Development of a Pefloxacin Disk Diffusion Method for Detection of Fluoroquinolone-Resistant Salmonella enterica.
ABSTRACT: Fluoroquinolones (FQs) are among the drugs of choice for treatment of Salmonella infections. However, fluoroquinolone resistance is increasing in Salmonella due to chromosomal mutations in the quinolone resistance-determining regions (QRDRs) of the topoisomerase genes gyrA, gyrB, parC, and parE and/or plasmid-mediated quinolone resistance (PMQR) mechanisms including qnr variants, aac(6')-Ib-cr, qepA, and oqxAB. Some of these mutations cause only subtle increases in the MIC, i.e., MICs ranging from 0.12 to 0.25 mg/liter for ciprofloxacin (just above the wild-type MIC of ?0.06 mg/liter). These isolates are difficult to detect with standard ciprofloxacin disk diffusion, and plasmid-mediated resistance, such as qnr, is often not detected by the nalidixic acid screen test. We evaluated 16 quinolone/fluoroquinolone disks for their ability to detect low-level-resistant Salmonella enterica isolates that are not serotype Typhi. A total of 153 Salmonella isolates characterized for the presence (n = 104) or absence (n = 49) of gyrA and/or parC topoisomerase mutations, qnrA, qnrB, qnrD, qnrS, aac(6')-Ib-cr, or qepA genes were investigated. All isolates were MIC tested by broth microdilution against ciprofloxacin, levofloxacin, and ofloxacin and by disk diffusion using EUCAST or CLSI methodology. MIC determination correctly categorized all isolates as either wild-type isolates (MIC of ?0.06 mg/liter and absence of resistance genes) or non-wild-type isolates (MIC of >0.06 mg/liter and presence of a resistance gene). Disk diffusion using these antibiotics and nalidixic acid failed to detect some low-level-resistant isolates, whereas the 5-?g pefloxacin disk correctly identified all resistant isolates. However, pefloxacin will not detect isolates having aac(6')-Ib-cr as the only resistance determinant. The pefloxacin disk assay was approved and implemented by EUCAST (in 2014) and CLSI (in 2015).
Project description:Recent studies show that rectal colonization with low-level ciprofloxacin-resistant Escherichia coli (ciprofloxacin minimal inhibitory concentration (MIC) above the epidemiological cutoff point, but below the clinical breakpoint for resistance), i.e., in the range >?0.06-0.5 mg/L is an independent risk factor for febrile urinary tract infection after transrectal ultrasound-guided biopsy (TRUS-B) of the prostate, adding to the other risk posed by established ciprofloxacin resistance in E. coli (MIC?>?0.5 mg/L) as currently defined. We aimed to identify the quinolone that by disk diffusion best discriminates phenotypic wild-type isolates (ciprofloxacin MIC???0.06 mg/L) of E. coli from isolates with acquired resistance, and to determine the resistance genotype of each isolate. The susceptibility of 108 E. coli isolates was evaluated by ciprofloxacin, levofloxacin, moxifloxacin, nalidixic acid, and pefloxacin disk diffusion and correlated to ciprofloxacin MIC (broth microdilution) using EUCAST methodology. Genotypic resistance was identified by PCR and DNA sequencing. The specificity was 100% for all quinolone disks. Sensitivity varied substantially, as follows: ciprofloxacin 59%, levofloxacin 46%, moxifloxacin 59%, nalidixic acid 97%, and pefloxacin 97%. We suggest that in situations where low-level quinolone resistance might be of importance, such as when screening for quinolone resistance in fecal samples pre-TRUS-B, a pefloxacin (S???24 mm) or nalidixic acid (S???19 mm) disk, or a combination of the two, should be used. In a setting where plasmid-mediated resistance is prevalent, pefloxacin might perform better than nalidixic acid.
Project description:Detection of fluoroquinolone resistance in Salmonella enterica has become increasingly difficult due to evolving resistance mechanisms to this antimicrobial class in this organism. We evaluated two quinolone disks and five fluoroquinolone disks for their ability to act as a surrogate agent for the detection of fluoroquinolone resistance in a collection of 136 S. enterica isolates, including 111 with intermediate or resistant ciprofloxacin MICs mediated by a variety of resistance mechanisms. Ciprofloxacin, ofloxacin, and pefloxacin disks detected all isolates resistant to ciprofloxacin (0% very major error) and yielded false resistance (major error) in 8, 4, and 12% of susceptible isolates, respectively. Ciprofloxacin and pefloxacin provided clearer differentiation of susceptible and resistant isolates.
Project description:Turtle-borne Salmonella enterica owns significance as a leading cause in human salmonellosis. The current study aimed to determine the quinolone susceptibility and the genetic characteristics of 21 strains of S. enterica subsp. enterica isolated from pet turtles. Susceptibility of four antimicrobials including nalidixic acid, ciprofloxacin, ofloxacin, and levofloxacin was examined in disk diffusion and MIC tests where the majority of the isolates were susceptible to all tested quinolones. In genetic characterization, none of the isolates were positive for qnr or aac(6')-Ib genes and no any target site mutations could be detected in gyrA, gyrB, and parC quinolone resistance determining regions (QRDR). In addition, neighbor-joining phylogenetic tree derived using gyrA gene sequences exhibited two distinct clads comprising; first, current study isolates, and second, quinolone-resistant isolates of human and animal origin. All results suggest that studied strains of S. enterica subsp. enterica isolated from pet turtles are susceptible to quinolones and genetically more conserved with regards to gyrA gene region.
Project description:This study aimed to characterize novel conjugative plasmids that encode transferable ciprofloxacin resistance in Salmonella In this study, 157 nonduplicated Salmonella isolates were recovered from food products, of which 55 were found to be resistant to ciprofloxacin. Interestingly, 37 of the 55 CiprSalmonella isolates (67%) did not harbor any mutations in the quinolone resistance-determining regions (QRDR). Six Salmonella isolates were shown to carry two novel types of conjugative plasmids that could transfer the ciprofloxacin resistance phenotype to Escherichia coli J53 (azithromycin resistant [Azir]). The first type of conjugative plasmid belonged to the ?110-kb IncFIB-type conjugative plasmids carrying qnrB-bearing and aac(6')-Ib-cr-bearing mobile elements. Transfer of the plasmid between E. coli and Salmonella could confer a ciprofloxacin MIC of 1 to 2 ?g/ml. The second type of conjugative plasmid belonged to ?240-kb IncH1/IncF plasmids carrying a single PMQR gene, qnrS Importantly, this type of conjugative ciprofloxacin resistance plasmid could be detected in clinical Salmonella isolates. The dissemination of these conjugative plasmids that confer ciprofloxacin resistance poses serious challenges to public health and Salmonella infection control.
Project description:Infections with Salmonella enterica serovar Typhi isolates that have reduced susceptibility to ofloxacin (MIC ? 0.25 ?g/ml) or ciprofloxacin (MIC ? 0.125 ?g/ml) have been associated with a delayed response or clinical failure following treatment with these antimicrobials. These isolates are not detected as resistant using current disk susceptibility breakpoints. We examined 816 isolates of S. Typhi from seven Asian countries. Screening for nalidixic acid resistance (MIC ? 16 ?g/ml) identified isolates with an ofloxacin MIC of ?0.25 ?g/ml with a sensitivity of 97.3% (253/260) and specificity of 99.3% (552/556). For isolates with a ciprofloxacin MIC of ?0.125 ?g/ml, the sensitivity was 92.9% (248/267) and specificity was 98.4% (540/549). A zone of inhibition of ?28 mm around a 5-?g ofloxacin disc detected strains with an ofloxacin MIC of ?0.25 ?g/ml with a sensitivity of 94.6% (246/260) and specificity of 94.2% (524/556). A zone of inhibition of ?30 mm detected isolates with a ciprofloxacin MIC of ?0.125 ?g/ml with a sensitivity of 94.0% (251/267) and specificity of 94.2% (517/549). An ofloxacin MIC of ?0.25 ?g/ml and a ciprofloxacin MIC of ?0.125 ?g/ml detected 74.5% (341/460) of isolates with an identified quinolone resistance-inducing mutation and 81.5% (331/406) of the most common mutant (carrying a serine-to-phenylalanine mutation at codon 83 in the gyrA gene). Screening for nalidixic acid resistance or ciprofloxacin and ofloxacin disk inhibition zone are suitable for detecting S. Typhi isolates with reduced fluoroquinolone susceptibility.
Project description:Non-typhoidal salmonellosis remains a pressing public health problem worldwide. Quinolones, particularly fluoroquinolones, are widely used to treat various infections, including non-typhoidal salmonellosis, which can be a serious illness. The emergence of fluoroquinolone-resistant Salmonella has resulted in treatment failure and high mortality rates. In this study, we estimated the presence of plasmid-mediated quinolone resistance (PMQR) genes in Salmonella enterica isolated from human salmonellosis patients in South Korea from 2016 to 2019. We evaluated the association of these genes with fluoroquinolone susceptibility. Antimicrobial susceptibility tests for Salmonella isolates were performed using the Vitek II system, and the minimum inhibitory concentrations (MIC) of ciprofloxacin and levofloxacin were determined using the E-test method. Plasmid-mediated quinolone resistance (PMQR) genes were detected by PCR amplification and quinolone resistance-determining regions (QRDRs) of the gyrA and parC genes were analyzed following Sanger sequencing of the PCR products. Thirty-four Salmonella strains with reduced susceptibility to fluoroquinolones (ciprofloxacin MIC ≥ 0.125 µg/mL and levofloxacin MIC ≥ 0.25 µg/mL) were selected from 208 human clinical Salmonella isolates. Among them, 22 Salmonella strains harbored one PMQR gene (qnrA, qnrB, or qnrS), and three Salmonella strains carried two PMQR genes (qnrS and aac(6')-Ib-cr or qnrA and qnrB). qnrS was the most common PMQR gene. Serotyping revealed that Salmonella 4,12:i:- (32.4%, 11/34) and Salmonella Typhimurium (29.4%, 10/34) were the two most predominant serovars, and Multi-locus sequence typing (MLST) showed that ST19 and ST34 were the most frequent sequence types. In conclusion, qnr gene-positive Salmonella 4,,12:i:- and Salmonella Typhimurium were the main serovars responsible for reduced susceptibility to fluoroquinolones. Therefore, our findings suggest that PMQR-positive Salmonella strains, which can be isolated from various samples including human, food, and the environment, should be carefully monitored.
Project description:Since its discovery, qnrA has been found in most common Enterobacteriaceae. Ciprofloxacin MICs conferred by different qnrA-positive plasmids could range from 0.1 microg/ml to 2 microg/ml in Escherichia coli J53. The reasons for different ciprofloxacin MICs conferred by qnrA have not been fully clarified. Five hundred forty-one consecutive gram-negative clinical strains that were resistant or intermediate to ciprofloxacin and that were isolated in Shanghai in 2005 were screened for qnrA by PCR. For qnrA-positive isolates, the transferability of quinolone resistance was determined by conjugation and mutations within the quinolone resistance-determining region (QRDR) of gyrA and parC. aac(6')-Ib-cr was detected and qnrA RNA expression was determined using real-time reverse transcription-PCR for transconjugants with different ciprofloxacin MICs. The qnrA gene was detected in 7 of the 541 clinical isolates. Quinolone resistance was transferred in four strains by conjugation. Mutations in the QRDR of gyrA and parC were detected in five qnrA-positive clinical strains with higher ciprofloxacin MICs. Of four qnrA-bearing plasmids in E. coli J53, pHS4 and pHS5 conferred ciprofloxacin MICs of 0.094 to 0.125 microg/ml; pHS3, which harbored the aac(6')-Ib-cr gene as well, conferred a ciprofloxacin MIC of 0.25 microg/ml, and pHS6, which had both the aac(6')-Ib-cr gene and a high expression level of qnrA, had a ciprofloxacin MIC of 1.0 microg/ml. The prevalence of qnrA appeared to be higher in Enterobacter cloacae than in other Enterobacteriaceae. The coexistence of qnrA and aac(6')-Ib-cr in a single plasmid and increased qnrA expression can account for the different levels of ciprofloxacin resistance seen in transconjugants.
Project description:<h4>Background and aim</h4>Recently, the extensive use of quinolones led to increased resistance to these antimicrobial agents, with different rates according to the organism and the geographical region. The aim of this study was to detect the resistance rate of <i>Klebsiella pneumoniae</i> Iraqi isolates toward quinolone antimicrobial agents, to determine genetic mutations in <i>gyrA</i> and <i>parC</i>, to screen for efflux-pump activity, and to screen the presence of plasmid-mediated quinolone resistance (PMQR) genes.<h4>Methods</h4>Forty-three <i>K. pneumoniae</i> isolates were confirmed phenotypically and genotypically by Vitek 2 system and species specific primers by PCR using the targeting <i>rpo</i> gene followed by sequencing. Antibiotic susceptibility test was carried out using disc diffusion method. Quinolone resistant isolates were subjected to ciprofloxacin MIC testing, and cartwheel method to screen for efflux pump activity. The presence of the plasmid mediated quinolone resistance genes <i>qepA, qnrB, qnrS</i>, and <i>aac(6)Ib</i> was tested by PCR. Sequencing of <i>gyr</i>A and <i>par</i>C was performed.<h4>Results</h4>We observed a high rate of resistance to ceftriaxone, gentamicin ciprofloxacin, and levofloxacin. Low rate of resistance was detected against amikacin and azithromycin. Ciprofloxacin MIC results revealed that 96.1% of the isolates had MICs >256 µg/mL, 83.4% had MICs >512 µg/mL while 34.6% had MIC >1024 µg/mL. Testing of isolates against ciprofloxacin mixed with EtBr at various concentrations resulted in decreased resistant. Sequencing results showed that Ser83Leu was the most common mutation in <i>gyr</i>A that was observed in all quinolone resistant isolates, followed by Asp87Asn. Ser80Ile mutation in <i>par</i>C was observed in 77.7% of the tested isolates. The prevalence of PMQR genes was 92.5% <i>aac (6)-Ib</i>, 51.8% <i>qnr</i>B, 40.7% <i>qep</i>A, and 37% <i>qnr</i>S.<h4>Conclusion</h4>Quinolone resistance is common in <i>K. pneumoniae</i> isolates in Baghdad. The frequent mutation in <i>gyr</i>A and <i>par</i>C, and the presence of PMQR genes is alarming.
Project description:We studied a collection of 105 clinical enterobacteria with unusual phenotypes of quinolone susceptibility to analyze the occurrence of plasmid-mediated quinolone resistance (PMQR) and oqx genes and their implications for quinolone susceptibility. The oqxA and oqxB genes were found in 31/34 (91%) Klebsiella pneumoniae and 1/3 Klebsiella oxytoca isolates. However, the oqxA- and oqxB-harboring isolates lacking other known quinolone resistance determinants showed wide ranges of susceptibility to nalidixic acid and ciprofloxacin. Sixty of the 105 isolates (57%) harbored at least one PMQR gene [qnrB19, qnrB10, qnrB2, qnrB1, qnrS1, or aac(6')-Ib-cr)], belong to 8 enterobacterial species, and were disseminated throughout the country, and most of them were categorized as susceptible by the current clinical quinolone susceptibility breakpoints. We developed a disk diffusion-based method to improve the phenotypic detection of aac(6')-Ib-cr. The most common PMQR genes in our collection [qnrB19, qnrB10, and aac(6')-Ib-cr] were differentially distributed among enterobacterial species, and two different epidemiological settings were evident. First, the species associated with community-acquired infections (Salmonella spp. and Escherichia coli) mainly harbored qnrB19 (a unique PMQR gene) located in small ColE1-type plasmids that might constitute its natural reservoirs. qnrB19 was not associated with an extended-spectrum ?-lactamase phenotype. Second, the species associated with hospital-acquired infections (Enterobacter spp., Klebsiella spp., and Serratia marcescens) mainly harbored qnrB10 in ISCR1-containing class 1 integrons that may also have aac(6')-Ib-cr as a cassette within the variable region. These two PMQR genes were strongly associated with an extended-spectrum ?-lactamase phenotype. Therefore, this differential distribution of PMQR genes is strongly influenced by their linkage or lack of linkage to integrons.
Project description:OBJECTIVE:To establish the relative importance of Salmonella enterica serovar Typhi with non-classical quinolone resistance. METHODS:Eight 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. RESULTS:Eighty isolates with a nalidixic acid MIC of <32 mg/L (NALS) and a ciprofloxacin MIC of >0.064 mg/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 3-12 mg/L and CIP MIC of 0.064-0.5 mg/L.; and Group C, mutation in gyrA at codon 83 (n = 2) NAL MIC of 16 mg/L and CIP MIC of 0.25-0.38 mg/L. Group B isolates were found in different strain backgrounds as defined by MLVA. CONCLUSION:The use of nalidixic acid to screen for reduced susceptibility to fluoroquinolones in S. Typhi misses CIPI-NALS isolates, an established phenotype in India.