Quinolone Resistance Determinants Research Articles

Molecular characterization of fluoroquinolone-resistant Mycobacterium tuberculosis clinical isolates from Shanghai, China

China is one of the countries with the highest prevalence of fluoroquinolone-resistant (FQr) Mycobacterium tuberculosis. Nevertheless, knowledge on the molecular characterization of the FQrM. tuberculosis strains of this region remains very limited. This study was performed to investigate the frequencies and types of mutations present in FQrM. tuberculosis clinical isolates collected in Shanghai, China. A total of 206 FQrM. tuberculosis strains and 21 ofloxacin-sensitive (FQs) M. tuberculosis strains were isolated from patients with pulmonary tuberculosis in Shanghai. The phenotypic drug susceptibilities were determined by the proportion method, and the mutations inside quinolone resistance–determining region (QRDR) of gyrA and gyrB genes were identified by DNA sequence analyses. Among 206 FQrM. tuberculosis strains, 44% (90/206) were multidrug-resistant isolates and 39% (81/206) were extensively drug–resistant isolates. Only 9% (19/206) were monoresistant to ofloxacin. In total, 79.1% (163/206) of FQr isolates harboured mutations in either gyrA or gyrB QRDR. Mutations in gyrA QRDR were found in 75.7% (156/206) of FQr clinical isolates. Among those gyrA mutants, a majority (75.6%) harboured mutations at amino acid position 94, with D94G being the most frequent amino acid substitution. Mutations in gyrA QRDR showed 100% positive predictive value for FQrM. tuberculosis in China. Mutations in gyrB were observed in 15.5% (32/206) of FQr clinical isolates. Ten novel mutations were identified in gyrB. However, most of them also harboured mutations in gyrA, limiting their contribution to FQr resistance in M. tuberculosis. Our findings indicated that, similar to other geographic regions, mutations in gyrA were shown to be the major mechanism of FQr resistance in M. tuberculosis isolates. The mutations in gyrA QRDR can be a good molecular surrogate marker for detecting FQrM. tuberculosis in China.

Complete nucleotide sequence of pVQS1 containing a quinolone resistance determinant from Salmonella enterica serovar Virchow associated with foreign travel

Nalidixic acid-resistant Salmonella enterica serovars Kentucky (n = 5) and Virchow (n = 6) cultured from individuals were investigated for the presence of plasmid-mediated quinolone resistance (PMQR) determinants. PMQR markers and mutations within the quinolone resistance-determining regions of the target genes were investigated by PCR followed by DNA sequencing. Conjugation, plasmid profiling and targeted PCR were performed to demonstrate the transferability of the qnrS1 gene. Subsequently, a plasmid was identified that carried a quinolone resistance marker and this was completely sequenced. A Salmonella Virchow isolate carried a qnrS1 gene associated with an IncN incompatibility group conjugative plasmid of 40 995 bp, which was designated pVQS1. The latter conferred resistance to ampicillin and nalidixic acid and showed sequence similarity in its core region to plasmid R46, whilst the resistance-encoding region was similar to pAH0376 from Shigella flexneri and pINF5 from Salmonella Infantis and contained an IS26 remnant, a complete Tn3 structure, a truncated IS2 element and a qnrS1 marker, followed by IS26. In contrast to pINF5, IS26 was identified immediately downstream of the qnrS1 gene. This is the first known report of a qnrS1 gene in Salmonella spp. in Switzerland. Analysis of the complete nucleotide sequence of the qnrS1-containing plasmid showed a novel arrangement of this antibiotic resistance-encoding region.

Plasmid-mediated quinolone resistance determinant qnrB19 in non-typhoidal Salmonella enterica strains isolated in Venezuela

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Transfer of quinolone resistance gene qnrA1 to Escherichia coli through a 50 kb conjugative plasmid resulting from the splitting of a 300 kb plasmid

To analyse the in vitro transfer of the qnrA1 gene by a 50 kb (pSZ50) self-transferable plasmid that derives from a 300 kb plasmid (pSZ300) and to determine the complete nucleotide sequence of plasmid pSZ50. Extended-spectrum β-lactamase (ESBL) and plasmid-mediated quinolone resistance (PMQR) genes of an Escherichia coli clinical isolate were analysed. Plasmid analysis included conjugation and selection on seven antibiotics examined by antimicrobial susceptibility testing, RFLP comparison, Southern hybridization, incompatibility group identification and shotgun sequencing. The E. coli 5509 isolate carries the genes encoding the ESBL CTX-M-15 and the quinolone resistance determinants qnrA1, qnrB2 and aac(6')-Ib-cr on a 300 kb plasmid. Seven transfer resistances were analysed by conjugation under two conditions (30 and 37°C), leading to two distinct transconjugant phenotypes with different resistances. Transconjugants of phenotype A harboured a 300 kb plasmid named pSZ300 that conferred resistance to eight antibiotics and harboured the qnrA1, aac(6')-Ib-cr and bla(CTX-M-15) genes. Transconjugants of phenotype B were resistant to three antibiotics and they harboured the qnrA1 gene on an ≈ 50 kb plasmid named pSZ50. Both plasmids were self-transferable at a frequency of 1 × 10(-3). Plasmid pSZ300 was typed to be both an IncF and IncN plasmid, whereas pSZ50 corresponded only to type IncN. Fingerprinting and Southern hybridization showed that plasmid pSZ50 derived from pSZ300. The complete nucleotide sequence of plasmid pSZ50 was determined (51556 bp) and 55 open reading frames were predicted. The qnrA1 gene was identified in a tandem duplicate inside a sul1-type integron structure. The plasmid pSZ300 represented a fusion of two replicons (IncF and IncN), and our observations suggest that the plasmid pSZ50 (IncN) may split and transfer antibiotic resistance determinants. This mechanism could be advantageous in the dissemination of antibiotic resistance genes.

Prevalence of plasmid-mediated quinolone resistance determinants qnr and aac(6′)-Ib-cr in Escherichia coli and Klebsiella pneumoniae producing extended-spectrum β-lactamases in Spain

The presence of the plasmid-mediated quinolone resistance determinants qnrA, qnrB, qnrS and aac(6′)-Ib-cr was evaluated in a collection of 382 isolates of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli and Klebsiella pneumoniae collected between February and March 2006 for the nationwide Spanish GEIH-ESBL 2006 project. In total, 14 isolates (3.7%) were positive for qnr genes (3 qnrA1, 5 qnrB-like and 6 qnrS1) and 62 isolates (16.2%) were positive for the mutant variant of aac(6′)-Ib-cr. The Aac(6′)-Ib-cr enzyme was the most prevalent plasmid-mediated mechanism of quinolone resistance in Spain. Most of the Aac(6′)-Ib-cr-producing E. coli isolates (94.2%) carried two mutations in gyrA and two in parC, whilst only 57.2% of K. pneumoniae harbouring this enzyme were gyrA and/or parC mutants. Most qnr plasmids were transferable, but only four were conjugative. Plasmid incompatibility groups were identified for only four plasmids, belonging to FIA, HI2 and I1γ. The most prevalent ESBLs associated with qnr plasmids belonged to the SHV and CTX-M families. The present study highlights the broad geographical spread of qnr-like determinants in Spain and their association with the SHV-12 and CTX-M-9 ESBLs in human clinical isolates.

Molecular characterization of NDM-1 producing Enterobacteriaceae isolates in Singapore hospitals

In this study, we molecularly characterized 12 NDM-1 producing clinical Enterobacteriaceae (Klebsiella pneumoniae, Escherichia coli, Enterobacter cloacae) isolates that were part of a collection of non-carbapenem susceptible isolates obtained during a one-year period. These isolates were obtained from four local general hospitals in Singapore. Polymerase chain reaction (PCR) assays and sequencing was used to determine the presence of β-lactamase encoding genes (bla) including bla NDM-1 and plasmid-mediated quinolone and aminoglycoside resistance determinants. Conjugation experiments were performed to determine the transferability of bla NDM-1. Isolate relatedness was determined by multilocus sequence typing (MLST). The isolates were completely resistant to the second- and third-generation cephalosporins tested as well as carbapenems. Susceptibility profiling of the isolates indicated that 100% retained susceptibility to tigecycline while 11/12 (91.7%) were susceptible to colistin. The bla NDM-1 gene was encoded on plasmids that were easily transferable. None of the patients had a travel history to countries where NDM-1 has been reported. The isolates appear clonally unrelated with MLST, revealing a diversity of clonal types among the Klebsiella pneumoniae and Escherichia coli isolates. The ease of NDM-1 plasmid transmissibility may help their dissemination among the Enterobacteriaceae. Although it appears that the isolates are clonally unrelated, epidemiological links cannot be fully excluded without further research.

Molecular epidemiology of extended-spectrum- -lactamase-producing Klebsiella pneumoniae over a 10 year period in Calgary, Canada

A study was designed to investigate the molecular epidemiology of extended-spectrum β-lactamase (ESBL)-producing Klebsiella pneumoniae isolated in a centralized region over a 10 year period (2000-09). Molecular characterization was done using isoelectric focusing, PCR and sequencing for bla(CTX-M), bla(TEM) and bla(SHV) genes and plasmid-mediated quinolone resistance determinants. Genetic relatedness was determined with PFGE using XbaI and multilocus sequencing typing. A total of 89 patients with incident infections were identified; the majority presented with hospital-onset urinary tract infections. The absolute number of ESBL-producing isolates remained very low until 2003, increased slightly in 2004, remained stable until 2008 and then in 2009 there was an abrupt increase in the numbers of ESBL producers identified. The majority of K. pneumoniae produced CTX-M-14 and -15, and have replaced SHV-12-producing isolates since 2005. We identified four different major sequence types (STs) among 32% of isolates (i.e. ST17, ST20, and the new ST573 and ST575) and provided insight into their clinical and molecular characteristics. The ST isolates were more likely to produce community-onset infections, were associated with bla(CTX-M) and emerged during the latter part of the study period. ST17 produced CTX-M-15 and SHV-12, and was more likely to be positive for qnrB; ST20 produced CTX-M-14 and was positive for qnrS. The multiresistant ST575 that produced CTX-M-15 appeared in 2009. Our study highlights the importance of molecular epidemiology in providing insight into the emergence, characteristics and distribution of STs among ESBL-producing K. pneumoniae.

Evaluation of quinolone resistance–determining region mutations and efflux pump expression in Neisseria meningitidis resistant to fluoroquinolones

Three Neisseria meningitidis serotype B strains displaying elevated ciprofloxacin MIC values (0.06 and 0.25 μg/mL) and a GyrA alteration at T91I have been described in the United States by Wu et al. [Wu et al., (2009) Emergence of ciprofloxacin-resistant Neisseria meningitidis in North America. N. Engl. J. Med. 360:886–892] and were further evaluated here by reference broth microdilution against fluoroquinolones (FQ) and other antimicrobial agents. Additional quinolone resistance–determining region (QRDR) mutations and alterations in structure and expression of the mtrCDE efflux system, including its intergenic regions, were also analyzed. Two strains showed ciprofloxacin and levofloxacin MIC values at 0.25 μg/mL, and 1 strain had a ciprofloxacin MIC at 0.06 μg/mL. In addition to T91I, the 2 strains displaying higher FQ MIC values also possessed a T173A alteration on GyrA. All components of the efflux pump mtrCDE (also associated with rifampin resistance) were intact, excluding the presence of insertions/deletions within the pump operon. The promoter region (mtrR) was fully sequenced and was distinct from FQ-susceptible controls. Isolates with higher ciprofloxacin MIC values had identical promoter region, whereas the isolate displaying a lower ciprofloxacin MIC value possessed a different sequence. Expression experiments showed discrepancies of the mRNA in pump components. The most remarkable difference was for the outer membrane protein encoded by mtrE that was hyperexpressed (>3600× elevated compared to control) in the strain displaying a ciprofloxacin MIC value of 0.06 μg/mL. These results confirm that T91I alteration on GyrA had an important role in elevating ciprofloxacin MIC values; however, additional resistance determinants, including other gyrA mutations, also contribute to higher FQ MIC levels. Alterations in expression of mtrCED pump appear to have minimal influence on ciprofloxacin resistance, and this finding was supported by low rifampin susceptible-level results (MIC, ≤0.008 to 0.03 μg/mL).

Coastal Seawater Bacteria Harbor a Large Reservoir of Plasmid-Mediated Quinolone Resistance Determinants in Jiaozhou Bay, China

Diversity and prevalence of plasmid-mediated quinolone resistance determinants were investigated in environmental bacteria isolated from surface seawater of Jiaozhou Bay, China. Five qnr gene alleles were identified in 34 isolates by PCR amplification, including qnrA3 gene in a Shewanella algae isolate, qnrB9 gene in a Citrobacter freundii isolate, qnrD gene in 22 Proteus vulgaris isolates, qnrS1 gene in 1 Enterobacter sp. and 4 Klebsiella spp. isolates, and qnrS2 gene in 1 Pseudomonas sp. and 4 Pseudoalteromonas sp. isolates. The qnrC, aac(6')-Ib-cr, and qepA genes could not be detected in this study. The 22 qnrD-positive Proteus vulgaris isolates could be differentiated into four genotypes based on ERIC-PCR assay. The qnrS1 and qnrD genes could be transferred to Escherichia coli J53 Azi(R) or E. coli TOP10 recipient strains using conjugation or transformation methods. Among the 34 qnr-positive isolates, 30 had a single point mutation in the QRDRs of GyrA protein (Ala67Ser, Ser83Ile, or Ser83Thr), indicating that cooperation of chromosome- and plasmid-mediated resistance contributed to the spread and evolution of quinolone resistance in this coastal bay. Eighty-five percent of the isolates were also found to be resistant to ampicillin, and bla(CMY), bla(OXY), bla(SHV), and bla(TEM) genes were detected in five isolates that also harbored the qnrB9 or qnrS1 gene. Our current study is the first identification of qnrS2 gene in Pseudoalteromonas and Pseudomonas strains, and qnrD gene in Proteus vulgaris strains. High prevalence of diverse qnr genes in Jiaozhou Bay indicates that coastal seawater may serve as an important reservoir, natural source, and dissemination vehicle of quinolone resistance determinants.

Asociación en un mismo plásmido de blaVIM-1 y qnrS2 en Klebsiella pneumoniae y Klebsiella oxytoca aisladas en Sevilla

Combined resistance to quinolones and β-lactams is common in Enterobacteriaceae. The appearance in enterobacteria coding for metallo-β-lactamases and determinants of plasmid-mediated quinolone resistance are an emerging problem in our country. The susceptibility was determined by E-test. The resistance genes were detected by PCR and the corresponding plasmids were characterised. This study describes 2 strains (1 Klebsiella oxytoca, 1 Klebsiella pneumoniae) carrying the genes qnrS2 and blaVIM-1 in a transferable plasmid of 70-Kb isolated in surveillance cultures at the University Hospital Virgen Macarena in Seville. This is the first combination of qnrS2 and bla(VIM-1) on the same non-typeable plasmid isolated in our centre.

Plasmid-mediated quinolone resistance determinant qepA1 and extended-spectrum β-lactamase gene bla CTX-M-14 co-located on the same plasmid in two Escherichia coli strains from China

Plasmid-mediated quinolone resistance determinant qepA1 and extended-spectrum β-lactamase gene bla CTX-M-14 co-located on the same plasmid in two Escherichia coli strains from China

Mechanisms of reduced susceptibility to ciprofloxacin in Escherichia coli isolates from Canadian hospitals.

To determine whether plasmid-mediated quinolone resistance (PMQR) determinants play a role in the increasing resistance to fluoroquinolones among Escherichia coli isolates in Canadian hospitals, and to determine the mechanisms of reduced susceptibility to ciprofloxacin in a recent collection of 190 clinical E coli isolates. E coli isolates (n=1702) were collected as part of the 2007 Canadian Hospital Ward Antibiotic Resistance Surveillance (CANWARD) study. Antimicrobial susceptibility testing was performed by Clinical and Laboratory Standards Institute (CLSI) broth microdilution. Using a representative subset of isolates (n=190), the mechanisms of reduced susceptibility to ciprofloxacin were detected by polymerase chain reaction and sequencing of the quinolone resistance-determining regions (QRDR) of chromosomal gyrA and parC genes, and by polymerase chain reaction for the PMQR genes: qnr, aac(6') Ib-cr and qepA. 2.1% and 1.1% of E coli harboured aac(6')Ib-cr and qnrB, respectively. Single amino acid substitutions in the QRDR of gyrA were observed among isolates with ciprofloxacin minimum inhibitory concentrations as low as 0.12 μg/mL. As the ciprofloxacin minimum inhibitory concentration increased to 1 μg/mL (which is still considered to be susceptible by the CLSI), the vast majority of isolates demonstrated both gyrA and parC mutations. PMQR determinants and QRDR mutants among clinical E coli isolates with reduced susceptibility to ciprofloxacin demonstrates the need for increased surveillance and the need to re-evaluate the current CLSI breakpoints to prevent further development of fluoroquinolone resistance.

Molecular characterization of VIM-producing Klebsiella pneumoniae from Scandinavia reveals genetic relatedness with international clonal complexes encoding transferable multidrug resistance

VIM-producing Klebsiella pneumoniae (VPKP) has been identified as a source of hospital outbreaks and is prevalent particularly in the Mediterranean region. In this study we have characterized eight VPKP isolates identified in Scandinavia during 2005–2008. With the exception of one isolate, all were from patients with recent history of hospitalization abroad (Greece, n = 6; Turkey, n = 1). Multilocus sequence typing (MLST) resulted in five sequence types (STs), ST36 (n = 1), ST147 (n = 4), ST272 (n = 1), ST273 (n = 1) and ST383 (n = 1), which except for ST272 were part of putative international clonal complexes. All were multidrug resistant due to the presence of other resistance determinants, including extended-spectrum β-lactamases (CTX-M-3, SHV-5 and SHV-12), 16S rRNA methylases (ArmA) and plasmid-mediated quinolone resistance determinants (QnrS). One isolate harboured a novel VIM-variant (VIM-26) while VIM-1 and VIM-19 were detected in six and one isolate, respectively. Two different genetic structures surrounding the blaVIM gene were identified in four isolates. In two isolates blaVIM-1 and blaVIM-26 were located in an integron similar to In-e541 (intI1;blaVIM-1/-26;aacA7; dhfrI;aadA1;3’CS) while in the other two isolates blaVIM-1 was located in an integron lacking 3’CS but with an IS26 element in the 3’end (intI1;blaVIM-1;aac(6’)-Ib;IS26), as identified in the IncN plasmid pKOX105. The blaVIM-genes were located on transferable plasmids ranging from -40 to -240 kb and associated with Tn21 in four isolates. PCR-based replicon typing indicated association of blaVIM with IncN (n = 3) and A/C (n = 1) broad-host-range plasmids but also with unknown replicons (n = 4). In conclusion, Scandinavian VPKP is associated with importation and genetically related to international clones encoding transferable plasmid-mediated multidrug resistance.

Rapid evolution of fluoroquinolone-resistant Escherichia coli in Nigeria is temporally associated with fluoroquinolone use

BackgroundAntibiotic resistance has necessitated fluoroquinolone use but little is known about the selective forces and resistance trajectory in malaria-endemic settings, where selection from the antimalarial chloroquine for fluoroquinolone-resistant bacteria has been proposed.MethodsAntimicrobial resistance was studied in fecal Escherichia coli isolates in a Nigerian community. Quinolone-resistance determining regions of gyrA and parC were sequenced in nalidixic acid resistant strains and horizontally-transmitted quinolone-resistance genes were sought by PCR. Antimicrobial prescription practices were compared with antimicrobial resistance rates over a period spanning three decades.ResultsBefore 2005, quinolone resistance was limited to low-level nalixidic acid resistance in fewer than 4% of E. coli isolates. In 2005, the proportion of isolates demonstrating low-level quinolone resistance due to elevated efflux increased and high-level quinolone resistance and resistance to the fluoroquinolones appeared. Fluoroquinolone resistance was attributable to single nucleotide polymorphisms in quinolone target genes gyrA and/or parC. By 2009, 35 (34.5%) of isolates were quinolone non-susceptible with nine carrying gyrA and parC SNPs and six bearing identical qnrS1 alleles. The antimalarial chloroquine was heavily used throughout the entire period but E. coli with quinolone-specific resistance mechanisms were only detected in the final half decade, immediately following the introduction of the fluoroquinolone antibacterial ciprofloxacin.ConclusionsFluoroquinolones, and not chloroquine, appear to be the selective force for fluoroquinolone-resistant fecal E. coli in this setting. Rapid evolution to resistance following fluoroquinolone introduction points the need to implement resistant containment strategies when new antibacterials are introduced into resource-poor settings with high infectious disease burdens.

Molecular Epidemiology of Ciprofloxacin-Resistant Extended-Spectrum β-Lactamase–ProducingKlebsiella pneumoniaein Taiwan

Fluoroquinolone resistance in extended-spectrum β-lactamases (ESBL)-producing isolates results in very few antimicrobial treatment options. In Taiwan's Surveillance of Antimicrobial Resistance (TSAR) III program, 124 (52.8%) cases of ESBL-producing Klebsiella pneumoniae (ESBL-KP) were resistant to ciprofloxacin. The prevalence of plasmid-mediated quinolone resistance (PMQR) determinants and chromosomal quinolone resistance-determining regions (QRDR) of gyrA and parC genes among ESBL-KP isolates was assessed via PCR sequencing. Chromosomal QRDR mutations were present in most of the 123 (96.8%) cases of ciprofloxacin-resistant ESBL-KP isolates. Sixty-six (53.2%) isolates had at least one PMQR gene. qnrB2, qnrB4, and qnrS1 were detected in 26, 19, and 13 isolates, respectively, whereas qnrA, qnrC, and qnrD were not detected. ESBL genes were transferable via conjugation with either aac(6')Ib-cr or qnrB in 63.6% of the isolates carrying PMQR genes. QnrB was associated with either CTX-M-15 or SHV-12, and aac(6')Ib-cr was linked to CTX-M-3 or CTX-M-14 in plasmids. qnrS did not co-transfer with ESBL genes. Clonal spread of PMQR genes harboring ESBL-KP isolates was observed in three hospitals. QnrA, which is common in Asia, was unexpectedly absent in ESBL-KP in Taiwan. Aside from transmission via clonal spread for ciprofloxacin-resistant ESBL-KP, concomitant transference of PMQR genes with either bla(CTX-M) or bla(SHV) via plasmid was common.

First characterisation in Tunisia of plasmid mediated AmpC bta-lactamase DHA-1 coexpressed TEM-24 and QnrA6 in a multidrug resistant Proteus mirabilis clinical strain

The aim of the study was to investigate the coresistance of qnrA6 determinants and DHA-1, TEM-24 β-lactamases in a multidrug-resistant Proteus mirabilis SM18575 strainisolated from Tunisia with reduced succeptibility to oximinocephalosporins. The strain with an inducible phenotype was detected in March 2007 in a Tunisian hospital. The isolate was tested for broth minimum inhibitory concentrations, isoelectric focusing (IEF), polymerase chain reaction (PCR) for SHV, TEM, and DHA-derived β-lactamases, and nucleotide sequencing. A multiplex PCR was performed for the screnning of qnrA, qnrB and qnrS. IEF, PCR and sequence analysis revealed that the isolate possessed a blaTEM-1, blaTEM-24 and a blaDHA-1 gene and produced quinolone resistance determinants qnrA6.The inducible cephalosporin resistance was transferred and the transconjugantsexpressed non susceptibility to third generation cephalosporins. To the best of our knowledge, this is the first detection of DHA-1 and TEM-24 β-lactamases in a Tunisian hospital. Keys words: β-Lactamase, coresistance, qnr determinants.

Molecular characterization of strains of fluoroquinolone-resistant Salmonella enterica serovar Schwarzengrund carrying multidrug resistance isolated from imported foods

To determine the fluoroquinolone resistance determinants in Salmonella enterica serovar Schwarzengrund from imported foods. Antibiotic susceptibility of Salmonella Schwarzengrund to 16 antibiotics was examined using disc agar diffusion and Etest. Quinolone resistance determinants were examined by sequence analysis of gyrA, gyrB, parC and parE, PCR amplification of qnrA, qnrB and qnrS, and expression of acrB, ramA, marA, soxS and rob using quantitative RT-PCR. The contribution of efflux pump activities to antibiotic resistance was determined by the addition of carbonyl cyanide m-chlorophenylhydrazone (CCCP). The effect of ramR deletion on ciprofloxacin resistance was determined by complementing with wild-type ramR. Salmonella strains 30 and 487 were susceptible to ciprofloxacin and had a single mutation in gyrA as compared with strain 75, which was highly resistant to ciprofloxacin and had a double mutation in gyrA. Increased expression of ramA was associated with high resistance to ciprofloxacin. Strain 75 had a deletion of 315 bp in the ramR gene, which regulates ramA expression. Overexpression of ramA was possibly related to a loss of ramR. Introduction of ramR decreased the MIC of ciprofloxacin from 48 to 24 mg/L. The addition of CCCP did not reduce antibiotic resistance. To our knowledge, this study reports for the first time the natural deletion of ramR in Salmonella Schwarzengrund. This study indicates that fluoroquinolone-resistant Salmonella are prevalent in imported food. Double mutations in gyrA and a loss of ramR were associated with high-level quinolone resistance in multidrug-resistant Salmonella Schwarzengrund strain 75.

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.

Coexistence of blaOXA48andaac(6′)-Ib-crGenes inKlebsiella PneumoniaeIsolates from Istanbul, Turkey

This study evaluated the presence of carbapenem hydrolysing β-lactamase genes and plasmid-mediated quinolone resistance (PMQR) determinants in 22 Klebsiella pneumoniae isolates collected from the Istanbul Medical Faculty, Turkey, which reduced the susceptibility or resistance to carbapenem. The VITEK(®) 2 system and E-tests were used to determine the minimum inhibitory concentrations needed to inhibit bacterial growth. Genes were screened by polymerase chain reaction, and gene transferability was evaluated by transconjugation. Strain clonality was investigated by pulsed-field gel electrophoresis (PFGE). All strains were OXA-48 β-lactamase producers and three (13.6%) were also positive for the aac(6')-Ib-cr gene. Most of the strains harboured other β-lactamase (bla) genes such as bla(TEM), bla(SHV), bla(CTX-M) and bla(VEB-1). The transconjugants mostly harboured bla(OXA-48) and other β-lactamases separately. PFGE revealed eight pulsotypes among the isolates. The coexistence of bla(OXA-48) and PMQR in K. pneumoniae isolates may present a significant threat to health, especially in the nosocomial setting.

The characteristics of VIM-1-producingKlebsiella pneumoniaefrom South Africa

A study was designed to characterize a carbapenem-resistant Klebsiella pneumoniae (KPSA01) isolated from a patient in Gauteng, South Africa without recent travel outside South Africa. Molecular characterization was done using isoelectric focusing, polymerase chain reaction and sequencing for bla(VIM), bla(IMP), bla(NDM), bla(CTX-Ms), bla(OXAs), bla(TEMs), and bla(SHV), plasmid-mediated quinolone resistance determinants, multilocus sequencing typing, plasmid replicon typing, and addiction factors. KPSA01 produced VIM-1 and belonged to the newly described sequence type ST569. The plasmid that harboured bla(VIM) typed within the narrow host range IncF replicon group, contained the aadA1 gene cassette, and tested positive for the vagCD and ccdAB addiction systems. This is the first report of VIM-1-producing K. pneumoniae outside Europe. It is important that surveillance studies be undertaken in Africa to determine if VIM-1-producing K. pneumoniae are present in significant numbers.