Resistance Determinants of Pseudomonas Species from Aquaculture in Australia

Members of the genus Pseudomonas are a ubiquitous group of Gram-negative, motile, rod-shaped bacteria known for their metabolic versatility with genetic and physiologic capabilities that allow them to flourish in environments hostile to many other bacteria. It is one of the most diverse bacterial genera, containing over 60 validly described species [1]. Some species of Pseudomonad particularly P. aeruginosa are opportunistic pathogens that can cause serious and life-threatening infections in immunocompromised and cystic fibrosis patients [2,3]. In aquaculture, P. aeruginosa and P. fluorescens especially are the most frequently isolated opportunistic pathogenic species [4,5] although other species may also be serious opportunistic pathogens, including P. anguilliseptica in eels, Anguilla japonica [6] P. chlororaphis in amago trout, Oncorhynchus rhodurus [7], P. plecoglossicida in ayu, P. altivelis [8] and more recently P. putida in rainbow trout, Oncorhynchus mykiss [9].


Introduction
Members of the genus Pseudomonas are a ubiquitous group of Gram-negative, motile, rod-shaped bacteria known for their metabolic versatility with genetic and physiologic capabilities that allow them to flourish in environments hostile to many other bacteria. It is one of the most diverse bacterial genera, containing over 60 validly described species [1]. Some species of Pseudomonad particularly P. aeruginosa are opportunistic pathogens that can cause serious and life-threatening infections in immunocompromised and cystic fibrosis patients [2,3]. In aquaculture, P. aeruginosa and P. fluorescens especially are the most frequently isolated opportunistic pathogenic species [4,5] although other species may also be serious opportunistic pathogens, including P. anguilliseptica in eels, Anguilla japonica [6] P. chlororaphis in amago trout, Oncorhynchus rhodurus [7], P. plecoglossicida in ayu, P. altivelis [8] and more recently P. putida in rainbow trout, Oncorhynchus mykiss [9].
The objective of this study was to identify integrons and genes coding for multidrug efflux pumps such as mexAB-oprM which is known to contribute greatly to the natural resistance seen in Pseudomonas spp. These pumps have broad substrate specificity and may act synergistically with the permeability barrier to result in significant intrinsic resistance to many antimicrobials including β-lactams, β-lactamase inhibitors, quinolones, chloramphenicol, tetracycline, trimethoprim, sulphamethoxazole and novobiocin [21,22]. Although 12 potential efflux systems of this family have been identified in the P. aeruginosa genome [23], four of them are best characterised as antibiotic transporters [24]. This study also determined the mechanisms of intrinsic antibiotic resistance and resistance to cadmium, one of the heavy metals tested. Several mechanisms have been identified within bacteria that confer cadmium resistance. These include sequestration by metallothionein-like proteins and ATPase (cad operon) and non-ATPase (czc operon) efflux pumps. Metallothionein-like proteins that sequester cadmium have been identified in the Cyanobacterium Synechococcus and P. putida, whereas the czc resistance operon has been found in Alcaligenes spp [25,26] and P. aeruginosa [27].

Bacterial strains and plasmids
One hundred and twenty nine Pseudomonas spp. isolated from healthy fish and sediments from nine freshwater trout farms in Victoria (Australia) between August and December 2004 were used in this study. Isolation, identification, heavy metal and antibiotic sensitivity testing have been previously described [28]. The positive controls used were Escherichia coli strains containing integron plasmids; pR388, a class 1 integron with a two cassette array dfrB2 and orfA and pR1033 that contains Tn1496 which in turn contains a class 1 integron with four cassettes, aac, orfE, aadA2 and cmlA, (kindly provided by Hatch Stokes, Macquarie University, Sydney, Australia) and an E. coli strain with the Int2 gene. Escherichia coli strain JIR5431 (Burdett 1996) carrying a tetM gene (kindly provided by Julian Rood, Monash University, Melbourne, Australia) and Aeromonas strains WA13 (tetA), WA14 (tetD) and WA18 (tetE) were from the culture collection of our laboratory (University of south Australia).

Total and plasmid DNA extraction
DNA was extracted by inoculating isolates into Tryptone Soy Broth (Oxoid CM0129) and incubating overnight at 30°C. A 1.5-ml amount was dispensed into Eppendorf tubes and centrifuged at 12 662 g for 5 min. The supernatant was discarded and cell pellets re-suspended in 200 µl TE buffer, and they were then heated in the heating block at 95ºC for 10 min. The samples were placed on ice to cool and then centrifuged at 12 662 g for 5 min, and the supernatants were transferred into fresh Eppendorf tubes and these served as DNA templates for polymerase chain reaction (PCR). For plasmid DNA extraction, cell pellets were extracted by alkaline lysis of cells [32].

PCR amplification of 16S rRNA
16S rRNA gene universal primers were used for the PCR amplification of the Pseudomonas spp. to confirm the identification obtained by the biochemical method. The primers used for PCR are listed in Table 1. The reaction mixture (final volume 25 μL) contained 10× PCR buffer (1.5 mm MgCl 2 , 10 mm Tris-HCl (pH 8.8 at 25°C), 50 mm KCl and 0.1% Triton X-100), 200 μm of each deoxynucleotide triphosphate, 0.5 μm L −1 of each oligonucleotide primer and 0.25 μL (0.5 U μL −1 ) of Taq polymerase (Promega) and 200ng µl −1 of Bovine serum albumin. A 5 μL volume of DNA template was added to each reaction tube. The cycling conditions were as follows: 95°C for 5 min, 30 cycles of denaturation 94°C for 30 s, annealing at 68°C for 60 s and extension at 72°C for 60 s and a final single cycle of extension at 72°C for 10 min.
The PCR products were analysed by electrophoresis on 1.5 % agarose gels, stained with ethidium bromide and visualised using the Biorad Gel Documentation system (GelDoc).

Integron PCR
Detection of integrons and the resistant gene cassette was performed using PCR amplification with the specific primers listed in Table 1. All PCR amplification was carried out in a BioRad MyCycler thermal cycler. Integrase genes Int1, Int2 and Int3 were targeted in a multiplex PCR as previously described [29] using primers Int1F-Int1R [30], Int2F-Int2R and Int3F-Int3R [31]. Assays were carried out in 25 μL volumes containing 2.5 μL of 10× PCR buffer [100 mM Tris-HCl (pH 8.8), 500 mM KCl, 15 mM MgCl 2 and 1% Triton X-100], 0.5 μL of 10 mM DNTP, 1 μL of each primer stock solution (25 pmol μL −1 )] and 0.25 μL of BioTaq polymerase (5 U μL −1 ). Three microlitres of the prepared DNA extract was added to provide the DNA template. A volume of 16.75 μL sterile milliQ water was added to make up the volume to 25 μL. All PCRs were subjected to an initial denaturation step at 94°C for 5   (30 cycles). PCR amplicons were analysed by electrophoresis on 1.5 % agarose gels and a 100-bp ladder was used as the molecular size marker (New England Biolabs).

Detection of beta lactamase resistance genes
The PCR for the amplification of beta lactamase genes bla TEM and bla SHV was carried out using primers listed in Table 1. The reaction mixture was the same as those described for integron PCR but 1 µl of 10 mM DNTP was used. The cycling conditions used were denaturation at 96°C for 5 min; 35 cycles of 96°C for 1 min, 58°C for 1 min, and 72°C for 1 min; and a final extension period of 72°C for 10 min. Electrophoresis was performed as described for integron PCR.

Detection of mexA, mexB, and oprM genes
PCR reaction mixture was as described above. PCR reactions were subjected to an initial denaturation for 5 min, then 30 cycles with 95°C for 30 s, followed by 54°C for 30 s and 72°C for 45 s with an additional elongation at 72°C for 5minutes. Electrophoresis was performed as described for integron PCR.

Detection of cadmium resistance genes
PCR reaction mixture was as described above. The cycling condition used for cadA was an initial denaturation step at 95°C for 4 min; then 35 cycles with 95°C for 30 s, followed by 61.5°C (cadA1) or 55°C (cadA2 and czrABC) for 30 s and 72°C for 80 s with an additional elongation at 72°C for 5minutes. Electrophoresis was performed as described for integrin PCR.

Sequencing
The PCR products of the gene cassette and other resistance genes were purified using an Ultraclean DNA purification kit (MO-BIO Laboratories, Inc.) and sequenced using the BigDye Terminator v3.1 1 cycle sequencing kit and analysed with an ABI 3100 Genetic Analyser (Flinders Medical Centre, South Australia). Primer sets used to sequence the genes are those listed in Table 1. Database similarity searches for the nucleotide sequences were carried out with BLAST at the National Centre of Biotechnology Information website (http://blast. ncbi.nlm.nih.gov/Blast.cgi).

Southern hybridization
In order to check for the co-location of cadA and aadA gene, the PCR products were digoxigenin (DIG)-labelled as probes as described by the manufacturer (Boehringer, Mannheim, Germany) and used in subsequent southern hybridisation procedures on plasmids [32].

Detection of integrase, streptomycin and β-lactamase resistance genes
Class 1 integrons were detected in 30 of the 129 (23%) strains investigated whereas Class 2 and class 3 integrons were not detected.
β-lactamase resistance genes bla TEM and bla SHV were investigated however, the genes were not detected in any of the isolates tested. Suggesting a different mechanism for the ticarcillin resistance observed.
The presence of streptomycin resistance genes was investigated in representative isolates (with ≥ 16 µg/ml from previous study) using primers specific for aadA gene which is normally associated with integrons and codes for resistance to streptomycin and spectinomycin; aadA gene was detected in 28 of the isolates.

Detection of mexA, mexB, and oprM genes
mexB was detected in 85 of the 129 (66 %) isolates tested. Neither mexA nor oprM could be amplified with the primers used. No PCR product was obtained when the primers that amplifies the whole operon was used. Fifty-five of the Pseudomonas isolates positive for mexB, with a PCR product of 732 bp ( Figure 2) were from sediments and thirty were from fish.
Some of the mexB genes obtained by PCR were sequenced to confirm the gene identity. The sequence analysis of our gene showed similarities to the Resistance-Nodulation-Division family (RND) multidrug efflux transporters including mexB and its different homolog TtgB, so called because in addition to multidrug efflux it is also able to transport toluene; which have been described in Pseudomonas spp.
The sequences obtained have been deposited in the Genbank under the nucleotide sequence accession numbers listed in Table 2.

Detection of cadmium resistance genes
The Pseudomonas strains with cadmium Minimum Inhibitory Concentration (MIC) of ≥ 200 μg/ml were investigated for the presence of cadmium resistance gene. The cadA gene which was a PCR product M 1 2 3 4 5 6 7 8 9 10  (Figure 3) was detected in 59 of the 92 (64%) isolates tested. The homolog of the czc genes, called czr, was not detected in any of the isolates. Thirty-five of the Pseudomonas strains carrying cadA were from sediments and twenty-four were from fish.
Plasmid DNA was extracted from 129 Pseudomonas isolates and 55 of them were found to possess plasmids. It was found that 29 of the 59 (49%) cadA positive isolates possessed plasmids.
The southern hybridization of plasmids with the cadA as probe did not reveal the presence of cadA on the plasmid, suggesting that cadA is likely to be chromosomally located. Some of the cadA genes obtained were sequenced to confirm the gene identity. The sequence analysis confirms similarities to the cadmium translocating P-type ATPases, cadA of various Pseudomonas spp. The sequences obtained have been deposited in the Genbank under the nucleotide sequence accession numbers and are listed in Table 2.

Discussion
The association of integrons with various resistance gene cassettes in aquatic environments has been well documented [33][34][35][36][37], however there are few reports of integron and associated resistance genes in Pseudomonas spp. There is limited information on the occurrence of integrons and other resistance genes in bacteria of aquaculture origin in Australia [38,39]. Integrons are known to have the capacity to carry many antibiotic resistance genes and so the resistant Pseudomonas strains were investigated for the presence or absence of integrase genes for class 1, class 2 and class 3 integrons. In this study, Class 1 integron was detected in 30 of the 129 strains investigated. Streptomycin resistance gene aadA was detected in 28 of the 59 integrase positive strains; the primers used were specific for the detection of the aadA1a gene cassette and other closely related aadA gene cassettes (with the exception of aadA4 and aadA5). The presence of aadA and other resistance gene cassettes in different aquatic environments has been reported [33,35,37]; it is therefore not surprising to detect these genes in the isolates tested.
It is a well known fact that the members of the family Pseudomonadaceae show significant intrinsic resistance to a wide variety of structurally unrelated compounds [40,41]. The intrinsic resistance previously attributed only to non-specific impermeability of the outer membrane [42] is now known to result from a synergy with drug efflux pumps with a wide spectrum of activity [43].
One of the efflux system, mexAB-oprM that was investigated in this study encodes a tripartite pump is able to exports a wide range of antibiotics including chloramphenicol and β-lactams [21,44,45]. The detection of mexB, the major integral inner membrane efflux component of the mexAB-oprM pump in a large number of our isolates clearly suggests that some of the natural/intrinsic resistance of these isolates is partly as a result of this efflux system. A similar mechanism has been described in clinical P. aeruginosa strains which were meropenem resistant; where mexB was detected in all blaIMP, bla SPM and bla VIM negative isolates [46]. The sequence analysis of isolates from this study demonstrated similarity to the RND multidrug efflux transporter mexB and its homologue TtgB which in addition to multidrug efflux also transports toluene out of the cell and have been described in P. putida [47,48], and in P. fluorescens Pf-5 genome sequence. No PCR product was obtained when the primers that amplifies the whole operon mexAB-oprM was used, this could be that it was too large to amplify. β-lactamase resistance genes bla TEM and bla SHV were investigated however, the genes were not detected in any of the isolates tested suggesting a different mechanism for the ticarcillin resistance observed.
In addition to some antibiotic resistance mechanisms, we also report the incidence of the heavy metal resistance determinant, cadA gene in 59 of the 92 strains tested. A well characterised cadmium resistance system in Gram-negative bacteria is the cadmium, zinc and cobalt (czc) resistance determinant of Alcaligenes eutrophus where the czcC, czcB, and czcA proteins comprise an active efflux mechanism driven by antiporter, rather than a cation transporting ATPase [49]. A homolog of the czc gene, called czr, which confers cadmium and zinc resistance, have been identified in the chromosome of P. aeruginosa and appear to be highly conserved in environmental isolates of that species [27]; however this resistance determinant was not detected in our isolates. Instead we detected cadA which is mostly found in Gram-positive bacteria [50][51][52][53]. The genome sequences of several Gram-negative bacteria have revealed homologs of cadA [54,55] and there are some reports of cadA-encoded cadmium resistance being identified in some Pseudomonas species [56,57]. The cadA gene detected in this study is believed to be chromosomally located since southern hybridisation did not detect the presence of the gene on the plasmids in any of the isolates tested. Chromosomally located cadA have also been reported in P. putida [57].
There are reports of correlation between heavy metal contamination  and antimicrobial resistance [58,59] and this is a potential public health concern. Pseudomonas spp. carrying integrons, efflux gene and cadmium resistance genes are present in farm-raised fish and sediments even though no antibiotics were licensed for use in Australian aquaculture at the time of the study.

Conclusion
Int1 of Class one integrons and aadA genes were detected in addition to mexB, the major integral inner membrane efflux component of the mexAB-oprM pump which would account for some of the intrinsic resistance. However, this pump can also mediate resistance to many antibiotics. Cadmium resistance gene cadA was also detected in the isolates. These results suggest that bacteria from farmed raised fish and environments in Australia are potential sources of antibiotic and heavy metal resistance genes in the environment. Established environmental reservoirs of resistance genes can lead to contamination of food and human water sources and this has great implications for public health.