Molecular analysis of antimicrobial resistance in Yersinia ruckeri strains isolated from rainbow trout (Oncorhynchus mykiss) grown in commercial fish farms in Turkey

In this study, molecular characterization of antimicrobial resistance of 116 Yersinia ruckeri strains isolated from rainbow trout (Oncorhynchus mykiss) with enteric redmouth disease (ERM) in commercial fish farms in the Northern and Eastern regions of Turkey was performed. The putative Y. ruckeri strains isolated were confirmed by PCR assays specific to the 16S rRNA gene of bacterium Y. ruckeri. Eighty-six (74.1%) strains were identified to belong to serovar O1 by agglutination test in which type O1 antibody was used. No strains carried antimicrobial gene casse es inserted into integrons. Neither TEMnor SHV-type ß-lactamase gene was found in ampicillin-resistant strains (33/116). tetA and tetB genes were screened in 41/116 oxytetracycline-resistant strains by PCR, and it was found that 21 (51.2%) carried a tetA and/or tetB gene. We conclude that the antimicrobial resistant Y. ruckeri strains may act as a reservoir of antimicrobial resistance genes within fish farm environments. Introduction Yersinia ruckeri is the causative agent of enteric redmouth disease (ERM), a serious infectious disease of the farm fish, which leads to significant economical losses in salmonid commercial aquaculture worldwide. (Furones et al., 1993). Infections caused by bacteria resistant to multiple antimicrobial agents have been a major concern and constraint especially in hospitals during the last two decades (Hunter et al., 2010). A ention has been paid to the use of antimicrobial agents in aquaculture and resistance development in * Corresponding author’s email: [email protected] fish pathogenic bacteria and the indigenous bacteria exposed to these agents (Alderman and Smith, 2001). Resistance genes are o en located on transferable genetic elements such as plasmids or transposons which enable the acquired resistance to disseminate (Aoki, 1997). It has been reported that class 1 and class 2 integrons comprise a site-specific recombination systems capable of integrating and expressing antimicrobial resistance genes contained in casse e-like structures (Hall and Collis, 1998), which resulted in the Bull. Eur. Ass. Fish Pathol., 30(6) 2010, 212 spread of those genes among enterobacterial species (Fluit and Schmitz, 2004). Although phenotypic and genetic homogeneity of Y. ruckeri strains isolated from farmed rainbow trout has been investigated in Turkey (Ozer et al., 2008), li le information is available on the status and molecular mechanisms of resistance to antimicrobials (De Grandis and Stevenson, 1985; Schmidt et al., 2000) of this pathogen in spite of the economic significance of the disease. We have studied for the first time the molecular mechanisms of resistance to antimicrobials of Y. ruckeri strains isolated from rainbow trout with ERM in Turkey. Materials and methods Sample collection and bacterial identification Between the years 2001 and 2008, 1200 rainbow trout were sampled from 37 commercial fish farms in the cities (16 in Trabzon, 18 in Rize, 1 in Erzurum, 1 in Erzincan and 1 in Artvin) located in Northern and Eastern regions of Turkey. Live trout showing symptoms of ERM were transported to the laboratory. A er aseptically dissecting, diseased tissues from the organs (kidney, spleen, liver or brain) were homogenized and streaked on Trypticase Soy Agar (TSA) (Difco, England) plates with incubation at 20±2°C for 48 hours. Putative Yersinia-like bacterial colonies were subcultured onto Sho s-Waltman Agar (SWA) plates and incubated at 20±2°C for 48 hours to isolate purified strains (Waltman and Sho s, 1984). Bacteria were identified to the species level by API 20E strips (BioMerieux, France). All identified strains were subsequently confirmed by PCR using the Y. ruckeri-specific 16S rRNA primers YER3 and YER4 (Table 1). Serotyping Pure colonies derived from the strains were inoculated on TSA and incubated at 20±1°C for 48 hours. They were checked by a slide agglutination test using rabbit anti-Y. ruckeri Serotype O1 serum (kindly provided by Dr. H. Çagirgan from the Ege University, Fisheries Faculty, Department of Aquaculture, Urla-Izmir, Turkey). A dense suspension of Y. ruckeri colonies was made in 5 μls of isotonic saline on to separate glass slides. Then, 10 μl of O1 antiserum was added to one of the suspensions and observed for agglutination. A negative control slide was checked for the absence of auto-agglutination. Antimicrobial susceptibility testing The susceptibility tests were carried out by the standard disk diffusion method, and the results were interpreted (Table 2) as described in the Clinical Laboratory Standards Institute guidelines for the family Enterobacteriaceae and Gram negative bacteria in human and veterinary medicine, including those used in aquaculture (CLSI, 2003; CLSI, 2004). The following antibiotic disks (Oxoid, Basingstoke, UK) were used: oxytetracycline (30μg), oxolinic acid (2μg), sulfamethoxazole (25μg), ampicillin (10μg), florfenicol (30μg), streptomycin (10μg), trimethoprimsulfamethoxazole (25μg), enrofloxacin (5μg). Reference strain Escherichia coli ATCC 25922 was used as quality control in the antimicrobial susceptibility tests. DNA isolation and PCR for antimicrobial resistance genes To prepare DNA templates for PCR assays, the strains were inoculated into 3 ml Luria-Bertani broth (1% tryptone, 0.5% sodium chloride, Bull. Eur. Ass. Fish Pathol., 30(6) 2010, 213 Table 2. Antimicrobial susceptibility test breakpoints used in the study. Antimicrobial agent Disc content of antimicrobial (μg) Diameter of zone of inhibition (mm) Resistant Intermediate Susceptible Reference Oxytetracycline 30 ≤14 15-18 ≥ 19 CLSI, 2004 Oxolinic acid 2 ≤10 11-12 ≥13 CLSI, 2004 Sulfamethoxazole 25 ≤12 13-16 ≥17 CLSI, 2003 Ampicillin 10 ≤13 14-16 ≥17 CLSI, 2003 Florfenicol 30 ≤12 13-17 ≥18 CLSI, 2004 Streptomycin 10 ≤11 12-14 ≥15 CLSI, 2003 Enrofloxacin 5 ≤17 18-21 ≥22 CLSI, 2004 Trimethoprimsulfamethoxazole 1.25 μg/23.75 ≤10 11-15 ≥16 CLSI, 2003 Table 1. Oligonucleotide primers used in the PCR amplifications. Primer Target Nucleotide sequence Reference OT1 OT2 blaTEM (intragenic gene) 5’-TTGGGTGCACGAGTGGGTTA-3’ 5’-TAATTGTTGCCGGGAAGCTA-3’ Arlet and Philippon, (1991) OS1 OS2 blaSHV (intragenic gene) 5’-TCGGGCCGCGTAGGCATGAT-3’ 5’-AGCAGGGCGACAATCCCGCG-3’ Arlet and Philippon, (1991) tetA-1 tetA-2 tetA gene 5’-GTAATTCTGAGCACTGTCGC-3’ 5’-CTGCCTGGACAACATTGCTT-3’ Guardabassi et al., (2000) tetB-1 tetB-2 tetB gene 5’-CTCAGTATTCCAAGCCTTTG-3’ 5’-ACTCCCCTGAGCTTGAGGGG-3’ Guardabassi et al., (2000) 5’-CS 3’-CS Class 1 integron variable regions 5’-GGCATCCAAGCAGCAAG-3’ 5’-AAGCAGACTTGACCTGA-3’ Lévesque et al., (1995) hep51 hep74 Class 2 integron variable regions 5’-GATGCCATCGCAAGTACGAG-3’ 5’-CGGGATCCCGGACGGATGCACGATTTGTA-3’ White et al., (2001) YER3 YER4 Y. ruckeri 16S rRNA gene 5’-CGAGGAGGAAGGGTTAAGT-3’ 5’-AAGGCACCAAGGCATCTCT-3’ Gibello et al., (1999) Bull. Eur. Ass. Fish Pathol., 30(6) 2010, 214 0.5% yeast extract, pH 7.4) and incubated for 20 h at 20±2°C with shaking. Bacterial cells from 1.5 ml of the culture were harvested by micro-centrifugation. A er decanting the supernatant, the pellet was re-suspended in 500 μl of sterile deionized water. Bacterial cells were lysed by boiling for 10 min. A er the debris was removed by centrifugation, a 1-μl of supernatant was used as template for all PCRs. TEM and SHV type ß-lactamase genes were screened in ampicillin-resistant strains by using the intragenic oligonucleotide primers shown in Table 1. Reaction mixture and cycling parameters were as previously described (Arlet and Philippon, 1991). The presence of integrons were screened by PCR with specific primers amplifying the variable regions of class 1 and class 2 integrons. Reaction composition and cycling parameters used were as in the procedures described for class 1 integrons (Lévesque et al., 1995) and for class 2 integrons (White et al., 2001). As positive control of class 1 integron-specific PCRs, pCEm1 plasmid DNA (kindly provided with Andreas Schlueter, Fakultat fur Biologie, Lehrstuhl fur Genetik, Universitat Bielefeld, Bielefeld, Germany) containing dfrA17 aadA5 array inserted into class 1 integron was used (Tennstedt et al., 2003). As positive control of class 2 integron-specific PCRs, DNA from E. coli strain KD27 harboring dfrA1 sat1 aadA1 array inserted into a class 2 integron (GenBank accession number EU339237) was used. Tetracycline-resistant strains were screened for the tetracycline resistance determinants, tetA and tetB genes, using the primer pairs shown in Table 1. The reaction compositions and the cycling parameters were carried out according to the method as previously described (Guardabassi et al., 2000). The PCR products were then electrophoresed on 1% agarose gel containing 0.5 μg/ml ethidium bromide (Sigma, St. Louis, MO) and visualized with UV light. Results and discussion A total of 116 Y. ruckeri were isolated from the 1200 fish samples. Eighty-six (74.1%) of 116 strains were serotype O1. The remaining 30 strains were not screened for other serotypes. Of 116 strains 19 (16.3%) were resistant to three or more of eight antimicrobial drugs, and were classified as multi-drug resistant (data not shown). Intermediate-resistant strains were regarded as sensitive in the calculation of resistance levels. The highest incidence of resistance was to oxytetracycline (35.3%) followed by ampicillin (29%), oxolinic acid (11.1%), streptomycin (10.2%), sulfamethoxazole (9.4%), trimethoprimsulfamethoxazole (9.4%) and florfenicol (4.2%), and the lowest resistance was for the quinolone and enrofloxacin (2.3%). Trimethoprim-sulfamethoxazole has been used to control ERM outbreaks in the sampling fish farms. Although resistance frequency of trimethoprim-sulfamethoxazole is higher than florfenicol and enrofloxacin, trimethoprimsulfamethoxazole is commonly used to treat bacterial infections of fishes in the sampling region because it is cheaper than the others. This treatment protocol was really effective to control the ERM outbreaks according to the feedback of the fish farms in the region (data not shown). Y. ruckeri has been reported to be sensitive to ß-lactam antibiotics, including amoxicillin Bull. Eur. Ass. Fish Pathol., 30(6) 2010, 215 and certain cephalosporins, indicating the absence or low-level expression of ß-lactamase enzymes (Stock et al., 2002). Interestingly, we found that 29% of the strains were resistant to the ß-lactam antibiotic, ampicillin. The strains were screened for carriage of blaTEM or blaSHV genes (class A enzymes) by PCR, but no strains harbored these genes. However, Schiefer et al. (2005) have recently shown that Y. ruckeri expresses AmpC-type enzyme at a low level, which may explain the mechanism for resistance to ampicillin observed in our strains although the exact molecular mechanism could not be determined. The presence of class 1 and 2 integrons was studied, but no integron gene casse es were amplified by PCR applied to variable regions of class 1 and class 2 integrons. We do not know whether the integrons are completely absent or just empty because the presence of the integrase genes of class 1 and class 2 integrons (intI1 and intI2, respectively) were not investigated in this study. Although there are gene casse es encoding for resistance to sulfamethoxazole, trimethoprimsulfamethoxazole or streptomycin (which are known to be harbored by class 1 and class 2 integrons) (Lévesque et al., 1995), no integron structures were found in the current study. That’s why we concluded that resistance to the above antimicrobial agents were not integron-mediated traits. We also have not yet encountered any publications demonstrating gene casse es encoding for resistance to oxolinic acid, florfenicol and enrofloxacin inserted into class 1 or class 2 integron structures. Tetracyclines are widely used for therapy or prophylaxis of bacterial infections in both human and veterinary medicine, including aquaculture. A variety of tet genes conferring resistance to tetracyclines have been described in the family Enterobacteriaceae. The most frequently reported types of tet genes in Enterobacteriaceae belong to classes A to E (Chopra and Roberts, 2001). Guardabassi et al. (2000) have shown that tetAand tetB-mediated tetracycline resistances were widespread in clinical and aquatic Acinetobacter baumannii strains. Moreover, we previously reported that tetA and tetB genes were predominant in fecal coliforms isolated from public drinking waters in the Northern region of Turkey (Ozgumus et al., 2007). In addition, we also have detected tetracycline resistance mediated by tetB gene product in tetracycline-resistant coliform bacteria of anthropogenic origin in sea water collected in the region where the current study has been conducted (unpublished data). The tetB gene has been shown to be prevalent in infantile colonic flora in Korea, whereas the tetD gene has been shown to be the common tetracycline resistance determinant in bacteria from mariculture environments in China (Dang et al., 2007) and in other countries (Lanz et al., 2003; Bryan et al., 2004). A total of 44 oxytetracycline-resistant strains (Table 3) were analyzed for the presence of genetic determinants, tetA and tetB. More than half of the oxytetracycline-resistant strains (24 of 44 strains) were serotype O1 (Table 3). Resistance to oxytetracycline was mediated by tetA in 16 (36.3%) strains and by tetB in two (4.5%) strains. The presence of both tetA and tetB was detected in three (6.8%) Y. ruckeri strains. No tetA or tetB genes were detected in the remaining 23 (52.2%) Bull. Eur. Ass. Fish Pathol., 30(6) 2010, 216 Table 3. Epidemiological properties of oxytetracycline-resistant Yersinia ruckeri strains isolated from rainbow trout. Strain Place Source Date* Antibiotic resistance profile** Serotype O1*** tet PCR 1 Rize Kidney 14.06.07 OTC unclassified 37 Trabzon Kidney 05.05.07 OTC + tetA 49 Trabzon Kidney 29.03.04 OTC + tetA 53 Rize Kidney 18.07.03 OTC + tetA 59 Trabzon Kidney 01.05.03 OTC + unclassified 68 Trabzon Kidney 23.10.02 OTC unclassified 69 Trabzon Kidney 04.06.01 OTC + tetA 70 Trabzon Kidney 19.05.05 OTC, SUL, AMP, FFN, SXT tetA 80 Rize Kidney 19.06.07 OTC, OA, AMP + unclassified 83 Trabzon Spleen 08.05.05 OTC + tetA 84 Trabzon Kidney 08.03.05 OTC + tetA 85 Rize Kidney 12.07.07 OTC + tetA 86 Rize Spleen 12.06.07 OTC + tetA 98 Rize Kidney 19.07.05 OTC + unclassified 99 Rize Kidney 20.06.05 OTC unclassified 115 Erzincan Kidney 08.06.05 OTC, STR tetA, tetB 120 Trabzon Kidney 31.07.02 OTC, AMP, STR unclassified 121 Rize Kidney 21.07.07 OTC + unclassified 122 Trabzon Kidney 23.01.03 OTC, OA, AMP, STR tetA 139 Rize Kidney 26.03.04 OTC, OA, STR + unclassified 142 Rize Kidney 29.02.04 OTC, OA, SUL, STR, SXT unclassified 150 Trabzon Kidney 22.05.01 OTC, AMP, SUL, SXT + unclassified 190 Rize Kidney 29.02.04 OTC, OA unclassified 192 Rize Kidney 31.07.02 OTC, AMP unclassified 197 Rize Spleen 31.07.07 OTC, SUL, SXT tetA 198 Rize Liver 31.07.07 OTC, OA, STR unclassified 201 Rize Kidney 01.07.05 OTC, SUL, AMP, SXT unclassified 211 Erzurum Kidney 07.12.05 OTC, OA, AMP unclassified 216 Rize Kidney 02.03.07 OTC, SUL, AMP, FFN, SXT + unclassified 219 Rize Brain 29.02.04 OTC, OA, SUL, EN unclassified 221 Rize Kidney 02.03.07 OTC, SUL, SXT unclassified 222 Rize Kidney 21.05.05 OTC, AMP unclassified 225 Trabzon Kidney 05.07.06 OTC, OA + unclassified 226 Rize Kidney 15.06.06 OTC + tetB 227 Rize Kidney 01.07.06 OTC, AMP, SUL, SXT tetB 230 Trabzon Kidney 07.12.05 OTC, SUL, AMP, FFN, SXT tetA, tetB 238 Rize Kidney 07.12.05 OTC, OA tetA, tetB 240 Trabzon Kidney 17.10.07 OTC, OA, SUL, AMP, FFN, SXT + unclassified 245 Rize Kidney 02.11.07 OTC, SUL, AMP, FFN, STR, SXT, EN + unclassified 246 Artvin Kidney 17.11.07 OTC + tetA 438 Rize Kidney 27.06.08 OTC + tetA 454 Rize Kidney 28.06.07 OTC + tetA 711 Trabzon Kidney 10.05.07 OTC + tetA 712 Trabzon Kidney 04.05.07 OTC + tetA * Day/Month/Year ** OTC, oxytetracycline; OA, oxolinic acid; SUL, sulfamethoxazole; AMP, ampicillin; FFN, florfenicol; STR, streptomycin; SXT, trimethoprim-sulfamethoxazole; EN, enrofloxacin. *** Result of agglutination for serotype O1 Bull. Eur. Ass. Fish Pathol., 30(6) 2010, 217 strains. The remaining strains most likely harbored alternative resistance mechanisms. Both tetA and tetB genes encode efflux mechanisms for tetracycline resistance, and have been considered as the most common tetracycline resistance genes in E. coli of both human and animal origin (Dominguez et al., 2002; Lanz et al., 2003). tetB has been reported to provide additional resistance to doxycycline in contrast to tetA, which confer resistance to tetracycline, oxytetracycline, and chlortetracycline (Chopra and Roberts, 2001). In fish farming environments, the spread of determinants for tetracycline resistance and integron-associated antimicrobial resistance genes have been demonstrated among fish bacterial pathogens such as the aeromonads (Schmidt et al., 2001). However, in the current study no integron gene casse es were found in the antimicrobial resistant Y. ruckeri strains. Epidemiological data indicates that tetracycline resistance determinants conferring resistance to oxytetracycline are common among Y. ruckeri strains isolated from rainbow trout grown in this region (Table 3). It is noteworthy that multi drug resistant Y. ruckeri were isolated in all years (2001-2008) (Table 3), suggesting that the continuity of predisposing factors contributed to the persistent emergence of antimicrobial resistant Y. ruckeri in the examined fish farms. The present study has not demonstrated the transferable nature of the observed tetracycline resistance determinants. Yet, it should be noted that, as suggested by other authors (Chopra and Roberts, 2001; Schmidt et al., 2001) the antibiotic resistant Y. ruckeri strains may act as a reservoir of antimicrobial resistance genes within fish farm environments. 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