Real-time PCR detection of Pseudomonas aeruginosa in clinical and municipal wastewater and genotyping of the ciprofloxacin-resistant isolates

Real-time quantification of Pseudomonas aeruginosa was performed in various wastewater systems including clinical, municipal wastewaters and inflow from a wastewater treatment plant. The highest concentrations of P. aeruginosa-specific targets were detected in clinical wastewaters. Limitations of the detection system resulting from inhibition or cross-reaction were identified. Ciprofloxacin-resistant P. aeruginosa strains were isolated after specific enrichment from clinical and municipal wastewaters. In some cases they were also cultivated from effluent of a wastewater treatment plant, and from its downstream river water. A total of 119 isolates were phenotypically characterized as ciprofloxacin-resistant via antibiogram testing. Subsequently, the fluoroquinolone-resistance-mediating mutations in the genes gyrA codon positions 83 and 87, gyrB codon position 466 and parC codon positions 87 and 91 were determined by mini-sequencing. Ciprofloxacin resistance was mainly associated with mutations in gyrA codon position 83 and parC mutation in codon positions 87 or 91 of the bacterial gyrase and topoisomerase II genes. All ciprofloxacin-resistant P. aeruginosa strains were compared with genotypes from clinical data of fluoroquinolone-resistant P. aeruginosa infections. The results were in agreement with data from clinical analyses, with the exception that no gyrA 87 and no gyrB mutations were found in ciprofloxacin-resistant P. aeruginosa wastewater isolates.     

Characterization of Pseudomonas aeruginosa isolates from dogs and cats in Japan: current status of antimicrobial resistance and prevailing resistance mechanisms

Seventy-three Pseudomonas aeruginosa isolates were collected from dogs and cats in Japan to investigate antimicrobial susceptibility and resistance mechanisms to anti-pseudomonal agents. Resistance rates against orbifloxacin, enrofloxacin, ciprofloxacin, cefotaxime, aztreonam and gentamicin were 34.2, 31.5, 20.5, 17.8, 12.3 and 4.1%, respectively. The degree of resistance to cefotaxime, orbifloxacin, and enrofloxacin was greatly affected by efflux pump inhibitors, indicating overexpression of efflux pump contributes to these resistances. Notably, orbifloxacin and enrofloxacin resistance was observed even in isolates without mutations in the target sites. This is the first report on cephalosporin- and fluoroquinolone-resistant isolates of P. aeruginosa from Japanese companion animals.     

High-level fluoroquinolone resistance in Pseudomonas aeruginosa due to interplay of the MexAB-OprM efflux pump and the DNA gyrase mutation

Fluoroquinolone resistance in Pseudomonas aeruginosa is mainly attributable to the constitutive expression of the xenobiotic efflux pump and mutation in DNA gyrase or topoisomerase IV. We constructed cells with a double-mutation in gyrA and mexR encoding DNA gyrase and repressor for the mexAB-oprM operon, respectively. The mutant showed 1,024 times higher fluoroquinolone resistance than cells lacking the MexAB-OprM. Cells with a single mutation in gyrA and producing a wild-type level of the MexAB-OprM efflux pump showed 128 times higher fluoroquinolone resistance than cells lacking the MexAB-OprM. In contrast, a single mutation in gyrA or mexR caused only 4 and 64 times higher resistance, respectively. These findings manifested the interplay between the MexAB-OprM efflux pump and the target mutation in fluoroquinolone resistance.     

结核分枝杆菌耐氟喹诺酮类药物的分子机制研究进展

结核病是由结核分枝杆菌(Mycobacterium tuberculosis)通过空气传播引起人类感染的慢性传染病,耐药结核分枝杆菌的流行是目前结核病防治的世界难题。氟喹诺酮类药物是人工合成药物,应用于耐药结核的临床治疗中,在治疗中起着核心的作用。但近年来,氟喹诺酮类药物的抗性菌株不断出现,愈发增加了结核病治疗的困难与治疗失败风险。在临床中氟喹诺酮药物的靶点比较清楚,是结核分枝杆菌的DNA旋转酶。目前发现结核分枝杆菌耐氟喹诺酮类药物的机制主要包括药物靶点DNA旋转酶的关键氨基酸改变、药物外排泵系统、细菌细胞壁厚度的增加以及喹诺酮抗性蛋白MfpA介导的DNA旋转酶活性调控。其中在氟喹诺酮靶标DNA旋转酶功能活性改变的耐药机制方面,编码DNA旋转酶基因突变一直是研究的热点,但近年来发现DNA旋转酶的调控蛋白MfpA以及DNA旋转酶的修饰在细菌耐药性中起着重要的作用,相关机制还亟待发现。本文综述了当前结核分枝杆菌耐氟喹诺酮类药物的作用机制,旨在为研发精准诊断技术和药物发掘提供科学理论基础和参考。     

A monoclonal antibody that inhibits mycobacterial DNA gyrase by a novel mechanism

DNA gyrase is a DNA topoisomerase indispensable for cellular functions in bacteria. We describe a novel, hitherto unknown, mechanism of specific inhibition of Mycobacterium smegmatis and Mycobacterium tuberculosis DNA gyrase by a monoclonal antibody (mAb). Binding of the mAb did not affect either GyrA-GyrB or gyrase-DNA interactions. More importantly, the ternary complex of gyrase-DNA-mAb retained the ATPase activity of the enzyme and was competent to catalyse DNA cleavage-religation reactions, implying a new mode of action different from other classes of gyrase inhibitors. DNA gyrase purified from fluoroquinolone-resistant strains of M.tuberculosis and M.smegmatis were inhibited by the mAb. The absence of cross-resistance of the drug-resistant enzymes from two different sources to the antibody-mediated inhibition corroborates the new mechanism of inhibition. We suggest that binding of the mAb in the proximity of the primary dimer interface region of GyrA in the heterotetrameric enzyme appears to block the release of the transported segment after strand passage, leading to enzyme inhibition. The specific inhibition of mycobacterial DNA gyrase with the mAb opens up new avenues for designing novel lead molecules for drug discovery and for probing gyrase mechanism.     

DNA gyrase and DNA topoisomerase of Bacillus subtilis: expression and characterization of recombinant enzymes encoded by the gyrA,gyrB and parC,parE genes

Bacillus subtilis Bs gyrA and gyrB genes specifying the DNA gyrase subunits, and parC and parE genes specifying the DNA topoisomerase IV subunits, have been separately cloned and expressed in Escherichia coli as hexahistidine (his6)-tagged recombinant proteins. Purification of the gyrA and gyrB subunits together resulted in predominantly two bands at molecular weights of 94 and 73kDa; purification of the parC and parE subunits together resulted in predominantly two bands at molecular weights of 93 and 75kDa, as predicted by their respective sequences. The ability of the subunits to complement their partner was tested in an ATP-dependent decatenation/supercoiling assay system. The results demonstrated that the DNA gyrase and the topoisomerase IV subunits produce the expected supercoiled DNA and relaxed DNA products, respectively. Additionally, inhibition of these two enzymes by fluoroquinolones has been shown to be comparable to those of the DNA gyrases and topoisomerases of other bacterial strains. In sum, the biological and enzymatic properties of these products are consistent with their authenticity as DNA gyrase and DNA topoisomerase IV enzymes from B. subtilis.     

Structural insights into quinolone antibiotic resistance mediated by pentapeptide repeat proteins: conserved surface loops direct the activity of a Qnr protein from a gram-negative bacterium

Quinolones inhibit bacterial type II DNA topoisomerases (e.g. DNA gyrase) and are among the most important antibiotics in current use. However, their efficacy is now being threatened by various plasmid-mediated resistance determinants. Of these, the pentapeptide repeat-containing (PRP) Qnr proteins are believed to act as DNA mimics and are particularly prevalent in gram-negative bacteria. Predicted Qnr-like proteins are also present in numerous environmental bacteria. Here, we demonstrate that one such, Aeromonas hydrophila AhQnr, is soluble, stable, and relieves quinolone inhibition of Escherichia coli DNA gyrase, thus providing an appropriate model system for gram-negative Qnr proteins. The AhQnr crystal structure, the first for any gram-negative Qnr, reveals two prominent loops (1 and 2) that project from the PRP structure. Deletion mutagenesis demonstrates that both contribute to protection of E. coli DNA gyrase from quinolones. Sequence comparisons indicate that these are likely to be present across the full range of gram-negative Qnr proteins. On this basis we present a model for the AhQnr:DNA gyrase interaction where loop1 interacts with the gyrase A 'tower' and loop2 with the gyrase B TOPRIM domains. We propose this to be a general mechanism directing the interactions of Qnr proteins with DNA gyrase in gram-negative bacteria.     

DNA gyrase (Topoisomerase II) from Pseudomonas aeruginosa

DNA gyrase (Topoisomerase II) has been purified from Pseudomonas aeruginosa strain PAO. This enzyme is inhibited by novobiocin and nalidixic acid. DNA gyrase from P. aeruginosa is resistant to a much higher level of nalidixic acid than is Escherichia coli DNA gyrase. This increased level of resistance may explain, at least in part, the higher levels of natural resistance exhibited by P. aeruginosa toward nalidixic acid.     

Nucleotide sequence of the Staphylococcus aureus gyrB-gyrA locus encoding the DNA gyrase A and B proteins.

We have determined the nucleotide sequence of a 5.3-kb segment of the Staphylococcus aureus chromosome that includes the gyrA and gyrB genes coding for both subunits of DNA gyrase, the enzyme that catalyzes ATP-dependent DNA supercoiling. The gene order at this locus, dnaA-dnaN-recF-gyrB-gyrA, is similar to that found in the Bacillus subtilis replication origin region. S. aureus recF, gyrB, and gyrA genes are closely spaced, occupy the same reading frame, and may be coordinately expressed. The S. aureus gyrB and gyrA genes encode 640- and 889-residue proteins, respectively, that share strong homology with other bacterial gyrase subunits, notably those from B. subtilis. These results are discussed in regard to the mechanism of DNA gyrase and its role as a target for the 4-quinolones and other antistaphylococcal agents.     

Measurement of Pseudomonas aeruginosa multidrug efflux pumps by quantitative real-time polymerase chain reaction

Multidrug efflux pumps contribute to multiple antibiotic resistance in Pseudomonas aeruginosa. Pump expression usually has been quantified by Western blotting. Quantitative real-time polymerase chain reaction has been developed to measure mRNA expression for genes of interest. Whether this method correlates with pump protein quantities is unclear. We devised a real-time PCR for mRNA expression of MexAB-OprM and MexXY-OprM multidrug efflux pumps. In laboratory strains differing in MexB and MexY expression and in several clinical isolates, protein and mRNA expression correlated well. Quantitative real-time PCR should be a useful alternative in quantitating expression of multidrug efflux pumps by P. aeruginosa isolates in clinical laboratories.     

Comparison of the metabolic activities of four wild-type <Emphasis Type="Italic">Clostridium perfringens</Emphasis> strains with their gatifloxacin-selected resistant mutants

The production of short-chain fatty acids, reductive enzymes, and hydrolytic enzymes by four gatifloxacin-selected, fluoroquinolone-resistant, mutant strains of C. perfringens, with stable mutations either in DNA gyrase or in both DNA gyrase and topoisomerase IV, was compared with that produced by the wild-type parent strains to investigate the effect of mutations associated with the selection of gatifloxacin resistance on bacterial metabolic activities. The mutants differed from their respective wild-type parent strains in the enzymatic activities of azoreductase, nitroreductase, and β-glucosidase and in the ratio of butyric acid to acetic acid production. Microarray analysis of one wild type and the corresponding mutant revealed different levels of mRNA expression for the enzymes involved in short-chain fatty acid (SCFA) synthesis and for β-glucosidase and oxidoreductases. In addition to mutations in the target genes, selection of resistance to gatifloxacin resulted in strain-specific physiological changes in the resistant mutants of C. perfringens that affected their metabolic activities.     

Characterization of the antiseptic-resistance gene qacEΔ1 isolated from clinical and environmental isolates of Vibrio parahaemolyticus and Vibrio cholerae non-O1

The nucleotide sequence and mechanism of action were examined on the antiseptic-resistance gene qacE delta 1 that had been isolated from Pseudomonas aeruginosa, Vibrio parahaemolyticus and Vibrio cholerae non-O1. The nucleotide sequences of qacE delta 1 genes isolated from environmental isolates of V. cholerae non-O1 and V. parahaemolyticus differed by one base from that of the gene from P. aeruginosa. Escherichia coli C600 that harbored qacE delta 1 genes from several strains of Vibrio spp. exhibited low-level resistance to intercalating dyes. The resistance of E. coli cells with these genes to intercalating dyes, such as ethidium bromide, was mediated by an efflux system. Moreover, the activity of QacE delta 1 was inhibited in the presence of calcium channel blockers but not of calmodulin inhibitors. These results indicate that the qacE delta 1 gene can be function in E. coli and that the gene mediates resistance in a similar manner to the antiseptic-resistance gene smr.     

Diversity of tet resistance genes in tetracycline-resistant bacteria isolated from a swine lagoon with low antibiotic impact

Tetracycline resistance has been extensively studied and shown to be widespread. A number of previous studies have clearly demonstrated that a variety of tetracycline resistance genes are present in swine fecal material, treatment lagoons, and the environments surrounding concentrated animal feeding operations (CAFOs). The diversity of tetracycline resistance within a swine lagoon located at a CAFO that used only bacitricin methylene disalicylate as an antibiotic was evaluated by screening 85 tetracycline-resistant isolates for the presence of 18 different genes by performing PCR with primers that target tetracycline efflux genes of Gram-negative bacteria and ribosomal protection proteins. In addition, partial 16S rRNA sequences from each of these isolates were sequenced to determine the identity of these isolates. Of the 85 isolates examined, 17 may represent potential novel species based on BLAST results. Greater than 50% of the isolates (48 out of 85) were found to not contain targeted tet efflux genes. Though minimum inhibitory concentrations ranged widely (16 - >256 mg/L), these values did not give an indication of the tet genes present. Ten new genera were identified that contain at least one tet efflux gene. Five other genera possessed tet efflux genes that were not found in these organisms previously. Interestingly, none of the isolates possessed any of the selected ribosomal protection protein genes. Though tetracycline resistance was found in bacteria isolated from a swine CAFO lagoon, it appears that the limited antibiotic use at this CAFO might have impacted the presence and diversity of tetracycline resistance genes.     

Modulation of DNA supercoiling activity of Escherichia coli DNA gyrase by F plasmid proteins. Antagonistic actions of LetA (CcdA) and LetD (CcdB) proteins.

The letA (ccdA) and letD (ccdB) genes of F plasmid contribute to stable maintenance of the plasmid in Escherichia coli cells; a product of the latter has a lethal effect on the host cell and that of the former neutralizes functions of the letD. In cells that overproduce the LetD (CcdB) protein, the plasmid DNA is extensively relaxed. Correspondingly, DNA supercoiling activity in a cell-free extract of the overproducing strain decreases to a level of less than 1% of that seen in normal cells. However, the extract does not inhibit DNA gyrase reconstituted from purified subunits, thereby indicating that the intrinsic DNA gyrase is inactivated in the overproducing strain. Upon addition of purified LetA (CcdA) protein to the extract of LetD overproducing cells, the DNA supercoiling activity was fully restored. Using this rejuvenation as an assay, we purified the "inactivated gyrase" and obtained evidence that the LetD protein formed an isolable complex with the A subunit of DNA gyrase. Thus, the LetD and the LetA proteins constitute an opposing pair in modulating the DNA supercoiling activity of gyrase, probably by direct interaction.     

Identification and molecular characterization of class 1 integrons in multiresistant Escherichia coli isolates from poultry litter

This study describes the prevalence of arrays of class 1 integron cassettes and Qnr determinants (A, B, and S) in 19 fluoroquinolone-resistant Escherichia coli isolates from chicken litter. qnrS and qnrA were the predominant genes in these fluoroquinolone-resistant isolates, and an uncommon array of aacA4-catB3-dfrA1 gene cassettes from a class1 integron was found. Additionally, aadA1 and dfrA1 gene cassettes, encoding resistance to streptomycin and trimethoprim, constituted the most common genes identified and was located on megaplasmids as well on the chromosome. Antibiotic resistance, pulsed-field gel electrophoresis (PFGE), and plasmid data suggest a genetically diverse origin of poultry E. coli isolates.     

Identification of a gene cluster, czr, involved in cadmium and zinc resistance in Pseudomonas aeruginosa

Pseudomonas aeruginosa CMG103 was isolated from a metal-polluted river in Pakistan and displayed a high level of Zn and Cd resistance. An omega-Km transposon mutant of strain CMG103, which showed a substantial decrease in resistance to Zn and Cd, was obtained. A 12.8 kb region determining Zn and Cd resistance in strain CM103 was cloned by complementing the mutant strain, and its nt sequence was determined. Five genes, czrSRCBA, involved in Zn and Cd resistance, were identified. The predicted gene products of czrCBA show a significant similarity with the proteins encoded by the plasmid borne metal resistant determinants czc, cnr and ncc of Ralstonia strains, which determine a chemiosmotic cation-antiporter efflux system. The predicted CzrS and CzrR proteins show a significant similarity to the sensor and regulatory protein, respectively, of two component regulatory systems, such as CopS/CopR and PcoS/PcoR involved in the regulation of plasmid-borne Cu-resistant determinants, and CzcS/CzcR involved in the regulation of czc. The cloned czr region contained downstream of czrCBA additional ORFs whose predicted gene products are similar to proteins involved in catabolism of aromatic compounds. DNA-DNA hybridization indicated strong conservation of czr in other environmental P. aeruginosa isolates and in the P. aeruginosa type strain PAO1, a clinical isolate. This was confirmed by a comparison of the sequence of the CMG103 czr region with the currently available genome sequence of strain PAO1. A high sequence identity (till 99% at the nt level) and organizatory conservation of the czr region of CMG103 was found in PAO1 as well regarding coding sequences as intervening sequences between ORFs. The czr locus was localized between coordinates 2400 and 2550 kb on the physical map of the chromosome of PAO1.