Beta-lactam and metronidazole resistance in the Bacteroides fragilis group

Sammanfattning: The Bacteroides fragilis group organisms are anaerobic gram-negative bacilli. Ten species are currently included in this group. Members of the B. fragilis group are the major constituents of the normal colonic microflora and the most commonly encountered anaerobic bacteria in clinical specimens. beta-Lactam antibiotics and 5-nitroimidazoles have been extensively used against anaerobic bacteria. However, antibiotic resistance is increasingly common among anaerobic gram-negative bacilli. The classical mechanisms of resistance to beta-lactams are 1) production of beta-lactamases, 2) alteration of penicillinbinding proteins (PBPs), and 3) changes in outer membrane permeability to beta-lactams. The 5nitroimidazole molecule is a prodrug whose activation depends upon reduction of the nitro group in the absence of oxygen. Decreased uptake and altered reduction are believed to be responsible for metronidazole resistance. Five nim genes (A, B, C, D and E) have been identified in the B. fragilis group that confer resistance to 5-nitroimidazole antibiotics. Knowledge of the status and the mechanisms of resistance is critical for both the selection of antimicrobial therapy and the design of new antimicrobial agents. The purpose of this investigation was to study the mechanisms for and the prevalence of beta-lactam and metronidazole resistance in strains belonging to the B. fragilis group. PCR fingerprinting technique was used for characterization of beta-lactam-resistant B. fragilis strains. It was found that the imipenem-resistant B. fragilis strains presented unique PCR fingerprints. These strains produced high amounts of Zn++ dependent beta-lactamases, which were inactivated by EDTA but not by clavulanic acid. The ccrA gene coding for the metallo-beta-lactamase was detected by PCR. The purified PCR products of ccrA from two imipenem-resistant strains were sequenced. The nucleotide sequences of ccrA from these two strains shared >98% identity with the metallo-beta-lactamase gene from a control strain. Production of the metallo-beta-lactamases has been the most perplexing problem over the past 18 years since the first cases of such resistance appeared. These enzymes are capable of hydrolysing all beta-lactam agents except the monobactams, which have no antibacterial activity against Bacteroides, and are not inhibited by the available beta-lactamase inhibitors. The appearance of these enzymes has caused great concern in the clinic. To study the imipenem and metronidazole resistance profiles of B. fragilis group strains in faecal samples and to detect the corresponding resistance genes (ccrA and nim), 925 faecal samples were examined. It was noted that the incidences of imipenem-resistant and metronidazole-resistant B. fragilis group strains were low in the investigated diarrhoea patients. Simultaneous resistance to imipenem and metronidazole was detected, which is of great concern in clinical medicine. The proposed PCR assays may be useful in epidemiological studies on distribution of resistance genes in clinical isolates and in intestinal rnicrofiora. To investigate the mechanisms involved in beta-lactam resistance, two resistant mutants (238m and 1186m) of B. thetaiotaomicron were obtained from clinical isolates (238 and 1186) by selection with increasing concentrations of cefoxitin. The mutant strains showed decreased susceptibilities not only to cefoxitin but also to other beta-lactam antibiotics. Alterations in both penicillin-binding proteins (PBPs) and outer membrane proteins (OMPs) were observed in the mutants as compared to their parent strains. Further investigation found that the growth pattems and the morphological changes induced by cefoxitin were associated with the properties of PBPs The resistant mutants with deficiency in PBPs grew slower than the susceptible parent strains, and cefoxitin caused filamentation at sub-MIC in B. thetaiotaomicron.