Genetic analysis of an ambler class A extended-spectrum beta-lactamase from Capnocytophaga ochracea

A beta-lactamase gene (cfxA3, 966 bp) was isolated from a beta-lactam-resistant Capnocytophaga ochracea clinical isolate and amplified using primers from the cfxA gene of Bacteroides vulgatus. The MICs of third-generation cephalosporins were much higher than those of the transconjugant Escherichia coli strain. The deduced protein sequence, by comparison with CfxA2 of Prevotella intermedia, had a Y239D substitution and possessed the characteristics of a class A, group 2e beta-lactamase.     

Annual incidence, epidemiology, and comparative in vitro susceptibilities to cefoxitin, cefotetan, cefmetazole, and ceftizoxime of recent community-acquired isolates of the Bacteroides fragilis group.

The six species of the Bacteroides fragilis group are potent pathogens and commonly have different susceptibility patterns. We determined the relative annual isolation rate of anaerobic bacteria and the susceptibility of B. fragilis group species isolated during 1987 at two community hospitals. The relative frequencies of isolation of 261 strains were as follows: B. fragilis, 61%; B. thetaiotaomicron, 17%; B. distasonis, 7%; B. vulgatus, 6%; B. ovatus, 5%; and B. uniformis, 4%. A total of 234 recent clinical isolates were tested against cefmetazole, cefotetan, cefoxitin, ceftizoxime, clindamycin, imipenem, and piperacillin by a brucella agar dilution method. Imipenem was the most active agent tested with all but three isolates (two B. vulgatus and one B. distasonis) susceptible to less than 2 micrograms/ml. Of the cephalosporins tested, cefoxitin, cefotetan, and cefmetazole were relatively equal against B. fragilis, with 93 to 98% of strains susceptible to 32 micrograms/ml. Ceftizoxime was less active, with an MIC for 90% of strains tested of 128 micrograms/ml and only 75% of isolates susceptible to 32 micrograms/ml. Against B. ovatus, B. vulgatus, B. thetaiotaomicron, and B. uniformis, cefoxitin showed a two- to threefold-superior activity compared with that of cefotetan and cefmetazole. In general, ceftizoxime was much less active, except against B. distasonis, for which 78% of isolates were susceptible to 32 micrograms/ml compared with 68% for cefoxitin, 19% for cefmetazole, and 16% for cefotetan. Clindamycin and piperacillin showed activity similar to that of cefoxitin, except piperacillin was less active versus B. vulgatus and B. distasonis. We therefore suggest that clinical laboratories determine the species of B. fragilis group isolates as well as perform susceptibility studies on the isolates. Clinicians should be aware that while B. fragilis is the most frequent isolate, 38% of isolates are from other, more resistant B. fragilis group species.     

Encapsulation of Bacteroides species

Capsules were detected by the India ink method in cultures of Bacteroides fragilis, B. vulgatus, B. thetaiotaomicron, and B. ovatus. No capsules were found in the five strains of B. distasonis examined.     

Differentiation of Bacteroides fragilis species by gas chromatographic detection of phenylacetic acid.

Of 382 strains tested, 261 strains of the species Bacteroides fragilis, B. thetaiotaomicron, B. ovatus, and B. distasonis produced phenylacetic acid; the remaining strains, belonging exclusively to the species B. vulgatus, failed to do so. This differentiation characteristic may be useful in routine clinical bacteriology.     

Superoxide dismutase in Bacteroides fragilis and related Bacteroides species.

Superoxide dismutase (SOD) activity was demonstrated in cell-free extracts of Bacteroides fragilis, Bacteroides vulgatus, Bacteroides distasonis, Bacteroides ovatus, and Bacteroides thetaiotaomicron. The strains were grown under anaerobic conditions in Trypticase soy broth, and the specific activity of SOD in the extracts was, in most strains, higher than in cell-free extracts of Escherichia coli B grown under anaerobic conditions. Isoelectric focusing of the extracts in polyacrylamide gel demonstrated distinct forms of SOD in the different species.     

Post-antibiotic effect in Bacteroides fragilis group

Post-antibiotic effect (PAE) is the transient suppression of bacterial growth after brief antimicrobial exposure. While numerous reports have described PAE with aerobic and facultative microorganisms, virtually no studies have been conducted with anaerobic isolates. Intraabdominal isolates of the Bacteroides fragilis group were exposed for one hour to antibiotic (cefoxitin, cefotetan, and imipenem) concentrations two to four times the minimal inhibitory concentration (MIC). Post-antibiotic effect was described as the difference between the time required for microbial growth in the test versus the control to increase one Log10 above the quantitation observed immediately after drug removal. Bacteroides fragilis, B. ovatus, B. thetaiotaomicron and B. vulgatus exhibit PAEs for all test compounds. The time intervals for the PAEs were strain variable and ranged from six to 50 hours. No PAE was demonstrated with B. distasonis strains by the broth dilution technique. The results suggest that brief high dose exposure of some members of the B. fragilis group to anaerobe active beta-lactams produces a prolonged suppression in growth. In theory, a prolonged PAE could influence the dosage regimentation of selective antibiotics.     

Detection of beta-lactamases in the Bacteroides fragilis group

One hundred and thirty three strains of Bacteroides fragilis group isolated from clinical specimen were tested for beta-lactamase activity against five beta-lactam antibiotics, ampicillin, cefaloridin, cefuroxime, cefotaxime and cefoxitin. The Minimal Inhibitory Concentration of antibiotics was determined using the agar dilution method. Beta-lactamase production was detected by a microbiological method in 128 of the 133 (96%) strains. The detected beta-lactamases had a broad-spectrum activity, hydrolyzing both penicillins and cephalosporins (101 strains). Some strains had a wide activity against beta-lactam antibiotics, including cefoxitin (7 strains); among these strains, 3 were found hydrolyzing imipenem.     

Pathogenicity of the Bacteroides fragilis group

The Bacteroides fragilis group is one of the most important pathogens in polymicrobial infections. The distribution of the different members of the B. fragilis group in clinical infections varies. Bacteroides fragilis accounts for 63 percent of all the group isolates, Bacteroides thetaiotaomicron for 14 percent, Bacteroides vulgatus and Bacteroides ovatus for seven percent each, Bacteroides distasonis for six percent and Bacteroides uniformis for two percent. All members of the group induced bacteremia that was associated with an average mortality of 27 percent. The B. fragilis group resist beta lactam antibiotics by producing the enzyme beta-lactamase. This enzyme can be detected in abscess fluid, and can interfere with the eradication of other bacteria that are susceptible to penicillins and cephalosporins. All members of the B. fragilis group can become encapsulated during an inflammatory process as was demonstrated in a subcutaneous abscess model in mice. Non-encapsulated strains can become encapsulated with the assistance of their aerobic counterparts. These encapsulated strains are more virulent to the host than non-encapsulated strains. This increased virulence can be demonstrated by a higher rate of induction of bacteremia, and a greater enhancement of growth of other bacteria in mixed infection. Antimicrobial therapy directed only at the eradication of the aerobic bacteria did not prevent encapsulation, or reduce the number of Bacteroides species. The virulence of all members of the B. fragilis group highlights the need to direct antimicrobial therapy against all members of this group.     

Bactericidal activity of a granule extract from human polymorphonuclear leukocytes against Bacteroides species

The microbicidal activity of an acetate extract of human polymorphonuclear leukocyte granules was tested against Bacteroides fragilis, Bacteroides vulgatus, Bacteroides distasonis and Bacteroides thetaiotaomicron. All strains tested were killed by the extract, and there were no significant differences between the different Bacteroides species.     

An investigation of beta-lactamases from clinical isolates of Bacteroides species.

Among a group of 116 clinically significant isolates of Bacteroides spp., 24 exhibited beta-lactamase activity greater than the basal level characteristic of most Bacteroides strains. Investigation of specific enzyme activity, iso-electric point and enzyme inhibition profiles revealed that the beta-lactamases involved could be divided into four groups, some showing similarity to those described in previous studies. Seven of the enzymes were able to hydrolyse cefoxitin, latamoxef or imipenem, and eight enzymes degraded penicillin in the presence of clavulanic acid. Five strains showed reduced susceptibility to cefoxitin, latamoxef or imipenem which was not associated with beta-lactamase activity.     

Detection of cfxA and cfxA2, the beta-lactamase genes of Prevotella spp., in clinical samples from dentoalveolar infection by real-time PCR

While most bacteria involved in dentoalveolar infection are highly susceptible to penicillin, some Prevotella strains exhibit resistance to this agent through the production of beta-lactamase. The production of beta-lactamase by Prevotella spp. is in turn associated with the expression of the genes cfxA and cfxA2. The aim of the present study was to determine the prevalence of cfxA and cfxA2 in Prevotella strains by use of real-time PCR and to assess the performance of this molecular method for the direct detection of the genes in 87 clinical samples (pus and root canal exudates) from dentoalveolar infection. Production of beta-lactamase by each isolate was determined using a nitrocefin disk. beta-Lactamase production was seen in 31% of Prevotella isolates, while all isolates of other species were beta-lactamase negative. The penicillin resistance of isolates strongly correlated with the production of beta-lactamase. Real-time PCR was found to detect the cfxA and cfxA2 genes from at least five cells per reaction mixture (5 x 10(3) CFU/ml of pus). Using real-time PCR, the presence of cfxA and cfxA2 was evident for all 48 beta-lactamase-positive Prevotella strains. In contrast, neither beta-lactamase-negative Prevotella (n = 91) or non-Prevotella (n = 31) strains were positive for the genes. In this study, 31 of the 87 samples yielded beta-lactamase-positive Prevotella results, and cfxA and cfxA2 were detected in all 31 samples. Of the 56 culture-negative samples, 8 (14%) were positive for cfxA and cfxA2 by the real-time PCR. This sensitive and specific molecular method offers a rapid clinical test for aiding in the selection of an appropriate antibiotic for treatment of dentoalveolar infection. Although penicillin remains largely effective in the treatment of dentoalveolar infection, beta-lactamase-stable antibiotics should be considered in cases in which beta-lactamase-positive Prevotella strains are involved.     

Specificity of immunoglobulin M antibodies in normal human serum that participate in opsonophagocytosis and intracellular killing of Bacteroides fragilis and Bacteroides thetaiotaomicron by by human polymorphonuclear leukocytes

Studies were performed to determine the specificity of immunoglobulin M (IgM) antibodies in normal human serum that participate in opsonophagocytosis and intracellular killing of Bacteroides fragilis 1365 and Bacteroides thetaiotaomicron 1343 by human polymorphonuclear leukocytes. Purified normal human IgM was adsorbed with washed heat-killed cells of the homologous strains and heterologous strains of B. fragilis, B. thetaiotaomicron, Bacteroides vulgatus, Bacteroides distasonis, and Bacteroides asaccharolyticus and with erythrocytes coated with outer membrane complex prepared from the homologous strains. Hypogammaglobulinemic serum was supplemented with the adsorbed IgM preparations, and the ability of the supplemented sera to support opsonophagocytosis and killing of B. fragilis 1365 and B. thetaiotaomicron 1343 by human polymorphonuclear leukocytes was measured in vitro under anaerobic conditions. Normal IgM adsorbed with heat-killed cells of B. fragilis 1365 and B. thetaiotaomicron 1343 or with erythrocytes coated with outer membrane complex prepared from these strains failed to restore the ability of hypogammaglobulinemic serum to support opsonophagocytosis and intracellular killing of the homologous strain. In contrast, adsorption of normal IgM with heat-killed cells of the heterologous strains did not alter its opsonophagocytosis-promoting activity for either test strain. These results indicated that the IgM antibodies in normal human serum that participate in opsonophagocytosis and intracellular killing of B. fragilis 1365 and B. thetaiotaomicron 1343 are directed against strain-specific antigenic determinants contained in the outer membrane complex.     

RNA polymerase β-subunit gene (rpoB) sequence analysis for the identification of Bacteroides spp.

Partial rpoB sequences (317 bp) of 11 species of Bacteroides, two Porphyromonas spp. and two Prevotella spp. were compared to delineate the genetic relationships among Bacteroides and closely related anaerobic species. The high level of inter-species sequence dissimilarities (7.6-20.8%) allowed the various Bacteroides spp. to be distinguished. The position of the Bacteroides distasonis and Bacteriodes merdae cluster in the rpoB tree was different from the position in the 16S rRNA gene tree. Based on rpoB sequence similarity and clustering in the rpoB tree, it was possible to correctly re-identify 80 clinical isolates of Bacteroides. In addition to two subgroups, cfiA-negative (division I) and cfiA-positive (division II), of Bacteroides fragilis isolates, two distinct subgroups were also found among Bacteroides ovatus and Bacteroides thetaiotaomicron isolates. Bacteroides genus-specific rpoB PCR and B. fragilis species-specific rpoB PCR allowed Bacteroides spp. to be differentiated from Porphyromonas and Prevotella spp., and also allowed B. fragilis to be differentiated from other non-fragilisBacteroides spp. included in the present study.     

Encapsulated strains ofBacteroides fragilis in clinical specimens

Studies were made of the presence of capsules in 294 strains of theBacteroides fragilis group of bacteria isolated from clinical infections and in 30 strains from the fecal flora using the Indian ink and ruthenium red staining methods. A total of 77% of theB. fragilis strains were encapsulated; higher figures were observed in isolates from blood and wound infections. Of theB. ovatus, B. distasonis, B. vulgatus, B. uniformis, andB. thetaiotaomicron isolates from clinical infections, 17% were encapsulated. The percentage of encapsulatedBacteroides strains isolated from feces was approximately the same as in the strains isolated from clinical infections.     

Effect of ceftazidime on enterobacteria and Pseudomonas producers of beta-lactamases

The activity of cephalotin, cefoxitin, cefamandole, cefoperazone, cefotaxime, latamoxef, thienamycin, azthreonam, cefsulodine and ceftazidime against beta-lactamase producing strains of Enterobacteriaceae and Pseudomonas was compared by determination of 99% inhibitory concentrations. The isogenic strains studied differed by a single genetic event: mutation, gene amplification, acquisition of a high or low copy-number plasmid or of a transposon and were representative of the major known mechanisms of resistance toward beta-lactams. The results obtained indicate an overall excellent activity of ceftazidime, in particular against Pseudomonas.     

Predicting the susceptibility of anaerobes to cefoperazone, cefotaxime, and cefoxitin with the thioglycolate broth disk procedure.

A variety of clinical anaerobic isolates were tested against cefoperazone (216 strains), cefoxitin (120 strains), and cefotaxime (120 strains) by the thioglycolate anaerobic broth disk method, and the results were compared with the National Committee for Clinical Laboratory Standards reference agar dilution method. The broth disk and reference breakpoint concentrations were as follows: cefoperazone, 60 and 64 or 30 and 32 micrograms/ml; cefotaxime, 30 and 32 micrograms/ml; cefoxitin, 18 and 16 micrograms/ml, respectively. Discrepant results were retested to obtain a mode. There was 99% agreement between the broth disk and reference methods for cefotaxime, 98% for cefoperazone with 60- and 64-micrograms/ml breakpoints and 91% with 30- and 32-micrograms/ml breakpoints, and 75% for cefoxitin. All but one of the strains that produced false susceptibility results by broth disk were members of the Bacteroides fragilis group, 1 with cefoperazone using the 60-micrograms/ml concentration, 14 with cefoperazone at the 30-micrograms/ml concentration, and 27 with cefoxitin. One strain of Clostridium difficile produced false susceptibility results to cefoperazone at the 30-micrograms/ml concentration. The lack of agreement between the broth disk and reference methods with cefoxitin may be a reflection of the number of isolates at the 16-micrograms/ml level and that the broth disk breakpoint was slightly higher than this concentration. Increased incubation time did not improve the results significantly.     

Beta-lactamases of type culture strains of the Bacteroides fragilis group and of strains that hydrolyse cefoxitin, latamoxef and imipenem

Susceptibilities to beta-lactam antibiotics and beta-lactamase content of two groups of Bacteroides strains were compared. Type cultures produced low levels of beta-lactamase and were susceptible to cefoxitin, latamoxef, imipenem and the combination of benzylpenicillin and clavulanic acid. Other Bacteroides strains that produced higher levels of beta-lactamase were generally less susceptible to these antibiotics; this resistance was more closely related to enzyme type than to the amount of enzyme present. The beta-lactamases produced by the test strains fell into three broad groups on the basis of antibiotic degradation and inhibitor profiles: those that inactivated benzylpenicillin, but not cefoxitin, latamoxef or imipenem, and were susceptible to inhibition by beta-lactamase inhibitors; those that hydrolysed benzylpenicillin, cefoxitin and latamoxef, but not imipenem, and which were less susceptible to inhibition by beta-lactamase inhibitors; an enzyme that inactivated all the antibiotics and was not inhibited by beta-lactamase inhibitors.     

Susceptibility to beta-lactam antibiotics and production of beta-lactamase inBacteroides fragilis

Using the agar dilution technique, 231 strains of Bacteroides fragilis, isolated during a 2-year period from human infections, were identified at subspecies level and were tested for susceptibility to 13 beta-lactam antibiotics. The penicillins were benzylpenicillin, ampicillin, carbenicillin, cloxacillin, and the recently described penicillin derivatives cyclacillin, ticarcillin, and PC-904. The following cephalosporin derivatives were tested: cephaloridine, cephalothin, cephalexin, cefamandole and cefuroxime. The cephamycin C derivative cefoxitin was also included in the study. Cefoxitin was the most effective drug tested since more than 80% of the strains were inhibited by 8 microgram/ml or less, and no strain had a minimal inhibitory concentration (MIC) of more than 64 microgram/ml. There was no marked difference in sensitivity among the subspecies with exception of subspecies vulgatus, which was slightly more sensitive to all antibiotics tested. The size of the inoculum was an important factor for obtaining reproducible results in the sensitivity tests. Increased inocula resulted in markedly higher MICs for cephaloridine and cefuroxime. Production of beta-lactamase was performed on all isolates by a chromogenic cephalosporin substrate and about 90% of the strains were found to be beta-lactamase producers.     

Antimicrobial susceptibility of Bacteroides fragilis group isolates in Europe

Objective  To evaluate the activity of old and newer antianaerobic drugs against clinical isolates of Bacteroides fragilis group strains from different parts of Europe.
Methods  Bacteroides fragilis group isolates from 37 laboratories in 19 countries were biochemically characterized. The MICs of seven antimicrobial agents were determined by the agar dilution method as recommended by the NCCLS. Production of beta-lactamase was detected by nitrocefin.
Results  There were 1284 B. fragilis group isolates included in the study. Abdominal infections and wounds were the most common sources of isolation and B. fragilis was the dominating species. Ninety-nine percent of the strains were resistant to ampicillin (breakpoint 2 mg/L), 6% to cefoxitin (64 mg/L), 15% to clindamycin (8 mg/L) and 9% to moxifloxacin (8 mg/L). Less than 1% were resistant to imipenem (16 mg/L), piperacillin-tazobactam (128 mg/L) and metronidazole (32 mg/L). Ninety-six percent of the isolates were beta-lactamase producers.
Conclusions  Antimicrobial resistance among the B. fragilis group is increasing.     

Cefoxitin sensitivity as a marker for inducible beta-lactamases

Inducibility of beta-lactamase activity by cefoxitin was examined in 626 gram-negative clinical isolates selected for amoxycillin and cephalothin resistance. The results indicated that precise identification and cefoxitin sensitivity or resistance could be used to predict the inducibility of beta-lactamase. Of 326 organisms from species capable of beta-lactamase induction, induction was shown in 68% and was predictable from the cefoxitin-sensitivity and identification data. No induction of beta-lactamase occurred in the remaining species. A comparison of beta-lactamase activities against cefotaxime, cefoperazone and latamoxef showed that induction of enzyme activity against cefotaxime and cefoperazone occurred at similar rates. Induction of activity against latamoxef did not occur or was minimal with three bacterial species. The data show that of 119 strains of Enterobacteriaceae displaying inducible beta-lactamase, 113 would have been reported as unequivocally sensitive to cefotaxime, 109 as sensitive to cefoperazone and 116 as sensitive to latamoxef if the disk-diffusion technique alone had been used. The majority of Pseudomonas strains examined produced inducible enzyme and they were more resistant to the three cephalosporins tested than were the Enterobacteriaceae.