MICs Of Nalidixic Acid Research Articles

Antimicrobial Resistance and Whole-Genome Characterisation of High-Level Ciprofloxacin-Resistant Salmonella Enterica Serovar Kentucky ST 198 Strains Isolated from Human in Poland.

Background: Salmonella Kentucky belongs to zoonotic serotypes that demonstrate that the high antimicrobial resistance and multidrug resistance (including fluoroquinolones) is an emerging problem. To the best of our knowledge, clinical S. Kentucky strains isolated in Poland remain undescribed. Methods: Eighteen clinical S. Kentucky strains collected in the years 2018–2019 in Poland were investigated. All the strains were tested for susceptibility to 11 antimicrobials using the disc diffusion and E-test methods. Whole genome sequences were analysed for antimicrobial resistance genes, mutations, the presence and structure of SGI1-K (Salmonella Genomic Island and the genetic relationship of the isolates. Results: Sixteen of 18 isolates (88.9%) were assigned as ST198 and were found to be high-level resistant to ampicillin (>256 mg/L) and quinolones (nalidixic acid MIC ≥ 1024 mg/L, ciprofloxacin MIC range 6–16 mg/L). All the 16 strains revealed three mutations in QRDR of GyrA and ParC. The substitutions of Ser83 → Phe and Asp87 → Tyr of the GyrA subunit and Ser80→Ile of the ParC subunit were the most common. One S. Kentucky isolate had qnrS1 in addition to the QRDR mutations. Five of the ST198 strains, grouped in cluster A, had multiple resistant determinants like blaTEM1-B, aac(6′)-Iaa, sul1 or tetA, mostly in SGI1 K. Seven strains, grouped in cluster B, had shorter SGI1-K with deletions of many regions and with few resistance genes detected. Conclusion: The results of this study demonstrated that a significant part of S. Kentucky isolates from humans in Poland belonged to ST198 and were high-level resistant to ampicillin and quinolones.

Subinhibitory concentrations of nalidixic acid alter bacterial physiology and induce anthropogenic resistance in a commensal strain of Escherichia coli invitro.

The human gut houses a complex group of bacterial genera, including both opportunistic pathogens and commensal micro-organisms. These are regularly exposed to antibiotics, and their subinhibitory concentrations play a pivotal role in shaping the microbial responses. This study was aimed to investigate the effects exerted by sub-MICs of nalidixic acid (NA) on the growth rate, bacterial motility, biofilm formation and expression of outer membrane proteins (OMPs) in a commensal strain of E.coli. The NA-sensitive strain was sequentially passaged under sub-MICs of NA. E-test was used to determine the MIC values of NA. Results indicated significant changes in the growth profile of commensal E.coli upon exposure to NA at sub-MICs. Differential expression of OMPs was observed in cells treated with sub-MICs of NA. Bacterial motility was reduced under 1/2 MIC of NA. Interestingly, successive passaging under 1/2 MIC of NA led to the emergence of resistant E.coli with an increased MIC value of 64 µg ml-1 in just 24 days. The NA-resistant variant was confirmed by comparing its 16S rRNA sequence to that of the sensitive commensal strain. Mutations in the Quinolone Resistance-Determining Regions (QRDRs) of chromosomal gyrA, and Topoisomerase IV-encoding parC genes were detected in NA-resistant E.coli. Our results demonstrate how antibiotics play an important role as signalling molecules or elicitors in driving the pathogenicity of commensal bacteria invitro.

Occurrence of a novel class 1 integron harboring qnrVC4 in Salmonella Rissen

We described qnrVC4 in S. Rissen 166ANSS50, a swine isolate, which was detected in the study on quinolone resistance mechanisms of nontyphoidal Salmonella in Thailand. The isolate was found to harbor a ̴17-kb non-conjugative plasmid carrying qnrVC4 within 8.91kb of a novel In4-like class 1 integron (In805). It contained the multi-drug resistance gene cassettes of qnrVC4-qacH4-aacA4-cmlA7-blaOXA-10-aadA1-dfrA14 and unusual 3′-CS of mobC-IS6100. This 1014-bp qnrVC4 cassette included with promoter (PqnrVC4: –35 TTGAGA and −10 TAGTCT) showed high homology with qnrVC4 in superintegron of V. cholerae O1 El Tor. The qnrVC4 recombinant plasmid resulted in 4-, 8-, and 16-fold increase in the MICs of nalidixic acid (2–8μg/mL), ciprofloxacin (0.015–0.125μg/mL), and norfloxacin (0.03–0.5μg/mL), respectively. In addition, the backbone plasmid revealed a novel replicon belonging to the MOBQ1 group from the broad-host-range mobilisable IncQ1 plasmid RFS1010 based on relaxase sequences. This is the first known report of qnrVC in Salmonella enterica. The qnrVC4 gene was co-transferred with other resistance genes via a novel plasmid-borne In805. This allowed the spread of this resistance gene to Enterobacteriaceae.

Revisit of fluoroquinolone and azithromycin susceptibility breakpoints for Salmonella enterica serovar Typhi.

In recent years, increase in occurrence of fluoroquinolone (FQ)-resistant S almonella Typhi isolates has caused considerable inconvenience in selecting appropriate antimicrobials for treatment of typhoid. The World Health Organization (WHO) recommends azithromycin for the empirical treatment option of uncomplicated typhoid. The CLSI updated the breakpoints of disc diffusion (DD) and MIC results of FQs and azithromycin for Salmonella Typhi in 2015, but DD breakpoints of ofloxacin and levofloxacin were not included. In this study, the inhibition zone diameters and MICs of nalidixic acid, ciprofloxacin, ofloxacin, levofloxacin and azithromycin were determined in Salmonella Typhi Kolkata isolates (n =146) over a 16-year period (1998 to 2013) and the data were compared with the available CLSI breakpoints. Very major error and major error (ME) of FQs were not observed in the study isolates, but the minor error of ciprofloxacin (15.8 %) and ME of azithromycin (3.5 %) exceeded the acceptable limit. A positive correlation between MICs of FQ and mutations in the quinolone-resistance-determining region (QRDR) showed the reliability of MIC results to determine FQ susceptibility of Salmonella Typhi (n =74). Isolates showing decreased ciprofloxacin susceptibility (MIC 0.125-0.5 µg ml-1) were likely to have at least one mutation in the QRDR region. The results on DD breakpoints of ofloxacin (resistant, ≤15 mm; intermediate, 16-24 mm, and susceptible, ≥25 mm) and levofloxacin (resistant, ≤18 mm; intermediate, 19-27 mm, and susceptible, ≥28 mm) corroborated those of earlier studies. In view of the emerging FQ- and azithromycin-resistant Salmonella Typhi isolates, DD and MIC breakpoints of those antimicrobials should be revisited routinely.

Quinolone resistance mechanisms among extended-spectrum beta-lacta- mase (ESBL)-producing Escherichia coli isolated from farm animals in Switzerland.

Sixty extended-spectrum b-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-69-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-b-naphthylamide (PAbN). 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 Ser83RLeu was detected in GyrA followed by Asp87RAsn in GyrA, Ser80RIle in ParC, Glu84RVal in ParC and Ser458RAla in ParE. Plasmidmediated quinolone resistance mechanisms were found in twenty isolates bearing QnrS1 (4/20), AAC-69-Ib-cr (15/20) and QepA (1/20) determinants, respectively. No qnrA, qnrB, qnrC and qnrD were found. In the presence of PAbN, the MICs of nalidixic acid were decreased 4to 32-fold. (Fluoro) quinolone resistance is due to various mechanisms frequently associated with ESBL-production in E. coli from surface waters in Switzerland. Citation: Zurfluh K, Abgottspon H, Hachler H, Nuesch-Inderbinen M, Stephan R (2014) Quinolone Resistance Mechanisms among Extended-Spectrum BetaLactamase (ESBL) Producing Escherichia coli Isolated from Rivers and Lakes in Switzerland. PLoS ONE 9(4): e95864. doi:10.1371/journal.pone.0095864 Editor: Asad U. Khan, Aligarh Muslim University, India Received October 5, 2013; Accepted April 1, 2014; Published April 22, 2014 Copyright: 2014 Zurfluh et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The authors thank the Swiss Federal Office for Public Health, Division Communicable Diseases, for partial financial support. No additional funding received for this study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: stephanr@fsafety.uzh.ch

Characterization of non-classical quinolone resistance in Salmonella enterica serovar Typhi: Report of a novel mutation in gyrB gene and diagnostic challenges

ObjectiveTo establish the relative importance of Salmonella enterica serovar Typhi with non-classical quinolone resistance. MethodsEight hundred and ninety-one isolates of S. Typhi, isolated between 2004 and 2011, were tested for antibiotic susceptibility determination using disc diffusion and E-test. The mechanisms of fluoroquinolone resistance were studied in a sub-set of the NALS (nalidixic acid susceptible) isolates by wave nucleic acid fragment analysis of PCR products from gyrA, gyrB, parC and parE and from the plasmid borne determinants: qnrA,B,S; aac(6′)-Ib-cr and qepA. To assess genetic relatedness multi-locus variable number tandem repeat analysis was carried out using five loci. ResultsEighty isolates with a nalidixic acid MIC of <32mg/L (NALS) and a ciprofloxacin MIC of >0.064mg/L CIPI (ciprofloxacin reduced susceptibility) were found. In 36 NALS CIPI isolates two distinct genotypes were identified when compared with 16 susceptible controls: Group B (n=34), mutation in gyrB at codon 464, NAL MIC of 3–12mg/L and CIP MIC of 0.064–0.5mg/L.; and Group C, mutation in gyrA at codon 83 (n=2) NAL MIC of 16mg/L and CIP MIC of 0.25–0.38mg/L. Group B isolates were found in different strain backgrounds as defined by MLVA. ConclusionThe use of nalidixic acid to screen for reduced susceptibility to fluoroquinolones in S. Typhi misses CIPI-NALS isolates, an established phenotype in India.

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.

Molecular characterization of the antimicrobial resistance of Riemerella anatipestifer isolated from ducks

The antimicrobial susceptibility of 103 Riemerella anatipestifer isolates obtained from ducks during 2008 and 2010 in Southern China, to 23 antimicrobial agents was investigated using the agar dilution method. The MIC50 and MIC90 values of streptomycin, kanamycin, gentamicin, apramycin, amikacin, neomycin, nalidixic acid and sulfadimidine were high (32–≥128μg/ml) among the 103 R. anatipestifer isolates. However, relatively low MIC90 values (8μg/ml) of ampicillin and florfenicol were observed among these isolates. The presence of resistance genes and integrons was determined using PCR. The genes blaTEM-1, aph(3′)-VII, aadA1, aadA2, aac(3′)-IV, aac(3′)-IIc, aac(6′)-Ib, cat2, cmlA, floR, tet(A), tet(B), tet(C), sul1, and sul2 were detected in 1, 2, 24, 35, 11, 4, 67, 16, 26, 10, 6, 1, 9, 36 and 2 isolates, respectively. Twenty isolates contained one or two class 1 integrons carrying aadA2 or aac(6′)-II-catB3-aadA1 gene cassette(s). Mutation analysis of the quinolone resistance-determining regions (QRDRs) of 43 R. anatipestifer isolates with nalidixic acid MICs≥32μg/ml, showed that the most prevalent mutations in gyrA were those resulting in the amino acid exchanges Ser83-Ile (n=37), followed by Asp87-His (n=7) and Ser83-Arg (n=5). Point mutations in parC (Arg120-Glu) were observed in 5 isolates with a ciprofloxacin MIC of >16μg/ml. No plasmid-mediated quinolone resistance gene was detected. PFGE analysis showed that the clonal spread of multi-drug resistant R. anatipestifer isolates occurred in the same farm or between different farms. Our results reported, for the first time, the mechanism of quinolone resistance in R. anatipestifer.

English

The quinolone-resistance determining-region of&nbsp;gyrA&nbsp;was PCR-amplified and sequenced in seven fluoroquinolone-resistant&nbsp;Pseudomonas aeruginosa&nbsp;(Nalidixic acid MICs ranging 8 to128 &micro;g/ml, ciprofloxacin MICs ranging 8 to 32 &micro;g/ml and&nbsp; levofloxacin MICs ranged from 32 to 64 &micro;g/ml) isolated from poultry that died of septicaemic clinical diseases in Ibadan, Oyo State, South Western Nigeria. The seven isolates were also multidrug resistant to various combinations of the commonly used antibiotics like streptomycin, ampicillin, tetracycline, kanamycin, neomycin and chloramphenicol. The entire seven isolates possessed the&nbsp;gyrA&nbsp;mutation encoding the histidine to tyrosine conversion at amino acid 150 (H150Y). Additional substitutions observed included: aspartic acid to tyrosine substitution at amino acid 87 (D87Y) in two isolates, aspartic acid to glycine D87G substitution in three isolates, another simultaneous seven substitutions: aspartic acid to alanine, D87A; alanine to proline, A62P; tyrosine to isoleucine, Y83I; methionine to leucine, M92L; leucine to methionine, L98M; L128M; and aspartic acid to proline D148P&nbsp; in one of the isolate, whereas one of the seven isolates possessed only the H150Y substitution. The H150Y is typical of the quinolone resistant&nbsp;P. aeruginosa&nbsp;isolated from septic poultry in Nigeria, regardless of other resistance- patterns exhibited to other commonly used antibiotics.&nbsp; This study associate&nbsp;gyrA&nbsp;mutations with fluoroquinolone resistance in&nbsp;P. aeruginosa&nbsp;isolated from septicaemic poultry in Nigeria, where fluoroquinolone use in livestock is not strictly regulated and misuse/abuse of most antibiotics is rampart. &nbsp; Key words:&nbsp;Pseudomonas aeruginosa,&nbsp;resistance, fluoroquinolone, poultry, Oyo state, Nigeria.

Mechanisms of Fluoroquinolone Resistance in Escherichia coli Isolates from Food-Producing Animals

Eleven multidrug-resistant Escherichia coli isolates (comprising 6 porcine and 5 bovine field isolates) displaying fluoroquinolone (FQ) resistance were selected from a collection obtained from the University Veterinary Hospital (Dublin, Ireland). MICs of nalidixic acid and ciprofloxacin were determined by Etest. All showed MICs of nalidixic acid of >256 μg/ml and MICs of ciprofloxacin ranging from 4 to >32 μg/ml. DNA sequencing was used to identify mutations within the quinolone resistance-determining regions of target genes, and quantitative real-time PCR (qRT-PCR) was used to evaluate the expression of the major porin, OmpF, and component genes of the AcrAB-TolC efflux pump and its associated regulatory loci. Decreased MIC values to nalidixic acid and/or ciprofloxacin were observed in the presence of the efflux pump inhibitor phenylalanine-arginine-β-naphthylamide (PAβN) in some but not all isolates. Several mutations were identified in genes coding for quinolone target enzymes (3 to 5 mutations per strain). All isolates harbored GyrA amino acid substitutions at positions 83 and 87. Novel GyrA (Asp87 → Ala), ParC (Ser80 → Trp), and ParE (Glu460 → Val) substitutions were observed. The efflux activity of these isolates was evaluated using a semiautomated ethidium bromide (EB) uptake assay. Compared to wild-type E. coli K-12 AG100, isolates accumulated less EB, and in the presence of PAβN the accumulation of EB increased. Upregulation of the acrB gene, encoding the pump component of the AcrAB-TolC efflux pump, was observed in 5 of 11 isolates, while 10 isolates showed decreased expression of OmpF. This study identified multiple mechanisms that likely contribute to resistance to quinolone-based drugs in the field isolates studied.

The prevalence of fluoroquinolone resistance mechanisms in colonizing Escherichia coli isolates recovered from hospitalized patients.

Fluoroquinolones are the most commonly prescribed antimicrobials. The epidemiology of fecal colonization with Escherichia coli demonstrating reduced susceptibility to fluoroquinolones remains unclear. During a 3-year period (15 September 2004 through 19 October 2007), all patients hospitalized for >3 days were approached for fecal sampling. All E. coli isolates with reduced susceptibility to fluoroquinolones (minimum inhibitory concentration [MIC] of levofloxacin, 0.125 microg/mL) were identified. We characterized gyrA and parC mutations and organic solvent tolerance. Isolates were compared using pulsed-field gel electrophoresis. Of 353 patients colonized with E. coli demonstrating reduced fluoroquinolone susceptibility, 300 (85.0%) had 1 gyrA mutation, 161 (45.6%) had 1 parC mutation, and 171 (48.6%) demonstrated organic solvent tolerance. The mean numbers of total mutations (ie, gyrA and parC) for E. coli isolates with a levofloxacin MIC of 8 microg/mL versus <8.0 microg/mL were 2.70 and 0.82 (P < .001). Of the 136 E. coli isolates with a levofloxacin MIC of 8 microg/mL, 90 (66.2%) demonstrated a nalidixic acid MIC of 16 microg/mL. Significant differences were found over time in the proportion of E. coli isolates demonstrating gyrA mutation, parC mutation, and organic solvent tolerance. There was little evidence of clonal spread of isolates. Conclusions. Gastrointestinal tract colonization with E. coli demonstrating reduced susceptibility to levofloxacin is common. Although 40% of study isolates exhibited a levofloxacin MIC of <8 microg/mL (and would thus be missed by current Clinical and Laboratory Standards Institute breakpoints), nalidixic acid resistance may be a useful marker for detection of such isolates. Significant temporal changes occurred in the proportion of isolates exhibiting various resistance mechanisms.

Detection of gyrA Mutation Among Clinical Isolates of Campylobacter jejuni Isolated in Egypt by MAMA-PCR

Campylobacter spp are the major cause of enteritis in humans and more than 90% of reported infections are caused by Campylobacter jejuni. Fluoroquinolones such as ciprofloxacin are the antibiotics of choice for treatment. An increase in the frequency of ciprofloxacin-resistant Campylobacter has been reported globally due to a single base mutation (C-257 to T) in codon 86 of the quinolone resistance determining region (QRDR) of the gyrA gene altering the amino acid sequence from threonine at position 86 to isoleucine (Thr-86 to Ile). Campylobacter spp (n = 118) were selected from a collection of Egyptian isolates spanning 1998 to 2005. The presence of C. jejuni gyrA gene was confirmed in each isolate by a PCR assay amplifying 368 bp portion of the gyrA gene. C to T alteration was detected by the mismatch amplification mutation assay MAMA PCR. The MIC of nalidixic acid (NA) and ciprofloxacin (CIP) was determined by E-test. C. jejuni gyrA gene was detected in 100 of the Campylobacter spp studied; the other 18 isolates were found to be Campylobacter coli by lpxA PCR. The mutation was detected in 89 C. jejuni resistant isolates with MIC values (NA; 8 - >256 μg/ml) and (CIP; 4 - >32 μg/ml). The other 11 sensitive C. jejuni isolates with MIC values (NA; 0.38 - 3 µg/ml) and (CIP; 0.03 - 0.125 µg/ml) were not amplified by the MAMA primers. There was 100% congruence with MAMA PCR, MIC results and gyrA gene sequence analysis. In Egypt the main mechanism for resistance to fluoroquinolones is an alteration in the gyrA QRDR. MAMA PCR provides an economical and rapid means for screening fluoroquinolone resistance.

Novel genetic environment of plasmid-mediated quinolone resistance gene qnrB2 in Salmonella Bredeney from poultry

Novel genetic environment of plasmid-mediated quinolone resistance gene qnrB2 in Salmonella Bredeney from poultry

Plasmid-Mediated Quinolone Resistance in &lt;i&gt;Salmonella&lt;/i&gt; Isolated from Patients with Overseas Travelers&lt;i&gt;'&lt;/i&gt; Diarrhea in Japan

Twenty-eight of the 302 Salmonella strains isolated from patients with overseas travelers' diarrhea who were examined at Kansai Airport Department Station during the 6-year period between 2001 and 2007 showed decreased susceptibility to both nalidixic acid (NA) and ciprofloxacin (CPFX) (MIC of NA, 16-64 μg/mL; MIC of CPFX, 0.064-2.0 μg/mL). These 28 strains revealed no variations in the quinolone resistance-determining region; however, 25 of the strains showed retention of the qnr gene. The qnr-retaining Salmonella belonged to 6 serotypes, and 21 and 4 of the 25 strains showed qnrS1 and qnrS2, respectively. The most common serovar was S. Corvallis (17 strains).

Detection of mutations in the gyrA of clinical Salmonella spp.

The high prevalence of resistance to nalidixic acid and reduced susceptibility to ciprofloxacin of Salmonella spp. obtained from stool samples of neonates presenting with acute diarrhea in 2001 at the King Edward VIII hospital in Durban, South Africa, prompted this study to determine if there were any mutations in the QRDR of these isolates and to search for the qnrA gene. All isolates with nalidixic acid MICs > 48 μg/ml had the single mutation D87N, or D87G in the QRDR of the gyrA gene, and only 2 strains had an additional mutation; S83L and S83F respectively. The mutation S83T was present in only one isolate with the nalidixic acid MIC of 10 μg/ml whilst the 6 other strains with nalidixic acid MICs gyrA gene. The qnrA gene was not found. These findings indicate that there are mutations in the gyrA of Salmonella isolates which could contribute to resistance to nalidixic acid with reduced susceptibility to ciprofloxacin and there is the co-expression of quinolone and extended-spectrum s-lactam resistance among Salmonella spp.

Plasmid-mediated quinolone resistance in Aeromonas allosaccharophila recovered from a Swiss lake

To search for plasmid-mediated qnr genes among waterborne environmental Aeromonas spp. recovered from Switzerland. Isolates presenting MICs of nalidixic acid or ciprofloxacin > or = 1 mg/L were screened for qnr genes by a multiplex PCR approach followed by sequencing. Plasmids were transferred by transformation, and further analysis of the genetic structures surrounding the qnrS2 gene was carried out by PCR and sequencing. A qnrS2 gene was identified from a single Aeromonas allosaccharophila isolate (Lugano lake, Lugano), as part of a mobile insertion cassette located on a broad host range IncU-type plasmid. This plasmid co-harboured a class 1 integron containing the aac(6')-Ib-cr, bla(OXA-1), catB3 and arr-3 gene cassettes. These findings strengthen further the role of Aeromonas spp. as a reservoir of antimicrobial resistance determinants in the environment.

HumanSalmonellaand Concurrent Decreased Susceptibility to Quinolones and Extended-Spectrum Cephalosporins

The National Antimicrobial Resistance Monitoring System monitors susceptibility among Enterobacteriaceae in humans in the United States. We studied isolates exhibiting decreased susceptibility to quinolones (nalidixic acid MIC >32 microg/mL or ciprofloxacin MIC > or =0.12 microg/mL) and extended-spectrum cephalosporins (ceftiofur or ceftriaxone MIC > or =2 microg/mL) during 1996-2004. Of non-Typhi Salmonella, 0.19% (27/14,043) met these criteria: 11 Senftenberg; 6 Typhimurium; 3 Newport; 2 Enteridis; and 1 each Agona, Haifa, Mbandaka, Saintpaul, and Uganda. Twenty-six isolates had gyrA mutations (11 at codon 83 only, 3 at codon 87 only, 12 at both). All Senftenberg isolates had parC mutations (S801 and T57S); 6 others had the T57S mutation. The Mbandaka isolate contained qnrB2. Eight isolates contained bla(CMY-2); 1 Senftenberg contained bla(CMY-23). One Senftenberg and 1 Typhimurium isolate contained bla(SHV-12); the Mbandaka isolate contained bla(SHV-30). Nine Senftenberg isolates contained bla(OXA-1) contained bla(OXA-9). Further studies should address patient outcomes, risk factors, and resistance dissemination prevention strategies.

Prevalence and diversity of qnr alleles in AmpC-producing Enterobacter cloacae, Enterobacter aerogenes, Citrobacter freundii and Serratia marcescens: a multicentre study from Korea

To investigate the prevalence of qnr determinants, their influence on quinolone susceptibility and their association with other plasmid-mediated genes in AmpC-producing Enterobacteriaceae without any selection criteria. A total of 644 consecutive, non-duplicate isolates of Enterobacter cloacae (186), Enterobacter aerogenes (154), Citrobacter freundii (138) and Serratia marcescens (166) were examined. We performed antimicrobial susceptibility testing and PCR for qnr determinants (qnrA, qnrB and qnrS), extended-spectrum beta-lactamase (ESBL) (bla(TEM), bla(SHV) and bla(CTX-M)), orf513, orf1005 and bla(DHA-1.) To differentiate qnr subtypes, restriction enzyme analysis and sequencing was performed. The prevalence of qnr determinants was high in C. freundii (38.4%) and E. cloacae (28.5%), but low in E. aerogenes (3.2%) and S. marcescens (2.4%). qnrA1 was most frequent in E. cloacae, and qnrB was prevalent in C. freundii. All the qnrA- and qnrB4-positive isolates showed ciprofloxacin MICs > or = 0.5 mg/L and nalidixic acid MICs > or = 16 mg/L. However, the B1 and B2 subtypes showed a wide range of quinolone MICs. In relation to ESBLs, we found that qnrA1, qnrB2 and qnrB4 producers were significantly more frequent among ESBL producers (P < 0.05). Twelve of 13 qnrB4 producers harboured bla(DHA-1). orf513 was detected in 43 isolates of the 47 isolates with co-resident qnr and ESBL genes. None of the qnr producers harboured orf1005. The prevalence of qnrA and qnrB was high among C. freundii and E. cloacae in Korea and there were characteristics unique to the qnr subtypes. Quinolones should be used cautiously in these species, especially when they are ESBL producers.

Vibrio splendidus as the Source of Plasmid-Mediated QnrS-Like Quinolone Resistance Determinants

Plasmid-mediated quinolone resistance due to Qnr-like proteins is increasingly reported worldwide (5, 9) since the first report from the United States in 1998 (4). Six variants of both QnrA and QnrB determinants and two of QnrS have been described so far (9). QnrS1 was firstly identified in a Shigella flexneri isolate from Japan (3) and then from several other countries (2, 6), whereas the QnrS2 variant (92% identity with QnrS1) was only identified from a mobilizable IncQ-related plasmid from Germany (1) and also reported from a single non-Typhi Salmonella isolate from the United States (2). Whereas the origin of the qnrA gene was identified as being Shewanella algae (7), the reservoirs of the qnrB and qnrS genes remain unknown. However, we have already considered that they might be close to some species of the Vibrionaceae family which possess chromosome-encoded Qnr-like determinants (40% to 67% identity with QnrA, QnrB, and QnrS determinants) conferring resistance to quinolones (8). By in silico analysis of the recently released genome sequence of Vibrio splendidus strain 12B01 (accession no. {type:entrez-nucleotide,attrs:{text:NZ_AAMR01000029,term_id:84391733,term_text:NZ_AAMR01000029}}NZ_AAMR01000029, we identified an open reading frame (V12B01_17906) coding for a 218-amino-acid protein sharing 84% and 88% amino acid identity with QnrS1 and QnrS2, respectively. Therefore, DNA fragments corresponding to the entire sequences of the qnrVS1 and qnrVS2 genes were PCR amplified from V. splendidus strains 12B01 (seawater, United States) and LGP32 (oyster, France) (D. Mazel, personal collection), respectively, were cloned into the kanamycin resistance pCR-BluntII-TOPO plasmid (Invitrogen, Life Technologies, Cergy-Pontoise, France) as previously described (8), together with the qnrS1 and qnrS2 genes as controls. When overexpressed in Escherichia coli TOP10 (Invitrogen), recombinant plasmids with the qnrVS1 (pVS1) and qnrVS2 (pVS2) genes conferred an 8-fold increase in the MIC of nalidixic acid and 4- to 16-fold increases in the MICs of fluoroquinolones compared to those for E. coli TOP10 (Table ​(Table1),1), as previously reported for other species of the Vibrionaceae family (8). In addition, MICs of quinolones for E. coli TOP10 harboring recombinant plasmid pVS1 or pVS2 were very similar to those obtained with E. coli with recombinant plasmids harboring the qnrS1 (pS1) and qnrS2 (pS2) genes (Table ​(Table11). TABLE 1. MICs of quinolones and fluoroquinolones for V. splendidus isolates and E. coli recombinant clones The QnrVS1 and QnrVS2 protein sequences were almost identical, with only four amino acid substitutions, corresponding to 98.1% amino acid identity. In addition, the QnrVS1 and QnrVS2 determinants shared 83.9% and 83.1% amino acid identity, respectively, with the plasmid-mediated QnrS1 determinant and 87.6% and 87.1% identity, respectively, with plasmid-mediated QnrS2. In silico analysis showed that chromosome-encoded Qnr-like proteins were identified in other species of the Vibrionaceae family (V. angustrum, V. alginolyticus, V. fischeri, V. vulnificus, V. parahaemolyticus, V. salmonicida, Photobacterium profundum) and shared no more than 64% amino acid identity with any Qnr determinants, including the QnrS2 determinant. In addition, the G+C contents of the qnrVS1 (45.2%) and qnrVS2 (45.1%) genes are close to those of qnrS1 and qnrS2 (both are 43.8%). These results underline that gram-negative bacterial species of the aquatic environment may be the reservoir of plasmid-mediated Qnr-like determinants. Although the exact progenitor of the plasmid-encoded QnrS determinant remains unknown, it should be closely related to V. splendidus.

Changing Epidemiology of Shigellosis and Emergence of Ciprofloxacin-Resistant Shigellae in India

Changing Epidemiology of Shigellosis and Emergence of Ciprofloxacin-Resistant Shigellae in India