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.

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.

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.

Impact of imipenem/cilastatin therapy on normal fecal flora.

The impact of parenteral imipenem/cilastatin therapy on the bowel flora of six patients was evaluated. Stool samples were collected before and during therapy and qualitative and quantitative bacteriologic studies were performed. Imipenem had no effect on total microorganism counts. Two patients acquired Candida albicans during therapy, and three patients acquired Proteus species. Pseudomonas species in one patient acquired resistance. Imipenem appears to have a relatively modest effect on the bowel flora and apparently does not readily induce resistance in the resident flora as compared with other agents.

Current perspectives on anaerobic infections: diagnostic approaches.

This article discusses when to look for anaerobes, anaerobic infections as clues to other problems in patients, and underlying clinical conditions as clues to the nature of anaerobic infections. Diagnostic approaches, identification methods, and susceptibility testing are reviewed.

In vitro activity of grepafloxacin (OPC-17116) against anaerobic bacteria.

In vitro activity of the quinolone grepafloxacin (OPC-17116) was compared with that of ciprofloxacin, fleroxacin, clindamycin, imipenem, and metronidazole by using the NCCLS-approved Brucella-base-laked blood agar dilution method and breakpoints, when available. Clindamycin, metronidazole, and imipenem inhibited > or = 98% of Bacteroides fragilis at the breakpoint; grepafloxacin, ciprofloxacin, and fleroxacin inhibited 83%, 6%, and 0, respectively, at 2 micrograms/ml. Grepafloxacin inhibited 39% of other B. fragilis group species isolated (80) at breakpoint (< or = 2 micrograms/ml) compared with 100% for metronidazole and imipenem, 83% for clindamycin, 6% for ciprofloxacin, and 1% for fleroxacin. Grepafloxacin demonstrated substantially better activity against B. fragilis than did ciprofloxacin or fleroxacin; overall activity against anaerobes was marginally better than that of ciprofloxacin or fleroxacin.

Six-year retrospective survey of the resistance of Bacteroides fragilis group species to clindamycin and cefoxitin.

Two hundred and forty-six strains of the Bacteroides fragilis group, all clinical isolates, collected at the Wadsworth Veterans Administration Medical Center from 1977 to 1982, were tested for susceptibility to clindamycin and cefoxitin. There was no significant change in resistance to either clindamycin or cefoxitin over the time period tested for any individual species, nor for the B. fragilis group in toto. Striking differences in susceptibility to the two drugs were seen among species of the B. fragilis group. B. fragilis displayed resistance to cefoxitin (32 micrograms/ml) and clindamycin (8 micrograms/ml) of 0.0% and 0.8%, respectively, whereas B. thetaiotaomicron showed resistances of 12.7% to cefoxitin (32 micrograms/ml) and 9% to clindamycin (8 micrograms/ml). B. thetaiotaomicron, B. distasonis, and B. ovatus are distinctly more resistant to cefoxitin than B. fragilis and B. vulgatus. Similarly B. thetaiotaomicron and B. distasonis are much more resistant to clindamycin than are the other B. fragilis group species. It is apparent that determination of species within the B. fragilis group is important in evaluating a potential therapeutic regimen.

The isolation and characterisation of a major outer-membrane protein from Bacteroides distasonis.

An outer-membrane protein (OMP) was isolated from a clinical strain of Bacteroides distasonis. Changes in growth media did not appreciably affect the appearance of this protein in crude outer-membrane preparations examined by SDS-PAGE. However, the proportion of the protein relative to other OMPs was greater in 24-h cultures than in 48-h cultures. The protein could not be readily solubilised by various conventional detergent extraction techniques but treatment of the insoluble material at 100 degrees C with SDS released the protein, as did overnight extraction at 37 degrees C with SDS. This OMP was heat-modifiable, and thus was similar to the OmpA protein of Escherichia coli, with a faster mobility on SDS-PAGE when solubilised at 25 degrees C than at 100 degrees C. The critical temperature for conversion was between 80 degrees C and 90 degrees C. Because of the characteristic heat-modifiability, the protein was called B. distasonis HMP-1 (heat modifiable protein-1). Overnight exposure to EDTA or NaCl at 37 degrees C favoured conversion of the 25 degrees C form to the 100 degrees C form. In intact cells, the protein was labelled by a cell-surface radio-iodination procedure, and thus is at least partially exposed at the cell surface. No reactions between the B. distasonis HMP-1 and antibodies to either E. coli OmpA or E. coli porin were found by Western blot analysis. A B. distasonis OM preparation containing predominantly HMP-1 had pore-forming ability in a liposome assay. This study is the first report of the isolation and characterisation of a heat-modifiable OMP in Bacteroides, and it is the first description of pore-forming activity in a Bacteroides OM fraction.

In vitro activity of selected antibiotics against anaerobes.

Testing antibiotics for their activity against microorganisms is fraught with problems. The various methods and media yield different results, and controversy exists as to which is the most reliable. The technique used in our laboratory has shown wide differences in the susceptibility patterns of Bacteroides strains and other anaerobes to different antibiotics. Particularly with respect to the third-generation cephalosporins, reliable clinical data are needed to determine which in vitro tests most accurately predict clinical efficacy.

Media- and method-dependent variation in MIC values of ceftizoxime for clinical isolates of the Bacteroides fragilis group.

Although ceftizoxime has been used effectively in several clinical trials for infections involving anaerobic bacteria, reports of its in vitro activity against anaerobes are contradictory and confusing. In an effort to clarify the discrepant reports, we tested 90 strains of Bacteroides fragilis group organisms from patients with perforated or gangrenous appendicitis using eight different susceptibility testing procedures. The minimal inhibitory concentration values were dependent on the technique used; agar dilution values were often four twofold dilutions higher than microbroth dilution values. Agar techniques (including spiral gradient end point) gave values of 36% to 61% susceptible at breakpoint (depending on the technique), while the microbroth dilution techniques gave values of 84% to 92% susceptible. When a 64 microgram/ml breakpoint for agar dilution testing was used, the methods were more comparable, with the agar methods giving values of 64% to 83% susceptible. The results of the broth disk elution procedure were difficult to read and often did not agree with other values.

Antibiotic susceptibility testing of anaerobic organisms using the agar dilution method: comparison of three techniques.

Three currently used anaerobic susceptibility testing methods were compared: (1) the technique used at the Wadsworth Microbial Diseases Research Laboratory, (2) the technique listed as the reference standard by the National Committee on Clinical Laboratory Standards, and (3) the technique used at the Tufts New England Medical Center. Four-hundred-seventy anaerobic microorganisms, isolated from clinical specimens, were tested against cefoxitin, cefotetan, ceftizoxime, cefotaxime, ceftazidime, imipenem, and clindamycin. Significant differences were noted in mean inhibitory concentrations and percent susceptible at breakpoint among the three techniques used and varied with the antimicrobial agent and species tested.

Effect of inoculum size and medium on activity of seven antimicrobial agents against Bacteroides fragilis strains.

Antimicrobial activity of cefoxitin, cefotetan, cefotaxime, ceftizoxime, ceftazidime, imipenem, and clindamycin against four inocula of Bacteroides fragilis strains was determined on three different media. The inoculum sizes were 10(4), 10(5), 10(6), and 10(7) colony forming units (CFU) per spot. On all three media, substantial effects of inoculum size on minimal inhibitory concentrations (MIC) of cefotaxime and ceftizoxime were found: the doubled dilution differences in MICs between inocula of 10(7) and 10(4) CFU/spot were 2.2, 2.3, and 2.1 micrograms/ml of cefotaxime and 1.8, 4.4, and 4.0 micrograms/ml of ceftizoxime on Brucella base-laked blood agar, Wilkins-Chalgren agar, and a brain-heart infusion medium, respectively. An inoculum difference found on all three media with ceftazidime may also be of practical significance. There was evidence of larger differences between inocula on the Wilkins-Chalgren agar and brain-heart infusion than on the Brucella agar.

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.

Media- and method-dependent variation in MIC values of ceftizoxime for clinical isolates of the Bacteroides fragilis group.

Although ceftizoxime has been used effectively in several clinical trials for infections involving anaerobic bacteria, reports of its in-vitro activity against anaerobes are contradictory and confusing. In an effort to clarify the discrepant reports, we tested 90 strains of Bacteroides fragilis group organisms from patients with perforated or gangrenous appendicitis using eight different susceptibility testing procedures. The minimal inhibitory concentration values were dependent on the technique used; agar dilution values were often four twofold dilutions higher than microbroth dilution values. Agar techniques (including spiral gradient end point) gave values of 36% to 61% susceptible at breakpoint (depending on the technique), while the microbroth dilution techniques gave values of 84% to 92% susceptible. When a 64 micrograms/ml breakpoint for agar dilution testing was used, the methods were more comparable, with the agar methods giving values of 64% to 83% susceptible. The results of the broth disk elution procedure were difficult to read and often did not agree with other values.

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.

Biochemical and polyacrylamide gel electrophoretic analyses of vaginosis-associated anaerobic curved rods.

Morphologic, biochemical, and sodium-dodecyl sulfate polyacrylamide gel electrophoresis characteristics of twenty-six anaerobic curved rods isolated from vaginal discharge or transcervical uterine cultures were compared with each other and with those of five known control strains of anaerobic bacilli. The curved rods could be divided into two distinct groups, based on shared morphological characteristics and similar patterns of major cell protein bands. The protein band pattern exhibited by Group I organisms, which were short, Gram-variable, comma-shaped bacilli, did not resemble the protein band pattern of Group II organisms, which were seen as long, thin, Gram-negative curved rods. Polyacrylamide gel electrophoretic analysis of protein band patterns of these curved rods failed to show any strong similarities to the patterns of the five known bacterial strains that were tested. Although the role of these organisms in the etiology of bacterial vaginosis is not known at present, characterization studies such as this should facilitate the further recognition and identification of these bacilli as a prerequisite to determining their significance in human infection.

Susceptibility testing of anaerobic bacteria: myth, magic, or method?

The demand for susceptibility testing of anaerobes has increased, yet consensus as to procedure and interpretation in this area has not been achieved. While routine testing of anaerobic isolates is not needed, certain isolates in specific clinical settings should be tested. Also, laboratories may monitor their local antibiograms by doing periodic surveillance batch testing. The National Committee for Clinical Laboratory Standards has published a protocol of methods approved for susceptibility testing of anaerobic bacteria. Both agar and broth microdilution are included; however, the broth disk elution method is no longer approved by the National Committee for Clinical Laboratory Standards because of method-related interpretive errors. A number of newer methods are undergoing evaluation and seem promising. Clinicians and microbiologists reviewing susceptibility reports should be aware of sources of variability in the test results. Variables in susceptibility testing of anaerobes include the media and methods used, organisms chosen for testing, breakpoints chosen for interpretation, antibiotic, and determination of endpoint. Clustering of MICs around the breakpoint may lead to significant variability in test results. Adherence of testing laboratories to approved methods and careful descriptions of the method and the breakpoints used for interpretation would facilitate interlaboratory comparisons and allow problems of emerging resistance to be noted. A variety of resistance mechanisms occurs in anaerobic bacteria, including the production of beta-lactamase and other drug-inactivating enzymes, alteration of target proteins, and inability of the drug to penetrate the bacterial wall. Antimicrobial resistance patterns in the United States and abroad are described.

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.

Susceptibility testing of anaerobic bacteria--the state of the art.

Demand for susceptibility testing of anaerobes has increased, but no consensus on procedure and interpretation has been achieved. The need for reliable methods for testing anaerobic bacteria extends from small hospital laboratories to large research centers. Agar dilution testing is too costly and labor intensive for many clinical laboratories. Microbroth dilution is more convenient; however, some fastidious anaerobes do not grow well enough in this system, and the choice of antimicrobial agents may not reflect the hospital formulary. Disapproval of the broth disk elution system leaves fewer options open to clinical laboratories and emphasizes the need for more convenient and reliable techniques. Some newer methods are undergoing evaluation. Variables in susceptibility testing of anaerobes include the media and methods used, organisms chosen, breakpoints chosen, and endpoint determination. This latter variable is probably the most problematic since no endpoint based on interaction of organism and antimicrobial agent rather than on subjective observation has been defined. Also, clustering of MICs around the breakpoint may lead to significant variability in reported methods. A more accurate MIC measurement is needed. Adherence of laboratories to approved methods and careful reporting of methods and the interpretive breakpoints would facilitate interlaboratory comparisons and the identification of emerging resistance.