Microbial Ecology

Concurrence of cat and tet Genes in Multiple Antibiotic-Resistant Bacteria Isolated from a Sea Cucumber and Sea Urchin Mariculture Farm in China
Hongyue Dang1, Linsheng Song2, Mingna Chen1,3 and Yaqing Chang4
(1) (2) (3) (4) Key Laboratory of Marine Geology and Environment, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China Experimental Marine Biology Laboratory, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China Graduate School, Chinese Academy of Sciences, Beijing 100039, China Key Lab of Mariculture and Biotechnology, Dalian Fisheries University, Dalian 116023, China

Received: 26 January 2006 / Accepted: 8 April 2006 / Online publication: 15 August 2006

Abstract

A basic understanding of abundance and diversity of antibiotic-resistant microbes and their genetic determinants is necessary for nding a way to prevent and control the spread of antibiotic resistance. For this purpose, chloramphenicol and multiple antibiotic-resistant bacteria were screened from a mariculture farm in northern China. Both sea cucumber and sea urchin rearing ponds were populated with abundant antibiotic-resistant bacteria, especially marine vibrios. Sixty-ve percent chloramphenicol-resistant isolates from sea cucumber harbored a cat gene, either cat IV or cat II, whereas 35% sea urchin isolates harbored a cat gene, actually cat II. The predominant resistance determinant cat IV gene mainly occurred in isolates related to Vibrio tasmaniensis or Pseudoalteromonas atlantica, and the cat II gene mainly occurred in Vibrio splendidus-like isolates. All the cat-positive isolates also harbored one or two of the tet genes, tet(D), tet(B), or tet(A). As no chloramphenicol-related antibiotic was ever used, coselection of the cat genes by other antibiotics, especially oxytetracycline, might be the cause of the high incidence of cat genes in the mariculture farm studied.

Introduction

Both beche-de-mer (trepang or dried sea cucumber) and sea urchin roe have long been considered delicacies in Asia and Europe, and these marine animals have been extensively overshed for decades globally. Mariculture is becoming the major resort for valuable seafood producCorrespondence to: Linsheng Song; E-mail: lshsong@ms.qdio.ac.cn

tion and natural resources protection [5, 17]. Temperate water Apostichopus japonicus (Selenka) has traditionally been regarded to be of high nutritional and medicinal values. It has become the predominant cultured species of sea cucumbers in China because of the success of the techniques used in commercial hatcheries [5]. On the other hand, the Chinese currently cultured sea urchin species, Strongylocentrotus intermedius, was introduced from Japan in 1989 [15, 37]. The ever-growing domestic and global seafood market stimulates the rapid development of Chinese maricultural industry, which ranks no. 1 currently in the world in the light of annual production. However, farmed marine animal infectious diseases break out frequently, causing devastating mariculture failure and huge economical loss during the last decade. Bacteria are often the causative agents of infectious diseases, such as skin ulcer disease and bacterial ulceration syndrome during A. japonicus breeding, aestivation and outdoor cultivation stages [36], and black mouth disease and red spotting disease during S. intermedius cultivation [20, 35]. Many antibiotics have been used as therapeutic and preventive agents for battling microbial diseases. As a consequence, antibiotic-resistant bacteria prevail in maricultural environments. Chloramphenicol was once extensively and intensely used in China. The resistance to chloramphenicol is mainly caused by drug inactivation mediated by chloramphenicol acetyltransferases (CAT). CATs produced by R plasmids in gram-negative bacteria are encoded by four types of resistance genes, cat I, cat II, cat III, or cat IV [3]. Determining the identities and distribution of the cat genes is helpful in understanding the origins and their dissemination pathways in mariculture and surrounding environments.
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DOI: 10.1007/s00248-006-9091-3

& Volume 52, 634643 (2006) & *

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Although chloramphenicol has been banned in China for mariculture use since 1999, chloramphenicol-resistant pathogenic or environmental bacteria might have remained prevalent, either because of the use of orfenicol, a derivative of chloramphenicol, in mariculture farms or because of the phenomenon that chloramphenicolresistant genes derived from limited sources could be transferred among aquatic microorganisms without a high selective pressure [39]. In the latter case, cross-resistance or coresistance caused by cross-selection or coselection might be the real mechanism involved [7]. In our previous study, we found that the occurrence of the bacterial cat genes in multiple antibiotic-resistant strains in the studied mariculture farm was coincident to the occurrence of the tet(B) gene, which encodes the resistance to antibiotics in the tetracycline family [9]. Because of its broad-spectrum activity and low toxicity, oxytetracycline is currently the most frequently used antibiotic in China in aquaculture systems to treat and prevent bacterial diseases. Among the 39 tet and otr genes encoding for oxytetracycline resistance [1, 28], genes encoding cell membrane active efux pumps, such as tet(A)(E) and tet(G), are the predominant resistance determinants found in marine gram-negative bacteria [16]. It is possible that the high incidence of the cat genes could be a result of oxytetracycline coselection effect in the mariculture environment studied. For nding more evidence to support this postulation, chloramphenicolresistant bacteria were isolated from the same sea cucumber and sea urchin rearing ponds, and resistance to multiple antibiotics, including oxytetracycline, was assayed. The cat and the major efux type tet genes were screened by the multiplex polymerase chain reaction (PCR) methods described previously [16, 39]. The current results indicated that all the occurrence of the cat genes was coincident to the occurrence of one or two of the tet genes screened, implicating that coselection of the cat genes by the selective pressure of oxytetracycline might be the cause of the high incidence of chloramphenicol-resistant bacteria in the mariculture environment studied, in line with the seemingly contradictory fact that no chloramphenicol-related antibiotic has ever been used during the last few years in this mariculture farm.
Materials and Methods

weights were 0.210 g, and the sea urchins test diameters were 25 mm. Sea cucumbers were fed with articial feed, and sea urchins were fed with kelp Laminaria japonica in excess every day. Uneaten food and feces were removed from the pond once in every 2 days. Water was exchanged once or twice every day with a replacement of 50 to 100% with a temperature difference usually less than 1-C. The antibiotics oxytetracycline, penicillin, streptomycin, enrooxacin, and furazolidone were used for disease prevention frequently at 0.53 ppm. Water samples were kept on ice in a cooler before they were transferred to the laboratory. Subsamples for microscopy total microbial number counts were xed with formaldehyde with a nal concentration of 2% and stored at 4-C in the dark.
Microbial Number Counts. For total microbial number counts, the samples xed in 2% formaldehyde were vortexed tempestuously after being mixed with 0.09% Tween 80 (Sigma, USA) then stained with 40 -6diamidino-2-phenylindole (Sigma) for 10 min in the dark at a nal concentration of 0.1 mg/mL. Samples were ltered onto black 0.22-mm pore-sized Poretics polycarbonate disk lters (Osmonics Inc., USA) before they were mounted on an epiuorescence microscope (Axioplan Zeiss, Germany) for total microbial cell counts under ultraviolet excitation. For total culturable bacteria number counts, 1-mL seawater samples were serially diluted in sterilized articial seawater (25 g NaCl, 5 g MgSO4, 1 g CaCl2, and 1 g KCl in 1 L of distilled water), plated in triplicate on 2216 marine agar plates (Difco formula). Colony forming units were counted after 24-h incubation at 25-C. For chloramphenicol-resistant bacteria counts, serially diluted seawater samples were plated in triplicate on tryptic soy agar (Difco formula) plates supplemented with 3% (w/v) NaCl and 5 mg/mL chloramphenicol (CP5) for 24-h incubation at 25-C. Then the CP5-resistant isolates were further screened with 10 mg/mL chloramphenicol (CP10) based on the method described by Yoo et al. [39] for antibiotic susceptibility tests. Only CP10-resistant isolates were counted as chloramphenicolresistant bacteria. Chloramphenicol-Resistant Bacteria Isolation and After the CP5Multiple Antibiotic Resistance Assay.

Rearing water samples were collected from two indoor aquaculture ponds, each of 40 m3, for A. japonicus and S. intermedius cultivations, respectively, from a mariculture farm located in Dalian, northern China, on September 15, 2004. The rearing water temperature and salinity were 1820-C and 31.0, respectively. The ponds received articial illumination of 5002000 lx. The sea cucumbers body
Sample Collection and Processing.

resistant colonies were tested for CP10 resistance, bacteria resistant to CP10 were duplicated from the CP5 plates of the second highest positive dilutions, as the highest positive dilution plates had too few colonies growing. About one third of the colonies growing on each of the three replicative plates were randomly selected, and the three subsets were pooled, resulting in 68 isolates from the sea cucumber sample and 84 from the sea urchin sample. Basically, these isolates should stand well for the

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dominant CP10-resistant bacteria in the maricultural environment studied. They were further determined of higher level resistance to chloramphenicol (30 and 100 mg/ mL, CP30 and CP100) and other antibiotics, e.g., oxytetracycline (30 mg/mL, OTC30), streptomycin (30 mg/mL, SM30), erythromycin (15 mg/mL, EM15), ampicillin (10 mg/mL, AMP10), and nalidixic acid (30 mg/mL, NA30), based on the method described by Jun et al. [16] and Yoo et al. [39] for antibiotic susceptibility tests. As none of the isolates has ever been proven to be pathogenic to either A. japonicus or S. intermedius, minimum inhibitory concentrations were not determined.
Molecular Determination of Phylogenetic Afliations CP10-resistant bacteria of Selected Resistant Bacteria.

(20 from each of the sea cucumber and sea urchin isolates, respectively) were randomly selected for molecular determination of their phylogenetic afliations. A simple boiling method was used for rapid bacterial genomic DNA extraction [10]. The primers used for amplication of the 16S rDNA were 27F (50 -AGAGTTTGATCCTGGCTCAG30 ) and 785R (50 -CTACCAGGGTATCTAATCC-30 ) or 926R (50 -CCGTCAATTCATTTGAGT-30 ). The standard PCR reaction mixture contained 100 mM dNTP, 1.5 mM MgCl2, 0.05 mM each primer, and 1.5 U Taq DNA polymerase (TaKaRa, Japan). PCR amplications were performed with a PTC-100 i programmable thermal controller (MJ Research Inc., USA). The standard program was as follows: the initial denaturation was at 94-C for 5 min, followed by 30 thermal cycles including denaturation at 94-C for 1 min, annealing at 54-C for 1 min, and extension at 72-C for 1 min and 20 s, and a nal extension step at 72-C for 10 min. PCR products were examined by electrophoresis on 1% agarose gel. Primer 27F was also used for sequencing with puried PCR products as templates. The DNA fragments sequenced were usually about 600800 bp long and covered at least the V1V3 hypervariable regions of bacterial 16S rDNA. Bioinformatic determination of sequence afliations followed the standard methods [8]. For each sequence, a query was made by the on-line Basic Local Alignment Search Tool search program to the GenBank database maintained by the American National Center for Biotechnology Information (NCBI) for an initial determination of the nearest phylogenetic neighbor sequences [2]. Sequences were aligned by using the ClustalX program (version 1.8) [31]. Phylogenetic trees were constructed using DNADIST and NEIGHBOR programs within the PHYLIP package (version 3.6) [12].
Multiplex PCR Determinations of cat and Major Efux Type tet Genes in Chloramphenicol-Resistant The above 40 CP10-resistant bacterial isoIsolates.

developed by Yoo et al. [39]. Once the cat gene was conrmed, these cat -positive strains were further screened for the tet genes by the multiplex PCR method originally developed by Jun et al. [16], as all the catpositive bacterial isolates were also OTC30-resistant based on our multiple antibiotic resistance assay. The multiplex PCR primers for cat and tet genes detection were listed in Table 1. The detailed experimental procedures and PCR amplication conditions described originally in the papers of Yoo et al. [39] and Jun et al. [16] were followed, only with a minor modication; for example, a simple boiling procedure was used for total DNA (including genomic and plasmid DNA) extraction from bacterial isolates [10]. Representative multiplex PCR products were sequenced for conrmation of their identities as cat or tet genes.
Nucleotide Sequence Accession Numbers. The 16S rDNA sequences determined in this study have been deposited in the NCBI GenBank database under accession nos. DQ313646 DQ313685, and the two representative cat gene sequences determined in this study are under accession numbers DQ313686 and DQ313687; the three representative tet gene sequences determined in this study are under accession numbers DQ313688, DQ313689, and DQ313690.

Results

The total counts of microorganisms determined using the epiuorescence microscopy technique were (2.58 T 0.22) 107 and (1.48 T 0.17) 107 cells/mL for sea cucumber and sea urchin rearing water, respectively. The total culturable microbial counts determined by 2216 marine agar plating technique were (1.82 T 0.14) 105 and (4.74 T 0.35) 105 cells/mL, and the CP10-resistant microbial counts
Microbial Number Counts.
Table 1. Multiplex PCR primers for cat and tet genes detection (from Jun et al. [16] and Yoo et al. [39])

Primer C-R C-1 C-2 C-3 C-4 TETF TAR TBR TCR TDR TER TGR

Target and position Common antisense primer for cat genes cat I, 245264 cat II, 1535 cat III, 307326 cat IV, 122141 Common sense primer for tet genes tet(A), 534517 tet(B), 318301 tet(C), 778761 tet(D), 631613 tet(E), 393377 tet(G), 950933

Expected amplicon size (bp)

349 567 275 451 387 171 631 484 246 803

lates randomly selected for 16S rDNA sequencing were screened for cat genes by the multiplex PCR method

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Table 2. Percentage of antibiotic resistant microbes in CP10-resistant isolates

Sample Sea cucumber Sea urchin

CP30 97.1 100

CP100 80.9 65.5

OTC30 100 83.3

AMP10 72.1 94.0

SM30 77.9 33.3

NA30 29.4 14.3

EM15 30.9 4.8

CP30 = chloramphenicol, 30 mg/mL; CP100 = chloramphenicol, 100 mg/mL; OTC30 = oxytetracycline, 30 mg/mL; AMP10 = ampicillin, 10 mg/mL; SM30 = streptomycin, 30 mg/mL; NA30 = nalidixic acid, 30 mg/mL; EM15 = erythromycin, 15 mg/mL.

were (6.60 T 1.15) 104 and (7.17 T 0.76) 104 cells/mL for sea cucumber and sea urchin rearing water, respectively. CP10-resistant microbes accounted for 36.2 and 15.1% of the total culturable microbes of the sea

cucumber and the sea urchin rearing water, respectively. Although the percentage of CP10-resistant bacteria was lower for the sea urchin pond, the absolute numbers were similar for the two types of culture ponds.

Table 3. Bacterial 16S rDNA sequence afliations, antibiotics resistance spectra, and cat and tet genes detected

Strain

Closest sequence match (accession number and similarity) Vibrio splendidus strain 636 (AY620972, 98%) Vibrio splendidus strain 636 (AY620972, 99%) Pseudoalteromonas sp. RE1-12a (AF539781, 99%) Vibrio tasmaniensis strain 236.10 (AY620969, 99%) Unidentied bacterium 4c (AF293974, 99%) Vibrio tasmaniensis strain 236.10 (AY620969, 99%) Pseudoalteromonas atlantica (AB100883, 99%) Vibrio tasmaniensis strain 236.10 (AY620969, 100%) Vibrio splendidus strain 636 (AY620972, 100%) Vibrio tasmaniensis strain 236.10 (AY620969, 100%) Pseudoalteromonas atlantica (AB100883, 99%) Vibrio splendidus strain 636 (AY620972, 100%) Mucus bacterium 5 (AY654794, 98%) Vibrio tasmaniensis strain 236.10 (AY620969, 100%) Vibrio splendidus strain 636 (AY620972, 100%) Vibrio tasmaniensis strain 236.10 (AY620969, 99%) Pseudoalteromonas atlantica (AB049728, 100%) Vibrio tasmaniensis strain 236.10 (AY620969, 99%) Mucus bacterium 5 (AY654794, 99%) Vibrio splendidus strain 636 (AY620972, 99%) Vibrio splendidus strain 636 (AY620972, 100%) Vibrio splendidus strain 636 (AY620972, 99%) Pseudoalteromonas haloplanktis (AF214729, 99%) Mucus bacterium 59 (AY654788, 99%) Vibrio splendidus strain 636 (AY620972, 99%) Vibrio splendidus strain 636 (AY620972, 99%) Vibrio splendidus strain 636 (AY620972, 99%) Vibrio splendidus strain 636 (AY620972, 100%) Mucus bacterium 5 (AY654794, 99%) Mucus bacterium 5 (AY654794, 99%) Pseudoalteromonas atlantica (AB049728, 99%) Mucus bacterium 5 (AY654794, 99%) Vibrio splendidus strain 636 (AY620972, 100%) Vibrio tasmaniensis strain 236.10 (AY620969, 100%) Pseudoalteromonas atlantica (AB100883, 99%) Vibrio splendidus strain 636 (AY620972, 100%) Vibrio splendidus strain 636 (AY620972, 100%) Vibrio splendidus strain 636 (AY620972, 100%) Pseudoalteromonas sp. SW08 (U85861, 99%) Vibrio splendidus strain 636 (AY620972, 99%)

Antibiotics resistance prole CP100, CP100, CP100, CP100, CP100, CP100, CP100, CP100, CP100, CP100, CP100, CP100, CP100, CP100, CP100, CP100, CP100, CP100, CP100, CP100, CP100, CP100, CP100, CP100, CP100, OTC30, OTC30, OTC30, OTC30, OTC30, OTC30, OTC30, OTC30, OTC30, OTC30, OTC30, OTC30, OTC30, OTC30, OTC30, OTC30, OTC30, OTC30, OTC30, OTC30, OTC30, OTC30, OTC30, OTC30, OTC30, AMP10 AMP10, SM30, NA30 SM30, EM15 SM30, EM15 AMP10 AMP10, SM30, EM15 AMP10, EM15, NA30 AMP10, SM30 SM30, EM15, NA30 SM30, EM15, NA30 SM30 AMP10, NA30 AMP10, NA30 AMP10, SM30 AMP10, NA30 AMP10, SM30, EM15 SM30, SM30, EM15, NA30 AMP10, NA30 AMP10, NA30 AMP10, SM30 AMP10 AMP10, SM30, EM15, NA30 AMP10, NA30 AMP10, NA30

cat gene cat II NO cat IV cat IV cat IV cat IV

tet gene tet(D) ND tet(B) tet(B) tet(D) tet(A), tet(B) ND ND ND tet(B) tet(B) ND ND tet(D) tet(D) tet(A), tet(B) tet(D) tet(B) tet(D) ND tet(D) tet(D) ND ND tet(B), tet(D) ND ND ND ND ND ND ND tet(D) tet(D) ND tet(D) tet(D) ND ND ND

CCH-01 CCH-24 CCH-26 CCH-27 CCH-29 CCH-31 CCH-33 CCH-34 CCH-40 CCH-44 CCH-45 CCH-48 CCH-49 CCH-53 CCH-56 CCH-59 CCH-63 CCH-64 CCH-67 CCH-68 UCH-03 UCH-04 UCH-20 UCH-23 UCH-25 UCH-34 UCH-35 UCH-41 UCH-45 UCH-48 UCH-51 UCH-54 UCH-56 UCH-59 UCH-68 UCH-69 UCH-79 UCH-83 UCH-84 UCH-85
a b c

NO NO NO cat IV cat IV NO NO cat II, cat IV cat II cat IV cat IV cat IV cat IV NO cat II cat II NO NO cat II NO NO NO NO NO NO NO cat II cat II NO cat II cat II NO NO NO

CP100, OTC30, AMP10, NA30 CP30, OTC30, AMP10, NA30 CP100, OTC30, AMP10 CP100, OTC30, AMP10, NA30 CP100, OTC30, AMP10 CP100, SM30, NA30 CP100, OTC30, AMP10 CP100, OTC30, AMP10 CP100, OTC30, AMP10, SM30 CP100, OTC30, AMP10, SM30, EM15, NA30 CP100, OTC30, AMP10 CP100, OTC30, AMP10, SM30 CP100, OTC30, AMP10 CP100, OTC30, AMP10, SM30 CP100, OTC30, AMP10, SM30

Sea cucumber isolates were labeled initially with CCH, and sea urchin isolates with UCH. NO means no cat gene could be detected for this strain. ND means the tet genes were not screened for this strain.

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Of the randomly selected 152 CP10-resistant strains (68 from the sea cucumber isolates and 84 from the sea urchin isolates), 150 (98.7%) were also resistant to CP30; the only two CP30-sensitive isolates were from the sea cucumber rearing pond. More than 65% of the CP10resistant isolates were also resistant to CP100 (Table 2). These data could be translated to 5.33 104 and 4.69 104 cells/mL of CP100-resistant bacteria in the sea cucumber and sea urchin rearing ponds, respectively. This high incidence of chloramphenicol-resistant bacteria in both culture ponds was apparently not a result of direct selective pressure of the specic type of antibiotic, as no chloramphenicol-related drug was ever used before and during our sampling period in this mariculture farm. All the 68 CP10-resistant sea cucumber isolates were also resistant to at least two other types of the antibiotics

tested in this study, 62 (91.2%) isolates were resistant to at least three other types of antibiotics, and 13 (19.1%) isolates were resistant to four other types of antibiotics. All the 84 CP10-resistant sea urchin isolates were also resistant to at least one other type of antibiotics, 81 (96.4%) isolates were resistant to at least two other types of antibiotics, 19 (22.6%) isolates were resistant to at least three other types of antibiotics, 6 (7.1%) isolates were resistant to at least four other types of antibiotics, and 3 (3.6%) isolates were resistant to all the antibiotics tested. Resistance to OTC30 or AMP10 occurred frequently in both sea cucumber and sea urchin CP10-resistant isolates (Table 2). The sea cucumber isolates also had high incidence of SM30 resistance. For antibiotics NA30 and EM15, the percentages of resistant microbes were

Figure 1. Phylogenetic tree of the 40 selected CP10-resistant strains constructed based on partial 16S rDNA sequences in a 647-bp frame length using neighbor-joining method with the Kimura two-parameter model for nucleotide change. The tree branch distances represent nucleotide substitution rate, and the scale bar represents the expected number of changes per homologous nucleotide position. Bootstrap values greater than 70% of 100 resamplings are shown near nodes. (a) As 16S rDNA sequences of strains CCH-24 and UCH-25 are identical for the region used for tree construction, the evolutional positions and phylogenetic afliations of these strains should be identical. (b) CCH-40, CCH-48, CCH-56, CCH-67, CCH68, UCH-03, UCH-34, UCH-41, UCH-45, UCH-48, UCH-54, UCH56, UCH-69, UCH-79, and UCH83 are identical in the 16S rDNA region analyzed; they should have the same evolutional position and phylogenetic afliation. (c) CCH27 and CCH-31 are identical in the 16S rDNA region analyzed; they should have the same evolutional position and phylogenetic afliation. (d) CCH-26, CCH-34, CCH44, CCH-53, and CCH-59 are identical in the 16S rDNA region analyzed; they should have the same evolutional position and phylogenetic afliation.

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Figure 2. Electrophoretic analyses of the cat genes amplied with the multiplex PCR method developed by Yoo et al. [39] from selected

CP10-resistant strains isolated from sea cucumber (a) and sea urchin (b) rearing ponds, respectively (the strains that did not harbor any of the cat genes screened were not shown). The strains with positive cat screening results were labeled by their strain numbers on the gel images. M: Molecular weight standard DL2000 (2000, 1000, 750, 500, 250, and 100 bp, respectively). E: Escherichia coli strain TOP10 was _ used to serve as negative control for multiplex PCR reactions, as this strain did not harbor chloramphenicol resistance. ( ): Negative control PCR reaction without template DNA.

quite different between the sea cucumber and the sea urchin samples. The differences in specic antibiotic resistance and between the two rearing ponds were probably because of the status and history of antibiotic usage (detailed data were not available from the farm). Higher incidence of resistance to a variety of antibiotics (except AMP10) in the sea cucumber pond probably reected the fact that A. japonicus was more susceptible to diseases caused by pathogenic microbes than S. intermedius was; thus, more drugs were probably used for prevention and treatment in the sea cucumber culture pond.
16S rDNA Determination of Phylogenetic Afliations Based on 16S rDNA of Selected Resistant Strains.

sequences, most of the 40 randomly selected CP10resistant isolates were associated with the marine Vibrio groups and a few with the marine Pseudoalteromonas group (Table 3). Marine bacteria closely related to Vibrio tasmaniensis and V. splendidus were the most abundant resistant strains in the sea cucumber rearing pond, whereas V. splendidus-like strains were dominant in the sea urchin rearing pond. Phylogenetic tree constructed based on neighbor joining method further veried most of the afliations of these antibiotic-resistant isolates (Fig. 1). Although only 40 CP10-resistant bacterial isolates were sequenced for phylogenetic afliation determination, our result indicates that the resistant microbe diversity was probably captured, as the distribution of resistant species is quite narrow. All the isolates belonged to either Vibrio or Pseudoalteromonas groups of microorganisms in the g-

Proteobacteria subdivision. In the Vibrio group, all the isolates were closely related to either V. splendidus or V. tasmaniensis. Of the 20 sea cucumber CP10-resistant bacteria analyzed, 9 isolates were closely related to V. splendidus, 8 isolates to V. tasmaniensis, and the remaining 3 isolates to Pseudoalteromonas atlantica. Of the 20 sea urchin CP10-resistant bacteria analyzed, 15 isolates were closely related to V. splendidus, 1 isolate to V. tasmaniensis, and the remaining 4 isolates to marine Pseudoalteromonas. As the source for rearing water exchange of both culture ponds was the same, similar background bacterial community structure, diversity, and abundance could be assumed. The difference in resistant bacteria species distribution and diversity between the sea cucumber and the sea urchin samples was probably a result of the difference of the status and history of antibiotics usage in the two mariculture ponds, although chloramphenicol per se could not be the culprit. The best-matched GenBank sequence of isolate CCH-26 was classied as Pseudoalteromonas (accession number AF539781; see Table 3). However, according to the 16S rDNA phylogenetic tree constructed in this study (Fig. 1), the strain Pseudoalteromonas sp. RE1-12a should be classied as a Vibrio species. A phylogenetic tree constructed by using almost full-length 16S rDNA sequences downloaded from GenBank database further conrmed our conclusion (data not shown).
Molecular Determination of cat Genes. The cat genes encoding for CAT were screened by multiplex PCR method for the 40 CP10-resistant isolates to determine the molecular mechanisms of chloramphenicol resistance.

Table 4. Representative cat and tet gene sequences determined in the current study

Strain CCH-44 CCH-53 CCH-59 CCH-64 UCH-56

Closest sequence match Vibrio anguillarum cat IV Escherichia coli plasmid pSa cat II Vibrio cholerae SXT tet(A) Vibrio sp. TC68 tet(B) Pasteurella piscicida R plasmid pSP9351 tet(D)

Sequence length (similarity) 399/399 499/500 337/337 124/125 437/439 (100%) (99%) (100%) (99%) (99%)

Accession number S48276 L06822 AB114188 AB089593 D16172

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Figure 3. Electrophoretic analyses of PCR products obtained by the amplication of tet genes with the multiplex PCR method developed

by Jun et al. [16] from the cat-positive strains isolated from sea cucumber (a) and sea urchin (b) rearing ponds, respectively (the strains that did not harbor any of the tet genes tested were not shown). The strains with positive tet screening results were labeled by their strain numbers on the gel images. M: Molecular weight standard DL2000. E: E. coli strain TOP10 was used to serve as negative control for _ multiplex PCR reactions. ( ): Negative control PCR reaction without template DNA.

Two types of cat genes, cat II and cat IV, were identied from some of the sea cucumber strains. Of the 20 selected CP10-resistant sea cucumber isolates, 10 isolates carried a single cat IV gene, 2 isolates carried a single cat II gene, and 1 isolate carried both the cat II and the cat IV genes (Table 3 and Fig. 2a). All the cat-positive V. tasmaniensis-like strains harbored the cat IV gene, and strain CCH-53 also harbored the cat II gene. Half of the cat-positive V. splendidus-like strains harbored the cat IV gene, and half harbored the cat II gene. Both the catpositive P. atlantica-like strains harbored the cat IV gene. Only the cat II gene was detected in 7 of the 20 sea urchin strains, 6 of which were related to V. splendidus and one was to V. tasmaniensis (Table 3 and Fig. 2b). Some of the sea cucumber and sea urchin CP10-resistant strains could not be detected to harbor any of the cat genes screened in this study; other or even novel chloramphenicol resistance genetic determinant might exist. The cat gene identities were veried by DNA sequencing of representative PCR products resulted from the multiplex PCR reactions (Table 4). Both sequences had more than 99% similarity to the best-match known cat genes retrieved from the NCBI GenBank database, and they also had more than 99% similarity to the bestmatch known partial CAT protein sequences retrieved from GenBank after they were translated into conceptual amino acid sequences. The multiplex PCR method for cat gene screening and classication was very effective and reliable [39]. As all the 20 cat-positive isolates determined in the above step were also resistant to OTC30, multiplex PCR method was used to screen for tet(A)(E) and tet(G) genes to understand molecular mechanisms of oxytetracycline resistance in these isolates. All the 20 resistant strains harbored at least one of the tet genes screened. Three types of tet genes, tet(A), tet(B), and tet(D), were detected in the 13 sea cucumber cat-positive strains (see Table 3 for strain
Molecular Determination of tet Genes.

information), six isolates were found to carry a single tet(D) gene, ve isolates harbored a single tet(B) gene, and two isolates harbored both tet(A) and tet(B) genes (Table 3 and Fig. 3a). All the cat-positive V. splendiduslike strains harbored a single tet(D) gene. Of the seven V. tasmaniensis-like resistant strains, four isolates harbored a single tet(B) gene, one harbored a single tet(D) gene, and the rest harbored both tet(A) and tet(B) genes. Of the two cat-positive marine P. atlantica-like isolates, one harbored the tet(B) gene, and the other harbored the tet(D) gene. Interestingly, all the seven cat-positive sea urchin isolates harbored the tet(D) gene, and the V. splendiduslike strain UCH-25 also carried the tet(B) gene (Table 3 and Fig. 3b). The tet gene identities were veried by DNA sequencing of representative PCR products resulted from the multiplex PCR reactions (Table 4). All the three representative sequences had more than 99% similarity to the best-match known tet genes retrieved from the NCBI GenBank database, and they also had more than 97% similarity to the best-match known partial efux type tetracycline resistance protein sequences retrieved from GenBank after they were translated into conceptual amino acid sequences. The multiplex PCR method for tet gene screening and classication was very effective and reliable [16].
Discussion

Sea cucumber and sea urchin farming has been becoming an important mariculture industry in the world. However, farmed sea cucumbers and sea urchins are susceptible to many infectious bacterial diseases. Frequent disease outbreaks resulted in extensive and intensive use and misuse of antibiotics. As a harmful side effect, antibiotic-resistant bacteria are abundant and persistent in mariculture and surrounding environments. These microorganisms, especially those harboring multiple antibiotics resistance, may cause even higher level of difculty and failure in aquacul-

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tural disease control and treatment. Furthermore, via food chain and human maritime activities, antibiotic resistance could be transferred to and spread in healthy marine and terrestrial environments. Some bacterial pathogens of marine animals are zoonotic [30]. The spread of marine pathogens could be achieved at extremely rapid rates because of the lack of barriers to dispersal in marine environment and because of the potential for long-term survival of pathogens outside the host [21]. Thus, antibiotic-resistant microbes may pose a severe threat in the coastal areas, putting human health and well-being at great risk. Maricultural environments, as a reservoir of antibiotic resistance, call for global attention and investment of research efforts. However, because of certain degree of ignorance, only very limited information about antibiotic resistance in aquaculture environments is available in China. The lack of resistance research is disproportional to the current status of Chinese mariculture industry. The lack of systematic resistance research also imparts a hindrance to healthy and sustainable development of Chinese mariculture industry. Furthermore, without accurate monitoring and prompt action, resistant microbes in mariculture environments may develop into a severe threat to human health and marine animals. Based on these considerations, a series of surveys of antibiotic resistance were carried out. This article focuses on the abundance and diversity of chloramphenicol-resistant bacteria and the likely molecular mechanism for the high incidence of these strains in a mariculture environment previously studied for oxytetracycline resistance [9].
Antibiotic-Resistant Marine Vibrios. Marine vibrios present serious threat to cultured marine animals, as some are opportunist pathogens [23]. They also have long been recognized as reservoirs and vehicles of antibiotic resistance because of their abundance in coastal waters, their ability to readily develop and acquire antibiotic resistance in response to selective pressure, and their ability to spread resistance by horizontal genetic material exchange. Our current results showed that marine vibrios were the most dominant chloramphenicol and multiple antibiotic-resistant strains in the mariculture farm studied. Interestingly, V. tasmaniensis-related resistant strains were very abundant in the sea cucumber rearing pond, but they were very scarce in the sea urchin rearing pond. However, both mariculture ponds harbored abundant V. splendidus-like resistant strains. The contradiction of the distribution of marine vibrio species between the different marine animal rearing ponds is actually consistent to our previous study result on oxytetracycline-resistant bacteria isolated from the same farm [9]. V. splendidus is a common coastal planktonic bacterium [24], usually predominant in the culturable microbial populations [34]. Recent work also showed that the diversity of V. splendidus is quite high in coastal areas

[32]. Our data indicate that this might also be true in maricultural environments, although the microbes here are under articial antibiotics selective pressures most of the time. Certain V. splendidus strains were found to be pathogenic to several cultured marine animals, such as bivalves, shrimp, and sh [4, 13, 14, 22, 27, 29, 33]. Although V. splendidus has not been reported to be pathogenic to A. japonicus or S. intermedius, a study indicated that some strains could establish intimate associations with the purple sea urchin Strongylocentrotus purpuratus [11]. We also found that some of the strains in our isolates might associate with A. japonicus or S. intermedius intimately as they could attach to tested articial surfaces to form biolm. Thus, the association of V. splendidus with farmed marine animals might facilitate antibiotic resistance transmission among marine microbes because of colocalization. Although resistant V. splendidus might not be pathogenic to sea cucumbers or sea urchins, once released to the surrounding environment, they might serve as resistance transmission vehicles, forming a serious threat to other marine animals. Our data indicate that V. tasmaniensis might also form a sizable bacterial antibiotic resistance population, contributing to resistance dissemination in marine environment.
Diversity and Distribution of Chloramphenicol Our current multiplex PCR Acetyltransferase Genes.

results indicated that both cat II and cat IV genes existed in the mariculture environment studied. The frequency and type of the cat genes in the sea cucumber and the sea urchin rearing ponds were quite different. Sixty-ve percent sea cucumber CP10-resistant isolates harbored one or two cat genes, whereas only 35% sea urchin CP10resistant isolates harbored a cat gene. Interestingly, the cat-positive strains isolated from the sea urchin rearing pond harbored only the cat II gene, whereas the strains from the sea cucumber rearing pond harbored mainly the cat IV gene. The difference in cat gene type and frequency between the two culture ponds seems to be related to bacterial species. The V. splendidus-like strains harbored mainly the cat II gene, whereas the V. tasmaniensis-like strains harbored mainly the cat IV gene. In our previous study of oxytetracycline and multiple antibiotic-resistant bacteria isolated from the same two culture ponds, we also found that the V. tasmaniensislike strains harbored predominantly the cat IV gene [9]. Multiple antibiotic-resistant sh pathogens isolated in Korea were found to carry only cat II or cat IV gene [39], consistent to the results we obtained in this study. The cat IV gene was originally found on the R plasmid detected in sh pathogenic Vibrio anguillarum strains isolated in Japan before 1977; the cat gene of the R plasmid from V. anguillarum strains isolated in Japan between 1989 and 1991 was classied as cat II [3]. The

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prevalence of the two types of cat genes in Vibrio species in Korea and China at the present time could aid in the speculation about the origin and spread of drug resistance genes among the three neighboring countries. The P. atlantica-like strains CCH-45 and CCH-63 isolated from the sea cucumber rearing pond harbored the cat IV gene. Both of the strains were psychrotrophic; they could be active in winter once they were released into the surrounding coastal environment via water exchange from the culture ponds. As the most serious diseases of farmed sea cucumbers usually occur in wintertime in northern China, antibiotic-resistant psychrotrophic P. atlantica-like bacteria might contribute to direct pathogenesis or to the spread of antibiotic resistance among bacterial pathogens. In the current study, no cat gene could be detected by the multiplex PCR method for the other ve marine Pseudoalteromonas-like bacterial strains. There might be other or even novel genetic determinants for chloramphenicol resistance for these strains.
Possible Genetic Mechanism for the Prevalence of The high incidence of chloramphenicolcat Genes.

resistant strains in the sea cucumber and sea urchin rearing ponds was surprising, as no chloramphenicol-related antibiotics have ever been used in the mariculture farm studied. It has been found that the use of one antibiotic can increase levels of resistance not only to that specic drug but also to many others, even those using very different modes of antibacterial action [18]. The high frequency of resistance to oxytetracycline and ampicillin in the sea cucumber and sea urchin chloramphenicol-resistant isolates implied that these antibiotics might be effective for coselection of chloramphenicol resistance (see Table 2). However, as all the cat-positive isolates detected in the current study were also resistant to OTC30, but not all of them were resistant to AMP10 (see Table 3), the effect of ampicillin as the major coselective agent of cat genes could not be justied. In our previous study, we found that the occurrence of the cat genes were highly coincident to the occurrence of the tet(B) gene in the studied mariculture environment [9]. Although the current data indicate that the relationship of the cat genes and the tet genes was more complicated than we originally detected, all the catpositive strains identied in the current study did harbor one or more of the efux type tet genes. Results from both studies imply the coselective effect of oxytetracycline on cat genes in the mariculture environment studied. Besides tet(B), the tet(D) gene was even more predominant and might have tighter association with the cat genes, especially for the sea urchin isolates in the current study (see Table 3). The reason that slightly different relationships of the cat and tet genes were found in our successive studies might be due to the initial types of antibiotics and their concentrations used for resistant bacteria isolation. In our previous study, OTC10 was used

for initial screening of resistant bacteria, whereas in the current study, CP5 was used for initial resistant bacteria isolation. Different antibiotics and their concentrations might select different subpopulations within the entire resistant bacterial population. Environmental bacterial strains with different physiological status might react to the same cultivation condition (i.e., the same culture medium and physical condition) differently in the existence of different antibiotic selection pressure. The concurrence of chloramphenicol and tetracycline resistances has been found frequently in both gramnegative and gram-positive bacteria [26, 38]. It has been found that long-term use of tetracycline selects not only for tetracycline-resistant bacteria but also for multiple antibiotic-resistant strains [19]. The tet genes are often found on the same mobile genetic elements as other antibiotic resistance genes [6]. Thus, the co-occurrence of the cat and tet genes in the resistant strains isolated in the current study could be a result of the possibility that these two types of resistance genes were located on the same genetic element, such as plasmids, transposons, or integrons. Research is being carried out to test this hypothesis in our laboratories. Interestingly, the two cat IV-positive P. atlantica-like strains harbored different tet genes. Strain CCH-45 harbored tet(B), whereas CCH-63 harbored tet(D). Some species of marine Pseudoalteromonas, such as Pseudoalteromonas piscicida, was reported to be pathogenic to sh [25]. The impacts of resistant Pseudoalteromonas strains on mariculture and natural environment, on cultured and wild marine animals, and on human beings health and public safety are waiting to be investigated.

Acknowledgments

The authors would like to express our appreciation to the anonymous reviewers. With the kind critiques and suggestions from reviewer 1, our manuscript was improved greatly in both scientic accuracy and writing style. This work was nancially supported by the National Natural Science Foundation of China grants (NSFC grant nos. 40476058 and 40576069), the Pilot Projects of Knowledge Innovation Project of Chinese Academy of Sciences grants (CAS grant nos. KZCX3-SW-223 and KZCX3-SW-233), and the grant from Institute of Oceanology, Chinese Academy of Sciences (IOCAS grant no. LSC2-3-1).

References
1. Agerso, Y, Guardabassi, L (2005) Identication of Tet 39, a novel class of tetracycline resistance determinant in Acinetobacter spp. of environmental and clinical origin. J Antimicrob Chemother 55: 566 569

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ffer, AA, Zhang, JH, Zhang, Z, 2. Altschul, SF, Madden, TL, Scha Miller, W, Lipman, DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25: 33893402 3. Aoki, T (2000) Transferable drug resistance plasmids in sh-pathogenic bacteria. In: Arthur, JR, Lavilla-Pitogo, CR, Subasinghe, RP (Eds.) Use of Chemicals in Aquaculture in Asia, SEAFDEC Aquaculture Department, Tigbauan, Iloilo, Philippines, pp 3133 4. Balebona, MC, Zorilla, I, Morinigo, MA, Borrego, JJ (1998) Survey of bacterial pathologies affecting farmed gilt-head sea bream (Sparus aurata L.) in southwestern Spain. Aquaculture 166: 1935 5. Chen, J (2004) Present status and prospects of sea cucumber industry in China. In: Lovatelli, A, Conand, C, Purcell, S, Uthicke, S, Hamel, J, Mercier, A (Eds.) Advances in Sea Cucumber Aquaculture and Management, FAO Fisheries Technical Paper, No. 463, FAO, Rome, Italy, pp 2538 6. Chopra, I, Roberts, M (2001) Tetracycline antibiotics: mode of action, applications, molecular biology, and epidemiology of bacterial resistance. Microbiol Mol Biol Rev 65: 232260 7. Courvalin, P, Trieu-Cuot, P (2001) Minimizing potential resistance: the molecular view. Clin Infect Dis 33: S138S146 8. Dang, H, Lovell, CR (2000) Bacterial primary colonization and early succession on surfaces in marine waters as determined by amplied rRNA gene restriction analysis and sequence analysis of 16S rRNA genes. Appl Environ Microbiol 66: 467475 9. Dang, H, Zhang, X, Song, L, Chang, Y, Yang, G (2006) Molecular characterizations of oxytetracycline resistant bacteria and their resistance genes from mariculture waters of China. Mar Poll Bull (in press) 10. De Medici, D, Croci, L, Delibato, E, Di Pasquale, S, Filetici, E, Toti, L (2003) Evaluation of DNA extraction methods for use in combination with SYBR green I real-time PCR to detect Salmonella enterica serotype enteritidis in poultry. Appl Environ Microbiol 69: 34563461 11. Doroudgar, S (2005) Intimate association of Vibrio splendidus with the purple sea urchin Strongylocentrotus purpuratus. Saltman Q 2: 4144 12. Felsenstein, J (1989) PHYLIPPhylogeny Inference Package (Version 3.2). Cladistics 5: 164166 13. Gay, M, Renault, T, Pons, AM, Le Roux, F (2004) Two Vibrio splendidus related strains collaborate to kill Crassostrea gigas: taxonomy and host alterations. Dis Aquat Org 62: 6574 14. Gomez-Leon, J, Villamil, L, Lemos, ML, Novoa, B, Figueras, A (2005) Isolation of Vibrio alginolyticus and Vibrio splendidus from aquacultured carpet shell clam (Ruditapes decussatus) larvae associated with mass mortalities. Appl Environ Microbiol 71: 98104 15. Jia, JS, Chen, JX (2001) Sea farming and sea ranching in China. FAO Fisheries Technical Paper, No. 418. FAO, Rome, Italy 16. Jun, LJ, Jeong, JB, Huh, MD, Chung, JK, Choi, DL, Lee, CH, Jeong, HD (2004) Detection of tetracycline-resistance determinants by multiplex polymerase chain reaction in Edwardsiella tarda isolated from sh farms in Korea. Aquaculture 240: 89100 17. Keesing, JK, Hall, KC (1998) Review of harvests and status of world sea urchin sheries points to opportunities for aquaculture. J Shellsh Res 17: 15971604 18. Kummerer, K (2004) Resistance in the environment. J Antimicrob Chemother 54: 311320 19. Levy, SB (1992) The Antibiotic Paradox: How Miracle Drugs are Destroying the Miracle. Plenum Press, New York, NY 20. Li, T, Xu, S, Wang, R, Xu, S, Su, X (2000) Preliminary studies on the black mouth disease of sea urchin, Strongylocentrotus intermedius (Strongylocentrotidae Echinoidea). Mar Sci 24: 4143 (in Chinese) 21. McCallum, H, Harvell, D, Dobson, A (2003) Rates of spread of marine pathogens. Ecol Lett 6: 10621067

22. Miranda, CD, Rojas, R (1996) Vibriosis in the ounder Paralichthys adspersus (Steindachner, 1867) in captivity. Rev Biol Mar 31: 19 23. Muroga, K (2001) Viral and bacterial diseases of marine sh and shellsh in Japanese hatcheries. Aquaculture 202: 2344 24. Nealson, KH, Wimpee, B, Wimpee, C (1993) Identication of Vibrio splendidus as a member of the planktonic luminous bacteria from the Persian Gulf and Kuwait region with luxA probes. Appl Environ Microbiol 59: 26842689 25. Nelson, EJ, Ghiorse, WC (1999) Isolation and identication of Pseudoalteromonas piscicida strain Cura-d associated with diseased damselsh (Pomacentridae) eggs. J Fish Dis 22: 253260 26. Padilla, C, Lobos, O, Brevis, P, Abaca, P, Hubert, E (2004) In vitro antibacterial activity of the peptide PsVP-10 against antimicrobialresistant Enterococcus faecalis isolated from clinical samples. J Antimicrob Chemother 53: 390 392 27. Prayitno, SB, Latchford, JW (1995) Experimental infections of crustaceans with luminous bacteria related to Photobacterium and Vibrio spp. effect of salinity and pH on infectiosity. Aquaculture 132: 105112 28. Roberts, MC (2005) Update on acquired tetracycline resistance genes. FEMS Microbiol Lett 245: 195203 29. Sugumar, G, Nakai, T, Hirata, Y, Matsubara, D, Muroga, K (1998) Vibrio splendidus biovar II as the causative agent of bacillary necrosis of Japanese oyster Crassostrea gigas larvae. Dis Aquat Org 33: 111118 30. Tantillo, GM, Fontanarosa, M, Di Pinto, A, Musti, M (2004) Updated perspectives on emerging vibrios associated with human infections. Lett Appl Microbiol 39: 117126 31. Thompson, JR, Gibson, TJ, Plewniak, F, Jeanmougin, F, Higgins, DG (1997) The ClustalX windows interface: exible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 24: 4876 4882 32. Thompson, JR, Pacocha, S, Pharino, C, Klepac-Ceraj, V, Hunt, DE, Benoit, J, Sarma-Rupavtarm, R, Distel, DL, Polz, MF (2005) Genotypic diversity within a natural coastal bacterioplankton population. Science 307: 13111313 33. Thomson, R, Macpherson, HL, Riaza, A, Birkbeck, TH (2005) Vibrio splendidus biotype 1 as a cause of mortalities in hatcheryreared larval turbot, Scophthalmus maximus (L.). J Appl Microbiol 99: 243250 34. Urakawa, H, Kita-Tsukamoto, K, Ohwada, K (1999) 16S rDNA restriction fragment length polymorphism analysis of psychrotrophic vibrios from Japanese coastal water. Can J Microbiol 45: 10011007 35. Wang, B, Li, Y, Li, X, Qu, J, Zhao, X (2005) Pathogenic mechanism of the causative vibrio found in Bred spotting^ diseased sea urchin Strongylocentrotus intermedius. J Dalian Fish Univ 20: 1115 (in Chinese) 36. Wang, Y, Zhang, C, Rong, X, Chen, J, Shi, C (2004) Diseases of cultured sea cucumber, Apostichopus japonicus, in China. In: Lovatelli, A, Conand, C, Purcell, S, Uthicke, S, Hamel, J, Mercier, A (Eds.) Advances in Sea Cucumber Aquaculture and Management, FAO Fisheries Technical Paper, No. 463, FAO, Rome, Italy, pp 297310 37. Wang, ZC, Chang, YQ (1997) Research prospects for mariculture of sea urchin. Mar Sci 2: 2224 (in Chinese) 38. Wright, JG, Tengelsen, LA, Smith, KE, Bender, JB, Frank, RK, Grendon, JH, Rice, DH, Thiessen, AM, Gilbertson, CJ, Sivapalasingam, S, Barrett, TJ, Besser, TE, Hancock, DD, Angulo, FJ (2005) Multidrug-resistant Salmonella typhimurium in four animal facilities. Emerg Infect Dis 11: 12351241 39. Yoo, MH, Huh, MD, Kim, EH, Lee, HH, Jeong, HD (2003) Characterization of chloramphenicol acetyltransferase gene by multiplex polymerase chain reaction in multidrug-resistant strains isolated from aquatic environments. Aquaculture 217: 1121