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Characterization of transferable tetracycline resistance genes in Enterococcus faecalis isolated from raw food
FEMS Microbiology Letters 243 (2005) 15–19 www.fems-microbiology.org
Characterization of transferable tetracycline resistance genes in Enterococcus faecalis isolated from raw food Andrea Wilcks, Sigrid Rita Andersen, Tine Rask Licht
*
Department of Microbiological Food Safety, Danish Institute for Food and Veterinary Research, Moerkhoej Bygade 19, DK-2860 Soeborg, Denmark Received 17 August 2004; received in revised form 16 November 2004; accepted 16 November 2004 First published online 7 December 2004 Edited by W. Kneifel
Abstract The prevalence of tetracycline resistance, and of specific genetic determinants for this resistance was investigated in 1003 strains of Enterococcus faecalis isolated from various raw food products originating from five categories including chicken meat, other poultry meat, beef, pork, and ÔotherÕ. For the 238 resistant isolates identified, the ability to transfer the resistant phenotype to a given recipient in vitro was investigated. New and interesting observations were that the tet(L) resistance determinant was more readily transferred than tet(M), and that the presence of Tn916-like elements known to encode tet(M) did not correlate with increased transferability of the resistant phenotype. 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords: Tetracycline; Resistance; Food; Enterococcus faecalis; Horizontal gene transfer
1. Introduction In recent time, a lot of attention has been drawn to Enterococci as reservoirs and vehicles of antibiotic resistance [1,2]. This is due to their abundance in the intestine of most mammals, their ability to readily develop antimicrobial resistance in response to selective pressure, and the fact that they are identified as the cause of a continuously increasing number of hospital-acquired infections [3]. Tetracycline resistance in Enterococci can be conferred by various genes and mechanisms including efflux mechanisms encoded by tet(K) and tet(L), ribosomal protection encoded by tet(M), tet(O), and tet(S), as well
*
Corresponding author. Tel.: +45 72 34 71 86; fax: +45 72 34 76 98. E-mail address: [email protected] (T.R. Licht).
as an unknown mechanism encoded by tet(U) [4]. The tet(M) gene is often associated with the Tn916–Tn1545 family of conjugative transposons [5]. These transposons are very abundant in fecal Enterococci from food animals [6], have an extraordinarily broad host range, and are transferred at rather low frequencies among Enterococci on solid surfaces in vitro [7]. Other transferable elements carrying tetracycline resistance genes include pheromone-inducible plasmids found solely in Enterococcus faecalis and known to transfer very efficiently in vitro in liquid media [8]. While a number of studies have addressed the prevalence of genetic tetracycline resistance determinants within Enterococci from fecal samples of animals in Danish husbandry [9,10], only few reports exist on the occurrence of such genes in Danish food [11]. In the present study, we have investigated the occurrence of tetracycline resistance and of specific genetic determinants within a large number of E. faecalis isolated from
0378-1097/$22.00 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.femsle.2004.11.028
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A. Wilcks et al. / FEMS Microbiology Letters 243 (2005) 15–19
raw food samples. Also putative transfer of the resistant phenotype from these isolates to a given recipient strain under different transfer conditions was investigated.
2. Materials and methods 2.1. Bacterial stains and media The E. faecalis food isolates all originated from raw food samples collected from Danish retail outlets in the period from 1996 to 2002 as part of the national resistance surveillance program DANMAP [12]. The isolation procedure did not include the use of any antibiotics. One strain of E. faecalis from each food sample was used for susceptibility testing. Susceptibility testing was performed according to NCCLS guidelines [13] and identity of the species was verified by PCR for all tetracycline resistant isolates [14]. E. faecalis OG1RF [15], which is resistant to rifampicin and fusidic acid was used as recipient in all mating experiments. The two strains E. faecalis OG1SS:Tn916 alias SF8583 [16] and E. faecalis OG1SS/pCF10 [17,18], both carrying resistance to streptomycin and spectinomycin, as well as transferable elements encoding tetracycline resistance, were used as positive controls in the mating experiments. Brain heart infusion (BHI) broth (Oxoid, Hampshire, England) was used for liquid as well as solid cultures of all strains. Donor isolates were always grown in the presence of tetracycline, while the recipient strain was grown in the presence of rifampicin and fusidic acid. Selection for transconjugants was carried out on BHI containing tetracycline, rifampicin and fusidic acid. Antibiotics (Sigma) were used in the following concentrations: tetracycline hydrochloride, 10 lg ml 1; rifampicin, 25 lg ml 1; fusidic acid, 25 lg ml 1. 2.2. Characterization of tetracycline determinants by PCR For the 238 E. faecalis isolated from food, as well as for all transconjugants resulting from the mating experiments, PCR was carried out in order to characterize the tetracycline resistance determinants encoded by these strains. DNA from donors and transconjugants was extracted by boiling a bacterial colony for 10 minutes, after centrifugation the supernatant was used for PCR amplification. All PCR amplifications were run in a Peltier Thermal Cycler PTC-225 (MJ Research). 2.2.1. Multiplex PCR for detection of tet-genes Positive control strains for the multiplex PCR were the ones formerly described and kindly provided by Yvonne Agersø [19]. Primers for the detection of tet(K), tet(L), tet(M), tet(O), and tet(S) were described by Ng et al. [20]. The
PCR reaction mixture consisted of 30 ll Eppendorf MasterMix (2.5·) (Radiometer, Denmark), 10 ll DNA and 10 ll primer-mix with the concentration of primers as previously described by Ng et al. [20]. The following conditions for multiplex PCR were used: An initial denaturation step at 95 C for 5 min, followed by 35 cycles at 94 C for 30 s, 53 C for 30 s, 72 C for 1.5 min, and a final extension step at 72 C for 7 min. 2.2.2. PCR for detection of int genes of the Tn916–Tn1545 family The tet(M) positive strains were analysed for the presence of Tn916/Tn1545-like sequences using primers Int-FW and Int-RV [21,22]. The PCR reaction mixture (25 ll) consisted of one Ready-To-Go PCR Bead (Amersham Pharmacia Biotech), 5 ll of DNA and 10 pmol of each primer. Cycling parameters were denaturation at 94 C for 3 min, followed by 35 cycles at 94 C for 40 s, 53 C for 40 s, 72 C for 1.5 min, and a final extension at 72 C for 10 min. 2.3. Mating assays Overnight cultures of each donor isolate as well as cultures of the two positive control donor strains were washed in buffered saline, resuspended in broth media without tetracycline, and mixed with equal amounts of a similarly washed overnight culture of the recipient strain. Drops of 20 ll of these mixtures were placed on agar plates containing no antibiotics. Also, aliquots of 1 ml of the donor–recipient mixtures diluted 1:10 in broth were made. Solid as well as liquid mating mixtures were incubated over night at 37 C. Mating ÔdropsÕ were scraped of the agar plate and resuspended in buffered saline. The mating mixtures were spread on plates selective for transconjugants. Prior to mixing of donor and recipient strains, it was verified, that neither donor strains nor recipient strain were capable of growth on transconjugant-selective plates. 2.4. Statistics The observed prevalence of the tet(L) gene among the potential donor isolates was 81/238 = 0.34. Assuming that the prevalence of the tet(L) gene is the same among the transconjugants as among the original isolates, the expected number of tet(L) genes among the 27 transconjugants is thus 27 · 0.34 = 9 ± sqrt(9). The real number of transconjugants encoding tet(L) was 19. This differs from the expected average of 9 with more that three SD intervals of sqrt(9) = 3, meaning that there is 99% probability (P > 0.99) that the prevalence of the tet(L) gene among transconjugants was different from the prevalence of the tet(L) gene among the potential donor isolates.
A. Wilcks et al. / FEMS Microbiology Letters 243 (2005) 15–19
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3.2. Prevalence of tetracycline determinants and Tn916-like elements
tet genes previously reported in this genus [4]. As shown in Table 2 the resistance determinants tet(L), tet(M) tet(O) and tet(S) are together responsible for 93% of the tetracycline resistant phenotype, while tet(K) was not found. The tet(O) gene was detected only in isolates recovered from meat of chicken and other poultry. This observation fits well with the finding that tet(O) mediated resistance in E. faecalis is present in the intestinal tract of broilers, but not in pigs [9]. The tet(L) gene was predominant within the E. faecalis strains originating from poultry products other than chicken (59%), while the tet(M) gene was predominant in all other food categories including chicken meat (Table 2). The second observation is coherent with a previous report showing that tet(M) is the most prevalent tetracycline resistance determinant in pigs as well as in broilers [9]. Transposable elements belonging to the Tn916-family of conjugative transposons, which carries tet(M) [5] were present in 90% of the E. faecalis isolates that carried tet(M) (data not shown). A large part of the tet(M) determinants present in the food samples was therefore likely to be attributed to the presence of Tn916-like conjugative transposons. This is consistent with a number of previous communications [4,5,23].
The genetic basis of the observed tetracycline resistant phenotypes was tested by multiplex PCR of five
3.3. Transferability of tetracycline genes from food isolates
3. Results and discussions 3.1. Prevalence of tetracycline resistance among food categories A total number of 1003 E. faecalis strains were isolated from raw food samples collected at food retail outlets in Denmark in the period from 1996 to 2002 [12]. Hereof, 238 isolates (24%) were resistant to tetracycline (Table 1). This is in concordance with results from a recently published study concentrating on food isolates [23]. Other reports suggest prevalences as high as 45% tetracycline resistant E. faecalis in food [24]. In spite of the fact that more tetracycline is used for pigs than for broilers in Denmark [12], most of the tetracycline resistant E. faecalis were isolated from chicken meat or other poultry products, whereas the prevalence of resistance was lower in pork and beef (Table 1). This is consistent with another study looking at Swedish and Danish pork and chicken [11], while studies on food isolates from the US report very high prevalences of resistance in pork [25,26].
Out of the 238 tetracycline resistant E. faecalis isolates, a total of 27 (11%) transferred the resistant phenotype to the recipient strain E. faecalis OG1RF after mating on a solid agar surface (Table 3). No transfer of tetracycline resistance from any of the isolates was detected after mating in liquid media, indicating that no transfer events were mediated by pheromone-inducible plasmids [8]. In all cases of transfer on solid media, the genetic determinant of tetracycline resistance detected in the transconjugants was identical to the determinant present in the donor isolate that had produced the transconjugant. Three of the donor isolates capable of transfer carried both tet(M) and tet (L). In all three cases, both of
Table 1 Prevalence of tetracycline resistant E. faecalis in various food categories Tetracycline resistant (%)a Chicken meat (n = 266) Other poultry meatb (n = 96) Beef (n = 180) Pork (n = 275) Otherc (n = 186) Total (n = 1003)
35 55 15 16 12 24
a
MIC-values P 16 lg/ml. Mainly turkey and duck meat. c Includes fish, (n = 12), vegetables (n = 5), dairy products (n = 2), lamb (n = 2) and chicken mayo salad (n = 1). b
Table 2 Prevalence of genetic tetracycline determinants within food categories
Chicken meat (n = 92) Other poultry meatb (n = 53) Beef (n = 27) Pork (n = 44) Otherc (n = 22) Total (n = 238) a b c
N.i.a (%)
La (%)
LM (%)
M (%)
MO (%)
O (%)
S (%)
7 2 7 7 18 7
30 53 15 16 18 30
4 6 7 2 0 4
40 26 67 73 64 48
1 0 0 0 0 0
17 13 0 0 0 10
0 0 4 2 0 1
N.i., not identified. The tet(L) gene is indicated by ÔLÕ, a combination of the tet(L) and the tet(M) gene by ÔLMÕ and so forth. Mainly turkey and duck meat. Includes fish, (n = 12), vegetables (n = 5), dairy products (n = 2), lamb (n = 2) and chicken mayo salad (n = 1).
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A. Wilcks et al. / FEMS Microbiology Letters 243 (2005) 15–19
Table 3 Transferability of tetracycline genes from food isolates by mating on solid media
Transferable (%)
N.i. (n = 16)
L (n = 71)
LM (n = 10)
M (n = 115)
MO (n = 1)
O (n = 23)
S (n = 2)
Total (n = 238)
0
23
30
6
0
4
0
11
N.i., not identified. The tet(L) gene is indicated by ÔLÕ, a combination of the tet(L) and the tet(M) gene by ÔLMÕ and so forth. The recipient used in transfer studies was E. faecalis OG1RF.
the two genes were transferred to the transconjugants, suggesting that they were encoded on the same transferable element, or that one mobilized the other. There was a significant (P > 99%) preference for transfer of the tet(L) gene, which was present in 19 of the 27 transconjugants (70%), but only in 81 of the 238 potential donors (34%). Only one other recent study by Huys et al. [23] indicates that tet(L) can be transferred in vitro from food isolates of E. faecalis. This Belgian group find that acquisition of tet(L) by the recipient strain E. faecalis JH2-2 is associated with the acquisition of one or more large plasmids. However, in strict contrast to our findings (Table 2), Huys and coworkers find that tet(M) is transferred much more frequently than tet(L). Putative explanations for this difference might be that different recipient strains were used in the two investigations, and that the isolates studied by Huys et al. originated primarily from European cheeses, while isolates from other food categories dominated the present investigation (Table 1). Furthermore, the investigated number of tet(M) and tet(L) encoding food isolates in the present study were 125 and 81, respectively, while the corresponding numbers of investigated isolates in the Belgian study were 32 and 13. The observed large mobility of the tet(L) gene in the present study was surprising since it was expected that a large part of the transfer events would be attributed to the conjugative transposons belonging to the Tn916 family encoding tet(M), as has also been shown by 23. However, of the 10 transconjugants carrying tet(M), only three carried Tn916-like elements (data not shown), even though such elements were present in 90% of the E. faecalis donor isolates shown to carry tet(M), as described above. There was thus no correlation between the presence of Tn916-like elements and the mobility of the tet(M) genes in food isolates.
number of E. faecalis isolates (Table 2). Except for one single reported isolate [23], this resistance determinant has not previously been found in Enterococci from raw food or from Danish pigs and broilers, but is common in samples of human faeces [9]. This may indicate that the few tet(S) isolates originate from humans processing the beef and pork. Our observation of the high in vitro transferability of the tet(L) determinant from food isolates (Table 3) is new and might be of great importance to the understanding of the mechanisms causing horizontal spread of resistance genes within the food chain. The same is true for the observation that the presence of conjugative transposons belonging to the Tn916–Tn1545 family did not – as expected – lead to an increased prevalence of transfer events to a defined recipient within the host range of these elements. Finally, the attention is drawn to the fact that tet(L), which was observed to be more readily transferred than the other tetracycline resistance determinants (Table 3), was most common in the food category Ôother poultry meatÕ, and that within this category also the highest prevalence of resistant isolates was found. We therefore speculate, that transfer of resistance genes might contribute to the high prevalence observed.
Acknowledgements We thank Rikke Kubert and Kate Vibefeldt Nielsen for technical assistance, and Yvonne Agersø for kindly providing us with positive control strains. The collection and identification of food isolates was part of T he Danish Integrated Antimicrobial Resistance Monitoring and Research Programme (DANMAP), funded jointly by the Ministry of Food, Agriculture and Fisheries and the Ministry of the Interior and Health.
3.4. Concluding remarks To our knowledge, this is the first investigation of resistance in food borne bacteria having such a large number of isolates at its disposal. The described distribution of tetracycline determinants among different food categories correlate to some extent with the distribution observed among food animals [9], indicating that many resistant bacteria in food products originate from the animals used for production of the food. A new observation was the identification of tet(S) in a limited
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A. Wilcks et al. / FEMS Microbiology Letters 243 (2005) 15–19 [4] Chopra, I. and Roberts, M. (2001) Tetracycline antibiotics: mode of action, applications, molecular biology, and epidemiology of bacterial resistance. Microbiol. Mol. Biol. Rev. 65, 232–260. [5] Clewell, D.B., Flannagan, S.E. and Jaworski, D.D. (1995) Unconstrained bacterial promiscuity: the Tn916–Tn1545 family of conjugative transposons. Trends Microbiol. 3, 229–236. [6] Haack, B.J. and Andrews, R.E.J. (2000) Isolation of Tn916-like conjugal elements from swine lot effluent. Can. J. Microbiol. 46, 542–549. [7] Marra, D., Pethel, B., Churchward, G.G. and Scott, J.R. (1999) The frequency of conjugative transposition of Tn916 is not determined by the frequency of excision. J. Bacteriol. 181, 5414– 5418. [8] Dunny, G.M., Leonard, B.A. and Hedberg, P.J. (1995) Pheromone-inducible conjugation in Enterococcus faecalis: interbacterial and host-parasite chemical communication. J. Bacteriol. 177, 871–876. [9] Aarestrup, F.M., Agerso, Y., Gerner-Smidt, P., Madsen, M. and Jensen, L.B. (2000) Comparison of antimicrobial resistance phenotypes and resistance genes in Enterococcus faecalis and Enterococcus faecium from humans in the community, broilers, and pigs in Denmark. Diagn. Microbiol. Infect. Dis. 37, 127–137. [10] Sengelov, G., Halling-Sorensen, B. and Aarestrup, F.M. (2003) Susceptibility of Escherichia coli and Enterococcus faecium isolated from pigs and broiler chickens to tetracycline degradation products and distribution of tetracycline resistance determinants in E. coli from food animals. Vet. Microbiol. 95, 91–101. [11] Quednau, M., Ahrne, S., Petersson, A.C. and Molin, G. (1998) Antibiotic-resistant strains of Enterococcus isolated from Swedish and Danish retailed chicken and pork. J. Appl. Microbiol. 84, 1163–1170. [12] DANMAP 2002. (2003) Use of antimicrobial agents and occurrence of antimicrobial resistance from food animals, foods and humans in Denmark. ISSN 1600-2032. [13] National Committee for Clinical Laboratory Standards (1997) Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. Appoved Standard M7-A4, National Committee for Clinical Laboratory Standards, Wayne, PA. [14] Dutka-Malen, S., Evers, S. and Courvalin, P. (1995) Detection of glycopeptide resistance genotypes and identification to the species level of clinically relevant enterococci by PCR. J. Clin. Microbiol. 33, 24–27. [15] Dunny, G.M. and Clewell, D.B. (1975) Transmissible toxin (hemolysin) plasmid in Streptococcus faecalis and its mobilization
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Characterization of transferable tetracycline resistance genes in Enterococcus faecalis isolated from raw food Andrea Wilcks, Sigrid Rita Andersen, Tine Rask Licht
*
Department of Microbiological Food Safety, Danish Institute for Food and Veterinary Research, Moerkhoej Bygade 19, DK-2860 Soeborg, Denmark Received 17 August 2004; received in revised form 16 November 2004; accepted 16 November 2004 First published online 7 December 2004 Edited by W. Kneifel
Abstract The prevalence of tetracycline resistance, and of specific genetic determinants for this resistance was investigated in 1003 strains of Enterococcus faecalis isolated from various raw food products originating from five categories including chicken meat, other poultry meat, beef, pork, and ÔotherÕ. For the 238 resistant isolates identified, the ability to transfer the resistant phenotype to a given recipient in vitro was investigated. New and interesting observations were that the tet(L) resistance determinant was more readily transferred than tet(M), and that the presence of Tn916-like elements known to encode tet(M) did not correlate with increased transferability of the resistant phenotype. 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords: Tetracycline; Resistance; Food; Enterococcus faecalis; Horizontal gene transfer
1. Introduction In recent time, a lot of attention has been drawn to Enterococci as reservoirs and vehicles of antibiotic resistance [1,2]. This is due to their abundance in the intestine of most mammals, their ability to readily develop antimicrobial resistance in response to selective pressure, and the fact that they are identified as the cause of a continuously increasing number of hospital-acquired infections [3]. Tetracycline resistance in Enterococci can be conferred by various genes and mechanisms including efflux mechanisms encoded by tet(K) and tet(L), ribosomal protection encoded by tet(M), tet(O), and tet(S), as well
*
Corresponding author. Tel.: +45 72 34 71 86; fax: +45 72 34 76 98. E-mail address: [email protected] (T.R. Licht).
as an unknown mechanism encoded by tet(U) [4]. The tet(M) gene is often associated with the Tn916–Tn1545 family of conjugative transposons [5]. These transposons are very abundant in fecal Enterococci from food animals [6], have an extraordinarily broad host range, and are transferred at rather low frequencies among Enterococci on solid surfaces in vitro [7]. Other transferable elements carrying tetracycline resistance genes include pheromone-inducible plasmids found solely in Enterococcus faecalis and known to transfer very efficiently in vitro in liquid media [8]. While a number of studies have addressed the prevalence of genetic tetracycline resistance determinants within Enterococci from fecal samples of animals in Danish husbandry [9,10], only few reports exist on the occurrence of such genes in Danish food [11]. In the present study, we have investigated the occurrence of tetracycline resistance and of specific genetic determinants within a large number of E. faecalis isolated from
0378-1097/$22.00 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.femsle.2004.11.028
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A. Wilcks et al. / FEMS Microbiology Letters 243 (2005) 15–19
raw food samples. Also putative transfer of the resistant phenotype from these isolates to a given recipient strain under different transfer conditions was investigated.
2. Materials and methods 2.1. Bacterial stains and media The E. faecalis food isolates all originated from raw food samples collected from Danish retail outlets in the period from 1996 to 2002 as part of the national resistance surveillance program DANMAP [12]. The isolation procedure did not include the use of any antibiotics. One strain of E. faecalis from each food sample was used for susceptibility testing. Susceptibility testing was performed according to NCCLS guidelines [13] and identity of the species was verified by PCR for all tetracycline resistant isolates [14]. E. faecalis OG1RF [15], which is resistant to rifampicin and fusidic acid was used as recipient in all mating experiments. The two strains E. faecalis OG1SS:Tn916 alias SF8583 [16] and E. faecalis OG1SS/pCF10 [17,18], both carrying resistance to streptomycin and spectinomycin, as well as transferable elements encoding tetracycline resistance, were used as positive controls in the mating experiments. Brain heart infusion (BHI) broth (Oxoid, Hampshire, England) was used for liquid as well as solid cultures of all strains. Donor isolates were always grown in the presence of tetracycline, while the recipient strain was grown in the presence of rifampicin and fusidic acid. Selection for transconjugants was carried out on BHI containing tetracycline, rifampicin and fusidic acid. Antibiotics (Sigma) were used in the following concentrations: tetracycline hydrochloride, 10 lg ml 1; rifampicin, 25 lg ml 1; fusidic acid, 25 lg ml 1. 2.2. Characterization of tetracycline determinants by PCR For the 238 E. faecalis isolated from food, as well as for all transconjugants resulting from the mating experiments, PCR was carried out in order to characterize the tetracycline resistance determinants encoded by these strains. DNA from donors and transconjugants was extracted by boiling a bacterial colony for 10 minutes, after centrifugation the supernatant was used for PCR amplification. All PCR amplifications were run in a Peltier Thermal Cycler PTC-225 (MJ Research). 2.2.1. Multiplex PCR for detection of tet-genes Positive control strains for the multiplex PCR were the ones formerly described and kindly provided by Yvonne Agersø [19]. Primers for the detection of tet(K), tet(L), tet(M), tet(O), and tet(S) were described by Ng et al. [20]. The
PCR reaction mixture consisted of 30 ll Eppendorf MasterMix (2.5·) (Radiometer, Denmark), 10 ll DNA and 10 ll primer-mix with the concentration of primers as previously described by Ng et al. [20]. The following conditions for multiplex PCR were used: An initial denaturation step at 95 C for 5 min, followed by 35 cycles at 94 C for 30 s, 53 C for 30 s, 72 C for 1.5 min, and a final extension step at 72 C for 7 min. 2.2.2. PCR for detection of int genes of the Tn916–Tn1545 family The tet(M) positive strains were analysed for the presence of Tn916/Tn1545-like sequences using primers Int-FW and Int-RV [21,22]. The PCR reaction mixture (25 ll) consisted of one Ready-To-Go PCR Bead (Amersham Pharmacia Biotech), 5 ll of DNA and 10 pmol of each primer. Cycling parameters were denaturation at 94 C for 3 min, followed by 35 cycles at 94 C for 40 s, 53 C for 40 s, 72 C for 1.5 min, and a final extension at 72 C for 10 min. 2.3. Mating assays Overnight cultures of each donor isolate as well as cultures of the two positive control donor strains were washed in buffered saline, resuspended in broth media without tetracycline, and mixed with equal amounts of a similarly washed overnight culture of the recipient strain. Drops of 20 ll of these mixtures were placed on agar plates containing no antibiotics. Also, aliquots of 1 ml of the donor–recipient mixtures diluted 1:10 in broth were made. Solid as well as liquid mating mixtures were incubated over night at 37 C. Mating ÔdropsÕ were scraped of the agar plate and resuspended in buffered saline. The mating mixtures were spread on plates selective for transconjugants. Prior to mixing of donor and recipient strains, it was verified, that neither donor strains nor recipient strain were capable of growth on transconjugant-selective plates. 2.4. Statistics The observed prevalence of the tet(L) gene among the potential donor isolates was 81/238 = 0.34. Assuming that the prevalence of the tet(L) gene is the same among the transconjugants as among the original isolates, the expected number of tet(L) genes among the 27 transconjugants is thus 27 · 0.34 = 9 ± sqrt(9). The real number of transconjugants encoding tet(L) was 19. This differs from the expected average of 9 with more that three SD intervals of sqrt(9) = 3, meaning that there is 99% probability (P > 0.99) that the prevalence of the tet(L) gene among transconjugants was different from the prevalence of the tet(L) gene among the potential donor isolates.
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3.2. Prevalence of tetracycline determinants and Tn916-like elements
tet genes previously reported in this genus [4]. As shown in Table 2 the resistance determinants tet(L), tet(M) tet(O) and tet(S) are together responsible for 93% of the tetracycline resistant phenotype, while tet(K) was not found. The tet(O) gene was detected only in isolates recovered from meat of chicken and other poultry. This observation fits well with the finding that tet(O) mediated resistance in E. faecalis is present in the intestinal tract of broilers, but not in pigs [9]. The tet(L) gene was predominant within the E. faecalis strains originating from poultry products other than chicken (59%), while the tet(M) gene was predominant in all other food categories including chicken meat (Table 2). The second observation is coherent with a previous report showing that tet(M) is the most prevalent tetracycline resistance determinant in pigs as well as in broilers [9]. Transposable elements belonging to the Tn916-family of conjugative transposons, which carries tet(M) [5] were present in 90% of the E. faecalis isolates that carried tet(M) (data not shown). A large part of the tet(M) determinants present in the food samples was therefore likely to be attributed to the presence of Tn916-like conjugative transposons. This is consistent with a number of previous communications [4,5,23].
The genetic basis of the observed tetracycline resistant phenotypes was tested by multiplex PCR of five
3.3. Transferability of tetracycline genes from food isolates
3. Results and discussions 3.1. Prevalence of tetracycline resistance among food categories A total number of 1003 E. faecalis strains were isolated from raw food samples collected at food retail outlets in Denmark in the period from 1996 to 2002 [12]. Hereof, 238 isolates (24%) were resistant to tetracycline (Table 1). This is in concordance with results from a recently published study concentrating on food isolates [23]. Other reports suggest prevalences as high as 45% tetracycline resistant E. faecalis in food [24]. In spite of the fact that more tetracycline is used for pigs than for broilers in Denmark [12], most of the tetracycline resistant E. faecalis were isolated from chicken meat or other poultry products, whereas the prevalence of resistance was lower in pork and beef (Table 1). This is consistent with another study looking at Swedish and Danish pork and chicken [11], while studies on food isolates from the US report very high prevalences of resistance in pork [25,26].
Out of the 238 tetracycline resistant E. faecalis isolates, a total of 27 (11%) transferred the resistant phenotype to the recipient strain E. faecalis OG1RF after mating on a solid agar surface (Table 3). No transfer of tetracycline resistance from any of the isolates was detected after mating in liquid media, indicating that no transfer events were mediated by pheromone-inducible plasmids [8]. In all cases of transfer on solid media, the genetic determinant of tetracycline resistance detected in the transconjugants was identical to the determinant present in the donor isolate that had produced the transconjugant. Three of the donor isolates capable of transfer carried both tet(M) and tet (L). In all three cases, both of
Table 1 Prevalence of tetracycline resistant E. faecalis in various food categories Tetracycline resistant (%)a Chicken meat (n = 266) Other poultry meatb (n = 96) Beef (n = 180) Pork (n = 275) Otherc (n = 186) Total (n = 1003)
35 55 15 16 12 24
a
MIC-values P 16 lg/ml. Mainly turkey and duck meat. c Includes fish, (n = 12), vegetables (n = 5), dairy products (n = 2), lamb (n = 2) and chicken mayo salad (n = 1). b
Table 2 Prevalence of genetic tetracycline determinants within food categories
Chicken meat (n = 92) Other poultry meatb (n = 53) Beef (n = 27) Pork (n = 44) Otherc (n = 22) Total (n = 238) a b c
N.i.a (%)
La (%)
LM (%)
M (%)
MO (%)
O (%)
S (%)
7 2 7 7 18 7
30 53 15 16 18 30
4 6 7 2 0 4
40 26 67 73 64 48
1 0 0 0 0 0
17 13 0 0 0 10
0 0 4 2 0 1
N.i., not identified. The tet(L) gene is indicated by ÔLÕ, a combination of the tet(L) and the tet(M) gene by ÔLMÕ and so forth. Mainly turkey and duck meat. Includes fish, (n = 12), vegetables (n = 5), dairy products (n = 2), lamb (n = 2) and chicken mayo salad (n = 1).
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A. Wilcks et al. / FEMS Microbiology Letters 243 (2005) 15–19
Table 3 Transferability of tetracycline genes from food isolates by mating on solid media
Transferable (%)
N.i. (n = 16)
L (n = 71)
LM (n = 10)
M (n = 115)
MO (n = 1)
O (n = 23)
S (n = 2)
Total (n = 238)
0
23
30
6
0
4
0
11
N.i., not identified. The tet(L) gene is indicated by ÔLÕ, a combination of the tet(L) and the tet(M) gene by ÔLMÕ and so forth. The recipient used in transfer studies was E. faecalis OG1RF.
the two genes were transferred to the transconjugants, suggesting that they were encoded on the same transferable element, or that one mobilized the other. There was a significant (P > 99%) preference for transfer of the tet(L) gene, which was present in 19 of the 27 transconjugants (70%), but only in 81 of the 238 potential donors (34%). Only one other recent study by Huys et al. [23] indicates that tet(L) can be transferred in vitro from food isolates of E. faecalis. This Belgian group find that acquisition of tet(L) by the recipient strain E. faecalis JH2-2 is associated with the acquisition of one or more large plasmids. However, in strict contrast to our findings (Table 2), Huys and coworkers find that tet(M) is transferred much more frequently than tet(L). Putative explanations for this difference might be that different recipient strains were used in the two investigations, and that the isolates studied by Huys et al. originated primarily from European cheeses, while isolates from other food categories dominated the present investigation (Table 1). Furthermore, the investigated number of tet(M) and tet(L) encoding food isolates in the present study were 125 and 81, respectively, while the corresponding numbers of investigated isolates in the Belgian study were 32 and 13. The observed large mobility of the tet(L) gene in the present study was surprising since it was expected that a large part of the transfer events would be attributed to the conjugative transposons belonging to the Tn916 family encoding tet(M), as has also been shown by 23. However, of the 10 transconjugants carrying tet(M), only three carried Tn916-like elements (data not shown), even though such elements were present in 90% of the E. faecalis donor isolates shown to carry tet(M), as described above. There was thus no correlation between the presence of Tn916-like elements and the mobility of the tet(M) genes in food isolates.
number of E. faecalis isolates (Table 2). Except for one single reported isolate [23], this resistance determinant has not previously been found in Enterococci from raw food or from Danish pigs and broilers, but is common in samples of human faeces [9]. This may indicate that the few tet(S) isolates originate from humans processing the beef and pork. Our observation of the high in vitro transferability of the tet(L) determinant from food isolates (Table 3) is new and might be of great importance to the understanding of the mechanisms causing horizontal spread of resistance genes within the food chain. The same is true for the observation that the presence of conjugative transposons belonging to the Tn916–Tn1545 family did not – as expected – lead to an increased prevalence of transfer events to a defined recipient within the host range of these elements. Finally, the attention is drawn to the fact that tet(L), which was observed to be more readily transferred than the other tetracycline resistance determinants (Table 3), was most common in the food category Ôother poultry meatÕ, and that within this category also the highest prevalence of resistant isolates was found. We therefore speculate, that transfer of resistance genes might contribute to the high prevalence observed.
Acknowledgements We thank Rikke Kubert and Kate Vibefeldt Nielsen for technical assistance, and Yvonne Agersø for kindly providing us with positive control strains. The collection and identification of food isolates was part of T he Danish Integrated Antimicrobial Resistance Monitoring and Research Programme (DANMAP), funded jointly by the Ministry of Food, Agriculture and Fisheries and the Ministry of the Interior and Health.
3.4. Concluding remarks To our knowledge, this is the first investigation of resistance in food borne bacteria having such a large number of isolates at its disposal. The described distribution of tetracycline determinants among different food categories correlate to some extent with the distribution observed among food animals [9], indicating that many resistant bacteria in food products originate from the animals used for production of the food. A new observation was the identification of tet(S) in a limited
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