ABSTRACT
Mutations in the gyrA gene contribute considerably to quinolone resistance in Escherichia coli. Mechanisms for quinolone resistance in anaerobic bacteria are less well studied. The Bacteroides fragilis group are the anaerobic organisms most frequently isolated from patients with bacteremia and intraabdominal infections. Forty-four clinafloxacin-resistant and-susceptible fecal and clinical isolates of the B. fragilis group (eight Bacteroides fragilis, three Bacteroides ovatus, five Bacteroides thetaiotaomicron, six Bacteroides uniformis, and 22 Bacteroides vulgatus) and six ATCC strains of the B. fragilis group were analyzed as follows: (i) determination of susceptibility to ciprofloxacin, levofloxacin, moxifloxacin, and clinafloxacin by the agar dilution method and (ii) sequencing of the gyrA quinolone resistance-determining region (QRDR) located between amino acid residues equivalent to Ala-67 through Gln-106 in E. coli. Amino acid substitutions were found at hotspots at positions 82 (n = 15) and 86 (n = 8). Strains with Ser82Leu substitutions (n = 13) were highly resistant to all quinolones tested. Mutations in other positions of gyrA were also frequently found in quinolone-resistant and -susceptible isolates. Eight clinical strains that lacked mutations in their QRDR were susceptible to at least two of the quinolones tested. Although newer quinolones have good antimicrobial activity against the B. fragilis group, quinolone resistance in B. fragilis strains can be readily selected in vivo. Mutational events in the QRDR of gyrA seem to contribute to quinolone resistance in Bacteroides species.
Subject(s)
Anti-Infective Agents/pharmacology , Aza Compounds , Bacteroides fragilis/genetics , DNA Topoisomerases, Type II/genetics , Fluoroquinolones , Quinolines , Amino Acid Substitution , Bacteroides fragilis/drug effects , Ciprofloxacin/pharmacology , DNA Gyrase , DNA Topoisomerases, Type II/physiology , Drug Resistance, Microbial/genetics , Humans , Levofloxacin , Microbial Sensitivity Tests , Moxifloxacin , Mutation , Ofloxacin/pharmacologyABSTRACT
Enterococcus faecium has six penicillin-binding proteins (PBP), where PBP5 seems to be the main target for beta-lactam antibiotics. The PBP profiles of three imipenem-resistant, ampicillin-sensitive E. faecium strains, isolated from the same patient, were studied using biotinylated ampicillin and chemiluminescence detection. Imipenem resistance in these strains was found to be associated with hyperproduction of PBP5 compared to the ampicillin- and imipenem-susceptible strain ATCC 19434. PBP5 in the imipenem-resistant strains (S1, B2) exhibited a selectively decreased affinity for imipenem. An 854 bp DNA fragment, corresponding to the penicillin-binding domain of pbp5fm, was studied in the resistant strains and the reference strain. Four amino acid substitutions were observed in the resistant strains compared to the susceptible one. The contribution of these substitutions to the increased production of PBP5 in these strains is unclear since the substitution was observed also in a strain without increased production of PBP5. Our results suggest that the moderate imipenem resistance observed in these strains is associated with increased production of PBP5 with relatively decreased affinity for imipenem, and that evolution of imipenem resistance in E. faecium is dinstinct from that of the other beta-lactams such as ampicillin.
Subject(s)
Ampicillin/pharmacology , Bacterial Proteins , Enterococcus faecium/drug effects , Hexosyltransferases , Imipenem/pharmacology , Peptidyl Transferases , Ampicillin Resistance/genetics , Binding Sites/genetics , Binding, Competitive , Carrier Proteins/chemistry , Carrier Proteins/genetics , Carrier Proteins/metabolism , DNA, Bacterial/genetics , Drug Resistance, Bacterial/genetics , Enterococcus faecium/genetics , Enterococcus faecium/metabolism , Humans , In Vitro Techniques , Molecular Weight , Muramoylpentapeptide Carboxypeptidase/chemistry , Muramoylpentapeptide Carboxypeptidase/genetics , Muramoylpentapeptide Carboxypeptidase/metabolism , Mutation , Penicillin-Binding ProteinsABSTRACT
In a retrospective study, 45 clinical isolates of non-tuberculous mycobacteria were identified to the species level by biochemical profile, gas liquid chromatography and partial sequence analysis of 16S rRNA, and were found to represent 13 different species. The results of sequence analysis showed 100% identity with conventional tests for 34 isolates (76%) and could identify species such as M. bohemicum which are difficult to characterise with conventional methods. Most of the discrepant results for the remaining 11 isolates resulted in species of the same group of mycobacteria. Based on these findings. we concluded that direct sequence analysis of amplified 16S rRNA gene is a promising rapid and accurate method for species determination of non-tuberculous mycobacteria.
Subject(s)
Nontuberculous Mycobacteria/genetics , RNA, Bacterial/analysis , RNA, Ribosomal, 16S/analysis , Bacterial Typing Techniques , Chromatography, Gas , Genotype , Humans , Nontuberculous Mycobacteria/chemistry , Nontuberculous Mycobacteria/classification , Polymerase Chain Reaction , Retrospective Studies , Sequence Analysis, RNA/classification , Species SpecificityABSTRACT
High-level quinolone resistance in Enterococcus faecium was associated with mutations in both gyrA and parC genes in 10 of 11 resistant strains. On low-level resistant strain without such mutations may instead possess an efflux mechanism or alterations in the other subunits of the gyrase or topoisomerase IV genes. These findings are similar to those for other gram-positive bacteria, such as Enterococcus faecalis.
