Pone.0008388 1.9

High Prevalence of Multidrug-Tolerant Bacteria andAssociated Antimicrobial Resistance Genes Isolated fromOrnamental Fish and Their Carriage Water David W. Verner-Jeffreys1*, Timothy J. Welch2, Tamar Schwarz1,3, Michelle J. Pond1, Martin J.
Woodward4, Sarah J. Haig1,3, Georgina S. E. Rimmer1, Edward Roberts1, Victoria Morrison4, Craig 1 Centre for Environment, Fisheries and Aquaculture Sciences, Weymouth Laboratory, Weymouth, Dorset, United Kingdom, 2 United States Department of Agriculture/ Agricultural Research Service, National Center for Cool and Cold Water Aquaculture, Kearneysville, West Virginia, United States of America, 3 Division of Infection and Immunity, University of Glasgow, Glasgow, United Kingdom, 4 Veterinary Laboratories Agency, Addlestone, Surrey, United Kingdom Background: Antimicrobials are used to directly control bacterial infections in pet (ornamental) fish and are routinely addedto the water these fish are shipped in to suppress the growth of potential pathogens during transport.
Methodology/Principal Findings: To assess the potential effects of this sustained selection pressure, 127 Aeromonas spp.
isolated from warm and cold water ornamental fish species were screened for tolerance to 34 antimicrobials. Representativeisolates were also examined for the presence of 54 resistance genes by a combination of miniaturized microarray andconventional PCR. Forty-seven of 94 Aeromonas spp. isolates recovered from tropical ornamental fish and their carriagewater were tolerant to $15 antibiotics, representing seven or more different classes of antimicrobial. The quinolone andfluoroquinolone resistance gene, qnrS2, was detected at high frequency (37% tested recent isolates were positive by PCR).
Class 1 integrons, IncA/C broad host range plasmids and a range of other antibiotic resistance genes, including floR,blaTEM21, tet(A), tet(D), tet(E), qacE2, sul1, and a number of different dihydrofolate reductase and aminoglycoside transferasecoding genes were also detected in carriage water samples and bacterial isolates.
Conclusions: These data suggest that ornamental fish and their carriage water act as a reservoir for both multi-resistantbacteria and resistance genes.
Citation: Verner-Jeffreys DW, Welch TJ, Schwarz T, Pond MJ, Woodward MJ, et al. (2009) High Prevalence of Multidrug-Tolerant Bacteria and AssociatedAntimicrobial Resistance Genes Isolated from Ornamental Fish and Their Carriage Water. PLoS ONE 4(12): e8388. doi:10.1371/journal.pone.0008388 Editor: Stefan Bereswill, Charite´-Universita¨tsmedizin Berlin, Germany Received October 5, 2009; Accepted October 15, 2009; Published December 21, 2009 Copyright: ß 2009 Crown. This is an open-access article distributed under the terms of the Re-use of Public Sector Information Regulations 2005, which permitunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was funded by The UK government’s Department for Environment Food and Rural Affairs through projects FC1178, FB001 and a sandwichplacement studentship for T.S. (Project FC1172). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of themanuscript Competing Interests: The authors have declared that no competing interests exist.
* E-mail: david.verner-jeffreys@cefas.co.uk human and animal health [7]. Resistance can either arise frommutations in genes native to the chromosome of the bacterial The trade in ornamental (pet) fish is greater then 1 billion species in which they are found, or by acquisition of transferable animals per year globally [1]. More than 45 million fish per year genetic elements (e.g. plasmids and/or resistance gene encoding are imported into the United Kingdom (UK) alone from a wide integrons) [8]. It is known that either process can lead to the clonal range of countries, in particular those in South East Asia. An expansion of resistant pathogens that affect humans and farmed estimated 14% of all UK households have an aquarium or, Antibiotic resistance research has typically been very disease- Antimicrobials are used by owners and retailers to directly focused, likely contributing to our limited understanding regarding control bacterial infections [3]. They are also routinely added to the ecology, evolution and dissemination of antibiotic resistance in the water these fish are transported in to suppress the growth of the environment [9]. However, studies have established that potential pathogens during transport [4]. Trust and Whitby [5] plasmids, integrons [10] and associated antimicrobial resistance noted that this practice was widespread more than thirty years ago.
(AMR) genes of bacteria recovered from the aquatic environment As a consequence, antibiotic tolerant bacteria have likely been can share very high sequence homology to clinically important selected for and proliferate in the trade [3,5].
Currently, microbial resistance to antibiotics spans all known This suggests that there is resistance gene flow between aquatic classes of natural and synthetic drug agents [6], and bacterial and anthropogenic sources. The direction of this route of transfer is resistance to antibiotics continues to pose a serious threat to unknown, as are the potential health risks arising from this transfer.
December 2009 | Volume 4 | Issue 12 | e8388 Despite the increasing body of evidence regarding the role of resistance-determining regions of these two genes. Apart from transferable genetic elements in the dissemination of antibiotic ampicillin, to which A. hydrophila is intrinsically resistant [23], no resistance in pathogens that affect farmed fish, there is a relative antibiotics were included in any of the isolation media used.
paucity of data concerning their role in the development andtransfer of resistance in pet fish. A recent Australian study also noted a possible a link between the ownership of ornamental fish Antimicrobial susceptibility was determined for 94 Aeromonas and a limited number of multidrug resistant (MDR) Salmonella Java isolates from warmwater species against 34 antibiotics. Methods for disk-diffusion and broth-microdilution assays followed guide- It has been recommended that the risks associated with the lines from the Clinical and Laboratory Standards Institute [24,25].
transfer of antibiotic resistant bacteria through direct contact SensititreTM panels (Trek Diagnostic systems, UK) were used for exposure to ornamental fish should be determined [19]. It is also broth microdilution tests, and antibiotic discs (Abtek Biologicals important for the ornamental fish industry to recognize the extent Ltd, Liverpool, UK) for disc-diffusion tests. The MIC values for a to which the bacteria associated with ornamental fish have subset of isolates to six antimicrobials (Table S4) were also determined using laboratory prepared broth-microdilution assays, Towards these overall aims, a characterization of antibiotic as recommended by CLSI [25]. The antimicrobials used in testing tolerance in Aeromonas spp. present in ornamental fish and carriage were representative of those commonly used to control diseases water samples was undertaken. Aeromonas spp. were selected as caused by Gram negative bacteria in human and veterinary some species, e.g. A. hydrophila, include pathotypes of clinical medicine, including those used in aquaculture. A total of 33 significance for both fish and humans [20] and are also ubiquitous, isolates recovered by Cefas between 1992–2004 from coldwater representative members of the aquatic microbial community. As species, goldfish (Carassius auratus; 12/33 tested isolates), koi carp well as determining their tolerance to a range of antimicrobials, (Cyprinus carpio; 15/33 tested isolates) and other species (6/33), the presence of select resistance genes in isolates was determined were also included in the analysis. These species are typically using a miniaturized microarray. To give an indication of the reared at temperatures less then 20uC. All historical Aeromonas extent to which the whole microbial communities present in isolates were recovered aseptically from the Cefas Bacterial carriage water may be enriched for genes conferring resistance to Culture Collection (BCC) held at 280uC in ProtectTM vials, antimicrobials, a culture-independent characterization of class 1 freeze dried cultures or in liquid nitrogen.
integron diversity in water samples was also undertaken.
In the absence of published resistance breakpoints for Aero- monads, tolerance to the antimicrobials tested was determined by examination of the frequency distribution of minimum inhibitoryconcentration (MIC) and disc diffusion diameter values for all the 127 isolates examined. The boundaries of the populations of isolates A total of 25 consignments, each containing different varieties showing clearly increased tolerance (non wild type phenotype) and and species of warm water ornamental fish, were sampled between those with higher susceptibility were then defined, with those in February and April 2008. Fish species and associated carriage between determined as of intermediate (I) susceptibility. Two waters sampled included, guppies (Poecilia reticulata Peters), control strains, E. coli ATCC 25922, recommended by both CLSI threadfin rainbow (Iriatherina werneri Meinken), celebes rainbow guidelines, and A. hydrophila NCIMB 9240T, were also included in (Telmatherina ladigesi Ahl), neon gold barb (Puntius semifasciolatus parallel in all testing. The range of concentrations of antibiotics and Gu¨nther), harlequin rasbora (Trigonostigma heteromorpha Duncker), the epidemiological cut-off tolerance values used for both MIC and neon tetra species (Paracheirodon innesi Myers and Hyphessobrycon disc diffusion testing are shown in Tables S1 and S2. All tests were herbertaxelrodi Ge´ry), red wag platy (Xiphophorus maculates), kuhli performed at 2262uC and results read after between 44–48 h loach (Pangio kuhlii Valenciennes) and silver molly (Poecilia shenops).
These species are typically reared at temperatures between 24 and 30 uC. The fish were predominantly shipped to the UK fromSingapore (19/25 samples), although wild caught species shipped Detection of Antibiotic Resistance Genes Using the from Columbia, Guyana and Brazil were also included. Bags were Identibac AMR-veTM Miniaturised Micro-Array either intercepted at the UK’s London Heathrow Airport en route A total of 23 isolates were analysed for the presence of 54 different to distributors, or the morning after they arrived in the UK from a antimicrobial resistance genes using the Identibac AMR-veTM local wholesale distributor (Weymouth, Dorset). Fish were kept miniaturised micro-array (http://www.identibac.com/identibac_ and transported to the laboratory in their original carriage water amr.php). Isolates were analysed following manufacturers instruc- in sealed bags in boxes, with enough oxygen to survive 24–48 h tions as previously described [26], with minor modifications. Isolates before sampling. Samples (10 ml) of carriage water and whole fish were grown overnight at 22uC on Tryptone Soya Agar. Lysates homogenised in phosphate buffered saline (PBS) were seeded onto were prepared by suspending a loopful of culture in 400 ml lysis solid Aeromonas media (Oxoid, Basingstoke UK). Resultant buffer (0.1M Tris HCl, 0.005% Tween 20, Proteinase K). This was presumptive colonies were subcultured and confirmed as Aeromonas incubated at 65uC for 2 h with regular vortexing, followed by spp., based on phenotypic testing criteria (Gram negative, heating to 95uC for 15 min. Approximately two micrograms of cytochrome oxidase and catalase positive, motile, rods able to resultant genomic DNA released from the cells were linearly ferment and oxidize glucose, with API 20NE system (Biomerieux, amplified using the set of antisense primers provided and France) biochemical test profiles typical of Aeromonas spp.). A subset simultaneously biotin labeled. Single-stranded labeled amplified of 41 isolates, including all those described in Table 1, were further products were hybridised to the arrays and a signal intensity value confirmed as Aeromonas spp., based on partial 16S rRNA gene was determined for each spot on the array by calculating the sequencing [21]. The gyrA and gyrB genes of a further four of these quantitative staining value using IconoClust software (version 2; isolates were partially sequenced, using previously described CLONDIAG). The mean signal value for the three replicate spots methods [22]. This was to confirm 16S rRNA gene based per probe was used for analysis with a signal intensity greater than identifications and to identify potential mutations in the quinolone 0.3 considered positive, and a signal intensity lower than 0.1 as December 2009 | Volume 4 | Issue 12 | e8388 December 2009 | Volume 4 | Issue 12 | e8388 negative. Those with an intensity value between 0.1 and 0.3 were M13F and M13R primers. Clones that contained inserts were cryopreserved in 50% glycerol. Colonies were then lifted with asterile wooden pick, and stabbed into ampicillin-supplemented PCR Detection of Antibiotic Resistance Genes, Class 1 lauria agar wells on a 96 well plate. The plate was then incubated overnight at 37uC prior to transfer to GATC Biotech (Germany)for plasmid extraction and sequencing using M13F and M13R Water bacterial community DNA samples and isolates were also directly screened for a range of resistance genes, class 1 integronsand incompatability group (Inc) A/C and IncN plasmids, usingpublished primers and PCR protocols (Table S3). Template DNA for use in PCR procedures was prepared from isolates by heating The DNA sequences of a number of the class 1 integrons, tet(A), colonies suspended in 100 ml molecular grade water at 94uC for tet(D), tet(E) genes and partial 16S rRNA genes obtained in this 5 min and/or DNAzolTM (Invitrogen) based DNA extraction, study were deposited in EMBL under the following accession following the manufacturer’s instructions. The presence of floR, IncN and IncA/C markers were initially assessed by multiplexPCR utilizing the HotStarTaq Plus Master Mix Kit (Qiagen) and the primer sets listed in Table S3. PCR conditions were as follows: 5 min activation step at 95uC followed by 35 cycles of 94uC for Half (47/94) of the isolates recovered from warmwater species 1 min, 55uC for 1 min, 72uC for 1 min, and then a final 10 min in 2008 were individually tolerant to $15 different antibiotics (Table 2). This multi-drug tolerance (MDT) was broad ranging,with 64% of the isolates shown to be individually tolerant to General PCR-Conditions and DNA Sequencing antimicrobials from seven or more different structural classes of PCR reaction mixtures (50 ml) generally contained sterile antimicrobial (Figure 1). Many of the isolates recovered from molecular-grade water, 1x reaction buffer, 1.5 mM magnesium coldwater species, were also shown to be MDT, with 27% chloride, 1.25 U (0.25 ml) Go Taq polymerase (Promega, UK), individually tolerant to antimicrobials from $3 structural classes (Figure 1). There were some antimicrobials that most bacteria 50 pmol of each primer. 2.5 ml of template was then added to tested were highly susceptible to; these included third and fourth the reaction mixture and samples heated at 94u C for 5 min in a generation cephalosporins (ceftriaxone, ceftazidime, cefpodoxime, PTC-225 Peltier thermocycler (MJ Research Inc., Massachusetts, cefepime and moxalactam) and the carbapenems, imipenem and USA.). Cycling consisted of 35 cycles of 94uC for 1 min, annealing meropenem (Table 2). However, some isolates were tolerant to temperature as indicated for 1 min, 72uC for 1 min, and then a these antimicrobials, including an A. punctata-like isolate recovered final 10 min extension at 72uC. All isolates positive for the from a Singapore guppy sample (Table 1; isolate 08063). This presence of bla_TEM, florR, qnrS, tet(A), tet(E), tet(D) genes and IncA/ organism was tolerant to 28 of the antimicrobials tested, including C plasmids were reconfirmed by repeat amplication, in parallel moxalactam, piperacillin, cefpodixime and imipenem (Table 1).
with previously negative isolates. For isolates positive for floR and The organism was also determined to have heightened tolerance IncA/C plasmid markers in the triplex PCR, this was confirmed to aztreonam (MIC 16 mg l21) and cefepime (MIC 8 mg l21). The using separate single target PCR. The identity of a number of the MIC values for six of the antimicrobials were also determined for resistance genes was also determined by sequencing the resultant 27 isolates, including all those listed in Table 1 (Table S4). Isolates PCR amplicons. In some cases (for class 1 integrons obtained from were shown to grow in concentrations of up to 384 mg L21 the isolates and amplicons generated using qnrS primers; Table ciprofloxacin (3/27 isolates) and oxytetracycline (14/27 isolates). A S3), PCR products were cloned using the Promega pGEM-T number of isolates grew in 768 mg L21 of nalidixic acid (15/27 system (Promega, UK). Sequencing was performed either at the isolates) and oxolinic acid (7/27 isolates). Isolate 08063 also grew Cefas Weymouth Laboratory, using an ABI 3700 DNA analyser, in the highest concentrations tested (768 mgL21) for chloram- or by GATC Biotech Germany (see below).
phenicol and streptomycin (1024 mg L21). A total of 16/27 Sequence data was assembled and initially analysed using the isolates grew in the highest concentration of suplhadiazine/ Sequencher program (Gene Codes Corp., Ann Arbor, MI, USA).
trimethoprim tested (.730/38.4 mgL21). In total 11/27 of theisolates had MIC values for chloramphenicol of at least 96 mgL21.
Culture-Independent Cloning of Partial Class 1 Integronsfrom Carriage Water Microbial Communities Miniaturised Microarray and PCR Detection of Antibiotic For this, approximately 300 ml of each carriage water sample was Resistance Genes, Class 1 Integrons and Plasmid Markers vacuum filtered through 0.45 mm (Difco) membranes until saturated (3–5 filters per sample). Filters were then placed in 25 ml of A total of 23 isolates were analysed for the presence of 54 molecular grade water (VWR, Leics, UK) in 50 ml falcon tubes different antimicrobial resistance genes using the Identibac AMR- (Alpha laboratories, UK). These were then vortexed to resuspend the veTM miniaturised micro-array. DNA probes for a range of bacteria. The filters were removed with sterile forceps and the different resistance genes hybridized with DNA prepared from the suspension was centrifuged at 3000 g for 15 min. The supernatant Aeromonas isolates (Table 1). Positive probes included those directed was discarded and the pellet was resuspended in 200 ml of molecular at genes mediating resistance to tetracyclines, with samples positive grade water by vortexing thoroughly. Template DNA was then by this method for the presence of tet(A), tet(C), tet(D), tet(E) and extracted using DNAzolTM (Invitrogen) following the manufacturer’s tet(G). The isolates were also tested in parallel using conventional instructions. Extractions were stored at 220uC. Partial copies of class PCR-based detection for tet A-F (Table S3). The presence of tet(A), 1 integrons were PCR-amplified from the carriage water metage- tet(D) and tet(E) was also confirmed by both PCR and DNA nomic DNA samples using the primers 5CS/3CS (Table S3).
sequencing in a number of isolates. However there were some PCR amplicons were cloned as described above. Resultant discrepancies, with a number of isolates positive for the presence of clones were screened for the presence of inserts by PCR using tet genes by miniaturised microarray, even though these genes December 2009 | Volume 4 | Issue 12 | e8388 Table 2. Proportions (%) of warm water and historical cold water species isolates showing atolerance to 34 differentantimicrobials.
% tolerant isolates (% intermediate tolerant) % tolerant isolates (% intermediate tolerant) 94 warm water species isolates and 33 coldwater species isolates were tested in total.
aTesting was done in compliance with CLSI guidelines (CLSI 2004a; CLSI 2004b). Range of concentrations of antimicrobials tested and interpretative tolerance criteria bSXT = sulphamethoxazole/trimethoprim.
doi:10.1371/journal.pone.0008388.t002 Figure 1. Proportions (%) of isolates recovered from warm water and coldwater species showing tolerance to numbers of differentstructural classes of antimicrobial. Resistance was seen to representatives of the following eleven structural classes: aminoglycocides, second,third and fourth generation cephalosporins, carbapenems, foliate pathway inhibitors, nitrofurans, phenicols, quinolones, fluoroquinolones andtetracyclines. Note, all isolates also displayed expected wild type resistance to penicillins/first generation cephalosporins (not included in figure).
doi:10.1371/journal.pone.0008388.g001 December 2009 | Volume 4 | Issue 12 | e8388 could not be detected by PCR (Table 1). These isolates were also different gene cassettes, including; dihydrofolate reductase types nearly all tolerant to tetracycline and oxytetracycline (Tables 1 dfrA1, dfrA17, dfrA5, dfrA21, dfrA22, dfrA23; the aminoglycoside adenyltransferase types aadA1, aadA2; and the quaternary Genes mediating resistance to betalactams (bla_OXA7 and bla_TEM1) were detected in many of the isolates by microarray (Table 3). The 19 other inserts sequenced contained other cloned and, in the case of bla_TEM1, PCR. A number of isolates were also sections of microbial community DNA (partial copies of bacterial positive for the presence of qnrS using the miniaturized DNA encoding polymerase genes and other bacterial DNA; data miocroarray. All these isolates and a number of other isolates were also tested in parallel by PCR for qnrS by PCR, with 47 of 94recently isolated bacteria from warmwater species, shown to be positive by this alternative method. One organism originallyisolated in 1998 (isolate 98013; Table 1) was also positive. Out of Other studies have also reported high levels of resistance in these 48 amplicons, 24 were double-digested with the restriction bacteria isolated from warmwater ornamental species [3,5].
enzymes HhaI and RsaI and shown to share the same restriction Tolerance to many of these antimicrobials has likely been driven profile. Four amplicons were sequenced and shown to share 100% by their use in the pet fish trade. In particular, oxytetracycline, identity with a qnrS2 sequence (EU439941), derived from an nitrofurans (e.g. furazolidone), potentiated suphonamides, and Aeromonas sp. isolated from the river Seine in France [27].
oxolinic acid, which many of the tested organisms showed high The florfenicol and chloramphenicol resistance gene floR [28] tolerance to (Table 2 and Table S4), have been used for many was detected by miniaturized microarray in three out of 23 years [3]; Cefas Fish Health Inspectorate Staff, Personal isolates. Follow up PCR analysis confirmed the presence of this Observations). Two small-scale surveys in the USA in the early gene in 16 out of 93 recent bacterial isolates from ornamental fish 1990’s also showed a difference in relative tolerance between species. It was not detected in any of the historical coldwater isolates recovered from warmwater and coldwater species [3,30].
isolates. Additionally, the floR amplicon was detected in 18/21 Tolerance to tetracyclines was particularly widespread across all of the carriage water microbial community DNA samples (not screened isolates (Table 2). It is well established that transferable tet genes are widely disseminated in the aquatic fish farming Correlating with observed resistance to aminoglycocides, environment [11–14,31], and similar genes were identified in aminoglycoside transferase genes (aadA1, aadA2, aac61b) were some of the isolates in this study by both PCR and miniaturized detected in isolates.(e.g. isolates 93024, 08041, 08049, 08063, microarray. There were also a number of isolates containing DNA 08094 and 08095; Table 1) Miniaturized microarray analysis also that hybridized with tet probes in the microarray, but were identified the likely presence of dihydrofolate reductase (dfrA1, otherwise negative for these and similar genes by PCR. One dfrA12, dfrA13), as well as sul1, that mediates resistance to explanation could be that these isolates contained novel variants of sulmethoprim, in a range of isolates that were resistant to known tet genes that hybrized with the probes used for sulphamethoxazole/trimethoprim (Table 1). Fifty percent (56/ miniaturized microarray analysis, but which were not comple- 112) of the isolates tested were also confirmed as positive for class 1 mentary to the PCR primer sets used. More detailed genetic integrases by PCR. Additionally, DNA sequencing of class 1 characterization, that was beyond the scope of this study (e.g.
integron PCR amplicons from five example isolates identified whole genome sequencing or tetracycline-directed cloning), is antibiotic resistance gene cassettes (Table 1). These included likely required to accurately determine the genetic basis to confirming the presence of genes also detected by microarray in tetracycline resistance in these isolates. It should be borne in these isolates (dfrA1 and dfrA12, aadA1 and aadA2), a gene encoding mind that the design of the probes on the array was biased toward a quaterinary ammonium drug pump qacE2, arr2 that mediates known sequences of Gram negative organisms of human health rafampacin resistance, as well as the expected int1 gene(Table 1).
Two gene cassettes that encode proteins of unknown function, that There were also high levels of tolerance observed to all the have also been identified by other workers in class 1 integrons from quinolones and fluoroquinolones, particularly in the organisms a variety of clinical bacterial isolates, were also identified, orfF and isolated from warmwater species (Table 2; Table S4). It is orfC. Three of the isolates were also shown to be positive for the interesting to note that Dixon et al. [3] only reported relatively low IncA/C plasmid marker by PCR, but none of the isolates were tolerance to the fluoroquinolone, sarafloxacin, included in that positive for IncN plasmid markers (Fig. S1). Plasmid markers were study. Although care should be taken in directly comparing results detected by PCR in 8/21 (IncN) and 11/21 (IncA/C) carriage from limited surveys generated using different methodologies, it is water microbial community DNA samples (not shown).
possible that the tolerance of pet fish associated bacteria to thefluoroquinolones has increased since they were first introduced for Culture Independent Cloning of Partial Class 1 Integrons widespread use in clinical and veterinary medicine in the 1980s.
from Carriage Water Microbial Communities Such tolerance is often mediated by mutations to chromosomal The class 1 integrons and associated gene cassettes present in genes [32], with resistance in a number of aquatic bacterial species the microbial communities in selected water samples were also linked to changes in the genes coding for DNA gyrase and examined using a culture-independent approach. Copies of partial topoisomerase IV enzymes [22,33–34]. The quinolone resistance class 1 integrons were directly PCR-amplified from water samples, determining regions of the gyrA and gyrB genes of five cloned into E. coli and sequenced. The inserts from a total of 58 representative isolates were sequenced (isolates 08020, 08030, clones obtained from five carriage water samples were sequenced 08033, 08043 and 08094; Table 1), with no coding mutations and shown to be between 103 and 808 bases in length. Initial noted, suggesting other mechanisms may be responsible. Trans- BLAST comparisons [29] with GenBank deposited DNA ferable, plasmid-mediated resistance is increasingly recognized sequences determined that 39 of these inserts contained copies [35]. The finding of qnrS2 [27] at such high prevalence, in of sections of class 1 integrons. Further comparison with class 1 historical and more recent Aeromonas isolates recovered from integrons in the Integrall database (http://integrall.bio.ua.pt/) ornamental fish suggests it may be ubiquitous in bacteria in the showed that these partial class 1 integrons contained a number of ornamental fish trade. Its role in observed tolerance to quinolones December 2009 | Volume 4 | Issue 12 | e8388 Table 3. Resistance gene cassettes identified in copies of class 1 integrons obtained by PCR directly from samples of carriagewater microbial community DNA and cloned into E. coli.
aadA1 (2) aadA2 (2) dfrA5 dfrA17 dfrA27 qacE2 (4) qacE2 (3) aadA1 (3) dfrA21 dfrA22 (3) dfrA23 Table 3 footnotes.
aResistance gene cassettes were identified by comparison with sequences in the integrall database http://integrall.bio.ua.pt/. The sequences of nine of the 38 partial class 1 integron DNA sequences obtained were deposited in EMBL under the accession numbers FM957877 to FM957885.
baad, aminoglycoside adenylyltransferase, encoding streptomycin-spectinomycin resistance protein; dfr, dihydrofolate reductase genes mediating trimethoprim resistance; qacE2, gene encoding a quaternary ammonium resistance compound protein (multidrug pump); intI1, integrase: site specific recombination (attI and attCsite).
and fluoroquinolones in isolates carrying the gene is equivocal as gene cassettes characterized was associated with antibiotic and this gene typically mediates only low level resistance to these biocide resistance. This suggests that the carriage water microbial communities examined were enriched for class 1 integrons Chloramphenicol and florfenicol tolerance was also observed containing antimicrobial resistance gene cassettes.
for many of the organisms associated with warmwater species The detection of IncA/C plasmids in three of the isolates (Tables 1 and 2). It was also shown that a relatively high recovered from warm water species was noteworthy. Recent work proportion of the isolates were also positive for floR. Dissemination [15–17] has shown that IncA/C plasmids are responsible for self- of genes conferring resistance to florfenicol is of concern, as in transmissible antibiotic resistance in North American aquaculture many countries, including the UK, it has only relatively recently pathogens, as well as being increasingly important in veterinary been licensed for use in animals, including fish, destined for and human medicine [15,40–42]. Preliminary conjugal transfer human consumption. Many of the isolates also contained genes experiments, using tetracycline resistance for selection, showed coding for Beta-lactamases, with blaTEM21 and blaOXA-7 both successful transfer of IncA/C plasmid markers associated with one detected (Table 1). Apart from the A. salmonicida isolate tested of the three positive isolates (isolate 08020; Table 1) to Yersinia (08078), the Aeromonas spp that were found to carry these two genes ruckeri and subsequently to E. coli ATCC25922 (using methodology are typically considered intrinsically resistant to the first generation described in [15]). In the context of Aeromonas spp. as reservoirs of cephalosporins and narrow spectrum penicillins they mediate this clinically important class of plasmids, it is noteworthy that the resistance to. It is possible that these genes were acquired in original IncA/C reference plasmid pRA1 was recovered from a association with functionally more useful genes, coded by plasmids fish-pathogenic A. hydrophila isolate in 1971 [42,43]. Work assessing or other transferable elements. As some tolerance was also noted the importance of plasmid-mediated resistance in the identified to third generation cephalosporins (Tables 1 and 2), it is suggested that this be investigated further to determine if this tolerance was Isolates were identified here that exhibited tolerance to agents mediated by transferable elements as transfer of these elements from a number of different structural classes, synthetic (i.e.
nalidixic acid, sulphamethoxazole/trimethorprim) as well as A total of 50% of the isolates were positive for class 1 integrons, naturally derived agents, and to relatively new antimicrobials similar to the levels reported in a survey of motile Aeromonads recently introduced in human medicine (i.e. ciprofloxacin) (Table recovered from freshwater fish farms [12] and the 35% reported S4). Of these resistant isolates, many demonstrated resistance to for isolates recovered from a slaughterhouse wastewater treatment multiple antibiotics in the hundreds of mg per liter range, (Table plant [36]. The proportions of bacteria that were positive for class S40. These observations suggest a ‘superbug’ phenomenon, 1 integrons appear much lower in the other aquatic environments whereby multi-antibiotic resistant isolates also demonstrate higher that have so far been sampled [37–39].
overall resistance levels. Enne et al. [44] postulate that low fitness Carriage of dfr, sul and aad genes may have contributed to the costs are associated with multi-antibiotic resistance in E. coli. The high levels of resistance noted to both foliate pathway inhibitors authors noted that, once established, combinatorial resistances and aminoglycocides seen, particularly in the organisms associated (particularly facilitated via mobile genetic elements such as with warmwater species (Table 1). Comparisons with sequences in plasmids) might be difficult to eliminate through reduction in the Integrall database of integron sequences (http://integrall.bio.
prescribing alone. These results imply that this process may not be ua.pt/) showed that very similar arrangements of resistance gene restricted to established pathogenic or opportunistic bacteria, but a cassettes to those found in bacterial isolates and water microbial phenomenon common in environmental bacteria, or bacteria with communities have previously been described in class 1 integrons established environmental reservoirs.
found in other human, fish and terrestrial animal pathogens.
These included those associated with clinical and environmental Aeromonas isolates from Taiwan [20].
A surprisingly high level of antimicrobial tolerance was All 39 of the class 1 integrons identified in the constructed clone identified in bacteria associated with warmwater ornamental library contained gene cassettes that have also been recovered species and ornamental fish carriage water. The significance of from human and veterinary clinical isolates. All but one of the 40 these tolerant bacteria from ornamental fish in acting as a December 2009 | Volume 4 | Issue 12 | e8388 potential reservoir for mobilisable antibiotic resistance should be Interpretative tolerance cut offs and range of systematically assessed. This should help prevent the potential concentrations of discs used for disc diffusion testing [25].
spread of resistance to pathogens of human and animal health Found at: doi:10.1371/journal.pone.0008388.s003 (0.03 MB importance, and improve fish welfare and treatment. Antibiotic use for prophylactic purposes should also ideally be replaced by Primers and PCR conditions used in study.
better husbandry and transport conditions and the use of Found at: doi:10.1371/journal.pone.0008388.s004 (0.03 MB MIC values (IˆJg/ml) determined for selected isolates Multiplex detection of the florfenicol resistance gene, Found at: doi:10.1371/journal.pone.0008388.s005 (0.06 MB floR, and markers for the IncA/C and IncN plasmids.
Found at: doi:10.1371/journal.pone.0008388.s001 (0.46 MB PPT) Tolerance cut off values and range of concentrations of antimicrobials used in broth microdilution testing [24]. Also Jennifer Harper provided valuable and skillful technical assistance.
shown are the ranges in MIC values recorded for the two controlstrains included in parallel during testing, E. coli NCIMB 25922 Conceived and designed the experiments: DWVJ TJW TS MJP MW CBA.
Found at: doi:10.1371/journal.pone.0008388.s002 (0.05 MB Performed the experiments: DWVJ TJW TS MJP GSER SJH ER VM.
Analyzed the data: DWVJ TJW TS MJP MW GSER SJH ER VM CBA.
Wrote the paper: DWVJ TJW TS CBA.
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