Topoisomerase Genes Research Articles

Multiple transmissible genes encoding fluoroquinolone and third-generation cephalosporin resistance co-located in non-typhoidal Salmonella isolated from food-producing animals in China

The aim of this study was to identify genes conferring resistance to fluoroquinolones and extended-spectrum β-lactams in non-typhoidal Salmonella (NTS) from food-producing animals in China. In total, 31 non-duplicate NTS were obtained from food-producing animals that were sick. Isolates were identified and serotyped and the genetic relatedness of the isolates was determined by pulsed-field gel electrophoresis of XbaI-digested chromosomal DNA. Antimicrobial susceptibility was determined using Clinical and Laboratory Standards Institute methodology. The presence of extended-spectrum β-lactamase (ESBL) and fluoroquinolone resistance genes was established by PCR and sequencing. Genes encoded on transmissible elements were identified by conjugation and transformation. Plasmids were typed by PCR-based replicon typing. The occurrence and diversity of numerous different transmissible genes conferring fluoroquinolone resistance [qnrA, qnrD, oqxA and aac(6′)-Ib-cr] and ESBLs (CTX-M-27 and CTX-M-14), and which co-resided in different isolates and serovars of Salmonella, were much higher than in European countries. Furthermore, different plasmids encoded fluoroquinolone resistance (ca. 6kb) and β-lactam resistance (ca. 63kb) and these co-resided in isolates with mutations in topoisomerase genes (gyrA and parC) giving very resistant Salmonella. The presence of multidrug-resistant bacteria in food-producing animals in countries that export foodstuffs suggests that global transfer of antibiotic resistances from country to country on food is possible.

Prevalence and Molecular Characterization of Fluoroquinolone Resistance inEscherichia coliIsolates from Dairy Cattle with Endometritis in China

Fluoroquinolones are frequently used to treat infectious disease that is caused by Escherichia coli in dairy cattle. However, fluoroquinolone resistance occurs and is due either to chromosomal mutations in the bacterial topoisomerase genes and/or to plasmid-mediated resistance genes. The purpose of this study was to determine the prevalence and molecular characteristics of fluoroquinolone resistance determinants in E. coli strains (n=148) isolated from dairy cattle with bovine endometritis in Inner Mongolia (China). Analysis of the mutations in the quinolone resistance-determining regions of resistant E. coli isolates confirmed previously reported substitutions in the GyrA and ParE. However, we identified additional substitutions in the ParC and GyrB that have not been reported earlier. No plasmid-mediated quinolone resistance genes in any of the isolates were found. The number of point mutations found per isolate correlated with an increase in the minimum inhibitory concentration of ciprofloxacin. Overall, 45.5% of the isolates were positive for the class I integrase gene along with four gene cassettes that were responsible for resistance to trimethoprim (dfr1 and dfrA17) and aminoglycosides (aadA1 and aadA5), respectively. The prevalence of extended-spectrum β-lactamases (ESBLs) was 100%, and the blaTEM gene was predominant in all of the isolates. In conclusion, our results identify the mechanism of quinolone resistance for the first time and reveal the prevalence of integron and ESBLs in E. coli isolates from dairy cattle with bovine endometritis in China after 20 years of quinolone usage in cattle.

ISOLATION AND CHARACTERIZATION OF A CERVIDPOXVIRUS FROM A GOITERED GAZELLE (GAZELLA SUBGUTTUROSA) FROM A ZOOLOGIC PARK IN MINNESOTA

Deerpox virus (DPV) is the sole member of the newly ratified Cervidpoxvirus genus in the subfamily Chordopoxvirinae. Presented here is the first diagnostic report of isolation of DPV from a goitered gazelle (Gazella subgutturosa). A tissue homogenate was submitted by a zoologic park to the Minnesota Veterinary Diagnostic Laboratory at the University of Minnesota for poxvirus diagnostic investigation and then referred to Plum Island Foreign Animal Disease Diagnostic Laboratory for confirmation. Poxviral infection was confirmed using electron microscopy. The virus was cultured in vero cells and subjected to further diagnoses for characterization. Polymerase chain reaction targeting the major envelope (B2L) protein and RNA polymerase of parapoxviruses, and the poly-A polymerase gene of capripoxviruses, were all negative. Degenerative pan-poxvirus primers that target the DNA polymerase (DNApol) and DNA topoisomerase (DNAtopo) genes, however, successfully amplified poxviral DNA fragments. Amplification of the DNApol and DNAtopo genes yielded fragments of 543 and 344 base pairs, respectively. DNA sequence and phylogenetic analysis of each gene fragment from the gazelle isolate showed >97% identity in BLAST searches with two DPV virus strains (W848-83 and W-1170-84) isolated from North American mule deer (Odocoileus hemionus) in 1983-1984. Neighbor-joining trees indicate that the isolate is a member of the Cervidpoxvirus genus and shows a more-distant relationship to other ruminant poxviruses, namely the Capripoxvirus genus consisting of lumpy skin disease, sheeppox, and goatpox viruses. This report documents the premiere finding of DPV, a recently characterized virus, in gazelles and demonstrates the need for broadened investigation when diagnosing poxvirus infections in ruminants.

Role of Topoisomerases in Pediatric High Grade Osteosarcomas: TOP2A Gene Is One of the Unique Molecular Biomarkers of Chemoresponse

Currently, the treatment of pediatric high-grade osteosarcomas systematically includes one topoisomerase IIα inhibitor. This chemotherapy is usually adapted to the response to the neo-adjuvant therapy after surgery. The current and unique marker of chemoresponsiveness is the percentage of viable residual cells in the surgical resection. This late patient management marker has to be evaluated earlier in the therapeutic history of the patients on initial biopsy. Therefore, new biomarkers, especially those involved in the topoisomerase IIα inhibitor response might be good candidates. Therefore, our study was designed to target TOP1, TOP2A and TOP2B genes in 105 fresh-frozen diagnostic biopsies by allelotyping and real-time quantitative PCR. Our analyses in those pediatric osteosarcomas, homogeneously treated, highlighted the frequent involvement of topo-isomerase genes. The main and most important observation was the statistical link between the presence of TOP2A amplification and the good response to neo-adjuvant chemotherapy. Compared to adult cancers, the 17q21 amplicon, including TOP2A and ERBB2 genes, seems to be differentially implicated in the osteosarcoma chemoresponsiveness. Surprisingly, there is no ERBB2 gene co-amplification and the patients harboring TOP2A amplification tend to show a worse survival, so TOP2A analyses remain a preliminary, but a good molecular approach for the evaluation at diagnosis of pediatric osteosarcoma chemoresponsiveness.

Chromosomal and plasmid-mediated fluoroquinolone resistance mechanisms among broad-spectrum-cephalosporin-resistant Escherichia coli isolates recovered from companion animals in the USA

To determine the prevalence of plasmid-mediated quinolone resistance (PMQR) determinants and investigate mutations in gyrase and topoisomerase genes that may contribute to increased fluoroquinolone resistance in canine and feline Escherichia coli isolates in the USA that displayed reduced susceptibility to extended-spectrum cephalosporins. This study was undertaken because previous epidemiological studies identified a potential correlation between extended-spectrum cephalosporins and fluoroquinolone resistance. Isolates (n = 54) with reduced susceptibility to ceftazidime or cefotaxime were screened by PCR for the presence of PMQR determinants and gyrase and topoisomerase genes were sequenced. Isolates were further characterized by conjugation and phylogenetic analyses. PMQR determinants aac(6')-Ib-cr, qnrS and qepA were identified in 30, 23 and 5 isolates, respectively. Multiple mutations were identified in the quinolone resistance-determining region, including the novel substitutions of Glu-84 → Ala and Leu-88 → Gln in ParC and Arg-432 → Ser and Glu-460 → Val in ParE. The isolate that exhibited the highest level of enrofloxacin resistance (MIC > 256 mg/L) had a double mutation in gyrA (Ser-83 → Leu and Asp-87 → Asn) and a triple mutation in parC (Ser-80 → Ile, Glu-84 → Gly and a novel mutation, Leu-88 → Gln). The presence of PMQR genes increased the ciprofloxacin MIC values 4-fold to 8-fold in transconjugants relative to the recipient strain. Approximately 39% of the isolates belonged to phylogenetic group D and 30% to group B2, which typically contain an increased number of virulence determinants compared with other groups. Novel mutations in topoisomerase genes and PMQR determinants aac(6')-Ib-cr, qnrS and qepA genes were detected among extended-spectrum β-lactamase-producing E. coli in the USA.

Plasmid-mediated quinolone resistance (PMQR) and mutations in the topoisomerase genes of Salmonella enterica strains from Brazil

The objective of this study was to identify mutations in the Quinolone Resistance Determining sources Regions (QRDR) of the gyrA, gyrB, parC, and parE genes and to determine if any of the qnr variants or the aac(6′)-Ib-cr variant were present in strains of Salmonella spp. isolated in Brazil. A total of 126 Salmonella spp. strains from epidemic (n = 114) and poultry (n = 12) origin were evaluated. One hundred and twelve strains (88.8%) were resistant to nalidixic acid (NAL) and 29 (23.01%) showed a reduced susceptibility to ciprofloxacin (Cip). The mutations identified were substitutions limited to the QRDR of the gyrA gene in the codons for Serine 83, Aspartate 87 and Alanine 131. The sensitivity to NAL seems to be a good phenotypic indication of distinguishing mutated and non-mutated strains in the QRDR, however the double mutation in gyrA did not cause resistance to ciprofloxacin. The qnrA1 and qnrB19 genes were detected, respectively, in one epidemic strain of S. Enteritidis and one strain of S. Corvallis of poultry origin. Despite previous detection of qnr genes in Brazil, this is the first report of qnr gene detection in Salmonella, and also the first detection of qnrB19 gene in this country. The results alert for the continuous monitoring of quinolone resistance determinants in order to minimize the emergence and selection of Salmonella spp. strains showing reduced susceptibility or resistance to quinolones.

Characterization of Recombinant Fluoroquinolone-Resistant Pneumococcus-Like Isolates

Fourteen fluoroquinolone-resistant streptococcal isolates with recombinant DNA topoisomerase genes, preliminarily identified as pneumococci, were further characterized using phenotypic and genotypic approaches. Phenotypic tests classified them as atypical pneumococci. Phylogenetic relationships were analyzed by using the sequences of seven housekeeping alleles from these isolates and from isolates of Streptococcus pneumoniae, Streptococcus mitis, Streptococcus oralis, and Streptococcus pseudopneumoniae. Four isolates grouped with S. pneumoniae, seven grouped with S. pseudopneumoniae, and three grouped with S. mitis. These results generally agreed with those obtained with an optochin susceptibility test and with the organization of the atp operon chromosomal region, encoding the F(o)F(1) H(+)-ATPase (the target of optochin). All seven isolates grouping with S. pseudopneumoniae share the same spr1368-atpC-atpA gene order; all four grouping with S. pneumoniae share the spr1368-IS1239-atpC-atpA order, and two out of the three grouping with S. mitis share the spr1284-atpC-atpA order. In addition, evidence for recombination within the seven housekeeping alleles of the S. pseudopneumoniae population was provided by several methods: the index of association (0.4598, P < 0.001), the pairwise homoplasy index, and the split-decomposition method. This study confirms the existence of pneumococci among the alpha-hemolytic streptococci with DNA topoisomerase genes showing a mosaic structure and reveals a close relationship between atypical pneumococci and S. pseudopneumoniae.

Prevalence of quinolone resistance and mutations in the topoisomerase genes in Salmonella enterica serotype Enteritidis isolates from Serbia

The prevalence of quinolone resistance was studied in Salmonella enterica serotype Enteritidis isolates collected during 2005–2010 in Southern Bačka County, Serbia. A total of 878 clinical isolates were examined, among which 19 (2.2%) nalidixic acid (NAL)-resistant S. Enteritidis were detected by selection on agar plates containing 64mg/L NAL. Antimicrobial susceptibility of the isolates was tested by the agar dilution method. According to Clinical and Laboratory Standards Institute (CLSI) breakpoints, ciprofloxacin (CIP) resistance was not present in the strains. Multiple drug resistance was rare, and resistance to NAL was most often present as a single resistance property. All but one NAL-resistant S. Enteritidis showed reduced susceptibility to CIP [i.e. minimum inhibitory concentration (MIC)≥0.125mg/L]. This isolate of human origin had a CIP MIC of 0.064mg/L and DNA sequencing revealed that it contained an Asp87Gly gyrA mutation. Most of the remaining isolates had MICs for NAL and CIP of 256mg/L and 0.256mg/L, respectively. Mutations in the Asp87 codon resulted in substitutions to Asn in most of the isolates, but Asp87Gly and Ser83Phe exchanges were also detected. No mutations were present in the gyrB, parC or parE genes. Although CIP resistance was absent, reduced susceptibility characterised by mutations in gyrA was apparent among S. Enteritidis isolates from Serbia.

Molecular characterization of quinolone-insensitive Streptococcus pneumoniae isolates from Japanese patients

Fluoroquinolone resistance in Streptococcus pneumoniae has become a growing concern. Using S. pneumoniae isolates (n=61) for which the minimum inhibitory concentration (MIC) of levofloxacin was not less than 1μg/ml, we investigated the susceptibility of S. pneumoniae isolates to other fluoroquinolones (ciprofloxacin, pazufloxacin, moxifloxacin, garenoxacin, and sitafloxacin) and sequenced the quinolone resistance-determining regions of two topoisomerase genes, parC and gyrA, in these isolates to identify mutations. As the number of missense mutations increased, the MIC values for each drug increased. However, moxifloxacin, garenoxacin, and sitafloxacin showed potent activities against the isolates, while the MICs of ciprofloxacin and pazufloxacin were higher than the MICs of levofloxacin.

Increase in fluoroquinolone non-susceptibility among clinical Streptococcus pyogenes in Belgium during 2007-10

To study the temporal evolution of fluoroquinolone non-susceptibility among Streptococcus pyogenes during 2007-10 in Belgium. S. pyogenes (n = 4690) recovered from patients with tonsillopharyngitis or skin, wound or invasive infections were screened for fluoroquinolone non-susceptibility. A selection of fluoroquinolone-non-susceptible strains was investigated for resistance mechanisms: reserpine-sensitive efflux and mutations in topoisomerase genes parC and gyrA. Clonality was determined by emm typing. Fluoroquinolone non-susceptibility (ciprofloxacin MIC ≥2 mg/L) was identified in 535 (11.4%) of 4690 S. pyogenes recovered during 2007-10 in Belgium. The proportion of fluoroquinolone-non-susceptible S. pyogenes increased significantly from 4.3% (2008) to 10.9% (2009) to 21.6% (2010) and coincided with a significant increase in emm6 strains among fluoroquinolone-non-susceptible S. pyogenes. Ciprofloxacin MICs of 2-8 mg/L correlated with first-step ParC substitutions. Two high-level fluoroquinolone-resistant S. pyogenes strains (ciprofloxacin MICs 32 mg/L) showed second-step substitutions in GyrA (Ser-81→Phe or Tyr) in addition to first-step mutations in parC. Reserpine-sensitive efflux was not observed. We report an unprecedented increase in fluoroquinolone-non-susceptible S. pyogenes in Belgium, a country with high quinolone use, as well as emergence of two high-level fluoroquinolone-resistant S. pyogenes strains with second-step mutations in gyrA, warning us of the need for more prudent use of fluoroquinolones and for continued resistance surveillance.

Emerging fluoroquinolone resistance in Streptococcus agalactiae in South Korea

The goal of this study was to determine the prevalence of fluoroquinolone resistance in Streptococcus agalactiae and the serotype distribution of this resistant bacterium. S. agalactiae strains collected from 221 asymptomatic pregnant women (35-37weeks of gestation) and 838 patients with S. agalactiae infection in Korea, from 2006 to 2008, were tested for susceptibility to four fluoroquinolones. Rates of resistance of S. agalactiae to ciprofloxacin, levofloxacin, and moxifloxacin were 9.3%, 9.5%, and 0.8%, respectively; greater than 94% of S. agalactiae strains were resistant to norfloxacin. Resistance to ciprofloxacin and levofloxacin increased between 2006 and 2008. All strains were susceptible to penicillin. Resistance to ciprofloxacin and levofloxacin was higher in the clinical strains of S. agalactiae isolated from infections than in colonizing strains isolated from pregnant women. Mutations in the quinolone resistance-determining regions of gyrase and topoisomerase genes were detected in strains resistant to ciprofloxacin, levofloxacin, and moxifloxacin; no such mutations were found in strains resistant only to norfloxacin. There was a strong correlation between the minimum inhibitory concentrations and the presence of mutations in gyrase and topoisomerase genes. In conclusion, the prevalence of fluoroquinolone resistance was unexpectedly high. Strain serotypes were not associated with susceptibility to fluoroquinolones.

Model for MLL translocations in therapy-related leukemia involving topoisomerase IIβ-mediated DNA strand breaks and gene proximity

Topoisomerase poisons such as the epipodophyllotoxin etoposide are widely used effective cytotoxic anticancer agents. However, they are associated with the development of therapy-related acute myeloid leukemias (t-AMLs), which display characteristic balanced chromosome translocations, most often involving the mixed lineage leukemia (MLL) locus at 11q23. MLL translocation breakpoints in t-AMLs cluster in a DNase I hypersensitive region, which possesses cryptic promoter activity, implicating transcription as well as topoisomerase II activity in the translocation mechanism. We find that 2-3% of MLL alleles undergoing transcription do so in close proximity to one of its recurrent translocation partner genes, AF9 or AF4, consistent with their sharing transcription factories. We show that most etoposide-induced chromosome breaks in the MLL locus and the overall genotoxicity of etoposide are dependent on topoisomerase IIβ, but that topoisomerase IIα and -β occupancy and etoposide-induced DNA cleavage data suggest factors other than local topoisomerase II concentration determine specific clustering of MLL translocation breakpoints in t-AML. We propose a model where DNA double-strand breaks (DSBs) introduced by topoisomerase IIβ into pairs of genes undergoing transcription within a common transcription factory become stabilized by antitopoisomerase II drugs such as etoposide, providing the opportunity for illegitimate end joining and translocation.

Characteristics of Klebsiella pneumoniae harboring QnrB32, Aac(6')-Ib-cr, GyrA and CTX-M-22 genes.

Quinolone resistance in members of the Enterobacteriaceae family is mostly due to mutations in the quinolone resistance-determining regions of topoisomerase genes. CTX-M-22 is a member of the CTX-M family which can reduce extended-spectrum β-lactamase (ESBL) production and modulate antibiotic resistance, resulting in low ceftazidime minimum inhibitory concentrations (MICs). There are four different genes in Klebsiella pneumoniae (KP4707) including qnrB32 (novel qnr allele gene, HQ704413), aac(6')-Ib-cr (novel aac(6')-Ib allele gene, HQ680690), gyrA (novel gyrA allele gene, HQ680691) and CTX-M-22 gene. Five point amino acid mutations Arn(N)27 → Leu(L), Val(V)129 → Ala(A), Iie(I)142 → Met(M), Gly(G)188 → Arg(R), Val(V)212 → Iie(I) were observed in the qnr32 gene when compared to qnrB1. Of all qnrB alleles, a novel variant of the qnrB32 gene, with qnrB31, had the highest amino acid homology. Three point amino acid mutations including Trp(W)105 → Arg(R), Asp(D)182 → Tyr(Y) and Val(V)201 → Asp(D) were observed in aac(6')-Ib-cr gene, when compared to GenBank number AF479774. New variants of qnr32, aac(6')-Ib-cr, gyrA and CTX-M-22 or other genotype determinants continuously appear in different genomic sites and also outside the Enterobacteriaceae family.

Xer Recombinase and Genome Integrity in Helicobacter pylori, a Pathogen without Topoisomerase IV

In the model organism E. coli, recombination mediated by the related XerC and XerD recombinases complexed with the FtsK translocase at specialized dif sites, resolves dimeric chromosomes into free monomers to allow efficient chromosome segregation at cell division. Computational genome analysis of Helicobacter pylori, a slow growing gastric pathogen, identified just one chromosomal xer gene (xerH) and its cognate dif site (difH). Here we show that recombination between directly repeated difH sites requires XerH, FtsK but not XerT, the TnPZ transposon associated recombinase. xerH inactivation was not lethal, but resulted in increased DNA per cell, suggesting defective chromosome segregation. The xerH mutant also failed to colonize mice, and was more susceptible to UV and ciprofloxacin, which induce DNA breakage, and thereby recombination and chromosome dimer formation. xerH inactivation and overexpression each led to a DNA segregation defect, suggesting a role for Xer recombination in regulation of replication. In addition to chromosome dimer resolution and based on the absence of genes for topoisomerase IV (parC, parE) in H. pylori, we speculate that XerH may contribute to chromosome decatenation, although possible involvement of H. pylori's DNA gyrase and topoisomerase III homologue are also considered. Further analyses of this system should contribute to general understanding of and possibly therapy development for H. pylori, which causes peptic ulcers and gastric cancer; for the closely related, diarrheagenic Campylobacter species; and for unrelated slow growing pathogens that lack topoisomerase IV, such as Mycobacterium tuberculosis.

Temporal Interplay between Efflux Pumps and Target Mutations in Development of Antibiotic Resistance in Escherichia coli

The emergence of resistance presents a debilitating change in the management of infectious diseases. Currently, the temporal relationship and interplay between various mechanisms of drug resistance are not well understood. A thorough understanding of the resistance development process is needed to facilitate rational design of countermeasure strategies. Using an in vitro hollow-fiber infection model that simulates human drug treatment, we examined the appearance of efflux pump (acrAB) overexpression and target topoisomerase gene (gyrA and parC) mutations over time in the emergence of quinolone resistance in Escherichia coli. Drug-resistant isolates recovered early (24 h) had 2- to 8-fold elevation in the MIC due to acrAB overexpression, but no point mutations were noted. In contrast, high-level (≥ 64× MIC) resistant isolates with target site mutations (gyrA S83L with or without parC E84K) were selected more readily after 120 h, and regression of acrAB overexpression was observed at 240 h. Using a similar dosing selection pressure, the emergence of levofloxacin resistance was delayed in a strain with acrAB deleted compared to the isogenic parent. The role of efflux pumps in bacterial resistance development may have been underappreciated. Our data revealed the interplay between two mechanisms of quinolone resistance and provided a new mechanistic framework in the development of high-level resistance. Early low-level levofloxacin resistance conferred by acrAB overexpression preceded and facilitated high-level resistance development mediated by target site mutation(s). If this interpretation is correct, then these findings represent a paradigm shift in the way quinolone resistance is thought to develop.

Mutations in the quinolone resistance-determining regions of gyrA and parC in Enterobacteriaceae isolates from Brazil.

Mutations in the quinolone resistance-determining regions (QRDR) in chromosomal gyrA and parC genes and fluoroquinolone susceptibility profiles were investigated in quinolone-resistant Enterobacteriaceae isolated from community and hospitalized patients in the Brazilian Southeast region. A total of 112 nalidixic acid-resistant enterobacterial isolates collected from 2000 to 2005 were investigated for mutations in the topoisomerases genes gyrA and parC by amplifying and sequencing the QRDR regions. Susceptibility to fluoroquinolones was tested by the agar dilution method. Amongst the 112 enterobacterial isolates, 81 (72.3%) were resistant to ciprofloxacin and 5 (4.5%) showed reduced susceptibility. Twenty-six (23.2%) were susceptible to ciprofloxacin. Several alterations were detected in gyrA and parC genes. Escherichia coli isolates (47.7%) showed double mutations in the gyrA gene and a single one in the parC gene. Two unusual aminoacid substitutions are reported, an Asp87-Asn in a Citrobacter freundii isolate with reduced susceptibility to fluoroquinolones and a Glu84-Ala in one E. coli isolate. Only a parC gene mutation was found in fluoroquinolone-susceptible Enterobacter aerogenes. None of the isolates susceptible to ciprofloxacin presented mutations in topoisomerase genes. This comprehensive analysis of QRDRs in gyrA and parC genes, covering commonly isolated Enterobacteriaceae in Brazil is the largest reported up to now.

Prevalence and characteristics of quinolone resistance in Escherichia coli in veal calves

Quinolone resistance is studied and reported increasingly in isolates from humans, food-producing animals and companion animals. Resistance can be caused by chromosomal mutations in topoisomerase genes, plasmid-mediated resistance genes, and active transport through efflux pumps. Cross sectional data on quinolone resistance mechanisms in non-pathogenic bacteria from healthy veal calves is limited. The purpose of this study was to determine the prevalence and characteristics of quinolone resistance mechanisms in Escherichia coli isolates from veal calves, after more than 20 years of quinolone usage in veal calves. MIC values were determined for all isolates collected as part of a national surveillance program on antimicrobial resistance in commensal bacteria in food-producing animals in The Netherlands. From the strains collected from veal calves in 2007 (n=175) all isolates with ciprofloxacin MIC≥0.125mg/L (n=25) were selected for this study, and screened for the presence of known quinolone resistance determinants. In this selection only chromosomal mutations in the topoisomerase type II and IV genes were detected. The number of mutations found per isolate correlated with an increasing ciprofloxacin MIC. No plasmid-mediated quinolone resistance genes were found. The contribution of efflux pumps varied from no contribution to a 16-fold increase in susceptibility. No correlation was found with the presence of resistance genes of other antimicrobial classes, even though all quinolone non-wild type isolates were resistant to 3 or more classes of antibiotics other than quinolones. Over twenty years of quinolone usage in veal calves in The Netherlands did not result in a widespread occurrence of plasmid-mediated quinolone resistance, limiting the transmission of quinolone resistance to clonal distribution.

Fluoroquinolone Resistance Mechanisms in Urinary Tract PathogenicEscherichia coliIsolated During Rapidly Increasing Fluoroquinolone Consumption in a Low-Use Country

Resistance to ciprofloxacin in Escherichia coli from urinary tract infections (UTI) in Denmark is increasing parallel to increased use of fluoroquinolones both in Denmark and in other European countries. The objective was to investigate the occurrence of ciprofloxacin resistance mechanisms, phenotypic coresistance, and if ciprofloxacin resistance was caused by clonal spread or to individual mutational events in a collection of consecutively obtained E. coli submitted to a clinical microbiology department at a Danish hospital. One hundred four UTI-related E. coli resistant toward nalidixic acid by disc diffusion were typed by Pulsed Field Gel Electrophoresis (PFGE) with XbaI. One isolate representing each PFGE type and only one patient (n = 77) were investigated for point mutations in sequenced PCR amplicons of the four topoisomerase genes; qnr genes by use of PCR; aac(6')-Ib-cr by BtsCI restriction of PCR products; and efflux using efflux pump inhibitors in a broth dilution assay. Minimal inhibitory concentration (MIC) was determined for 21 antibacterial agents, including ciprofloxacin. Of the 77 isolates, the majority were resistant to ciprofloxacin (91%) and multiresistant (resistant to ≥ 3 antimicrobial classes, 83%). Ciprofloxacin-resistant isolates showed at least one target mutation. A significant, positive correlation was found regarding MIC of ciprofloxacin and the number of target mutations. Efflux was found as a resistance mechanism in 77% of isolates tested (n = 60). The aac(6')-Ib-cr gene was detected on plasmids from five isolates showing ciprofloxacin MICs >512 mg/L. No overall clonal relationship among isolates was found according to PFGE. Target modification is the dominating fluoroquinolone resistance mechanism often found in combination with efflux and sometimes aac(6')-Ib-cr. In Denmark, increasing ciprofloxacin resistance in E. coli is mainly due to mutational events and not to spread of clones.

Sequence analysis of topoisomerase gene of pseudocowpoxvirus isolates from camels ( Camelus dromedarius)

Topoisomerase gene of pseudocowposvirus from Indian dromedarian camel was amplified by PCR using the primers of PCPV from Finnish reindeer and cloned into pGEM-T for sequence analysis. Analysis of amino acid identity revealed that Indian PCPV of camel shared 95.9–96.8 with PCPV of reindeer, 96.2–96.5 with ORFV and 87.5 with BPSV.

Mutations in gyrase and topoisomerase genes associated with fluoroquinolone resistance in Salmonella serovars from retail meats

Mutations in gyrase and topoisomerase genes (gyrA, gyrB, parC and parE) were examined among 30 multidrug-resistant (MDR) Salmonella isolates recovered from retail meats using PCR and DNA sequencing analysis. Six isolates had a single gyrA mutation (Ser83→Phe or Ser83→Tyr or Asp87→Asn) with ciprofloxacin (Cip) minimum inhibitory concentrations (MICs) ranging from 0.125 to 0.5μg/ml except 1 with 16μg/ml. Three isolates had both a gyrA mutation (Ser83→Tyr or Ser83→Phe) and a parC mutation (Ser80→Arg) (Cip MICs, 8 to 16μg/ml). Fifteen isolates had both double mutations in gyrA (Ser83→Phe and Asp87→Gly or Asp87→Asn) and a single mutation (Ser80→Arg) in parC (Cip MICs, 8 to ≥16μg/ml). Three had double gyrA mutations (Ser83→Phe, Asp87→Gly or Asp87→Asn), one parC mutation (Ser80→Arg), and 1 parE mutation (Lys428→Gln or Gly442→Ser) (Cip MICs, 8 to ≥16μg/ml). One had double gyrA mutations (Ser83→Phe, Asp87→Gly or Asp87→Asn), one parC mutation (Ser80→Arg), and 2 parE mutations (Lys441→Ile and Asp494→Asn) (Cip MICs, 8 to ≥16μg/ml). Isolates with single gyrA mutations were less resistant to quinolone and fluoroquinolones than those together with additional parC and/or parE mutations in Salmonella. Our findings confirmed the importance of point mutations in gyrase and topoisomerase in reduced susceptibility to quinolone and fluoroquinolones. Mutations in gyrA confer low-level quinolone and fluoroquinolone resistance, while additional gyrA mutation(s) together with parC and/or parE mutations increase the resistance to a high level.