Antibiotic resistance in anaerobic bacteria

[摘要] English: Anaerobic bacteria are important human pathogens capable of causing serious debilitating infections ranging from abscesses to life threatening infections and warrant more attention than they are currently receiving. Anaerobes are often present in mixed infections in association with other anaerobes/facultative anaerobes and aerobic bacteria and it is imperative to administer correct antimicrobial therapy ab initio. Antibiotic resistance development in anaerobic bacteria has a tremendous impact on selection of effective antimicrobial agents for empiric therapy. β-Lactam antibiotics are frequently used and have for many years been the first choice in the treatment and prophylaxis of anaerobic and mixed aerobic/anaerobic infections. Current knowledge of β-lactam resistance progression and resistance mechanisms in anaerobic bacteria is, however, limited. Metronidazole is often used empirically against suspected anaerobic infections, but anaerobes can no longer be considered to be universally susceptible as resistance has been noted in strains of Bacteroides fragilis, clostridia and peptostreptococci. The objectives of the study were to: 1) assess antibiotic susceptibilities of anaerobic bacteria isolated in the Bloemfontein area to antimicrobial agents currently employed in empiric treatment, 2) compare the in vitro activity of currently employed antibiotics with new antimicrobial agents, and 3) conduct studies on antibiotic resistance development. Anaerobic bacteria were isolated from clinically significant infections from April 1996 to March 1997 from the Universitas and Pelonomi Hospitals, Bloemfontein. Infection sites traced for 302 of 378 of the isolates, were from blood, brain abscesses, liver abscesses, lung infection/abscesses, eustachian infection/sepsis, neoplasms, bone fracture/infection, post-operative/amputation sepsis, gunsho/stab wound infection/ sepsis, genital tract isolates, general abscesses, and intestinal tract infections. Isolates were identified in the routine diagnostic laboratory by presumptive and preliminary methods and results confirmed by the Rapid ID32A identification system. Minimum inhibitory concentrations (MICs) were determined by the National Committee for Clinical laboratory Standards (NCCLS) agar dilution method for the following 18 antimicrobial agents: six β-lactams (amoxicillin, ampicillin, penicillin, piperacillin, cefoxitin, cefepime and cefpirome), two carbapenems (imipenem and meropenem), metronidazole, clindamycin, chloramphenicol, ciprofloxacin, trovafloxacin, vancomycin, dalfopristin/quinupristin, linezolid and loracarbef. Screening for β-lactamase production was performed by employing nitrocefin and inhibition of β-lactamases determined using amoxicillin/clavulanic acid combination. For detection of carbapenernase/metallo-β-lactarnase production a biological assay was performed; cell extracts and imipenem being added to agar seeded with E. coli ATCC 25922. Detection of metallo-β-lactamase genes was undertaken with primers directed to cfiA, cphA and blalMP genes. PBP profiles and penicillin affinities were determined by labelling with [3H]penicillin, separation of proteins by SOS-PAGE and visualisation after fluorography. In PBP competition studies whole cell samples were initially preincubated with rupenern, piperacillin or ampicillin at different concentrations and post-labelled with [3H]penicillin, followed by SOS-PAGE and fluorography. Metronidazole MICs of 64 isolates were correlated with inhibitory concentrations (ICs) obtained with two batches of Etest strips (range 0.006 - 32 µg/ml and 0.016 - 256 µg/ml). Membrane proteins of parental and metronidazole mutant strains of a VeilIonelIa sp. and Peptostreptococcus prevotii were separated by SOS-PAGE and profiles compared. The prevalence of rdxA genes was investigated in 16 anaerobic/ facultative anaerobic bacteria with metronidazole MICs 1 µg/ml employing two sets of primers and fragments of approximately 937 bp and 491 bp sequenced. The prevalence of nim genes were investigated in 64 anaerobic/facultative anaerobic isolates with metronidazole MICs 0.5 µg/ml using a pair of universal mm gene primers. Amplification was performed at two annealing temperatures (52°C & 62°C) and fragments at approximately 458 bp recorded as presumptive positives and sequenced. Positive strains were subjected to plasmid extraction. MICs indicated overall susceptibility of Gram-positive anaerobic isolates to be higher than for the Gram-negative isolates. Reduced susceptibility to penicillin (MICs �? µg/ml) was found in 20 Peptostreptococcus strains and seven non-perfringens Clostridium spp. β-Lactamases hydrolysing both penicillins and cephalosporins were demonstrated in all Bacteroides and Prevotella isolates with ampicillin MICs> 4 Iµg/ml. Only 8% Peptostreptococcus spp. were resistant to piperacillin in contrast to 41% Bacteroides spp. and 68% Veillonella spp. Veillonella spp. exhibited selective β- Iactam resistance to piperacillin. Cefoxitin showed excellent activity against both Gram-positive and Gram-negative isolates, except for Bacteroides and Fusobacterium species. The majority of Gram-positive isolates were susceptible to cefepime and cefpirome, whereas < 50% Bacteroides spp. and < 70% Prevotella spp. were susceptible. High-level resistance to imipenem/meropenem (MICs> 128 µg/ml) was seen for 13/37 Fusobacterium spp. Two P. magnus isolates were resistant to metronidazole (MIC >128 µg/ml), three C. perfringens strains showed reduced susceptibility (MICs 4-8 µg/ml), while two Prevotella spp. had metronidazole MICs of 32 µg/ml. Eighty five percent of all isolates were susceptible to clindamycin. Oalfopristin/quinupristin exhibited excellent activity throughout the Gram-positive bacterial spectrum with only one Peptostreptococcus sp. showing reduced susceptibility (MIC 8 µg/ml), but poor activity against B. tragiIis group isolates. Trovafloxacin was effective against all the Gram-positive anaerobes except for two P. anaerobius strains (MICs 8 µg/ml), and demonstrated superior activity to ciprofloxacin against the Gram-negative isolates. Overall, chloramphenicol was the most effective antibiotic, with only two Clostridium spp. being resistant (MICs 16 µg/ml). PCR products of predicted size of cfiA genes were found in two strains of B. vulgatus, a B. capillosus and a P. loescheii strain and of cphA genes in three B. tragi/is strains and a strain of P. loescheii. None of the PCR products on sequencing, however, were seen to be positive for ctiA or cphA genes. No bla/MPgenes were amplified. PBP profiles were analysed with respect to identification based on the API Rapid ID 32A system. Seven major groups of fusobacteria PBPs could be identified, but although PBP profile/API agreement was evident for the majority of F. mortiterum strains, the API system did not lend itself to reliable identification of fusobacteria. PBP profiles were seen to distinguish species/subspecies of Clostridium species other than C. perfringens, while comparison with the API identification method showed some correlation, but not with most of the species investigated. Comparing PBP profiles of nine Veillonella spp. assigned three groups of species/subspecies. Commercial identification systems appeared not to be as reliable as promoted. The differentiation of anaerobic bacterial species by PBP profiling could certainly assist in situations of therapeutic failure. For F. mortiterum the PBP with the lowest affinity for penicillin and imipenem was the highest-molecular-weight PBP, 74 kDa. The PBP profile of an imipenem-resistant variant of F. varium (MIC >128 µg/ml) demonstrated an additional PBP (±69 kDa) when compared to that of the parental strain (apparent MIC 0.5 µg/ml). In two C. tertium strains, a C. sporogenes and a C. bitermentans strain low-molecularweight< 50 kDa) PBPs exhibited reduced affinity towards penicillin. In Veillonella isolates a PBP (66 kDa) that possessed the highest affinity for penicillin, was seen to exhibit the lowest affinity for piperacillin. Regression analysis revealed good correlation between metronidazole agar dilution MIC and Etest IC values. In the clinical setting, metronidazole Etests provide an important role in the susceptibility testing of anaerobes. Inducing metronidazole resistance in a P. prevotii and a VeilIonelIa strain produced mutants with only a twofold increase in metronidazole MICs increase, yet alterations to several membrane proteins were apparent. Such findings, as were also found with PBP analysis conducted in this study, complexes interpretation as to how antibiotic resistance has developed in anaerobic bacteria. Nim genes were demonstrated in 14/64 strains (MICs �?0.5 µg/ml) and identified as NimA genes in five propionibacteria, a P. bivia, a C. bitermentans and an A. odonto/yticus strain, with nimB genes identified in five isolates of B. tragi/is and a P. magnus strain. Sequence divergence was < 4% from the respective documented nimA and nimB gene sequences. Although the origin of nim genes is unknown, predominance of nimA genes in facultative anaerobes, propionibacteria, may indicate a transferable nimA gene source in anaerobic environments. To avert the clinical problem of untreatable anaerobic infections, it is necessary to continuously monitor for the emergence of antibiotic-resistant strains, conduct investigations into how resistance has developed, and understand conditions that foster inter- and intra-dissemination of resistance genes among anaerobic bacteria.

[发布机构] University of the Free State