High level of quinolone resistance in <Emphasis Type="Italic">Escherichia coli</Emphasis> from healthy chicken broilers

The development of resistance to quinolones (nalidixic acid, ciprofloxacin and enrofloxacin) in 2006–2008 was evaluated in 317 strains of Escherichia coli isolated from healthy chicken broilers from various farms. The isolates (2006/2007/2008) showed a high resistance to nalidixic acid (87/85/67 %), ciprofloxacin (CIP) (49/54/29 %) and enrofloxacin (ENR) (52/42/22 %). Nalidixic acid-resistant isolates with low level of MIC for CIP and ENR represented a single mutation; intermediary MIC for CIP and ENR were related to two mutations and high level resistance MIC for CIP (≥4 mg/L) and ENR (≥16 mg/L) represented three mutations (two in gyrA and one in parC). There was a correlation between the phenotype reading of high-level resistance and mutations in gyrA (Ser83Leu, Asp87Tyr or Asp87Asn) and parC (Ser80Ile) gene. Plasmid-mediated quinolone-resistance qnrS gene was detected in one Escherichia coli strain with a high level of ciprofloxacin resistance. Our results demonstrate the increase in occurrence of multiresistant E. coli strains with a high level of chromosomal and plasmid resistance to fluoroquinolones.     

Drug resistance mutations and susceptibility phenotypes of <Emphasis Type="Italic">Neisseria gonorrhoeae</Emphasis> isolates in Russia

Steady growth in the degree of antimicrobial resistance in Neisseria gonorrhoeae calls for the control of the spreading of resistance mutations. Here we present the data describing drug resistance mutations, the results of antimicrobial susceptibility tests, and molecular genotypes of 128 recent N. gonorrhoeae isolates collected across 9 regions of the Russian Federation. The mutations in chromosome genes penA, ponA, rpsJ, gyrA, parC, which determine the susceptibility of N. gonorrhoeae to penicillins, tetracyclines, and fluoroquinolones were detected by multiplex amplification followed by hybridization on a hydrogel microarray. The most frequent mutation was an insertion of an aspartate at position 345 of penA gene (76.6%), whereas mutations Leu421Pro in ponA gene, Val57Met in rpsJ gene, Ser91Phe in gyrA gene, Asp95Gly in gyrA gene, and Ser87Arg in parC gene were detected in 32.8–36.7% of strains. One third of studied N. gonorrhoeae isolates harbored multiple drug resistance mutations in bacterial chromosome, resulting in the bimodal distribution of mutation profiles and related patterns of antimicrobial susceptibility. The spread of multiple resistance could be explained by the vertical transfer of the mutations resulting in the clonality of the N. gonorrhoeae population.     

Development of Flouroquinolone (Ciprofloxacin) Resistance in Mycoplasma hominis in the Presence of HeLa Cells

The effect of cocultivation of eukaryotic HeLa cells and Mycoplasma hominis mycoplasma on the resistance of the latter to fluoroquinolones (ciprofloxacin) was examined. It was shown that cocultivation of the M. homonisand HeLa cells during 24 h with subsequent addition of ciprofloxacin resulted in an increase of the micoplasma resistance to this antimicrobial agent. In the M. hominis cells cultivated in the presence of HeLa cells and the increasing concentration of ciprofloxacin mutations in the parC gene were observed only at low concentrations of the antimicrobial agent, while mutations in the gyrA gene were never detected. A gradual elevation of ciprofloxacin concentration up to 10 g/ml resulted in the reversion of the parC mutations in mycoplasmas. Mycoplasma cells resistant to high flouroquinolone concentrations and isolated after cocultivation with the HeLa cells were characterized by the wild-type genotype in respect of the gyrA and parC genes. It was shown that infection of HeLa cells resulted in the appearance of genome rearrangements in M. hominis cells.     

In vitro selection of resistance in <Emphasis Type="Italic">Escherichia coli</Emphasis> and <Emphasis Type="Italic">Klebsiella</Emphasis> spp. at <Emphasis Type="Italic">in vivo</Emphasis> fluoroquinolone concentrations

Background  

Fluoroquinolones are potent antimicrobial agents used for the treatment of a wide variety of community- and nosocomial- infections. However, resistance to fluoroquinolones in Enterobacteriaceae is increasingly reported. Studies assessing the ability of fluoroquinolones to select for resistance have often used antimicrobial concentrations quite different from those actually acquired at the site of infection. The present study compared the ability to select for resistance of levofloxacin, ciprofloxacin and prulifloxacin at concentrations observed in vivo in twenty strains of Escherichia coli and Klebsiella spp. isolated from patients with respiratory and urinary infections. The frequencies of spontaneous single-step mutations at plasma peak and trough antibiotic concentrations were calculated. Multi-step selection of resistance was evaluated by performing 10 serial cultures on agar plates containing a linear gradient from trough to peak antimicrobial concentrations, followed by 10 subcultures on antibiotic-free agar. E. coli resistant strains selected after multi-step selection were characterized for DNA mutations by sequencing gyrA, gyrB, parC and parE genes.     

Quinolone-resistant mutations of the gyrA gene of Escherichia coli

Summary DNA fragments of 8.5 kb containing the gyrA gene were cloned from Escherichia coli KL-16 and from four spontaneous gyrA mutants which showed various levels of resistance to quinolones. The gyrA gene was situated at about 4 kb in front of the nrdA gene and transcribed counterclockwise on the E. coli chromosome. It encoded a polypeptide of 875 amino acids with a molecular weight of about 97000. The four gyrA mutations were located strikingly close to one another within a small region near the N-terminus of the gyrA polypeptide, i.e., nucleotide changes from C to T, from C to G, from G to T and from G to T at nucleotides 248, 248, 318 and 199, respectively, resulting in amino acid changes from Ser to Leu, from Ser to Trp, from Gln to His and from Ala to Ser at amino acids 83, 83, 106 and 67, respectively. These mutations were situated in the relatively hydrophilic regions of the GyrA polypeptide and close to Tyr at amino acid 122 which has been shown to be the site covalently bound to DNA.     

Analysis of the NH2-Terminal 87th Amino Acid of Escherichia coli GyrA in Quinolone-Resistance

The functional contributions of amino acid residue Asp87 of Escherichia coli gyrase A protein (GyrA) was analyzed by site-directed mutagenesis. We generated a series of mutants, in which Asp87 of GyrA was changed to Ala, Val, Phe, Asn, Ser, and Lys. By genetic analysis of gyrA genes in a gyrA temperature-sensitive (Ts) background, it was shown that all these mutations caused the quinolone-resistance. These results indicate that the 87th amino acid of E. coli GyrA must have negative charge in expressing the phenotype of quinolone sensitivity. These findings also suggest that the carboxyl group of Asp87 may interact with quinolone drugs.     

Analysis of the NH2-Terminal 83rd Amino Acid of Escherichia coli GyrA in Quinolone-Resistance

Artificial mutations of Gyrase A protein (GyrA) in Escherichia coli by site-directed mutagenesis were generated to analyze quinolone-resistant mechanisms. By genetic analysis of gyrA genes in a gyrA temperature sensitive (Ts) background, exchange of Ser at the NH₂-terminal 83rd position of GyrA to Trp, Leu, Phe, Tyr, Ala, Val, and Ile caused bacterial resistance to the quinolones, while exchange to Gly, Asn, Lys, Arg and Asp did not confer resistance. These results indicate that it is the most important for the 83rd amino acid residue to be hydrophobic in expressing the phenotype of resistance to the quinolones. These findings also suggest that the hydroxyl group of Ser would not play a major role in the quinolone-gyrase interaction and Ser83 would not interact directly with other amino acid residues.     

Quinolone Resistance Mechanisms among Extended-Spectrum Beta-Lactamase (ESBL) Producing Escherichia coli Isolated from Rivers and Lakes in Switzerland

Sixty extended-spectrum β-lactamase (ESBL)-producing Escherichia coli isolated from rivers and lakes in Switzerland were screened for individual strains additionally exhibiting a reduced quinolone susceptibility phenotype. Totally, 42 such isolates were found and further characterized for their molecular (fluoro)quinolone resistance mechanisms. PCR and sequence analysis were performed to identify chromosomal mutations in the quinolone resistance-determining regions (QRDR) of gyrA, gyrB, parC and parE and to describe the occurrence of the following plasmid-mediated quinolone resistance genes: qepA, aac-6′-Ib-cr, qnrA, qnrB, qnrC, qnrD and qnrS. The contribution of efflux pumps to the resistance phenotype of selected strains was further determined by the broth microdilution method in the presence and absence of the efflux pump inhibitor phe-arg-β-naphthylamide (PAβN). Almost all strains, except two isolates, showed at least one mutation in the QRDR of gyrA. Ten strains showed only one mutation in gyrA, whereas thirty isolates exhibited up to four mutations in the QRDR of gyrA, parC and/or parE. No mutations were detected in gyrB. Most frequently the amino-acid substitution Ser83→Leu was detected in GyrA followed by Asp87→Asn in GyrA, Ser80→Ile in ParC, Glu84→Val in ParC and Ser458→Ala in ParE. Plasmid-mediated quinolone resistance mechanisms were found in twenty isolates bearing QnrS1 (4/20), AAC-6′-Ib-cr (15/20) and QepA (1/20) determinants, respectively. No qnrA, qnrB, qnrC and qnrD were found. In the presence of PAβN, the MICs of nalidixic acid were decreased 4- to 32-fold. (Fluoro) quinolone resistance is due to various mechanisms frequently associated with ESBL-production in E. coli from surface waters in Switzerland.     

Distinguishing importation from diversification of quinolone-resistant <Emphasis Type="Italic">Neisseria gonorrhoeae</Emphasis> by molecular evolutionary analysis

Background  

Distinguishing the recent introduction of quinolone resistant gonococci into a population from diversification of resistant strains already in the population is important for planning effective infection control strategies. We applied molecular evolutionary analyses to DNA sequences from 9 housekeeping genes and gyrA, parC and porB of 24 quinolone resistant N. gonorrhoeae (QRNG) and 24 quinolone sensitive isolates collected in Israel during 2000–2001.     

Spontaneous mutation frequency and molecular mechanisms of Shigella flexneri fluoroquinolone resistance under antibiotic selective stress

The incidence of fluoroquinolone-resistant Shigella strains has risen rapidly, presumably in response to ciprofloxacin antibiotic stress. Understanding the molecular mechanisms underlying this resistance phenotype is critical to developing novel and effective therapeutic strategies. In this study, the frequency of ciprofloxacin-induced mutation was measured in antibiotic resistance genes (gyrA, gyrB, parC, parE, marOR, and marA) of Shigella flexneri. The S. flexneri 2a strain 301 was cultured on Luria–Bertani agar plates containing one of seven different ciprofloxacin concentrations (range: 0.03125–2 μg mL^?1). Resistant colonies were selected for gene-targeted sequencing analysis; the identified point mutations were subsequently confirmed by insertion into antibiotic cassette plasmids and growth under ciprofloxacin stress. The results demonstrated that the seven different ciprofloxacin concentrations produced dose-dependent frequencies of spontaneous mutations: 10^?8 (0.03125 and 0.0625 μg mL^?1), 10^?9 (0.125 μg mL^?1), and <10^?9 (0.25, 0.5, 1, 2 μg mL^?1). PCR sequencing of the ten randomly selected resistant colonies (minimum inhibitory concentrations (MICs) of 0.125 μg mL^?1, n = 5 and 0.25 μg mL^?1, n = 5) revealed that all colonies had mutations in the gyrA gene at either codon 83 (Ser83 → Leu) or 87 (Asp87 → Tyr or → Gly), both of which were confirmed at MIC of 0.125 μg mL^?1. None of the spontaneous mutation colonies exhibited gyrB, parC, parE, marOR, or marA mutations. In conclusion, S. flexneri is normomutable under ciprofloxacin antibiotic stress and fluoroquinolone resistance by spontaneous mutation occurs at a low rate. Codon mutations gyrA 83 and/or gyrA 87 cause a 4-fold increase in the ciprofloxacin MIC, and may represent the natural mechanism of fluoroquinolone resistance.     

Mutations in the gyrA and parC genes associated with fluoroquinolone resistance in clinical isolates of Citrobacter freundii

We determined partial sequences of the gyrA and parC genes of Citrobacter freundii type strain, and then examined 38 C. freundii clinical strains isolated from patients with urinary tract infections for the association of alterations in GyrA and ParC with susceptibility to fluoroquinolones. Our results suggest that in C. freundii DNA gyrase may be a primary target of quinolones, that an amino acid change at Thr-83 or Asp-87 in GyrA is sufficient to decrease susceptibility to fluoroquinolones, and that accumulation of changes in GyrA with the simultaneous presence of an alteration at Ser-80 or Glu-84 in ParC may be associated with the development of high-level fluoroquinolone resistance in C. freundii clinical isolates.     

Fluoroquinolone resistance in Helicobacter pylori: role of mutations at position 87 and 91 of GyrA on the level of resistance and identification of a resistance conferring mutation in GyrB

Background and Aims: Fluoroquinolone‐containing regimens have been suggested as an alternate to standard triple therapy for the treatment of Helicobacter pylori infections. To determine the relationship between fluoroquinolone resistance and mutations of GyrA and GyrB in H. pylori, we exchanged the mutations at positions 87and 91 of GyrA among fluoroquinolone‐resistant clinical isolates. GyrB of a strain with no mutations in GyrA was also analyzed to identify mechanisms of resistance to norfloxacin. Materials & Methods: Natural transformation was performed using the amplified fragment of the gyrA and gyrB gene as donor DNA. The amino acid sequences of GyrA and GyrB were determined by DNA sequencing of the gyrA and gyrB genes. Results: Norfloxacin‐resistant strains which had mutations at position 87 and 91 became susceptible when the mutations were converted to the wild type. When the mutation from Asp to Asn at position 91 was exchanged to the mutation from Asn to Lys at position 87, the MIC to levofloxacin, gatifloxacin, and sitafloxacin increased. Norfloxacin‐resistant strain TS132 with no mutations in GyrA but had a mutation at position 463 in GyrB. Transformants obtained by natural transformation using gyrB DNA of TS132 had a mutation at position 463 of GyrB and revealed resistant to norfloxacin and levofloxacin. Conclusion: Mutation from Asn to Lys at position 87 of GyrA confers higher resistance to levofloxacin and gatifloxacin than does mutation from Asp to Asn at position 91. We propose that mutation at position 463 in GyrB as a novel mechanism of fluoroquinolone resistance in H. pylori.     

Membrane glycerophospholipid biosynthesis in Neisseria meningitidis and Neisseria gonorrhoeae: identification, characterization, and mutagenesis of a lysophosphatidic acid acyltransferase

Lysophosphatidic acid (LPA) acyltransferases of Neisseria meningitidis and Neisseria gonorrhoeae were identified which share homology with other prokaryotic and eukaryotic LPA acyltransferases. In Escherichia coli, the conversion of LPA to phosphatidic acid, performed by the 1-acyl-sn-glycerol-3-phosphate acyltransferase PlsC, is a critical intermediate step in the biosynthesis of membrane glycerophospholipids. A Tn916-generated mutant of a serogroup B meningococcal strain was identified that exhibited increased amounts of capsular polysaccharide, as shown by colony immunoblots, and a threefold increase in the number of assembled pili. The single, truncated 3.8 kb Tn916 insertion in the meningococcal mutant was localized within a 771 bp open reading frame. The gonococcal equivalent of this gene was identified by transformation with the cloned meningococcal mutant gene. In N. gonorrhoeae, the mutation increased piliation fivefold. The insertions were found to be within a gene that was subsequently designated nIaA (n eisserial L PA acyltransferase). The predicted neisserial LPA acyltransferases were homologous (>20% identity,>40% amino acid similarity) to the family of PlsC protein homologues. A cloned copy of the meningococcal nIaA gene complemented in trans a temperature-sensitive E. coli PlsC^ts? mutant. Tn916 and Ω-cassette insertional inactivations of the neisserial nIaA genes altered the membrane glycerophospholipid compositions of both N. meningitidis and N. gonorrhoeae but were not lethal. Therefore, the pathogenic Neisseria spp. appear to be able to utilize alternative enzyme(s) to produce phosphatidic acid. This hypothesis is supported by the observation that, although the amounts of mature glycerophospholipids were altered in the meningococcal and the gonococcal nIaA mutants, glycerophospholipid synthesis was detectable at significant levels. In addition, acyltransferase enzymatic activity, while reduced in the gonococcal nIaA mutant, was increased in the meningococcal nIaA mutant. We postulate that the pathogenic Neisseria spp. are able to utilize alternate acyltransferases to produce glycerophospholipids in the absence of nIaA enzymatic activity.Implementation of these secondary enzymes results in alterations of glycerophospholipid composition that lead to pleiotropic effects on the cell surface components, including effects on capsule and piliation.     

pUB307 mobilizes resistance plasmids from Escherichia coli into Neisseria gonorrhoeae

Summary The plasmid pUB307, a derivative of RP1, is a conjugative, broad-host-range plasmid. We have shown that this element mobilizes gonococcal resistance plasmids from Escherichia coli to Neisseria gonorrhoeae, thus providing evidence that extrachromosomal elements can efficiently enter gonococci by conjugation. Furthermore, pUB307 can also be used as a helper element to mobilize the cloning vector pLES2 into N. gonorrhoeae. This finding significantly increases the usefulness of pLES2 as a shuttle vector between E. coli and gonococcus.     

肺炎克雷伯菌gyrA基因与parC基因的突变研究

目的探讨肺炎克雷伯菌对环丙沙星和左氧氟沙星的药物敏感性,及对喹诺酮敏感和耐药菌株中gyrA与parC基因的突变情况。方法收集肺炎克雷伯菌临床分离株231株,采用K-B纸片法测定肺炎克雷伯菌对环丙沙星和左氧氟沙星的敏感性,随机选取对环丙沙星和左氧氟沙星均耐药菌株4株和均敏感的菌株3株,分别PCR扩增gyrA基因和parC基因的耐药决定区,扩增片段长度分别为625、319bp,PCR扩增产物经纯化后测序并做序列分析。结果肺炎克雷伯菌对环丙沙星和左氧氟沙星的耐药率分别为51.1%（118/231）和45.9%（106/231）;gyrA和parC基因经序列分析显示,耐药株均有gyrA基因的突变,其中1株出现第83、87和27位氨基酸的改变,2株出现第83位氨基酸的改变,1株出现第47位点的改变;环丙沙星敏感株中未出现gyrA基因的突变。4株耐药株均有parC基因的突变,引起相应氨基酸Ser80→Arg的改变,2株环丙沙星敏感株也发生了同样的改变。结论哈尔滨地区肺炎克雷伯菌对环丙沙星和左氧氟沙星的耐药性显著,在喹诺酮耐药株中有gyrA和parC基因的同时突变,在敏感株中也发现了parC基因的突变。     

Genetical conditioning of fluoroquinolone resistance mechanisms of clinical enterococcus faecalis strains. II. The mutations present in the qrdrs of gyrA, gyrB, parC and parE genes

The analyze selected fluoroquinolone resistance mechanisms of clinical E. faecalis strains was presented. In the second part of the study of genetic polymorphisms and mutations in the QRDRs of gyrA, gyrB, parC and parE genes were analyzed. The MSSCP technique and DNA sequencing were used. The activity (MICs) of ciprofloxacin, sparfloxacin and moxifloxacin were determined against 180 tested strains. The MSSCP method allows rapid screening of the genetic polymorphisms analyze of gyrA, gyrB, parC i parE genes. The amino acid substitutions of GyrA, GyrB and ParC were observed. The results indicate that mutations present among clinical E. faecalis strains associated with high level resistance to fluoroquinolons.