bs_bs_banner

Environmental Microbiology (2014) 16(5), 1310–1320

doi:10.1111/1462-2920.12421

Antimicrobial resistance and antimicrobial resistance genes in marine bacteria from salmon aquaculture and non-aquaculture sites

Syed Q. A. Shah,1 Felipe C. Cabello,2 Trine M. L’Abée-Lund,1 Alexandra Tomova,2 Henry P. Godfrey,3 Alejandro H. Buschmann4 and Henning Sørum1* 1 Norwegian University of Life Sciences, Ullevålsvein 72, Oslo, Norway. Departments of 2Microbiology and Immunology and 3 Pathology, New York Medical College, Valhalla, NY 10595, USA. 4 Centro i-mar, Universidad de Los Lagos, Puerto Montt, Chile. Summary Antimicrobial resistance (AR) detected by disc diffusion and antimicrobial resistance genes detected by DNA hybridization and polymerase chain reaction with amplicon sequencing were studied in 124 marine bacterial isolates from a Chilean salmon aquaculture site and 76 from a site without aquaculture 8 km distant. Resistance to one or more antimicrobials was present in 81% of the isolates regardless of site. Resistance to tetracycline was most commonly encoded by tetA and tetG; to trimethoprim, by dfrA1, dfrA5 and dfrA12; to sulfamethizole, by sul1 and sul2; to amoxicillin, by blaTEM; and to streptomycin, by strA-strB. Integron integrase intl1 was detected in 14 sul1-positive isolates, associated with aad9 gene cassettes in two from the aquaculture site. intl2 Integrase was only detected in three dfrA1-positive isolates from the aquaculture site and was not associated with gene cassettes in any. Of nine isolates tested for conjugation, two from the aquaculture site transferred AR determinants to Escherichia coli. High levels of AR in marine sediments from aquaculture and nonaquaculture sites suggest that dispersion of the large amounts of antimicrobials used in Chilean salmon aquaculture has created selective pressure in areas

Received 1 October, 2013; revised 29 January, 2014; accepted 31 January, 2014. *For correspondence. E-mail henning.sorum@ nmbu.no; Tel. +47 22964770; Fax +47 22964818.

© 2014 Society for Applied Microbiology and John Wiley & Sons Ltd

of the marine environment far removed from the initial site of use of these agents. Introduction Chile is the second largest producer of farmed salmon and trout in the world after Norway (FAO, 2011). Infections by bacteria, parasites, fungi and viruses are a frequent cause of morbidity and mortality in intensive salmon aquaculture in Chile, with concomitant decreases in production and income (Asche et al., 2010; Cabello et al., 2013). Efforts to increase productivity by high-stocking densities favour the emergence of infections and facilitate the free dissemination of epizootic pathogens (Asche et al., 2010; Ibieta et al., 2011). While infection control in salmon aquaculture by vaccination and improved hygienic conditions provides the best long-term approach (Markestad and Grave, 1997; Berg et al., 2006; Bravo and Midtlyng, 2007; Midtlyng et al., 2011), antimicrobials are the current primary tools for prevention and treatment of bacterial infections (Grave et al., 1999; Sørum, 2006). In Chile, a lack of use of the few available vaccines, the continuous high prevalence of bacterial infections such as salmon rickettsial syndrome caused by Piscirickettsia salmonis, the presence of antimicrobial-resistant pathogens and the emergence of new pathogens has stimulated the use of large quantities of antimicrobials (Asche et al., 2010; Ibieta et al., 2011; Millanao et al., 2011; Cabello et al., 2013). Medicated feed and rarely, bath immersion, are commonly used in salmon farming to prevent bacterial infections (Sørum, 2006; Smith et al., 2009b). Some of the feed is not ingested, and its antimicrobial activity, along with that of metabolized and unmetabolized antimicrobials in fish urine and faeces, ends up in the surrounding environment (Cabello et al., 2013). Passage of antimicrobials into the environment can select for resistant bacteria and increase horizontal gene transfer (HGT) and genetic recombination of antimicrobial resistance genes (ARG). There appears to be an unimpeded flow of ARG between the resistome of environmental, animal and human bacteria, so that increased antimicrobial resistance (AR) in the aquatic environment has the potential to introduce AR determinants into the resistomes of piscine and human

Antimicrobial resistance and salmon aquaculture pathogens (Capone et al., 1996; Rhodes et al., 2000; Cabello, 2006; Sørum, 2006; Welch et al., 2007; Baquero et al., 2008). There is increasing evidence for at least some of these potential effects of excessive aquacultural use of antimicrobials (Sørum, 2008; Cabello et al., 2013). For example, in our previous study, we found significant increases in numbers of bacteria resistant to oxytetracycline, oxolinic acid and florfenicol (three antimicrobials commonly used in Chilean aquaculture) in sediments from an aquaculture site compared with those from a non-aquaculture control site (Buschmann et al., 2012). Interestingly, a limited examination of unselected marine bacteria from these two sites indicated that there were similar numbers of ARG isolated from both sites (Buschmann et al., 2012). Because of the limited nature of this initial assessment of AR and ARG and because of the importance of this knowledge to veterinary and potentially to human health, a more extensive examination of AR and ARG was undertaken. This more extended study involved examination of phenotypic resistance of a wider range of antimicrobials in 200 unselected marine bacteria isolated from sediments from aquaculture areas and from non-aquaculture areas as well as genotyping the ARG of these isolates for many more genes than had been previously examined.

Results AR in bacteria in marine sediments from salmon aquaculture and non-aquaculture sites Disc diffusion susceptibility testing of 124 isolates from marine sediments at a salmon aquaculture site and 76 isolates from a site 8 km distant with no aquacultural activities (‘control’ site) revealed that 162 isolates (81%) expressed phenotypic resistance against at least one of the tested antimicrobials, tetracycline, trimethoprim, streptomycin, amoxicillin, oxolinic acid, chloramphenicol, florfenicol and erythromycin (Table 1). Sediments from both aquaculture and non-aquaculture sites have been previously shown to be contaminated with traces of the quinolone antimicrobial flumequine (Buschmann et al., 2012). Thirteen percent of the isolates were resistant to a single antimicrobial, 3–13% were resistant to two to eight and 9% were resistant to all nine tested antimicrobials. Resistance to the three most commonly used antimicrobials in Chile, tetracycline, florfenicol and oxolinic acid, was 32%, 26% and 53% at the aquaculture site and 22%, 25% and 45% at the non-aquaculture site (Table 1). Levels of AR were not statistically different at the two sites. Southern hybridization with gene probes for different ARG (Supporting Information Table S1) did not detect additional AR strains not already detected by disc diffusion (Table 1).

1311

ARG in bacteria in marine sediments from salmon aquaculture and non-aquaculture sites Because phenotypic determination of AR did not show differences between the aquaculture and the non-aquaculture sites, we investigated whether there were differences in the genes responsible for expression of these resistances. As AR phenotypes can be encoded by a multiplicity of genes and mechanisms, we focused our investigation on genes that have been previously detected in aquatic environments (Husevåg et al., 1991; Buschmann et al., 2012; Shah et al., 2012b). Bacterial isolates that were phenotypically resistant by disc diffusion (Table 1) and/or had shown positive Southern hybridization with specific resistance determinant DNA probes (Table 1) were assayed by polymerase chain reaction (PCR) with primers for ARG (Supporting Information Table S2). Identity of the amplicons was confirmed by their size in agarose gel electrophoresis with molecular weight standards and by DNA sequencing of a few selected amplicons (Supporting Information Table S3). DNA sequence analysis confirmed the expected sequences of ARG (including tet, sul, dfrA, strA-B and blaTEM) in 42 amplicons from bacteria isolated from the aquaculture site and in 14 amplicons from bacteria isolated from the non-aquaculture site (Supporting Information Table S3). There were similarities and differences in the presence and frequency of ARG between bacteria from aquaculture and non-aquaculture sites; however statistical analysis did not show any significant differences in ARG between these sites (Table 1). Of the eight tetracycline genes tested, only tetA and tetG were present in the bacteria from the aquaculture site, and none was present in bacteria from the non-aquaculture site. Bacteria from the two sites also differed in the presence and frequency of sul genes. Both sul1 and sul2 were present in bacteria from the aquaculture site but only sul1 genes were present in bacteria from the non-aquaculture site and in much reduced frequency (Table 1). Similarly, the strA-B genes were only present in bacteria from the aquaculture site. In contrast, distribution of trimethoprim and β-lactamase genetic determinants was relatively homogenous between the two sites. Importantly, a large proportion of the phenotypically AR bacteria from the aquaculture and non-aquaculture sites did not contain most of the genes tested (Table 1). Detection of gene cassettes (GCs), integrons and conjugation of AR determinants To further explore the genetic basis of resistance and because integrons play an important role in the evolution of AR in aquatic environments (Mazel, 2006), selected AR isolates were screened for carriage of integrons and associated GCs (Supporting Information Table S4). All 14 sul1+

© 2014 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology, 16, 1310–1320

32.2

46.8

50.0

58.1 45.2

53.2 18.5 25.8 44.4

Antimicrobial

Tetracycline

Trimethoprim

Sulfamethizole

Streptomycin Amoxicillin

Oxolinic acid Chloramphenicol Florfenicol Erythromycin

ND ND ND ND

1.5 ND

5

7

2.5

Southern hybridizationc (%)

a. Total isolates from aquaculture site: 124. b. Total isolates from non-aquaculture site: 76. c. Genes detected by Southern hybridization. ND, not done.

Disc test (%)

0 0 0 0

tetA tetG dfrA1 dfrA5 dfrA12 sul1 sul2 strA-B blaTEM

PCR+ (%)

Aquaculture sitea

0 0 0 0

1.6 0.8 13.7 7.3 17.7 9.7 2.4 1.6 8.9 44.7 15.8 25.0 47.4

42.1 48.7

57.9

53.9

22.4

Disc test (%)

ND ND ND ND

0 ND

4

8

0

Southern hybridizationc (%)

0 0 0 0

0 blaTEM

dfrA1 dfrA5 dfrA12 sul1

0

PCR+ (%)

Non-aquaculture siteb

(18.4) (13.2) (18.4) (2.6)

0 0 0 0

0 8 (10.5)

14 10 14 2

0

aadA1, aadA2, strA-Bc blaTEM, blaSHV, blaOXA, CTX-M G-1, CTX-M G-2, CTX-M G-8, CTX-M G-9, CTX-M G-25 qnrA, qnrB, qnrS1 cat-1, cmlA1 floR mefA, ermB

sul1c, sul2c, sul3

dfrA1c, dfrA2d, dfrA3, dfrA5c, dfrA7c, dfrA8, dfrA9, dfrA10, dfrA12c, dfrA14, dfrA17, dfrA24, dfrA26

tetAc, tetBc, tetCc, tetDc, tetEc, tetGc, tetHc, tet31c

Genes tested

Table 1. Antimicrobial resistances and resistance genes in marine bacteria from an aquaculture and a non-aquaculture site in Chile.

1312 S. Q. A. Shah et al.

© 2014 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology, 16, 1310–1320

Antimicrobial resistance and salmon aquaculture

1313

Table 2. Class 1 and 2 integrons and gene cassettes in marine bacteria from aquaculture and non-aquaculture sites in Chile. Site

Gene

Integron

Gene cassette

Species

Aquaculturea

sul1 sul1 sul1 sul1 sul1 sul1 sul1 sul1 sul1 sul1 sul1 sul1 dfrA1 dfrA1 dfrA1 sul1 sul1

intl1 intl1 intl1 intl1 intl1 intl1 intl1 intl1 intl1 intl1 intl1 intl1 intl2 intl2 intl2 intl1 intl1

aadA9 aadA9 − − − − − − − − − − − − − − −

Arthrobacter bergeri isolate 1 Arthrobacter bergeri isolate 2 Unknown Bacillus aryabhattai Exiguobacterium sibiricum Marinobacter litoralis Psychrobacter pulmonis Stenotrophomonas maltophilia Thalassospiro xiamenensis Unknown Unknown Unknown Agarivorans albus Vibrio kanaloae Unknown Pseudoalteromonas mariniglutinosa Unknown

Non-aquacultureb

a. Twelve sul1-containing isolates were tested for intl1, 10/12 were positive for int1 (92% positive), and 2/10 intl1-positive isolates were also positive for aadA9 gene cassette (20% positive). Ten dfrA1-containing isolates were tested for intl2, 3/10 were positive (30% positive). b. Two sul1-containing isolates were tested for intl1, both were positive (100% positive); eight dfrA1-containing isolates were tested for intl2, none was positive (0% positive).

isolates, 12 from the aquaculture site and 2 from the non-aquaculture site, harboured the intl1 integrase gene, suggesting the presence of the sul1 gene within class 1 integrons. These 14 sul1 and intl1 bearing isolates were screened for the presence of GCs using primer pair L1 and RI (Supporting Information Table S4) located at the 5’ and 3’ conserved regions of the class 1 integron (Maguire et al., 2001). The streptomycin resistance gene aadA9 (encoding an aminoglycoside-3’-adenylyltransferase) was found as a GC in three of the isolates from the aquaculture site and in none of the isolates from the nonaquaculture site (Table 2). As class 2 integrons mostly carry dfrA1 as a GC, 10 dfrA1+ isolates (eight from the aquaculture site and two from the non-aquaculture site) were included in the screening for the presence of class 2 integrons. intl2 was found only in three isolates from the aquaculture site; GC were not demonstrated in any of these isolates. Class 3 integrons were not found in any of the screened isolates. The potential for HGT of the AR determinants present in some of these bacteria was assessed by conjugation either with Escherichia coli DH5α or E. coli HB101 depending on resistance profile of donor and recipient (Supporting Information Table S5). These studies used nine multiresistant isolates, six from the aquaculture site and three from the non-aquaculture site (Supporting Information Table S5). Two isolates from the aquaculture site, Marinobacter litoralis and Stenotrophomonas maltophilia, were able to transfer both tetA and sul2 genes into E. coli recipients. The presence of the genes in the recipients was confirmed by PCR and gene sequencing in one exconjugant recipient for each mating (data not shown).

Species of antimicrobial resistant bacteria in marine sediments in Chile 16S rDNA gene sequences in a randomly selected group of 27 multiple-resistant bacteria, 16 from the aquaculture site and 11 from the non-aquaculture site (Table 3) confirmed that the AR bacteria being studied were in fact marine bacteria. The aquatic bacteria Agarivorans albus, Arthrobacter spp., Bacillus aryabhattai, Exiguobacterium sibiricum, Halomonas venusta, Marinobacter litoralis, Pectobacterium carotovorum, Pseudomonas stutzeri, Psychrobacter pulmonis, Psychrobacter marincola, Shewanella algidipiscicoIa, Sporosarcina saromensis, Stenotrophomonas maltophilia and Vibrio kanaloae were found in sediments from the aquaculture site, while Exiguobacterium aurantiacum, Pectobacterium carotovorum, Providencia sp., Pseudoalteromonas mariniglutinosa, Psychrobacter submarinus, Serratia grimesii, Serratia proteamaculans, Thalassospira xiamenensis, Vibrio cyclitrophicus, Vibrio lentus and Vibrio splendidus were found in sediments from the non-aquaculture sites.

Discussion We have found that levels of AR in bacterial isolates collected from Chilean marine sediments were much higher than in other marine farming regions of the world. Frequencies of bacterial resistance against tetracycline, sulfonamides, trimethoprim and amoxicillin in Chilean sediments were 32%, 50%, 47% and 45% respectively (Table 1). For example, in marine sediment of one salmon aquaculture sites in Puget Sound, Washington, with an

© 2014 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology, 16, 1310–1320

1314 S. Q. A. Shah et al. Table 3. Bacterial species identified by 16S rDNA sequences.

Site

Bacteria

Similarity to GeneBank (%)

Antibiotic resistances found against (n) number of antibiotics

Aquaculture (16)

Agarivorans albus Arthrobacter bergeri (isolate 1) Arthrobacter bergeri (isolate 2) Bacillus aryabhattai Exiguobacterium sibiricum Halomonas venusta Marinobacter litoraIis Pectobacterium carotovorum Pseudomonas stutzeri Psychrobacter pulmonis Psychrobacter marincola Shewanella algidipiscicoIa Sporosarcina saromensis Stenotrophomonas maltophilia Vibrio kanaloae (isolate 1) Vibrio kanaloae (isolate 2) Exiguobacterium aurantiacum Providencia spp. Pseudoalteromonas marinigIutinosa Psychrobacter submarinus Serratia grimesii Serratia proteamaculans Thalassospira xiamenensis (isolate 1) Thalassospira xiamenensis (isolate 2) Vibrio cyclitrophicus Vibrio lentus Vibrio splendidus

97 99 99 98 99 99 99 99 99 99 99 99 99 99 99 99 97 99 99 98 99 99 97 97 99 99 99

5 6 8 2 7 4 9 9 5 0 1 5 0 9 8 9 0 3 2 5 8 4 0 2 4 5 9

Non-aquaculture (11)

annual use of 450 kg of antimicrobials, 3–9% of the bacteria were resistant to tetracycline, 7–12% were resistant to sulfonamides/trimethoprim and 6–14% were resistant to amoxicillin (Herwig and Gray, 1997; Herwig et al., 1997). In a study assessing the impact of tetracycline on marine sediments at aquaculture farms in Norway, bacteria in the sediment were 100% tetracycline resistant immediately after application of this medication; this resistance subsequently fluctuated between 10% and 50% over the next 18 months (Samuelsen et al., 1992). In this latter study, less than 1% of bacteria in the control site were tetracycline resistant (Samuelsen et al., 1992). In a Danish study of antimicrobial usage in fresh water rainbow trout farms, 4.8% of bacteria collected from water, sediments and fish were resistant to tetracycline, 4.7% to sulfonamides and trimethoprim, 15.8% to oxolinic acid, 14.5% to amoxicillin and 9.5% to florfenicol (Schmidt et al., 2000). The high frequency of resistance among Chilean bacterial isolates in the present study confirms many previous reports on the role antimicrobial use plays in selecting AR bacteria in water and aquatic sediments (Björklund et al., 1991; Hansen et al., 1992; Herwig and Gray, 1997; Chelossi et al., 2003; Akinbowale et al., 2007; Dang et al., 2009; Hellweger et al., 2011). Many previous studies have showed that increases in AR in human medicine, agriculture and aquaculture are directly related to the amounts of antimicrobials used

(Levy et al., 1976; Møller et al., 1977; Capone et al., 1996; Chelossi et al., 2003; Aarestrup, 2005; van de Sande-Bruinsma et al., 2008; Brolund et al., 2010; Dang et al., 2011; Gullberg et al., 2011; Buschmann et al., 2012). The higher frequency of AR bacteria in Chile appears to be directly related to the present and past use of much larger amounts of antimicrobials in Chilean salmon aquaculture than in aquaculture in other regions (Asche et al., 2010; Fernandez-Alarcon et al., 2010; Contreras and Miranda, 2011; Ibieta et al., 2011; Millanao et al., 2011; Cabello et al., 2013). In contrast to previous studies that showed significant differences in the proportion of AR bacteria between aquaculture and control sites (Husevåg et al., 1991; Herwig and Gray, 1997; Herwig et al., 1997; Tamminen et al., 2011), we found no significant difference between these sites (Table 1). A lack of differences regarding carriage of ARG between bacteria from the aquaculture and non-aquaculture/control sites might be a result of the limited number of genes tested in the present study and from our focus on culturable bacteria, a group known to constitute only 1% of the microbiome of marine sediments (Bissett et al., 2006). However, this lack of difference in AR and carriage of ARG between aquaculture and nonaquaculture sites more probably results from the much larger amounts of antimicrobials used in aquaculture in Chile than in other countries and diffusion of antimicrobi-

© 2014 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology, 16, 1310–1320

Antimicrobial resistance and salmon aquaculture als from salmon farming sites as a result of leaching and dispersal by currents (Contreras and Miranda, 2011; Cabello et al., 2013). This is supported by the detection of residues of flumequine in sediments from the site chosen as a control site (Buschmann et al., 2012). This contamination was unknown to us when the study was initiated. It was unexpected because the site was at an appreciable distance from the aquaculture site and offshore the only coastal area with no salmon farms in this region. This fact made it impossible to sample a control site not impacted by fish farming activities related to antimicrobials in this aquacultural region. Our data thus suggest that the impact zone of antimicrobial use in aquaculture can extend far beyond the licensed farming sites in regions with high density of salmon farms and high levels of application of these agents (Buschmann et al., 2009). This collection of strains also displayed high frequencies of resistance to sulfamethizole (50%), streptomycin (58%), amoxicillin (45%) and erythromycin (44%). This might indicate that these antimicrobials are also used extensively in salmon aquaculture or that resistance for them has been selected by the modular nature of the genetic elements responsible for antimicrobial resistance including plasmids, integrative and conjugative elements (ICE), genomic islands and integrons (Herwig and Gray, 1997; Herwig et al., 1997; Tamminen et al., 2011; Miranda, 2012; Cabello et al., 2013). An alternative explanation for the higher levels of AR in the aquaculture and non-aquaculture sites in these experiments could be the presence of high levels of natural/intrinsic resistance, but this does not appear to be the case as background AR in bacteria from pristine marine sediments fluctuates between 1–2% and 5% (Nygaard et al., 1992; Kerry et al., 1996). Class 1 and class 2 integrons and conjugative plasmids were present in the small number of bacteria tested. This suggests that these mobile and recombinatable genetic structures may play a role in the evolution of AR in these sediments. Conditions exist in this environment for stimulation of capture of GCs and generation of new arrangements of ARG within these integrons as well as HGT stimulated by residual antimicrobials and mediated by the SOS system of the bacterial cells (Rowe-Magnus and Mazel, 1999; Baharoglu and Mazel, 2011). That HGT may take place in these sediments is demonstrated by the transfer of the tetracycline resistance gene tetA and the sulfonamide resistance gene sul2 from Marinobacter litoralis and Stenotrophomonas maltophilia to E. coli (Supporting Information Table S5). Failure to transfer AR determinants by conjugation from the other seven strains tested may be the result of the location of these determinants on non-conjugative plasmids, of their presence in the chromosome or alternatively, to lack of compatibility for HGT between the recipient and donor, i.e., restriction

1315

systems, surface exclusion and incompatibility (L’Abée-Lund and Sørum, 2001; Roberts, 2012). We were unable to find the plasmid-borne quinolone resistance genes qnrA, qnrB, and qnrS in quinoloneresistant bacteria and floR in florfenicol-resistant bacteria despite previous detection of some of these genes in some strains of this collection (Buschmann et al., 2012). This may be the result of PCR screening done on the basis of inhibition zone diameter (IZD), which excluded isolates with larger IZD. Low levels of resistance to quinolones, evidenced by larger IZD in these quinolone-resistant isolates, are likely due to the presence of qnr plasmids alone in the absence of chromosomal mutations in the DNA gyrase and topoisomerase IV genes (Mammeri et al., 2005; Cavaco and Aarestrup, 2009; Karah et al., 2010; Shah et al., 2012a). Exclusion of strains with larger IZD may have eliminated these strains. Such strains could be frequent in the marine environment and a possible source of qnr genes (Cavaco and Aarestrup, 2009; Poirel et al., 2012; Cabello et al., 2013). We have also observed that after a few passages in solid and liquid media some of these strains often appear to lose their qnr determinants (A. Tomova and F. C. Cabello, unpublished). Elimination of strains with larger IZD for florfenicol may have similarly biased against strains containing only floR resistance genes and lacking other florfenicol resistance genes (Tao et al., 2012). A large proportion of the AR bacteria from the aquaculture and the non-aquaculture sites did not contain most of the tested genes (Table 1). This indicates that a large proportion of the resistance genes in use among these bacteria are unknown at least in terms of the primers used. This in turn points to the marine ecosystem as an important source of unknown ARG (Roberts, 2012). The present study confirms and extends the conclusions of earlier reports finding that high prevalence of AR bacteria and ARG in salmon farming facilities and in nearby marine sediments is correlated with the large amounts of antimicrobials used in this activity in Chile (Contreras and Miranda, 2011; Buschmann et al., 2012). Aquatic and terrestrial bacteria, including fish and human pathogens, may belong to the same taxa, e.g., Aeromonas spp., Kluyvera spp., Vibrio spp. and Yersinia spp. (Sarria et al., 2001; Heuer et al., 2009; Cabello et al., 2013), and animal and human pathogens may contaminate the aquatic environment (Silva et al., 1987). Contaminated native fish with antimicrobials consumed by local communities may contribute to increase AR and ARG of human pathogens (Fortt et al., 2007). Such conditions can be expected to facilitate and increase the flow of ARG and mobile genetic structures between the aquatic and the terrestrial environments leaving an increasing number of AR bacterial infections in fish, animals and human beings in their wake (Sørum, 2006; Baquero et al., 2008; Taylor et al., 2011; Finley et al., 2013).

© 2014 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology, 16, 1310–1320

1316 S. Q. A. Shah et al. Experimental procedures Bacterial isolates from marine sediments Bacterial isolates were collected in the Calbuco Archipelago (41°48′ S, 73°11′ W), Los Lagos Region in Southern Chile in 2008 and 2009 from sediments close to a commercial salmon farm and a site 8 km distant without aquacultural activity, which served as a non-aquaculture/control site (Buschmann et al., 2012). Aquacultural activity in this region is intense, and this site is in the most isolated locality from salmon farms in the region. Despite the isolated nature of this site, traces of flumequine were detected in sediments taken from it (Buschmann et al., 2012). A 10-fold dilution series from sediment suspensions (undiluted to 10−5) was plated on antibioticfree Marine agar (MA) plates (Difco, Detroit, MI, USA). Isolated colonies of 200 bacteria including 124 from the aquaculture site and 76 from the non-aquaculture site growing on MA plates were selected and stored frozen at −80°C in 96-well microtitre plates in 36% glycerol for later study. These bacterial cultures were obtained from −80°C and transported in soft MA in 1.5 ml Eppendorf microtubes (Hauppauge, NY, USA) from Puerto Montt, Chile to Oslo, Norway. Recipient strains and strains used as controls in PCR tests, DNA hybridizations and conjugations were obtained from the collection of the Norwegian School of Veterinary Science (Supporting Information Tables S1, S2, S5).

Determination of antibiotic susceptibility of marine sedimental bacteria Isolates from aquaculture and non-aquaculture sites (200 in total) were tested for their susceptibility to antimicrobials by disc diffusion in 2% NaCl fortified Müller–Hinton (MH) agar media (CLSI, 2006) against tetracycline (80 μg), trimethoprim (5.2 μg), sulfamethizole (240 μg), amoxicillin (30 μg), oxolinic acid (10 μg), streptomycin (100 μg) chloramphenicol (60 μg), florfenicol (30 μg) and erythromycin (78 μg) (NeoSensitabs Rosco, Taastrup, Denmark). As there are no specific established IZD breakpoints for aquatic bacteria, we calculated breakpoints according to the guidelines of Smith and colleagues (2009a) and Kronvall and colleagues (2011). IZD of < 40 mm was selected as a breakpoint between resistant and susceptible isolates (data not shown). To verify the significance level of IZD, 95% confidence interval of expected neighbouring IZD values were constructed by using the theory of simple binomial sequences (Agresti, 2002).

sul2), streptomycin resistance (strA-strB), trimethoprim resistance (dfrA1, dfrA5, dfrA7 and dfrA12) and integrase (intl1). DNA probes were labelled in vitro by [α-32P] dCTP (Hartmann Analytic, Braunschweig, Germany) using Ready-to-go DNA beads (GE Healthcare, Little Chalfont, UK) according to the manufacturer’s instructions. All membranes were treated as described previously; resistant colonies were identified by autoradiography (Shah et al., 2012b).

Identification of ARG, integrons and GCs by PCR The presence of several ARG was determined in AR bacteria to each antimicrobial by PCR using primers listed in Supporting Information Table S2. Bacterial isolates with IZD < 40 mm and positive on Southern hybridization were selected for PCR screening. Although we could not select more potential bacterial isolates for PCR screening by Southern hybridization, we used this method as a parallel technique for selection of potential AR bacteria. Total DNA of the bacteria was extracted using DNeasy Blood and Tissue Kit (Qiagen, Sollentuna, Sweden) according to the manufacturer’s instructions and stored at −20°C. PCR amplifications were performed in a total reaction volume of 25 μl using Taq polymerase (VWR International, Arlington Heights, IL, USA). Multiplex PCR was used for screening of trimethoprim resistance genes (Grape et al., 2007) and β-lactam resistance genes (Colom et al., 2003; Woodford et al., 2006). PCR products were identified on the basis of expected amplicon size after agarose gel electrophoresis (Bartlett and Stirling, 2003) (Supporting Information Tables S1, S2). Randomly selected PCR products for ARG were sequenced (39 of 94 from the aquaculture site and 14 of 50 from the non-aquaculture site) (Supporting Information Table S4). PCR products were purified using the QIAquick PCR purification kit (Qiagen, Duesseldorf, Germany) and sequenced by GATC Biotech AG, Konstanz, Germany. Confirmation of the identity of sequences was carried out by comparison with DNA sequences in the GenBank using BLAST. Class 1 and Class 2 integrons were detected by amplifying the respective integrase gene with multiplex PCR (Ishikawa, 2011) with specific primers (Su et al., 2006) (Supporting Information Table S4). Class 1 integron GCs were detected using primer pair L1 and R1 located at the 5’ and 3’ conserved regions of the class 1 integron respectively (Maguire et al., 2001). Potential GCs carried by class 2 integrons were screened by primer pair TiF and TiB (Su et al., 2006). All PCR products were sequenced for confirmation of conserved regions of integrons and identification of GCs.

Identification of ARG by Southern hybridization Bacteria growing as isolated colonies were transferred to Hybond-N + nylon filters (Amersham Biosciences, Buckinghamshire, UK) from freshly grown blood agar (with 2% NaCl) plates and were allowed to grow for two days at 15°C on the filter on blood agar plates containing 2% NaCl. Nylon filter membranes were treated as described earlier (Sambrook et al., 1989; Shah et al., 2012b). All 200 isolates were screened by Southern hybridization using DNA probes generated using primer pairs listed in Supporting Information Table S1 for genes for tetracycline resistance (tetA, tetB, tetC, tetD, tetE, tetG, tetH, tet31), sulfonamide resistance (sul1 and

Species identification of selected multiple antimicrobial-resistant isolates Twenty-seven AR bacterial isolates resistant to tetracycline, trimethoprim, sulfamethizole, amoxicillin, oxolinic acid, streptomycin, erythromycin, chloramphenicol and/or florfenicol were taxonomically identified by phenotypic characteristics (colony morphology and biochemical tests) and by 16S rDNA gene amplification and sequencing using primer pair 27f and 1392r (Mao et al., 2012) (Supporting Information Table S2). Partial 16S rDNA gene sequences were compared with sequences available in the GenBank using NCBI BLAST.

© 2014 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology, 16, 1310–1320

Antimicrobial resistance and salmon aquaculture Conjugative transfer of AR genes Nine multi-resistant isolates (six from the aquaculture site, three from the non-aquaculture site) were selected for conjugation experiments (Supporting Information Table S5). Isolates were grown at 15°C in Luria broth (LB) with 2% NaCl for 3 days, diluted 1:20 and incubated at 15°C for 2 days. Escherichia coli DH5α or E. coli HB101 grown in LB were used as recipients. The same volume (0.5 ml) of donors and recipient at the exponential phase of growth were mixed and allowed to conjugate overnight at 15°C and at room temperature (L’Abée-Lund and Sørum, 2001). Presumptive transconjugants were selected by plating 100 μl of undiluted mating mixtures on selective LB plates with 10 μg ml−1 of tetracycline, 50 μg ml−1 of trimethoprim, 100 μg ml−1 of sulfamethoxazole, or 150 μg ml−1 of ampicillin, and the counter selective antimicrobials 25 μg ml−1 of nalidixic acid or 50 μg ml−1 of streptomycin depending on the donor and the recipient strains (Supporting Information Table S6). Matings with Aeromonas salmonicida subsp. salmonicida containing conjugative plasmid pRAS1 were used as a positive mating control.

Statistical analysis Results are expressed in percentage with 95% confidence intervals. The confidence intervals are constructed by using the theory of simple binomial sequences (Agresti, 2002). The significance level for differences between groups was set at P ≤ 0.05. Comparison of groups with regard to categorical variables was performed by using contingency table analysis (Agresti, 2002).

Acknowledgements We thank Professor Stig Larsen, the Norwegian School of Veterinary Science, for his help with statistical analysis of the results. This work has been supported by a grant from the Lenfest Ocean Program/Pew Charitable Trusts to F. C. Cabello and A. H. Buschmann.

References Aarestrup, F.M. (2005) Veterinary drug usage and antimicrobial resistance in bacteria of animal origin. Basic Clin Pharmacol Toxicol 96: 271–281. Agresti, A. (2002) Categorical Data Analysis, 2nd edn. Hoboken, NJ, USA: John Wiley & Sons. Akinbowale, O.L., Peng, H., and Barton, M.D. (2007) Diversity of tetracycline resistance genes in bacteria from aquaculture sources in Australia. J Appl Microbiol 103: 2016–2025. Asche, F., Hansen, H., Tveterås, R., and Tveterås, S. (2010) The salmon disease crisis in Chile. Mar Resour Econ 24: 405–411. Baharoglu, Z., and Mazel, D. (2011) Vibrio cholerae triggers SOS and mutagenesis in response to a wide range of antibiotics: a route towards multiresistance. Antimicrob Agents Chemother 55: 2438–2441. Baquero, F., Martinez, J.L., and Canton, R. (2008) Antibiotics and antibiotic resistance in water environments. Curr Opin Biotechnol 19: 260–265.

1317

Bartlett, J.M.S., and Stirling, D. (2003) A short history of the polymerase chain reaction. In PCR Protocols. Bartlett, J.M.S., and Stirling, D. (eds). Totowa, NJ, USA: Humana Press, pp. 3–6. Berg, A., Rødseth, O.M., Tangerås, A., and Hansen, T. (2006) Time of vaccination influences development of adhesions, growth and spinal deformities in Atlantic salmon Salmo salar. Dis Aquat Organ 69: 239–248. Bissett, A., Bowman, J., and Burke, C. (2006) Bacterial diversity in organically-enriched fish farm sediments. FEMS Microbiol Ecol 55: 48–56. Björklund, H.V., Råbergh, C.M.I., and Bylund, G. (1991) Residues of oxolinic acid and oxytetracycline in fish and sediments from fish farms. Aquaculture 97: 85–96. Bravo, S., and Midtlyng, P.J. (2007) The use of fish vaccines in the Chilean salmon industry 1999–2003. Aquaculture 270: 36–42. Brolund, A., Sundqvist, M., Kahlmeter, G., and Grape, M. (2010) Molecular characterization of trimethoprim resistance in Escherichia coli and Klebsiella pneumoniae during a two year intervention on trimethoprim use. PLoS ONE 5: 1–5. Buschmann, A.H., Cabello, F.C., Young, K., Carvajal, J., Varela, D.A., and Henríquez, L. (2009) Salmon aquaculture and coastal ecosystem health in Chile: analysis of regulations, environmental impacts and bioremediation systems. Coastal Ocean Manage 52: 243–249. Buschmann, A.H., Tomova, A., Lopez, A., Maldonado, M.A., Henriquez, L.A., Ivanova, L., et al. (2012) Salmon aquaculture and antimicrobial resistance in the marine environment. PLoS ONE 7: e42724. Cabello, F.C. (2006) Heavy use of prophylactic antibiotics in aquaculture: a growing problem for human and animal health and for the environment. Environ Microbiol 8: 1137– 1144. Cabello, F.C., Godfrey, H.P., Tomova, A., Ivanova, L., Dölz, H., Millanao, A., and Buschmann, A.H. (2013) Antimicrobial use in aquaculture re-examined: its relevance to antimicrobial resistance and to animal and human health. Environ Microbiol 15: 1917–1942. Capone, D.G., Weston, D.P., Miller, V., and Shoemaker, C. (1996) Antibacterial residues in marine sediments and invertebrates following chemotherapy in aquaculture. Aquaculture 145: 55–75. Cavaco, L.M., and Aarestrup, F.M. (2009) Evaluation of quinolones for use in detection of determinants of acquired quinolone resistance, including new transmissible resistance mechanisms qnrA, qnrB, qnrS and aaa(6’)Ib-cr, in Escherichia coli and Salmonella enterica and determinations of wild-type distributions. J Clin Microbiol 47: 2751– 2758. Chelossi, E., Vezzulli, L., Milano, A., Branzoni, M., Fabiano, M., Riccardi, G., and Banat, I.M. (2003) Antibiotic resistance of benthic bacteria in fish-farm and control sediments of the Western Mediterranean. Aquaculture 219: 83–97. CLSI (2006) Performance Standards for Antimicrobial Disk Susceptibility Tests; Approved Standard. M2-A9. Wayne, PA, USA: CLSI Standards Centre. Colom, K., Perez, J., Alonso, R., Fernandez-Aranguiz, A., Larino, E., and Cisterna, R. (2003) Simple and reliable

© 2014 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology, 16, 1310–1320

1318 S. Q. A. Shah et al. multiplex PCR assay for detection of blaTEM, blaSHV and blaOXA-1 genes in Enterobacteriaceae. FEMS Microbiol Lett 223: 147–151. Contreras, S., and Miranda, C. (2011) INFORME FINAL. Vigilancia de la resistencia bacteriana en la salmonicultura. FIP no. 2008-65. [Survey of bacterial resistance in salmon aquaculture]. URL http://www.fip.cl/Archivos/Hitos/ Informes/INFORME%20HITO%20FINAL1039Adjunto1 .pdf. Dang, H.Y., Zhao, J.Y., Song, L.S., Chen, M.N., and Chang, Y.Q. (2009) Molecular characterizations of chloramphenicol- and oxytetracycline-resistant bacteria and resistance genes in mariculture waters of China. Mar Pollut Bull 58: 987–994. Dang, S.T.T., Petersen, A., Van Truong, D., Chu, H.T.T., and Dalsgaard, A. (2011) Impact of medicated feed on the development of antimicrobial resistance in bacteria at integrated pig-fish farms in Vietnam. Appl Environ Microbiol 77: 4494–4498. FAO (2011) The State of the World Fisheries and Aquaculture 2010. Rome, Italy: UNO. Fernandez-Alarcon, C., Miranda, C.D., Singer, R.S., Lopez, Y., Rojas, R., Bello, H., et al. (2010) Detection of the floR gene in a diversity of florfenicol resistant Gram-negative bacilli from freshwater salmon farms in Chile. Zoonoses Public Health 57: 181–188. Finley, R.L., Collignon, P., Larsson, D.G.J., McEwen, S.A., Li, X.Z., Gaze, W.H., et al. (2013) The scourge of antibiotic resistance: the important role of the environment. Clin Infect Dis 57: 704–710. Fortt, Z.A., Cabello, F.C., and Buschmann, R.A. (2007) Residues of tetracycline and quinolones in wild fish living around a salmon aquaculture centre in Chile. Rev Chil Infectol 24: 14–18. Grape, M., Motakefi, A., Pavuluri, S., and Kahlmeter, G. (2007) Standard and real-time multiplex PCR methods for detection of trimethoprim resistance dfr genes in large collection of bacteria. Clin Microbiol Infect 13: 1112– 1118. Grave, K., Lingaas, E., Bangen, M., and Rønning, M. (1999) Surveillance of the overall consumption of antibacterial drugs in humans, domestic animals and farmed fish in Norway in 1992 and 1996. J Antimicrob Chemother 43: 243–252. Gullberg, E., Cao, S., Berg, O.G., Ilback, C., Sandegren, L., Hughes, D., and Andersson, D.I. (2011) Selection of resistant bacteria at very low antibiotic concentrations. PLoS Pathog 7: e1002158. Hansen, P.K., Lunestad, B.T., and Samuelsen, O.B. (1992) Effects of oxytetracycline, oxolinic acid, and flumequine on bacteria in an artificial marine fish farm sediment. Can J Microbiol 38: 1307–1312. Hellweger, F.L., Ruan, X., and Sanchez, S. (2011) A simple model of tetracycline antibiotic resistance in the aquatic environment (with application to the Poudre River). Int J Environ Res Public Health 8: 480–497. Herwig, R.P., and Gray, J.P. (1997) Microbial response to antibacterial treatment in marine microcosms. Aquaculture 152: 139–154. Herwig, R.P., Gray, J.P., and Weston, D.P. (1997) Antibacterial resistant bacteria in surficial sediments near salmon

net-cage farms in Puget Sound, Washington. Aquaculture 149: 263–283. Heuer, O.E., Kruse, H., Grave, K., Collignon, P., Karunasagar, I., and Angulo, F.J. (2009) Human health consequences of use of antimicrobial agents in aquaculture. Clin Infect Dis 49: 1248–1253. Husevåg, B., Lunestad, B.T., Johannessen, P.J., Enger, Ø., and Samuelsen, O.B. (1991) Simultaneous occurrence of Vibrio salmonicida and antibiotic-resistant bacteria in sediments at abandoned aquaculture sites. J Fish Dis 14: 631–640. Ibieta, P., Tapia, V., Venegas, C., Hausdorf, M., and Takle, H. (2011) Chilean salmon farming on the horizon of sustainability: Review of the development of a highly intensive production, the ISA crisis and implemented actions to reconstruct a more sustainable aquaculture industry. In Aquaculture and the Environment – a Shared Destiny. Sladonja, B. (ed.). Rijeka, Croatia: INTECH, pp. 215–246. Ishikawa, S. (2011) Simultaneous PCR detection of multiple classes of integron integrase genes for determining the presence of multidrug-resistant bacteria in environmental samples. Curr Microbiol 62: 1677–1681. Karah, N., Poirel, L., Begtsson, S., Sundqvist, M., Kahlmeter, G., Nordmann, P., et al. (2010) Plasmid-mediated quinolone resistance determinants qnr and aac(6’)-Ib-cr in Escherichia coli and Klebsiella spp. from Norway and Sweden. Diagn Microbiol Infect Dis 66: 425–431. Kerry, J., Coyne, R., Gilroy, D., Hiney, M., and Smith, P. (1996) Spatial distribution of oxytetracycline and elevated frequencies of oxytetracycline resistance in sediments beneath a marine salmon farm following oxytetracycline therapy. Aquaculture 145: 31–39. Kronvall, G., Giske, C.G., and Kahlmeter, G. (2011) Setting interpretive breakpoints for antimicrobial susceptibility testing using disk diffusion. Int J Antimicrob Agents 38: 281–290. L’Abée-Lund, T.M.L., and Sørum, H. (2001) Class 1 integrons mediated antibiotic resistance in the fish pathogenic Aeromonas salmonicida worldwide. Microb Drug Resist 7: 263–272. Levy, S.B., FitzGerald, G.B., and Macone, A.B. (1976) Changes in intestinal flora of farm personnel after introduction of a tetracycline-supplemented feed on a farm. N Engl J Med 295: 583–588. Maguire, A.J., Brown, D.F.J., Gray, J.J., and Desselberger, U. (2001) Rapid screening technique for class 1 integrons in Enterobacteriaceae and non fermenting gram-negative bacteria and its use in molecular epidemiology. Antimicrob Agents Chemother 45: 1022–1029. Mammeri, H., Van De Loo, M., Poirel, L., Martinez-Martinez, L., and Nordmann, P. (2005) Emergence of plasmidmediated quinolone resistance in Escherichia coli in Europe. Antimicrob Agents Chemother 49: 71–76. Mao, D.P., Zhou, Q., Chen, C.Y., and Quan, Z.X. (2012) Coverage evaluation of universal bacterial primers using the metagenomic datasets. BMC Microbiol 12: 66. Markestad, A., and Grave, K. (1997) Reduction of antibacterial drug use in Norwegian fish farming due to vaccination. Dev Biol Stand 90: 365–369. Mazel, D. (2006) Integrons: agents of bacterial evolution. Nat Rev Microbiol 4: 608–620.

© 2014 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology, 16, 1310–1320

Antimicrobial resistance and salmon aquaculture Midtlyng, P.J., Grave, K., and Horsberg, T.E. (2011) What has been done to minimize the use of antibacterial and antiparasitic drugs in Norwegian aquaculture. Aquac Res 42: 28–34. Millanao, B., Arrientos, H., Gomez, C., Tomova, A., Buschmann, A.H., Dölz, H., and Cabello, F.C. (2011) Uso inadecuado y excesivo de antibioticos: salud publica y salmonicultura en Chile [Injudicious and excessive use of antibiotics: public health and salmon aquaculture in Chile]. Rev Med Chile 139: 107–118. Miranda, C.D. (2012) Antimicrobial resistance associated with salmonid farming. In Antimicrobial Resistance in the Environment. Keen, P.L., and Montforts, M.H.H.M. (eds). Hoboken, NJ, USA: Wiley-Blackwell, pp. 423–451. Møller, J.K., Bak, A.L., Stenderup, A., Zachariae, H., and Afzelius, H. (1977) Changing patterns of plasmid-mediated drug resistance during tetracycline therapy. Antimicrob Agents Chemother 11: 388–391. Nygaard, K., Lunestad, B.T., Hektoen, H., Berge, J.A., and Hormazabal, V. (1992) Resistance to oxytetracycline, oxolinic acid and furazolidone in bacteria from marine sediments. Aquaculture 104: 31–36. Poirel, L., Cattoir, V., and Nordmann, P. (2012) Plasmidmediated quinolone resistance; interactions between human, animal, and environmental ecologies. Front Microbiol 3: 24. Rhodes, G., Huys, G., Swings, J., Mcgann, P., Hiney, M., Smith, P., and Pickup, R.W. (2000) Distribution of oxytetracycline resistance plasmids between aeromonads in hospital and aquaculture environments: implication of Tn1721 in dissemination of the tetracycline resistance determinant Tet A. Appl Environ Microbiol 66: 3883–3890. Roberts, M.C. (2012) Mechanisms of bacterial antibiotic resistance and lessons learned from environmental tetracycline resistant bacteria. In Antimicrobial Resistance in the Environment. Keen, P.L., and Montforts, M.H.H.M. (eds). Hoboken, NJ, USA: Wiley-Blackwell, pp. 93–121. Rowe-Magnus, D.A., and Mazel, D. (1999) Resistance gene capture. Curr Opin Microbiol 2: 483–488. Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989) Molecular Cloning. A Laboratory Manual. Woodbury, NY, USA: Cold Spring Harbor Laboratory Press. Samuelsen, O.B., Torsvik, V., and Ervik, A. (1992) Longrange changes in oxytetracycline concentration and bacterial resistance toward oxytetracycline in a fish farm sediment after medication. Sci Total Environ 114: 125–126. van de Sande-Bruinsma, N., Grundmann, H., Verloo, D., Tiemersma, E., Monen, J., Goossens, H., and Ferech, M. (2008) Antimicrobial drug use and resistance in Europe. Emerg Infect Dis 14: 1722–1730. Sarria, J.C., Vidal, A.M., and Kimbrough, R.C. (2001) Infections caused by Kluyvera species in humans. Clin Infect Dis 33: e69–e74. Schmidt, A.S., Brunn, M.S., Dalsgaard, I., Pedersen, K., and Larsen, J.L. (2000) Occurrence of antimicrobial resistance in fish-pathogenic and environmental bacteria associated with four Danish rainbow trout farms. Appl Environ Microbiol 66: 4908–4915. Shah, S.Q.A., Karatas, S., Nilsen, H., Steinum, T.M., Colquhoun, D.J., and Sørum, H. (2012a) Characterization and expression of the gyrA gene from quinolone resistant

1319

Yersinia ruckeri strains isolated from Atlantic salmon (Salmo salar L.) in Norway. Aquaculture 350–353: 37– 41. Shah, S.Q.A., Colquhoun, D.J., Nikuli, H.L., and Sørum, H. (2012b) Prevalence of antibiotic resistance genes in the bacterial flora of integrated fish farming environments of Pakistan and Tanzania. Environ Sci Technol 46: 8672– 8679. Silva, J., Zemelman, R., Mandoca, M.A., Henriquez, M., Merino, C., and Gonzalez, C. (1987) Antibiotic-resistant gram negative bacilli isolated from sea water and shellfish. Possible epidemiological implications. Rev Latinoam Microbiol 29: 165–169. Smith, P., Douglas, I., McMurray, J., and Carroll, C. (2009a) A rapid method of improving the criteria being used to interpret disc diffusion antimicrobial susceptibility test data for bacteria associated with fish diseases. Aquaculture 290: 172–178. Smith, P.R., Le Breton, A., Horsberg, T.E., and Corsin, F. (2009b) Guidelines for antimicrobial use in aquaculture. In Guide to Antimicrobial Use in Animals. Guardabassi, L., Jensen, L.B., and Kruse, H. (eds). Oxford: Blackwell Publishing, pp. 207–218. Sørum, H. (2006) Antimicrobial drug resistance in fish pathogens. In Antimicrobial Resistance in Bacteria of Animal Origin. Aarestrup, F.M. (ed.). Washington, DC, USA: ASM Press, pp. 213–238. Sørum, H. (2008) Antibiotic resistance associated with veterinary drug use in fish farms. In Improving Farmed Fish Quality and Safety. Lie, Ø. (ed.). Cambridge, UK: Woodhead Publishing, pp. 157–182. Su, J., Shi, L., Yang, L., Xiao, Z., Li, X., and Yamasaki, S. (2006) Analysis of integrons in clinical isolates of Escherichia coli in China during the last six years. FEMS Microbiol Lett 254: 75–80. Tamminen, M., Karkman, A., Lohmus, A., Muziasari, W.I., Takasu, H., Wada, S., et al. (2011) Tetracycline resistance genes persist at aquaculture farms in the absence of selection pressure. Environ Sci Technol 45: 386–391. Tao, W., Lee, M.H., Wu, J., Kim, N.H., Kim, J.C., Chung, E., et al. (2012) Inactivation of chloramphenicol and florfenicol by a novel chloramphenicol hydrolase. Appl Environ Microbiol 78: 6295–6301. Taylor, N.G., Verner-Jeffreys, D.W., and Baker-Austin, C. (2011) Aquatic systems: maintaining, mixing and mobilizing antimicrobial resistance? Trends Ecol Evol 26: 278– 284. Welch, T.J., Fricke, W.F., McDermott, P.F., White, D.G., Rosso, M.L., Rasko, D.A., et al. (2007) Multiple antimicrobial resistance in plague: an emerging public health risk. PLoS ONE 2: e309. Woodford, N., Fagan, E.J., and Ellington, M.J. (2006) Multiplex PCR for rapid detection of genes encoding CTX-M extended-spectrum β-lactamases. J Antimicrob Chemother 57: 154–155.

Supporting information Additional Supporting Information may be found in the online version of this article at the publisher’s web-site:

© 2014 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology, 16, 1310–1320

1320 S. Q. A. Shah et al. Table S1. Primers, amplicon sizes (bp), annealing temperatures (Tm) and positive control bacteria used to generate DNA probes for Southern hybridization detection of antimicrobial resistance genes. Table S2. Primers and bacterial isolates used as positive controls for PCR detection of antimicrobial resistance genes.

Table S3. Antimicrobial resistance genes amplified from isolates from aquaculture and non-aquaculture sites. Table S4. Primers used for screening of integrons and resistance gene cassettes by PCR. Table S5. Phenoypic and genotypic characteristics of donor marine bacteria from aquaculture and non-aquaculture sites tested in conjugation.

© 2014 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology, 16, 1310–1320