Characterization of a mobilizable megaplasmid carrying multiple resistance genes from a clinical isolate of Pseudomonas aeruginosa.

Introduction: The horizontal transfer of antibiotic resistance genes mediated by plasmids seriously hinders the effectiveness of modern medical treatment, and thus has attracted widespread attention. Additionally, the co-selection mechanism of antibiotic resistance genes (ARGs) and heavy metal resistance genes (MRGs) on mobile elements may further exacerbate the horizontal transfer of resistance genes. Methods: In this study, a multidrug-resistant Pseudomonas aeruginosa strain, termed BJ86 (CHPC/NPRC1.4142), was isolated from a patient's sputum specimen. In vitro tests for antimicrobial susceptibility, conjugation, whole-genome sequencing, and bioinformatics analysis were used to explore the potential mechanisms of resistance and its spread. Results and discussion: Sequencing analysis indicates that P. aeruginosa BJ86 carries an amazing 522.5 kb-length megaplasmid, pBJ86, which contained a 93.5 kb-length multiple resistance region (MRR); 18 kinds of genes were identified as ARGs in this region, including tmexCD-oprJ, blaDIM-1, qnrVC6 that mediate resistance to multiple antibiotics and the operons mer that mediates heavy metal mercury resistance. In addition, there is also an 80 kb variable region (VR) on the plasmid pBJ86, and the genes encoding relaxase and type IV coupling protein (T4CP) were determined in this region, both of which are related to the conjugation and transfer ability of the plasmid. Bioinformatics analysis shows that many functional genes have insertion sequences and transposases on their flanks, which may have accumulated in the plasmid pBJ86 after multiple acquisition events. Conjugated transfer and in vitro tests for antimicrobial susceptibility verified the mobility and plasmid pBJ86-mediated resistance. To our knowledge, we are the first to report a mobilizable megaplasmid that simultaneously carried tmexCD-oprJ, blaDIM-1, qnrVC6, and the operons mer and can be transferred with frequencies of 6.24 × 10-7 transconjugants per donor cell.

Quinolone Resistance Genes and Their Contribution to Resistance in Vibrio cholerae Serogroup O139.

BACKGROUND: Quinolones are commonly used for reducing the duration of diarrhea, infection severity, and limiting further transmission of disease related to Vibrio cholerae, but V. cholerae susceptibility to quinolone decreases over time. In addition to mutations in the quinolone-resistance determining regions (QRDRs), the presence of qnr and other acquired genes also contributes to quinolone resistance. RESULTS: We determined the prevalence of quinolone resistance related genes among V. cholerae O139 strains isolated in China. We determined that eight strains carried qnrVC, which encodes a pentapeptide repeat protein of the Qnr subfamily, the members of which protect topoisomerases from quinolone action. Four qnrVC alleles were detected: qnrVC1, qnrVC5, qnrVC12, and qnrVC9. However, the strains carrying qnrVC1, qnrVC5, and qnrVC12 were ciprofloxacin (CIP)-sensitive. Contrastingly, the strain carrying qnrVC9 demonstrated high CIP resistance. qnrVC9 was carried by a small plasmid, which was conjugative and contributed to the high CIP resistance to the receptor V. cholerae strain. The same plasmid was also detected in V. vulnificus. The qnrVC1, qnrVC5, and qnrVC12 were cloned into expression plasmids and conferred CIP resistance on the host V. cholerae O139 strain. CONCLUSIONS: Our results revealed the contribution of quinolone resistance mediated by the qnrVC9 carried on the small plasmid and its active horizontal transfer among Vibrio species. The results also suggested the different effects of qnrVC alleles in different V. cholerae strains, which is possibly due to differences in sequences of qnrVC alleles and even the genetic characteristics of the host strains.

High prevalence of qnrVC variants in Vibrio spp. isolated from food samples in South China.

Phenotypic resistance to fluoroquinolones due to mutational changes in the gyrA and parC genes is common among clinical Vibrio strains; the plasmid-mediated quinolone resistance (PMQR) qnrVC genes were also suggested to play a role in enhancing resistance development. This study investigated the prevalence of qnrVC genes in foodborne Vibrio strains collected in Shenzhen, China, during the period August 2015 and April 2017. A total of 1811 foodborne Vibrio strains were collected, mostly (73.8%) from shrimp samples and 20.2% of these strains were resistant to ciprofloxacin. Investigation of resistance mechanisms showed that mutations in the gyrA and parC genes were commonly associated with ciprofloxacin resistance. The presence of qnrVC genes was shown to enhance ciprofloxacin MIC in Vibrio strains and 69.7% of Vibrio strains that harbored target mutations also carried qnrVC genes, yet only 27.5% of the isolates not harboring such mutations carried the qnrVC genes. A total of 141 strains were found to carry the qnrVC alleles, with qnrVC5 and qnrVC1 being the most common types. Fourteen qnrVC variant genes that contained novel mutations were detectable, with 12 (85.7%) involving qnrVC5-like alleles. For the first time, we found a variant that was likely formed by the recombination of qnrVC1 and qnrVC5. The genetic context of the qnrVC genes found in this study was highly variable, with most being accompanied by mobile genetic elements and other resistance genes. The increasing prevalence of qnrVC genes in Vibrio and its contribution on mediating the development of ciprofloxacin resistance need to be further investigated.

qnrVC occurs in different genetic contexts in Klebsiella and Enterobacter strains isolated from Brazilian coastal waters.

OBJECTIVES: In contrast to other qnr families, qnrVC has been reported mainly in Vibrio spp. and inserted in class 1 integrons. This study aimed to identify the variants of qnrVC genes detected in Klebsiella pneumoniae carbapenemase-2-producing Enterobacter and Klebsiella strains isolated from Brazilian coastal waters and the genetic contexts associated with their occurrence. METHODS: qnrVC variants were identified by Sanger sequencing. Stains were typified by pulsed-field gel electrophoresis. Antimicrobial susceptibility testing, conjugation assays, and whole genome sequencing (WGS) were applied to identify the strains' antimicrobial resistance profile, qnrVC and blaKPC-2 co-transference, and qnrVC genetic context. RESULTS: qnrVC1 was identified in 15 Enterobacter and 3 Klebsiella, and qnrVC4 in 2 Enterobacter strains. Pulsed-field gel electrophoresis revealed 12 clonal profiles of Enterobacter and one of Klebsiella. Strains were resistant to aminoglycosides, beta-lactams, fosfomycin, quinolones, and sulfamethoxazole-trimethoprim. Co-transference of qnrVC and blaKPC-2 were obtained from five representative Enterobacter strains, which showed resistance to ampicillin and amoxicillin-clavulanate, and reduced susceptibility to extended-spectrum cephalosporins, meropenem, and ciprofloxacin. WGS analysis from representative strains revealed one K. quasipneumoniae subsp. similipneumoniae, one E. soli, four E. kobei, and seven isolates belonging to Enterobacter Taxon 3. Long-read WGS showed qnrVC and blaKPC-2 were carried by the same replicon on Klebsiella and Enterobacter strains, and the qnrVC association with not previously described genetic environments composed of insertion sequences and truncated genes. These contexts occurred in small- and high-molecular-weight plasmids belonging to IncFII, IncP6, pKPC-CAV1321, and IncU groups. CONCLUSION: Our results suggest that the dissemination of qnrVC among Enterobacterales in Brazilian coastal waters is associated with several genetic recombination events.

Emergence of Haitian variant genotype and altered drug susceptibility in Vibrio cholerae O1 El Tor-associated cholera outbreaks in Solapur, India.

It is evident from previous cholera epidemics/outbreaks in India, Africa and America that isolates of the seventh pandemic Vibrio cholerae El Tor (7PET) with Haitian cholera toxin (HCT) genotype were associated with increased mortality. The present study highlights the emergence of 7PET-HCT isolates causing two cholera outbreaks in Walsang and Wagdari (Solapur, India) in 2016. Molecular analyses revealed that 7PET strains from earlier outbreaks (2010 and 2012) were progenitors of the current 7PET-HCT isolates. Isolates from the 2016 outbreaks carried qnrVC and floR genes and showed reduced susceptibility to tetracycline, ciprofloxacin and azithromycin, drugs recommended by the World Health Organization (WHO) for the treatment of cholera. Remarkably, protein profiling and mass spectrometry revealed disappearance of the outer membrane protein U (OmpU) porin in 7PET-HCT isolates from the second outbreak in 2016. Downregulation of ompU gene expression was also confirmed at the transcriptional level. Strains with downregulated OmpU showed reduced minimum inhibitory concentrations (MICs) for polymyxin B, which is a pore-forming antimicrobial agent. A multipronged approach is of utmost importance to prevent further spread of circulating 7PET-HCT strains. There is a pressing need for the formulation and implementation of international policies to closely monitor the effective use of antibiotics in order to prevent the further rise and spread of antimicrobial resistance.

Acquired qnrVC1 and blaNDM-1 resistance markers in an international high-risk Pseudomonas aeruginosa ST773 clone.

A multidrug-resistant Pseudomonas aeruginosa PS1 isolated from urine clinical sample was investigated in this study. The strain exhibited resistance to piperacillin/tazobactam, ciprofloxacin, imipenem, ceftazidime but it was susceptible to colistin. Analysis of whole-genome sequencing data by ResFinder detected various resistance determinants including qnrVC1 and blaNDM-1. The multiresistant P. aeruginosa isolate belonged to ST773 high-risk clone. The qnrVC1 and blaNDM-1 determinants were incorporated into different integrons. Expression of blaNDM-1 was fourfold and qnrVC1 was twofold increased, compared to that of rpsL housekeeping gene. Mutations in gyrA Thr83Leu and parC Ser87Leu were detected and additionally qnrVC1 expression indicates protective effect of QnrVC1 pentapeptid protein on gyrase and topoisomerase. High-risk P. aeruginosa clones integrate various carbapenemase and other resistance determinants into their genomes that facilitates further dissemination of multiresistance among clinical isolates. We report blaNDM-1 and qnrVC1 genes in P. aeruginosa ST773 international high-risk clone.

Complete Sequence of a Novel Multidrug-Resistant Pseudomonas putida Strain Carrying Two Copies of qnrVC6.

This study aimed at identification and characterization of a novel multidrug-resistant Pseudomonas putida strain Guangzhou-Ppu420 carrying two copies of qnrVC6 isolated from a hospital in Guangzhou, China, in 2012. Antimicrobial susceptibility was tested by Vitek2™ Automated Susceptibility System and Etest™ strips, and whole-genome sequencing facilitated analysis of its multidrug resistance. The genome has a length of 6,031,212 bp and an average G + C content of 62.01%. A total of 5,421 open reading frames were identified, including eight 5S rRNA, seven 16S rRNA, and seven 23S rRNA, and 76 tRNA genes. Importantly, two copies of qnrVC6 gene with three ISCR1 around, a blaVIM-2 carrying integron In528, a novel gcu173 carrying integron In1348, and six antibiotic resistance genes were identified. This is the first identification of two copies of the qnrVC6 gene in a single P. putida isolate and a class 1 integron In1348.

Molecular Characterization of qnrVC Genes and Their Novel Alleles in Vibrio spp. Isolated from Food Products in China.

This study reports the prevalences of qnrVC genes in 74 ciprofloxacin-resistant Vibrio sp. isolates. Two novel functional qnrVC alleles, qnrVC8 and qnrVC9, sharing 98% and 99% nucleotide similarity with qnrVC6 and qnrVC7, respectively, were identified. Our findings suggested that carriage of qnrVC alleles, together with target mutations in gyrA and parC genes, may contribute to the development of fluoroquinolone resistance in Vibrio species, posing a serious threat to public health.

Detection of qnrVC6, within a new genetic context, in an NDM-1-producing Citrobacter freundii clinical isolate from Uruguay.

OBJECTIVES: The objective of this study was to characterise the mechanisms underlying quinolone and oxyimino-cephalosporin resistance in a Citrobacter freundii clinical isolate obtained from the ICU in a university hospital in Uruguay. METHODS: Citrobacter freundii strain CF638 was isolated from a urine culture. Identification was performed using a VITEK®2 system, and antimicrobial susceptibility was established by MIC determination and disk diffusion assay. Resistance genes and mobile genetic elements were identified by PCR and sequencing. Plasmid transfer was assessed by conjugation and the plasmid size was estimated by S1-PFGE. Plasmid incompatibility (Inc) group and toxin-antitoxin systems were sought by PCR. RESULTS: Strain CF638 showed a multidrug-resistant profile, including resistance to carbapenems and quinolones. Transconjugant TcCF638, harbouring an ca. 200-kb IncA/C plasmid, also showed resistance to all ß-lactams (except aztreonam) and diminished susceptibility to ciprofloxacin. PCR was positive for blaNDM-1 and qnrVC in CF638 and TcCF638. Two different class 1 integrons were detected (In127 and In907). In127 featured the genetic array aadA2-ltr2. Conversely, complex In907 featured two variable regions (VRs); VR-1 consisted of aadB-blaOXA-10-aadA1cc, whereas VR-2 featured a qnrVC6 gene 108bp downstream from ISCR1 and 45bp upstream from qacEΔ1. Expression of qnrVC6 was due to a putative promoter region, detected using the Neural Network Promoter Prediction program. CONCLUSION: To the best of our knowledge, this constitutes the first report of qnrVC within a complex class 1 integron, as well as the first report of the occurrence of such a gene in an NDM-1-producing enterobacterial clinical isolate.

Complete sequence of pBM413, a novel multidrug resistance megaplasmid carrying qnrVC6 and blaIMP-45 from pseudomonas aeruginosa.

This study aimed to characterise a novel multidrug resistance megaplasmid carrying qnrVC6 and blaIMP-45 from Pseudomonas aeruginosa strain Guangzhou-Pae617 isolated from a patient hospitalised in Guangzhou, China, in 2012. The plasmid pBM413 has a length of 423 017 bp and an average G + C content of 56.41%. A qnrVC6 gene flanked by two copies of insertion sequence (IS) elements ISCR1, a multiresistance class 1 integron In786 containing aacA4-blaIMP-45-blaOXA-1-catB3 cassettes, an armA gene, and an aphA7 gene flanked by two copies of IS26 were identified. To our knowledge, this is the first identification of a qnrVC6 gene in P. aeruginosa.

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

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

Co-resistance to different classes of antibiotics among ESBL-producers from aquatic systems.

In this study we investigated the co-occurrence of resistance to non-beta-lactams among cefotaxime-resistant extended-spectrum beta-lactamase (ESBL) producers (ESBL(+)) versus non-ESBL producers (ESBL(-)), from aquatic environments. Higher prevalence of resistance to tetracycline, fluoroquinolones and aminoglycosides were observed in ESBL(+). Among ESBL(+) resistant to tetracycline (n = 18), tet(A) was detected in 88.9% and tet(B) in 16.7%. Among fluoroquinolone-resistant-ESBL(+) (n = 15), aacA4-cr and qnrVC4 were identified in 26.6% and 40% strains, respectively. The qnrVC4 gene was detected for the first time in Pseudomonas sp. and Escherichia coli. Class 1 integrase genes were detected in 56.41% of ESBL(+) and in 27.67% ESBL(-). Gene cassette arrays identified conferred resistance to aminoglycosides (aadA-type genes and aacA4), trimethoprim (dfrA17), chloramphenicol (catB8), fluoroquinolones (qnrVC4) and beta-lactams (blaOXA-10). Conjugation experiments were performed with CTX-M-producers. Transconjugants showed multiresistance to 3 or more classes of antibiotics, and conjugative plasmids were assigned to IncF, IncK and IncI1 replicons. Results obtained showed that co-selection of resistance to aminoglycosides, quinolones and tetracyclines is prevalent among ESBL-producers and that these features are successfully mobilized by IncF, IncK and IncI1 conjugative plasmids. This study reinforces the importance of natural aquatic systems as reservoir of mobile genetic platforms carrying multiple resistance determinants. Moreover, to the best of our knowledge, this constitutes the first observation of IncK::CTX-M-3 in Aeromonas hydrophila and the first report of IncK plasmids in Portugal.

Molecular characterisation of a multidrug resistance conjugative plasmid from Vibrio parahaemolyticus.

Vibrio parahaemolyticus is a major causative agent of gastroenteritis and is the leading cause of food-borne illness in Hong Kong. Recent studies of resistance to extended-spectrum ß-lactams and fluoroquinolones in V. parahaemolyticus have caused huge concern. This work reports the characterisation of a multidrug resistance conjugative plasmid in V. parahaemolyticus isolated from shrimp samples from Hong Kong. The plasmid is ca. 200 kb and carries multidrug resistance genes, including a novel plasmid-mediated quinolone resistance gene qnrVC6 surrounded by several known and novel insertion sequence (IS) elements, an extended-spectrum ß-lactamase gene bla(PER-1) mediated by ISCR1, and a ca. 3-kb four-gene cassette (aacA3, catB2, dfrA1 and aadA1) class 1 integron. Transmission of this multidrug resistance conjugative plasmid among Vibrio spp. would compromise the effectiveness of Vibrio infection control and pose a huge threat to public health.