Overexpression of the novel MATE fluoroquinolone efflux pump FepA in Listeria monocytogenes is driven by inactivation of its local repressor FepR.
ABSTRACT: Whereas fluoroquinolone resistance mainly results from target modifications in gram-positive bacteria, it is primarily due to active efflux in Listeria monocytogenes. The aim of this study was to dissect a novel molecular mechanism of fluoroquinolone resistance in this important human pathogen. Isogenic L. monocytogenes clinical isolates BM4715 and BM4716, respectively susceptible and resistant to fluoroquinolones, were studied. MICs of norfloxacin and ciprofloxacin were determined in the presence or in the absence of reserpine (10 mg/L). Strain BM4715 was susceptible to norfloxacin (MIC, 4 mg/L) and ciprofloxacin (MIC, 0.5 mg/L) whereas BM4716 was highly resistant to both drugs (MICs 128 and 32 mg/L, respectively). Reserpine was responsible for a 16-fold decrease in both norfloxacin and ciprofloxacin MICs against BM4716 suggesting efflux associated resistance. Whole-genome sequencing of the strains followed by comparative genomic analysis revealed a single point mutation in the gene for a transcriptional regulator, designated fepR (for fluoroquinolone efflux protein regulator) belonging to the TetR family. The frame-shift mutation was responsible for the introduction of a premature stop codon resulting in an inactive truncated protein. Just downstream from fepR, the structural gene for an efflux pump of the MATE family (named FepA) was identified. Gene expression was quantified by qRT-PCR and demonstrated that fepA expression was more than 64-fold higher in BM4716 than in BM4715. The clean deletion of the fepR gene from BM4715 was responsible for an overexpression of fepA with resistance to norfloxacin and ciprofloxacin, confirming the role of FepR as a local repressor of fepA. In conclusion, we demonstrated that overexpression of the new MATE efflux pump FepA is responsible for fluoroquinolone resistance in L. monocytogenes and secondary to inactivation of the FepR repressor.
Project description:The effective elimination of <i>Listeria monocytogenes</i> through cleaning and sanitation is of great importance to the food processing industry. Specifically in fresh produce operations, the lack of a kill step requires effective cleaning and sanitation to mitigate the risk of cross-contamination from the environment. As facilities rely on sanitizers to control <i>L. monocytogenes</i>, reports of the development of tolerance to sanitizers and other antimicrobials through cross-resistance is of particular concern. We investigated the potential for six <i>L. monocytogenes</i> isolates from fresh produce handling and processing facilities and packinghouses to develop cross-resistance between a commercial sanitizer and antibiotics. Experimental adaptation of isolates belonging to hypervirulent clonal complexes (CC2, CC4, and CC6) to a commercial quaternary ammonium compound sanitizer (cQAC) resulted in elevated minimum inhibitory concentrations (2-3 ppm) and minimum bactericidal concentrations (3-4 ppm). Susceptibility to cQAC was restored for all adapted (qAD) isolates in the presence of reserpine, a known efflux pump inhibitor. Reduced sensitivity to 7/17 tested antibiotics (chloramphenicol, ciprofloxacin, clindamycin, kanamycin, novobiocin, penicillin, and streptomycin) was observed in all tested isolates. qAD isolates remained susceptible to antibiotics commonly used in the treatment of listeriosis (i.e., ampicillin and gentamicin). The whole genome sequencing of qAD strains, followed by comparative genomic analysis, revealed several mutations in <i>fepR</i>, the regulator for FepA fluoroquinolone efflux pump. The results suggest that mutations in <i>fepR</i> play a role in the reduction in antibiotic susceptibility following low level adaptation to cQAC. Further investigation into the cross-resistance mechanisms and pressures leading to the development of this phenomenon among <i>L. monocytogenes</i> isolates recovered from different sources is needed to better understand the likelihood of cross-resistance development in food chain isolates and the implications for the food industry.
Project description:Thirty-four ciprofloxacin-resistant (MIC > or = 2 microg/ml) and 12 ciprofloxacin-susceptible clinical isolates of Streptococcus pneumoniae were divided into four groups based upon susceptibility to norfloxacin and the effect of reserpine (20 microg/ml). The quinolone-resistance-determining regions of parC, parE, gyrA, and gyrB of all ciprofloxacin-resistant clinical isolates were sequenced, and the activities of eight other fluoroquinolones, acriflavine, ethidium bromide, chloramphenicol, and tetracycline in the presence and absence of reserpine were determined. Despite a marked effect of reserpine upon the activity of norfloxacin, there were only a few isolates for which the activity of another fluoroquinolone was enhanced by reserpine. For most isolates the MICs of acriflavine and ethidium bromide were lowered in the presence of reserpine despite the lack of effect of this efflux pump inhibitor on fluoroquinolone activity. The strains that were most resistant to the fluoroquinolones were predominantly those with mutations in three genes. Expression of the gene encoding the efflux pump PmrA was examined by Northern blotting (quantified by quantitative competitive reverse transcriptase PCR) and compared with that of S. pneumoniae R6 and R6N. Within each group there were isolates that had high-, medium-, and low-level expression of this gene; however, increased expression was not exclusively associated with those isolates with a phenotype suggestive of an efflux mutant. These data suggest that there is another reserpine-sensitive efflux pump in S. pneumoniae that extrudes ethidium bromide and acriflavine but not fluoroquinolones.
Project description:Antibiotic efflux is observed in both eukaryotic and prokaryotic cells, modulating accumulation and resistance. The present study examines whether eukaryotic and prokaryotic fluoroquinolone transporters can cooperate in the context of an intracellular infection. We have used (i) J774 macrophages (comparing a ciprofloxacin-resistant cell line overexpressing an MRP-like transporter with wild-type cells with basal expression), (ii) Listeria monocytogenes (comparing a clinical isolate [CLIP21369] displaying ciprofloxacin resistance associated with overexpression of the Lde efflux system with a wild-type strain [EGD]), (iii) ciprofloxacin (substrate of both Lde and MRP) and moxifloxacin (nonsubstrate), and (iv) probenecid and reserpine (preferential inhibitors of MRP and Lde, respectively). The ciprofloxacin MICs for EGD were unaffected by reserpine, while those for CLIP21369 were decreased approximately fourfold (and made similar to those of EGD). Neither probenecid nor reserpine affected the moxifloxacin MICs against EGD or CLIP21369. In dose-response studies (0.01x to 100x MIC) in broth, reserpine fully restored the susceptibility of CLIP21369 to ciprofloxacin (no effect on EGD) but did not influence the activity of moxifloxacin. In studies with intracellular bacteria, reserpine, probenecid, and their combination increased the activity of ciprofloxacin in wild-type and ciprofloxacin-resistant macrophages in parallel with an increase in ciprofloxacin accumulation in macrophages for EGD and an increase in accumulation and decrease in MIC (in broth) for CLIP21369. Moxifloxacin accumulation and intracellular activity were consistently not affected by the inhibitors. A bacterial efflux pump may thus actively cooperate with a eukaryotic efflux transporter to reduce the activity of a common substrate (ciprofloxacin) toward an intracellular bacterial target.
Project description:The purpose of this study was to investigate the clinical distribution and genotyping of Stenotrophomonas maltophilia, its resistance to antimicrobial agents, and the possible mechanisms of this drug resistance.S. maltophilia isolates were collected from clinical specimens in a university hospital in Northwestern China during the period between 2010 and 2012, and were identified to the species level with a fully automated microbiological system. Antimicrobial susceptibility testing was performed for S. maltophilia with the Kirby-Bauer disc diffusion method. The minimal inhibitory concentrations (MICs) of norfloxacin, ofloxacin, chloramphenicol, minocycline, ceftazidime, levofloxacin and ciprofloxacin against S. maltophilia were assessed using the agar dilution method, and changes in the MIC of norfloxacin, ciprofloxacin and ofloxacin were observed after the addition of reserpine, an efflux pump inhibitor. Fluoroquinolone resistance genes were detected in S. maltophilia using a polymerase chain reaction (PCR) assay, and the expression of efflux pump smeD and smeF genes was determined using a quantitative fluorescent (QF)-PCR assay. Pulsed-field gel electrophoresis (PFGE) was employed to genotype identified S. maltophilia isolates.A total of 426 S. maltophilia strains were isolated from the university hospital from 2010 to 2012, consisting of 10.1% of total non-fermentative bacteria. The prevalence of norfloxacin, ciprofloxacin and ofloxacin resistance was 32.4%, 21.9% and 13.2% in the 114 S. maltophilia isolates collected from 2012, respectively. Following reserpine treatment, 19 S. maltophilia isolates positive for efflux pump were identified, and high expression of smeD and smeF genes was detected in two resistant isolates. gyrA, parC, smeD, smeE and smeF genes were detected in all 114 S. maltophilia isolates, while smqnr gene was found in 25.4% of total isolates. Glu-Lys mutation (GAA-AAA) was detected at the 151th amino acid of the gyrA gene, while Gly-Arg mutation (GGC-CGC) was found at the 37th amino acid of the parC gene. However, no significant difference was observed in the prevalence of gyrA or parC mutation between fluoroquinolone-resistant and -susceptible isolates (p> 0.05). The smqnr gene showed 92% to 99% heterogenicity among the 14 S. maltophilia clinical isolates. PFGE of 29 smqnr gene-positive S. maltophilia clinical isolates revealed 25 PFGE genotypes and 28 subgenotypes.Monitoring the clinical distribution and antimicrobial resistance of S. maltophilia is of great significance for the clinical therapy of bacterial infections. Reserpine is effective to inhibit the active efflux of norfloxacin, ciprofloxacin and ofloxacin on S. maltophilia and reduce MIC of fluoroquinolones against the bacteria. The expression of efflux pump smeD and smeF genes correlates with the resistance of S. maltophilia to fluoroquinolones.
Project description:Stenotrophomonas maltophilia exhibits wide spectrum of fluoroquinolone resistance using different mechanisms as multidrug efflux pumps and Smqnr alleles. Here, the role of smeDEF, smeVWX efflux genes and contribution of Smqnr alleles in the development of fluoroquinolone resistance was assessed. Ciprofloxacin, levofloxacin and moxifloxacin resistance were found in 10.9%, 3.5%, and 1.6% of isolates, respectively. More than four-fold differences in ciprofloxacin MICs were detected in the presence of reserpine and smeD, F, V expression was significantly associated with ciprofloxacin resistance (p = 0.017 for smeD, 0.003 for smeF, and 0.001 for smeV). Smqnr gene was found in 52% of the ciprofloxacin-resistant isolates and Smqnr8 was the most common allele detected. Fluoroquinolone resistance in S. maltophilia clinical isolates was significantly associated with active efflux pumps. There was no correlation between the Smqnr alleles and ciprofloxacin resistance; however, contribution of the Smqnr genes in low-level levofloxacin resistance was revealed.
Project description:Streptococcus suis is an emerging zoonotic pathogen. With the lack of an effective vaccine, antibiotics remain the main tool to fight infections caused by this pathogen. We have previously observed a reserpine-sensitive fluoroquinolone (FQ) efflux phenotype in this species. Here, SatAB and SmrA, two pumps belonging to the ATP binding cassette (ABC) and the major facilitator superfamily (MFS), respectively, have been analyzed in the fluoroquinolone-resistant clinical isolate BB1013. Genes encoding these pumps were overexpressed either constitutively or in the presence of ciprofloxacin in this strain. These genes could not be cloned in plasmids in Escherichia coli despite strong expression repression. Finally, site-directed insertion of smrA and satAB in the amy locus of the Bacillus subtilis chromosome using ligated PCR amplicons allowed for the functional expression and study of both pumps. Results showed that SatAB is a narrow-spectrum fluoroquinolone exporter (norfloxacin and ciprofloxacin), susceptible to reserpine, whereas SmrA was not involved in fluoroquinolone resistance. Chromosomal integration in Bacillus is a novel method for studying efflux pumps from Gram-positive bacteria, which enabled us to demonstrate the possible role of SatAB, and not SmrA, in fluoroquinolone efflux in S. suis.
Project description:Fluoroquinolone resistance can cause major clinical problems. Here, we investigated fluoroquinolone resistance mechanisms in a clinical Escherichia coli isolate, HUE1, which had no mutations quinolone resistance-determining regions (QRDRs) of DNA gyrase and topoisomerase IV. HUE1 demonstrated MICs that exceeded the breakpoints for ciprofloxacin, levofloxacin, and norfloxacin. HUE1 harbored oqxAB and qnrS1 on distinct plasmids. In addition, it exhibited lower intracellular ciprofloxacin concentrations and higher mRNA expression levels of efflux pumps and their global activators than did reference strains. The genes encoding AcrR (local AcrAB repressor) and MarR (MarA repressor) were disrupted by insertion of the transposon IS3-IS629 and a frameshift mutation, respectively. A series of mutants derived from HUE1 were obtained by plasmid curing and gene knockout using homologous recombination. Compared to the MICs of the parent strain HUE1, the fluoroquinolone MICs of these mutants indicated that qnrS1, oqxAB, acrAB, acrF, acrD, mdtK, mdfA, and tolC contributed to the reduced susceptibility to fluoroquinolone in HUE1. Therefore, fluoroquinolone resistance in HUE1 is caused by concomitant acquisition of QnrS1 and OqxAB and overexpression of AcrAB-TolC and other chromosome-encoded efflux pumps. Thus, we have demonstrated that QRDR mutations are not absolutely necessary for acquiring fluoroquinolone resistance in E. coli.
Project description:We characterized the mechanism of fluoroquinolone-resistance in two isolates of Streptococcus pseudopneumoniae having fluoroquinolone-efflux as unique mechanism of resistance. Whole genome sequencing and genetic transformation experiments were performed together with phenotypic determinations of the efflux mechanism. The PatAB pump was identified as responsible for efflux of ciprofloxacin (MIC of 4 ?g/ml), ethidium bromide (MICs of 8-16 ?g/ml) and acriflavine (MICs of 4-8 ?g/ml) in both isolates. These MICs were at least 8-fold lower in the presence of the efflux inhibitor reserpine. Complete genome sequencing indicated that the sequence located between the promoter of the patAB operon and the initiation codon of patA, which putatively forms an RNA stem-loop structure, may be responsible for the efflux phenotype. RT-qPCR determinations performed on RNAs of cultures treated or not treated with subinhibitory ciprofloxacin concentrations were performed. While no significant changes were observed in wild-type Streptococcus pneumoniae R6 strain, increases in transcription were detected in the ciprofloxacin-efflux transformants obtained with DNA from efflux-positive isolates, in the ranges of 1.4 to 3.4-fold (patA) and 2.1 to 2.9-fold (patB). Ciprofloxacin-induction was related with a lower predicted free energy for the stem-loop structure in the RNA of S. pseudopneumoniae isolates (-13.81 and -8.58) than for R6 (-15.32 kcal/mol), which may ease transcription. The presence of these regulatory variations in commensal S. pseudopneumoniae isolates, and the possibility of its transfer to Streptococcus pneumoniae by genetic transformation, could increase fluoroquinolone resistance in this important pathogen.
Project description:Five Listeria monocytogenes isolates (CLIP 21369, CLIP 73298, CLIP 74811, CLIP 75679, and CLIP 79372) were found to be resistant to fluoroquinolones during the screening for antibiotic resistance of 488 L. monocytogenes isolates from human cases of listeriosis in France. On the basis of a fourfold or greater decrease in the ciprofloxacin MIC in the presence of reserpine, fluoroquinolone resistance was attributed to active efflux of the drugs. The lde gene (Listeria drug efflux; formerly lmo2741) encodes a 12-transmembrane-segment putative efflux pump belonging to the major facilitator superfamily of secondary transporters that displayed 44% identity with PmrA from Streptococcus pneumoniae. Insertional inactivation of the lde gene in CLIP 21369 indicated that the corresponding protein was responsible for fluoroquinolone resistance and was involved in the level of susceptibility to dyes such as ethidium bromide and acridine orange.
Project description:Listeria monocytogenes is an important food-borne pathogen that can persist in food processing environments and thus contaminate food products. Benzalkonium chloride (BC) is a common disinfectant widely used in food industry. Selective pressure associated with exposure to BC may result in adaptation to this agent in L. monocytogenes. In this study, the effect of BC adaptation on susceptibility to antimicrobial agents and tolerance to environmental stresses, as well as the role of efflux pumps in BC adaptation were investigated in Listeria monocytogenes. Exposure of L. monocytogenes to progressively increasing concentrations of BC led to adaptation not only to BC but also to several other antimicrobial agents with different modes of action, including cefotaxime, cephalothin, ciprofloxacin, and ethidium bromide (EtBr), indicating that the disinfectant BC has the ability to select for antibiotic resistance. Reserpine, an efflux pump inhibitor, reduced minimum inhibitory concentrations (MICs) of cephalosporins, ciprofloxacin, and EtBr in BC adapted strains, indicating that efflux pumps are involved in cross-adaptation to these antimicrobial agents. Our results showed that expression levels of the efflux pump MdrL in the BC adapted strains increased significantly relative to the corresponding wild-type strains (P < 0.05), with the highest increase in one BC adapted strain named HL06BCA. Moreover, the knockout mutant HL06BCAΔmdrL showed impaired growth compared to that of HL06BCA when exposed to 2 μg/ml of BC. It suggests that efflux pump MdrL is associated with BC adaptation in L. monocytogenes. However, we did not find mdrL to be associated with cross-adaptation to cephalosporins, ciprofloxacin, and EtBr in HL06BCA. Additionally, increased sensitivity to acid, alkali, osmotic, ethanol, and oxidative stresses was observed in most strains after repeated exposure to BC. These results suggest rotation of different disinfectant is helpful to maintain high effectiveness of BC toward L. monocytogenes and ethanol and hydrogen peroxide are at least the appropriate candidates.