Resistance Characterization,Virulence Factors,and ERIC-PCR Fingerprinting of Aeromonas veronii Strains Isolated from Diseased Trionyx sinensis

Abstract

Aeromonas veronii (AV) is an important pathogen causing severe diseases in aquaculture. Fifteen A. veronii strains isolated from diseased Trionyx sinensis from four aquafarms was characterized by enterobacterial repetitive intergenic consensus–polymerase chain reaction (ERIC-PCR), antibiotic susceptibility testing, and identification of virulence factors. A. veronii strains were grouped into nine ERIC types with discriminatory ability (D) of 91.43 based on 90% similarity. The virulence genes were detected for 73% (aer), 80% (ast), 13% (alt), and 40% (act) of the strains, respectively, and five antibiotic resistance patterns with D of 0.829 were observed by antibiotic susceptibility testing. Furthermore, some AV isolates (AV1, AV2, AV3, and AV4) show the same characterization (the same ERIC types, presence of virulence genes in genomes, and antibiotics resistance).

Introduction

I n aquaculture, Aeromonas is the major factor causing hemorrhagic diseases (Nawaz et al., 2006; Jiang et al., 2010). The U.S. Environmental Protection Agency (USEPA) has placed Aeromonas spp. on the contaminant candidate list and has actively monitored water supplies for aeromonads since 2002 (Nawaz et al., 2006). Virulence genes of Aeromonas spp. have been detected and studied (Sen and Rodgers, 2004; Aguilera-Arreola et al., 2005; Nagar et al., 2011). Furthermore, virulence genes in A. veronii isolated from catfishes have also been detected (Nawaz et al., 2010, Sreedharan et al., 2011).

To our knowledge, little is known about the traits of A. veronii strains from diseased Trionyx sinensis, and no special attentions have focused on genetic characterization of A. veronii by enterobacterial repetitive intergenic consensus–polymerase chain reaction (ERIC-PCR) fingerprinting. In this study, A. veronii strains isolated from diseased Trionyx sinensis were characterized by identification of virulence genes, antibiotic susceptibility testing, and ERIC-PCR.

Materials and Methods

Identification of A. veronii and DNA extraction

Twenty-five Trionyx sinensis samples with typical symptoms, including caverned disease and red-neck disease, were collected from four aquafarms (Bengbu1, Bengbu2, Shouxian1, and Shouxian2), and A. veronii were isolated by Aeromonas chromogenic agar (Haibo, Qingdao) and full 16S rRNA gene sequencing. Genomic DNA was extracted by a universal extraction kit (Sangon, Shanghai) and was stored at −20°C for further use. The universal primers: 5’-AGGAGGTGATCCAACCGCA-3’ and 5’-AGAGTTTGATCATGGCTCAG-3’ were used to amplify the full 16S rRNA gene that was sequenced (Sangon, China). PCR procedure was performed as described by Nawaz et al. (2006).

ERIC-PCR of A. veronii isolates

For ERIC-PCR, the primers ERIC-1R (5’-ATGTAAGCTCCTGGGGATTCAC-3') and ERIC2 (5’-AAGTAAGTGACTGGGGTGAGCG-3’) (Versalovic et al., 1991) were used. PCR reaction was performed in 25 μL of solution containing 1 μM of primers for each, 2.5 μL of 10×buffers, 200 μm deoxynucleotide triphosphates (dNTPs), 2.5 mM MgCl₂, and 3.0 U of Taq DNA polymerase (Sangon, Shanghai). PCR conditions were as follows: one cycle at 95°C for 5 min, followed by 30 cycles of 1 min at 94°C, 45 s at 48°C, and 4 min at 72°C; and the last extension at 72°C for 8 min. ERIC-PCR fingerprinting was analyzed by Statistical Package for the Social Sciences (SPSS) 15.0 using average linkage and rescaled distance. Similarity of ERIC-PCR fingerprinting over 90% was considered as the same DNA patterns (Fig. 1).

FIG. 1.

Characterizations of Aeromonas veronii strains (enterobacterial repetitive intergenic consensus–polymerase chain reaction [ERIC-PCR] fingerprinting, antibiotic susceptibility testing, presence of virulence genes and sources). Ce, cephalothin; te, tetracycline; va, vancomycin; am, ampicillin; ni, nitrofurantoin; ch, chloramphenicol; st, streptomycin; no, norfloxacin; r, resistance; i, intermediate; s, sensitivity.

Antibiotics susceptibility testing of A. veronii isolates

The disk diffusion assay was performed according to the instructions of the Clinical and Laboratory Standards Institute (CLSI, 2011). Antibiotic discs used contained ampicillin (10 μg), kanamycin (30 μg), vancomycin (30 μg), norfloxacin (10 μg), tetracycline (30 μg) and chloromycetin (30 μg), cephalotin (30 μg), and gentamicin (10 μg). A. veronii was incubated on Luria-Bertani agar at 32°C for 12–16 h, then were suspended in 2 mL of Mueller-Hinton broth (Oxoid, Basingstoke Hampshire, UK) to obtain suspensions with an absorbance of 0.08–0.10 at 625 nm. The suspension was spread onto the Mueller-Hinton agar plates and plates were dried for 5–10 min. Then, four or five antibiotic disks were placed on each plate, which were incubated at 32°C for 12–16 h under aerobic conditions. Results were interpreted according to CLSI guidelines for Enterobacteriaceae or Enterococcus (CLSI, 2011). The D index was calculated as described by Hunter and Gaston (1988).

Detection of virulence genes from A. veronii

Virulence genes, including alt, ast, aer, and act, in A. veronii were detected as described by Sen and Rodgers (2004) and Jiang et al. (2010). The primers were as follows: ast (F: 5’-TCTCCATGCTTCCCTTCCACT-3’; R: 5’-GTGTAGGGATTGAAGAAGCCG-3’), act (F: 5’-AGAAGGTGACCACCAA GAACA-3’, R: 5’-AACTGACATCGGCCTTGAACTC-3’), aer (F: 5’-CGCCTTGTCCTTGTA-3’; R: AACCGAACTCTCCAT), alt (F: 5’-TGACCCAGTCCTGGCACGGC-3’; R: 5’-GGTGATCGATCACCACCAGC-3’). The total mixture of 25 μL consisted of 10×buffer 2.5 μL, 30 ng genomic DNA, 1 μM of primers for each, 200 μM dNTP, 2.5 mM MgCl₂, and 3.0 U Taq DNA polymerase. PCR was performed as follows: 5 min at 95°C, followed by 35 cycles at 95°C for 45 s, 52°C for 45 s, 72°C for 45 s, and a final extension at 72°C for 8 min. The PCR product was detected by electrophoresis on 1.0% agarose gel with ethidium bromide staining (0.006%, v/v).

Results and Discussion

The genetic and phenotypic characterization of A. veronii strains isolated from diseased animals could be helpful for the establishment of control measures of this disease (Sen and Rodgers, 2004). In this study, 15 A. veronii strains were isolated from 25 diseased Trionyx sinensis. Furthermore, 16S rRNA gene sequencing indicated strains have 98–99% similarity to A. veronii in Genbank in the National Center for Biotechnology Information by BLAST. All strains were grouped into nine ERIC types with D of 0.9143. A. veronii isolates (AV1, AV2, AV3, and AV4) from the Bengbu1 aquafarm showed the same ERIC types. Meanwhile, AV5 and AV12 from different aquafarms (Bengbu1 and Shouxian2) have closely ERIC types, but show different resistance patterns and virulence factors. Furthermore, AV10 and AV13 from Shouxian2 aquafarm showed identical ERIC fingerprinting, distinct resistance patterns, and virulence factors.

Five resistance patterns were found with D of 0.829, and all strains were resistant to ampicillin. In addition, 73.3% of A. veronii strains showed two or more antibiotic resistances. The distinct resistance characterization might due to environmental pressure or overuse of antibiotics in aquaculture. For example, Nawaz et al. (2006) tested the biochemical and molecular characterization of A. veronii isolates that were resistant to tetracycline; only four strains were intermediate resistant to tetracycline in this study.

Virulence genes were present in 73%, 80%, 13%, and 40% for aer, ast, alt, and act from 15 A. veronii isolates, respectively. We also found that 80% of A. veronii had at least two virulence genes in genomes. Interestingly, a good correlation between resistance patterns, ERIC-PCR fingerprinting, and the presence of virulence genes was observed in AV1, AV2, AV3, and AV4 from Bengbu1 aquafarm, which indicated that analyses of genetic and phenotypic characterizations are reliable for tracing the origins of A. veronii.

Footnotes

Acknowledgments

We acknowledge the support of Key Subject of Anhui Academy of Agricultural Sciences.

Disclosure Statement

No competing financial interests exist.

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