In vitro surveys of antimicrobial resistance among clinically important anaerobes are an important source of information that can be used for clinical decisions in the choice of empiric antimicrobial therapy. This study surveyed the susceptibilities of 556 clinical anaerobic isolates from four large medical centers using a broth microdilution method. Piperacillin-tazobactam was the only antimicrobial agent to which all the isolates were susceptible. Similarly, imipenem, meropenem, and metronidazole were highly active (resistance, <0.5%), whereas the lowest susceptibility rates were noted for penicillin G, ciprofloxacin, and clindamycin. For most antibiotics, blood isolates were less susceptible than isolates from intra-abdominal, obstetric-gynecologic, and other sources. All isolates of the Bacteroides fragilis group were susceptible to piperacillin-tazobactam and metronidazole, while resistance to imipenem and meropenem was low (<2%). For these same isolates, resistance rates (intermediate and resistant MICs) to ampicillin-sulbactam, cefoxitin, trovafloxacin, and clindamycin were 11, 8, 7, and 29%, respectively. Among the individual species of the B. fragilis group, the highest resistance rates were noted among the following organism-drug combinations: for clindamycin,Bacteroides distasonis and Bacteroides ovatus; for cefoxitin, Bacteroides thetaiotaomicron, B. distasonis, and Bacteroides uniformis; for ampicillin-sulbactam,B. distasonis, B. ovatus, and B. uniformis; and for trovafloxacin, Bacteroides vulgatus. For the carbapenens, imipenem resistance was noted among B. fragilis and meropenem resistance was seen among B. fragilis, B. vulgatus, and B. uniformis. With few exceptions all antimicrobial agents were highly active against isolates of Prevotella, Fusobacterium, Porphyromonas, andPeptostreptococcus. These data further establish and confirm that clinically important anaerobes can vary widely in their antimicrobial susceptibilities. Fortunately most antimicrobial agents were active against the test isolates. However, concern is warranted for what appears to be a significant increases in resistance to ampicillin-sulbactam and clindamycin.

Two linezolid-resistant Enterococcus faecium isolates (MICs, 8 g/ml) from unique patients of a medical center in New Orleans were included in this study. Isolates were initially investigated for the presence of mutations in the V domain of 23S rRNA genes and L3, L4, and L22 ribosomal proteins, as well as cfr. Isolates were subjected to pulsed-field gel electrophoresis (just one band difference), and one representative strain was submitted to whole-genome sequencing. Gene location was also determined by hybridization, and cfr genes were cloned and expressed in a Staphylococcus aureus background. The two isolates had one out of six 23S rRNA alleles mutated (G2576T), had wild-type L3, L4, and L22 sequences, and were positive for a cfr-like gene. The sequence of the protein encoded by the cfr-like gene was most similar (99.7%) to that found in Peptoclostridium difficile, which shared only 74.9% amino acid identity with the proteins encoded by genes previously identified in staphylococci and non-faecium enterococci and was, therefore, denominated Cfr(B). When expressed in S. aureus, the protein conferred a resistance profile similar to that of Cfr. Two copies of cfr(B) were chromosomally located and embedded in a Tn6218 similar to the cfr-carrying transposon described in P. difficile. This study reports the first detection of cfr genes in E. faecium clinical isolates in the United States and characterization of a new cfr variant, cfr(B). cfr(B) has been observed in mobile genetic elements in E. faecium and P. difficile, suggesting potential for dissemination. However, further analysis is necessary to access the resistance levels conferred by cfr(B) when expressed in enterococci.

Multidrug-resistant Pseudomonas aeruginosa with combined decreased susceptibility to ceftazidime, ciprofloxacin, imipenem, and piperacillin is increasingly being found as a cause of nosocomial infections. It is important to look for combinations of drugs that might be synergistic. Ciprofloxacin resistance by P. aeruginosa is mediated in part by an efflux pump mechanism. Gatifloxacin, an 8-methoxyfluoroquinolone, inhibits a staphylococcal efflux pump. An earlier in vitro study using an Etest synergy method and time-kill assay suggested synergy of ciprofloxacin and gatifloxacin against P. aeruginosa. Synergy testing was performed by Etest and time-kill assay for 31 clinically unique, plasmid DNA distinct, U.S. P. aeruginosa isolates. Etest MICs for ciprofloxacin were 4 to >32 g/ml, and for gatifloxacin they were >32 g/ml. Ciprofloxacin plus gatifloxacin showed synergy by the Etest method for 6 (19%) of the 31 P. aeruginosa isolates using a summation fractional inhibitory concentration of <0.5 for synergy. Synergy was demonstrated for 13/31 (42%) of isolates by time-kill assay. No antagonism was detected. The remaining isolates were indifferent to the combination. The Etest method and time-kill assay were 65% (20/31) concordant. The mechanism of the in vitro synergy may include P. aeruginosa ciprofloxacin efflux pump inhibition by gatifloxacin.It is now accepted that bacterial multidrug antimicrobial resistance is a worldwide problem, in large part related to antimicrobial use in humans and other animals. Serious infections with these resistant bacteria are commonplace. Therapy for Pseudomonas aeruginosa, a major cause of life-threatening nosocomial infection, is problematic because of the propensity for multiple-drug resistance (19). Some P. aeruginosa strains are only susceptible to the polymyxins (18). Mechanisms of resistance of P. aeruginosa are dependent mainly on impermeability and multidrug efflux pumping (9). This raises the MICs of penicillins, cephalosporins, quinolones, tetracyclines, and chloramphenicol. Carbapenem resistance is associated with metallo-beta-lactamases (20). The development of vaccines and new antimicrobial agents has not kept pace with resistance; therefore, the search for other methods of therapy such as synergistic combinations is necessary. Many antimicrobial combinations have been studied for synergy in vitro and in vivo against P. aeruginosa (3-5, 14).Ciprofloxacin (CIP) is the most in vitro-active anti-P. aeruginosa fluoroquinolone, but increasing resistance (now 25% in most of the United States) frequently precludes its use (7). The mechanism of the resistance includes efflux pumping. Gatifloxacin (GAT) is an 8-methoxyfluoroquinolone with similar substituent configuration to reserpine, a known efflux pump inhibitor. Perhaps related to this, it resists efflux pumping by gram-positive bacteria and has low MICs for these bacteria. Gatifloxacin is inherently not as active in vitro against Pseudomonas aeruginosa. If gatifloxacin were to interfere with the efflux pumping of ciprofloxa...

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In vitro synergy testing of daptomycin plus rifampin was performed against 24 unique isolates of Enterococcus faecium resistant to both linezolid and vancomycin. Synergy testing showed that 21/24 (88%) were synergistic and 3/24 (12%) were indifferent by the Etest method. Time-kill assays revealed synergy for 18/24 (75%) and indifference for 6/24 (25%).