1394 Journal of Food Protection, Vol. 77, No. 8, 2014, Pages 1394-1401 doi: 10.4315/0362-028X.JFP-13-510 Copyright © , International Association for Food Protection

Research Note

Characterization of Antibiotic Resistance in Escherichia coli Isolated from Shrimps and Their Environment KANJANA CHANGKAEW,1 FUANGFA UTRARACHKIJ,2 KANOKRAT SIRIPANICHGON,2 CHIE NAKAJIM A,1 ORASA SUTHIENKUL,2* and YASUHIKO SUZUKI1* 1Division of Global Epidemiology, Research Centerfor Zoonosis Control, Hokkaido University, Sapporo, Japan; and 2Department of Microbiology, Faculty of Public Health, Mahidol University, Bangkok, Thailand MS 13-510: Received 26 November 2013/Accepted 7 February 2014

ABSTRACT Antimicrobial resistance in bacteria associated with food and water is a global concern. To survey the risk, 312 Escherichia coli isolates from shrimp farms and markets in Thailand were examined for susceptibility to 10 antimicrobials. The results showed that 17.6% of isolates (55 of 312) were resistant to at least one of the tested drugs, and high resistance rates were observed to tetracycline (14.4%; 45 of 312), ampicillin (8.0%; 25 of 312), and trimethroprim (6.7%; 21 of 312); 29.1% (16 of 55) were multidrug resistant. PCR assay of the tet(A), tet(B), tet(C), tet(D), tet(E), and tet(G) genes detected one or more of these genes in 47 of the 55 resistant isolates. Among these genes, tet(A) (69.1%; 38 of 55) was the most common followed by tet(B) (56.4%; 31 of 55) and tet(C) (3.6%; 2 of 55). The resistant isolates were further investigated for class 1 integrons. Of the 55 resistant isolates, 16 carried class 1 integrons and 7 carried gene cassettes encoding trimethoprim resistance (dfrA12 or dfrA17) and aminoglycosides resistance (aadA2 or aadA5). Two class 1 integrons, In54 (dfrA17-aadA5) and In27 (dfrA12-oifF-aadA2), were found in four and three isolates, respectively. These results indicate a risk of drug-resistant E. coli contamination in shrimp farms and selling places. The occurrence of multidrug-resistant E. coli carrying tet genes and class 1 integrons indicates an urgent need to monitor the emergence of drug-resistant E. coli to control the dissemination of drug-resistant strains and the further spread of resistance genes to other pathogenic bacteria.

Food contamination with antimicrobial-resistant bacte­ ria is a public health concern because the resistant organisms can transfer to humans through the consumption of contaminated food and can thus compromise human health. Antimicrobial use in aquaculture results in a risk of the evolution of antimicrobial-resistant bacteria in aquatic animals and the environment (14). In shrimp fanning industries, large amounts of antimicrobial agents are used as feed additives and thrown into the pond water as therapeutic and prophylactic agents for shrimp. Consequently, antimi­ crobials persisting in shrimp, water, and sediment in the shrimp pond may contribute to the acquisition of drug resistance among shrimp pathogens and the intestinal flora of the shrimp. In recent studies in various countries, the prevalence of antimicrobial-resistant bacteria in shrimp farms and shrimp products has been reported (8, 19, 29, 31, 35, 36). The microbiological condition of shrimp is mainly dependent on the transportation, handling, and processing conditions (30). Antimicrobial-resistant bacteria can contaminate shrimp products through improper han­ dling of shrimp between harvesting and marketing, which is

* Authors for correspondence. Tel: +81-11-706-9503; Fax: + 81-11-7067310; E-mail: [email protected].. (Y.S.). Tel: +66-2354 8543-9, Ext 6609; Fax: +66-2354 8538; E-mail: [email protected] (O.S.).

a particular problem in markets where raw shrimp are sold and handled by sellers and customers (23, 30). The horizontal transfer of resistance genes, especially mobile genetic elements, has led to the rapid emergence of multidrug-resistant bacteria in the aquaculture environment, which may disseminate to humans. Integrons are gene­ capturing systems located on the bacterial chromosome or a plasmid that incorporate gene cassettes and make them functional. Integrons play an important role in the carriage and dissemination of antimicrobial resistance genes (9). Class 1 integrons are most frequently found in multidrugresistant gram-negative bacteria and have been identified in bacterial strains recovered from aquatic animals, their products, and the environment (15, 19). Escherichia coli is commonly used as an indicator of fecal contamination in water and food quality analyses (11). E. coli also has been introduced as an indicator species in surveillance programs to analyze the antimicro­ bial resistance status of the enteric microflora of both farm animals and humans. However, little is known about the phenotypic drug resistance and the genetic mechanisms of resistance in E. coli isolates from shrimps in Thailand. The objectives of this study were to investigate drug resistance profiles and characterize class 1 integrons in E. coli isolates from shrimps and their environment. Resistance determinants for the major resistance phenotype were also identified.

DRUG-RESISTANT E. COU IN SHRIMP

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TABLE 1. Oligonucleotide primers used in this study PCR conditions Primer name

Primer sequence (5'—3')

Target

Denaturing

tetAF (643-662) tetAR (882-863) tetB (F) tetB (R) tetC (F) tetC (R) tetD (F) tetD (R) tetE (F) tetE (R) tetG (F) tetG (R) INT-1U INT-1D 5'CS2 (F) 3'CS2 (R) Sull (R) Sul2 (R) Sul3 (R)

GGCGGTCTTCTTCATCATGC AAGCAGGATGTAGCCTGTGC TTGGTTAGGGGCAAGTTTTG GTAATGGGCCAATAACACCG CTTGAGAGCCTTCAACCCAG ATGGTCGTCATCTACCTGCC AAACCATTACGGCATTCTGC GACCGGATACACCATCCATC AAACCACATCCTCCATACGC AAATAGGCCACAACCGTCAG GCTCGGTGGTATCTCTGCTC AGCAACAGAATC GGGAACAC GTTCGGTCAAGGTTCTG GCCAACTTTCAGCACATG TGTACAGTCTATGCCTCGGGCATCC AGCAGACTTGACCTGATAGTTTGGC CTAGGCATGATCTAACCCTCGGTCT CGAATTCTTGCGGTTTCTTTCAGC CATCTGCAGCTAACCTAGGGCTTTGGA

tet(A)

95°C, 30 s

55°C, 30 s 72°C, 15 s"

This work

tet(B)

98°C, 5 s

55°C, 15 s 72°C, 60 sh

22

tet(C)

98°C, 5 s

55°C, 15 s 72°C, 60 s*

22

tet( D)

98°C, 5 s

55°C, 15 s 72°C, 60 s*

22

tet(E)

98°C, 5 s

55°C, 15 s 72°C, 60 sft

22

tet(G)

98°C, 5 s

55°C, 15 s 72°C, 60 sh

22

intll

94°C, 30 s

50°C, 30 s 72°C, 2 minc 22

5'CS 3'CS sull sul2 sul3

98°C, 10 s

60°C, 10 s 72°C, 5 min'* This This This This This

“ After h After c After d After

30 35 30 30

cycles, cycles, cycles, cycles,

final final final final

extension extension extension extension

wasconducted at wasconducted at wasconducted at wasconducted at

72°C 72°C 72°C 72°C

Annealing

Extension

Reference

work work work work work

for 5 min. for 10 min. for 7 min. for 10 min.

MATERIALS AND METHODS E. coli isolates and DNA preparation. A total of 312 E. coli isolates were collected from shrimp farms (149 isolates) and markets (163 isolates) in a province in southern Thailand from February 2007 to February 2008. From shrimp farms, 27,54, 55,11, and 2 isolates were from shrimp (Penaeus vannamei), surface water, shrimp pond waste, shrimp pond sediment, and shrimp feed, respectively. From markets, 37 and 126 isolates were from shrimp and water, respectively. The isolates were revived, and their identity was reconfirmed with biochemical tests (20). One loopful of E. coli stock was streaked directly on MacConkey agar and incubated at 37°C for 24 h. One lactose-positive colony was picked and used for the IMViC (indole, methyl red, Voges-Proskauer, and citrate) test was performed. Isolates with an IMViC pattern of positive-positive­ negative-negative was considered an E. coli strain. Drug-resistant strains were cultured in Luria broth at 37°C for 24 h for DNA extraction. DNA was prepared by phenol-chloroform extraction based on the procedure of Maniatis et al. (26). Antimicrobial susceptibility tests. Antimicrobial suscepti­ bility tests were carried out using the Kirby-Bauer agar disk diffusion method in accordance with the standard and interpretive criteria recommended by the Clinical and Laboratory Standards Institute (7). The following commercially available antimicrobial disks (Oxoid, Basingstoke, UK) were used: ampicillin (AMP), 10 pg; cefotaxime (CTX), 30 pg; ceftriazone (CRO), 30 pg; ceftazidime (CAZ), 30 pg; streptomycin (STR), 10 pg; kanamycin (KAN), 30 pg; tetracycline (TET), 30 pg; nalidixic acid (NAL), 30 pg; chloramphenicol (CHL), 30 pg; and trimethoprim (TMP), 5 pg. E. coli ATCC 25922 was used to verily the quality and accuracy of the tests. Characterization of tetracycline resistance genes. Tetra­ cycline efflux genes were amplified by PCR according to Kumai et

al. (22). The primer sequences and PCR conditions are presented in Table 1. PCR products were analyzed by electrophoresis on a 2% agarose gel with 0.5 pg/ml ethidium bromide in 1 x Tris-acetateEDTA buffer at 50 mA for 25 min, followed by visualization under UV light (ATTO Co., Tokyo, Japan). The sizes of the PCR products were calculated based on a 50-bp DNA marker. Identification of class 1 integrons. Resistant isolates were tested for the presence of class 1 integrase genes by PCR using primers INT-1U and INT-1D (22). The PCR mixture (total 20 pi) consisted of 1 pi of DNA template, 1 x Green GoTaq reaction buffer, 0.2 mM concentrations of each deoxynucleoside triphos­ phate, 0.4 pM concentrations of each primer, and 0.5 U of GoTaq DNA polymerase. PCR products were analyzed by electrophoresis on a 1% agarose gel, and the sizes of the PCR products were determined based on a 100-bp DNA ladder and a lambda ffindlH digest as size markers. Characterization of gene cassettes in integrons. All class 1 integrase PCR-positive strains were further analyzed for their gene cassette pattern by amplifying the variable region between the 5'conserved segment (5'CS) and 3'-conserved segment (3'CS) or sul genes. Four sets of one forward primer and one of the following four reverse primers were designed and used: 5'CS2 (F) with 3'CS2 (R), Sull (R), Sul2 (R), or Sul3 (R) (Table 1). The PCR mixture (total 20 pi) consisted of 1 pi of DNA template, 1 x LA PCR buffer (Mg2+ free), 0.375 mM concentrations of each deoxynucleoside triphosphate, 2.5 mM MgCl2 , 0.4 pM concentra­ tions of each primer, and 1 U of LA Taq (Takara Bio, Shiga, Japan). After agarose gel electrophoretic separation, PCR products were purified with Wizard SV Gel and PCR Clean-Up System Kits (Promega, Madison, WI). The concentration and quality of the purified products were measured with ND-1000 V3.1.0 (Thermo Fisher Scientific, Waltham, MA). The purified products were

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CHANGKAEW ET AL.

J. Food Prot., Vol. 77, No. 8

TABLE 2. Oligonucleotide primers used for primer walking Primer name 5'CS-RV (R) aadA2(202-182) (R) aadA5 (25-2) (F) dfrl7 (274-287) (F) dfr 17-Cap (606-625) (F) Sull (284-263) (R) Sul2 (179-160) (R) Sul2-5'side noncode-1 (R) Sul2-5'side (244) Sul2-5'side (230) QacE deltal-Cap (272-292) (R)

Primer sequence (5'— 3')

Target

CTTTGTTTTAGGGCGACTG TGAGCAATGCTCGCCGCGTCG AACGATTGAGAGAATCTTGCGTTG GATCATGTATATGTCTCTGGCGGG TCCGCCACGACATCCTTTCC CGCTTGAGCGCATAGCGCTGGG GAAACAGGCGCGGCGTCGGG GATTTTATTCCCGCGCCTCG GCAGGGAATACTCAGCAAGC ACCTCCCGTGCTGTACTGTC CCCTCGCTAGATTTTAATGCG

5'CS aadA2 aadA5 dfr17 dfr17 sull sul2 sul2 sul2 sul2 qacEAl

sequenced by primer walking using the BigDye terminator cycle sequencing ready reaction kit and analyzed using the 3130 Genetic Analyzer (Applied Biosystems, Foster City, CA). Contiguous sequences were analyzed with the BLAST search engine (National Center for Biotechnology Information, http://blast.ncbi.nlm.nih. gov/Blast.cgi) and compared with those registered in the GenBank database. The primer sets designed in this study and used for primer walking are shown in Table 2. Data analysis. The prevalence of antimicrobial-resistant E. coli isolates in various samples from shrimp farms and markets was recorded as percentages. Antimicrobial resistance rates were compared with a chi-square test. Differences were considered significant at P < 0.05. RESULTS Antimicrobial susceptibility of E. coli isolates. The results indicate that 17.6% of the isolates (55 of 312) were resistant to at least one antimicrobial agent (Table 3). High resistance rates were observed for TET (14.4%; 45 of 312 isolates), AMP (8.0%; 25 of 312), and TMP (6.7%; 21 of 312). Resistance to NAL (2.9%; 9 of 312), CHL (2.2%; 7 of 312), KAN (1.6%; 5 of 312), STR (1.3%; 4 of 312), CTX (0.3%; 1 of 312), CRO (0.3%; 1 of 312), and CAZ (0.3%; 1 of 312) was less common. Twenty-two resistance patterns were found among the 55 drug-resistant isolates (Table 3). The rate of resistance to two drugs was the highest at 38.2% (21 of 55 isolates), followed by resistance to one drug (32.7%; 18 of 55), three drugs (14.5%; 8 of 55), four drugs (9.1%; 5 of 55), and five drugs (5.5%; 3 of 55). The prevalence of multidrug-resistant isolates (i.e., resistant to three or more than drugs) was 29.1% (16 of 55). Regarding the source of the samples, the resistant isolates were found in shrimp (29.6%, 8 of 27), pond water (7.4%; 4 of 54), and pond waste (25.5%, 14 of 55) on the farms and in shrimp (13.5%, 5 of 37) and storage water (19.0%; 24 of 126) in the markets. The resistance rate of E. coli isolates from storage water in the markets (19%; 24 of 126 isolates) was significantly higher than that in the shrimp farm pond water (7.4%; 4 of 54) (P < 0.05). Conversely, no significant differences were observed in resistant isolate rates between shrimp from the farm and shrimp from the market. The multidrug-resistant isolate ratio was highest in shrimp on the

farms (62.5%, 5 of 8); however, the ratio for isolates resistant to more than three drugs was predominantly higher in shrimp storage water in the markets (25%, 6 of 24) than in other samples. Characterization of tetracycline resistance genes. Tetracycline resistance genes were amplified by PCR with six sets of primers targeting efflux genes. Among the 55 resistant isolates, 47 were positive for the tet genes (Table 4). The most common tet gene identified was tet(A) (69.1%; 38 of 55 isolates). The second common gene was tet{B), which was found in 31 isolates (56.4%), and tet(C), which was found in 2 isolates (3.6%). The tet(D), tet(E), and tet(G) genes were not detected. Fifteen resistant isolates carried only tet{A), 7 carried only tet(B), 23 carried both tet(A) and tet(B), and 2 carried ref(B) and tet(C). Class 1 integron detection and characterization. All 55 resistant isolates were tested for the presence of the class 1 integrase gene (intll) by PCR amplification. Sixteen isolates (11 from water in the markets, 4 from shrimp pond waste, and 1 from shrimp in a farm) carried the intll gene (Table 4) and were further examined for gene cassettes. Gene cassettes were successfully detected in seven isolates; however, detection failed in the remaining nine isolates presumably because of alterations in the primer binding site. Two integrons (In54 and In27) were defined by their structural features (28). In54 was amplified from four isolates (N24B1, N230B3, N237B3, and N244B3) with the primer set 5'CS2 plus Sul2. This integron harbored dfrA17, aadA5, and sul2. DNA fragments of 2.0 and 3.7 kb were amplified with the primer sets 5'CS2 plus 3'CS2 and 5'CS2 plus Sull, respectively, from three isolates (N361B4, N369B4 and N370B4). Both fragments were derived from the same integron consisting of dfrA12, orfF, aadA2, qacEAl, and sull (In27). The promoter region in each integron was also analyzed and is listed in Table 4. DISCUSSION Antimicrobials are used for the treatment of infectious diseases in humans and for nonhuman applications. The emergence of antimicrobial-resistant organisms is a major

TABLE 3. Antimicrobial resistance patterns ofE. coli isolates from shrimp farms and markets

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of the culture pond), and shrimp feed. In markets, E. coli isolates were obtained from samples of shrimp and water (ice water in shrimp container).

ceftazidime. On shrimp farms, E. coli isolates were obtained from samples of shrimp, surface water, pond waste (accumulated waste in the center of the culture pond), pond sediment (pond soil around the edge

AMP, ampicillin; NAL, nalidixic acid; STR, streptomycin; TET, tetracycline; TMP, trimethoprim; CHL, chloramphenicol; KAN, kanamycin; CRO, ceftriazone; CTX, cefotaxime; CAZ,

J. Food Prot., Vol. 77, No. 8 DRUG-RESISTANT E. COLI IN SHRIMP 1397

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CHANGKAEW ET AL.

J. Food Prot., Vol. 77, No. 8

TABLE 4. Phenotypes and genotypes of antimicrobial-resistant E. coli isolates from shrimp and their environments Antimicrobial resistance profile0

Source Farm

Sample Shrimp

Shrimp pond waste

Water

Market

Shrimp

Water

Area

Code

BA

N52B1

BI

N347B4

BI BI AI BA BI AI AI

N362B4 N230B3 N373B4 N53B1 N222B3 N37B1 N93B1

AI AI AI AI AA AA AA AI AI AI AI AI AI AA AA AA AI BI Cl CA Cl Cl CA

N246B3 N244B3 N245B3 N247B3 N270B3 N274B3 N275B3 N266B3 N267B3 N268B3 N276B3 N277B3 N278B3 N21B1 N71B1 N218B3 N23B1 N321B4 N13B1 N16B1 N171B2 N173B2 N220B3

CA

N106B2

CA

N369B4

CA

N370B4

CA

N360B4

CA

N33B1

Cl CA CA Cl CA CA CA CA CA Cl CA CA

N229B3 N219B3 N361B4 N28BI N48B1 N100B1 N104B2 N166B2 N150B2 N85B1 N368B4 N24B1

Resistant STR, KAN, CHL, TMP, TET AMP, KAN, TET AMP, KAN, TET AMP, CHL, TET AMP, NAL, TET CHL, TMP TET AMP AMP, CRO, CTX, CAZ, TET NAL, TMP, TET TMP, TET TMP, TET TMP, TET TET TET TET TET TET TET TET TET TET AMP, TET AMP, TET TMP, TET TET AMP, NAL, TET AMP, TMP NAL, TMP NAL STR AMP, KAN, CHL, TMP, TET AMP, NAL, CHL, TMP AMP, NAL, TMP, TET AMP, NAL, TMP, TET AMP, CHL, TMP, TET AMP, CHL, TMP, TET AMP, STR, TET KAN, TMP, TET AMP, TMP AMP, TMP AMP, TET AMP, TET AMP, TET AMP, TET AMP, TET AMP, TET TMP, TET TMP, TET

Intermediate

CRO, NAL, TMP NAL STR

NAL

AMP, AMP, AMP, AMP, STR STR

STR STR STR STR

STR, NAL

STR, TET

inm PCR

Integron*

Promoter (Pc)

tet gene

_

A

-

A

+ — — + + — — — + + — — —

In54

PcW

A, B A, B B B, C A, B

In54

PcHl

A, B A A A, B A, B A A, A, A, A, A, A,

B B B B B B

A A A, B A B B, C

STR

+

A, B

STR

-

A

+

In27

PcW TGN-10

A

+

In27

PcW TGN-10

A

—

A, B

-

STR, CHL STR STR, TET

NAL

+ + + + + +

In27

In54

PcW TGN-10

A, B A, B B

PcHl

B A A A, B A, B B A A, B

DRUG-RESISTANT E. COLI IN SHRIMP

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TABLE 4. Continued Antimicrobial resistance profile”

intll Source

Sample

Area

Code

Resistant

CA Cl CA CA CA CA

N237B3 N116B2 N165B2 N350B4 N348B4 N366B4

TMP, TET STR, TET TET TET TET NAL

Intermediate

PCR

Integron*

Promoter (Pc)

tet gene

+ + —

In54

PcHl

A, B B A, B A, B A B

a STR, streptomycin; KAN, kanamycin; CHL, chloramphenicol; TMP, trimethoprim; TET, tetracycline; AMP, ampicillin; CRO, ceftriazone; NAL, nalidixic acid; CTX, cefotaxime; CAZ, ceftazidime.

h In54 consists o f dfrA17 and aadA5. In27 consists of ddfrA12, orfF, and aadA2.

public health concern because these organisms may be circulating in the food chain, including shrimp farms and markets. In our study, high resistance rates were found for tetracycline, ampicillin, and trimethoprim. In Thai shrimp farms, quinolones, tetracyclines, and sulfonamides are used to prevent and treat Vibrio infections (13). Similar resistance phenotypes have been previously detected in Salmonella and Vibrio strains from shrimp farms in Thailand (19, 20). Similar results have been published for sea bass farms in Turkey and shrimp farms in Brazil and the Philippines (27, 31, 34, 36). These presence of antimicrobial-resistant organisms appeared to be due to the uncontrolled use of tetracycline, ampicillin, and trimethoprim and the spread of antibiotic-resistant E. coli strains in the aquatic environment. Tetracyclines have been used commonly as growth promoters, and their residues have been carried over to animal husbandry and aquaculture (12). Long-term lowlevel exposure to tetracyclines can increase the spread of resistant bacterial strains, which can occur in shrimp farms and markets where tetracyclines are regularly used, as indicated by the high prevalence of tetracycline-resistant E. coli strains in this study. In aquaculture farms in different regions of the world, tet(A), tet(B), and tet(E) are prevalent (3,10). Two of these genes, tet(A) and tet(B), were found in the present study. The genetic background of tetracycline resistance among bacteria from this aquatic environment appeared to be diverse locally, probably because of differences in the drag resistance gene pool in the environment. In some tetracycline-resistant isolates, no genes for efflux proteins were detected. This resistance phenotype may have been caused by an alternative mechanism of tetracycline resistance, such as ribosomal protection or target mutation (5, 6). The high prevalence of ampicillin-resistant strains may reflect (i) the widespread clinical use of penicillins in both humans and animals, which results in selective pressure on bacterial populations in the environment, or (ii) some illegal use of penicillins in shrimp farms and markets, because 13lactams are not officially used in shrimp farming (2). Alternatively, the wide distribution of penicillin resistance could be due to coselection of antibiotics, as suggested by the frequent presence of several kinds of drug resistance genes on the same mobile element. In our study, 29% of

resistant isolates were resistant to more than three drugs. Multidmg resistance has been observed in bacteria from aquaculture environments in association with the use of various drugs (1,34, 36). Akinbowale et al. (1) found that use of one or more antibiotics in aquaculture provided selection pressure for the emergence of multidrug-resistant bacteria. Class 1 integrons were detected in 16 resistant isolates. Of these isolates, seven carried gene cassettes that included dihydrofolate reductase genes (dfrA12 or dfrA17) confer­ ring trimethoprim resistance and aminoglycoside adenyltransferase A genes (aadA2 or aadA5) conferring strepto­ mycin resistance. Class 1 integrons are widely distributed among bacteria isolated from shrimp, fish, and shellfish (19, 32, 35). Five isolates in out study containing In54 were found in samples from different areas (AI, BI, and CA) (Table 4), suggesting they are widely spread in southern Thailand. Kang et al. (17) reported that the In54 integron is commonly found in E. coli isolates from humans in Korea. In Thailand, a study of class 1 integrons among Salmonella enterica isolates from poultry and swine revealed that the gene cassette array dfrA12-otfE-aadA2 (In27) was the most prevalent among these isolates (18). Wannaprasat et al. (37) also reported that Salmonella isolated from pork and humans in northern Thailand carried class 1 integrons and that the most frequently found profile was In27. In our work, E. coli isolates possessing In27 were detected from samples obtained in the markets, indicating that In27 is widespread among humans and food animals in Thailand. Among the isolates carrying the integrons with the dfrA gene cassette, six were resistant to trimethoprim and one was susceptible to this agent. All isolates carrying the integrons with the aadA gene cassette were not resistant to streptomycin; however, four isolates expressed a strepto­ mycin intermediate phenotype (Table 4). The MICs of isolates with the aadA gene cassette were 4 mg/liter (six isolates) and 8 mg/liter (one isolate). A similar result was reported by Sunde and Norstrom (33), who suggested that E. coli strains carrying aadA gene cassettes could have MICs far below the breakpoint values normally used to classify a strain as susceptible to streptomycin. Zhao et al. (38) reported that the integron-bome aadA gene was silent

1400

CHANGKAEW ET AL.

in E. coli 0 1 1 1:NM but became fully expressed when it was transferred to Hafnia alvei. This silent aadA gene can become expressed when transferred to a new host, and horizontal transfer may enhance the expression of a resistance gene. Variable expression of gene cassettes in integrons can be caused by several factors, e.g., whether the integron is located on a high-copy-number plasmid or the position of the cassette on the integron is near the 3' end (4). Several versions of the integron promoters located in the 5'conserved segment of the integron cause differences in the strength of the promoter (25). Five predominant gene cassette promoter (Pc) variants possessing different expres­ sion efficacies have been identified (16) (weakest to the strongest): PcW, PcHl, PcWTGN_10, PcH2, and PcS. In the present study, three different Pc variants were found: PcW, PcHl, and PcWTGN_10 (Table 4). One isolate carrying In54 harbored PcW (N230B3), the weakest promoter, and was susceptible to trimethoprim although it also carried a dfrAl 7 cassette. In contrast, three other isolates carrying In54 with PcHl (N244B3, N24B1 and N237B3) were resistant to trimethoprim (Table 4). Our results suggest that gene cassettes of class 1 integrons may be differently expressed depending on the Pc promoter variant. The presence of E. coli in water or food is an indicator of fecal contamination and suggests the possibility of exposure of consumers to potential pathogens. We postu­ lated that drug-resistant E. coli strains on farms and in markets might come from a variety of sources, including workers’ hands, animal waste, shrimp feed, and water. The highest rate of antibiotic resistance was found in shrimp samples. However, the prevalence of highly drug-resistant E. coli (i.e., resistant to more than four drugs) was significantly higher in shrimp storage water (ice water) in markets than that on shrimp farms. Contamination by microorganisms might occur after harvesting shrimps, during transportation and sale in the markets. Our findings are in agreement with reports from various countries, in which drug-resistant E. coli contamination of shrimp and associated materials was also found in selling places, e.g., fresh markets, supermarkets, and retail stores (21, 23, 30, 35). Previous studies have indicated that water used for washing seafood and ice used for chilling seafood may be heavily contaminated with dmg-resistant organisms that can grow on shrimps (24, 30). The environment on shrimp farms and in selling places is the main source of drug-resistant E. coli strains that contaminate the shrimp. Therefore, the improvement of hygiene practices along the food chain and pmdent use of antimicrobials on shrimp farms are essential. The occur­ rence of multidrug-resistant E. coli isolates carrying tet genes and class 1 integrons in this study (16 of 312 isolates; 5.1%) suggests that multidrug-resistant pathogenic bacterial strains other than E. coli may occur in shrimp and associated materials and could be a significant public health concern. This multidrug resistance is a result of the adaptation of bacteria to antimicrobials in the environment and/or of transfer of mobile genetic elements from resistant bacteria; hence, resistance genes transferring from farm to fork can compromise human health.

J. Food Prot., Vol. 77, No. 8

ACKNOWLEDGMENTS This work was supported in part by a Department of Microbiology grant from the China Medical Board (Faculty of Public Health, Mahidol University) and the National Development of Science and Technology (Ministry of Science and Technology, Bangkok, Thailand) to O. Suthienkul, in part by J-GRID (the Japan Initiative for Global Research Network on Infectious Diseases, Ministry of Education, Culture, Sports, Science, and Technology, Japan [MEXT]), and in part by the Global Center of Excellence program, “ Establishment of International Collaboration Centers for Zoonosis Control” from MEXT to Y. Suzuki.

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