- Email: [email protected]
Comparison of ESBL contamination in organic and conventional retail chicken meat
International Journal of Food Microbiology 154 (2012) 212–214
Contents lists available at SciVerse ScienceDirect
International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro
Short Communication
Comparison of ESBL contamination in organic and conventional retail chicken meat James Cohen Stuart a,⁎, Thijs van den Munckhof a, Guido Voets a, Jelle Scharringa a, Ad Fluit a, Maurine Leverstein-Van Hall a, b a b
Department of Medical Microbiology, University Medical Center Utrecht, The Netherlands Centre for Infectious Disease Control, National Institute for Public Health and the Environment (RIVM), Bilthoven, The Netherlands
a r t i c l e
i n f o
Article history: Received 14 September 2011 Received in revised form 7 November 2011 Accepted 26 December 2011 Available online 3 January 2012 Keywords: ESBL Meat Organic
a b s t r a c t Contamination of retail chicken meat by Extended Spectrum Beta-Lactamase (ESBL) producing bacteria likely contributes to the increasing incidence of infections with these bacteria in humans. This study aimed to compare the prevalence and load of ESBL positive isolates between organic and conventional retail chicken meat samples, and to compare the distribution of ESBL genes, strain genotypes and co-resistance. In 2010, 98 raw chicken breasts (n = 60 conventional; n = 38 organic) were collected from 12 local stores in the Netherlands. Prevalence of ESBL producing micro-organisms was 100% on conventional and 84% on organic samples (p b 0.001). Median loads of ESBL producing micro-organisms were 80 (range b 20–1360) in conventional, and b 20 (range 0–260) CFU/25 g in organic samples (p = 0.001). The distribution of ESBL genes in conventional samples and organic samples was 42% versus 56%, respectively (N.S.), for CTX-M-1, 20% versus 42% (N.S.) for TEM-52, and 23% versus 3% (p b 0.001) for SHV-12. CTX-M-2 (7%), SHV-2 (5%) and TEM-20 (3%) were exclusively found in conventional samples. Co-resistance rates of ESBL positive isolates were not different between conventional and organic samples (co-trimoxazole 56%, ciprofloxacin 14%, and tobramycin 2%), except for tetracycline, 73% and 46%, respectively, p b 0.001). Six of 14 conventional meat samples harbored 4 MLST types also reported in humans and 5 of 10 organic samples harbored 3 MLST types also reported in humans (2 ST10, 2 ST23, ST354). In conclusion, the majority of organic chicken meat samples were also contaminated with ESBL producing E. coli, and the ESBL genes and strain types were largely the same as in conventional meat samples. © 2012 Elsevier B.V. All rights reserved.
1. Introduction
2. Methods
Contamination of retail chicken meat by Enterobacteriaceae producing Extended Spectrum Beta-Lactamases (ESBLs) has been reported in several countries (Dhanji et al. 2010; Doi et al. 2010; Lavilla et al. 2008; Leverstein-van Hall et al. 2011; Randall et al. 2011), and likely contributes to the increased incidence of infections with these bacteria in humans (Leverstein-van Hall et al. 2011; Overdevest et al., 2011). We hypothesized that the ESBL prevalence and the load of ESBL producing micro-organisms is lower in organic meat, because antibiotic use is lower in organic chicken farms than in conventional farms. The aims of this study were to compare the prevalence and load of ESBL positive isolates between organic and conventional retail chicken meat samples, and to compare the distribution of ESBL genes, strain genotypes and degree of co-resistance.
In 2010, 98 raw chicken breasts were collected from 12 stores in Utrecht, a city in the centre of the Netherlands. Eighty percent of the meat samples were collected from 9 stores from 5 supermarket chains with a combined Dutch market share of approximately 90%, and 20% of the meat samples were collected from organic butcheries. Sixty meat samples were sold as “conventional” and 38 meat samples as organic (30 “EKO”, 8 “free ranging”). The antibiotic policies for these 3 different categories of poultry rearing are as follows: 1) Conventional rearing: 37 d.d.d. per year per animal, i.e. 4 treatment days per chicken per 42 day cycle (95% CI 3–6), with preventive and therapeutic use of antibiotics (Anonymous, 2011). 2) Organic (Type “EKO”): Antibiotic use limited to one therapeutic course of antibiotics and is controlled by SKAL (Stichting EKO keurmerk controle), the inspection and certification body for organic production in the Netherlands.3) Organic (Type “Free Ranging”): use of antibiotics not specified. Information on the country of origin was available on the package of only 30 supermarket conventional meat samples (27% indicated as from the Netherlands, 73 from the Benelux) and for none of the samples collected from the organic butcheries. Phenotypic detection of ESBL producing micro-organisms on the meat samples was performed by homogenizing the meat, followed
⁎ Corresponding author at: Department of Medical Microbiology, G04-614, University Medical Centre Utrecht, Heidelberglaan 100, 3584CX, Utrecht, The Netherlands. Tel.: + 31 6 21277988; fax: + 31 30 2541770. E-mail address: [email protected] (J. Cohen Stuart). 0168-1605/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2011.12.034
by culturing the homogenate on an agar plate, which is selective for ESBL producing gram negative micro-organisms. Per meat sample, 25 g was homogenized with 225 ml peptone water in a stomacher. For quantitative cultures, an ESBL selective plate (ESBL Brilliance, Thermofisher, Basingstoke, UK) was inoculated with 0.1 ml of a 1:1000 dilution of the homogenate. The lower limit of quantification was 20 colony forming units (CFU) per 25 g of meat. For qualitative culturing, an overnight pre-enrichment was performed with 225 ml of the homogenate, followed by inoculation of an ESBL selective plate with 0.1 ml of the pre-enriched homogenate. In case of growth on the ESBL selective plate, species identification of each morphologically different colony on the ESBL selective plate was performed by MALDI-TOF (Bruker Daltonics, Germany). Phenotypic ESBL production was determined using ESBL Etests on all (n = 163) isolates (Biomerieux, Marcy L'etoile, Lyon, France) growing on the selective plates. ESBL gene detection by PCR and sequencing was performed on a random 50% of those isolates (n = 81). Minimum inhibitory concentrations (MICs) of antibiotics were determined by broth microdilution (Merlin, Germany) using EUCAST breakpoints (www. eucast.org). Strain typing was performed on isolates from a random 24 meat samples (14 conventional, 10 organic) using multi locus sequence typing (MLST) (www.mlst.net). Comparisons of frequencies were performed using the Chi-square test (SPSS for windows 15.0, IBM Inc., Armonk, NY, USA)
3. Results In total, 163 isolates were isolated from 98 chicken meat samples. From these, 92 (94%) harbored at least one E. coli isolate with an ESBL phenotype (Fig. 1). One sample also harbored an ESBL producing Escherichia fergusonii, and another sample also an ESBL producing Klebsiella pneumoniae. The average number of (morphologically different) E. coli isolates per meat sample was 2; (range 1–4 isolates). The prevalence of ESBL producing micro-organisms was 100% (95% confidence interval 94–100%) on conventional samples, versus 84% (95% CI 70–93%%) on organic samples (p b 0.001; Fig. 1). The ESBL prevalence on the organic “EKO” type samples was 90% (95% CI 74– 97%) and organic “free ranging” type was 63% (95CI 31–86%), but this difference was not statistically significant (p = 0.09). To be able to detect ESBL producing micro-organisms, the pre-enrichment step was required in 47% of the organic meat samples versus 29% of the conventional samples (p b 0.001). Median loads of ESBL producing micro-organisms were 80 CFU/25 g (range b20–1360) in
Fig. 1. Percentage of ESBL positive meat samples per rearing method.*The difference of ESBL prevalence between the “EKO” and “Free ranging” samples was not statistically significant.
% in cfu-category
J. Cohen Stuart et al. / International Journal of Food Microbiology 154 (2012) 212–214 50% 45% 40% 35% 30% 25% 20% 15% 10% 5% 0%
213
47%
conventional organic
40%
29% 21% 16% 13%
11% 8%
8% 5% 2%
0
1-19*
20-49
50-99
100-499
500-999
>=1000
cfu's ESBL E. coli per 25 gram Fig. 2. Distribution of load of ESBL producing micro-organisms.*ESBL only detected after pre-enrichment.
conventional meat samples versus b20 CFU/25 g (range 0–260) in organic samples (p = 0.001) (Fig. 2). Sequencing of the ESBL genes showed that the distribution of ESBL genes in conventional samples and organic samples was 42% versus 56% for CTX-M-1 (N.S.), 20% versus 42% for TEM-52 (N.S.), and 23% versus 3% for SHV-12 (p b 0.001), respectively. The ESBL genes CTXM-2 (7%), SHV-2 (5%) and TEM-20 (3%) were exclusively found in conventional samples. In 12 (12%) samples (7 organic, 5 conventional) CMY-2 was detected, an ampC gene previously reported in isolates from humans (Simner et al. 2011). Co-resistance rates of ESBL positive isolates were: co-trimoxazole 56%, ciprofloxacin 14%, and tobramycin 2% (no significant differences between organic and conventional isolates). However, tetracycline co-resistance was more prevalent in conventional than in organic samples (73% versus 46%, p b 0,001). Six of the 14 conventional meat samples harbored 4 MLST types also reported in humans ( 2 ST10, 2 ST23, ST57, ST117) and 5 of 10 organic samples harbored 3 MLST types also reported in humans (2 ST10, 2 ST23, ST354) (Leverstein-van Hall et al. 2011). 4. Discussion This is the first study comparing ESBL contamination of conventional and organic chicken retail meat samples. It was shown that all conventional retail chicken meat samples from supermarkets representative for the Netherlands were contaminated with ESBL producing E. coli. However, the majority (84%) of organic chicken meat samples was also contaminated. The high contamination rate of organic meat is surprising because of the limited use of antibiotics during the organic rearing process, which is considered the most important reason for the presence of ESBL positive strains in chickens. A possible explanation is the introduction of ESBL colonised one-dayold broilers into the organic farms, as it was reported that ESBLs have been found in all levels of the broiler production chain in the Netherlands (Anonymous, 2011), i.e. also in the parent and grand-parent animals. Alternatively, cross contamination may occur between conventional and organic flocks during the rearing process or in the slaughter plants, as has been reported for Salmonella (Rasschaert et al. 2007), or at the retail level. Finally, contamination via ESBL contaminated environment (soil, surface water) may play a role (Blaak et al., 2011). The finding that conventional and organic meat samples have a different distribution of ESBL genes indicates that the routes of broiler colonisation or meat contamination are not completely identical. However, because the predominant ESBL genes are the same in conventional and organic meat samples, and because the E. coli strain types are largely similar, this also indicates a common source. In accordance with the study hypothesis, the median bacterial load per contaminated sample was higher on conventional poultry meat than on organic meat. The lower co-resistance rate to the tetracyclines of the ESBL E. coli isolated from organic meat, an antibiotic group frequently used in Dutch poultry farming (Anonymous, 2011), supports
214
J. Cohen Stuart et al. / International Journal of Food Microbiology 154 (2012) 212–214
the statement of lower antibiotic use in organic farms. A longer antibiotic abstinence interval before slaughter may also explain this load difference (>96 h in organic production versus >48 h in conventional production), allowing susceptible E. coli to out-compete the ESBL producers. The relevance of the different load of ESBL positive E. coli in both types of meat for the probability of food borne transmission of ESBL genes to humans is unknown. For food borne transmissions of related micro-organisms, minimum infectious doses have been reported between b10–100 CFU (enterohemorrhagic E. coli and Shigella) and 10 5–10 8 CFU (Salmonella, Enterotoxigenic E. coli (ETEC) (Todd et al. 2008), but the minimum dose of ingested ESBL producing organisms required for ESBL transmission from food to humans is still unknown. Funding Part of study has been funded by an unrestricted grant from Stichting Wakker Dier. Transparency declarations None to declare. References Anonymous, 2011. MARAN-2009-Monitoring of Antimicrobial Resistance and Antibiotic usage in animals in the Netherlands in 2009. http://edepot.wur.nl/165958. Blaak, H., Schets, F.M., Italiaander, R., Schmitt, H., de Roda Husman, A.M., 2011. Antibiotic resistant bacteria in surface water in an area with a high density of animal farms in the Netherlands. http://www.rivm.nl/bibliotheek/rapporten/703719031.pdf.
Dhanji, H., Murphy, N.M., Doumith, M., Durmus, S., Lee, S.S., Hope, R., Woodford, N., Livermore, D.M., 2010. Cephalosporin resistance mechanisms in Escherichia coli isolated from raw chicken imported into the UK. Journal of Antimicrobial Chemotherapy 65, 2534–2537. Doi, Y., Paterson, D.L., Egea, P., Pascual, A., Lopez-Cerero, L., Navarro, M.D., ms-Haduch, J.M., Qureshi, Z.A., Sidjabat, H.E., Rodriguez-Bano, J., 2010. Extended-spectrum and CMY-type beta-lactamase-producing Escherichia coli in clinical samples and retail meat from Pittsburgh, USA and Seville, Spain. Clinical Microbiology and Infection 16, 33–38. Overdevest, I., Willemsen, I., Rijnsburger, M., Eustace, A., Xu, L., Hawkey, P., Heck, M., Savelkoul, P., Vandenbroucke-Grauls, C., Van der Zwaluw, K., Huijsdens, X., Kluytmans, J., 2011. Extended-Spectrum Beta-Lactamase Genes of Escherichia coli in Chicken Meat and Humans, the Netherlands. Emerging Infectious Diseases 17, 1216–1222. Lavilla, S., Gonzalez-Lopez, J.J., Miro, E., Dominguez, A., Llagostera, M., Bartolome, R.M., Mirelis, B., Navarro, F., Prats, G., 2008. Dissemination of extended-spectrum betalactamase-producing bacteria: the food-borne outbreak lesson. Journal of Antimicrobial Chemotherapy 61, 1244–1251. Leverstein-van Hall, M.A., Dierikx, C.M., Cohen Stuart, J., Voets, G.M., van den Munckhof, M.P., van Essen-Zandbergen, A., Platteel, T., Fluit, A.C., van de Sande-Bruinsma, N., Scharinga, J., Bonten, M.J., Mevius, D.J., 2011. Dutch patients, retail chicken meat and poultry share the same ESBL genes, plasmids and strains. Clinical Microbiology and Infection 17, 873–880. Randall, L.P., Clouting, C., Horton, R.A., Coldham, N.G., Wu, G., Clifton-Hadley, F.A., Davies, R.H., Teale, C.J., 2011. Prevalence of Escherichia coli carrying extendedspectrum beta-lactamases (CTX-M and TEM-52) from broiler chickens and turkeys in Great Britain between 2006 and 2009. Journal of Antimicrobial Chemotherapy 66, 86–95. Rasschaert, G., Houf, K., De, Z.L., 2007. Impact of the slaughter line contamination on the presence of Salmonella on broiler carcasses. Journal of Applied Microbiology 103, 333–341. Simner, P.J., Zhanel, G.G., Pitout, J., Tailor, F., McCracken, M., Mulvey, M.R., LagaceWiens, P.R., Adam, H.J., Hoban, D.J., 2011. Prevalence and characterization of extended-spectrum beta-lactamase- and AmpC beta-lactamase-producing Escherichia coli: results of the CANWARD 2007–2009 study. Diagnostic Microbiology and Infectious Disease 69, 326–334. Todd, E.C., Greig, J.D., Bartleson, C.A., Michaels, B.S., 2008. Outbreaks where food workers have been implicated in the spread of foodborne disease. Part 4. Infective doses and pathogen carriage. Journal of Food Protection 71, 2339–2373.
Contents lists available at SciVerse ScienceDirect
International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro
Short Communication
Comparison of ESBL contamination in organic and conventional retail chicken meat James Cohen Stuart a,⁎, Thijs van den Munckhof a, Guido Voets a, Jelle Scharringa a, Ad Fluit a, Maurine Leverstein-Van Hall a, b a b
Department of Medical Microbiology, University Medical Center Utrecht, The Netherlands Centre for Infectious Disease Control, National Institute for Public Health and the Environment (RIVM), Bilthoven, The Netherlands
a r t i c l e
i n f o
Article history: Received 14 September 2011 Received in revised form 7 November 2011 Accepted 26 December 2011 Available online 3 January 2012 Keywords: ESBL Meat Organic
a b s t r a c t Contamination of retail chicken meat by Extended Spectrum Beta-Lactamase (ESBL) producing bacteria likely contributes to the increasing incidence of infections with these bacteria in humans. This study aimed to compare the prevalence and load of ESBL positive isolates between organic and conventional retail chicken meat samples, and to compare the distribution of ESBL genes, strain genotypes and co-resistance. In 2010, 98 raw chicken breasts (n = 60 conventional; n = 38 organic) were collected from 12 local stores in the Netherlands. Prevalence of ESBL producing micro-organisms was 100% on conventional and 84% on organic samples (p b 0.001). Median loads of ESBL producing micro-organisms were 80 (range b 20–1360) in conventional, and b 20 (range 0–260) CFU/25 g in organic samples (p = 0.001). The distribution of ESBL genes in conventional samples and organic samples was 42% versus 56%, respectively (N.S.), for CTX-M-1, 20% versus 42% (N.S.) for TEM-52, and 23% versus 3% (p b 0.001) for SHV-12. CTX-M-2 (7%), SHV-2 (5%) and TEM-20 (3%) were exclusively found in conventional samples. Co-resistance rates of ESBL positive isolates were not different between conventional and organic samples (co-trimoxazole 56%, ciprofloxacin 14%, and tobramycin 2%), except for tetracycline, 73% and 46%, respectively, p b 0.001). Six of 14 conventional meat samples harbored 4 MLST types also reported in humans and 5 of 10 organic samples harbored 3 MLST types also reported in humans (2 ST10, 2 ST23, ST354). In conclusion, the majority of organic chicken meat samples were also contaminated with ESBL producing E. coli, and the ESBL genes and strain types were largely the same as in conventional meat samples. © 2012 Elsevier B.V. All rights reserved.
1. Introduction
2. Methods
Contamination of retail chicken meat by Enterobacteriaceae producing Extended Spectrum Beta-Lactamases (ESBLs) has been reported in several countries (Dhanji et al. 2010; Doi et al. 2010; Lavilla et al. 2008; Leverstein-van Hall et al. 2011; Randall et al. 2011), and likely contributes to the increased incidence of infections with these bacteria in humans (Leverstein-van Hall et al. 2011; Overdevest et al., 2011). We hypothesized that the ESBL prevalence and the load of ESBL producing micro-organisms is lower in organic meat, because antibiotic use is lower in organic chicken farms than in conventional farms. The aims of this study were to compare the prevalence and load of ESBL positive isolates between organic and conventional retail chicken meat samples, and to compare the distribution of ESBL genes, strain genotypes and degree of co-resistance.
In 2010, 98 raw chicken breasts were collected from 12 stores in Utrecht, a city in the centre of the Netherlands. Eighty percent of the meat samples were collected from 9 stores from 5 supermarket chains with a combined Dutch market share of approximately 90%, and 20% of the meat samples were collected from organic butcheries. Sixty meat samples were sold as “conventional” and 38 meat samples as organic (30 “EKO”, 8 “free ranging”). The antibiotic policies for these 3 different categories of poultry rearing are as follows: 1) Conventional rearing: 37 d.d.d. per year per animal, i.e. 4 treatment days per chicken per 42 day cycle (95% CI 3–6), with preventive and therapeutic use of antibiotics (Anonymous, 2011). 2) Organic (Type “EKO”): Antibiotic use limited to one therapeutic course of antibiotics and is controlled by SKAL (Stichting EKO keurmerk controle), the inspection and certification body for organic production in the Netherlands.3) Organic (Type “Free Ranging”): use of antibiotics not specified. Information on the country of origin was available on the package of only 30 supermarket conventional meat samples (27% indicated as from the Netherlands, 73 from the Benelux) and for none of the samples collected from the organic butcheries. Phenotypic detection of ESBL producing micro-organisms on the meat samples was performed by homogenizing the meat, followed
⁎ Corresponding author at: Department of Medical Microbiology, G04-614, University Medical Centre Utrecht, Heidelberglaan 100, 3584CX, Utrecht, The Netherlands. Tel.: + 31 6 21277988; fax: + 31 30 2541770. E-mail address: [email protected] (J. Cohen Stuart). 0168-1605/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2011.12.034
by culturing the homogenate on an agar plate, which is selective for ESBL producing gram negative micro-organisms. Per meat sample, 25 g was homogenized with 225 ml peptone water in a stomacher. For quantitative cultures, an ESBL selective plate (ESBL Brilliance, Thermofisher, Basingstoke, UK) was inoculated with 0.1 ml of a 1:1000 dilution of the homogenate. The lower limit of quantification was 20 colony forming units (CFU) per 25 g of meat. For qualitative culturing, an overnight pre-enrichment was performed with 225 ml of the homogenate, followed by inoculation of an ESBL selective plate with 0.1 ml of the pre-enriched homogenate. In case of growth on the ESBL selective plate, species identification of each morphologically different colony on the ESBL selective plate was performed by MALDI-TOF (Bruker Daltonics, Germany). Phenotypic ESBL production was determined using ESBL Etests on all (n = 163) isolates (Biomerieux, Marcy L'etoile, Lyon, France) growing on the selective plates. ESBL gene detection by PCR and sequencing was performed on a random 50% of those isolates (n = 81). Minimum inhibitory concentrations (MICs) of antibiotics were determined by broth microdilution (Merlin, Germany) using EUCAST breakpoints (www. eucast.org). Strain typing was performed on isolates from a random 24 meat samples (14 conventional, 10 organic) using multi locus sequence typing (MLST) (www.mlst.net). Comparisons of frequencies were performed using the Chi-square test (SPSS for windows 15.0, IBM Inc., Armonk, NY, USA)
3. Results In total, 163 isolates were isolated from 98 chicken meat samples. From these, 92 (94%) harbored at least one E. coli isolate with an ESBL phenotype (Fig. 1). One sample also harbored an ESBL producing Escherichia fergusonii, and another sample also an ESBL producing Klebsiella pneumoniae. The average number of (morphologically different) E. coli isolates per meat sample was 2; (range 1–4 isolates). The prevalence of ESBL producing micro-organisms was 100% (95% confidence interval 94–100%) on conventional samples, versus 84% (95% CI 70–93%%) on organic samples (p b 0.001; Fig. 1). The ESBL prevalence on the organic “EKO” type samples was 90% (95% CI 74– 97%) and organic “free ranging” type was 63% (95CI 31–86%), but this difference was not statistically significant (p = 0.09). To be able to detect ESBL producing micro-organisms, the pre-enrichment step was required in 47% of the organic meat samples versus 29% of the conventional samples (p b 0.001). Median loads of ESBL producing micro-organisms were 80 CFU/25 g (range b20–1360) in
Fig. 1. Percentage of ESBL positive meat samples per rearing method.*The difference of ESBL prevalence between the “EKO” and “Free ranging” samples was not statistically significant.
% in cfu-category
J. Cohen Stuart et al. / International Journal of Food Microbiology 154 (2012) 212–214 50% 45% 40% 35% 30% 25% 20% 15% 10% 5% 0%
213
47%
conventional organic
40%
29% 21% 16% 13%
11% 8%
8% 5% 2%
0
1-19*
20-49
50-99
100-499
500-999
>=1000
cfu's ESBL E. coli per 25 gram Fig. 2. Distribution of load of ESBL producing micro-organisms.*ESBL only detected after pre-enrichment.
conventional meat samples versus b20 CFU/25 g (range 0–260) in organic samples (p = 0.001) (Fig. 2). Sequencing of the ESBL genes showed that the distribution of ESBL genes in conventional samples and organic samples was 42% versus 56% for CTX-M-1 (N.S.), 20% versus 42% for TEM-52 (N.S.), and 23% versus 3% for SHV-12 (p b 0.001), respectively. The ESBL genes CTXM-2 (7%), SHV-2 (5%) and TEM-20 (3%) were exclusively found in conventional samples. In 12 (12%) samples (7 organic, 5 conventional) CMY-2 was detected, an ampC gene previously reported in isolates from humans (Simner et al. 2011). Co-resistance rates of ESBL positive isolates were: co-trimoxazole 56%, ciprofloxacin 14%, and tobramycin 2% (no significant differences between organic and conventional isolates). However, tetracycline co-resistance was more prevalent in conventional than in organic samples (73% versus 46%, p b 0,001). Six of the 14 conventional meat samples harbored 4 MLST types also reported in humans ( 2 ST10, 2 ST23, ST57, ST117) and 5 of 10 organic samples harbored 3 MLST types also reported in humans (2 ST10, 2 ST23, ST354) (Leverstein-van Hall et al. 2011). 4. Discussion This is the first study comparing ESBL contamination of conventional and organic chicken retail meat samples. It was shown that all conventional retail chicken meat samples from supermarkets representative for the Netherlands were contaminated with ESBL producing E. coli. However, the majority (84%) of organic chicken meat samples was also contaminated. The high contamination rate of organic meat is surprising because of the limited use of antibiotics during the organic rearing process, which is considered the most important reason for the presence of ESBL positive strains in chickens. A possible explanation is the introduction of ESBL colonised one-dayold broilers into the organic farms, as it was reported that ESBLs have been found in all levels of the broiler production chain in the Netherlands (Anonymous, 2011), i.e. also in the parent and grand-parent animals. Alternatively, cross contamination may occur between conventional and organic flocks during the rearing process or in the slaughter plants, as has been reported for Salmonella (Rasschaert et al. 2007), or at the retail level. Finally, contamination via ESBL contaminated environment (soil, surface water) may play a role (Blaak et al., 2011). The finding that conventional and organic meat samples have a different distribution of ESBL genes indicates that the routes of broiler colonisation or meat contamination are not completely identical. However, because the predominant ESBL genes are the same in conventional and organic meat samples, and because the E. coli strain types are largely similar, this also indicates a common source. In accordance with the study hypothesis, the median bacterial load per contaminated sample was higher on conventional poultry meat than on organic meat. The lower co-resistance rate to the tetracyclines of the ESBL E. coli isolated from organic meat, an antibiotic group frequently used in Dutch poultry farming (Anonymous, 2011), supports
214
J. Cohen Stuart et al. / International Journal of Food Microbiology 154 (2012) 212–214
the statement of lower antibiotic use in organic farms. A longer antibiotic abstinence interval before slaughter may also explain this load difference (>96 h in organic production versus >48 h in conventional production), allowing susceptible E. coli to out-compete the ESBL producers. The relevance of the different load of ESBL positive E. coli in both types of meat for the probability of food borne transmission of ESBL genes to humans is unknown. For food borne transmissions of related micro-organisms, minimum infectious doses have been reported between b10–100 CFU (enterohemorrhagic E. coli and Shigella) and 10 5–10 8 CFU (Salmonella, Enterotoxigenic E. coli (ETEC) (Todd et al. 2008), but the minimum dose of ingested ESBL producing organisms required for ESBL transmission from food to humans is still unknown. Funding Part of study has been funded by an unrestricted grant from Stichting Wakker Dier. Transparency declarations None to declare. References Anonymous, 2011. MARAN-2009-Monitoring of Antimicrobial Resistance and Antibiotic usage in animals in the Netherlands in 2009. http://edepot.wur.nl/165958. Blaak, H., Schets, F.M., Italiaander, R., Schmitt, H., de Roda Husman, A.M., 2011. Antibiotic resistant bacteria in surface water in an area with a high density of animal farms in the Netherlands. http://www.rivm.nl/bibliotheek/rapporten/703719031.pdf.
Dhanji, H., Murphy, N.M., Doumith, M., Durmus, S., Lee, S.S., Hope, R., Woodford, N., Livermore, D.M., 2010. Cephalosporin resistance mechanisms in Escherichia coli isolated from raw chicken imported into the UK. Journal of Antimicrobial Chemotherapy 65, 2534–2537. Doi, Y., Paterson, D.L., Egea, P., Pascual, A., Lopez-Cerero, L., Navarro, M.D., ms-Haduch, J.M., Qureshi, Z.A., Sidjabat, H.E., Rodriguez-Bano, J., 2010. Extended-spectrum and CMY-type beta-lactamase-producing Escherichia coli in clinical samples and retail meat from Pittsburgh, USA and Seville, Spain. Clinical Microbiology and Infection 16, 33–38. Overdevest, I., Willemsen, I., Rijnsburger, M., Eustace, A., Xu, L., Hawkey, P., Heck, M., Savelkoul, P., Vandenbroucke-Grauls, C., Van der Zwaluw, K., Huijsdens, X., Kluytmans, J., 2011. Extended-Spectrum Beta-Lactamase Genes of Escherichia coli in Chicken Meat and Humans, the Netherlands. Emerging Infectious Diseases 17, 1216–1222. Lavilla, S., Gonzalez-Lopez, J.J., Miro, E., Dominguez, A., Llagostera, M., Bartolome, R.M., Mirelis, B., Navarro, F., Prats, G., 2008. Dissemination of extended-spectrum betalactamase-producing bacteria: the food-borne outbreak lesson. Journal of Antimicrobial Chemotherapy 61, 1244–1251. Leverstein-van Hall, M.A., Dierikx, C.M., Cohen Stuart, J., Voets, G.M., van den Munckhof, M.P., van Essen-Zandbergen, A., Platteel, T., Fluit, A.C., van de Sande-Bruinsma, N., Scharinga, J., Bonten, M.J., Mevius, D.J., 2011. Dutch patients, retail chicken meat and poultry share the same ESBL genes, plasmids and strains. Clinical Microbiology and Infection 17, 873–880. Randall, L.P., Clouting, C., Horton, R.A., Coldham, N.G., Wu, G., Clifton-Hadley, F.A., Davies, R.H., Teale, C.J., 2011. Prevalence of Escherichia coli carrying extendedspectrum beta-lactamases (CTX-M and TEM-52) from broiler chickens and turkeys in Great Britain between 2006 and 2009. Journal of Antimicrobial Chemotherapy 66, 86–95. Rasschaert, G., Houf, K., De, Z.L., 2007. Impact of the slaughter line contamination on the presence of Salmonella on broiler carcasses. Journal of Applied Microbiology 103, 333–341. Simner, P.J., Zhanel, G.G., Pitout, J., Tailor, F., McCracken, M., Mulvey, M.R., LagaceWiens, P.R., Adam, H.J., Hoban, D.J., 2011. Prevalence and characterization of extended-spectrum beta-lactamase- and AmpC beta-lactamase-producing Escherichia coli: results of the CANWARD 2007–2009 study. Diagnostic Microbiology and Infectious Disease 69, 326–334. Todd, E.C., Greig, J.D., Bartleson, C.A., Michaels, B.S., 2008. Outbreaks where food workers have been implicated in the spread of foodborne disease. Part 4. Infective doses and pathogen carriage. Journal of Food Protection 71, 2339–2373.