Plasmid 66 (2011) 79–84
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Complete nucleotide sequence of a quinolone resistance gene (qnrS2) carrying plasmid of Aeromonas hydrophila isolated from fish Tanmay Majumdar a, Bhabatosh Das b,1, Rupak K. Bhadra b, Bomba Dam c, Shibnath Mazumder a,⇑,2 a
Immunobiology Laboratory, School of Life Sciences, Visva-Bharati University, Santiniketan 731 235, India Infectious Diseases and Immunology Division, Indian Institute of Chemical Biology (CSIR), 4, Raja S.C. Mullick Road, Kolkata 700 032, India c Microbiology Laboratory, School of Life Sciences, Visva-Bharati University, Santiniketan 731 235, India b
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Article history: Received 8 April 2010 Accepted 27 May 2011 Available online 12 June 2011 Communicated by Dr. W. Klimke Keywords: Aeromonas hydrophila Plasmid Quinolone qnrS Fish
a b s t r a c t Aeromonas hydrophila strain AO1 isolated from an infected fish was found to be resistant to several quinolones. A plasmid isolated from the strain AO1, termed pBRST7.6, was cloned and sequenced and shown to be 7621 bp in length with a GC content of 60%. Further analysis confirmed that it contained a gene with 100% identity to qnrS2 genes described in plasmids associated with other Aeromonas species, the product of which usually confers increased resistance to quinolones. The plasmid backbone contained a replication initiation module (repA repC) belonging to the IncQ-family and two genes (mobC and mobB), the products of which are putatively involved in plasmid mobilization. Putative iteron-based origin of replication and characteristic oriT like sequences were also present in the plasmid. The result suggests that Aeromonas spp. carrying plasmids with quinolone resistance genes are potential reservoirs of antimicrobial resistance determinants in the environment. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction Quinolones are among the most prescribed antibacterial drugs in human and veterinary medicine. However, indiscriminate use of these drugs has resulted in the development of resistance in many bacterial species. Quinolone resistance is mainly described as a chromosomal trait, being mediated either through mutations mainly in the gyrA gene (encoding DNA gyrase A subunit) or gyrA along with parC gene (encoding ParC subunit of the topoisomerase IV), the targets of quinolone; or by activating multidrug resistance efflux pumps and porins, which affect intracellular concentrations (Ruiz, 2003). Plasmid medi-
⇑ Corresponding author. Fax: +91 11 27667985. E-mail address:
[email protected] (S. Mazumder). Present address: Centre de Genetique Moleculaire, Batiment 26, CNRS UPR2167, Avenue de la Terrasse 91198, Gif Sur Yvette, France. 2 Present address: Immunobiology Laboratory, Department of Zoology, University of Delhi, Delhi 110 007, India. 1
0147-619X/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.plasmid.2011.05.001
ated quinolone resistance (PMQR) was first reported from a clinical isolate of Klebsiella pneumoniae and since then several quinolone resistant transferable plasmids has been isolated worldwide (Li, 2005; Robicsek et al., 2006). The PMQR determinants are broadly classified as: the Qnr-type pentapeptide proteins (QnrA, QnrB, QnrC, and QnrS) that protect DNA gyrase from binding to quinolones; the AAC (60 )-Ib-cr aminoglycoside acetyltransferases that possess two specific amino acid substitutions facilitating acetylation of ciprofloxacin and norfloxacin; and the QepA protein serving as an efflux pump to extrude several quinolones (Picao et al., 2008). Aeromonas hydrophila is a Gram-negative facultative bacterium. A. hydrophila and its close relative are ubiquitous, mostly harmless, members of the fresh water microbial communities. However, A. hydrophila is also associated with sporadic diarrhea and opportunistic wound infections in humans. It is also the major etiological agent of ulcerative disease syndrome (UDS) in fish (Pal and Pradhan, 1990). Plasmid mediated quinolone resistance
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in A. hydrophila has not been reported though reports of such resistance are available in several other members of the species including A. allosaccharophila (Picao et al., 2008), A. punctata subsp. punctata (Cattoir et al., 2008), A. media (Cattoir et al., 2008), A. veronii (Sanchez-Cespedes et al., 2008), and A. caviae (Arias et al., 2010), thereby raising concerns on the continued usage of quinolones in aquaculture and human infections. Here we report the complete nucleotide sequence of a quinilone resistance gene carrying plasmid, termed pBRST7.6, which was isolated from an A. hydrophila strain recovered from an infected fish and discuss the data with respect to current spread of quinolone resistance by mobile genetic elements. 2. Materials and methods 2.1. Bacterial strains, plasmid isolation, curing and transformation studies The wild-type A. hydrophila strain AO1 (Wt-AO1) used in this study was isolated from UDS-affected Channa punctatus. The processes for isolation, identification and maintenance of the isolate were as described earlier (Majumdar et al., 2009). A. hydrophila Strain 646 was obtained from the Microbial Type Culture Collections (MTCC), India. The AO1 isolates were grown to mid-log phase in brain–heart infusion broth (BHI, Hi-Media, India) under shaking condition at 37 °C, harvested and the plasmid DNA (pDNA) was extracted using a commercial midi plasmid extraction kit (Catologue No. 12143, Qiagen, Germany) following manufacturer’s instructions. This plasmid was designated pBRST7.6. For plasmid curing, an aliquot of actively growing Wt-AO1 at 37 °C was transferred to fresh BHI maintained at 42 °C and left overnight without shaking. The process was repeated twice; the bacterial culture was diluted and plated on nutrient agar without any antimicrobials. The single colonies obtained on heat treatment were checked individually for the presence of plasmid and screened for susceptibility to nalidixic acid (NA, 20 lg/ml) by replica plating onto nutrient agar plates. The plasmidcured isolates, designated C-AO1, were made competent by the calcium chloride method, followed by transformation with the plasmid DNA pBRST7.6, as described earlier in Majumdar et al. (2009). For selection of transformants, termed T-AO1, nutrient agar plates containing NA (75 lg/ ml) was used. Plasmid DNA was isolated from T-AO1 cells and isolation of plasmid was confirmed by gel electrophoresis. The identity of C-AO1 and T-AO1 isolates were confirmed by routine biochemical and serological testing methods (Majumdar et al., 2009). The Strain 646 was also transformed with pBRST7.6 and the identity of the transformed isolates was determined as described above. 2.2. Antimicrobial profile and minimum inhibitory concentration (MIC) testing
with 0.1 ml of an 18 h old culture of test bacterium in glucose supplemented (1%) nutrient broth. Antimicrobialimpregnated discs were placed on the solid medium and the plates were incubated at 30 °C for 24 h. When the bacteria gave a zone with a diameter less than 13 mm in the presence of an antimicrobial it was interpreted as resistant (R), when the zone had a diameter of 15–16 mm, the bacteria were considered to have intermediate sensitivity (I) and a clear zone with diameter of 17 mm or more indicated a high degree of sensitivity towards that antimicrobial (S) (Saha and Pal, 2002; Majumdar et al., 2009). The minimal inhibitory concentration (MIC) for each quinolone or fluroquinolone was determined in 96-well microtiter plates by the twofold standard broth micro-dilution method in MH broth (Hi-Media) using an inoculum of 1 104– 1 105 CFU per ml in all experiments keeping all other conditions unchanged. 2.3. Sequencing strategy The pBRST7.6 pDNA was initially separately digested with 15 different restriction endonucleases [New England Biolabs (NEB), USA], to identify suitable restriction fragments that could be generated for cloning purpose. When pBRST7.6 was digested with the enzyme BglII followed by electrophoresis in a 1% agarose gel and staining with ethidium bromide (0.5 lg/ml), two restriction fragments approximately 4.0 and 3.5 kb with equal intensity could be observed (data not shown). Each of these fragments was gel eluted, followed by ligation into a similarly digested calf intestinal phosphatase (NEB) treated cloning vector pDrive (Ampr, Kanr; Qiagen, Germany). Each ligation mixture was transformed into E. coli DH5a cells following manufacturer’s instructions. Transformant containing desired recombinant plasmid was selected by blue/white screening on X-Gal/IPTG plates in the presence of ampicillin (100 lg/ml) and kanamycin (40 lg/ml). The desired recombinant plasmids were designated as pVB1 (insert size 4.0 kb) and pVB2 (insert size 3.5 kb). For initial sequence information, cycle sequencing reactions were carried out using the recombinant plasmid pVB1 or pVB2 and the vector-specific universal primers M13 forward (M13F) or M13 reverse (M13R). Nucleotide sequence was determined by cycle sequencing method using the BigDyeÒ Terminator V3.1 Cycle Sequencing Kit as recommended by the manufacturer [Applied Biosystems Inc. (ABI), USA]. Results were analyzed using the software DNA Sequencing Analysis V5.1 (ABI). The initial sequence information obtained from the pVB1/pVB2 recombinant clones was used to design sequence specific primers. Using these primers, the complete nucleotide sequence of the plasmid pBRST7.6 was determined by the ‘primer walking’ method. The complete nucleotide sequence of the plasmid pBRST7.6 has been deposited in the NCBI GeneBank database under the accession number EU925817. 2.4. Sequence analysis and homology predictions of pBRST7.6
The antimicrobial sensitivity test discs (Hi-Media) were applied on the bacterial culture plates and incubated at 30 °C for 24 h. Antimicrobial sensitivity was tested in Mueller–Hinton (MH) agar plates which were inoculated
Sequence analyses and translations were done with the online search engines at ExPASy proteomics server (http:// www.expasy.org), JustBio (http://www.justbio.com/) and
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the Sequence Manipulation Suite: version 2 http:// www.bioinformatics.org/sms2/index.html). Annotation was performed using the GenBank CDS translations/PDB/ SwissProt/PIR/PRF protein databases and the DDBJ/EMBL/ GenBank DNA databases with the BLAST program (BLASTN, BLASTP, and BLASTX) (http://www.ncbi.nlm.nih.gov/ BLAST/) (Altschul et al., 1997). Homologues and protein coding sequences (CDSs) were identified using BLASTP analysis, with cut off values of
