Isolation and characterization of multidrug-resistant Klebsiella spp. isolated from shrimp imported from Thailand

International Journal of Food Microbiology 155 (2012) 179–184

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Isolation and characterization of multidrug-resistant Klebsiella spp. isolated from shrimp imported from Thailand Mohamed Nawaz a,⁎, S.A. Khan a, Q. Tran a, K. Sung a, A.A. Khan a, I. Adamu b, R.S. Steele a a b

Division of Microbiology, National Center for Toxicological Research, US Food and Drug Administration, Jefferson, AR 72079, USA University of Central Arkansas, Conway, AR 72035, USA

a r t i c l e

i n f o

Article history: Received 10 August 2011 Received in revised form 30 January 2012 Accepted 2 February 2012 Available online 9 February 2012 Keywords: Klebsiella spp. Fluoroquinolone Tetracycline resistance Imported shrimp

a b s t r a c t A study was undertaken to isolate and characterize tetracycline and nalidixic acid-resistant Klebsiella spp. in farm-raised, imported shrimp sold in the United States. Sixty-seven multiple antibiotic-resistant Klebsiella spp. strains were isolated from imported shrimp samples. Using morphological and biochemical methods, fifty-three strains were tentatively identified as Klebsiella pneumoniae and fourteen as K. oxytoca. Although all isolates were resistant to tetracycline, only 8 were resistant to nalidixic acid. These 8 isolates were further screened by PCR for quinolone resistance genes (qnrA, B, S, gyrA, B and parC). PCR protocols failed to amplify any qnr genes. The purified PCR amplicons of gyrA, gyrB and parC were sequenced and analyzed for point mutations that confer resistance to fluoroquinolone antibiotics. Analysis of the sequences of the gyrA amplicons from nalidixic acid-resistant Klebsiella spp. indicated two point mutations in gyrA at positions 83 (Ser → Phe) and 87 (Asp → Ala). Sequence analysis of the parC amplicons indicated an amino acid change at position 80 (Ser → Ile). No mutations were detected in gyrB. Template DNA from all isolates was screened for tetracycline resistance genes (tetA–E). Oligonucleotide primers specifically targeting a 305-bp region of tetB and a 477-bp region of tetD successfully amplified sequences from 91.0 and 44.0% of the isolates, respectively. None of the isolates contained tetA, tetC or tetE genes. Plasmids (2.0–16.0 kb) were found in 23 of the 67 isolates. XbaI-PFGE identified 32 distinct macro restriction patterns (mrps) among the 61 multiple drugresistant Klebsiella spp. that were typable. Our results indicate that imported shrimp is a reservoir for multidrug resistant Klebsiella spp. and potential health risks posed by such strains should not be underestimated. Published by Elsevier B.V.

1. Introduction Shrimp aquaculture is a highly profitable, rapidly growing industry in many developing Asian countries. These countries produce 80% of the world's farmed shrimp output (Csavas, 1994; Biao and Kaijin, 2007). The black tiger shrimp (Penaeus monodon) and the Pacific white shrimp (P. vannamei) are the most widely cultured for export (Bailey-Brock and Moss, 1992; Csavas, 1994). The United States (US) imported about 1.3 billion pounds (589,670 metric tons) of farmed shrimp with a market value of more than $6 billion from Asia (United States Department of Agriculture, 2010). The increased global demand and high rate of return in shrimp aquaculture have led to the development and implementation of technologies that improve production and profits (Csavas, 1994). Diseases are known to curtail shrimp production by up to 70%. Antibiotics are broadly used in shrimp ponds to stimulate growth and to retard the incidence of diseases caused by overcrowded, factory farm conditions (Bondad-Reantaso et al., 2005; Holmstrom et al., 2003; Park et al., 1994). Although the United States

⁎ Corresponding author. Tel.: + 1 870 543 7586. E-mail address: [email protected] (M. Nawaz). 0168-1605/$ – see front matter. Published by Elsevier B.V. doi:10.1016/j.ijfoodmicro.2012.02.002

Food and Drug Administration (USFDA) has not approved any antibiotic for use in shrimp aquaculture in the US, a variety of antibiotics such as tetracyclines, fluoroquinolones, macrolides, sulfonamides and trimethoprims are used in shrimp aquaculture elsewhere (Holmstrom et al., 2003). The residues of many of these antibiotics were found incorporated in shrimp tissues (Love et al., 2011; Johnston et al., 2002; Dang et al., 2010). The indiscriminate use of antibiotics in shrimp farms may select bacteria resistant to multiple antibiotics (Midtved and Lingaas, 1992; Nawaz et al., 2001). Similarly, the inadvertent abuse of fluoroquinolones and tetracycline in shrimp aquaculture could result in the selection of fluoroquinolone and tetracycline resistant Klebsiella spp. in shrimp ecosystem. Since fluoroquinolones and tetracyclines are recommended (Hooper and Wolfson, 1985; Hopkins et al., 2005) for the treatment of a variety of human infections (Zherebtsova et al., 2007; Savas et al., 2006; Krcmery et al., 1998; Castro et al., 2010) caused by Klebsiella spp., the inadvertent ingestion of multiple antibiotic resistant Klebsiella spp. in shrimp may reduce the efficacy of these drugs in clinical treatment of these diseases. In addition such bacteria may transfer antibiotic resistance determinants to pathogenic bacteria (Nawaz et al., 2001; Griffin et al., 2003). Numerous public health agencies in the United States want to limit the prevalence of antibiotic-

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resistant bacteria in food-producing ecosystems (Nawaz et al., 2001). In this report we describe the isolation and characterization of 67 multiple antibiotic-resistant Klebsiella spp. from imported shrimp sold in supermarkets.

ciprofloxacin (5 μg) and ofloxacin (5 μg) were used. The susceptibility or resistance of each isolate were determined as per the manufacturer's instructions and by the criteria of the CLSI (2002). 2.3. Determination of minimum inhibitory concentration (MIC)

2. Materials and methods

The MICs for the antibiotic nalidixic acid were determined by the broth dilution method using Mueller–Hinton broth (CLSI, 2002). The concentration ranges used were 30.0–512 μg mL − 1.

2.1. Isolation, characterization and identification Unless otherwise stated, bacteria were isolated from 330 farmraised, frozen imported shrimp (P. monodon) samples purchased between March, 2008 and July, 2009 from 6 different retail grocery stores in Little Rock, AR. The frozen shrimp was imported from Thailand. Typically, 2 g of thawed shrimp sample was homogenized with 10 mL of Luria broth (LB) in a stomacher (Tekmar Co., Cincinnatti, OH, USA) for 5 min. The homogenate was then incubated overnight at 37 °C. Enriched samples were streaked on MacConkey agar plates supplemented with 10 μg/mL of nalidixic acid and incubated at 37 °C overnight. Presumptive colonies of Klebsiella spp. were biochemically characterized (Table 1) and identified by the Vitek GNI + card with VTK-R07-01 software (bioMerieux Vitek, Hazelwood, MO, USA) and by fatty acid methyl ester analysis (MIDI, Newark, DE, USA). All isolates were stored in LB containing 20% glycerol at −70 °C. These isolates were grown at 37 °C on trypticase soy agar (TSA) plates supplemented with 5% sheep blood. 2.2. Antibiotic susceptibility testing by disk diffusion Antibiotic susceptibility of each Klebsiella isolate was determined by disk diffusion (Bauer et al., 1966) by the criteria specified by the Clinical and Laboratory Standards Institute (CLSI, 2002). Disks of ampicillin (30 μg), bacitracin (10 U), penicillin (10 U), rifampicin (5 μg), sulfamethoxazole:trimethoprim (23:75; 1.25 μg), tetracycline (30 μg), chloramphenicol (30 μg), streptomycin (10 μg), nalidixic acid (30 μg),

Table 1 Morphological and biochemical properties of fluoroquinolone-resistant Klebsiella spp. isolated from imported shrimp. Characteristics

Species according to Vitek GNI ± Card K. pneumoniae

K. oxytoca

Morphological characters Gram reaction Shape

− Rod

− Rod

Biochemical characters Glucose utilization Arginine dehydrolase Lysine decarboxylase Ornithine decarboxylase Voges Proskauer H2S production Citrate utilization Indole production Inulin fermentation

+ − + − + − + − −

+ − + − + − + + +

Acids from L-Arabinose Lactose D-Rhamnose D-Sorbitol Sucrose m-Inositol D-Mannitol Raffinose

+ + + + + + + +

+ + + + + + + +

+, positive reaction, −, negative reaction. Number of isolates identified as each species by Vitek was: 37, Klebsiella pneumonia, 12, Klebsiella oxytoca.

2.4. Genomic DNA extraction Genomic DNA was extracted from cells grown overnight at 37 °C with the QIAamp DNA Mini kit (Qiagen, Valencia, CA, USA). 2.5. Primer design and detection of fluoroquinolone-resistant genes by PCR The presence of quinolone-resistance genes (qnrA, B, S, gyrA, B and parC) were detected in the template DNA by PCR (Table 2). The Tm of the primers was calculated by the 2(A + T) + 4(G + C) formula. PCR amplification of quinolone-resistance genes was carried out in a reaction volume of 25 μL by using a PCR Kit (Applied Biosystems, Foster City, CA, USA). The thermal cycling conditions consisted of an initial denaturation of 94 °C for 2 min followed by a total of 35 cycles of amplification. Each cycle consisted of 94 °C denaturation for 30 s, annealing for 60 s at 1 °C below the lowest Tm of a given primer pair, and 72 °C extension for 10 min. PCR was started with an initial denaturation at 94 °C for 2 min. The amplified PCR products were maintained at 4 °C. A reagent blank contained all the components of the reaction mixture except template DNA, for which sterile distilled water was substituted. The PCR products were subjected to electrophoresis on 1.2% agarose gels in 1× Tris–borate–EDTA (TBE) buffer, stained with ethidium bromide (1 μg/mL), visualized with UV, and photographed using the Eagle Eye II gel documentation system (Stratagene, La Jolla, CA, USA). 2.6. Detection of mutations in the quinolone resistance determining region (QRDR) The target genes (gyrA–B and parC) were amplified by PCR and purified by the QIAquick PCR purification kit (Qiagen). Both strands of the purified PCR amplicons were sequenced by the primers used for amplification for the detection of mutations in the QRDR.

Table 2 Oligonucleotide primers used in the amplification of quinolone-resistance genes from Klebsiella spp. isolated from imported shrimp. Primers

Nucleotide sequence

Amplicon size (bp)

qnrAF qnrAR qnrBF qnrBR qnrSF qnrSR gyrAF gyrAR gyrBF gyrBR parCF parCR

5′-TCA GCA AGA GGA TTT CTC A-3′ 5′-GGC AGC ACT ATT ACT CCC A-3′ 5′-ATG ACG CCA TTA CTG TAT AA-3′ 5′-CTA ACC AAT CAC CGC GAT GCC-3′ 5′-ACC TTC ACC GCT TGC ACA TT-3′ 5′-AAT CAG TTC TTG CTG CCA GGC T-3′ 5′-CGA CCT TGC GAG AGA AAT-3′ 5′-GTT CCA TCA GCC CTT CAA -3′ 5′-GCG CGT GAG ATG ACC CGC CGT-3′ 5′-CTG GCG GTA GAA GAA GGT CAG-3′ 5′-CTT TGC GCT ACA TGA ATT TA-3′ 5′-CAG GTT ATG CGG TGG AAT AT-3′

608 681 557 626 390 535

Primers for the amplification of the quinolone resistant genes (qnrA, B, S) were detailed in an earlier study (Soge et al., 2006). The primers for the amplification of gyrA, gyrB and parC were designed by using a primer selection module of the Lasergene program (DNASTAR, Inc., Madison, WI, USA) and synthesized by Eurofins MWG Operon, Inc. (Huntsville, AL, USA).

M. Nawaz et al. / International Journal of Food Microbiology 155 (2012) 179–184

analyzed using the BioNumerics Software (Applied Maths, Kortrijk, Belgium).

2.7. Detection of tetracycline-resistance (tet) genes by PCR The five tet genes (tetA–E) were individually amplified by PCR with oligonucleotide primers (Table 3) as detailed by Fluit et al. (2005). The universal PCR amplification included 35 thermal cycles of 30 s at 94 °C, 59 s at 55 °C, and 60 s at 72 °C, with an additional extension in the last cycle for 300 s at 72 °C. The amplified PCR products were maintained at 4 °C. A reagent blank contained all components of the reaction mixture except template DNA, for which sterile distilled water was substituted. The PCR products were subjected to electrophoresis on 1.2% agarose gels in 1× Tris–borate–EDTA (TBE) buffer. 2.8. Plasmid isolation Plasmid DNA was isolated from bacterial cultures grown overnight at 37 °C in LB broth with a QIAprep Spin Miniprep kit (Qiagen, Valencia, CA, USA). The plasmids were separated on 1.0% agarose gels. Supercoiled plasmid DNA (Invitrogen, Carlsbad, CA, USA) ladder was used as a molecular weight marker for size determination of unknown plasmid. 2.9. Pulsed-field gel electrophoresis (PFGE) Genomic DNA samples of the isolates were subjected to PFGE (Nawaz et al., 2006; Tenover et al., 1995) after restriction digestion with XbaI. DNA plugs were digested with 20 U of XbaI (New England Biolabs, Ipswich, MA, USA) at 37 °C for 5 h. The digested DNA was separated on 1.0% SeaKem Gold (FMC Corp., Philadelphia, PA, USA) agarose gel with Chef-Mapper III PFGE (BioRad Laboratories, Hercules, CA, USA) system for 20 h. The gels were stained for 30 min with ethidium bromide, destained with distilled water, and photographed using the Eagle Eye II gel documentation system. The Salmonella ser. Braenderup strain H9812 Pulsenet standard was used as molecular weight marker after digestion with XbaI. The genetic relationship among the fluoroquinolone-resistant Klebsiella spp. isolates was

Table 3 Oligonucleotide primers used in the amplification of tetracycline-resistance (tet) genes and integrons from Klebsiella spp. isolated from imported shrimp. Primers Nucleotide sequence

PCR conditions Denaturing Anneal Extension

tetAF tetAR tetBF tetBR tetCF tetCR tetDF tetDR tetEF tetER intF intR

5′-TCT CTT GGA TCA ATT TGC TG-3′ 5′-CCA TCA GTG ATA TCG GCA AT-3′ 5′-GTC TTG CCA ACG TTA TTA CG-3′ 5′-GAG AAG CTG AGG TGG TAT CG-3′ 5′-ATC TAA CAA TGC GCT CAT CG-3′ 5′-CAT TAG GAA GCA GCC CAG TA-3′ 5′-GCT GGT GAT TAC ACT GCT GG-3′ 5′-AGT ATT GCC GCA ATG ACA AA-3′ 5′-CAC TGT GAT GAT GGC ACT GG-3′ 5′-GCC TGT AAC GAA AGT TGA CC-3′ 5′-CAA GGT TCT GGA CCA GTT GC-3′ 5′-CAG CAC ATG CGT GTA AAT CA-3′

181

Target Size (bp)

94 °C, 30 s

55 °C, 60 s

72 °C, 60 s

tetA

354

94 °C, 30 s

56 °C, 60 s

72 °C,60 s tetB

305

94 °C, 45 s

56 °C, 60 s

72 °C, 60 s

tetC

538

94 °C, 30 s

55 °C, 60 s

72 °C, 60 s

tetD

477

94 °C, 30 s

55 °C, 60 s

72 °C, 60 s

tetE

468

94 °C, 30 s

56 °C, 60 s

72 °C, 60 s

Int

930

tet genes were amplified after an initial denaturation at 94 °C for 3 min (Fluit et al., 2005). The integrons were amplified after an initial denaturation of 94 °C for 5 min. PCR was performed with 35 cycles and after the completion of the 35 cycles, a final extension step of 10 min at 72 °C was included in all protocols.

3. Results 3.1. Isolation and identification of Klebsiella spp. from imported shrimp samples Sixty-seven of the 312 bacterial isolates that fermented lactose on MacConkey agar were picked for further characterization and identification. The colonies of these isolates were mucoid and purplish pink in color. These colonies were restreaked on trypticase soy agar plates (TSA with 5% blood) to obtain pure cultures. The pure cultures were rod-shaped and Gram-negative (Table 1). All isolates were positive for lysine decarboxylase but not for ornithine decarboxylase or arginine dehydrolase. All isolates were able to utilize glucose, citrate, arabinose, lactose, rhamnose, sorbitol, sucrose, inositol, mannitol and raffinose. Fifty three (79.0%) were tentatively identified as Klebsiella pneumoniae and fourteen (21.0%) were tentatively identified as K. oxytoca. Results from the Vitek database indicated that the percent probabilities of identification of these strains as K. pneumoniae and K. oxytoca were 99.0%. 3.2. Antibiotic resistance profiles of Klebsiella spp. All 67 Klebsiella spp. were resistant to penicillin, ampicillin, tetracycline and bacitracin. Thirty-two (47%) of the isolates were resistant to 8 of the 11 antibiotics tested (penicillin, ampicillin, rifampicin, streptomycin, tetracycline, bacitracin, trimethoprim/sulfamethaxazole and chloramphenicol). Eight of the isolates (12.0%) were resistant to 7 (nalidixic acid, penicillin, ampicillin, streptomycin, tetracycline, bacitracin and trimethoprim/sulfamethaxazole). Twenty seven (40%) of the isolates were resistant to 5 of the antibiotics tested (penicillin, ampicillin, rifampicin, tetracycline and bacitracin). The MICs of the isolates resistant to nalidixic acid were determined. Four of the eight isolates were resistant to 30 μg/mL and three were resistant to 60 μg/mL of nalidixic acid. One isolate was resistant to 512 μg/mL of nalidixic acid. All isolates were susceptible to ciprofloxacin and ofloxacin. 3.3. Amplification of the gyrA, gyrB and parC genes and analysis of gene sequences Oligonucleotide primers specific for the amplification of the 626-bp gyrA, 535-bp parC, and 390-bp gyrB genes amplified the respective PCR products from the template DNA of all 67 Klebsiella strains (data not shown). The purified PCR amplicons were sequenced and analyzed. No mutations were detected in any of the nalidixic acid susceptible strains of Klebsiella spp. Analysis of the gyrA amplicons (Fig. 1A) from 4/8 nalidixic-acid resistant Klebsiella spp. indicated two point mutations, one in gyrA encoding an amino acid change at position 83 (Ser → Phe) and another at position 87 (Asp→ Ala). No mutations were observed in 4 other nalidixic acid resistant Klebsiella strains. Sequence analysis of the parC amplicons (Fig. 1B) indicated a point mutation at position 80 of the parC gene (Ser→ Ile). Sequence analysis of the gyrB amplicons indicated no mutations in any of them. 3.4. PCR amplification of the tetracycline-resistance genes (tetA–E) from Klebsiella spp. The PCR analysis was performed with the template DNA of both K. pneumoniae and K. oxytoca. Oligonucleotide primers specific for the amplification of tetA, tetC and tetE failed to amplify the respective PCR amplicons from the template DNA of any of the 67 isolates. However, one pair of synthetic tetB-specific oligonucleotide primers, targeting a 305-bp region of tetB, amplified the 305-bp PCR amplicons from the template DNA of 49/53 (92.0%) of K. pneumoniae isolates (data not

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A

Query 188 SARVVGDVIGKYHPHGD F AVY A TIVRMAQPFSLRYMLVDGQGNFGSIDGDSAAAMRYTEI 367 SARVVGDVIGKYHPHGD + AVY + TIVRMAQPFSLRYMLVDGQGNFGSIDGDSAAAMRYTEI Subject 66 SARVVGDVIGKYHPHGD S AVY D TIVRMAQPFSLRYMLVDGQGNFGSIDGDSAAAMRYTEI 125

B Query 184 GDVLGKYHPHGD I ACYEAMVLMAQPFSYRYPLVDGQGNWGAPDDPKSFAAMRYTESRLSK 363 GDVLGKYHPHGD + ACYEAMVLMAQPFSYRYPLVDGQGNWGAPDDPKSFAAMRYTESRLSK Subject 68 GDVLGKYHPHGD S ACYEAMVLMAQPFSYRYPLVDGQGNWGAPDDPKSFAAMRYTESRLSK 127 Fig. 1. A–B: Sequence analysis of mutations in quinolone-resistant determining-regions (QRDR) of Klebsiella spp.. A. gyrA (Ser83Phe, Asp87Ala), B. parC (Ser80Ile).

shown). The primer amplified the 305-bp product from the template DNA of all 14 (100.0%) of K. oxytoca isolates. Primers specific for the amplification of the tetD gene successfully amplified a 477-bp region of the gene from the template DNA of 23/53 (43.0%) of the K. pneumoniae isolates (data not shown). The primers failed to amplify the gene from the template DNA of any K. oxytoca. Oligonucleotide primers and PCR protocols specific for the amplification of a 354-bp region of tetA, 538bp tetC and 468-bp tetE failed to detect these genes in the template DNA of any of the 67-tetracycline-resistant Klebsiella spp. 3.5. Isolation and characterization of plasmids from fluoroquinoloneresistant Klebsiella spp.

4.0

16.0 14.0 12.0 10.0 8.0 7.0 6.0 5.0

1

16.0 14.0 12.0 10.0 8.0 7.0 6.0 5.0

Kb

4.0

2

Kp1s504 — Kpt732 —

3 4

Kp1 — Kptis507 —

5

Kbt701 —

6

Kbt711 —

7

Kptis519 —

8

Kot885 —

9 10

Kp2 —

11

Kptis498 —

12

Kptis1 —

13

Kotis522 —

14

Kptis320 —

15

Kot704 —

16

Kot3 —

17 18

Kot051 —

Kb

Profitable shrimp aquaculture demands extensive usage of antibiotics and pesticides to inhibit infectious diseases and pests (Csavas, 1994; Bondad-Reantaso et al., 2005). However, the usage of antibiotics in these ecosystems may select for antibiotic-resistant microorganisms (Nawaz et al., 2001; Holstrom et al., 2003). The isolation and

3.0

4. Discussion

3.0

Six of the 67 multiple antibiotic-resistant Klebsiella spp. were untypable by the PFGE methodology used in this study. XbaI restriction digestion yielded 12–30 DNA fragments measuring between 20.0 and 668.9 kb (Fig. 3). XbaI-PFGE identified 32 distinct macrorestriction patterns (mrps) among the 61 multiple drug resistant Klebsiella spp. that were typable. Dendrogram analysis indicated that the XbaI-PFGE profiles of KptIS301 and KptIS509 (lanes 28–29) had the highest similarity index of 85.0%. The macrorestriction banding patterns of strains KptIS499 and Kp401 (lanes 22–23) had a similarity index of ca. 82.0%. Dendrogram analysis indicated that the mrps of strains KptIS500 and Kp3 (lanes 13–14), strains KptIS502 and kptIS503 (lanes 24–25), strains Kpt600 and Kbt702 (lanes 30–31) had a similarity index of 80.0%. The mrps of other strains had a similarity index of less than 80.0%.

2.0

3.6. Pulsed-field gel electrophoresis

2.0

Only 23 of the 67 strains of Klebsiella spp. contained plasmids. These plasmids varied in sizes ranging from 2.0 to 16.0 kb (Fig. 2). Strain Kpt504 and strain Kbt711 had similar sized plasmids measuring ca. 5.5 kb (Fig. 2, lanes 2 and 7). Strain Kpt732 (lane 3) and strain Kbt701 (lane 6) contained 3 plasmids of various sizes. Strain Kpt732 had plasmids measuring 5.5, 10 and 14.0 kb while strain Kbt701 (lane 6) contained plasmids measuring 4.5, 7.0 and 12.0 kb. Strains KptIS519 (lane 8) and KptIS498 (lane 11) had similar sized plasmids measuring 6.0 and 12.0 kb, respectively, and strain Kp2 (lane 10) had 3 plasmids measuring 6.0, 7.0 and 12.0 kb. Six of the 12 strains of K. oxytoca contained plasmids (Fig. 2, lanes 12–17). Strain KptIS1 and KptIS320 (lanes 12 and 14) contained single plasmids measuring ca. 3.5 and 4.0 kb, respectively. Strains KotIS522 (lane 13) and Kot704 (lane 15) contained 5 plasmids measuring 4.4, 4.6, and 10.0 kb and ca. 3.6, 3.7, 4.0, 5.0 and 9.0 kb, respectively. Strain Kot3 (lane 16) contained 3 plasmids measuring 2.5, 2.7 and 4.5 kb, and strain Kot051 (lane 17) contained 2 plasmids measuring 6.0 and ca. 14.0 kb.

characterization of multiple antibiotic-resistant bacteria from imported shrimp in this study indicate that shrimp aquaculture may play a role as a reservoir of multiple antibiotic-resistant microorganisms and these organisms may transfer their resistance determinants to other ecosystems and may have the potential to become a public health risk. Quinolones are broad-spectrum antimicrobial agents that have been widely used in clinical medicine. Currently, the United States Food and Drug Administration has withdrawn the approval of use of quinolones in many food producing animals (FDA, 1997). The isolation and characterization of nalidixic acid-resistant Klebsiella spp. from imported shrimp indicate that nalidixic acid and other fluoroquinolones have been used in shrimp aquaculture. Bacterial resistance to fluoroquinolones arises through point mutations in DNA gyrase and topoisomerase IV genes (Hopkins et al., 2005; Jacoby, 2005; Ruiz, 2003; Wiegel et

Fig. 2. Plasmid profiles of select multiple antibiotic-resistant Klebsiella spp. isolated from imported shrimp samples. Lanes 1 and 18 supercoiled plasmid DNA ladder.

33

78

173 138

244

336

452

668

1135

100

Kb

183

Strain Antibiotic Number Susceptibility KL25 PARSTBXC

MW

80

60

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KL19

PARTB

KL23

PARSTBXC

KL22

PARTB

KL24

PARTB

KL26

PARTB

KL34

PARSTBXC

KL27

PARTB

KL30

PARSTBXC

KL21

PARTB

KL28

PARSTBXC

KL31

PARTB

KL33

PARSTBXC

KL29

PARTB

KL13

PARSTBXC

KL11

PARSTBXC

KL10

PARSTBXC

KL07

PARTB

KL15

PARTB

KL17

PARSTBXC

KL12

PARTB

KL02

PARSTBXC

KL09

PARSTBXC

KL08

PARSTBXC

KL20

PASTBXN

KL05

PASTBXN

KL06

PARTB

KL01

PARTB PASTBXN

KL03

PARTB

KL04

PASTBXN

KL32

PARSTBXC

33

78

173 138

244

336

452

668

1135

Kb

MW

KL16

Fig. 3. XbaI-pulsed field gel electrophoresis of the genomic DNA of multiple antibiotic resistant Klebsiella spp. isolated from imported shrimp samples and dendrogram analysis of the macrorestriction patterns (mrps) by BioNumeric Software. Phenotypes abbreviations: A, ampicillin; B, bacitracin; C, chloramphenicol; P, penicillin; R, rifampicin; S, streptomycin; N, nalidixic acid; X, sulfamethaxazole:trimethoprim; T, tetracycline.

al., 1998), within a region known as quinolone resistance determining region (QRDR), and through efflux proteins (Mazzariol et al., 2002; Poole, 2000). Studies in clinical strains of Klebsiella pneumoniae have indicated that point mutations at position 83 (Ser → Tyr/Leu/Ile/Thr) or 87 (Asp →Asn) in gyrA and position 80 (Ser→Ile/Trp) within the parC subunit of topoisomerase may be responsible for mediating resistance to fluoroquinolones (Chen et al., 2003; Deguchi et al., 1977). Another study indicated that mutations at positions 83 (Thr → Ile) and 87 (Asp →Gly) in GyrA conferred resistance in 9 clinical strains of K. oxytoca to levofloxacin (Horii et al., 2008). Sequence analysis of the gyrA from Klebsiella spp. isolated from shrimp in our study indicated point mutations occurred at positions 83 and 87 of GyrA. These variations included amino acid substitutions at positions 83 (Ser→ Phe) and 87 (Asp→ Ala). The point mutation at position 83 in K. pneumoniae observed in the present study is similar to a mutation that confers fluoroquinolone resistance in Salmonella spp. (Hopkins et al., 2005). The molecular mechanism of tetracycline resistance (Chopra and Roberts, 2001; Yamaguchi, 1997) can be mediated by efflux, ribosomal protection, or chemical modifications. A variety of tetracycline resistance

(tet) genes encode these mechanisms. DePaola et al. (1988) isolated tetracycline resistant Gram-negative bacteria from catfish and reported that tetracycline-resistance was mediated by either tetD or tetE. Aeromonads from Danish rainbow trout harbor either tetA or tetE (Schmidt et al., 2001). Furushita et al. (2003) and Nawaz et al. (2006, 2008) reported that a majority of tetracycline-resistant bacteria isolated from tuna and catfish harbor tetB. Similarly, our investigations indicate that tetB was the dominant tetracycline determinant in Klebsiella spp. isolated from imported shrimp, followed by tetD. These studies indicate that the occurrence and prevalence of tet genes in bacteria may be species and ecosystem specific. Characterization of isolates by antibiograms, plasmid profiles, PCR and PFGE can provide invaluable data on the epidemiology of occurrence of multiple antibiotic-resistant bacteria (Nawaz et al., 2006). Indeed these methods have been useful in tracing the reservoirs of infectious pathogens and the rates of transmission and mechanisms of infectious diseases (Tenover et al., 1995). These methods also provide more unambiguous data on the prevalence of these isolates than data based only on phenotypic methods (bacterial identification and

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antibiograms). Results based on bacterial identification and antibiograms classified the 67 Klebsiella spp. into 2 and 3 groups respectively. Results based on plasmid profile classified the 67 isolates into 23 different groups. However, PFGE identified 32 distinct groups among the 67 isolates. The method identified 14 distinct mrps among 27 isolates that were resistant to penicillin, ampicillin, rifampicin, tetracycline and bacitracin. The method also identified 14 distinct mrps among 26 isolates that were resistant to penicillin, ampicillin, rifampicin, streptomycin, tetracycline, bacitracin, trimethoprim/sulfamethaxazole and chloramphenicol and 4 distinct mrps among the 8 isolates that were resistant to penicillin, ampicillin, rifampicin, streptomycin, tetracycline, bacitracin, trimethoprim/sulfamethaxazole and nalidixic acid. These results indicate heterogeneity among the 67 multidrug resistant Klebsiella spp. isolated from imported seafood. Results from our study indicate that imported shrimp may be a reservoir of antibiotic resistant bacteria. The widespread occurrence of multiple antibiotic resistance in seafood raises concerns, in part because many of these life saving drugs are indiscriminately used on the farms for growth promotion, disease prophylaxis or disinfection may select for bacterial resistance to critically important antibiotics that may be used in the treatment of clinical infections, which in turn would lead to increased difficulty in treating bacterial infections. In addition the potential health risks due to the consumption of improperly cooked shrimp harboring such bacteria should not be underestimated. Although adequate cooking should eliminate pathogenic bacteria, undercooking or cross contamination during preparation could be of concern. Thus, the occurrence of multiple antibiotic resistance in imported seafood could pose a potential threat to public health. Acknowledgements We thank Dr Carl E. Cerniglia, J.B. Sutherland and Steve Foley for critical review of the manuscript. This work was supported by the National Center for Toxicological Research, US Food and Drug Administration. Views presented here do not necessarily reflect those of the FDA. References Bailey-Brock, J.H., Moss, S.M., 1992. Penaeid taxonomy, biology and zoogeography. In: Fast, A.W., Lester, L.J. (Eds.), Marine Shrimp Culture: Principles and Practices: Developments in Aquaculture and Fishery Sciences, vol. 23. Elsevier Science Publisher, B.V., The Netherlands, pp. 9–27. Bauer, A.W., Kirby, W.M.M., Sherris, J.C., Truck, M., 1966. Antibiotic susceptibility testing by a standardized single disk method. American Journal of Clinical Pathology 45, 493–496. Biao, X., Kaijin, Y., 2007. Shrimp farming in China: operating characteristics, environmental impact and perspectives. Ocean and Coastal Management 50, 538–550. Bondad-Reantaso, M.G., Subasinghe, R.P., Arthur, J.R., Ogawa, K., Chinabut, S., Adlard, R., Tan, Z., Shariff, M., 2005. Disease and health management in Asian aquaculture. Veterinary Parasitology 132, 249–272. Castro, B., Montesinos, I., Fuster-Jorge, P., Delgado, T., Miguel-Gomez, M.A., Sierra, A., 2010. Epidemiology of Enterobacteriaceae causing bloodstream infections in neonataes intensive care unit patients. Enfermedades Infecciosas y Microbiología Clínica 28, 227–232. Chen, F.J., Lauderdale, T.L., Ho, M., Lo, H.J., 2003. The roles of mutations in gyrA, parC and OmpK35 in fluoroquinolone resistance in Klebsiella pneumoniae. Microbial Drug Resistance 9, 265–271. Chopra, I., Roberts, M., 2001. Tetracycline antibiotics: mode of action, applications, molecular biology, and epidemiology of bacterial resistance. Microbiology and Molecular Biology Reviews 65, 232–260. Clinical and Laboratory Standards Institute, 2002. Development of in vitro susceptibility testing criteria and quality control parameters for veterinary antimicrobials agents. Approved guidelines M37-A2. National Committee for Clinical Standards, Wayne, PA. Csavas, I., 1994. Important factors in the success of shrimp farming. World Aquaculture 25, 34–56. Dang, P.K., Degand, G., Danyi, S., Pierret, G., Delahaut, P., Ton, V.D., Maghuin-Rogister, G., Scippo, M.L., 2010. Validation of a two-plate microbiological method for screening antibiotic residues in shrimp tissue. Analytica Chimica Acta 672, 30–39. Deguchi, T., Fukuoka, A., Yasuda, M., Nakano, M., Ozeki, S., Kanematsu, E., Nishino, Y., Ishihara, S., Ban, Y., Kawada, Y., 1977. Alteration in the gyrA subunit of DNA gyrase and the parC subunit of topoisomerase IV in quinolone-resistant clinical isolates of Klebsiella pneumoniae. Antimicrobial Agents and Chemotherapy 41, 699–701. DePaola, A., Flynn, P.A., McPherson, R.M., Levy, S.B., 1988. Phenotypic and genotypic characterization of tetracycline and oxytetracycline resistant Aeromonas hydrophila

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