Dihydroartemisinin inhibits plasmid transfer in drug-resistant Escherichia coli via limiting energy supply.

Conjugative transfer of antibiotic resistance genes (ARGs) by plasmids is an important route for ARG dissemination. An increasing number of antibiotic and nonantibiotic compounds have been reported to aid the spread of ARGs, highlighting potential challenges for controlling this type of horizontal transfer. Development of conjugation inhibitors that block or delay the transfer of ARG-bearing plasmids is a promising strategy to control the propagation of antibiotic resistance. Although such inhibitors are rare, they typically exhibit relatively high toxicity and low efficacy in vivo and their mechanisms of action are inadequately understood. Here, we studied the effects of dihydroartemisinin (DHA), an artemisinin derivative used to treat malaria, on conjugation. DHA inhibited the conjugation of the IncI2 and IncX4 plasmids carrying the mobile colistin resistance gene ( mcr-1) by more than 160-fold in vitro in Escherichia coli, and more than two-fold (IncI2 plasmid) in vivo in a mouse model. It also suppressed the transfer of the IncX3 plasmid carrying the carbapenem resistance gene bla NDM-5 by more than two-fold in vitro. Detection of intracellular adenosine triphosphate (ATP) and proton motive force (PMF), in combination with transcriptomic and metabolomic analyses, revealed that DHA impaired the function of the electron transport chain (ETC) by inhibiting the tricarboxylic acid (TCA) cycle pathway, thereby disrupting PMF and limiting the availability of intracellular ATP for plasmid conjugative transfer. Furthermore, expression levels of genes related to conjugation and pilus generation were significantly down-regulated during DHA exposure, indicating that the transfer apparatus for conjugation may be inhibited. Our findings provide new insights into the control of antibiotic resistance and the potential use of DHA.

Sarcocheilichthys vittatus, a new species of gudgeon (Teleostei: Cyprinidae) from the Poyang lake basin in Jiangxi Province, South China.

Sarcocheilichthys vittatus, a new species of gudgeon, is described from the Gan-Jiang and Fu-He flowing into the Poyang Lake Basin in Jiangxi Province, South China. It is similar to S. parvus and S. caobangensis in having a longitudinal black band extending from the snout tip to the caudal-fin base, a character separating them from all other congeneric species, but distinct from S. parvus in having more lateral-line scales, no longitudinal yellowish stripe above the lateral black band on the flank and no barbels, and from S. caobangensis in possessing two shorter lateral lobes of the lower lip restricted only to the side of the lower jaw, no black spots along the dorsal-fin base and a stouter caudal peduncle. Its validity was also warranted by a molecular phylogenetic analysis based on the cyt b gene. Critical notes were provided on some currently recognized Chinese congeners.

Antibacterial Effect and Mode of Action of Flavonoids From Licorice Against Methicillin-Resistant Staphylococcus aureus.

Staphylococcus aureus is a bacterial pathogen that causes food poisoning, various infections, and sepsis. Effective strategies and new drugs are needed to control S. aureus associated infections due to the emergence and rapid dissemination of antibiotic resistance. In the present study, the antibacterial activity, potential mode of action, and applications of flavonoids from licorice were investigated. Here, we showed that glabrol, licochalcone A, licochalcone C, and licochalcone E displayed high efficiency against methicillin-resistant Staphylococcus aureus (MRSA). Glabrol, licochalcone A, licochalcone C, and licochalcone E exhibited low cytotoxicity without hemolytic activity based on safety evaluation. Glabrol displayed rapid bactericidal activity with low levels of resistance development in vitro. Meanwhile, glabrol rapidly increased bacterial membrane permeability and dissipated the proton move force. Furthermore, we found that peptidoglycan, phosphatidylglycerol, and cardiolipin inhibited the antibacterial activity of glabrol. Molecular docking showed that glabrol binds to phosphatidylglycerol and cardiolipin through the formation of hydrogen bonds. Lastly, glabrol showed antibacterial activity against MRSA in both in vivo and in vitro models. Altogether, these results suggest that glabrol is a promising lead compound for the design of membrane-active antibacterial agents against MRSA and can be used as a disinfectant candidate as well.

Natural Products That Target Virulence Factors in Antibiotic-Resistant Staphylococcus aureus.

The increase in the incidence of antibiotic-resistant Staphylococcus aureus (S. aureus) associated infections necessitates the urgent development of novel therapeutic strategies and antibacterial drugs. Antivirulence strategy is an especially compelling alternative strategy due to its low selective pressure for the development of drug resistance in bacteria. Plants and microorganisms are not only important food and medicinal resources but also serve as sources for the discovery of natural products that target bacterial virulence factors. This review discusses the mechanisms of the major virulence factors of S. aureus, including the accessory gene regulator quorum-sensing system, bacterial biofilm formation, α-hemolysin, sortase A, and staphyloxanthin. We also provide an overview of natural products isolated from plants and microorganisms with activity against the major virulence factors of S. aureus and their adjuvant effects on existing antibiotics to overcome antibiotic-resistant S. aureus. Finally, the limitations and solutions of these antivirulence compounds are discussed, which will help in the development of novel antibacterial drugs against antibiotic-resistant S. aureus.

Natural Flavones from Morus alba against Methicillin-Resistant Staphylococcus aureus via Targeting the Proton Motive Force and Membrane Permeability.

The emergence and rapid spread of methicillin-resistant Staphylococcus aureus (MRSA) critically requires alternative therapeutic options. New antibacterial drugs and strategies are urgently needed to combat MRSA-associated infections. Here, we investigated the antibacterial activity of flavones from Morus alba and the potential mode of action against MRSA. Kuwanon G, kuwanon H, mulberrin, and morusin displayed high efficiency in killing diverse MRSA isolates. On the basis of structure-activity analysis, the cyclohexene-phenyl ketones and isopentenyl groups were critical to increase the membrane permeability and to dissipate the proton motive force. Meanwhile, mechanistic studies further showed that kuwanon G displayed rapid bactericidal activity in vitrowith difficulty in developing drug resistance. Kuwanon G targeted phosphatidylglycerol and cardiolipin in the cytoplasmic membrane through the formation of hydrogen bonds and electrostatic interactions. Additionally, kuwanon G promoted wound healing in a mouse model of MRSA skin infection. In summary, these results indicate that flavones are promising lead compounds to treat MRSA-associated infections through disrupting the proton motive force and membrane permeability.

Liquid to liquid extraction and liquid chromatography-tandem mass spectrometry determination of hainanmycin in feed.

Hainanmycin is a new veterinary polyether antibiotic and has few sensitive analytical method in present days. In this study, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) relying on multiple reaction monitoring (MRM) detection was developed for analysis of hainanmycin in animal feed. Feed samples were extracted with ethyl acetate and purified by two steps of liquid-liquid extraction (LLE) to get rid of water solvable matrix and lipids one by one. The final simple was analyzed by LC-MS/MS. The LC mobile phase was composed of 0.1% aqueous formic acid and 0.1% formic acidified acetonitrile by gradient elution. Average recoveries ranged from 74.22% to 87.85%, as determined by spiking with 2.0 (LOQ) ∼2500µgkg-1 of hainanmycin. The inter-day and intra-day coefficient of variation was 9.21% to 11.77% and 7.67% to 13.49%, respectively. The limit of detection (LOD) and the limit of quantitation (LOQ) were 0.36µgkg-1 and 2.0µgkg-1, respectively.

Novel lnu(G) gene conferring resistance to lincomycin by nucleotidylation, located on Tn6260 from Enterococcus faecalis E531.

Objectives: To identify a novel putative lincosamide resistance gene determinant in a swine Enterococcus faecalis E531 exhibiting a lincosamide resistance/macrolide susceptibility (L R M S ) phenotype and to determine its location and genetic environment. Methods: The whole genomic DNA of E. faecalis E531, which tested negative for the known lincosamide nucleotidyltransferase genes, was sequenced. A putative lincosamide resistance gene determinant was cloned into an Escherichia coli - E. faecalis shuttle vector (pAM401) and transformed into E. faecalis JH2-2. The MICs were determined by the microbroth dilution method. Inactivity of lincomycin was examined by UPLC-MS/MS. Inverse PCR and primer walking were used to explore the genetic environment based on the assembled sequence. Results: A novel resistance gene, designated lnu (G), which encodes a putative lincosamide nucleotidyltransferase, was found in E. faecalis E531. The deduced Lnu(G) amino acid sequence displayed 76.0% identity to Lnu(B) in Enterococcus faecium . Both E. faecalis E531 and E. faecalis JH2-2 harbouring pAM401- lnu (G) showed a 4-fold increase in the MICs of lincomycin, compared with E. faecalis JH2-2 or E. faecalis JH2-2 harbouring empty vector pAM401 only. UPLC-MS/MS demonstrated that the Lnu(G) enzyme catalysed adenylylation of lincomycin. The genetic environment analysis revealed that the lnu (G) gene was embedded into a novel putative transposon, designated Tn 6260 , which was active. Conclusions: A novel lincosamide nucleotidyltransferase gene lnu (G) was identified in E. faecalis . The location of the lnu (G) gene on a mobile element Tn 6260 makes it easy to disseminate.

The role of GRIP1 and ephrin B3 in blood pressure control and vascular smooth muscle cell contractility.

Several erythropoietin-producing hepatocellular receptor B family (EPHB) and their ligands, ephrinBs (EFNBs), are involved in blood pressure regulation in animal models. We selected 528 single nucleotide polymorphisms (SNPs) within the genes of EPHB6, EFNB2, EFNB3 and GRIP1 in the EPH/EFN signalling system to query the International Blood Pressure Consortium dataset. A SNP within the glutamate receptor interacting protein 1 (GRIP1) gene presented a p-value of 0.000389, approaching the critical p-value of 0.000302, for association with diastolic blood pressure of 60,396 individuals. According to echocardiography, we found that Efnb3 gene knockout mice showed enhanced constriction in the carotid arteries. In vitro studies revealed that in mouse vascular smooth muscle cells, siRNA knockdown of GRIP1, which is in the EFNB3 reverse signalling pathway, resulted in increased contractility of these cells. These data suggest that molecules in the EPHB/EFNB signalling pathways, specifically EFNB3 and GRIP1, are involved blood pressure regulation.

Vanmanenia maculata, a new species of hillstream loach from the Chang-Jiang Basin, South China (Teleostei: Gastromyzontidae).

Vanmanenia maculata, new species, is described from the middle and lower Chang-Jiang basin in Hubei, Hunan, and Jiangxi provinces, South China. This new species, along with V. caldwelli, V. stenosoma, and V. striata, is distinguished from all other Chinese species of the genus by lacking secondary rostral barbels. It is distinct from V. caldwelli and V. striata in anus placement, rostral lobule shape, and body coloration, and from V. stenosoma in having a larger scaleless area on the ventral surface of the body and a shallower caudal-peduncle. Vanmanenia polylepis should be removed from the synonymy of V. pingchowensis and regarded as valid.

The development of a fluorescence polarization immunoassay for aflatoxin detection.

A fluorescence polarization immunoassay (FPIA) was developed for the analysis ofaflatoxins (AFs) using an anti-aflatoxin B1 (AFB1) monoclonal antibody and a novel fluorescein-labeled AFB1 tracer. The FPIA showed an IC50 value of 23.33 ng/mL with a limit of detection of 13.12 ng/mL for AFB1. The cross-reactivities of AFB1, AFB2, AFG1, AFG2, AFM1, and AFM2 with the antibody were 100%, 65.7%, 143%, 23.5%, 111.4%, and 2%, respectively. The group-specificity of anti-AFB1mAb indicated that the FPIA could potentially be used in a screening method for the detection of total AFs, albeit not AFG2 and AFM2. The total time required for analyzing 96 samples in one microplate was less than 5 min. This study demonstrates the potential usefulness of the FPIA as a rapid and simple technique for monitoring AFs.

Prevalence and molecular characterization of fluoroquinolone resistance in Escherichia coli isolates 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 ß-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.

[Macrozoobenthos community structure and water quality evaluation of Tian'e Zhou Oxbows].

A quarterly investigation of the macrozoobenthos community in Tian'e Zhou Yangtze Oxbows was conducted during January 2011 to October 2011. And water quality was assessed based on the benthic macroinvertebrate community structure. It shows that, a total of 30 macrozoobenthos species were found, among which, Insecta (14 species) , Mollusca (6 species), Oligochaeta (8 species) and others (2 species) accounted for 46.67%, 20.00%, 26.67%, and 6.67% of the total, respectively. The dominant species were Clinotanypus, Cryptochironomus digitatus, Limnodrilus claparedeianus, and Limnodrilus hoffmeisteri. The average annual density and biomass of macrozoobenthos were 558.37 ind · m(-2), and 14.03 g · m(-2), respectively. The density of macrozoobenthos was highest in the winter and lowest in the spring, the biomass was highest in the autumn and lowest in the spring. In ten sampling points, No. 8 had the highest density, 1986.00 ind · m(-2), No. 7 had the highest biomass, 50.22 g · m(-2), and No. 6 had the lowest density and biomass, 98.00 ind · m(-2) and 0.85 g · m(-2). The evaluation with Shannon-Wiener diversity index (H'), Margalef richness index (d), Family-level biotic index (FBI), and integrated pollution index (BI) indicated that the overall water quality of the Tian'e Zhou Oxbow was moderate-heavy pollution (III-IV). As compared to that in 2003-2004 (II), the water quality of Tian'e Zhou Oxbow was somewhat decreased.

Development of a rapid multi-residue assay for detecting ß-lactams using penicillin binding protein 2x*.

OBJECTIVE: To develop a rapid multi-residue assay for detecting 16 demanded by the European Union (EU). METHODS: A recombinant penicillin-binding protein (PBP) 2x* from Streptococcus pneumoniae R6 was expressed in vitro and six ß-lactams were conjugated to HRP by four methods. A rapid multi-residue assay for ß-lactams was established with PBP2x* and HRP-conjugate. RESULTS: PBP2x* was expressed and purified successfully and the ideal HRP-conjugate was identified. The multi-residue assay was developed. After optimization, penicillin G, ampicillin, amoxicillin, cloxacillin, dicloxacillin, oxacillin, nafcillin, cephalexin, ceftiofur, cefalonium, cefquinome, cefazolin, cefoperazone, cephacetrile, and cephapirin can be detected at levels below MRL in milk with simple pretreatment. CONCLUSION: This assay developed can detect all 16 ß-lactams demanded by the European Union (EU). The whole procedure takes only 45 min and can detect 42 samples and the standards with duplicate analysis.

Development of an enzyme-linked immunosorbent assay for determination of the furaltadone etabolite, 3-amino-5-morpholinomethyl-2-oxazolidinone (AMOZ) in animal tissues.

OBJECTIVE: To determine 3-amino-5-morpholinomethyl-2-oxazolidinone (AMOZ) residues released from protein bound AMOZ in animal tissues. METHODS: Polyclonal and monoclonal antibodies were produced in this study. A rapid, sensitive, and specific competitive direct enzyme-linked immunosorbent assay (cdELISA) was developed. RESULTS: Rabbit polyclonal antibodies were used in the optimized cdELISA method, and exhibited negligible cross-reactivity with other compounds structurally related to AMOZ. The IC(50) of the polyclonal antibody was 0.16 ng/mL. The method limit of detection in four different types of animal and fish tissues was less than 0.06 µg/kg. Recoveries ranged from 80% to 120% for fortified samples with the coefficient of variation values less than 15%. The results of the cdELISA method were in good agreement with the results from an established liquid chromatography-tandem mass spectrometry confirmatory method used for AMOZ residues. CONCLUSION: The cdELISA method developed in the present study is a convenient practical tool for screening large numbers of animal and fish tissue samples for the the detection of released protein bound AMOZ residues.

Co-carriage of qnrS1, floR, and bla(CTX-M-14) on a multidrug-resistant plasmid in Escherichia coli isolated from pigs.

Plasmid-mediated quinolone resistance (PMQR) determinants were widely distributed among Enterobacteriaceae. The objectives of the present study were to analyze PMQR-positive Escherichia coli isolates from pigs, and to investigate the association between these determinants and other resistant genes. A total of 129 porcine E. coli isolates were included in this study. The presence of PMQR, floR, bla(CTX-M-14), and bla(TEM-1) genes were detected by polymerase chain reaction (PCR) amplification and confirmed by subsequent sequencing. The PMQR-positive isolates were subjected to plasmid profiling, and transformation experiments were conducted to identify the quinolone resistance plasmids. The qnrS1 region of a quinolone resistance plasmid was cloned and sequenced. Among the 129 E. coli isolates, the positive rate for PMQR determinants was 42.6%, and the prevalence of qnr genes, aa(6')-Ib-cr, and qepA were 23.3%, 18.6%, and 0.8%, respectively. A qnrS1-carrying plasmid of 81 kb, named plasmid T078 (pT078), was detected from one multidrug-resistant isolate. Hybridization and PCR analysis confirmed that floR, bla(CTX-M-14), and bla(TEM-1) genes were also located on this plasmid. Sequence analysis identified the qnrS1 gene flanked by a truncated transposase gene. Moreover, complete tetracycline resistance genes tet(A) and tet(R) were found upstream of the qnrS1 gene, and floR gene was found downstream of the qnrS1 gene on the plasmid pT078. To our knowledge, this is the first study demonstrating the occurrence of qnrS1, floR, bla(CTX-M-14), bla(TEM-1), and tet(A) on one plasmid in E. coli isolated from food animals.

Simultaneous determination of thirty non-steroidal anti-inflammatory drug residues in swine muscle by ultra-high-performance liquid chromatography with tandem mass spectrometry.

An ultra-high-performance liquid chromatography with tandem mass spectrometric detection (UHPLC-MS/MS) method was established for the simultaneous determination of residues of thirty non-steroidal anti-inflammatory drugs (NSAIDs) in swine muscle. The samples were extracted with acetonitrile and phosphoric acid. The extracts were defatted with n-hexane, and then purified by HLB solid-phase extraction cartridge. Analysis was carried out on UHPLC-ESI-MS/MS working with multiple reaction monitoring mode with polarity switching. Limits of detection were between 0.4 µg/kg and 2.0 µg/kg, and limits of quantification were between 1.0 µg/kg and 5.0 µg/kg. The recoveries of NSAIDs were between 61.7% and 125.7% at spiked levels of 1.0-500 µg/kg. The repeatability was less than 8% and the within-laboratory reproducibility was not more than 12.3%. The method was reliable, convenient and sensitive.

A survey of ß-lactamase and 16S rRNA methylase genes among fluoroquinolone-resistant Escherichia coli isolates and their horizontal transmission in Shandong, China.

The prevalence of ß-lactamase, 16S rRNA methylase genes, and plasmid-mediated fluoroquinolone-resistance (PMQR) determinants (qnrC and qnrD) was determined by polymerase chain reaction in fluoroquinolone-resistant Escherichia coli isolated from a chicken farm, a pig farm, and a hospital in Shandong, China in 2007. The bla(TEM) and bla(CTX-M) were the most prevalent ß-lactamase genes in isolates from chickens (88.4%, 175/198 and 81.3%, 161/198) and hospitalized patients (87.8%, 122/139 and 69.1%, 96/139). The bla(TEM) was the most prevalent ß-lactamase gene observed in isolates from pigs (98.5%, 135/137). The gene bla(CMY-2) was also predominant among isolates from chickens (20.2%, 40/198). The bla(LAP-1) gene was first detected in one strain from chickens and humans (pig farm workers) in China. Only one strain from hospitalized patients was found to possess bla(SHV). The rmtB was the most prevalent 16S rRNA methylase gene detected in isolates from chickens (19.7%, 39/198) and hospitalized patients (15.8%, 22/139). To our knowledge, this is the first report of the detection of the qnrD gene in E. coli from chickens and pigs in China. The qnrC and bla(KPC) genes were not detected in any of the isolates. Results of southern hybridization revealed that PMQR determinants, ß-lactamases, and 16S rRNA methylase genes were located on the same plasmid in E. coli strains derived from patients. Also, PMQR determinants and ß-lactamase genes were localized on the same plasmid in an E. coli strain of animal origin. Results of conjugation experiments revealed that all of these plasmid-based resistance genes can be transferred by conjugation through horizontal transmission.

Detection of the staphylococcal multiresistance gene cfr in Proteus vulgaris of food animal origin.

OBJECTIVES: To investigate the presence and the genetic environment of the multiresistance gene cfr in naturally occurring Gram-negative bacteria of pigs. METHODS: A total of 391 bacterial isolates with florfenicol MICs of ≥16 mg/L, obtained from 557 nasal swabs of individual pigs, were screened by PCR for the known florfenicol resistance genes. The species assignment of the cfr-carrying isolate was based on the results of Gram's staining, colony morphology, 16S rDNA sequencing and biochemical profiling. The location of the cfr and floR genes was determined by Southern blotting and the regions flanking the cfr gene were sequenced by a modified random primer walking strategy. RESULTS: A single Proteus vulgaris isolate, which carried the genes floR and cfr, was detected in this study. A cfr-carrying segment of 7 kb with homology to a staphylococcal plasmid was found to be inserted into the chromosomal fimD gene of P. vulgaris. This segment was flanked by two IS26 elements located in the same orientation, which are believed to have played a role in this integration process. Stability testing via inverse PCR approaches showed that this integrate is not entirely stable, but the cfr-carrying centre region plus one IS26 copy can be looped out via IS26-mediated recombination. CONCLUSIONS: To the best of our knowledge, this is the first report of the cfr gene in a naturally occurring Gram-negative bacterium. Surveillance and monitoring of the cfr gene in Gram-negative bacteria are warranted with respect to food safety and consumer protection.

Single and dual mutations at positions 2058, 2503 and 2504 of 23S rRNA and their relationship to resistance to antibiotics that target the large ribosomal subunit.

OBJECTIVES: To study mutations at positions A2058, A2503 and U2504 (Escherichia coli numbering) of 23S rRNA and their relationship to resistance to antibiotics that target the large ribosomal subunit. METHODS: Single and dual mutations at positions 2058, 2503 and 2504 of 23S rRNA were introduced into a Mycobacterium smegmatis strain with a single functional rRNA operon. MICs of macrolide, pleuromutilin, phenicol, lincosamide and oxazolidinone antibiotics were determined for the engineered mutants. The doubling times of the mutant strains were measured to investigate how the introduced mutations affected growth rate. RESULTS: Single mutations A2058G, A2503U and U2504G and double mutations A2058G-A2503U and A2058G-U2504G were successfully introduced. The A2058G mutation resulted in various levels of resistance to macrolides and clindamycin. The A2503U and U2504G mutations conferred resistance to valnemulin, chloramphenicol, florfenicol and linezolid. In addition, the A2503U mutant showed reduced susceptibility to the 16-membered macrolides tylosin, spiramycin and josamycin, and the U2504G mutant exhibited decreased susceptibility to spiramycin and josamycin. Moreover, the dual mutations A2058G-A2503U and A2058G-U2504G had co-effects on resistance to 16-membered macrolides. CONCLUSIONS: 23S rRNA mutations A2058G, A2503U and U2504G play key roles in resistance to clinically useful antibiotics that target the large ribosomal subunit. Furthermore, the double mutations A2058G-A2503U and A2058G-U2504G have combined effects on resistance to 16-membered macrolides.

Correlation of fluoroquinolone resistance with expression of qnrA gene in Kluyvera spp.

The objective of the present study was to examine whether the expression of qnrA may contribute to a high level of resistance among parent and induced strains of Kluyvera spp. Two clinical isolates of ciprofloxacin-resistant Kluyvera spp. were obtained from livers of diseased chickens, and upon induction with ciprofloxacin, six strains with increased resistance were produced. Point mutations in qnrA, aac(6')-Ib-cr, gyrA, gyrB, parC, and parE were investigated by polymerase chain reaction (PCR) amplification and DNA sequencing, and expression levels of acrAB and qnrA in all strains were investigated by quantitative real-time PCR (qRT-PCR). The induced strains contained the same mutations in quinolone resistance-determining region as those of the parent strains. qRT-PCR showed that the expression of the acrA gene was not detected in any strain and acrB gene expression was unchanged between induced and parental strains. However, difference in expression of qnrA was observed, which correlated well with the level of quinolone resistance in the parent and induced strains. The induced high resistance was not affected by mutations in qnrA and aac(6')-Ib-cr, by new mutations in the quinolone resistance-determining region of gyrA, gyrB, parC, and parE, or by the expression level of acrAB. These data suggest that the expression of qnrA may be a factor contributing to the high level of resistance among parent and induced strains of Kluyvera spp.