Genetic analysis of mechanisms of multidrug resistance in a clinical isolate of Bacteroides fragilis.

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.

Multilaboratory comparison of growth characteristics for anaerobes, using 5 different agar media.

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.

Multilaboratory comparison of anaerobe susceptibility results using 3 different agar media.

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.

Bacteriologic findings associated with chronic bacterial maxillary sinusitis in adults.

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.

In vitro activity of telithromycin (HMR 3647) against 502 strains of anaerobic bacteria.

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.

Short-term benefit from oral vancomycin treatment of regressive-onset autism.

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.

In vitro activities of MK-826 (L-749,345) against 363 strains of anaerobic bacteria.

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 vitro activity of levofloxacin against a selected group of anaerobic bacteria isolated from skin and soft tissue infections.

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.

Current susceptibility patterns of anaerobic bacteria.

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.

Adherence of Bilophila wadsworthia to cultured human embryonic intestinal cells.

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.

Pore-forming molecules in gram-negative anaerobic bacteria.

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.

In vitro susceptibility of anaerobes to quinolones in the United States.

The in vitro activity of early fluoroquinolone antibodies--including ciprofloxacin, ofloxacin, fleroxacin, pefloxacin, enoxacin, and lomefloxacin--against most anaerobes has been limited, a characteristic making them poor choices as antianaerobic agents. Newer fluoroquinolones, including levofloxacin, sparfloxacin, and grepafloxacin, have moderate activity against anaerobes, including the Bacteroides fragilis group as well as Clostridium, Peptostreptococcus, Prevotella, and Fusobacterium species. Fluoroquinolones that demonstrate the greatest activity against the B. fragilis group and other anaerobes include DU-6859a, clinafloxacin, and the related naphthyridone, trovafloxacin. There has been wide variation in the susceptibility results among different studies testing the same antibiotic; such variation may be due in part to the use of different methodologies, inoculum sizes, and testing media. In a direct comparison of susceptibility findings for ciprofloxacin, ofloxacin, and levofloxacin in three different media, we have determined that twofold dilution differences in minimum inhibitory concentration (MIC) values (MIC90, mode MIC, and geometric mean MIC) may occur in association with the choice of testing media. Thus, testing media should be considered when comparing results of different studies on the susceptibility of anaerobes to fluoroquinolones.

Present studies of therapy for anaerobic infections.

Therapeutic approaches to anaerobic infections are changing. Debridement, drainage, and other surgical approaches remain extremely important. Resistance to antimicrobial agents currently used for treatment of anaerobic infections is increasing. However, promising new agents are being introduced. We review the current status of therapy for anaerobic infections and discuss the potential role of these new agents. We stress an empirical approach to therapy that is based on the usual infecting flora in various types of infections.

In vitro activities of trovafloxacin against 557 strains of anaerobic bacteria.

The antimicrobial activity of trovafloxacin for 557 strains of anaerobic bacteria was determined by the National Committee for Clinical Laboratory Standards-approved Wadsworth agar dilution technique. The species tested included Bacteroides fragilis (n = 91), other members of the B. fragilis group (n = 130), Campylobacter gracilis (n = 15), other Bacteroides spp. (n = 16), Prevotella spp. (n = 49), Porphyromonas spp. (n = 15), Fusobacterium spp. (n = 62), Bilophila wadsworthia (n = 24), Sutterella wadsworthensis (n = 21), Clostridium spp. (n = 61), Peptostreptococcus spp. (n = 38), and gram-positive non-spore-forming rods (n = 35). Trovafloxacin inhibited all strains of B. fragilis at < or = 0.5 microgram/ml, 99% of other B. fragilis group species at < or = 2 micrograms/ml, and 96% of all anaerobes tested at < or = 2 micrograms/ml.

Comparison of spiral gradient endpoint and agar dilution methods for susceptibility testing of anaerobic bacteria: a multilaboratory collaborative evaluation.

A multilaboratory collaborative study was carried out to assess the utility of the spiral gradient endpoint (SGE) method for the determination of the antimicrobial susceptibilities of anaerobes and to evaluate the equivalence of the MICs obtained by the SGE method with those obtained by the reference agar dilution method of the National Committee for Clinical Laboratory Standards. The standard deviation of the MIC obtained by the SGE method for the five participating laboratories was +/- 0.26 of a twofold dilution, whereas it was +/- 1 twofold dilution by the reference method. The interlaboratory reproducibility of the results for two control strains tested with imipenem, chloramphenicol, and metronidazole indicated that 96% of the measurements fell within +/- 1 twofold dilution of the mode. The equivalence of the SGE method with the agar dilution method was assessed with a wide variety of anaerobic organisms. The MICs by both methods were within 1 doubling dilution in 93% of the measurements (n = 1,074). Discrepancies generally occurred with those organism-drug combinations that resulted in tailing endpoints (Fusobacterium nucleatum, 86% agreement) or in cases of light growth (Peptostreptococcus spp., 86% agreement).

Sutterella wadsworthensis gen. nov., sp. nov., bile-resistant microaerophilic Campylobacter gracilis-like clinical isolates.

Campylobacter gracilis (formerly Bacteroides gracilis) is an asaccharolytic, nitrate-positive, urease-negative organism that requires formate and fumarate or hydrogen as a growth additive and may pit agar media. Clinical isolates that were obtained primarily from appendiceal and peritoneal fluid specimens and initially were identified in our laboratory as B. gracilis were later found to include "unusual" strains that could be distinguished by biochemical and genetic criteria. These unusual C. gracilis strains were bile resistant, could not reduce tetrazolium chloride under aerobic conditions if formate and fumarate were added to the medium, and could grow in the presence of 2 or 6% oxygen if no blood was added to the medium. C. gracilis, other campylobacters, and the unusual strains produced distinctive dehydrogenase patterns when gels were incubated anaerobically. A cellular fatty acid analysis revealed that the cluster formed by the unusual organisms was distinct from the (separate) clusters formed by C. gracilis, Bacteroides ureolyticus, and other Campylobacter species. 16S rRNA sequence data indicated that these organisms are not related phylogenetically to either C. gracilis or other Campylobacter species; the most closely related taxa as determined by rRNA sequence analysis were unrelated aerobes (members of the genera Bordetella, Alcaligenes, Rhodocyclus, and Comamonas). DNA homology data confirmed that these taxa are separate groups. Our data indicate that the unusual organisms are members of a new genus and new species, for which we propose the name Sutterella wadsworthensis. The type strain of S. wadsworthensis is strain WAL 9799 (= ATCC 51579).