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Distribution and transferability of tetracycline resistance determinants in Escherichia coli isolated from meat and meat products
International Journal of Food Microbiology 145 (2011) 407–413
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International Journal of Food Microbiology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i j f o o d m i c r o
Distribution and transferability of tetracycline resistance determinants in Escherichia coli isolated from meat and meat products Hyon-Ji Koo, Gun-Jo Woo ⁎ Laboratory of Food Safety and Evaluation, Department of Food Bioscience and Technology, College of Life Sciences and Biotechnology, Korea University, Seoul 136-713, Republic of Korea
a r t i c l e
i n f o
Article history: Received 11 July 2010 Received in revised form 19 December 2010 Accepted 3 January 2011 Keywords: E. coli Antimicrobial resistance Tetracycline resistance tet PFGE
a b s t r a c t Escherichia coli is used to assess the hygienic quality of food products and the dissemination of antimicrobial resistance. In particular, tetracycline-resistant E. coli can be chosen as an indicator of antibiotic resistant bacteria because it has a high frequency of occurrence. The purpose of this study was to investigate the distribution and transfer of tetracycline resistance determinants in meatborne E. coli. A total of 121 tetracycline-resistant E. coli isolates were collected from meat and meat products (raw meat, fish, and processed foods) from 2004 to 2006 in Korea. Among these isolates, tet(A) (52.4%) was the most frequent tetracycline resistance determinant, followed by tet(B) (41.3%), whereas tet(C) (1.7%) and tet(D) (0.8%) were less frequently identified. Two isolates (1.6%) contained two tet genes simultaneously, tet(A) and tet(B). Minimal inhibitory concentrations (MICs) to tetracycline family antibiotics, such as tetracycline, minocycline, doxycycline, oxytetracycline, and chlortetracycline were higher for isolates carrying the tet(B) gene compared to isolates carrying tet(A) (P b 0.0001). Conjugation experiments were performed by the broth mating method; 119 isolates (98.3%) containing at least one of the tet genes were shown to transfer tetracycline resistance to recipient E. coli J53. Also, we observed high diversity of tetracycline-resistant E. coli isolates in meat and meat products in Korea by using XbaI pulsed-field gel electrophoresis (PFGE) typing. This study suggests that the high prevalence of tetracycline-resistant E. coli in meat may be due to the high transferability of tet determinants. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Antimicrobial-resistant Escherichia coli can be transferred between food-producing animals and humans through the food chain, and their plasmid-encoded resistant genes can be transferred to other pathogens, which potentially results in food poisoning that is more difficult to treat with conventional antimicrobial agents (Aslam and Service, 2006; Van Den Bogaard and Stobberingh, 2000). The contribution of food bacteria to the intestinal flora has been assessed by a study from France, which examined the same subjects when eating normal or sterilized food. Tetracycline resistance in the fecal flora was high in the group eating normal and non-sterilized food for 21 days, while it declined drastically when subjects consumed sterilized food for 17 days (Corpet, 1988). Indiscriminate use of antimicrobials while raising livestock causes a more extensive spread of antimicrobial resistance in bacteria. It is now widely accepted that there is a close relation between the imprudent and over-use of antimicrobial agents and the prevalence of resistance (Aarestrup, 1999).
⁎ Corresponding author. Tel.: + 82 232903021; fax: + 82 232904984. E-mail address: [email protected] (G.-J. Woo). 0168-1605/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2011.01.003
Tetracycline belongs to a family of broad-spectrum antibiotics. Its efficacy, low cost, and the lack of side effects make it the most popularly used antibiotic in livestock farming, including aquaculture. First-generation tetracyclines, such as tetracycline, chlortetracycline, and oxytetracycline, have been widely used as animal growth promoters for decades. Second-generation tetracyclines, such as minocycline and doxycycline, are commonly employed in the prophylactic and therapeutic treatment of human and animal infections. Such widespread use of tetracycline antibiotics has resulted in selection for resistant bacteria, and its imprudent use has caused a high prevalence of tetracycline resistance (Chopra and Roberts, 2001; Roberts, 1996, 2003). The major mechanisms of tetracycline resistance are known to be efflux pump activity, ribosomal protection, and enzymatic inactivation. Various tet genes confer resistance by these mechanisms. Among the forty tetracycline resistance genes discovered thus far (Thaker et al., 2010), the genes associated with an efflux mechanism, namely tet(A), tet(B), tet(C), tet(D), and tet(E) (Chopra and Roberts, 2001) confer tetracycline resistance in Escherichia spp. Most tetracycline resistance genes have been found on mobile elements, plasmids or transposons (Roberts, 1996). E. coli testing is used to assess the hygienic quality of food products, such as in meat (Brown et al., 2000). The level of resistance in commensal E. coli indicates the degree of selective pressure by
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antibiotics (Van Den Bogaard and Stobberingh, 2000). The prevalence of tetracycline resistance represents a useful marker to monitor resistance genes (Ozgumus et al., 2007), and it can provide a good model for ecological studies of antimicrobial resistance (Karami et al., 2006). Together, these results suggest that tetracycline-resistant E. coli can serve as a useful indicator for antibiotic resistant bacteria in the food chain due to its high occurrence. Several studies have demonstrated the prevalence and characterization of tetracyclineresistant E. coli from food-producing animals (Gow et al., 2008; Lanz et al., 2003; Sengeløv et al., 2003), humans (Karami et al., 2006; Schwaiger et al., 2010; Tuckman et al., 2007), companion animals (Costa et al., 2008), aquatic environments (Ozgumus et al., 2007), and raw seafood (Nawaz et al., 2009). However, very little data have been reported on the distribution and characterization of tetracyclineresistant E. coli isolated from meat and meat products. The purpose of this study was to investigate: i) the distribution of tetracycline resistance genes in meatborne E. coli, ii) the phenotypic characteristics of tetracycline-resistant isolates, and iii) the in vitro transfer and genetic relatedness of the isolates carrying tet genes.
Table 1 Primers used for PCR detection of tet genes. Target gene
Primer
Sequence (5 to 3 )
Amplicon size (bp)
Reference
tetA
TetA — F
CGCCTTTCCTTTGG GTTCTCTATATC CAGCCCACCG AGCACAGG GCCAGTCTTG CCAACGTTAT ATAACACCGG TTGCATTGGT TTCAACCCAG TCAGCTCCTT GGGAGGCAGAC AAGGTATAGG GAGCGTACC GCCTGGTTC TCTGATCAGCA GACAGATTGC TCCATACGCGA GATGATCTCC CGATTACAGCT GTCAGCTGGG CATTGCCCT GCTGATCG TTGGTGAGG CTTGTAAGC TTATGGTGGTTG TAGCTAGAAA AAAGGGTTAGA AACTCTTGAAA GAACGTCTCATT ACCTGATATTGC CAAACCCTGCT ACTGTTCCAA GTRAYGAACT TTACCGAATC ATCGYAGAA GCGGRTCAC AACTTAGGCAT TCTGGCTCAC TCCCACTGTTC CATATCGTCA ATTGCAGAAC TTGAAAAGGA CATTGGACCT CACCTTAAAA CCGCACTCA TTGTTGTCG TTTTCATCGCA AACAAGACC
182
This study
975
This study
560
This study
780
This study
442
Guillaume et al. (2000)
993
Tuckman et al. (2007)
348
Gevers et al. (2003)
728
This study
633
Guardabassi et al. (2000)
515
Ng et al. (2001)
589
This study
949
Tuckman et al. (2007)
TetA — R tetB
TetB — F TetB — R
tetC
TetC — F TetC — R
tetD
TetD — F TetD — R
tetE
TetE — F TetE — R
2. Materials and methods
tetG
2.1. Strains The E. coli strains isolated from meat and meat products were collected through the National Antimicrobial Resistance Management Program (NARMP) in Korea. A total of 121 tetracycline-resistant E. coli isolates were collected in Korea between March 2004 and October 2006, originating from pork (n = 18), beef (n = 26), poultry (n = 55), fish and fishery products (n = 17), and processed foods (n = 5). Samples were purchased from different retail stores. Tetracycline resistance was confirmed by plating on agar plates containing 8 μg of tetracycline per ml because the MIC value of tetracycline for E. coli was above or at the breakpoint (N16 μg/ml) for tetracycline resistance (CLSI, 2010).
TetG — R tetK
tetL
All the isolates of tetracycline-resistant strains were tested by single polymerase chain reaction (PCR) analyses for the presence of the tet(A), tet(B), tet(C), tet(D), tet(E), tet(G), tet(K), tet(L), tet(M), tet(O), tet(S), and tet(Y) genes. Bacterial DNA for PCR was obtained by suspending colonies of bacteria grown on MacConkey agar plates in 500 μl of ultrapure water and boiling at 100 °C for 10 min. The oligonucleotide primers used in this study are shown in Table 1, and the amplifications were carried out using an iCycler thermal cycler (Bio-Rad, Hercules, CA.). The reaction mixture (50 μl) contained 1.25 U of Taq polymerase (Takara Korea Biomedical, Seoul), 5 μl of 10× buffer, 10 mM of dNTPs, 20 ng template DNA, 10 pmol of each primer and 37 μl of ultrapure water. The reaction mixture was initially denatured at 94 °C for 5 min, followed by 35 cycles of 94 °C for 30 s, primer annealing at 55 °C for 30 s and extension at 72 °C for 30 s, with a final extension at 72 °C for 5 min. PCR products were resolved by electrophoresis of the products in
TetL — F TetL — R
tetM
TetM — F TetM — R
tetO
TetO — F TetO — R
tetS
2.3. Detection of tetracycline resistance genes
TetK — F TetK — R
2.2. Antimicrobial susceptibility testing The E. coli isolates were tested for susceptibility by the disk diffusion method in accordance with Clinical Laboratory Standards Institute guidelines (CLSI, 2010). The antibiotic discs (BD Diagnostic Systems, Sparks, MD, USA) analyzed in this study were ampicillin (AM, 10 μg), ampicillin-sulbactam (SAM, 10/10 μg), cephalothin (CF, 30 μg), streptomycin (S, 25 μg), chloramphenicol (C, 30 μg), gentamicin (GM, 10 μg), ciprofloxacin (CIP, 5 μg), trimethoprim/sulfamethoxazole (SXT, 1.25/23.75 μg) and tetracycline (TE, 30 μg). E. coli ATCC 25922 and Staphylococcus aureus ATCC 29213 were used as quality control strains for the susceptibility testing procedure.
TetG — F
TetS — F TetS — R
tetY
TetY — F TetY — R
ethidium bromide-stained 2% agarose gels (Promega, Madison, WI., USA) in 1× TAE buffer. The amplified PCR products were purified and sequenced at the Macrogen Inc. (Seoul, Korea). The sequence alignment was performed by using BLAST search in GenBank database, available at the National Center for Biotechnology Information website (http:// www.ncbi.nlm.nih.gov).
2.4. Determination of MICs of principal members of the tetracycline antibiotic family To investigate the phenotypic characteristics of tet genes, all tetpositive strains were tested by agar dilution to calculate MICs of principal members of the tetracycline antibiotics (CLSI, 2010), such as tetracycline, oxytetracycline, cholorotetracycline, doxycycline and minocycline. The concentrations of tetracycline, doxycycline, minocycline, and chlortetracycline tested were 2-fold dilutions from 0.06 to 512 μg/ml, except for oxytetracycline, which ranged from 0.06 to 1024 μg/ml. MIC tests were carried out three times for each tetpositive strain.
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2.5. Conjugation To determine the transferability of tetracycline resistance, conjugation experiments were carried out on all the 121 tetracyclineresistant isolates by a broth mating method. The E. coli J53-AR (resistant to sodium azide) strain was used as the recipient strain (Jacoby and Han, 1996) and tetracycline-resistant isolates served as donors. Overnight cultures of recipient and donor cells grown in Mueller–Hinton broth (BD Diagnostic Systems) at 37 °C in logarithmic phase were mixed with each other at 1:10 ratio (donor to recipient) and incubated for 3 h. Samples (0.2 ml) of this mixture were spread onto the surface of MacConkey agar plates with 8 mg/L tetracycline and 100 mg/L sodium azide to select for plasmid-encoded resistance, then incubated at 37 °C for 24 h. Transconjugants were selected on supplemented agar plates, and examined for antimicrobial susceptibility by disk diffusion and/or agar dilution. PCR was used to confirm that the transconjugants carried the same tet gene as their donors. 2.6. Analysis of genetic relatedness by PFGE To determine the genetic relationship between tetracyclineresistant meatborne E. coli, PFGE was carried out with the CHEFMapper system (Bio-Rad) in accordance with the PulseNet standardized protocol (Ribot et al., 2006). Genomic DNA was digested with 20 U XbaI (Roche Molecular Biochemicals, Indianapolis, IN) and separated on 1.0% pulsed-field certified agarose (Bio-Rad). Running conditions were 6.0 V/cm at 14 °C for 18 h with pulse times ramped from 2.2 to 54.2 s in 0.5× TBE buffer. A lambda DNA ladder (Bio-Rad) was used as the size marker. The genetic relationship between strains was analyzed using InfoQuest™ FP software, version 4.5 (Bio-Rad). Band-based dendrograms were produced by using Dice coefficients and an unweighted pair group method using arithmetic averages (UPGMA) with a position tolerance of 1% and an optimization of 1%.
409
tet(D), tet(E), tet(G), tet(K), tet(L), tet(M), tet(O), tet(S), and tet(Y). Of these, 119 (98.3%) isolates carried at least one of the tet genes examined, whereas two of the isolates (1.7%) did not contain any tetracycline resistance determinants. An examination of single tet determinants showed that 114 (94.2%) of all tetracycline-resistant isolates carried tet (A) or tet(B), with 64 (52.9%) isolates harboring only tet(A) and 50 (41.3%) isolates harboring only tet(B). The tet(C) and tet(D) were detected less frequently in only two (1.7%) and one (0.8%) isolates, respectively. The strains carrying tet(C) or tet(D) were isolated from beef. Only two (1.6%) isolates, from pork and chicken meat, contained two tet genes, tet(A) and tet(B) concurrently. The tet(E), tet(G), tet(K), tet(L), tet(M), tet(O), tet(S), or tet(Y) were not detected. There was no significant difference between the distribution of tet(A) and tet(B) genes among different meat sources (Chi-square test, PN 0.05) (Table 2). 3.2. Resistance phenotype All 121 isolates were resistant to tetracycline. Tetracycline resistance sometimes appeared in combination with resistance to streptomycin (67.8%), ampicillin (66.1%), ciprofloxacin (44.6%), trimethoprim/sulfamethoxazole (41.3%), chloramphenicol (26.4%), cephlothin (24%), and gentamicin (19%) (Table 3). Ninety-three isolates (76.9%) were shown to be multidrug-resistant (resistant at least to three different classes of antibiotic agents). The MIC distributions of tetracycline, minocycline, doxycycline, oxytetracycline, and chlortetracycline for each group of the isolates containing the same tetracycline resistance genes are presented in Table 4. Resistance to minocycline (MIC ≥ 16 μg/ml) was observed in 57 (47.1%) isolates, with 44 of these isolates encoding only tet(B). MICs for the isolates carrying the tet(B) determinant were significantly higher than those for the isolates carrying the tet(A) (Fig. 1). However, there was no statistically significant difference among the MIC values between different meat origins (Chi-square test, P N 0.05).
2.7. Statistical analysis 3.3. Transfer of tet genes by conjugation Data were analyzed using SAS/Statistics, version 9.2. The distribution of tetracycline determinants was analyzed using Chi-square (Fisher's exact) tests. To compare different tet gene and MIC values, survival analysis was carried out using Kaplan–Meier method and the curves were compared using long-rank test. Proportional hazard regression analysis was carried out to statistically assess the association between significant factors that influence resistance to tetracycline and other tetracycline family antibiotics. A P value of less than 0.05 was considered to be statistically significant. 3. Results 3.1. Distribution of tet genes A total of 121 tetracycline-resistant isolates were screened by PCR and sequencing for the 12 tetracycline determinants, tet(A), tet(B), tet(C),
All 119 isolates (98.3%) of meatborne E. coli containing at least one tet gene among tet(A) to tet(D) were found to transfer tetracycline resistance to the tetracycline-susceptible recipient strain, E. coli J53, in the conjugation experiments. Surprisingly, in all 117 isolates having one tet gene among tet(A) to tet(D), their transconjugants possessed phenotypic tetracycline resistance, which was provided by acquiring the corresponding tet gene from the donor strain by conjugation. Although the isolates ECO112-FC-KF06 and ECO153-FP-KF06 carried both tet(A) and tet(B), only tet(A) was transferred to the recipient strain. As shown in Table 5, MICs for the donor isolates (ECO112-FC-KF06, ECO153-FP-KF061) carrying both tet(A) and tet(B) were higher than for the transconjugants (trc ECO112-FC-KF06, trc ECO153-FP-KF06) that carry only tet(A). In two of the strains (ECO16-FP-KF04, ECO25-FCKF04), no tet gene was detectable, there was no transfer of tetracycline resistance to the recipient strain.
Table 2 Genotype distribution of tetracycline-resistant foodborne E. coli isolates from different meat sources. Origin
Pork Beef Poultry Fish and fishery products Processed food Total a
No. (%) of isolates
No. (%) of tetracycline resistance genes tet (A)
tet (B)
tet (C)
tet (D)
tet (A) + (B)
Not detectablea
18 (14.9) 26 (21.5) 55 (45.5) 17 (14) 5 (4.1) 121 (100)
8 (6.6) 12 (9.9) 32 (26.4) 8 (6.6) 4 (3.3) 64 (52.9)
8 11 21 9 1 50
– 2 (1.7) – – – 2 (1.7)
– 1 (0.8) – – – 1 (0.8)
1 (0.8) – 1 (0.8) – – 2 (1.7)
1 (0.8) – 1 (0.8) – – 2 (1.7)
(6.6) (9.1) (17.4) (7.4) (0.8) (41.3)
No determinant identified, tet(A), tet(B), tet(C), tet(D), tet(E), tet(G), tet(K), tet(L), tet(M), tet(O), tet(S), and tet(Y).
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Table 3 Phenotype distribution of resistance to tetracycline and other antibiotics from different meat origins. No. (%) of antimicrobial resistancea
Origin
Pork Beef Poultry Fish and fishery products Processed food Total a
AMP
GEM
STR
CEP
CIP
STX
CHL
12 (66.7) 10 (38.5) 46 (83.6) 9 (52.9) 3 (60) 80 (66.1)
2 1 17 2 1 23
9 (50) 14 (53.8) 45 (81.8) 9 (52.9) 5 (100) 82 (67.8)
4 (22.2) 3 (3.8) 15 (78.5) 6 (23.5) 0 29 (24)
2 (11.1) 1 (3.8) 43 (78.2) 4 (23.5) 4 (80) 54 (44.6)
5 (27.8) 5 (19.2) 35 (63.6) 2 (11.8) 3 (60) 50 (41.3)
6 (33.3) 7 (26.9) 14 (25.5) 4 (23.5) 1 (20) 32 (26.4)
(11.1) (3.8) (30.9) (11.8) (20) (19)
AMP, ampicillin; GEM, gentamicin; STR, streptomycin; CEP, cephalothin; CIP, ciprofloxacin; STX, trimethoprim/sulfamethoxazole; CHL, chloramphenicol.
3.4. PFGE The genetic relationships among the 121 tetracycline-resistant E. coli isolates collected over the three-year period were evaluated based on PFGE of XbaI-digested genomic DNA. Eighteen isolates could not be typed by PFGE as a result of nuclease activity. Among the remaining 103 tetracycline-resistant isolates, various PFGE patterns were observed. When using 85% similarity as a cut-off for the dendrogram, 95 (92.2%) distinct restriction patterns were observed (data not shown). 4. Discussion tet(A) and tet(B) genes were reported to be common in E. coli isolates from humans and animals in many countries (Lanz et al., 2003; Bryan et al., 2004; Costa et al., 2008; Schwaiger et al., 2010). In accordance with other reports, this study also demonstrated that tet(A) and tet(B) are widespread in meat sources, and a similar prevalence and pattern of tet gene expression could be found in previous studies of E. coli isolated from raw meat samples (Sunde and Norström, 2006; Van et al., 2008; Soufi et al., 2009). Schwaiger et al. (2010) and Maynard et al. (2004) found that tet(A) was common in E. coli isolates of animal origin. However, tet(B) was the most frequently observed in clinical isolates (Tuckman et al., 2007). tet(B) was found to be predominant in E. coli
Table 4 MICs of tetracycline family antibiotics according to different tetracycline resistance determinants in E. coli isolated from meat and meat products. Antibiotica
TET
MIN
DOX
OTC
CTC
a
Gene
tet(A) tet(B) tet(C) tet(D) tet(A), tet(A) tet(B) tet(C) tet(D) tet(A), tet(A) tet(B) tet(C) tet(D) tet(A), tet(A) tet(B) tet(C) tet(D) tet(A), tet(A) tet(B) tet(C) tet(D) tet(A),
MIC (μg/ml)
tet(B)
tet(B)
tet(B)
tet(B)
tet(B)
N
Median
Min
Max
64 50 2 1 2 64 50 2 1 2 63 50 2 1 2 64 50 2 1 2 64 50 2 1 2
128 256 24 512 384 6 32 1.5 8 24 16 64 5 64 48 256 512 32 512 384 64 128 12 128 96
64 128 16 512 256 1 8 1 8 16 4 32 2 64 32 32 128 32 512 256 8 12 8 128 64
512 512 32 512 512 32 64 2 8 32 64 128 8 64 64 512 1024 32 512 512 128 128 16 128 128
TET, tetracycline; MIN, minocycline; DOX, doxycycline; OTC, oxytetracycline; CTC, chlortetracycline.
O157:H7 isolates from humans and bovines (Wilkerson et al., 2004), as well as E. coli isolates from catfish in the U.S (Nawaz et al., 2009). The tet(B) confers a wider spectrum of resistance to tetracyclines (Roberts, 1996). When repeating the MIC measurements in this study, the presence of tet(B) in meatborne E. coli was associated with higher MICs for tetracyclines, as many researchers have already observed (Karami et al., 2006; Sengeløv et al., 2003; Tuckman et al., 2007). In the present study, the resistance level between tet genes could be differentiated based on the differences in MICs for tetracycline, minocycline, doxycycline, oxytetracycline and chlortetracycline. In a previous study of E. coli from a commercial beef processing plant, tet(C) was highly prevalent and was thought to be transferrable from meat processing workers to beef (Aslam et al., 2009). tet(C) was detected in the two strains isolated from beef in this study, and the possibility of tet(C) transfer from workers in a butchery or a beef processing company to our samples could not be excluded. Consistent with previous studies that reported tet(D) genes were too rare to be detected, even in E. coli isolated from animals (Lanz et al., 2003; Bryan et al., 2004), tet(D) was found only in one isolate in this study. In comparison to previous studies on E. coli isolated from meat, where only tet(A), tet(B), and tet(C) were detected, this study is the first report to detect tet(D) from the meat samples. It is important to note that tet(C) and tet(D) were each found to be carried by E. coli isolates from different beef samples. The frequency with which E. coli isolates from the meat have more than two tet genes is lower than in isolates from animals or humans (Bryan et al., 2004; Lanz et al., 2003; Sengeløv et al., 2003). In Korea, Cho and Kim (2008) reported that 40% of E. coli isolates from cows and pigs in slaughterhouse settings had two different tet genes (A+ B, A + C and A + D). Wu et al. (2009) reported that the prevalence of tetracycline resistance in E. coli generally declined at sequential stages of processing in the pig slaughterhouse indicating that some processing stages were effective in reducing tetracycline resistance in E. coli. This suggests that there is a need for further research to examine how the tet genes concerned might be lost during the process through slaughter and processing into retail meat products. Even though two of our isolates carried more than one tet gene in this study, they did not display higher MIC values. Bryan et al. (2004) explained this phenomenon with the theory that obtaining more than one tet gene is due to strong selective pressures rather than a selective advantage. Occurrence of each tet gene may be associated with different patterns of antibiotic use, due to co-selection (Chopra and Roberts, 2001; Lanz et al., 2003). It has been found that long-term use of tetracycline selects not only for tetracycline-resistant bacteria but also for multiple resistant strains because tet genes are present in the same mobile elements as other resistance genes (Levy, 1992). This study also found that tetracycline-resistant strains were frequently resistant to both tetracycline and other antimicrobials and had a relatively high multidrug resistance rate. This co-selection implies that tetracycline resistance is more important than simply surviving tetracycline treatment with respect to increasing the risk of inducing other resistances (Cox and Popken, 2010). The co-occurrence of tetracycline resistance with resistance to other antibiotics was strongly associated with the amount of antibiotics used for animals (KFDA, 2007).
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Fig. 1. Kaplan–Meier survival curves of isolates carrying tet(A) and isolates carrying tet(B) versus MIC values of the tetracycline family antibiotics. (A) tetracycline, (B) minocycline, (C) doxycycline, (D) oxytetracycline, and (E) chlortetracycline.
However, of the two studied isolates carrying both tet(A) and tet(B), only tet(A) was transferred to the recipient with a part of the donor's resistance. It seems likely that tet(A) and tet(B) are present in different conjugative plasmids. The tet(A) and tet(B) genes are known to be located on conjugative plasmids of different incompatibility group
(Jones et al., 1992). Gow et al. (2008) found a negative association between the presence of tet(A) and tet(B). In two strains out of 121 tetracycline-resistant isolates, no tet gene was detected in this study. Transfer of tetracycline resistance was not observed between these two strains and E. coli J53. These resistance
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Table 5 Resistance profiles of tetracycline-resistant E. coli isolates from meat and transconjugants. Strain
Donor strain
Origin
ECO112FC-KF06 ECO153FP-KF06 E. coli J53
Recipient strain Transtrc ECO112conjugant FC-KF06 trc ECO153FP-KF06 a
Year
Gene (s)
MIC (μg/ml)a TET MIN DOX OTC CTC
Chicken 2006 tet(A), 512 32 tet(B) Pork 2006 tet(A), 256 16 tet(B) 1 1
32
256
128
64
512
64
2
2
2
tet(A)
128
4
8
256
32
tet(A)
64
4
16
128
32
See Table 4.
phenotypes may be due to point mutations (Chopra and Roberts, 2001) rather than any tet gene, or they might carry an unidentified tetracycline resistance gene on non-mobile elements in E. coli. The transfer of conjugative plasmids is known to be the most common mechanism for genetic exchange between bacteria, as plasmid conjugation can occur at high frequency and is able to transfer resistance genes (Sunde and Norström, 2006). Our conjugation results showed that most tetracycline resistance genes found in our study were located on transferable elements such as conjugative plasmids. Also, we observed high diversity among the tetracycline-resistant E. coli isolates in meat and meat products in Korea by using XbaI PFGE typing. No clonal relationship was observed among most of the tetracycline-resistant E. coli (92.2%) in this study. These results suggest that the horizontal transfer of tet genes, rather than the dissemination of a specific clonal strain, led to widespread distribution of tetracycline resistance, as suggested in another study (Sawant et al., 2007). Taken together, our results imply that the high prevalence of tetracycline-resistant E. coli in meat may be due to selective pressure, as well as the high transferability of tet determinants (Chopra and Roberts, 2001; Roberts, 2003). To the best of our knowledge, this is the first report to study the distribution and transferability of tetracycline resistance genes in E. coli from meat and meat products in Korea. Of the antimicrobial agents used for livestock farming in Korea between 2002 and 2006, tetracyclines were the most common (43–51%), and most of these were used as growth promoters or disease prophylactics in the production of foodproducing animals (KFDA, 2007). Use of antibiotics as feed additives was partially limited in May 2005 and. antimicrobials such as oxytetracycline and chlortetracycline were entirely banned for non-human uses as of January 2009 in Korea. The observations of this study were collected before these regulations were implemented, so these findings can be used as reference data for comparison to antimicrobial resistance rate and risk assessment after the regulations are effective in the future. Acknowledgements This study was supported by a grant from the National Antimicrobial Resistance Management Program (08072NARMP150) of the Korea Food and Drug Administration. We would like to thank Professor Hyejung Chang (Kyung Hee University) for the statistical advice. We also thank the Korea University Food Safety Hall and Institute of Food and Biomedicine Safety for allowing the use of their equipment and facilities. References Aarestrup, F.M., 1999. Association between the consumption of antimicrobial agents in animal husbandry and the occurrence of resistant bacteria among food animals. International Journal of Antimicrobial Agents 12, 279–285. Aslam, M., Service, C., 2006. Antimicrobial resistance and genetic profiling of Escherichia coli from a commercial beef packing plant. Journal of Food Protection 69, 1508–1513.
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International Journal of Food Microbiology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i j f o o d m i c r o
Distribution and transferability of tetracycline resistance determinants in Escherichia coli isolated from meat and meat products Hyon-Ji Koo, Gun-Jo Woo ⁎ Laboratory of Food Safety and Evaluation, Department of Food Bioscience and Technology, College of Life Sciences and Biotechnology, Korea University, Seoul 136-713, Republic of Korea
a r t i c l e
i n f o
Article history: Received 11 July 2010 Received in revised form 19 December 2010 Accepted 3 January 2011 Keywords: E. coli Antimicrobial resistance Tetracycline resistance tet PFGE
a b s t r a c t Escherichia coli is used to assess the hygienic quality of food products and the dissemination of antimicrobial resistance. In particular, tetracycline-resistant E. coli can be chosen as an indicator of antibiotic resistant bacteria because it has a high frequency of occurrence. The purpose of this study was to investigate the distribution and transfer of tetracycline resistance determinants in meatborne E. coli. A total of 121 tetracycline-resistant E. coli isolates were collected from meat and meat products (raw meat, fish, and processed foods) from 2004 to 2006 in Korea. Among these isolates, tet(A) (52.4%) was the most frequent tetracycline resistance determinant, followed by tet(B) (41.3%), whereas tet(C) (1.7%) and tet(D) (0.8%) were less frequently identified. Two isolates (1.6%) contained two tet genes simultaneously, tet(A) and tet(B). Minimal inhibitory concentrations (MICs) to tetracycline family antibiotics, such as tetracycline, minocycline, doxycycline, oxytetracycline, and chlortetracycline were higher for isolates carrying the tet(B) gene compared to isolates carrying tet(A) (P b 0.0001). Conjugation experiments were performed by the broth mating method; 119 isolates (98.3%) containing at least one of the tet genes were shown to transfer tetracycline resistance to recipient E. coli J53. Also, we observed high diversity of tetracycline-resistant E. coli isolates in meat and meat products in Korea by using XbaI pulsed-field gel electrophoresis (PFGE) typing. This study suggests that the high prevalence of tetracycline-resistant E. coli in meat may be due to the high transferability of tet determinants. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Antimicrobial-resistant Escherichia coli can be transferred between food-producing animals and humans through the food chain, and their plasmid-encoded resistant genes can be transferred to other pathogens, which potentially results in food poisoning that is more difficult to treat with conventional antimicrobial agents (Aslam and Service, 2006; Van Den Bogaard and Stobberingh, 2000). The contribution of food bacteria to the intestinal flora has been assessed by a study from France, which examined the same subjects when eating normal or sterilized food. Tetracycline resistance in the fecal flora was high in the group eating normal and non-sterilized food for 21 days, while it declined drastically when subjects consumed sterilized food for 17 days (Corpet, 1988). Indiscriminate use of antimicrobials while raising livestock causes a more extensive spread of antimicrobial resistance in bacteria. It is now widely accepted that there is a close relation between the imprudent and over-use of antimicrobial agents and the prevalence of resistance (Aarestrup, 1999).
⁎ Corresponding author. Tel.: + 82 232903021; fax: + 82 232904984. E-mail address: [email protected] (G.-J. Woo). 0168-1605/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2011.01.003
Tetracycline belongs to a family of broad-spectrum antibiotics. Its efficacy, low cost, and the lack of side effects make it the most popularly used antibiotic in livestock farming, including aquaculture. First-generation tetracyclines, such as tetracycline, chlortetracycline, and oxytetracycline, have been widely used as animal growth promoters for decades. Second-generation tetracyclines, such as minocycline and doxycycline, are commonly employed in the prophylactic and therapeutic treatment of human and animal infections. Such widespread use of tetracycline antibiotics has resulted in selection for resistant bacteria, and its imprudent use has caused a high prevalence of tetracycline resistance (Chopra and Roberts, 2001; Roberts, 1996, 2003). The major mechanisms of tetracycline resistance are known to be efflux pump activity, ribosomal protection, and enzymatic inactivation. Various tet genes confer resistance by these mechanisms. Among the forty tetracycline resistance genes discovered thus far (Thaker et al., 2010), the genes associated with an efflux mechanism, namely tet(A), tet(B), tet(C), tet(D), and tet(E) (Chopra and Roberts, 2001) confer tetracycline resistance in Escherichia spp. Most tetracycline resistance genes have been found on mobile elements, plasmids or transposons (Roberts, 1996). E. coli testing is used to assess the hygienic quality of food products, such as in meat (Brown et al., 2000). The level of resistance in commensal E. coli indicates the degree of selective pressure by
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antibiotics (Van Den Bogaard and Stobberingh, 2000). The prevalence of tetracycline resistance represents a useful marker to monitor resistance genes (Ozgumus et al., 2007), and it can provide a good model for ecological studies of antimicrobial resistance (Karami et al., 2006). Together, these results suggest that tetracycline-resistant E. coli can serve as a useful indicator for antibiotic resistant bacteria in the food chain due to its high occurrence. Several studies have demonstrated the prevalence and characterization of tetracyclineresistant E. coli from food-producing animals (Gow et al., 2008; Lanz et al., 2003; Sengeløv et al., 2003), humans (Karami et al., 2006; Schwaiger et al., 2010; Tuckman et al., 2007), companion animals (Costa et al., 2008), aquatic environments (Ozgumus et al., 2007), and raw seafood (Nawaz et al., 2009). However, very little data have been reported on the distribution and characterization of tetracyclineresistant E. coli isolated from meat and meat products. The purpose of this study was to investigate: i) the distribution of tetracycline resistance genes in meatborne E. coli, ii) the phenotypic characteristics of tetracycline-resistant isolates, and iii) the in vitro transfer and genetic relatedness of the isolates carrying tet genes.
Table 1 Primers used for PCR detection of tet genes. Target gene
Primer
Sequence (5 to 3 )
Amplicon size (bp)
Reference
tetA
TetA — F
CGCCTTTCCTTTGG GTTCTCTATATC CAGCCCACCG AGCACAGG GCCAGTCTTG CCAACGTTAT ATAACACCGG TTGCATTGGT TTCAACCCAG TCAGCTCCTT GGGAGGCAGAC AAGGTATAGG GAGCGTACC GCCTGGTTC TCTGATCAGCA GACAGATTGC TCCATACGCGA GATGATCTCC CGATTACAGCT GTCAGCTGGG CATTGCCCT GCTGATCG TTGGTGAGG CTTGTAAGC TTATGGTGGTTG TAGCTAGAAA AAAGGGTTAGA AACTCTTGAAA GAACGTCTCATT ACCTGATATTGC CAAACCCTGCT ACTGTTCCAA GTRAYGAACT TTACCGAATC ATCGYAGAA GCGGRTCAC AACTTAGGCAT TCTGGCTCAC TCCCACTGTTC CATATCGTCA ATTGCAGAAC TTGAAAAGGA CATTGGACCT CACCTTAAAA CCGCACTCA TTGTTGTCG TTTTCATCGCA AACAAGACC
182
This study
975
This study
560
This study
780
This study
442
Guillaume et al. (2000)
993
Tuckman et al. (2007)
348
Gevers et al. (2003)
728
This study
633
Guardabassi et al. (2000)
515
Ng et al. (2001)
589
This study
949
Tuckman et al. (2007)
TetA — R tetB
TetB — F TetB — R
tetC
TetC — F TetC — R
tetD
TetD — F TetD — R
tetE
TetE — F TetE — R
2. Materials and methods
tetG
2.1. Strains The E. coli strains isolated from meat and meat products were collected through the National Antimicrobial Resistance Management Program (NARMP) in Korea. A total of 121 tetracycline-resistant E. coli isolates were collected in Korea between March 2004 and October 2006, originating from pork (n = 18), beef (n = 26), poultry (n = 55), fish and fishery products (n = 17), and processed foods (n = 5). Samples were purchased from different retail stores. Tetracycline resistance was confirmed by plating on agar plates containing 8 μg of tetracycline per ml because the MIC value of tetracycline for E. coli was above or at the breakpoint (N16 μg/ml) for tetracycline resistance (CLSI, 2010).
TetG — R tetK
tetL
All the isolates of tetracycline-resistant strains were tested by single polymerase chain reaction (PCR) analyses for the presence of the tet(A), tet(B), tet(C), tet(D), tet(E), tet(G), tet(K), tet(L), tet(M), tet(O), tet(S), and tet(Y) genes. Bacterial DNA for PCR was obtained by suspending colonies of bacteria grown on MacConkey agar plates in 500 μl of ultrapure water and boiling at 100 °C for 10 min. The oligonucleotide primers used in this study are shown in Table 1, and the amplifications were carried out using an iCycler thermal cycler (Bio-Rad, Hercules, CA.). The reaction mixture (50 μl) contained 1.25 U of Taq polymerase (Takara Korea Biomedical, Seoul), 5 μl of 10× buffer, 10 mM of dNTPs, 20 ng template DNA, 10 pmol of each primer and 37 μl of ultrapure water. The reaction mixture was initially denatured at 94 °C for 5 min, followed by 35 cycles of 94 °C for 30 s, primer annealing at 55 °C for 30 s and extension at 72 °C for 30 s, with a final extension at 72 °C for 5 min. PCR products were resolved by electrophoresis of the products in
TetL — F TetL — R
tetM
TetM — F TetM — R
tetO
TetO — F TetO — R
tetS
2.3. Detection of tetracycline resistance genes
TetK — F TetK — R
2.2. Antimicrobial susceptibility testing The E. coli isolates were tested for susceptibility by the disk diffusion method in accordance with Clinical Laboratory Standards Institute guidelines (CLSI, 2010). The antibiotic discs (BD Diagnostic Systems, Sparks, MD, USA) analyzed in this study were ampicillin (AM, 10 μg), ampicillin-sulbactam (SAM, 10/10 μg), cephalothin (CF, 30 μg), streptomycin (S, 25 μg), chloramphenicol (C, 30 μg), gentamicin (GM, 10 μg), ciprofloxacin (CIP, 5 μg), trimethoprim/sulfamethoxazole (SXT, 1.25/23.75 μg) and tetracycline (TE, 30 μg). E. coli ATCC 25922 and Staphylococcus aureus ATCC 29213 were used as quality control strains for the susceptibility testing procedure.
TetG — F
TetS — F TetS — R
tetY
TetY — F TetY — R
ethidium bromide-stained 2% agarose gels (Promega, Madison, WI., USA) in 1× TAE buffer. The amplified PCR products were purified and sequenced at the Macrogen Inc. (Seoul, Korea). The sequence alignment was performed by using BLAST search in GenBank database, available at the National Center for Biotechnology Information website (http:// www.ncbi.nlm.nih.gov).
2.4. Determination of MICs of principal members of the tetracycline antibiotic family To investigate the phenotypic characteristics of tet genes, all tetpositive strains were tested by agar dilution to calculate MICs of principal members of the tetracycline antibiotics (CLSI, 2010), such as tetracycline, oxytetracycline, cholorotetracycline, doxycycline and minocycline. The concentrations of tetracycline, doxycycline, minocycline, and chlortetracycline tested were 2-fold dilutions from 0.06 to 512 μg/ml, except for oxytetracycline, which ranged from 0.06 to 1024 μg/ml. MIC tests were carried out three times for each tetpositive strain.
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2.5. Conjugation To determine the transferability of tetracycline resistance, conjugation experiments were carried out on all the 121 tetracyclineresistant isolates by a broth mating method. The E. coli J53-AR (resistant to sodium azide) strain was used as the recipient strain (Jacoby and Han, 1996) and tetracycline-resistant isolates served as donors. Overnight cultures of recipient and donor cells grown in Mueller–Hinton broth (BD Diagnostic Systems) at 37 °C in logarithmic phase were mixed with each other at 1:10 ratio (donor to recipient) and incubated for 3 h. Samples (0.2 ml) of this mixture were spread onto the surface of MacConkey agar plates with 8 mg/L tetracycline and 100 mg/L sodium azide to select for plasmid-encoded resistance, then incubated at 37 °C for 24 h. Transconjugants were selected on supplemented agar plates, and examined for antimicrobial susceptibility by disk diffusion and/or agar dilution. PCR was used to confirm that the transconjugants carried the same tet gene as their donors. 2.6. Analysis of genetic relatedness by PFGE To determine the genetic relationship between tetracyclineresistant meatborne E. coli, PFGE was carried out with the CHEFMapper system (Bio-Rad) in accordance with the PulseNet standardized protocol (Ribot et al., 2006). Genomic DNA was digested with 20 U XbaI (Roche Molecular Biochemicals, Indianapolis, IN) and separated on 1.0% pulsed-field certified agarose (Bio-Rad). Running conditions were 6.0 V/cm at 14 °C for 18 h with pulse times ramped from 2.2 to 54.2 s in 0.5× TBE buffer. A lambda DNA ladder (Bio-Rad) was used as the size marker. The genetic relationship between strains was analyzed using InfoQuest™ FP software, version 4.5 (Bio-Rad). Band-based dendrograms were produced by using Dice coefficients and an unweighted pair group method using arithmetic averages (UPGMA) with a position tolerance of 1% and an optimization of 1%.
409
tet(D), tet(E), tet(G), tet(K), tet(L), tet(M), tet(O), tet(S), and tet(Y). Of these, 119 (98.3%) isolates carried at least one of the tet genes examined, whereas two of the isolates (1.7%) did not contain any tetracycline resistance determinants. An examination of single tet determinants showed that 114 (94.2%) of all tetracycline-resistant isolates carried tet (A) or tet(B), with 64 (52.9%) isolates harboring only tet(A) and 50 (41.3%) isolates harboring only tet(B). The tet(C) and tet(D) were detected less frequently in only two (1.7%) and one (0.8%) isolates, respectively. The strains carrying tet(C) or tet(D) were isolated from beef. Only two (1.6%) isolates, from pork and chicken meat, contained two tet genes, tet(A) and tet(B) concurrently. The tet(E), tet(G), tet(K), tet(L), tet(M), tet(O), tet(S), or tet(Y) were not detected. There was no significant difference between the distribution of tet(A) and tet(B) genes among different meat sources (Chi-square test, PN 0.05) (Table 2). 3.2. Resistance phenotype All 121 isolates were resistant to tetracycline. Tetracycline resistance sometimes appeared in combination with resistance to streptomycin (67.8%), ampicillin (66.1%), ciprofloxacin (44.6%), trimethoprim/sulfamethoxazole (41.3%), chloramphenicol (26.4%), cephlothin (24%), and gentamicin (19%) (Table 3). Ninety-three isolates (76.9%) were shown to be multidrug-resistant (resistant at least to three different classes of antibiotic agents). The MIC distributions of tetracycline, minocycline, doxycycline, oxytetracycline, and chlortetracycline for each group of the isolates containing the same tetracycline resistance genes are presented in Table 4. Resistance to minocycline (MIC ≥ 16 μg/ml) was observed in 57 (47.1%) isolates, with 44 of these isolates encoding only tet(B). MICs for the isolates carrying the tet(B) determinant were significantly higher than those for the isolates carrying the tet(A) (Fig. 1). However, there was no statistically significant difference among the MIC values between different meat origins (Chi-square test, P N 0.05).
2.7. Statistical analysis 3.3. Transfer of tet genes by conjugation Data were analyzed using SAS/Statistics, version 9.2. The distribution of tetracycline determinants was analyzed using Chi-square (Fisher's exact) tests. To compare different tet gene and MIC values, survival analysis was carried out using Kaplan–Meier method and the curves were compared using long-rank test. Proportional hazard regression analysis was carried out to statistically assess the association between significant factors that influence resistance to tetracycline and other tetracycline family antibiotics. A P value of less than 0.05 was considered to be statistically significant. 3. Results 3.1. Distribution of tet genes A total of 121 tetracycline-resistant isolates were screened by PCR and sequencing for the 12 tetracycline determinants, tet(A), tet(B), tet(C),
All 119 isolates (98.3%) of meatborne E. coli containing at least one tet gene among tet(A) to tet(D) were found to transfer tetracycline resistance to the tetracycline-susceptible recipient strain, E. coli J53, in the conjugation experiments. Surprisingly, in all 117 isolates having one tet gene among tet(A) to tet(D), their transconjugants possessed phenotypic tetracycline resistance, which was provided by acquiring the corresponding tet gene from the donor strain by conjugation. Although the isolates ECO112-FC-KF06 and ECO153-FP-KF06 carried both tet(A) and tet(B), only tet(A) was transferred to the recipient strain. As shown in Table 5, MICs for the donor isolates (ECO112-FC-KF06, ECO153-FP-KF061) carrying both tet(A) and tet(B) were higher than for the transconjugants (trc ECO112-FC-KF06, trc ECO153-FP-KF06) that carry only tet(A). In two of the strains (ECO16-FP-KF04, ECO25-FCKF04), no tet gene was detectable, there was no transfer of tetracycline resistance to the recipient strain.
Table 2 Genotype distribution of tetracycline-resistant foodborne E. coli isolates from different meat sources. Origin
Pork Beef Poultry Fish and fishery products Processed food Total a
No. (%) of isolates
No. (%) of tetracycline resistance genes tet (A)
tet (B)
tet (C)
tet (D)
tet (A) + (B)
Not detectablea
18 (14.9) 26 (21.5) 55 (45.5) 17 (14) 5 (4.1) 121 (100)
8 (6.6) 12 (9.9) 32 (26.4) 8 (6.6) 4 (3.3) 64 (52.9)
8 11 21 9 1 50
– 2 (1.7) – – – 2 (1.7)
– 1 (0.8) – – – 1 (0.8)
1 (0.8) – 1 (0.8) – – 2 (1.7)
1 (0.8) – 1 (0.8) – – 2 (1.7)
(6.6) (9.1) (17.4) (7.4) (0.8) (41.3)
No determinant identified, tet(A), tet(B), tet(C), tet(D), tet(E), tet(G), tet(K), tet(L), tet(M), tet(O), tet(S), and tet(Y).
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Table 3 Phenotype distribution of resistance to tetracycline and other antibiotics from different meat origins. No. (%) of antimicrobial resistancea
Origin
Pork Beef Poultry Fish and fishery products Processed food Total a
AMP
GEM
STR
CEP
CIP
STX
CHL
12 (66.7) 10 (38.5) 46 (83.6) 9 (52.9) 3 (60) 80 (66.1)
2 1 17 2 1 23
9 (50) 14 (53.8) 45 (81.8) 9 (52.9) 5 (100) 82 (67.8)
4 (22.2) 3 (3.8) 15 (78.5) 6 (23.5) 0 29 (24)
2 (11.1) 1 (3.8) 43 (78.2) 4 (23.5) 4 (80) 54 (44.6)
5 (27.8) 5 (19.2) 35 (63.6) 2 (11.8) 3 (60) 50 (41.3)
6 (33.3) 7 (26.9) 14 (25.5) 4 (23.5) 1 (20) 32 (26.4)
(11.1) (3.8) (30.9) (11.8) (20) (19)
AMP, ampicillin; GEM, gentamicin; STR, streptomycin; CEP, cephalothin; CIP, ciprofloxacin; STX, trimethoprim/sulfamethoxazole; CHL, chloramphenicol.
3.4. PFGE The genetic relationships among the 121 tetracycline-resistant E. coli isolates collected over the three-year period were evaluated based on PFGE of XbaI-digested genomic DNA. Eighteen isolates could not be typed by PFGE as a result of nuclease activity. Among the remaining 103 tetracycline-resistant isolates, various PFGE patterns were observed. When using 85% similarity as a cut-off for the dendrogram, 95 (92.2%) distinct restriction patterns were observed (data not shown). 4. Discussion tet(A) and tet(B) genes were reported to be common in E. coli isolates from humans and animals in many countries (Lanz et al., 2003; Bryan et al., 2004; Costa et al., 2008; Schwaiger et al., 2010). In accordance with other reports, this study also demonstrated that tet(A) and tet(B) are widespread in meat sources, and a similar prevalence and pattern of tet gene expression could be found in previous studies of E. coli isolated from raw meat samples (Sunde and Norström, 2006; Van et al., 2008; Soufi et al., 2009). Schwaiger et al. (2010) and Maynard et al. (2004) found that tet(A) was common in E. coli isolates of animal origin. However, tet(B) was the most frequently observed in clinical isolates (Tuckman et al., 2007). tet(B) was found to be predominant in E. coli
Table 4 MICs of tetracycline family antibiotics according to different tetracycline resistance determinants in E. coli isolated from meat and meat products. Antibiotica
TET
MIN
DOX
OTC
CTC
a
Gene
tet(A) tet(B) tet(C) tet(D) tet(A), tet(A) tet(B) tet(C) tet(D) tet(A), tet(A) tet(B) tet(C) tet(D) tet(A), tet(A) tet(B) tet(C) tet(D) tet(A), tet(A) tet(B) tet(C) tet(D) tet(A),
MIC (μg/ml)
tet(B)
tet(B)
tet(B)
tet(B)
tet(B)
N
Median
Min
Max
64 50 2 1 2 64 50 2 1 2 63 50 2 1 2 64 50 2 1 2 64 50 2 1 2
128 256 24 512 384 6 32 1.5 8 24 16 64 5 64 48 256 512 32 512 384 64 128 12 128 96
64 128 16 512 256 1 8 1 8 16 4 32 2 64 32 32 128 32 512 256 8 12 8 128 64
512 512 32 512 512 32 64 2 8 32 64 128 8 64 64 512 1024 32 512 512 128 128 16 128 128
TET, tetracycline; MIN, minocycline; DOX, doxycycline; OTC, oxytetracycline; CTC, chlortetracycline.
O157:H7 isolates from humans and bovines (Wilkerson et al., 2004), as well as E. coli isolates from catfish in the U.S (Nawaz et al., 2009). The tet(B) confers a wider spectrum of resistance to tetracyclines (Roberts, 1996). When repeating the MIC measurements in this study, the presence of tet(B) in meatborne E. coli was associated with higher MICs for tetracyclines, as many researchers have already observed (Karami et al., 2006; Sengeløv et al., 2003; Tuckman et al., 2007). In the present study, the resistance level between tet genes could be differentiated based on the differences in MICs for tetracycline, minocycline, doxycycline, oxytetracycline and chlortetracycline. In a previous study of E. coli from a commercial beef processing plant, tet(C) was highly prevalent and was thought to be transferrable from meat processing workers to beef (Aslam et al., 2009). tet(C) was detected in the two strains isolated from beef in this study, and the possibility of tet(C) transfer from workers in a butchery or a beef processing company to our samples could not be excluded. Consistent with previous studies that reported tet(D) genes were too rare to be detected, even in E. coli isolated from animals (Lanz et al., 2003; Bryan et al., 2004), tet(D) was found only in one isolate in this study. In comparison to previous studies on E. coli isolated from meat, where only tet(A), tet(B), and tet(C) were detected, this study is the first report to detect tet(D) from the meat samples. It is important to note that tet(C) and tet(D) were each found to be carried by E. coli isolates from different beef samples. The frequency with which E. coli isolates from the meat have more than two tet genes is lower than in isolates from animals or humans (Bryan et al., 2004; Lanz et al., 2003; Sengeløv et al., 2003). In Korea, Cho and Kim (2008) reported that 40% of E. coli isolates from cows and pigs in slaughterhouse settings had two different tet genes (A+ B, A + C and A + D). Wu et al. (2009) reported that the prevalence of tetracycline resistance in E. coli generally declined at sequential stages of processing in the pig slaughterhouse indicating that some processing stages were effective in reducing tetracycline resistance in E. coli. This suggests that there is a need for further research to examine how the tet genes concerned might be lost during the process through slaughter and processing into retail meat products. Even though two of our isolates carried more than one tet gene in this study, they did not display higher MIC values. Bryan et al. (2004) explained this phenomenon with the theory that obtaining more than one tet gene is due to strong selective pressures rather than a selective advantage. Occurrence of each tet gene may be associated with different patterns of antibiotic use, due to co-selection (Chopra and Roberts, 2001; Lanz et al., 2003). It has been found that long-term use of tetracycline selects not only for tetracycline-resistant bacteria but also for multiple resistant strains because tet genes are present in the same mobile elements as other resistance genes (Levy, 1992). This study also found that tetracycline-resistant strains were frequently resistant to both tetracycline and other antimicrobials and had a relatively high multidrug resistance rate. This co-selection implies that tetracycline resistance is more important than simply surviving tetracycline treatment with respect to increasing the risk of inducing other resistances (Cox and Popken, 2010). The co-occurrence of tetracycline resistance with resistance to other antibiotics was strongly associated with the amount of antibiotics used for animals (KFDA, 2007).
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Fig. 1. Kaplan–Meier survival curves of isolates carrying tet(A) and isolates carrying tet(B) versus MIC values of the tetracycline family antibiotics. (A) tetracycline, (B) minocycline, (C) doxycycline, (D) oxytetracycline, and (E) chlortetracycline.
However, of the two studied isolates carrying both tet(A) and tet(B), only tet(A) was transferred to the recipient with a part of the donor's resistance. It seems likely that tet(A) and tet(B) are present in different conjugative plasmids. The tet(A) and tet(B) genes are known to be located on conjugative plasmids of different incompatibility group
(Jones et al., 1992). Gow et al. (2008) found a negative association between the presence of tet(A) and tet(B). In two strains out of 121 tetracycline-resistant isolates, no tet gene was detected in this study. Transfer of tetracycline resistance was not observed between these two strains and E. coli J53. These resistance
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Table 5 Resistance profiles of tetracycline-resistant E. coli isolates from meat and transconjugants. Strain
Donor strain
Origin
ECO112FC-KF06 ECO153FP-KF06 E. coli J53
Recipient strain Transtrc ECO112conjugant FC-KF06 trc ECO153FP-KF06 a
Year
Gene (s)
MIC (μg/ml)a TET MIN DOX OTC CTC
Chicken 2006 tet(A), 512 32 tet(B) Pork 2006 tet(A), 256 16 tet(B) 1 1
32
256
128
64
512
64
2
2
2
tet(A)
128
4
8
256
32
tet(A)
64
4
16
128
32
See Table 4.
phenotypes may be due to point mutations (Chopra and Roberts, 2001) rather than any tet gene, or they might carry an unidentified tetracycline resistance gene on non-mobile elements in E. coli. The transfer of conjugative plasmids is known to be the most common mechanism for genetic exchange between bacteria, as plasmid conjugation can occur at high frequency and is able to transfer resistance genes (Sunde and Norström, 2006). Our conjugation results showed that most tetracycline resistance genes found in our study were located on transferable elements such as conjugative plasmids. Also, we observed high diversity among the tetracycline-resistant E. coli isolates in meat and meat products in Korea by using XbaI PFGE typing. No clonal relationship was observed among most of the tetracycline-resistant E. coli (92.2%) in this study. These results suggest that the horizontal transfer of tet genes, rather than the dissemination of a specific clonal strain, led to widespread distribution of tetracycline resistance, as suggested in another study (Sawant et al., 2007). Taken together, our results imply that the high prevalence of tetracycline-resistant E. coli in meat may be due to selective pressure, as well as the high transferability of tet determinants (Chopra and Roberts, 2001; Roberts, 2003). To the best of our knowledge, this is the first report to study the distribution and transferability of tetracycline resistance genes in E. coli from meat and meat products in Korea. Of the antimicrobial agents used for livestock farming in Korea between 2002 and 2006, tetracyclines were the most common (43–51%), and most of these were used as growth promoters or disease prophylactics in the production of foodproducing animals (KFDA, 2007). Use of antibiotics as feed additives was partially limited in May 2005 and. antimicrobials such as oxytetracycline and chlortetracycline were entirely banned for non-human uses as of January 2009 in Korea. The observations of this study were collected before these regulations were implemented, so these findings can be used as reference data for comparison to antimicrobial resistance rate and risk assessment after the regulations are effective in the future. Acknowledgements This study was supported by a grant from the National Antimicrobial Resistance Management Program (08072NARMP150) of the Korea Food and Drug Administration. We would like to thank Professor Hyejung Chang (Kyung Hee University) for the statistical advice. We also thank the Korea University Food Safety Hall and Institute of Food and Biomedicine Safety for allowing the use of their equipment and facilities. References Aarestrup, F.M., 1999. Association between the consumption of antimicrobial agents in animal husbandry and the occurrence of resistant bacteria among food animals. International Journal of Antimicrobial Agents 12, 279–285. Aslam, M., Service, C., 2006. Antimicrobial resistance and genetic profiling of Escherichia coli from a commercial beef packing plant. Journal of Food Protection 69, 1508–1513.
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