David W. Hecht

In vitro activities of 15 antimicrobial agents against 110 toxigenic clostridium difficile clinical isolates collected from 1983 to 2004.

ABSTRACT The incidence and severity of Clostridium difficile-associated disease (CDAD) is increasing, and standard treatment is not always effective. Therefore, more-effective antimicrobial agents and treatment strategies are needed. We used the agar dilution method to determine the in vitro susceptibility of the following antimicrobials against 110 toxigenic clinical isolates of C. difficile from 1983 to 2004, primarily from the United States: doripenem, meropenem, gatifloxacin, levofloxacin, moxifloxacin, OPT-80, ramoplanin, rifalazil, rifaximin, nitazoxanide, tizoxanide, tigecycline, vancomycin, tinidazole, and metronidazole. Included among the isolates tested were six strains of the toxinotype III, NAP1/BI/027 group implicated in recent U.S., Canadian, and European outbreaks. The most active agents in vitro were rifaximin, rifalazil, tizoxanide, nitazoxanide, and OPT-80 with MICs at which 50% of the isolates are inhibited (MIC50) and MIC90 values of 0.0075 and 0.015 μg/ml, 0.0075 and 0.03 μg/ml, 0.06 and 0.125 μg/ml, 0.06 and 0.125 μg/ml, 0.125 and 0.125 μg/ml, respectively. However, for three isolates the rifalazil and rifaximin MICs were very high (MIC of >256 μg/ml). Ramoplanin, vancomycin, doripenem, and meropenem were also very active in vitro with narrow MIC50 and MIC90 ranges. None of the isolates were resistant to metronidazole, the only agent for which there are breakpoints, with tinidazole showing nearly identical results. These in vitro susceptibility results are encouraging and support continued evaluation of selected antimicrobials in clinical trials of treatment for CDAD.

Rifampin and Rifaximin Resistance in Clinical Isolates of Clostridium difficile

ABSTRACT Rifaximin, a poorly absorbed rifamycin derivative, is a promising alternative for the treatment of Clostridium difficile infections. Resistance to this agent has been reported, but no commercial test for rifaximin resistance exists and the molecular basis of this resistance has not been previously studied in C. difficile. To evaluate whether the rifampin Etest would be a suitable substitute for rifaximin susceptibility testing in the clinical setting, we analyzed the in vitro rifaximin susceptibilities of 80 clinical isolates from our collection by agar dilution and compared these results to rifampin susceptibility results obtained by agar dilution and Etest. We found rifaximin susceptibility data to agree with rifampin susceptibility; the MICs of both antimicrobials for all isolates were either very low or very high. Fourteen rifaximin-resistant (MIC, ≥32 μg/ml) unique isolates from patients at diverse locations in three countries were identified. Molecular typing analysis showed that nine (64%) of these isolates belonged to the epidemic BI/NAP1/027 group that is responsible for multiple outbreaks and increased disease severity in the United Kingdom, Europe, and North America. The molecular basis of rifaximin and rifampin resistance in these isolates was investigated by sequence analysis of rpoB, which encodes the β subunit of RNA polymerase, the target of rifamycins. Resistance-associated rpoB sequence differences that resulted in specific amino acid substitutions in an otherwise conserved region of RpoB were found in all resistant isolates. Seven different RpoB amino acid substitutions were identified in the resistant isolates, which were divided into five distinct groups by restriction endonuclease analysis typing. These results suggest that the amino acid substitutions associated with rifamycin resistance were independently derived rather than disseminated from specific rifamycin-resistant clones. We propose that rifaximin resistance in C. difficile results from mutations in RpoB and that rifampin resistance predicts rifaximin resistance for this organism.

National Survey on the Susceptibility of Bacteroides fragilis Group: Report and Analysis of Trends in the United States from 1997 to 2004

ABSTRACT 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 β-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 μg/ml). In 2002, one isolate resistant to metronidazole (MIC = 64 μg/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.

Prevalence of Antibiotic Resistance in Anaerobic Bacteria: Worrisome Developments

Antibiotic-resistant anaerobic bacteria have become increasingly recognized as a confounding factor in the selection of therapeutic agents. The use of potent, broad-spectrum antibiotics as empirical therapy, along with appropriate adjunctive measures, has, in some ways, masked the magnitude of the antibiotic resistance problem that parallels that observed for nonanaerobic pathogens. The use of standardized testing methods that recognize resistance and an understanding of resistance mechanisms have become essential for the treatment of patients and the development of new agents.

Rifaximin Redux: Treatment of recurrent Clostridium difficile infections with Rifaximin immediately post-vancomycin treatment

We report our continued experience with rifaximin as a post-vancomycin treatment strategy in six patients with multiple recurrences of C. difficile infection (CDI). Four of the six patients (67%) had no further diarrhea episodes, but two patients failed shortly after or during the rifaximin treatment. C. difficile isolates from one of the two patients who failed treatment had an MIC of >256 microg/ml to rifampin. Serial therapy with vancomycin, followed by rifaximin remains an option for some patients with multiple CDI recurrences.

Lessons Learned from the Anaerobe Survey: Historical Perspective and Review of the Most Recent Data (2005–2007)

BACKGROUND The rationale and lessons learned through the evolution of the National Survey for the Susceptibility of Bacteroides fragilis Group from its initiation in 1981 through 2007 are reviewed here. The survey was conceived in 1980 to track emerging antimicrobial resistance in Bacteroides species. METHODS Data from the last 11 years of the survey (1997-2007), including 6574 isolates from 13 medical centers, were analyzed for in vitro antimicrobial resistance to both frequently used and newly developed anti-anaerobic agents. The minimum inhibitory concentrations of the antibiotics were determined using agar dilution in accordance with Clinical and Laboratory Standards Institute recommendations. RESULTS The analyses revealed that the carbapenems (imipenem, meropenem, ertapenem, and doripenem) and piperacillin-tazobactam were the most active agents against these pathogens, with resistance rates of 0.9%-2.3%. In the most recent 3 years of the survey (2005-2007), resistance to some agents was shown to depend on the species, such as ampicillin-sulbactam against Bacteroides distasonis (20.6%) and tigecycline against Bacteroides uniformis and Bacteroides eggerthii ( approximately 7%). Very high resistance rates (>50%) were noted for moxifloxacin and trovafloxacin, particularly against Bacteroides vulgatus. During that period of study, non-B. fragilis Bacteroides species had >40% resistance to clindamycin. Metronidazole-resistant Bacteroides strains were also first reported during that period. CONCLUSIONS In summary, resistance to antibiotics was greater among non-B. fragilis Bacteroides species than among B. fragilis and was especially greater among species with a low frequency of isolation, such as Bacteroides caccae and B. uniformis. The emergence of resistance among the non-B. fragilis Bacteroides species underscores the need for speciation of B. fragilis group isolates and for clinicians to be aware of associations between species and drug resistance.

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.

Association of Enterotoxigenic Bacteroides fragilis Infection with Inflammatory Diarrhea

BACKGROUND Diarrheal illnesses remain a leading cause of morbidity and mortality globally, with increasing recognition of long-term sequelae, including postinfectious irritable bowel syndrome and growth faltering, as well as cognitive deficits in children. Identification of specific etiologic agents is often lacking. In vitro and in vivo data suggest that enterotoxigenic Bacteroides fragilis (ETBF) may contribute to the burden of colonic inflammatory diarrheal disease. The study goal was to investigate the pathogenesis of ETBF diarrheal illnesses. METHODS We performed an observational study of children and adults with acute diarrheal illnesses in Dhaka, Bangladesh, from January 2004 through November 2005, to define the clinical presentation, intestinal inflammatory responses, and systemic and intestinal antibody responses to ETBF. Other enteric pathogens were also evaluated. RESULTS ETBF was identified to cause a clinical syndrome with marked abdominal pain and nonfebrile inflammatory diarrhea in both children (age, >1 year) and adults. Fecal leukocytes, lactoferrin, and proinflammatory cytokines (interleukin 8, tumor necrosis factor-alpha)-as well as B. fragilis toxin systemic antitoxin responses-increased rapidly in ETBF-infected patients. Evidence of intestinal inflammation often persisted for at least 3 weeks, despite antibiotic therapy. CONCLUSIONS ETBF infection is a newly recognized cause of inflammatory diarrhea in children and adults. Future studies are needed to evaluate the role of ETBF in persistent colonic inflammation and other morbid sequelae of acute diarrheal disease.

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.

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 β-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.