Trends in antimicrobial resistance among Bacteroides species and Parabacteroides species in the United States from 2010-2012 with comparison to 2008-2009.

The susceptibility trends for Bacteroides fragilis and related species against various antibiotics were determined using data from 3 years of surveillance (2010-2012) on 779 isolates referred by 7 medical centers. The antibiotic test panel included imipenem, ertapenem, meropenem, ampicillin-sulbactam, piperacillin-tazobactam, cefoxitin, clindamycin, moxifloxacin, tigecycline, linezolid, chloramphenicol and . MICs were determined using the agar dilution CLSI reference method. Carbapenem resistance remained low (range 1.1%-2.5%) and unchanged from 2008 to 9 through 2010-2012. Resistance also remained low to the beta-lactam/beta-lactamase inhibitor combinations (1.1%-4.4%). While resistance to clindamycin and moxifloxacin remained high; rates were lower for B. fragilis in 2010-12 (24% and 19% respectively) compared to the earlier time frame of 2008-9 (29% and 35% respectively for the earlier time frame). There were notable species and resistance associations which have been demonstrated previously. No resistance to metronidazole or chloramphenicol resistance was seen. These data demonstrate the continued variability in resistance among Bacteroides and Parabacteroides species, but do demonstrate that carbapenems and beta-lactam/beta-lactamase inhibitor combinations remain very active throughout the United States.

Susceptibility of Clostridium difficile isolates from a Phase 2 clinical trial of cadazolid and vancomycin in C. difficile infection.

OBJECTIVES: The aim of this study was to evaluate the susceptibilities of Clostridium difficile isolates to cadazolid, a novel antibiotic for the treatment of C. difficile infection. METHODS: Ribotyping and susceptibilities were determined for C. difficile isolates from a multicentre, double-blind, Phase 2 study of oral cadazolid in patients with C. difficile infection (NCT01222702, ClinicalTrials.gov; EudraCT 2010-020941-29, European Clinical Trials Database). Patients were randomized to receive 250, 500 or 1000 mg of cadazolid twice daily or 125 mg of vancomycin four times daily, for 10 days. MICs of cadazolid, vancomycin, fidaxomicin, linezolid and moxifloxacin were determined at baseline for all patients and post-baseline for patients with clinical failure or recurrence, using the agar dilution method. RESULTS: Seventy-eight of 84 patients had an evaluable toxigenic C. difficile isolate at baseline. The most frequent PCR ribotype was 027 (15.4%). Cadazolid MICs for baseline isolates (including epidemic strain 027) ranged from 0.06 to 0.25 mg/L. Baseline cadazolid MICs were similar to those of fidaxomicin and lower than those of vancomycin, linezolid and moxifloxacin. For each clinical outcome group (clinical cure, clinical failure, sustained clinical response and clinical failure or recurrence), the baseline cadazolid MIC range was 0.06-0.25 mg/L. Mean (min-max) cadazolid faecal concentration (µg/g) on day 5 was 884 (101-2710), 1706 (204-4230) and 3226 (1481-12 600) for the doses 250, 500 and 1000 mg, respectively. CONCLUSIONS: For all cadazolid doses, the faecal concentration was in excess of several thousand-fold the MIC90 for C. difficile. The MIC of cadazolid for all C. difficile isolates, including epidemic strains, was low and in the same narrow range regardless of treatment outcome.

Update on resistance of Bacteroides fragilis group and related species with special attention to carbapenems 2006-2009.

The susceptibility trends for the species of the Bacteroides fragilis group against various antibiotics were determined using data from 4 years [2006-2009] on 1957 isolates referred by 8 medical centers participating in a National Survey for the Susceptibility of B. fragilis. The antibiotic test panel included doripenem, ertapenem, imipenem, meropenem, ampicillin:sulbactam, piperacillin:tazobactam, cefoxitin, clindamycin, moxifloxacin, tigecycline, chloramphenicol and metronidazole. MICs were determined using agar dilution methods following CLSI recommendations. Genetic analysis of isolates from 2008 with elevated MICs (>2 µg/mL) to one or more of the carbapenems to detect presence of the cfiA gene was performed using PCR methodology. The results showed an increase in the resistance rates to the ß-lactam antibiotics. High resistance rates were seen for clindamycin and moxifloxacin (as high as 60% for clindamycin and >80% for moxifloxacin), with relatively stable low resistance (5.4%) for tigecycline. For carbapenems, resistance in B. fragilis was 1.1%-2.5% in 2008-9. One isolate resistant to metronidazole (MIC 32 µg/mL) was observed as well as isolates with elevated MICs to chloramphenicol (16 µg/mL). Genetic analysis indicated that the cfiA gene was present in some but not all of the isolates with high MICs to the carbapenems. These data indicate that there continue to be changes in susceptibility over time, and that resistance can be seen among the carbapenems. High antibiotic resistance rates tend to be associated with specific species.

National survey on the susceptibility of Bacteroides fragilis group: report and analysis of trends in the United States from 1997 to 2004.

The susceptibility trends for the species of the Bacteroides fragilis group against various antibiotics from 1997 to 2004 were determined by using data for 5,225 isolates referred by 10 medical centers. The antibiotic test panel included ertapenem, imipenem, meropenem, ampicillin-sulbactam, piperacillin-tazobactam, cefoxitin, clindamycin, moxifloxacin, tigecycline, chloramphenicol, and metronidazole. From 1997 to 2004 there were decreases in the geometric mean (GM) MICs of imipenem, meropenem, piperacillin-tazobactam, and cefoxitin for many of the species within the group. B. distasonis showed the highest rates of resistance to most of the beta-lactams. B. fragilis, B. ovatus, and B. thetaiotaomicron showed significantly higher GM MICs and rates of resistance to clindamycin over time. The rate of resistance to moxifloxacin of B. vulgatus was very high (MIC range for the 8-year study period, 38% to 66%). B. fragilis, B. ovatus, and B. distasonis and other Bacteroides spp. exhibited significant increases in the rates of resistance to moxifloxacin over the 8 years. Resistance rates and GM MICs for tigecycline were low and stable during the 5-year period over which this agent was studied. All isolates were susceptible to chloramphenicol (MICs < 16 microg/ml). In 2002, one isolate resistant to metronidazole (MIC = 64 microg/ml) was noted. These data indicate changes in susceptibility over time; surprisingly, some antimicrobial agents are more active now than they were 5 years ago.

Characterization of the Bacteroides fragilis pathogenicity island in human blood culture isolates.

Bacteroides fragilis is an important anaerobic pathogen accounting for up to 10% of bacteremias in adult patients. Enterotoxin producing B. fragilis (ETBF) strains have been identified as enteric pathogens of children and adults. In order to further characterize the B. fragilis pathogenicity island (BfPAI) and using PCR assays for bft- and mpII-metalloprotease genes, we determined the frequency of B. fragilis strains with pattern I (containing the BfPAI and its flanking region), pattern II (lacking both the BfPAI and the flanking region), and pattern III (lacking the BfPAI but containing the flanking region) in 63 blood culture isolates. The results were compared to 197 B. fragilis isolates from different clinical sources. We found 19% of blood culture isolates were pattern I (ETBF), 43% were pattern II (NTBF) and 38% were pattern III (NTBF). Comparatively, B. fragilis isolates from other clinical sources were 10% pattern I, 47% pattern II and 43% pattern III. This suggests that the pathogenicity island and the flanking elements may be general virulence factors of B. fragilis.

Clostridium clostridioforme: a mixture of three clinically important species.

Clostridium clostridioforme shows much variability in phenotypic and antimicrobial susceptibility tests, suggesting it may be more than a single species even though all strains share unique morphology. This study was designed to determine if there are multiple species and, if so, to demonstrate the differences that exist between them. A total of 107 strains of C. clostridioforme were investigated by sequencing of the 16S rRNA gene, phenotypic studies, and antimicrobial susceptibility testing. In addition, clinical data from patients whose infections yielded an organism identified as C. clostridioforme was reviewed. Data from the above studies revealed three principal species in what has been called C. clostridioforme: Clostridium bolteae, C. clostridioforme, and Clostridium hathewayi. Each species may be distinguished by certain phenotypic tests. All three species were involved in infections, including bacteremia. C. clostridioforme appears to be associated with more serious or invasive human infections than the other two species in the group. Resistance to penicillin G is common and is due to beta-lactamase production. Resistance to clindamycin and moxifloxacin is also seen. The three species differ in terms of virulence and antimicrobial resistance. "C. clostridioforme" actually represents three distinct species that are different in terms of 16S rRNA sequences, phenotypic characteristics, and antimicrobial susceptibility. It is important for microbiology laboratories to distinguish between these species and for clinicians to be aware of the differences between them.

In vitro activities of newer quinolones against bacteroides group organisms.

The activities of BMS-284576, clinafloxacin, moxifloxacin, sitafloxacin, trovafloxacin, imipenem, cefoxitin, and clindamycin against 589 Bacteroides fragilis group isolates were determined. The activity of BMS-284576 was comparable to that of trovafloxacin. Sitafloxacin and clinafloxacin were the most active quinolones, and moxifloxacin was the least active. B. fragilis was the most susceptible of the species, and Bacteroides vulgatus was the most resistant. Association of specific antibiotic resistance with Bacteroides species was noted for all quinolones.

National survey on the susceptibility of Bacteroides Fragilis Group: report and analysis of trends for 1997-2000.

The results of a multicenter US survey using the National Committee for Clinical Laboratory Standards currently recommended methodology for measuring in vitro susceptibility of 2673 isolates of Bacteroides fragilis group species were compared from 1997 to 2000. The test panel consisted of 14 antibiotics: 3 carbapenems, 3 beta-lactam-beta-lactamase inhibitors, 3 cephamycins, 2 fluoroquinolones, clindamycin, chloramphenicol, and metronidazole. Declines in the geometric mean minimum inhibitory concentrations were seen with imipenem, meropenem, ampicillin-sulbactam, and the cephamycins. Increased geometric means were observed with the fluoroquinolones and were usually accompanied by an increase in resistance rates. Bacteroides distasonis shows the highest resistance rates among beta-lactam antibiotics, whereas Bacteroides vulgatus shows the highest resistance levels among fluoroquinolones. B. fragilis shows the lowest resistance rates for all antibiotics. All strains were susceptible to chloramphenicol and metronidazole concentrations <8 microgram/mL. The data underscore the need for species identification and continued surveillance to monitor resistance patterns.

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.

Multicenter study of in vitro susceptibility of the Bacteroides fragilis group, 1995 to 1996, with comparison of resistance trends from 1990 to 1996.

Antimicrobial resistance, including plasmid-mediated resistance, among the species of the Bacteroides fragilis group is well documented. An analysis of the in vitro susceptibility of B. fragilis group species referred between 1995 and 1996 as well as during a 7-year (1990 to 1996), prospective, multicenter survey of over 4,000 clinical isolates of B. fragilis group species was undertaken to review trends in the percent resistance to and geometric mean MICs of the antibiotics tested. There was a trend toward a decrease in the geometric mean MICs of most beta-lactam antibiotics, while the percent resistance to most agents was less affected. Within the species B. fragilis, the geometric mean MICs showed significant (P < 0.05) decreases for piperacillin-tazobactam, ticarcillin-clavulanate, piperacillin, ticarcillin, ceftizoxime, cefotetan, and cefmetazole; a significant increase was observed for clindamycin and cefoxitin. For the non-B. fragilis species, a significant decrease in the geometric mean MICs was observed for meropenem, ampicillin-sulbactam, ticarcillin-clavulanate, piperacillin, ticarcillin, ceftizoxime, and cefmetazole; a significant increase was observed for cefoxitin. Significant increases in percent resistance were observed within the B. fragilis strains for ticarcillin and ceftizoxime and within the non-B. fragilis isolates for cefotetan. Significant increases in percent resistance among all B. fragilis group species were observed for clindamycin, while imipenem showed no significant change in resistance trends. The trend analysis for trovafloxacin was limited to 3 years, since the quinolone was tested only in 1994, 1995, and 1996. During the 7 years analyzed, there was no resistance to metronidazole or chloramphenicol observed. The data demonstrate that resistance among the B. fragilis group species has decreased in the past several years, the major exception being clindamycin. The majority of the resistance decrease has been for the beta-lactams in B. fragilis, compared to other species. The reasons for these changes are not readily apparent.

Bacteroides fragilis transfer factor Tn5520: the smallest bacterial mobilizable transposon containing single integrase and mobilization genes that function in Escherichia coli.

Many bacterial genera, including Bacteroides spp., harbor mobilizable transposons, a class of transfer factors that carry genes for conjugal DNA transfer and, in some cases, antibiotic resistance. Mobilizable transposons are capable of inserting into and mobilizing other, nontransferable plasmids and are implicated in the dissemination of antibiotic resistance. This paper presents the isolation and characterization of Tn5520, a new mobilizable transposon from Bacteroides fragilis LV23. At 4,692 bp, it is the smallest mobilizable transposon reported from any bacterial genus. Tn5520 was captured from B. fragilis LV23 by using the transfer-deficient shuttle vector pGAT400DeltaBglII. The termini of Tn5520 contain a 22-bp imperfect inverted repeat, and transposition does not result in a target site repeat. Tn5520 also demonstrates insertion site sequence preferences characterized by A-T-rich nucleotide sequences. Tn5520 has been sequenced in its entirety, and two large open reading frames whose predicted protein products exhibit strong sequence similarity to recombinase-integrase enzymes and mobilization proteins, respectively, have been identified. The transfer, mobilization, and transposition properties of Tn5520 have been studied, revealing that Tn5520 mobilizes plasmids in both B. fragilis and Escherichia coli at high frequency and also transposes in E. coli.

The Bacteroides fragilis BtgA mobilization protein binds to the oriT region of pBFTM10.

The Bacteroides fragilis conjugal plasmid pBFTM10 contains two genes, btgA and btgB, and a putative oriT region necessary for transfer in Bacteroides fragilis and Escherichia coli. The BtgA protein was predicted to contain a helix-turn-helix motif, indicating possible DNA binding activity. DNA sequence analysis of the region immediately upstream of btgA revealed three sets of inverted repeats, potentially locating the oriT region. A 304-bp DNA fragment comprising this putative oriT region was cloned and confirmed to be the functional pBFTM10 oriT by bacterial conjugation experiments using E. coli and B. fragilis. btgA was cloned and overexpressed in E. coli, and the purified protein was used in electrophoretic mobility shift assays, demonstrating specific binding of BtgA protein to its cognate oriT. DNase I footprint analysis demonstrated that BtgA binds apparently in a single-stranded fashion to the oriT-containing fragment, overlapping inverted repeats I, II, and III and the putative nick site.

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.

Comparison of activities of trovafloxacin (CP-99,219) and five other agents against 585 anaerobes with use of three media.

Agar dilution testing was used to compare the in vitro activities of trovafloxacin and ofloxacin, ciprofloxacin, imipenem, metronidazole, and clindamycin against 585 anaerobes. The activity of trovafloxacin was superior to those of ofloxacin and ciprofloxacin, with 94%, 91%, 96%, 100%, 90%, and 100% inhibition of Bacteroides fragilis, non-fragilis Bacteroides species, Peptostreptococcus magnus/Peptostreptococcus micros, Clostridium perfringens, Prevotella bivia/Prevotella disiens, and Fusobacterium species, respectively, at a breakpoint of 2 micrograms/mL. Trovafloxacin was more active than clindamycin against most anaerobes and slightly less active than imipenem and metronidazole. Different testing media, which were compared side by side, did affect the in vitro activities of trovafloxacin, ofloxacin, and ciprofloxacin but did not affect those of the other antibiotics. Testing with supplemented brain-heart infusion agar demonstrated lower minimum inhibitory concentrations than did testing with Wilkins-Chalgren agar and supplemented brucella agar. The activity of trovafloxacin was twofold lower when the pH of the testing media was adjusted to 6.0 than when the pH of the testing media was adjusted to 7.0 or 7.5.

Analysis of trends in antimicrobial resistance patterns among clinical isolates of Bacteroides fragilis group species from 1990 to 1994.

Antimicrobial resistance, including plasmid-mediated resistance, among Bacteroides fragilis group species is well documented. A 5-year (1990-1994) prospective, eight-center survey of 3,177 clinical isolates of Bacteroides species was undertaken to review trends in resistance, using the breakpoints for full and intermediate susceptibility established by the National Committee for Clinical Laboratory Standards. No documented resistance to either metronidazole or chloramphenicol was found in this survey. Among B. fragilis isolates virtually no resistance was seen to imipenem, meropenem, ampicillin/sulbactam, piperacillin/tazobactam, or ticarcillin/clavulanate. Significant increases in resistance among B. fragilis isolates to cefotetan, ceftizoxime, and clindamycin (p < .01) were noted. Resistance to cefoxitin remained unchanged. Among the non-fragilis species of the B. fragilis group, there was virtually no resistance to imipenem, meropenem, chloramphenicol, or metronidazole. The three beta-lactamase inhibitors had increasing levels of resistance, although 95%-98% of strains were susceptible (p < .05). There was a significant decline in cefoxitin, cefmetazole, and clindamycin activity over time against these strains (p <.01). There was a significant (P < .001) increase in geometric mean minimum inhibitory concentration for most drugs and species tested from 1990 to 1994. Clusters in the eight institutions could not account for this rise in resistance. This survey demonstrates that rates of resistance of B. fragilis and non-fragilis species of B. fragilis group are increasing.

Characterization and DNA sequence of the mobilization region of pLV22a from Bacteroides fragilis.

A 4.2-kb plasmid (pLV22a) native to Bacteroides fragilis LV22 became fused to a transfer-deficient Bacteroides spp.-Escherichia coli shuttle vector by an inverse transposition event, resulting in a transferrable phenotype. The transfer phenotype was attributable to pLV22a, which was also capable of mobilization within E. coli when coresident with the IncP beta R751 plasmid. Transposon mutagenesis with Tn1000 localized the mobilization region to a 1.5-kb DNA segment in pLV22a. The mobilization region has been sequenced, and five open reading frames have been identified. Mutants carrying disruptions in any of the three genes designated mbpA, mbpB, and mbpC and coding for deduced products of 11.3, 30.4, and 17.1 kDa, respectively, cannot be mobilized when coresident with R751. Mutations in all three genes can be complemented in the presence of the respective wild-type genes, indicating that the products of mbpA, mbpB, and mbpC have roles in the mobilization process and function in trans. The deduced 30.4-kDa MbpB protein contains a 14-amino-acid conserved motif that is also found in the DNA relaxases of a variety of conjugal and mobilizable plasmids and the conjugative transposon Tn4399. Deletion analysis and complementation experiments have localized a cis-acting region of pLV22a within mbpA.

Susceptibility results for the Bacteroides fragilis group: comparison of the broth microdilution and agar dilution methods.

The antimicrobial susceptibilities of members of the Bacteroides fragilis group were compared using the agar dilution and broth microdilution methods. A total of 455 B. fragilis group isolates were tested against 10 antibiotics. Significant disparity in susceptibility results for most antibiotics was observed between the two methods. Broth microdilution susceptibility results were most similar to agar dilution results when a twofold lower breakpoint was used. In addition, broth microdilution failed to detect resistance to some antibiotics when the recommended agar dilution breakpoint was used. MIC50, MIC90, and geometric mean MIC values for broth microdilution were consistently twofold to fourfold less than those for agar dilution. Susceptibilities of Bacteroides that are determined with use of broth microdilution may more accurately correspond to those determined with use of agar dilution if lower breakpoints are used for interpretation.

Effect of choice of medium on the results of in vitro susceptibility testing of eight antibiotics against the Bacteroides fragilis group.

The susceptibility of isolates of the Bacteroides fragilis group to eight antibiotics was determined by the agar dilution method on three media. At intermediate breakpoints established by the National Committee for Clinical Laboratory Standards (NCCLS), only minimal differences in the susceptibility of each species to any of the antibiotics were documented among the three media. At NCCLS susceptible breakpoints, greater differences among media were found for cefoxitin and cefotetan than for the other drugs tested. Only minimal differences among the three media were evident in comparisons of MIC50, MIC90, and geometric mean MIC values for all antibiotics tested. Thus, use of any one of these three media does not significantly affect agar-dilution MIC results for the B. fragilis group.

Plasmid labeling confirms bacterial translocation in pancreatitis.

To examine whether the gut is a source of infection in acute pancreatitis, bacterial translocation and alterations of intestinal microecology and morphology were studied in 16 dogs. Dogs were colonized with a strain of Escherichia coli (E. coli 6938K) bearing the plasmid pUC4K, which confers kanamycin resistance. In eight dogs (group I), pancreatitis was induced by sodium taurocholate/trypsin injection. Eight other dogs (group II) underwent laparotomy only. The pancreas, mesenteric lymph nodes, peritoneal fluid, liver, and spleen were harvested 7 days later for culturing and histologic analysis. Identification of E. coli 6938K was accomplished by plasmid DNA analysis. Group I dogs had severe pancreatitis and ischemic changes in small bowel mucosa. Group II dogs had no changes. Translocation to the pancreas occurred in five dogs and to mesenteric lymph nodes in six dogs with pancreatitis. No translocation occurred in group II dogs (p < 0.05). In addition to E. coli 6938K, other gram-negative kanamycin-resistant species were isolated, including E. coli (other than 6938K) and Enterobacter cloacae. Enteric origin of these strains was confirmed by antibiography and plasmid DNA analysis. No overgrowth of cecal gram-negative bacteria was found. This study suggests that the gut is a primary source of infection in pancreatitis and that ischemic damage of intestinal mucosa may promote bacterial translocation.