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In recent years, trade liberalisation has led to the spread of antibiotic-resistant bacteria (ARB) in food products. Because ARB has reportedly been found in imported foods, the spread of plasmid-mediated ARB through food products is a concern. Here, we report the complete genome sequences of ESBL-producing Vibrio vulnificus and V. alginolyticus strains harbouring a plasmid isolated from imported seafood. First, V. vulnificus and V. alginolyticus were isolated from purchased frozen and thawed Litopenaeus vannamei shrimp, and genome extraction and sequencing were performed. Hybrid genome assemblies were performed using Unicycler and annotated using DFAST. Then genome analysis was performed using BRIG. Plasmid comparisons showed that the plasmids carried by both Vibrios are remarkably similar and encode the same antibiotic-resistance genes. The 270–310 kb region specific to both Vibrios were isolated in this study and encodes the antibiotic-resistance genes blaCTX-M and qnr. Furthermore, the mobile genetic factors ISEc9, ISVch4, and ISVpa4 are located upstream and downstream of these genes. This is the first report of ESBL-producing V. vulnificus and V. alginolyticus harbouring a common plasmid encoding ISEc9 upstream of blaCTX-M-55 and qnrS2 isolated from imported seafood. [ABSTRACT FROM AUTHOR]

Abstract Background Quinolones are commonly used for treatment of infections by bacteria of the Enterobacteriaceae family. However, the rising resistance to quinolones worldwide poses a major clinical and public health risk. This study aimed to characterise a novel multiple resistance plasmid carrying three plasmid-mediated quinolone resistance genes in Escherichia coli clinical stain RJ749. Methods MICs of ceftriaxone, cefepime, ceftazidime, ciprofloxacin, and levofloxacin for RJ749 and transconjugant c749 were determined by the Etest method. Conjugation was performed using sodium azide-resistant E. coli J53 strain as a recipient. The quinolone resistance-determining regions of gyrA, gyrB, parC, and parE were PCR-amplified. Results RJ749 was highly resistant to quinolones, while c749 showed low-level resistance. S1-nuclease pulsed-field gel electrophoresis revealed that RJ749 and c749 both harboured a plasmid. PCR presented chromosomal mutation sites of the quinolone resistance-determining region, which mediated quinolone resistance. The c749 genome comprised a single plasmid, pRJ749, with a multiple resistance region, including three plasmid-mediated quinolone resistance (PMQR) genes (aac (6′)-Ib-cr, qnrS2, and oqxAB) and ten acquired resistance genes. One of the genes, qnrS2, was shown for the first time to be flanked by two IS26s. Three IS26-mediated circular molecules carrying the PMQR genes were detected. Conclusions We revealed the coexistence of three PMQR genes on a multiple resistance plasmid and a new surrounding genetic structure of qnrS2 flanked by IS26 elements. IS26 plays an important role in horizontal spread of quinolone resistance.

Background: Quinolones are commonly used for treatment of infections by bacteria of the Enterobacteriaceae family. However, the rising resistance to quinolones worldwide poses a major clinical and public health risk. This study aimed to characterise a novel multiple resistance plasmid carrying three plasmid-mediated quinolone resistance genes in Escherichia coli clinical stain RJ749. Methods: MICs of ceftriaxone, cefepime, ceftazidime, ciprofloxacin, and levofloxacin for RJ749 and transconjugant c749 were determined by the Etest method. Conjugation was performed using sodium azide-resistant E. coli J53 strain as a recipient. The quinolone resistance-determining regions of gyrA, gyrB, parC, and parE were PCR-amplified. Results: RJ749 was highly resistant to quinolones, while c749 showed low-level resistance. S1-nuclease pulsed-field gel electrophoresis revealed that RJ749 and c749 both harboured a plasmid. PCR presented chromosomal mutation sites of the quinolone resistance-determining region, which mediated quinolone resistance. The c749 genome comprised a single plasmid, pRJ749, with a multiple resistance region, including three plasmid-mediated quinolone resistance (PMQR) genes (aac (6′)-Ib-cr, qnrS2, and oqxAB) and ten acquired resistance genes. One of the genes, qnrS2, was shown for the first time to be flanked by two IS26s. Three IS26-mediated circular molecules carrying the PMQR genes were detected. Conclusions: We revealed the coexistence of three PMQR genes on a multiple resistance plasmid and a new surrounding genetic structure of qnrS2 flanked by IS26 elements. IS26 plays an important role in horizontal spread of quinolone resistance. [ABSTRACT FROM AUTHOR]

The dissemination of Klebsiella pneumoniae carbapenemases (KPCs) among Gram-negative bacteria is an important threat to global health. However, KPC-producing bacteria from environmental samples are rarely reported. This study aimed to elucidate the underlying resistance mechanisms of three carbapenem-resistant Aeromonas taiwanensis isolates recovered from river sediment samples. Pulsed-field gel electrophoresis (PFGE) and whole genome sequencing (WGS) analysis indicated a close evolutionary relationship among A. taiwanensis isolates. S1-PFGE, Southern blot and conjugation assays confirmed the presence of blaKPC–2 and qnrS2 genes on a non-conjugative plasmid in these isolates. Plasmid analysis further showed that pKPC-1713 is an IncP-6 plasmid with a length of 53,205 bp, which can be transformed into DH5α strain and mediated carbapenems and quinolones resistance. The plasmid backbone of p1713-KPC demonstrated 99% sequence identity to that of IncP-6-type plasmid pKPC-cd17 from Aeromonas spp. and IncP-6-type plasmid: 1 from Citrobacter freundii at 74% coverage. A 14,808 bp insertion sequence was observed between merT gene and hypothetical protein in p1713-KPC, which include the quinolone resistance qnrS2 gene. Emergence of plasmid-borned blaKPC and qnrS2 genes from A. taiwanensis isolates highlights their possible dissemination into the environment. Therefore, potential detection of such plasmids from clinical isolates should be closely monitored.

The dissemination of Klebsiella pneumoniae carbapenemases (KPCs) among Gram-negative bacteria is an important threat to global health. However, KPC-producing bacteria from environmental samples are rarely reported. This study aimed to elucidate the underlying resistance mechanisms of three carbapenem-resistant Aeromonas taiwanensis isolates recovered from river sediment samples. Pulsed-field gel electrophoresis (PFGE) and whole genome sequencing (WGS) analysis indicated a close evolutionary relationship among A. taiwanensis isolates. S1-PFGE, Southern blot and conjugation assays confirmed the presence of bla KPC–2 and qnrS2 genes on a non-conjugative plasmid in these isolates. Plasmid analysis further showed that pKPC-1713 is an IncP-6 plasmid with a length of 53,205 bp, which can be transformed into DH5α strain and mediated carbapenems and quinolones resistance. The plasmid backbone of p1713-KPC demonstrated 99% sequence identity to that of IncP-6-type plasmid pKPC-cd17 from Aeromonas spp. and IncP-6-type plasmid: 1 from Citrobacter freundii at 74% coverage. A 14,808 bp insertion sequence was observed between merT gene and hypothetical protein in p1713-KPC, which include the quinolone resistance qnrS2 gene. Emergence of plasmid-borned bla KPC and qnrS2 genes from A. taiwanensis isolates highlights their possible dissemination into the environment. Therefore, potential detection of such plasmids from clinical isolates should be closely monitored. [ABSTRACT FROM AUTHOR]

ABSTRACT A highly fluoroquinolone-resistant isolate of Aeromonas species was isolated from a wastewater treatment plant and found to possess multiple resistance mechanisms, including mutations in gyrA and parC, efflux pumps, and plasmid-mediated quinolone resistance (PMQR) genes. Complete sequencing of the IncU-type plasmid, pAC3, present in the strain revealed a circular plasmid DNA 15,872 bp long containing two PMQR genes [qnrS2 and aac(6′)-Ib-cr]. A mobile insertion cassette element containing the qnrS2 gene and a typical miniature inverted-repeat transposable element (MITE) structure was identified in the plasmid. The present study revealed that this MITE sequence appears in other Aeromonas species plasmids and chromosomes. Plasmid pAC3 was introduced into Escherichia coli, and its PMQR genes were expressed, resulting in the acquisition of resistance. Proteome analysis of the recipient E. coli strain harboring the plasmid revealed that aac(6′)-Ib-cr expression was constitutive and that qnrS2 expression was dependent upon fluoroquinolone stress through regulation by regulator of sigma D (Rsd). To the best of our knowledge, this is the first report to characterize a novel MITE sequence upstream of the PMQR gene within a mobile insertion cassette, as well as the regulation of qnrS2 expression. Our results suggest that this mobile element may play an important role in qnrS2 dissemination. IMPORTANCE In the present study, plasmid pAC3 isolated from a highly fluoroquinolone-resistant isolate of Aeromonas species was sequenced and found to contain two fluoroquinolone resistance genes, aac(6′)-Ib-cr and qnrS2. Comparative analyses of plasmid pAC3 and other Aeromonas sp. IncU-type plasmids revealed a mobile insertion cassette element with a unique structure containing a qnrS2 gene and a typical miniature inverted-repeat transposable element (MITE) structure. This study also revealed that this MITE sequence appears in other Aeromonas species plasmids and chromosomes. Our results also demonstrate that the fluoroquinolone-dependent expression of qnrS2 is associated with rsd in E. coli DH5α harboring plasmid pAC3. Our findings suggest that the mobile element may play an important role in qnrS2 dissemination and that Aeromonas species constitute an important reservoir of fluoroquinolone resistance determinants in the environment.

Abstract: Antimicrobial resistance patterns in a collection of 33 motile Aeromonas species were described in this study. Quinolone has been frequently employed for treatment of Aeromonas-related diseases, and prolonged use of antimicrobial compounds has led to development of resistant strains. In a sample of diseased fish and environmental water, we evaluated nalidixic acid (n = 19) and ciprofloxacin (n = 4) resistance via minimum inhibitory concentration (MIC) assays and the genetic basis was also investigated. Among the isolated Aeromonas spp., 17 strains encoded for chromosomal mutations of quinolone resistance-determining regions (QRDRs) in gyrA, 11 strains encoded for mutations of QRDRs in parC, 1 strain harbored plasmid-mediated quinolone resistance (PMQR) qnrS1-like gene and 4 strains harbored the PMQR qnrS2 gene. In particular, the new variant (qnrS1-like) differed from qnrS1 by 6 amino acid substitutions at positions 5 (Asn5→Arg5), 120 (Ser120→Thr120), 148 (Asn148→His148), 206 (Leu206→Glu206), 207 (Ile207→ Leu207), and 216 (Tyr216→Phe216), and the gene was designated qnrS5. These resistant strains may function as reservoirs of quinolone resistance. [Copyright &y& Elsevier]

Clin Microbiol Infect 2010; 16: 1005–1007 A qnrS2 gene was identified in an Aeromonas caviae isolate (MICs of ciprofloxacin, norfloxacin and ofloxacin >32 mg/L) from a stool sample collected from a patient with gastroenteritis. The analysis of the gyrA and parC genes revealed amino acid substitutions Ser83-Ile and Ser80-Thr, respectively. In addition, five out of 41 nalidixic acid-resistant Aeromonas isolates studied (26 identified as Aeromonas veronii bv sobria and 15 identified as A. caviae) showed ciprofloxacin resistance. The identification of plasmid-mediated qnr genes outside of the Enterobacteriaceae underlines a possible diffusion of these resistance determinants among Gram-negative rods. This emphasizes the importance of monitoring the emergence of these determinants as well as their dissemination among the Aeromonadaceae. [ABSTRACT FROM AUTHOR]

The dissemination of Klebsiella pneumoniae carbapenemases (KPCs) among Gram-negative bacteria is an important threat to global health. However, KPC-producing bacteria from environmental samples are rarely reported. This study aimed to elucidate the underlying resistance mechanisms of three carbapenem-resistant Aeromonas taiwanensis isolates recovered from river sediment samples. Pulsed-field gel electrophoresis (PFGE) and whole genome sequencing (WGS) analysis indicated a close evolutionary relationship among A. taiwanensis isolates. S1-PFGE, Southern blot and conjugation assays confirmed the presence of bla KPC- 2 and qnrS2 genes on a non-conjugative plasmid in these isolates. Plasmid analysis further showed that pKPC-1713 is an IncP-6 plasmid with a length of 53,205 bp, which can be transformed into DH5α strain and mediated carbapenems and quinolones resistance. The plasmid backbone of p1713-KPC demonstrated 99% sequence identity to that of IncP-6-type plasmid pKPC-cd17 from Aeromonas spp. and IncP-6-type plasmid: 1 from Citrobacter freundii at 74% coverage. A 14,808 bp insertion sequence was observed between merT gene and hypothetical protein in p1713-KPC, which include the quinolone resistance qnrS2 gene. Emergence of plasmid-borned bla KPC and qnrS2 genes from A. taiwanensis isolates highlights their possible dissemination into the environment. Therefore, potential detection of such plasmids from clinical isolates should be closely monitored., (Copyright © 2019 Hu, Yu, Shang, Xu, Guo, Liang, Kang, Song, Sun, Yue, Mao and Zheng.)

A highly fluoroquinolone-resistant isolate of Aeromonas species was isolated from a wastewater treatment plant and found to possess multiple resistance mechanisms, including mutations in gyrA and parC , efflux pumps, and plasmid-mediated quinolone resistance (PMQR) genes. Complete sequencing of the IncU-type plasmid, pAC3, present in the strain revealed a circular plasmid DNA 15,872 bp long containing two PMQR genes [ qnrS2 and aac ( 6' ) -Ib-cr ]. A mobile insertion cassette element containing the qnrS2 gene and a typical miniature inverted-repeat transposable element (MITE) structure was identified in the plasmid. The present study revealed that this MITE sequence appears in other Aeromonas species plasmids and chromosomes. Plasmid pAC3 was introduced into Escherichia coli , and its PMQR genes were expressed, resulting in the acquisition of resistance. Proteome analysis of the recipient E. coli strain harboring the plasmid revealed that aac ( 6' ) -Ib-cr expression was constitutive and that qnrS2 expression was dependent upon fluoroquinolone stress through regulation by regulator of sigma D (Rsd). To the best of our knowledge, this is the first report to characterize a novel MITE sequence upstream of the PMQR gene within a mobile insertion cassette, as well as the regulation of qnrS2 expression. Our results suggest that this mobile element may play an important role in qnrS2 dissemination. IMPORTANCE In the present study, plasmid pAC3 isolated from a highly fluoroquinolone-resistant isolate of Aeromonas species was sequenced and found to contain two fluoroquinolone resistance genes, aac ( 6' )- Ib-cr and qnrS2 . Comparative analyses of plasmid pAC3 and other Aeromonas sp. IncU-type plasmids revealed a mobile insertion cassette element with a unique structure containing a qnrS2 gene and a typical miniature inverted-repeat transposable element (MITE) structure. This study also revealed that this MITE sequence appears in other Aeromonas species plasmids and chromosomes. Our results also demonstrate that the fluoroquinolone-dependent expression of qnrS2 is associated with rsd in E. coli DH5α harboring plasmid pAC3. Our findings suggest that the mobile element may play an important role in qnrS2 dissemination and that Aeromonas species constitute an important reservoir of fluoroquinolone resistance determinants in the environment.