection strain no. ATCC25285 was searched for QS genes. The strain was grown to late exponential phase in the presence or absence of synthetic C6-HSL and C8-HSL or natural homoserine lactones from cell-free supernatants from spent growth cultures of other bacteria. Growth, susceptibility to antimicrobial agents, efflux pump gene (bmeB) expression, and biofilm formation were measured. Nine luxR and no luxI orthologues were found. C6-HSL and supernatants from Yersinia enterocolitica, Vibrio cholerae, and Pseudomonas aeruginosa caused a significant (1) reduction in cellular density and (2) increases in expression of four putative luxR genes, bmeB3, bmeB6, bmeB7, and bmeB10, resistance to various antibiotics, which was reduced by carbonyl cyanide-m-chlorophenyl hydrazone (CCCP, an uncoupler that dissipates the transmembrane proton gradient, which is also the driving force of resistance nodulation division efflux pumps) and (3) increase in biofilm formation. Susceptibility of ATCC25285 to C6-HSL was also reduced by CCCP. These data suggest that (1) B. fragilis contains putative luxR orthologues, which could respond to exogenous homoserine lactones and modulate biofilm formation, bmeB efflux pump expression, and susceptibility to antibiotics, and (2) BmeB efflux pumps could transport homoserine lactones.

OmpA proteins from Gram-negative anaerobes Porphyromonas asaccharolytica and Bacteroides fragilis induced release and expression of IL-1alpha, tumor necrosis factor (TNF)-alpha, IFN-gamma, IL-6, and IL-10 from murine splenocytes in vitro in a dose-dependent fashion. The release of the cytokines induced by B. fragilis Bf-OmpA was at much lower levels compared with P. asaccharolytica Omp-PA; Bf-OmpA did not induce release of IL-10. Omp-PA and Bf-OmpA were able to upregulate mRNA expression of the tested cytokines. The results obtained with refolded Bf-OmpA were similar to those with native Bf-OmpA. The data presented in this research demonstrate for the first time that Omps from anaerobic bacteria can induce the release of cytokines, suggesting that Omp-PA and Bf-OmpA may play important roles in the pathogenic processes of these bacteria.

Objectives: To determine the potential of active compounds (ACs) present in commonly used analgesics/antiseptics and cleaning agents (detergents and disinfectants) to induce multiple antibiotic resistance (MAR) in Bacteroides fragilis., Methods: B. fragilis ATCC 25285 untreated or pretreated with sublethal concentrations of ACs (n = 25) was grown for 12 h. Susceptibility of cells pre-treated with various ACs to antibiotics and expression of resistance nodulation division family (bmeB) efflux pumps and putative marA-like global activators (PGAs) were measured., Results: Twelve aromatic ACs containing benzene or its activated derivatives (salicylate, acetaminophen, gingerol, benzoate, phenol, chlorhexidine gluconate, capsaicin, juglone, cinnamaldehyde, benzene, ibuprofen and Triton X-100) induced MAR, which was reduced by carbonyl cyanide m-chlorophenylhydrazone. There was a positive correlation between the predicted degree of benzene activation and the level of induction. Deactivated benzene or non-aromatic ACs were either poor inducers or non-inducers. Efflux pumps bmeB1, 3, 4, 7 and two PGAs bfrA1 and bfrA2 were overexpressed. Expression of bfrA1 or bfrA2 in Escherichia coli caused a >2-fold increase in the MAR and overexpression of acrB, suggesting that they were putative marA orthologues., Conclusions: These data demonstrate (i) the presence of an MarA-like system(s) in B. fragilis and (ii) the propensity of benzene or its activated derivatives present in pharmaceutical products to induce MAR.

Summary: Bacteroides species are significant clinical pathogens and are found in most anaerobic infections, with an associated mortality of more than 19%. The bacteria maintain a complex and generally beneficial relationship with the host when retained in the gut, but when they escape this environment they can cause significant pathology, including bacteremia and abscess formation in multiple body sites. Genomic and proteomic analyses have vastly added to our understanding of the manner in which Bacteroides species adapt to, and thrive in, the human gut. A few examples are (i) complex systems to sense and adapt to nutrient availability, (ii) multiple pump systems to expel toxic substances, and (iii) the ability to influence the host immune system so that it controls other (competing) pathogens. B. fragilis, which accounts for only 0.5% of the human colonic flora, is the most commonly isolated anaerobic pathogen due, in part, to its potent virulence factors. Species of the genus Bacteroides have the most antibiotic resistance mechanisms and the highest resistance rates of all anaerobic pathogens. Clinically, Bacteroides species have exhibited increasing resistance to many antibiotics, including cefoxitin, clindamycin, metronidazole, carbapenems, and fluoroquinolones (e.g., gatifloxacin, levofloxacin, and moxifloxacin).

This study investigated whether B. fragilis from various human sites acquired stable traits enabling it to express certain efflux pumps (EPs), adopt a particular cell structure, and tolerate certain stressors. Isolates from blood, abscess, and stool (n = 11 each) were investigated. Bacteria from various sites portrayed different ultrastructres and EP expression. Blood isolates were tolerant to nutrient limitation and stool isolates to NaCl and bile salt stress. Stressors significantly increased EP expression. These data demonstrate that (1) B. fragilis acquires stable traits from various in vivo microenvironments; (2) that EPs are involved in stress responsiveness; and (3) that EP expression is tightly controlled and site dependent.

Bacteroides fragilis is the most common anaerobic bacterium isolated from human intestinal tract infections. Before B. fragilis interacts with the intestinal epithelial cells, it is exposed to bile salts at physiological concentrations of 0.1-1.3%. The aim of this study was to determine how pre-treatment with bile salts affected B. fragilis cells and their interaction with intestinal epithelial cells. B. fragilis NCTC9343 was treated with conjugated bile salts (BSC) or non-conjugated bile salts (BSM). Cellular ultrastructure was assessed by electron microscopy, gene expression was quantified by comparative quantitative real-time RT-PCR. Adhesion to the HT-29 human intestinal cell line and to PVC microtitre plates (biofilm formation) was determined. Exposure to 0.15% BSC or BSM resulted in overproduction of fimbria-like appendages and outer membrane vesicles, and increased expression of genes encoding RND-type efflux pumps and the major outer membrane protein, OmpA. Bile salt-treated bacteria had increased resistance to structurally unrelated antimicrobial agents and showed a significant increase in bacterial co-aggregation, adhesion to intestinal epithelial cells and biofilm formation. These data suggest that bile salts could enhance intestinal colonization by B. fragilis via several mechanisms, and could therefore be significant to host-pathogen interactions.

The RND-family efflux pump gene bmeB5 was previously shown to be overexpressed in metronidazole-resistant laboratory mutants of Bacteroides fragilis. In the present study, we characterized the bmeABC5 genes and an upstream putative TetR-family regulator gene (bmeR5). bmeR5 (645 bp) was located 51 bp upstream of bmeA5 and encoded a 24.9-kDa protein. Deletant strains lacking bmeB5 or bmeR5 were constructed from a wild-type B. fragilis strain ADB77. Strain antimicrobial susceptibility was determined and gene expression was quantified. bmeR5 was overexpressed in Escherichia coli using a 6x-His tag system; BmeR5-His6 was isolated from inclusion bodies and its binding to bmeABC5 promoter regions was determined. BmeR5-His6 bound specifically to the bmeR5-bmeC5 intergenic region (IT1). Deletion of bmeR5 (ADB77DeltabmeR5) resulted in a significant (p < 0.05) increase in expression of bmeA5, bmeB5, and bmeC5, and > two-fold increase in minimum inhibitory concentrations (MICs) of ampicillin, cefoxitin, cefoperazone, ciprofloxacin, imipenem, metronidazole, ethidium bromide, and sodium dodecyl sulfate (SDS). MICs were reduced by the efflux pump inhibitor carbonyl cyanide m-chlorophenyl hydrazone (CCCP). The MICs of ampicillin, cefoperazone, metronidazole, and SDS were reduced by approximately two-fold in ADB77DeltabmeB5. A multidrug (metronidazole)-resistant, nim-negative B. fragilis clinical isolate overexpressed bmeABC5 genes, had a G-->T point mutation in IT1, and significantly reduced binding to BmeR5-His6. These data demonstrate that BmeR5 is a local repressor of bmeABC5 expression and that mutations in IT1 can lead to a derepression and resistance to multiple antimicrobial agents, including metronidazole.

A single monomeric porin, Omp-PA (37kDa), was isolated from the outer membrane of the gram-negative anaerobic rod Porphyromonas asaccharolytica. Further characterization revealed that this porin consists of two different fractions: a heat-modifiable fraction which in its denatured form migrated on SDS-PAGE as a protein with a molecular weight of 41kDa and a heat-resistant fraction which did not change its migration on SDS-PAGE after boiling. A liposome swelling assay revealed that only the heat-resistant fraction was able to transport sugars after its incorporation into the liposomes, although it did not discriminate between differently sized sugars. We hypothesize that the heat-modifiable fraction corresponds to the "closed" conformer of Omp-PA, whereas the heat-resistant fraction corresponds to the "open" conformer of the protein. Cloning of the omp-PA gene revealed an open reading frame of 1161 bases, with a predicted protein sequence of 387 amino acids. The mature protein consists of 366 amino acids with a calculated MW of 41,102Da and an estimated pI of 7.24. The C-terminal domain of Omp-PA is homologous to the characteristic OmpA signature domain (71% similarity with the OmpA consensus domain). Sequence comparison with other anaerobes from the Bacteroides family demonstrated homology across the entire ORF. Digestion of the P. asaccharolytica outer membrane analysis of trypsin-digested Omp-PA yielded two proteins migrating with apparent molecular weights of 37 and 27kDa. These data fully supported our hypothesis that the C-terminal domain of the two-domain "closed" conformer of Omp-PA was digested by trypsin, whereas the single domain beta-barrel "open" conformer was inaccessible to trypsin.

This study investigated the mechanisms of multidrug resistance (MDR) in an isolate of Bacteroides fragilis (WI1) from a patient with anaerobic sepsis. The MDR of WI1 affected susceptibility to beta-lactams, clindamycin, fluoroquinolones, metronidazole and tetracycline. In addition to its 5.31-Mb chromosome, WI1 possessed two low-copy-number plasmids, pHagl (5.6 kb) and pHag2 (9.9 kb), that were absent from B. fragilis NCTC 9343. Restriction digestion with EcoRV, HindIII and SstI, combined with DNA sequencing, revealed that pHAG2 contained a tet(Q) gene at base position 3689 that resided on the conjugative transposon CTn341. Genes cfiA (encoding a metallo-beta-lactamase) and erm(F) (encoding a macrolide-lincosamide-streptogramin B resistance determinant) were also found in WI1, but were absent from B. fragilis NCTC 9343. Nitrocefin hydrolysis revealed that WI1 had high beta-lactamase activity. Sequencing of the gyrA quinolone resistance-determining region revealed a mutation causing a Ser82 --> Phe substitution, and comparative quantitative real-time RT-PCR revealed that the cfiA, erm(F) and tet(Q) genes were all expressed in WI1. In addition, the resistance-nodulation-division efflux pump genes bmeB9 and bmeB15 were significantly over-expressed (12.30 +/- 0.42-fold and 3541.1 +/- 95.4-fold, respectively), and the efflux pump inhibitors carbonyl cyanide m-chlorophenylhydrazone and reserpine significantly increased the susceptibility of the isolate to several unrelated antibiotics (p <0.005). These data suggested that WI1 was highly multidrug-resistant because of the additive effects of chromosome- and plasmid-encoded resistance determinants.

Bacteroides fragilis is an anaerobic commensal constituting only 1-2% of the micro-flora of the human gastrointestinal tract, yet it is the predominant anaerobic isolate in cases of intraabdominal sepsis and bacteremia. B. fragilis can play two roles in the host: in its role as friendly commensal, it must be able to establish itself in the host intestinal mucosa, to utilize and process polysaccharides for use by the host, and to resist the noxious effects of bile salts. In its role as pathogen, it must be able to attach itself to the site of infection, evade killing mechanisms by host defense, withstand antimicrobial treatment and produce factors that damage host tissue. The cell envelope of B. fragilis, likewise, must be able to function in the roles of aggressor, defender and strategist in allowing the organism to establish itself in the host--whether as friend or foe. Recent studies of the genomes and proteomes of the genus Bacteroides suggest that these organisms have evolved strategies to survive and dominate in the overcrowded gastrointestinal neighborhood. Analysis of the proteomes of B. fragilis and Bacteroides thetaiotaomicron demonstrates both a tremendous capacity to use a wide range of dietary polysaccharides, and the capacity to create variable surface antigenicities by multiple DNA inversion systems. The latter characteristic is particularly pronounced in the species B. fragilis, which is more frequently found at the mucosal surface (i.e., often the site of attack by host defenses). The B. fragilis cell envelope undergoes major protein expression and ultrastructural changes in response to stressors such as bile or antimicrobial agents. These agents may also act as signals for attachment and colonization. Thus the bacterium manages its surface characteristics to enable it to bind to its target, to use the available nutrients, and to avoid or evade hostile forces (host-derived or external) in its multiple roles.

Multidrug-resistant mutants of a wild-type Bacteroides fragilis strain (strain ADB77) and a quadruple resistance nodulation division family efflux pump deletion mutant (ADB77 Delta bmeB1 Delta bmeB3 Delta bmeB12 Delta bmeB15) were selected with antimicrobials. Ampicillin, doripenem, imipenem, levofloxacin, and metronidazole selected for mutants from both strains; cefoxitin selected for mutants from strain ADB77 only; and sodium dodecyl sulfate selected mutants from ADB77Delta bmeB1 Delta bmeB3 Delta bmeB12 Delta bmeB15 only. The mutants overexpressed one or more efflux pumps.

Objectives: The aim of the present study was to determine correlation between bmeB efflux pump overexpression and resistance to fluoroquinolones and beta-lactams in Bacteroides fragilis clinical isolates (n = 51) and the effects of broad-spectrum efflux pump inhibitors (EPIs) on the MICs of the test antibiotics., Methods: Susceptibility to garenoxacin, levofloxacin, moxifloxacin, cefoxitin and faropenem +/- EPIs (CCCP, MC-207,110, reserpine and verapamil) was determined. Expression of bmeB efflux pumps was measured, topoisomerase genes were sequenced and beta-lactamase production was determined., Results: Isolates were grouped into categories based on susceptibility patterns, topoisomerase sequence and efflux pump expression. Panel I isolates (19/51, 37.3%) were highly resistant to fluoroquinolones and cefoxitin (resistance to all agents was significantly reduced by EPIs, P < 0.05), had a point mutation in gyrA (C-->T) causing a Ser-82-->Phe substitution, and overexpressed bmeB4 and bmeB15. Panel II isolates (7/51; 13.7%) had intermediate-level resistance to fluoroquinolones and cefoxitin and a GyrA substitution. Panel IIIA isolates (21/51; 41.2%) had intermediate-level fluoroquinolone resistance and high-level cefoxitin resistance [resistance to all agents was significantly reduced by EPIs (P < 0.05)] and overexpressed bmeB4 and bmeB15. Panel IIIB isolates (4/51; 7.8%) had low-level fluoroquinolone resistance and high-level resistance to cefoxitin [cefoxitin resistance was significantly reduced by EPIs (P < 0.05)] and overexpressed bmeB4, bmeB6, bmeB10 and bmeB14. All isolates were beta-lactamase-positive., Conclusions: These data suggest that bmeB efflux pump overexpression can (i) cause low- to intermediate-level clinically relevant fluoroquinolone resistance; (ii) be coupled with GyrA substitutions to cause high-level fluoroquinolone resistance; (iii) contribute to high-level clinically relevant resistance to beta-lactams.

Objectives: To determine the prevalence of expression and function(s) of Bacteroides fragilis RND family efflux transport systems (bmeABC1-16)., Methods: The mRNA transcripts of bmeB efflux pump genes were detected in a wild-type strain ADB77 by RT-PCR and expression in different strains was quantified by comparative quantitative real-time RT-PCR. In order to determine independent or additive functions, BmeB 1, 3, 12 and 15 (the first efflux pumps identified) were deleted as singles, doubles, triples or quadruples by the double cross-over technique with pADB242 and antimicrobial susceptibility was assayed by the spiral gradient endpoint technique., Results: All efflux pumps except bmeB9 were expressed in the wild-type parental strain. Susceptibility to beta-lactams, fluoroquinolones, ethidium bromide, SDS and triclosan was increased in ADB77DeltabmeB3 (up to 3-fold) and ADB77DeltabmeB1DeltabmeB3DeltabmeB12 (up to 5-fold). Expression of bmeB9 was increased and that of bmeB11 repressed in the latter deletant. A quadruple deletant (ADB77DeltabmeB1DeltabmeB3DeltabmeB12DeltabmeB15) had similar changes as well as a 2-fold increase in expression of bmeB16 and norfloxacin resistance. Expression of bmeB3 was increased in two triple deletants ADB77DeltabmeB1DeltabmeB12DeltabmeB15-type I (2-fold) and ADB77DeltabmeB1DeltabmeB12DeltabmeB15-type II (5.8-fold). Antimicrobial MICs were also increased in the latter deletant; ampicillin (2.6-fold), cefoperazone (3.4-fold), cefoxitin (1.8-fold), tetracycline (36.4-fold), SDS (1.7-fold) and triclosan (2-fold)., Conclusions: These data demonstrate that constitutive bmeB expression is prevalent in B. fragilis. At least seven BmeB efflux pumps are functional in transporting antimicrobials and have overlapping substrate profiles, and at least four confer intrinsic resistance.

The in vitro activities of doripenem against 364 anaerobic isolates were measured and compared to those of ertapenem, imipenem, meropenem, ceftriaxone, and levofloxacin. All of the carbapenems were active against nearly all Bacteroides fragilis group isolates. Doripenem was either comparable to or slightly less active than imipenem and meropenem against most isolates but more active than the other penems against Clostridium difficile. Doripenem appears to have excellent activity against a broad range of anaerobes.

Sixteen homologs of multidrug resistance efflux pump operons of the resistance-nodulation-cell division (RND) family were found in the Bacteroides fragilis genome sequence by homology searches. Disruption mutants were made to the mexB homologs of the four genes most similar to Pseudomonas aeruginosa mexB. Reverse transcription-PCR was conducted and indicated that the genes were transcribed in a polycistronic fashion and that the promoter was upstream of bmeA (the mexA homolog). One of these disruption mutants (in bmeB, the mexB homolog) was more susceptible than the parental strain to certain cephems, polypeptide antibiotics, fusidic acid, novobiocin, and puromycin. The gene for this homolog and the adjacent upstream gene, bmeA, were cloned in a hypersensitive Escherichia coli host. The resultant transformants carrying B. fragilis bmeAB were more resistant to certain agents; these agents also had lower MICs for the B. fragilis bmeB disruption mutants than for the parental strain. The putative efflux pump operon is composed of bmeA, bmeB, and bmeC (a putative outer membrane channel protein homologous with OprM). Addition of the efflux pump inhibitors, carbonyl cyanide m-chlorophenylhydrazone (a proton conductor that eliminates the energy source) and Phe-Arg beta-naphthylamide (MC-207,110) (the first specific inhibitor described for RND pumps in P. aeruginosa), resulted in lowered MICs in the parental strain but not in the bmeB disruption mutant, indicating that the bmeB pump is affected by these inhibitors. This is the first description of RND type pumps in the genus Bacteroides.

Susceptibility of intestinal bacteria to various antimicrobial agents in vitro, together with levels of those agents achieved in the gut, provides information on the likely impact of the agents on the intestinal flora. Orally administered drugs that are poorly absorbed may be useful for treatment of intestinal infections and for certain other situations in which intestinal bacteria may play a role. The antimicrobial activity of ramoplanin (MDL 62,198) against 928 strains of intestinal anaerobic bacteria was determined using the NCCLS-approved Wadsworth brucella laked-blood agar dilution method. The activity of ramoplanin was compared with that of ampicillin, bacitracin, metronidazole, trimethoprim/sulfamethoxazole (TMP/SMX), and vancomycin. The organisms tested included Bacteroides fragilis group (n=89), other Bacteroides species (n=16), other anaerobic Gram-negative rods (n=56) anaerobic cocci (n=114), Clostridium species (n=426), and non-sporeforming anaerobic Gram-positive rods (n=227). The overall MIC(90)s of ramoplanin, ampicillin, bacitracin, metronidazole, and vancomycin were 256, 32, 128, 16, and 128 mcg/ml, respectively. Ramoplanin was almost always highly active vs. Gram-positive organisms and relatively poor in activity against Gram-negative organisms, particularly Bacteroides, Bilophila, Prevotella, and Veillonella. Vancomycin was quite similar to ramoplanin in its activity. Ampicillin was relatively poor in activity vs. organisms that often produce beta-lactamase, including most of the Gram-negative rods as well as Clostridium bolteae and C. clostridioforme. Bacitracin was relatively poor in activity against most anaerobic Gram-negative rods, but better vs. most Gram-positive organisms. Metronidazole was very active against all groups other than bifidobacteria and some strains of other types of non-sporeforming Gram-positive bacilli. TMP/SMX was very poorly active, with an MIC(90) of >2048 mcg/ml.

Ertapenem is a long-acting, 1beta-methyl parenteral Group 1 carbapenem antibiotic that has a broad antibacterial spectrum and once-a-day dosing supported by clinical studies. Ertapenem is active against both Gram-positive and Gram-negative bacteria, including Enterobacteriaceae, Streptococcus pneumoniae and most species of anaerobic bacteria. Isolates from a variety of infections (intra-abdominal infections, skin/soft-tissue infections, community-acquired pneumonia, pelvic infections and urinary tract infections) are inhibited by ertapenem. It has restricted activity against nosocomial pathogens such as Pseudomonas aeruginosa, Acinetobacter species, methicillin-resistant staphylococci and enterococci. Ertapenem has potent activity against the majority of anaerobic isolates from intra-abdominal infections, and against most of the aerobes isolated from these infections, with the exceptions of the nosocomial pathogens mentioned above. MIC(90)s for most species of Enterobacteriaceae were <1 mg/L, significantly lower than those of imipenem. MIC(90)s for most Bacteroides fragilis group isolates ranged from 1 to 4 mg/L, and MIC(90)s were species specific for Clostridium, ranging from 0.06 mg/L for Clostridium perfringens to 4 mg/L for Clostridium clostridioforme. Ertapenem was equivalent to or better than piperacillin-tazobactam in activity against most anaerobic species isolated from these infections, and was more potent than piperacillin-tazobactam and ceftriaxone against the most common skin pathogens (e.g. methicillin-susceptible Staphylococcus aureus). Ertapenem was highly active against most of the pathogens isolated from patients with community-acquired pneumonia, except for isolates of methicillin-resistant S. aureus (which are infrequent causes of community-acquired infection); these isolates were also resistant to ceftriaxone. Resistance to ertapenem is most commonly attributable to a variety of mechanisms including alterations in penicillin-binding proteins in Gram-positive organisms, and combinations of potent metallo-beta-lactamase enzymes, porin protein defects and efflux pumps in Gram-negative organisms.

The activity of faropenem, a new oral penem, was tested against 579 strains of anaerobic bacteria by using the NCCLS-approved reference method. Drugs tested included amoxicillin-clavulanate, cefoxitin, clindamycin, faropenem, imipenem, and metronidazole. Of the 176 strains of Bacteroides fragilis group isolates tested, two isolates had faropenem MICs of 64 micro g/ml and imipenem MICs of >32 micro g/ml. Faropenem had an MIC of 16 micro g/ml for an additional isolate of B. fragilis; this strain was sensitive to imipenem (MIC of 1 micro g/ml). Both faropenem and imipenem had MICs of < or=4 micro g/ml for all isolates of Bacteroides capillosus (10 isolates), Bacteroides splanchnicus (13 isolates), Bacteroides ureolyticus (11 isolates), Bilophila wadsworthia (11 isolates), Porphyromonas species (42 isolates), Prevotella species (78 isolates), Campylobacter species (25 isolates), Sutterella wadsworthensis (11 isolates), Fusobacterium nucleatum (19 isolates), Fusobacterium mortiferum/varium (20 isolates), and other Fusobacterium species (9 isolates). Faropenem and imipenem had MICs of 16 to 32 micro g/ml for two strains of Clostridium difficile; the MICs for all other strains of Clostridium tested (69 isolates) were < or =4 micro g/ml. Faropenem had MICs of 8 and 16 micro g/ml, respectively, for two strains of Peptostreptococcus anaerobius (MICs of imipenem were 2 micro g/ml). MICs were < or =4 micro g/ml for all other strains of gram-positive anaerobic cocci (53 isolates) and non-spore-forming gram-positive rods (28 isolates). Other results were as expected and reported in previous studies. No metronidazole resistance was seen in gram-negative anaerobes other than S. wadsworthensis (18% resistant); 63% of gram-positive non-spore-forming rods were resistant. Some degree of clindamycin resistance was seen in most of the groups tested.

A multilaboratory study compared the growth of 30 fastidious anaerobes, using 5 different agar media: Wilkins-Chalgren (WC), WC with either whole or laked sheep blood, and Brucella supplemented with vitamin K(1) and hemin and either laked or whole sheep blood. The media were compared for quality and quantity of growth. Experiments were conducted either entirely in an anaerobic chamber or inoculated in ambient air with anaerobic incubation. The results showed that (1) any medium plus whole or laked blood was better than unsupplemented WC, (2) whole blood and laked blood additives gave similar results, (3) supplemented Brucella with whole or laked blood was superior to WC and WC with whole or laked blood, and (4) anaerobic and aerobic inoculation with anaerobic incubation gave similar results. Brucella agar supplemented with whole or laked blood supports the growth of fastidious anaerobic species better than the WC agars do.

A 5-laboratory study was performed that used the National Committee for Clinical Laboratory Standards (NCCLS) reference agar dilution method with 3 media formulations to determine whether the use of different media would affect minimum inhibitory concentration (MIC) results. Wilkins-Chalgren, Brucella-based blood agar (BRU), and Wilkins-Chalgren agar plus blood (WCB) and 6 antibiotics (clindamycin, cefoxitin, ceftizoxime, piperacillin, metronidazole, and trovafloxacin) were evaluated with 58 isolates. The MIC values were compared, and a significant correlation of >0.80 was demonstrated for all media and each antibiotic/organism group. The cumulative rate of errors for all antibiotics was 0.1%. These data indicate that a change in the NCCLS reference medium for testing of anaerobic bacteria susceptibility to either BRU or WCB will not affect the MIC results for the antibiotics and organisms evaluated.

The outer-membrane proteins (OMPs) of bacteria function as the dynamic interface between the bacterium and its surroundings and are involved in maintenance of cell structure, binding a variety of substances, adhesion to other cells, and regulation of transport of both nutrients and bactericidal agents. There is a vast amount of information about aerobic OMPs and their roles in immunogenicity, virulence, and antimicrobial resistance. Knowledge about OMPs in anaerobic bacteria is much sparser. Genetic data present in data banks regarding aerobic porins are not readily helpful in identifying or analyzing anaerobic porins because of the large phylogenetic distance between the aerobic and anaerobic organisms. We recently identified and sequenced the genes for both a porin protein complex and an OmpA protein in Bacteroides fragilis, and the data are summarized here. Also, recent information is presented about similar OMPs found in other gram-negative anaerobic bacteria, including Bacteroides thetaiotaomicron, Bacteroides distasonis, Porphyromonas, and Fusobacterium.

An open-label, multicenter study was performed to assess bacteriologic findings associated with chronic bacterial maxillary sinusitis in adults. Seventy aerobic (52.2%) and 64 anaerobic (47.8%) pathogens were recovered from clinically evaluable patients at baseline (before therapy). The most commonly isolated anaerobes were Prevotella species (31.1%), anaerobic streptococci (21.9%), and Fusobacterium species (15.6%). The aerobes most frequently recovered included Streptococcus species (21.4%), Haemophilus influenzae (15.7%), Pseudomonas aeruginosa (15.7%), and Staphylococcus aureus and Moraxella catarrhalis (10.0% each). Recurrences of signs or symptoms of bacterial maxillary sinusitis associated with anaerobes were twice as frequent as were those associated with aerobes when counts of anaerobes were > or =10(3) cfu/mL. A pathogenic role for Granulicatella species in cases of chronic sinusitis was documented for the first time.

ABT-773 was tested against 317 fastidious isolates; it inhibited 99% of organisms at a concentration of 4.0 microg/ml. With ampicillin-sulbactam and levofloxacin, only 2 and 6% of these strains, respectively, were resistant at the breakpoint. With clindamycin, penicillin G, and metronidazole, 22, 26, and 58% of the strains, respectively, were resistant.

The high MW porin protein complex (Omp200, composed of Omp121 and Omp71) from Bacteroides fragilis ATCC 25285 was purified and tryptic peptide sequences were used to design degenerate oligonucleotide primers which were then used as a first step in amplification, identification and sequencing of the omp121 gene (GenBank Accession Number AF357210). Sequence analysis revealed an open reading frame of 3378 bases. The deduced amino acid sequence (which contained the experimentally determined peptide sequences) has 1125 or 1116 amino acids (depending on which start codon is used); the mature protein consists of 1096 amino acids, has a predicted MW of 121.4 and a theoretical pI of 6.32. It is preceded by a 29 or 18 amino acid signal peptide which includes a typical hydrophobic region near the N-terminus (VLVLVL). Hydropathy plots of the deduced amino acid sequence of B. fragilis Omp121 display striking similarity with those of Escherichia coli OmpC (a 16-stranded porin) and FepA (a 22-stranded ligand-gated transport protein). Three-dimensional modeling of B. fragilis Omp121 (based on 1D and 3D sequence profiles, coupled with secondary structure and solvation potential information) indicated that the closest homologues in terms in fold conservation were the E. coli 16-stranded porins (e.g. OsmA) and 22-stranded ligand gated transport proteins (e.g. FepA). The omp71 gene sequence was identified using the tryptic peptides to search the published Bacteroides genome data base. We found that omp71 is located immediately downstream of omp121 and confirmed this with PCR analysis. Omp71 has no known homologues but does share some characteristics with the Porphyromonas RagB antigen.

In a previous study, we compared HMR 3004 with azithromycin, clarithromycin, erythromycin and roxithromycin against 502 anaerobic bacteria using NCCLS-approved procedures. This report extends this study by reporting the activity of telithromycin (HMR 3647) against these strains. Telithromycin inhibited 10% of Bacteroides fragilis, 50% of other B. fragilis group organisms and 93% of other Bacteroides spp. Telithromycin inhibited all Porphyromonas spp. and 98% of Prevotella spp. Activity against Bilophila wadsworthia (85-96%) was excellent. Telithromycin was not active against the Fusobacterium mortiferum/varium group. Telithromycin inhibited 100% of Clostridium perfringens, 46-56% of Clostridium difficile and Clostridium ramosum and approximately 90% of non-spore-forming Gram-positive bacilli.

The activity of MK-826 was compared to the activities of cefoxitin, ceftriaxone, imipenem, and meropenem against 363 gram-negative and gram-positive anaerobes by using NCCLS procedures. At least 98% of the strains were susceptible to the carbapenems. All strains of Clostridium perfringens, Fusobacterium nucleatum, Peptostreptococcus, and Sutterella wadsworthensis were susceptible to all agents tested.

In most cases symptoms of autism begin in early infancy. However, a subset of children appears to develop normally until a clear deterioration is observed. Many parents of children with "regressive"-onset autism have noted antecedent antibiotic exposure followed by chronic diarrhea. We speculated that, in a subgroup of children, disruption of indigenous gut flora might promote colonization by one or more neurotoxin-producing bacteria, contributing, at least in part, to their autistic symptomatology. To help test this hypothesis, 11 children with regressive-onset autism were recruited for an intervention trial using a minimally absorbed oral antibiotic. Entry criteria included antecedent broad-spectrum antimicrobial exposure followed by chronic persistent diarrhea, deterioration of previously acquired skills, and then autistic features. Short-term improvement was noted using multiple pre- and post-therapy evaluations. These included coded, paired videotapes scored by a clinical psychologist blinded to treatment status; these noted improvement in 8 of 10 children studied. Unfortunately, these gains had largely waned at follow-up. Although the protocol used is not suggested as useful therapy, these results indicate that a possible gut flora-brain connection warrants further investigation, as it might lead to greater pathophysiologic insight and meaningful prevention or treatment in a subset of children with autism.

While antibiotic resistance among anaerobes continues to increase, the frequency of antimicrobial susceptibility testing for anaerobes is declining. Because anaerobic infections are often mixed and detailed bacteriology of the organisms involved may take some time, physicians must institute empiric therapy before susceptibility testing results are available. Also, economic realities and prudent use of resources mandate that careful consideration be given to the necessity for routine susceptibility testing of anaerobic bacteria. Determination of appropriate therapy can be based on published antibiograms; however, since patterns may vary within geographic regions and even within hospitals, it is strongly recommended that each hospital center periodically test their isolates to determine local patterns and detect any pockets of resistance. As a general guide, antibiograms from the last several years of susceptibility testing at the Wadsworth Anaerobe Laboratory are reported.

The in vitro activity of levofloxacin was compared to the activities of ofloxacin, ciprofloxacin, ampicillin-sulbactam (2:1), cefoxitin, and metronidazole for a selected group of anaerobes (n = 175) isolated from skin and soft tissue infections by using the National Committee for Clinical Laboratory Standards-approved Wadsworth method. Ampicillin-sulbactam and cefoxitin inhibited 99% of the strains of this select group, levofloxacin and ofloxacin inhibited 73 and 50%, respectively, at 2 microg/ml, and ciprofloxacin inhibited 51% at 1 microg/ml. The geometric mean MIC of levofloxacin was lower than those of ofloxacin and ciprofloxacin for every group except Veillonella.

Adherence of Bilophila wadsworthia to the cultured human embryonic intestinal cell line, Intestine 407 (Int 407), varied among the strains tested from strongly adherent (76-100% cells positive for one or more adherent bacteria) to non- or weakly adherent (0-25% positive cells). Although negative staining revealed that infrequent cells of an adherent strain, WAL 9077, the adherent type-strain, WAL 7959, and a non-adherent strain, WAL 8448, expressed loosely associated fimbrial structures, a role for these structures in adhesion could not be confirmed with either scanning or thin-section electron micrography. Ruthenium red staining of thin-section preparations and subsequent electron microscopy failed to reveal an extensive extracellular polysaccharide layer. SDS-PAGE analysis of crude outer membrane fractions of WAL 9077 and WAL 8448 demonstrated clear differences in their major and minor outer membrane protein components. Thus, we postulate that the adherence of B. wadsworthia to Int 407 cells is mediated by an outer membrane or cell wall component.

Little information is available about porin molecules in anaerobes. Porins from Bacteroides fragilis and Porphyromonas, Fusobacterium, and Campylobacter species have been described. A pore-forming outer membrane (OM) porin protein was isolated from B. fragilis (Omp-200); it is exposed at the cell surface and dissociated by boiling and application of reducing agents. Fusobacterium nucleatum FomA, an OM porin protein of 40 kD, had a deduced topology of FomA similar to that of established porins, despite the lack of sequence similarity. An OM preparation from Porphyromonas endodontalis (including a major protein with an apparent molecular mass of 31 kD and other proteins of 40.3-71.6 kD) formed pores in a liposome assay. A major outer membrane protein (MOMP) from Campylobacter jejuni (a microaerophile) is related to the family of trimeric bacterial porins, although little homology was seen with other porins. The development of antimicrobial resistance related to decreased permeability underlines the importance of identifying and characterizing the pore-forming molecules of anaerobes.