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Triclosan Resistance in a Bacterial Fish Pathogen, Aeromonas salmonicida subsp. salmonicida, is Mediated by an Enoyl Reductase, FabV
Triclosan Resistance in a Bacterial Fish Pathogen, Aeromonas salmonicida subsp. salmonicida, is Mediated by an Enoyl Reductase, FabV
Journal of Microbiology and Biotechnology. 2015. Apr, 25(4): 511-520
Copyright © 2015, The Korean Society For Microbiology And Biotechnology
  • Received : July 09, 2014
  • Accepted : November 04, 2014
  • Published : April 28, 2015
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About the Authors
Raees Khan
Department of Applied Biology, Dong-A University, Busan 604-714, Republic of Korea
Myung Hwan Lee
Department of Applied Biology, Dong-A University, Busan 604-714, Republic of Korea
Haejin Joo
Department of Applied Biology, Dong-A University, Busan 604-714, Republic of Korea
Yong-Hoon Jung
Department of Applied Bioscience, Dong-A University, Busan 604-714, Republic of Korea
Shabir Ahmad
Department of Applied Biology, Dong-A University, Busan 604-714, Republic of Korea
Jinhee Choi
Department of Applied Bioscience, Dong-A University, Busan 604-714, Republic of Korea
Seon-Woo Lee
Department of Applied Bioscience, Dong-A University, Busan 604-714, Republic of Korea
seonlee@dau.ac.kr

Abstract
Triclosan, the widely used biocide, specifically targets enoyl-acyl carrier protein reductase (ENR) in the bacterial fatty acid synthesis system. Although the fish pathogen Aeromonas salmonicida subsp. salmonicida exhibits triclosan resistance, the nature of this resistance has not been elucidated. Here, we aimed to characterize the triclosan resistance of A. salmonicida subsp. salmonicida causing furunculosis. The fosmid library of triclosan-resistant A. salmonicida subsp. salmonicida was constructed to select a fosmid clone showing triclosan resistance. With the fosmid clone showing triclosan resistance, a subsequent secondary library search resulted in the selection of subclone pTSR-1. DNA sequence analysis of pTSR-1 revealed the presence of a chromosomal-borne fabV -encoding ENR homolog. The ENR of A. salmonicida (FabVas) exhibited significant homology with previously known FabV, including the catalytic domain YX (8) K. fabVas introduction into E. coli dramatically increased its resistance to triclosan. Heterologous expression of FabVas might functionally replace the triclosan-sensitive FabI in vivo to confer E. coli with triclosan resistance. A genome-wide search for fabVas homologs revealed the presence of an additional fabV gene ( fabVas2 ) paralog in A. salmonicida strains and the fabVas orthologs from other gram-negative fish pathogens. Both of the potential FabV ENRs expressed similarly with or without triclosan supplement. This is the first report about the presence of two potential FabV ENRs in a single pathogenic bacterium. Our result suggests that triclosan-resistant ENRs are widely distributed in various bacteria in nature, and the wide use of this biocide can spread these triclosan-tolerant ENRs among fish pathogens and other pathogenic bacteria.
Keywords
Introduction
Antibiotic resistance and the growing number of multidrug-resistant bacteria has become a global public health concern [43] . One of the major concerns about antibiotic resistance is its rapid development over time, from resistance to single class of antibiotics to multidrug resistance, and finally to extreme drug resistance, raising a challenge for the development of more effective antibiotics [41] . Anthropogenic activities such as the use of biocides [12] and agricultural practices have significantly increased the emergence of antibiotic-resistant bacteria in the environment, with recent examples of multidrug-resistant genes from marine environments [1 , 23] , farms [49] , soils [5] , and wastewater treatment plants [34] .
Triclosan (5-chloro-2-(2,4-dichlorophenoxy)phenol) is a biocide used in hand soaps, toothpaste, body washes, household plastics, deodorants, fabrics, chopping boards, shampoos, and a variety of various personal care and other commercial products. It exhibits broad-spectrum activity against many microorganisms, including gram-positive and gram-negative bacteria, some fungi, and mycobacteria (to some extent) [36] . Triclosan specifically inhibits the bacterial fatty acid biosynthesis system (FAS) by binding to the enoyl substrate site of enoyl-acyl carrier protein (ACP) reductase (ENR). The detailed mechanism for ENR inhibition by triclosan includes ternary complex formation by accelerated binding of NAD + and triclosan to the enoyl substrate site of FabI, the triclosan target ENR [14] . However, triclosan-resistant bacteria are abundant in nature, and many mechanisms for triclosan resistance are known, namely (i) overexpression of ENR [46] ; (ii) presence of mutated and/or triclosan-tolerant ENR [26] ; (iii) the moderation of outer membrane [35] ; and (iv) the upregulation of efflux pumps [27 , 46] . More recently a number of other target genes from Escherichia coli ( pgsA, rcsA, gapC, ahpF, ascF ) conferring resistance to triclosan have been identified as potential targets for triclosan [47] . Triclosan has also been known to induce cross-resistance in various microorganisms such as Pseudomonas aeruginosa [7] , Salmonella enterica serovar Typhimurium [2] , Staphylococcus aureus [38] , Mycobacterium smegmatis [26] , Acinetobacter baumannii [6] , and E. coli [47] .
Most of the FAS-II enzymes are relatively conserved among bacteria [18] , except those catalyzing the last step of the fatty acid elongation cycle, namely, the ENR, resulting in the formation of a saturated acyl-ACP by an NAD(P)H-dependent reduction of the double bond of enoyl-ACP. Initially, this reaction was known to be catalyzed by the product fabI , considered a potent target of diazaborine compounds. This enzyme was later shown to be the target for triclosan [28] . A number of missense mutations were also reported from fabI , leading to triclosan resistance. So far, four ENR isozymes have been reported from bacteria, namely FabI, which is the only ENR in E. coli [4] , FabL [16] , FabV [25] , and FabK [15] . Except for FabK, which is a TIM barrel flavoprotein, all other ENRs are members of the short-chain dehydrogenase/reductase (SDR) superfamily [24] . Most of the bacterial groups contain a chromosomal fabI gene, whose products contain a conserved Tyr-156–(Xaa) 6 –Lys-163 (according to amino acids numbering in FabI of E. coli ) catalytic dyad, sharing about 40% similarity to E. coli FabI [25] . FabL, a moderately triclosan-resistant NADPH-dependent ENR, was identified in Bacillus subtilis . Although FabL shares a degree of sequence homology and a similar catalytic dyad with FabI, it is not a member of the FabI family [16] . Recently, a third class of ENR, FabV, was reported from Vibrio cholerae , which is completely refractory to triclosan inhibition. FabV is well conserved among a variety of organisms, including several clinically important pathogens such as P. aeruginosa , Yersinia pestis , and Burkholderia species [25] . This enzyme has a different catalytic domain (Tyr-Xaa 8 -Lys). Unlike FabI and FabL, which have a space of six amino acid residues between the active site tyrosine and lysine, this enzyme has a space of eight residues in the catalytic dyad. This catalytic domain differs from the other SDR superfamily, enclosing seven amino acid residues between tyrosine and lysine [25] ; moreover, this enzyme is 60% larger than the typical SDR family members [31] .
The genus Aeromonas comprises 14 bacterial species, mostly human pathogens and several fish pathogens, and is widespread in aquatic environments [32] . A. hydrophila , A. veronii biovar sobria , A. caviae , A. jandaei , A. veronii biovar veronii , A. schubertii , and A. trota have been associated with various human infections, including gastroenteritis, wound infections, and septicemia [11] . A. salmonicida is a nonmotile aeromonad, the etiological agent of furunculosis, a condition of bacterial septicemia in fish, and specifically salmonid fish [8] ; however, the atypical type of A. salmonicida subsp. salmonicida has a broad host range, including even non-salmonid fish [43] .
Genome analysis of A. salmonicida subsp. salmonicida A449 chromosome and two large plasmids [32] has revealed that this organism carries more than 25 genes for multidrug resistance and major facilitator efflux family proteins to counteract antimicrobials [9 , 33] . As triclosan from all sources finally makes it way to water (ultimately sea water), marine bacteria must have evolved certain mechanisms to tolerate this toxic biocide in their corresponding environment. In this study, we aimed to investigate the triclosan resistance mechanism of A. salmonicida . To our best knowledge, this is the first study describing the triclosan resistance mechanism specifically in A. salmonicida subsp. salmonicida , and possibly in most of the other known fish pathogenic bacteria.
Materials and Methods
- Bacterial Strains, Plasmids, and Culture Conditions
The bacterial strains and plasmids used in this study are described in Table 1 . E. coli strains DH5α and EPI-300 were routinely grown at 37℃ in Luria-Bertani (LB) broth or LB agar, while A. salmonicida subsp. salmonicida strain 14791 ( AS 14791) was grown at 25℃ on tryptone soya medium (Oxoid, UK) supplemented with the desired antibiotics. The antibiotic concentration used for susceptibility testing was as follows: ampicillin 100 µg/ml; tetracycline 20 µg/ml; chloramphenicol 30 µg/ml; and triclosan 1–100 µg/ml. Triclosan was purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Fosmid pCC1FOS (Epicentre, USA) was used to construct the genomic library, whereas pUC119 and pRK415 were used for further subcloning experiments. The AS 14791 strain was obtained from the Korean Agricultural Culture Collection. In order to examine its resistance to triclosan, this strain was grown on tryptone soya medium supplemented with 20 µg/ml of triclosan.
Strains and plasmids used in this work.
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aApr, ampicillin resistance; Cpr, chloramphenicol resistance; Tcr, tetracycline resistance.
- Determination of Minimum Inhibitory Concentration (MIC)
In order to determine the MIC of triclosan, E. coli EPI-300 cells were first grown to an OD 600 of 1.0, and this bacterial suspension was serially diluted up to 1 × 10 5 CFU/ml. The cell suspension (1 × 10 5 CFU/ml) was spread onto the LB agar medium containing triclosan in a range of 0.1–10 µg/ml. LB plates were incubated at 37℃ for 3 days and bacterial colony formation was examined at regular intervals of 24 h. The lowest concentration of triclosan that prevented the bacterial growth of E. coli EPI-300 was considered as the MIC for triclosan. This experiment included triplicates for each concentration of triclosan.
- General DNA Manipulations
Standard recombinant DNA techniques were followed using the method previously described [37] . DNA sequencing and primer synthesis were performed commercially at the DNA sequencing facility of MacroGen (Seoul, Korea). Sequence comparisons (nucleotides/amino acids) were performed using the BLAST and ORF finder online service provided by the National Center for Biotechnology Information (NCBI, http://blast.ncbi.nlm.nih.gov ). Multiple alignment analysis was performed using BioEdit software in combination with GeneDoc ( http://www.cris.com/~Ketchup/genedoc.shtml ).
- Construction of Genomic Library and Selection of Clones Showing Triclosan Resistance
The genomic library of AS 14791 was constructed in E. coli EPI-300 using the fosmid pCC1FOS (Epicentre). Genomic DNA was isolated with a Dokdo-prep bacterial genomic DNA purification kit according to the manufacturer’s instructions. The genomic DNA was size-fractionated in a 0.5% low-melting-point agarose gel and DNA fragments over 30 kb were collected for library construction. The purified genomic DNA was ligated into a linearized pCC1FOS vector. The ligation mixture was then packaged into lambda phages using packaging extracts, and subsequently introduced into E. coli EPI-300 to select transformants on LB agar supplemented with chloramphenicol. These genomic libraries in E. coli were stored at -80℃ in cryotubes as clone pools with over 500 clones in each pool.
To select triclosan-resistant clones from the fosmid library, the library pool stocks were diluted in a buffer (per liter: NaCl, 8.5 g; KH 2 PO 4 , 0.3 g; Na 2 HPO 4 , 0.6 g; MgSO 4 , 0.2 g; gelatin, 0.1 g), and genomic clones of AS 14791 were spread on LB agar containing 30 µg/ml of chloramphenicol and 10 µg/ml of triclosan. The number of clones per plate was adjusted to approximately 500 by dilution of the library stock, and at least 5-fold the number per initial stock was used to select triclosan-resistant clones. Growing E. coli colonies were picked on LB with triclosan and further tested at higher concentrations of triclosan up to 100 µg/ml. Pure cultures of triclosan-resistant clones were processed for fosmid isolation followed by Bam HI restriction digestion, and the unique clones were finally selected based on Bam HI restriction profiles.
Since all selected clones showed a similar restriction pattern, we chose one of the selected fosmid library clones carrying pTSR with a 30 kb insert; the pTSR was partially digested with Sau 3AI to subclone the triclosan-resistant gene. DNA fragments of 1–10 kb were gel purified, lig ated into the Bam HI site of pUC119, and a shotgun library was transformed into E. coli . Plasmid restriction profiling of transformants showing elevated levels of triclosan resistance was compared to select the subclone carrying the smallest DNA insert. In order to eliminate the high copy number effect of pUC119, the insert DNA was also transferred into the low-copy-number vector pRK415, resulting in pTSRK415. Triclosan resistance of E. coli carrying pTSRK415 was also investigated.
- RNA Extraction fromAeromonas salmonicidasubsp.salmonicidaand Reverse-Transcription Quantitative PCR (RT-qPCR)
The relative RNA expression levels of fabVas and fabVas2 were determined by RT-qPCR. Bacterial total RNA was isolated using the Hybrid-R RNA extraction kit (GeneAll Bio Inc., Seoul, Korea) from bacterial culture at the late exponential growth stage after 48 h incubation in the absence and presence of triclosan (5 µg/ml) at 25℃. The RNA was eluted in RNase-free water, and was directly used as template for cDNA synthesis by the Prime Script RT Master Mix kit (TaKaRa Biotech. Co., Japan), by following the manufacturer's instructions. Potential contamination of DNA in the RNA was excluded by DNaseI treatment prior to cDNA synthesis. The cDNA concentration was measured using a NanoDrop 2000 spectrophotometer (Thermo Scientific, Wilmington, USA). Quantitative PCR (qPCR) using the cDNA was conducted using a CFX384 Real Time System (Bio-Rad, Hercules, CA, USA). The qPCR components contained SYBR Premix Ex Taq mix (TaKaRa Biotech. Co., Japan), 1 µl of diluted cDNA template, 10 µM both forward and reverse primers ( Table 2 ), and RNase free water. Thermal cycling included two reaction steps; an initial preheat for 3 min at 95℃, followed by 39 cycles at 95℃ for 5 sec, 55℃ for 10 sec, and 72℃ for 35 sec. The qPCR data were displayed using the CFX Manager software ver. 3.1. The oligonucleotide primers were designed based on the A. salmonicida subsp. salmonicida A449 geonome sequence ( Table 2 ). RT-qPCR results of individual genes were evaluated using the iCycler iQ Real-Time PCR Detection System. The C(t) values of qPCR products of each gene were used to determine the target cDNA concentration based on the relative comparison with gyrA gene expression of A. salmonicida subsp. salmonicida as a reference gene. The cDNA copies for fabVas and fabVas2 of A. salmonicida subsp. salmonicida were compared with the number of cDNA copies of the gyrA gene. The RT-qPCR experiments included three replicates.
Oligonucleotides used for RT-qPCR.
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aAnnealing temperature for PCR amplification.
Results
- Candidate of Triclosan Resistance Gene forAeromonas salmonicidaA449
A. salmonicida subsp. salmonicida 14791 showed elevated levels of triclosan resistance when tested on tryptone soya medium containing 20 µg/ml of triclosan. In order to have an initial understanding of the mechanism of triclosan resistance in the A. salmonicida subsp. salmonicida , we searched for homologs of four already known ENR genes from the genome of A. salmonicida subsp. salmonicida A449, which was completely sequenced. The closest candidate was ABO89763, annotated as trans -2-enoyl-CoA reductase, exhibiting 70% similarity with FabV, a triclosan-tolerant ENR. Moreover, the FabV homolog from A. salmonicida subsp. salmonicida A449 contains the Tyr-(Xaa) 8 -Lys motif, first seen in V. cholerae FabV, indicating that this protein among aeromonad organisms might have ENR activity and triclosan tolerance.
- Selection of a Triclosan-Resistant Clone
In order to select a triclosan-resistant clone from the fosmid library of AS 14791 by functional screening, we first determined the MIC of triclosan for the host E. coli . The MIC of triclosan for E. coli EPI-300 was 0.2 µg/ml. The fosmid library construction of AS 14791 generated approximately 1,300 clones with 35 kb of average insert DNA (data not shown). The number of clones in the genomic library was able to cover the whole genome of AS 14791, which is approximately 4.7 Mb [32] . Genomic library screening of AS 14791 for triclosan resistance resulted in the selection of several candidate fosmid clones. All of the selected clones showed a similar restriction pattern by Bam HI, indicating that all of the clones might be overlapped clones spanning the same genomic DNA to carry the gene for triclosan resistance (data not shown). Fosmid clone pTSR, which showed elevated levels of resistance to triclosan ( Table 3 ), was selected and further used to isolate a gene for triclosan resistance. This fosmid clone, carrying a 30 kb insert, consistently complemented the growth of E. coli carrying FabI, the triclosan-sensitive target enzyme for fatty acid biosynthesis.
Triclosan susceptibility ofE. colistrains carrying various plasmids.
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Triclosan susceptibility of E. coli strains carrying various plasmids.
- Identification of a Gene Encoding FabV Enoyl Acyl Reductase Isoform
The pTSR fosmid clone carrying the 30 kb DNA fragment was digested with Bam HI, and resulting fragments were subcloned into pUC119 in order to obtain smaller high-copy-number complementing plasmids. One of these subclones was able to restore the growth of triclosan-sensitive EPI-300 in the medium containing triclosan. DNA sequence analysis and its comparison with the genome of the A. salmonicida subsp. salmonicida A449 strain revealed that this subclone carries a DNA fragment containing two open reading frames (ORFs), a similar genomic region of A. salmonicida subsp. salmonicida A449. One of these two ORFs was annotated as encoding trans -2-enoyl-CoA reductase (YP_001141511), whereas the second partial ORF (YP_001141513) was annotated as alanine aminotransferase.
In order to eliminate the multicopy effect of pUC119, we further subcloned the ORF encoding trans -2-enoyl-CoA reductase into a broad-host-range low-copy-number plasmid, pRK415. The originally triclosan-sensitive E. coli EPI-300 cells, harboring the ORF encoding trans -2-enoyl-CoA reductase in pRK415 (pTSRK415), showed significantly elevated levels of triclosan resistance ( Table 3 ).
- FabVasShares an Identical Catalytic Domain with Other FabV ENRs
Multiple alignments of FabVas were performed with all previously known ENRs (FabI, FabL, FabV, and FabK), which revealed significant homology with FabV. This analysis was further extended to multiple alignment and comparisons of deduced amino acid sequences of FabVas with the two detailed studies of FabV ENRs of V. cholerae O1 biovar El Tor. N16961 [25] and P. aeruginosa PAO1 [48] . fabVas encodes a 398 amino acid protein, and the deduced amino acid sequence analysis showed that FabVas was 74% identical to FabV of V. cholerae O1 biovar El Tor. N16961 and 60% identical to the FabV of P. aeruginosa PAO1. From multiple alignment analysis, FabVas was found to contain a Tyr-(Xaa) 8 -Lys motif ( Fig. 1 ), reported as the active site of the FabV ENR first seen in V. cholerae [25] and then in P. aeruginosa [48] . This suggests that the protein might have ENR activity.
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Multiple alignment of A. salmonicida FabV (FabVas) with V. cholerae (FabVvc) and P. aeruginosa (FabVpa) FabV. A. salmonicida FabV (top line) has 74% identity with FabV of V. cholerae, and 60% identity to that of P. aeruginosa. The active-site tyrosine and lysine residues are asterisked.
- Genome-Wide Analysis Revealed a Secondary FabV inA. salmonicidaGenome
Genome-wide searches of A. salmonicida subsp. salmonicida A449 for the presence of other SDRs and ENRs revealed an additional ENR (YP_001140005) termed as FabVas2. Like the YP_001141511 ORF, multiple alignment analysis of YP_001140005 with the available ENRs showed significant homology with FabV ( Fig. 2 ), indicating that the genome of this organism carries two paralogs of FabV. In addition, genome-wide searches revealed the presence of nine other different kinds of SDRs (YP_001140191, YP_001140383, YP_001140963, YP_001141579, YP_001141848, YP_001142856, YP_001142967, YP_001143327, and YP_001143116).
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Multiple alignment of A. salmonicida secondary FabV (FabVas2) ORF with V. cholerae FabV (FabVvc). FabVas2 (top line) has 49% identity and 69% similarity with V. cholerae. The active-site tyrosine and lysine residues are asterisked.
- Relative Gene Expression of FabVas and FabVas2 at Transcriptional Level
Gene expression for FabVas and FabVas2 of A. salmonicida subsp. salmonicida in the presence and absence of triclosan (5 µg/ml) was investigated by RT-qPCR. The gyrA gene of A. salmonicida subsp. salmonicida was used as a reference gene. The mRNA expression of fabVas and fabVas2 differed slightly for the control and treated samples ( Fig. 3 ). The fabVas2 gene was higly expressed relative to triclosan treatment as compared with the fabVas gene at the mRNA level. However, in the absence of triclosan, fabVas expression was found to be higher than that of fabVas2 .
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Relative gene expression of fabVas and fabVas2 in Aeromonas salmonicida subsp. salmonicida in the presence and absence of triclosan (5 µg/ml) by RT-qPCR. Total RNA from Aeromonas salmonicida subsp. salmonicida was isolated at 48 h incubation (late exponential growth stage). Expression levels of fabVas and fabVas2 for triclosan treated (48TS) and untreated samples (48) were estimated in comparison with gyrA as a reference gene. Vertical bars represent standard deviations of three replicates.
- Most of the Known Fish Pathogens Carry Similar ENR Homologs to FabVas
In order to extend a secondary hy pothesis that most of the fish pathogens might share the same mechanism to tolerate triclosan, a similarity search of FabVas was performed against 21 different fish pathogens whose complete genome sequence data are available. These were as follows: V. anguillarum 775 serotype O1, V. anguillarum 96F serotype O1, V. anguillarum RV22 serotype O2β, V. ordalii ATCC 33509, V. vulnificus YJ016 biotype 1, V. splendidus strain LGP32, Aliivibrio salmonicida strain LFI1238, Flavobacterium psychrophilum JIP02/86, F. branchiophilum FL-15, Edwardsiella tarda EIB202, V. harveyi ATCC BAA-1116, F. columnare ATCC 49512, E. ictaluri 93–146, A. hydrophila ATCC 7966, A. salmonicida A449, A. veronii strain B565, A. caviae Ae398, Renibacterium salmoninarum ATCC 33209, Streptococcus parauberis, Lactococcus garvieae UNIUD074, and M. marinum M. The analysis showed that most of these fish pathogens have FabVas homologs showing significant identity (56–97%) with FabVas enoyl reductase ( Table 4 ). This result indicated that these organisms might share the same FabV ENR for enoyl reductase activity in fatty acid biosynthesis.
Fish pathogens and FabVENR homologs in their genome.
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Fish pathogens and FabVENR homologs in their genome.
Discussion
The mechanism of triclosan resistance has been extensively studied in human pathogens; however, the resistance mechanisms of triclosan in fish pathogenic bacteria are largely uncharacterized. Several studies have reported toxic levels of triclosan (0.001–2,210 ng/l) from marine water around the globe [10 , 30 , 44] . Therefore, there must be a resistance mechanism among marine organisms to overcome the toxicity of triclosan. The discovery of a new class of ENRs from V. cholerae , denoted as FabV [25] , led to the conclusion that FabI [3] , FabL [16] , and FabK [15] are not the only types of ENRs catalyzing the last step of type II fatty acid biosynthesis. Moreover, homology searches against available databases for the presence of ENR homologs is not always the best option, the reason being that ENR annotation is sometimes problematic [22] . For example, many proteins that align well and share significant homology with S. pneumoniae FabK have been reported not to exhibit any ENR activity [24 , 28 , 22] . Another example is that of Enterococcus faecalis carrying two different ENRs, namely FabI and FabK, where the latter has been proved to play a minor role in fatty acid biosynthesis in E. faecalis [20] . Therefore, functional analysis of bacterial ENR homologs by forward and reverse genetics seems to be the proper option to confirm its authentic activity.
Genome analysis of previously known ENRs in A. salmonicida A449 and fosmid library screening of AS 14791 revealed the presence of a FabV homolog sharing the same catalytic domain Tyr-(Xaa) 8 -Lys with FabV of V. cholerae . This result is in perfect agreement with previous data from other gram-negative bacteria that are known to contain a triclosan-tolerant FabV ENR [17 , 21 , 25 , 48] . The triclosan resistance of recombinant E. coli EPI-300 containing pTSRK415 eliminated the possibility of resistance occurrence due to overexpression of genes in a high-copy-number plasmid, showing that the fabVas gene is capable of conferring resistance to triclosan when expressed normally. This result indicates that the triclosan resistance in A. salmonicida is due to the insensitivity of FabVas to the biocide in question. These results are in accordance with those of previous studies [21] reporting that triclosan is a rapid reversible inhibitor of Burkholderia mallei FabV (FabVbm), with a K i value of only 0.4 mM. At such a low concentration, triclosan would be almost unable to compete with the natural substrate of the enzyme in question [17] . It has been demonstrated that triclosan has an even lower (100-fold) affinity for Y. pestis FabV (FabVyp), indicating that FabV ENR is naturally insensitive to triclosan.
From the multiple alignment analysis, the ENR of this study that renders A. salmonicida subsp. salmonicida 14791 resistant to triclosan seems to be FabV, as it shares significant identity (74%) and a similar catalytic domain (Tyr-(Xaa) 8 -Lys) [31] with FabV enoyl ACP reductase of V. cholerae [25] . It has been extensively reported that FabV is the main cause of triclosan tolerance in four human pathogenic bacteria, namely P. aeruginosa [48] , V. cholerae [25] , B. mallei [21] , and Y. pestis [17] . However, this is the first report describing the presence of the same triclosan-tolerant enzyme from a fish pathogen.
Genome-wide searches of A. salmonicida subsp. salmonicida A449 revealed the presence of a secondary ENR that was presumably FabV ( Fig. 2 ). Moreover, the presence of nine different SDRs suggests that this organism might have two or more ENRs catalyzing the enoyl reduction step during fatty acid biosynthesis. Our result is in agreement with that of P. aeruginosa [48] with two different ENRs, one of which is triclosan-tolerant. A. salmonicida was known to have a number of plasmid-borne antibiotic resistance genes; namely streptomycin/spectinomycin, quaternary ammonia compounds, sulfonamides, tetracycline, and chloramphenicol [32] . Moreover, three chromosomal β-lactamase genes ( ampC , ampS , cphA ) and major facilitator efflux family proteins have been recently reported from this organism [32] . Thus, the presence of these genes, along with triclosanresistant fabVas and an additional FabV homolog in this study, indicates that this organism carries an array of genes to counteract antimicrobials and biocides.
Expression analysis in this study revealed that both the fabVas and fabVas2 genes were actively transcribed in the absence and presence of triclosan, indicating that these ENRs might be mutually involved in the fatty acid biosynthesis in A. salmonicida subsp. salmonicida . We do not have any clue if FabVas2 will play a certain role for triclosan resistance. Although fabVas2 was transcribed in the presence of triclosan, it is not clear if the mRNA will be translated into an active ENR to confer triclosan resistance to A. salmonicida subsp. salmonicida . In fact, FabK of E. faecalis carrying two different ENRs, FabI and FabK, was found to play a minor role in triclosan resistance [20] . Similarly, FabL, not FabI, was responsible to confer triclosan resistance in Bacillus subtilis with two different FabL and FabI ENRs [16] . To our best knowledge, this report is the first of the presence of two potential FabV ENRs, fabVas and fabVas2 , in a single pathogenic bacteria.
Genome-wide comparison of FabVas against most of the known fish pathogens revealed that most of these organisms might confer resistance to triclosan by sharing the same triclosan-resistant FabVas enzyme ( Table 4 ). This might be due to lateral gene transfer among bacterial strains with diverse FabI homologs [28] . In addition, the transfer of antibiotic-resistant genes, such as genes encoding resistance to oxytetracycline, trimethoprim, and sulfonamides, from fish pathogens to other bacteria has been recognized [33 , 38] . However, the lack of FabV in other fish pathogens indicates that those organisms might be susceptible to triclosan or have a different resistance mechanism to this biocide. Based on our results, we propose that triclosan resistance mediated by an alternative ENR may be widely distributed in bacterial systems in nature.
Acknowledgements
This study was carried out with the support of “Cooperative Research Program for Agricultural Science & Technology Development (Project No. PJ009465)”, Rural Development Administration, Republic of Korea.
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