Shiga-Toxin Genes and Genetic Diversity of Escherichia coli Isolated from Pasteurized Cow Milk in Brazil Simone Aparecida Hoffmann, Gabriella Giani Pieretti, Adriana Fiorini, Eliana Val´eria Patussi, Rosilene Fressatti Cardoso, and Jane Martha Graton Mikcha
This study evaluated the genetic similarity and prevalence of the stx1, stx2, eae, and ehxA genes in Escherichia coli isolated from pasteurized cow milk. Eighty-seven E. coli isolates from pasteurized cow milk from 22 dairies located in northwestern Paran´a state, Brazil, were analyzed. Genetic similarity was evaluated using enterobacterial repetitive intergenic consensus sequence polymerase chain reaction (ERIC-PCR) and repetitive extragenic palindromic sequence PCR (REP-PCR). E. coli isolates were also analyzed by PCR to investigate the presence of the stx1, stx2, eae, and ehxA genes. ERIC-PCR and REP-PCR clustered 87 bacterial isolates in 76 and 81 genomic profiles, respectively. Both techniques revealed high genetic diversity among the E. coli isolates, confirming the possibility of their use in epidemiological studies. The stx1, stx2, eae, and ehxA virulence genes were not detected in E. coli isolates, indicating a low prevalence of Shiga toxin-producing E. coli in milk produced in the region studied.
Abstract:
Keywords: enterobacterial repetitive intergenic consensus sequence polymerase chain reaction (ERIC-PCR), PCR
Knowledge about the presence of diarrheagenic Escherichia coli in pasteurized milk is important developing and implementing control measures in milk and dairy production.
Practical Application:
Introduction According to the Food and Agriculture Organization of the United Nations, the worldwide production of milk was estimated to be 760 million tons in 2012 (FAO 2013). To date, Brazil is the 6th largest milk producer in the world. In 2011, Brazilian milk production was estimated to be 31 billion liters, with 167 liters consumed per capita (MilkPoint 2012). Shiga toxin-producing Escherichia coli (STEC) is responsible for outbreaks of foodborne diseases in different parts of the world (Farrokh and others 2012). Several foods that originate from animals and vegetables have been incriminated in outbreaks caused by STEC (CDC 2012). Most food outbreaks caused by STEC have been associated with unpasteurized milk and its derivatives, but outbreaks related to the consumption of pasteurized milk have also been reported (Farrokh and others 2012). Various studies have investigated the virulence genes of STEC in unpasteurized milk and its derivatives (Vicente and others 2005; Paneto and others 2007; Timm and others 2009; Solomakos and others 2009; Altahi and Hassan 2009; Islam and others 2010; Rahimi and others 2011; Farzan and others 2012; Rantsiou and others 2012; D’Costa and others 2013). However, few studies have MS 20131814 Submitted 12/4/2013, Accepted 3/18/2014. Authors Hoffmann, Pieretti, and Mikcha are with Graduate Program in Food Sciences, Center of Agrarian Sciences, State Univ. of Maring´a, Colombo Ave. 5790, Block J-45, Maring´a, Paran´a, 87020–900, Brazil. Author Fiorini is with Federal Univ. of Paran´a, Pioneiro Street 2153, Palotina, Paran´a, CEP 85950–970, Brazil. Authors Patussi, Cardoso and Mikcha Dept. of Clinical Analyses and Biomedicine, State Univ. of Maring´a, Colombo Ave. 5790, Block T20, Maring´a, Paran´a, 87020–900, Brazil. Direct inquiries to author Mikcha (E-mail: [email protected]).
R C 2014 Institute of Food Technologists
doi: 10.1111/1750-3841.12477 Further reproduction without permission is prohibited
investigated STEC genes in pasteurized milk and its derivatives. Carneiro and others (2006) evaluated virulence attributes in E. coli isolated from pasteurized milk commercialized in Brazil and Manna and others (2006) evaluated the presence of E. coli O157 in foods of animal origin, including milk. Studies of the epidemiology of microorganisms, have been conducted using various molecular techniques, including enterobacterial repetitive intergenic consensus sequence polymerase chain reaction (ERIC-PCR) and repetitive extragenic palindromic sequences PCR (REP-PCR; Versalovic and others 1991). Studies have employed these techniques in E. coli from foods and the environment (Ling and others 2000; Ibenyassine and others 2006; Wenz and others 2006; Bhong and others 2008Chapaval and others 2010; Rantsiou and others 2012). However, to the best of our knowledge, no studies have used ERIC-PCR or REP-PCR to genotype E. coli in pasteurized, ready-to-consume cow milk. Therefore, the objective of this study was to evaluate the genetic similarity by ERIC and REP-PCR and identify the stx1, stx2, eae, and ehxA genes in E. coli isolated from pasteurized cow milk.
Methods Bacterial isolates Eighty-seven E. coli strains were isolated from pasteurized cow milk obtained from 22 dairies located in northeastern Parana state, Brazil, in a previous study (Zanella and others 2010). The bacterial isolates were kept at −20 ˚C in Brain and Heart Infusion (BHI; Difco, Becton Dickinson, Sparks, Md., U.S.A.) with glycerol 20% (w/v) in the Laboratory of Food Microbiology, Dept. of Clinical Analyses and Biomedicine, State Univ. of Maringa. Vol. 79, Nr. 6, 2014 r Journal of Food Science M1175
M: Food Microbiology & Safety
(polymerase chain reaction), repetitive extragenic palindromic sequence polymerase chain reaction (REP-PCR), Shiga toxin-producing Escherichia coli (STEC), virulence genes
Genotyping and virulence of E. coli . . .
M: Food Microbiology & Safety
DNA extraction Results and Discussion The genomic DNA of the isolates was extracted according to Swanenburg and others (1998). DNA quantification was per- ERIC-PCR and REP-PCR ERIC-PCR and REP-PCR applied to E. coli isolates provided formed using NanoDrop 2000 and the DNA concentration stana pattern of 3 to 20 bands with a size of approximately 100 to dardized to 100 ng/μL. 2000 bp. Considering a level of similarity of 98%, ERIC-PCR clustered ERIC-PCR and REP-PCR 87 bacterial isolates in 76 distinct genomic profiles. Sixty-six bacPCR reaction was performed by adding 1 μL of bacterial DNA at 100 ng/μL to a mixture of reagents that contained 11 μL terial isolates showed exclusive profiles, 18 isolates were included R (Fermentas GmbH, Germany), 1 μM of each in 9 genomic profiles that comprised 2 isolates each, and one Master Mix primer (ERIC1R and ERIC2; REP-IRDT and REP2-DT; Ver- profile comprised 3 isolates (Figure 1). Genomic profiles 3 and 5 salovic and others 1991), and purified DNase/RNase-free water comprised 2 E. coli isolates in each profile (5L10 and 7L10; 11L3 (Fermentas GmbH) to a final volume of 25 μL according to the and 12L3) from 2 milk samples processed in 2 dairies. Genomic manufacturer’s instructions. The amplification was performed in profile 7 comprised 3 isolates (5L9, 6L9, and 7L9) from a single an Eppendorf Thermocycler (Mastercycler gradient PCR, Ham- milk sample. Genomic profiles 1, 2, 4, 6, 8, 9, and 10 comprised burg, Germany). For ERIC-PCR, the following program was 14 E. coli isolates, 2 in each profile (3L3 and 4L3; 5L14 and 9L14; used: initial denaturing at 94 ˚C for 7 min, followed by 35 cy- 6L3 and 7L3; 3L16 and 4L16; 1L1 and 2L1; 4L8 and 6L8; 8L8 cles at 94 ˚C for 30 s, 52 ˚C for 1 min, and 72 ˚C for 8 min, and 11L8) from 13 milk samples processed in 5 dairies on different with a final extension at 72 ˚C for 16 min. For REP-PCR, the dates (Figure 1). Considering the 98% level of similarity in REP-PCR, the 87 following program was used: denaturing at 94 ˚C for 7 min, followed by 35 cycles at 94 ˚C for 30 s, 45 ˚C for 1 min, and 65 ˚C for E. coli isolates were grouped into 81 different genomic profiles. 8 min, with a final extension at 65 ˚C for 16 min. The amplicons Seventy-five isolates showed unique profiles, and 12 were grouped were analyzed by electrophoresis in 1.5% agarose gel (Amersham into 6 profiles with 2 isolates each. The E. coli isolates grouped in Pharmacia Biotech AB, Uppsala, Sweden) with 0.5 μg/mL ethid- genomic profiles 1 to 6 (1L7 and 2L7; 2L10 and 3L10; 9L3 and ium bromide at 7 to 10 V/cm2 for 2 h and visualized under 10L3; 2L19 and 3L19; 10L14 and 11L14; 6L9 and 7L9) were from ultraviolet transillumination. A 100-bp molecular weight marker 6 milk samples processed in 6 dairies (Figure 2). The molecular typing of different bacterial isolates from the was used (Ladder, Amersham Pharmacia Biotech, U.S.A.). The band-profile analysis was performed using BioNumerics software same milk sample was performed because a previous study in our (version 6.5, Applied Maths, Sint-Martens-Latem, Belgium) using laboratory demonstrated that these isolates showed different bioDice’s coefficient of similarity, and the phylogenetic distance was chemical profiles and antimicrobial resistance (Zanella and others determined based on the grouping coefficient of the unweighted 2010). In the combined ERIC-PCR and REP-PCR analyses, of the pair-group method, arithmetic averages algorithm. Isolates 87 E. coli isolates, 77 showed unique profiles, and 4 were grouped with ࣙ98% similarity were considered closely related. into 2 profiles with 2 isolates each (10L14 and 11L14; 6L9 and 7L9). Isolates 6L9 and 7L9 were considered to be identical by Discriminatory power of the methods The discriminatory power (D) of ERIC-PCR and REP-PCR ERIC-PCR and REP-PCR. Importantly, REP-PCR grouped only bacterial isolates from was calculated based on Simpson’s diversity index (Hunter and the same milk sample, whereas in ERIC-PCR, 14 isolates from Gaston 1988). different milk samples were considered identical. Neither of these Detection of virulence factors by PCR techniques grouped bacterial isolates from processed milk from The presence of genes that encode Shiga toxins 1 and 2 (stx1 different dairies. The ERIC-PCR profiles that grouped E. coli and stx2), intimine (eae), and enterohemolysin (ehxA) was detected isolates from milk samples processed on different dates indicated using the primers (Invitrogen, S˜ao Paulo, SP, Brazil) and condi- that milk contamination occurred by the same clone in the same tions previously described by Blanco and others (2004) and Paton dairy. This could be explained by the persistence of the source of and Paton (1998). The amplification reaction was performed by contamination for a long time or by a genotype from raw milk adding 1 μL of genomic DNA at 100 ng/μL to a mixture of repeatedly entering the dairy. These results indicate a failure in reagents that contained 11 μL Master Mix (Fermentas GmbH), processing and the need for control measures to minimize E. coli 1 μM of each primer, and DNase/RNase-free water (Fermentas contamination. GmbH) to a final volume of 25 μL according to the manufacERIC-PCR presented a discriminatory power (D = 0.9967) turer’s instructions. Amplification was performed in an Eppendorf that was slightly inferior to REP-PCR (D = 0.9983). The disThermocycler (Mastercycler gradient PCR), with the following criminatory power was 0.9994 when the ERIC-PCR and REPamplification conditions for the stx1, stx2, and ehxA genes: 95 ˚C PCR analyses were combined. According to Hunter and Gaston for 8 min, 35 cycles at 95 ˚C for 30 s, pairing temperature ac- (1988), an index greater than 0.90 is desirable for the genotyping cording to Blanco and others (2004) and Paton and Paton (1998) results to be interpreted with confidence. for 30 s, and 72 ˚C for 30 s, with a final extension at 72 ˚C for 1 The results showed high genetic diversity among the E. coli isomin. For the eae gene, the conditions were 94 ˚C for 2 min, 35 lates. Similar results have been reported. Wenz and others (2006) cycles at 94 ˚C for 1 min, 55 ˚C for 1 min, and 72 ˚C for 1 min, observed a high genotypic variability using ERIC-PCR to genowith a final extension at 72 ˚C for 1 min. After amplification, type E. coli from milk of cows with mastitis. Ling and others (2000) the PCR products were subjected to electrophoresis separation in also found high genetic heterogeneity in E. coli O157:H7 isolated agarose gel as previously described. E. coli O157:H7 (C7–88) was from chicken and cow hamburger using ERIC-PCR, in which used as a control for the stx1 gene, and E. coli O157:H7 (ATCC each isolate presented a unique genetic profile. E. coli isolates from 43889) was used as a control for the stx2, eae, and ehxA genes. mutton meat also showed high genetic diversity when typed using M1176 Journal of Food Science r Vol. 79, Nr. 6, 2014
Genotyping and virulence of E. coli . . .
M: Food Microbiology & Safety
Figure 1–Dendrogram of similarity of the ERIC-PCR electrophoretic profiles among E. coli isolated from pasteurized cow milk.
Vol. 79, Nr. 6, 2014 r Journal of Food Science M1177
Genotyping and virulence of E. coli . . . Figure 2–Dendrogram of similarity of the REP-PCR electrophoretic profiles among E. coli isolated from pasteurized cow milk.
M: Food Microbiology & Safety M1178 Journal of Food Science r Vol. 79, Nr. 6, 2014
Genotyping and virulence of E. coli . . .
Identification of virulence genes In this study, the stx1, stx2, eae, and ehxA virulence genes were not detected in the E. coli isolates. Similar results were obtained by Carneiro and others (2006), who reported that the stx1, stx2, and eae genes were not detected in E. coli isolates from pasteurized cow milk sold in Rio de Janeiro, Brazil. However, Manna and others (2006) reported the presence of the stx2 gene in E. coli O157 isolates from pasteurized milk in India. Most studies have reported the presence of STEC only in raw milk and its derivatives in different parts of the world. Farzan and others (2012) detected the stx1 and stx2 genes in 16 of 44 isolates (36.36%) and 35 of 44 isolates (79.54%), respectively. However, the eae gene was not detected, and the hlyA gene was present in only 5 of 44 (11.36%) of the E. coli isolates from raw cow milk. Altalhi and Hassan (2009) detected the presence of the eae gene in 9.1% of E. coli isolates from raw cow milk and the stx1 and stx2 genes in 3% and 6.1% of the isolates, respectively. Solomakos and others (2009) found that 33% of the E. coli isolates from raw cow milk had the stx1 or stx2, eae, and ehxA gene, belonging to the O157:H7 serotype. Costa and others (2013) showed that 14.29% of E. coli isolates from cow milk samples carried the stx1 gene alone, 3.89% possessed the stx2 alone and 14.29% carried both the stx1 and stx2 genes. Islam and others (2010) studied the prevalence of STEC in various foods, including raw milk, in which 10% of the samples had the stx gene but not the eae or ehxA gene. In Brazil, Vicente and others (2005) detected the stx1 and stx2 genes in 3.3% of E. coli isolates from milk samples collected during the milking process in S˜ao Paulo. However, similar to this study, the stx1, stx2, and eae genes were not found in samples of raw milk from southern Brazil (Timm and others 2009). Cattle are considered the main source of STEC (Farrokh and others 2012). In Brazil, some studies have reported a high prevalence of this bacteria in beef and dairy cattle (Irino and others 2005; Vicente and others 2005; Oliveira and others 2008). However, consistent with this study, other studies reported a low prevalence of STEC in milk, meat, and their derivatives (Vicente and others 2005; Carneiro and others 2006; Bergamini and others 2007; Paneto and others 2007; Timm and others 2009; Martins and others 2011). Despite the fact that we did not detect STEC genes, the presence of E. coli in pasteurized milk indicates and inadequate sanitary hygiene practices and does not exclude the possibility that other
pathotypes of E. coli are present that can present a health risk to consumers.
Conclusion ERIC-PCR and REP-PCR revealed high genetic diversity of E. coli isolates from bovine pasteurized milk, confirming the possibility of their use in epidemiological studies. The bacterial isolates did not have the stx1, stx2, eae, or ehxA genes that are normally present in STEC, indicating a low prevalence of this pathogen in milk produced in the region studied.
Acknowledgments This research was supported by Coordenac¸a˜ o de Aperfeic¸oamento de Pessoal de N´ıvel Superior (CAPES), Fundac¸a˜ o Arauc´aria, and the Graduate Program in Food Sciences of the State Univ. of Maring´a. Prof. Halha Ostrensky Saridakis and Prof. Jacinta Sanchez Pelayo of the Dept. of Microbiology, State Univ. of Londrina, who kindly provided E. coli isolates used as controls and Pedro Henrique Canezin for his help improving the figures.
Author Contributions Simone Aparecida Hoffmann:conducted the tests, interpreted the results and wrote the manuscript. Gabriella Giani Pieretti: helped conduct the tests, interpreted the results. Adriana Fiorini: perform Bionumerics analysis, interpreted the results. Eliana Val´eria Patussi: interpreted the results and helped write the manuscript. Rosilene Fressatti Cardoso: designed the study, interpreted the results and corrected the manuscript. Jane Martha Graton Mikcha: designed the study, interpreted the results and corrected the manuscript.
References Altalhi AD, Hassan SA. 2009. Bacterial quality of raw milk investigated by Escherichia coli and isolates analysis for specific virulence-gene markers. Food Control 20:913–7. Blanco JE, Blanco M, Alonso MP, Mora A, Dahbi G, Coira MA, Blanco J. 2004. Serotypes, virulence genes, and intimin types of Shiga toxin (verotoxin)-producing Escherichia coli isolates from human patients: prevalence in Lugo, Spain, from 1992 through 1999. J Clin Microbiol 42:311–9. Bergamini AMM, Sim˜oes M, Irino K, Gomes TAT, Guth BEC. 2007. Prevalence and characteristics of shiga toxin-producing Escherichia coli (STEC) strains in ground beef in S˜ao Paulo. Braz J Microbiol 38:553–6. Bhong CD, Brahmbhatt MN, Joshi CG, Rank DN. 2008. Detection of virulence determinants by real time PCR in E. coli isolated from mutton. Meat Sci 80:1129–31. Carneiro LAM, Lins MC, Garcia FRA, Silva APS, Mauller PM, Alves GB, Rosa ACP, Andrade JRC, Freitas-Ameida AC, Queiroz MLP. 2006. Phenotypic and genotypic characterization of Escherichia coli strains serogrouped as enteropathogenic E. coli (EPEC) isolated from pasteurized milk. Int J Food Microbiol 108:15–21. CDC (Centers for Disease Control and Prevention). 2012. Escherichia coli O157:H7 and other Shiga toxin-producing Escherichia coli (STEC). Available from: http://www.cdc.gov/ nczved/divisions/dfbmd/diseases/ecoli_o157h7/. Accessed 2012 February 12. Chapaval L, Olivindo CS, Souza FGC, Alves FSF, Frota IMA. 2010. Detecc¸a˜ o de Escherichia coli e Pseudomonas aeruginosa pela t´ecnica de REP-PCR no monitoramento da qualidade do leite de cabra em sala de ordenha. Comunicata Scientiae 1:49–56. D’Costa D, Bhosle SN, Dhuri RB, Doijad SP, Poharkar KV, Kalorey DR, Barbuddhe SB. 2013. Prevalence, serogroups, Shiga-toxin genes and Pulsed Field Gel Electrophoresis analyses of Escherichia coli isolated from bovine milk. Proc Natl Acad Sci, India, Sect B Biol Sci 83:423–29. FAO (Food and Agriculture Organization). 2013. Food outlook, global market analysis. Available from: http://www.fao.org/giews/english/fo/index.htm. Accessed 2012 March 20. Farrokh C, Jordan K, Auvray F, Glass K, Oppegaard H, Raynaud S, Thevenot D, Condron R, De Reu K, Alexander Govaris, Heggum K, Heyndrickx M, Hummerjohann J, Lindsay D, Miszczycha S, Moussiegt S, Verstraete K, Cerf O. 2012. Review of Shiga-toxin-producing Escherichia coli (STEC) and their significance in dairy production. Int J Food Microbiol 1–23. Farzan R, Rahimi E, Momtaz H. 2012. Virulence properties of shiga toxin-producing Escherichia coli isolated from iranian raw milk and dairy products. Slov Vet Res 49:159–66. Giammanco GM, Pignato S, Grimont F, Grimont PAD, Caprioli A, Morabito S, Giammanco G. 2002. Characterization of Shiga Toxin-Producing Escherichia coli O157:H7 Isolated in Italy and in France. J Clin Microbiol 40:4619–24. Hunter PR, Gaston MA. 1988. Numerical index of the discriminatory ability of typing systems: an application of Simpson’s index of diversity. J Clin Microbiol 26:2465–6. Ibenyassine K, AitMhand R, Karamoko Y, Cohen N. 2006. Use of repetitive DNA sequences to determine the persistence of enteropathogenic Escherichia coli in vegetables and in soil grown in fields treated with contaminated irrigation water. Appl Microbiol 43:528–33.
Vol. 79, Nr. 6, 2014 r Journal of Food Science M1179
M: Food Microbiology & Safety
ERIC-PCR and REP-PCR (Bhong and others 2008). REPPCR also efficiently compared E. coli isolates from goat milk and the milking environment, which contributed to the monitoring of the quality of goat milk (Chapaval and others 2010). In a study similar to the present one, Solomakos and others (2009)evaluated the genetic diversity of STEC obtained from unpasteurized dairy derivatives using ERIC-PCR. Considering a 70% coefficient of similarity, the authors obtained low discriminatory power. However, considering the same coefficient of similarity utilized in this study (98%), they observed high diversity among the isolates studied. The authors also found a correlation between the genomic groups and virulence genes. In contrast to the present results, ERIC-PCR did not successfully differentiate E. coli O157:H7 isolated from food, humans, and animals in a study by Giammanco and others (2002). The high genetic diversity observed in this study can be justified by several sources of contamination of cow milk by E. coli during the entire production process.
Genotyping and virulence of E. coli . . . Irino K, Kato MAMF, Vaz TMI, Ramos II, Souza MAC, Cruz AS, Gomes TAT Gomes Vieira MAM, Guth BEC. 2005. Serotypes and virulence markers of Shiga toxin-producing Escherichia coli (STEC) isolated from dairy cattle in S˜ao Paulo State, Brazil. Vet Microbiol 105:29–36. Islam MA, Mondol AS, Azmi IJ, Boer E, Beumer RR, Zwietering MH, Heuvelink AE, Talukder KA. 2010. Ocurrence and characterization of Shiga Toxin-Producing Escherichia coli in raw meat, raw milk, and street vended juices in Bangladesh. Foodborne Path Dis 7: 1381–85. Ling OW, Radu S, Rusul G, Karim MI, Purwati E, Lihan S. 2000. Enterobacterial Repetitive Intragenic Consensus (ERIC) genotyping of Escherichia coli O157:H7. Pakistan J Biol Sci 3:35–7. Manna SK, Brahmane MP, Das R, Manna C, Batabyal S. 2006. Detection of Escherichia coli O157 in foods of animal origin by culture and multiplex polymerase chain reaction. J Food Sci Technol 46:77–9. Martins RP, Nakazato L, Dutra V, Leite DS. 2011. Analysis of virulence genes in Escherichia coli isolated from grated cheese. Ciˆenc Tecnol Aliment 31:106–8. MilkPoint. 2012. Consumo de l´acteos tem expans˜ao. Available from: http://www.milkpoint. com.br/cadeia-do-leite/giro-lacteo/consumo-de-lacteos-tem-expansao-78049n.aspx. Accessed 2012 February 12. Oliveira MG, Brito JRF, Gomes TAT, Guth BECG, Vieira MAM, Naves ZVF, Vaz TMI, Irino K. 2008. Diversity of virulence profiles of Shiga toxin-producing Escherichia coli serotypes in food-producing animals in Brazil. Int J Food Microbiol 127: 139–46. Paneto BR, Schocken-Iturrino RP, Macedo C, Santo E, Marin JM. 2007. Occurrence of toxigenic Escherichia coli in raw milk cheese in Brazil. Arq Bras Med Vet Zootec 59: 508–12. Paton AW, Paton JC. 1998. Detection and characterization of Shiga toxigenic Escherichia coli by using multiplex PCR assays for stx1, stx2, eaeA, Enterohemorrhagic E. coli hlyA, rfbO111, and rfbO157. J Clinical Microbiol 11:598–602
M: Food Microbiology & Safety M1180 Journal of Food Science r Vol. 79, Nr. 6, 2014
Rahimi E, Chaleshtori SS, Parsaei P. 2011. Prevalence and antimicrobial resistance of Escherichia coli O157 isolated from traditional cheese, ice cream and yoghurt in Iran. African J Microbiol 5:3706–10. Rantsiou K, Alessandria V, Cocolin L. 2012. Prevalence of Shiga toxin-producing Escherichia coli in food products of animal origin as determined by molecular methods. Int J Food Microbiol 154:37–43. Solomakos N, Govaris A, Angelidis AS, Pournaras S, Burriel AR, Kritas SK, Papageorgiou DK. 2009. Occurrence, virulence genes and antibiotic resistance of Escherichia coli O157 isolated from raw bovine, caprine and ovine milk in Greece. Food Microbiol 26: 865–71. Swanenburg M, Urlings HAP, Keuzenkamp DA, Snijders JMA. 1998. Validation of ERIC-PCR as a tool in epidemiology research of Salmonella in slaughter pigs. J Ind Microbiol Biotechn 21:141–4. Timm CD, Conceic¸a˜ o FR, Menin A, Conceic¸a˜ o RCS, Dellagostin OA, Aleixo JAG. 2009. Prevalence of Shiga toxin-producing Escherichia coli in southern Brazil isolated from ground beef and raw milk. Ciˆenc Anim Brasi 10:641–9. Versalovic J, Koeuth T, Lupski JR. 1991. Distribution of repetitive DNA sequences in eubacteria and application to fingerprinting of bacterial genomes. Nucl Acids Res 19:6823–31. Vicente HIG, Amaral LA, Cerqueira AMF. 2005. Shigatoxigenic Escherichia coli serogroups O157, O111 and O113 in feces, water and milk samples from dairy farms. Brazilian J Microbiol 36:217–22. Zanella GN, Mikcha JMG, Bando E, Siqueira VLD, Machinski Jr. M. 2010. Occurrence, antibiotic resistance of coliforms and antimicrobial residues from cow’s milk in Brazil. J Food Prot 73 1684–7. Wenz JR, Barrington GM, Garry FB, Ellis RP, Magnuson RJ. 2006. Escherichia coli isolates’ serotypes, genotypes, and virulence genes and clinical coliform mastitis severity. J Dairy Sci 89: 3408–12.
This study evaluated the genetic similarity and prevalence of the stx1, stx2, eae, and ehxA genes in Escherichia coli isolated from pasteurized cow milk. Eighty-seven E. coli isolates from pasteurized cow milk from 22 dairies located in northwestern Paran´a state, Brazil, were analyzed. Genetic similarity was evaluated using enterobacterial repetitive intergenic consensus sequence polymerase chain reaction (ERIC-PCR) and repetitive extragenic palindromic sequence PCR (REP-PCR). E. coli isolates were also analyzed by PCR to investigate the presence of the stx1, stx2, eae, and ehxA genes. ERIC-PCR and REP-PCR clustered 87 bacterial isolates in 76 and 81 genomic profiles, respectively. Both techniques revealed high genetic diversity among the E. coli isolates, confirming the possibility of their use in epidemiological studies. The stx1, stx2, eae, and ehxA virulence genes were not detected in E. coli isolates, indicating a low prevalence of Shiga toxin-producing E. coli in milk produced in the region studied.
Abstract:
Keywords: enterobacterial repetitive intergenic consensus sequence polymerase chain reaction (ERIC-PCR), PCR
Knowledge about the presence of diarrheagenic Escherichia coli in pasteurized milk is important developing and implementing control measures in milk and dairy production.
Practical Application:
Introduction According to the Food and Agriculture Organization of the United Nations, the worldwide production of milk was estimated to be 760 million tons in 2012 (FAO 2013). To date, Brazil is the 6th largest milk producer in the world. In 2011, Brazilian milk production was estimated to be 31 billion liters, with 167 liters consumed per capita (MilkPoint 2012). Shiga toxin-producing Escherichia coli (STEC) is responsible for outbreaks of foodborne diseases in different parts of the world (Farrokh and others 2012). Several foods that originate from animals and vegetables have been incriminated in outbreaks caused by STEC (CDC 2012). Most food outbreaks caused by STEC have been associated with unpasteurized milk and its derivatives, but outbreaks related to the consumption of pasteurized milk have also been reported (Farrokh and others 2012). Various studies have investigated the virulence genes of STEC in unpasteurized milk and its derivatives (Vicente and others 2005; Paneto and others 2007; Timm and others 2009; Solomakos and others 2009; Altahi and Hassan 2009; Islam and others 2010; Rahimi and others 2011; Farzan and others 2012; Rantsiou and others 2012; D’Costa and others 2013). However, few studies have MS 20131814 Submitted 12/4/2013, Accepted 3/18/2014. Authors Hoffmann, Pieretti, and Mikcha are with Graduate Program in Food Sciences, Center of Agrarian Sciences, State Univ. of Maring´a, Colombo Ave. 5790, Block J-45, Maring´a, Paran´a, 87020–900, Brazil. Author Fiorini is with Federal Univ. of Paran´a, Pioneiro Street 2153, Palotina, Paran´a, CEP 85950–970, Brazil. Authors Patussi, Cardoso and Mikcha Dept. of Clinical Analyses and Biomedicine, State Univ. of Maring´a, Colombo Ave. 5790, Block T20, Maring´a, Paran´a, 87020–900, Brazil. Direct inquiries to author Mikcha (E-mail: [email protected]).
R C 2014 Institute of Food Technologists
doi: 10.1111/1750-3841.12477 Further reproduction without permission is prohibited
investigated STEC genes in pasteurized milk and its derivatives. Carneiro and others (2006) evaluated virulence attributes in E. coli isolated from pasteurized milk commercialized in Brazil and Manna and others (2006) evaluated the presence of E. coli O157 in foods of animal origin, including milk. Studies of the epidemiology of microorganisms, have been conducted using various molecular techniques, including enterobacterial repetitive intergenic consensus sequence polymerase chain reaction (ERIC-PCR) and repetitive extragenic palindromic sequences PCR (REP-PCR; Versalovic and others 1991). Studies have employed these techniques in E. coli from foods and the environment (Ling and others 2000; Ibenyassine and others 2006; Wenz and others 2006; Bhong and others 2008Chapaval and others 2010; Rantsiou and others 2012). However, to the best of our knowledge, no studies have used ERIC-PCR or REP-PCR to genotype E. coli in pasteurized, ready-to-consume cow milk. Therefore, the objective of this study was to evaluate the genetic similarity by ERIC and REP-PCR and identify the stx1, stx2, eae, and ehxA genes in E. coli isolated from pasteurized cow milk.
Methods Bacterial isolates Eighty-seven E. coli strains were isolated from pasteurized cow milk obtained from 22 dairies located in northeastern Parana state, Brazil, in a previous study (Zanella and others 2010). The bacterial isolates were kept at −20 ˚C in Brain and Heart Infusion (BHI; Difco, Becton Dickinson, Sparks, Md., U.S.A.) with glycerol 20% (w/v) in the Laboratory of Food Microbiology, Dept. of Clinical Analyses and Biomedicine, State Univ. of Maringa. Vol. 79, Nr. 6, 2014 r Journal of Food Science M1175
M: Food Microbiology & Safety
(polymerase chain reaction), repetitive extragenic palindromic sequence polymerase chain reaction (REP-PCR), Shiga toxin-producing Escherichia coli (STEC), virulence genes
Genotyping and virulence of E. coli . . .
M: Food Microbiology & Safety
DNA extraction Results and Discussion The genomic DNA of the isolates was extracted according to Swanenburg and others (1998). DNA quantification was per- ERIC-PCR and REP-PCR ERIC-PCR and REP-PCR applied to E. coli isolates provided formed using NanoDrop 2000 and the DNA concentration stana pattern of 3 to 20 bands with a size of approximately 100 to dardized to 100 ng/μL. 2000 bp. Considering a level of similarity of 98%, ERIC-PCR clustered ERIC-PCR and REP-PCR 87 bacterial isolates in 76 distinct genomic profiles. Sixty-six bacPCR reaction was performed by adding 1 μL of bacterial DNA at 100 ng/μL to a mixture of reagents that contained 11 μL terial isolates showed exclusive profiles, 18 isolates were included R (Fermentas GmbH, Germany), 1 μM of each in 9 genomic profiles that comprised 2 isolates each, and one Master Mix primer (ERIC1R and ERIC2; REP-IRDT and REP2-DT; Ver- profile comprised 3 isolates (Figure 1). Genomic profiles 3 and 5 salovic and others 1991), and purified DNase/RNase-free water comprised 2 E. coli isolates in each profile (5L10 and 7L10; 11L3 (Fermentas GmbH) to a final volume of 25 μL according to the and 12L3) from 2 milk samples processed in 2 dairies. Genomic manufacturer’s instructions. The amplification was performed in profile 7 comprised 3 isolates (5L9, 6L9, and 7L9) from a single an Eppendorf Thermocycler (Mastercycler gradient PCR, Ham- milk sample. Genomic profiles 1, 2, 4, 6, 8, 9, and 10 comprised burg, Germany). For ERIC-PCR, the following program was 14 E. coli isolates, 2 in each profile (3L3 and 4L3; 5L14 and 9L14; used: initial denaturing at 94 ˚C for 7 min, followed by 35 cy- 6L3 and 7L3; 3L16 and 4L16; 1L1 and 2L1; 4L8 and 6L8; 8L8 cles at 94 ˚C for 30 s, 52 ˚C for 1 min, and 72 ˚C for 8 min, and 11L8) from 13 milk samples processed in 5 dairies on different with a final extension at 72 ˚C for 16 min. For REP-PCR, the dates (Figure 1). Considering the 98% level of similarity in REP-PCR, the 87 following program was used: denaturing at 94 ˚C for 7 min, followed by 35 cycles at 94 ˚C for 30 s, 45 ˚C for 1 min, and 65 ˚C for E. coli isolates were grouped into 81 different genomic profiles. 8 min, with a final extension at 65 ˚C for 16 min. The amplicons Seventy-five isolates showed unique profiles, and 12 were grouped were analyzed by electrophoresis in 1.5% agarose gel (Amersham into 6 profiles with 2 isolates each. The E. coli isolates grouped in Pharmacia Biotech AB, Uppsala, Sweden) with 0.5 μg/mL ethid- genomic profiles 1 to 6 (1L7 and 2L7; 2L10 and 3L10; 9L3 and ium bromide at 7 to 10 V/cm2 for 2 h and visualized under 10L3; 2L19 and 3L19; 10L14 and 11L14; 6L9 and 7L9) were from ultraviolet transillumination. A 100-bp molecular weight marker 6 milk samples processed in 6 dairies (Figure 2). The molecular typing of different bacterial isolates from the was used (Ladder, Amersham Pharmacia Biotech, U.S.A.). The band-profile analysis was performed using BioNumerics software same milk sample was performed because a previous study in our (version 6.5, Applied Maths, Sint-Martens-Latem, Belgium) using laboratory demonstrated that these isolates showed different bioDice’s coefficient of similarity, and the phylogenetic distance was chemical profiles and antimicrobial resistance (Zanella and others determined based on the grouping coefficient of the unweighted 2010). In the combined ERIC-PCR and REP-PCR analyses, of the pair-group method, arithmetic averages algorithm. Isolates 87 E. coli isolates, 77 showed unique profiles, and 4 were grouped with ࣙ98% similarity were considered closely related. into 2 profiles with 2 isolates each (10L14 and 11L14; 6L9 and 7L9). Isolates 6L9 and 7L9 were considered to be identical by Discriminatory power of the methods The discriminatory power (D) of ERIC-PCR and REP-PCR ERIC-PCR and REP-PCR. Importantly, REP-PCR grouped only bacterial isolates from was calculated based on Simpson’s diversity index (Hunter and the same milk sample, whereas in ERIC-PCR, 14 isolates from Gaston 1988). different milk samples were considered identical. Neither of these Detection of virulence factors by PCR techniques grouped bacterial isolates from processed milk from The presence of genes that encode Shiga toxins 1 and 2 (stx1 different dairies. The ERIC-PCR profiles that grouped E. coli and stx2), intimine (eae), and enterohemolysin (ehxA) was detected isolates from milk samples processed on different dates indicated using the primers (Invitrogen, S˜ao Paulo, SP, Brazil) and condi- that milk contamination occurred by the same clone in the same tions previously described by Blanco and others (2004) and Paton dairy. This could be explained by the persistence of the source of and Paton (1998). The amplification reaction was performed by contamination for a long time or by a genotype from raw milk adding 1 μL of genomic DNA at 100 ng/μL to a mixture of repeatedly entering the dairy. These results indicate a failure in reagents that contained 11 μL Master Mix (Fermentas GmbH), processing and the need for control measures to minimize E. coli 1 μM of each primer, and DNase/RNase-free water (Fermentas contamination. GmbH) to a final volume of 25 μL according to the manufacERIC-PCR presented a discriminatory power (D = 0.9967) turer’s instructions. Amplification was performed in an Eppendorf that was slightly inferior to REP-PCR (D = 0.9983). The disThermocycler (Mastercycler gradient PCR), with the following criminatory power was 0.9994 when the ERIC-PCR and REPamplification conditions for the stx1, stx2, and ehxA genes: 95 ˚C PCR analyses were combined. According to Hunter and Gaston for 8 min, 35 cycles at 95 ˚C for 30 s, pairing temperature ac- (1988), an index greater than 0.90 is desirable for the genotyping cording to Blanco and others (2004) and Paton and Paton (1998) results to be interpreted with confidence. for 30 s, and 72 ˚C for 30 s, with a final extension at 72 ˚C for 1 The results showed high genetic diversity among the E. coli isomin. For the eae gene, the conditions were 94 ˚C for 2 min, 35 lates. Similar results have been reported. Wenz and others (2006) cycles at 94 ˚C for 1 min, 55 ˚C for 1 min, and 72 ˚C for 1 min, observed a high genotypic variability using ERIC-PCR to genowith a final extension at 72 ˚C for 1 min. After amplification, type E. coli from milk of cows with mastitis. Ling and others (2000) the PCR products were subjected to electrophoresis separation in also found high genetic heterogeneity in E. coli O157:H7 isolated agarose gel as previously described. E. coli O157:H7 (C7–88) was from chicken and cow hamburger using ERIC-PCR, in which used as a control for the stx1 gene, and E. coli O157:H7 (ATCC each isolate presented a unique genetic profile. E. coli isolates from 43889) was used as a control for the stx2, eae, and ehxA genes. mutton meat also showed high genetic diversity when typed using M1176 Journal of Food Science r Vol. 79, Nr. 6, 2014
Genotyping and virulence of E. coli . . .
M: Food Microbiology & Safety
Figure 1–Dendrogram of similarity of the ERIC-PCR electrophoretic profiles among E. coli isolated from pasteurized cow milk.
Vol. 79, Nr. 6, 2014 r Journal of Food Science M1177
Genotyping and virulence of E. coli . . . Figure 2–Dendrogram of similarity of the REP-PCR electrophoretic profiles among E. coli isolated from pasteurized cow milk.
M: Food Microbiology & Safety M1178 Journal of Food Science r Vol. 79, Nr. 6, 2014
Genotyping and virulence of E. coli . . .
Identification of virulence genes In this study, the stx1, stx2, eae, and ehxA virulence genes were not detected in the E. coli isolates. Similar results were obtained by Carneiro and others (2006), who reported that the stx1, stx2, and eae genes were not detected in E. coli isolates from pasteurized cow milk sold in Rio de Janeiro, Brazil. However, Manna and others (2006) reported the presence of the stx2 gene in E. coli O157 isolates from pasteurized milk in India. Most studies have reported the presence of STEC only in raw milk and its derivatives in different parts of the world. Farzan and others (2012) detected the stx1 and stx2 genes in 16 of 44 isolates (36.36%) and 35 of 44 isolates (79.54%), respectively. However, the eae gene was not detected, and the hlyA gene was present in only 5 of 44 (11.36%) of the E. coli isolates from raw cow milk. Altalhi and Hassan (2009) detected the presence of the eae gene in 9.1% of E. coli isolates from raw cow milk and the stx1 and stx2 genes in 3% and 6.1% of the isolates, respectively. Solomakos and others (2009) found that 33% of the E. coli isolates from raw cow milk had the stx1 or stx2, eae, and ehxA gene, belonging to the O157:H7 serotype. Costa and others (2013) showed that 14.29% of E. coli isolates from cow milk samples carried the stx1 gene alone, 3.89% possessed the stx2 alone and 14.29% carried both the stx1 and stx2 genes. Islam and others (2010) studied the prevalence of STEC in various foods, including raw milk, in which 10% of the samples had the stx gene but not the eae or ehxA gene. In Brazil, Vicente and others (2005) detected the stx1 and stx2 genes in 3.3% of E. coli isolates from milk samples collected during the milking process in S˜ao Paulo. However, similar to this study, the stx1, stx2, and eae genes were not found in samples of raw milk from southern Brazil (Timm and others 2009). Cattle are considered the main source of STEC (Farrokh and others 2012). In Brazil, some studies have reported a high prevalence of this bacteria in beef and dairy cattle (Irino and others 2005; Vicente and others 2005; Oliveira and others 2008). However, consistent with this study, other studies reported a low prevalence of STEC in milk, meat, and their derivatives (Vicente and others 2005; Carneiro and others 2006; Bergamini and others 2007; Paneto and others 2007; Timm and others 2009; Martins and others 2011). Despite the fact that we did not detect STEC genes, the presence of E. coli in pasteurized milk indicates and inadequate sanitary hygiene practices and does not exclude the possibility that other
pathotypes of E. coli are present that can present a health risk to consumers.
Conclusion ERIC-PCR and REP-PCR revealed high genetic diversity of E. coli isolates from bovine pasteurized milk, confirming the possibility of their use in epidemiological studies. The bacterial isolates did not have the stx1, stx2, eae, or ehxA genes that are normally present in STEC, indicating a low prevalence of this pathogen in milk produced in the region studied.
Acknowledgments This research was supported by Coordenac¸a˜ o de Aperfeic¸oamento de Pessoal de N´ıvel Superior (CAPES), Fundac¸a˜ o Arauc´aria, and the Graduate Program in Food Sciences of the State Univ. of Maring´a. Prof. Halha Ostrensky Saridakis and Prof. Jacinta Sanchez Pelayo of the Dept. of Microbiology, State Univ. of Londrina, who kindly provided E. coli isolates used as controls and Pedro Henrique Canezin for his help improving the figures.
Author Contributions Simone Aparecida Hoffmann:conducted the tests, interpreted the results and wrote the manuscript. Gabriella Giani Pieretti: helped conduct the tests, interpreted the results. Adriana Fiorini: perform Bionumerics analysis, interpreted the results. Eliana Val´eria Patussi: interpreted the results and helped write the manuscript. Rosilene Fressatti Cardoso: designed the study, interpreted the results and corrected the manuscript. Jane Martha Graton Mikcha: designed the study, interpreted the results and corrected the manuscript.
References Altalhi AD, Hassan SA. 2009. Bacterial quality of raw milk investigated by Escherichia coli and isolates analysis for specific virulence-gene markers. Food Control 20:913–7. Blanco JE, Blanco M, Alonso MP, Mora A, Dahbi G, Coira MA, Blanco J. 2004. Serotypes, virulence genes, and intimin types of Shiga toxin (verotoxin)-producing Escherichia coli isolates from human patients: prevalence in Lugo, Spain, from 1992 through 1999. J Clin Microbiol 42:311–9. Bergamini AMM, Sim˜oes M, Irino K, Gomes TAT, Guth BEC. 2007. Prevalence and characteristics of shiga toxin-producing Escherichia coli (STEC) strains in ground beef in S˜ao Paulo. Braz J Microbiol 38:553–6. Bhong CD, Brahmbhatt MN, Joshi CG, Rank DN. 2008. Detection of virulence determinants by real time PCR in E. coli isolated from mutton. Meat Sci 80:1129–31. Carneiro LAM, Lins MC, Garcia FRA, Silva APS, Mauller PM, Alves GB, Rosa ACP, Andrade JRC, Freitas-Ameida AC, Queiroz MLP. 2006. Phenotypic and genotypic characterization of Escherichia coli strains serogrouped as enteropathogenic E. coli (EPEC) isolated from pasteurized milk. Int J Food Microbiol 108:15–21. CDC (Centers for Disease Control and Prevention). 2012. Escherichia coli O157:H7 and other Shiga toxin-producing Escherichia coli (STEC). Available from: http://www.cdc.gov/ nczved/divisions/dfbmd/diseases/ecoli_o157h7/. Accessed 2012 February 12. Chapaval L, Olivindo CS, Souza FGC, Alves FSF, Frota IMA. 2010. Detecc¸a˜ o de Escherichia coli e Pseudomonas aeruginosa pela t´ecnica de REP-PCR no monitoramento da qualidade do leite de cabra em sala de ordenha. Comunicata Scientiae 1:49–56. D’Costa D, Bhosle SN, Dhuri RB, Doijad SP, Poharkar KV, Kalorey DR, Barbuddhe SB. 2013. Prevalence, serogroups, Shiga-toxin genes and Pulsed Field Gel Electrophoresis analyses of Escherichia coli isolated from bovine milk. Proc Natl Acad Sci, India, Sect B Biol Sci 83:423–29. FAO (Food and Agriculture Organization). 2013. Food outlook, global market analysis. Available from: http://www.fao.org/giews/english/fo/index.htm. Accessed 2012 March 20. Farrokh C, Jordan K, Auvray F, Glass K, Oppegaard H, Raynaud S, Thevenot D, Condron R, De Reu K, Alexander Govaris, Heggum K, Heyndrickx M, Hummerjohann J, Lindsay D, Miszczycha S, Moussiegt S, Verstraete K, Cerf O. 2012. Review of Shiga-toxin-producing Escherichia coli (STEC) and their significance in dairy production. Int J Food Microbiol 1–23. Farzan R, Rahimi E, Momtaz H. 2012. Virulence properties of shiga toxin-producing Escherichia coli isolated from iranian raw milk and dairy products. Slov Vet Res 49:159–66. Giammanco GM, Pignato S, Grimont F, Grimont PAD, Caprioli A, Morabito S, Giammanco G. 2002. Characterization of Shiga Toxin-Producing Escherichia coli O157:H7 Isolated in Italy and in France. J Clin Microbiol 40:4619–24. Hunter PR, Gaston MA. 1988. Numerical index of the discriminatory ability of typing systems: an application of Simpson’s index of diversity. J Clin Microbiol 26:2465–6. Ibenyassine K, AitMhand R, Karamoko Y, Cohen N. 2006. Use of repetitive DNA sequences to determine the persistence of enteropathogenic Escherichia coli in vegetables and in soil grown in fields treated with contaminated irrigation water. Appl Microbiol 43:528–33.
Vol. 79, Nr. 6, 2014 r Journal of Food Science M1179
M: Food Microbiology & Safety
ERIC-PCR and REP-PCR (Bhong and others 2008). REPPCR also efficiently compared E. coli isolates from goat milk and the milking environment, which contributed to the monitoring of the quality of goat milk (Chapaval and others 2010). In a study similar to the present one, Solomakos and others (2009)evaluated the genetic diversity of STEC obtained from unpasteurized dairy derivatives using ERIC-PCR. Considering a 70% coefficient of similarity, the authors obtained low discriminatory power. However, considering the same coefficient of similarity utilized in this study (98%), they observed high diversity among the isolates studied. The authors also found a correlation between the genomic groups and virulence genes. In contrast to the present results, ERIC-PCR did not successfully differentiate E. coli O157:H7 isolated from food, humans, and animals in a study by Giammanco and others (2002). The high genetic diversity observed in this study can be justified by several sources of contamination of cow milk by E. coli during the entire production process.
Genotyping and virulence of E. coli . . . Irino K, Kato MAMF, Vaz TMI, Ramos II, Souza MAC, Cruz AS, Gomes TAT Gomes Vieira MAM, Guth BEC. 2005. Serotypes and virulence markers of Shiga toxin-producing Escherichia coli (STEC) isolated from dairy cattle in S˜ao Paulo State, Brazil. Vet Microbiol 105:29–36. Islam MA, Mondol AS, Azmi IJ, Boer E, Beumer RR, Zwietering MH, Heuvelink AE, Talukder KA. 2010. Ocurrence and characterization of Shiga Toxin-Producing Escherichia coli in raw meat, raw milk, and street vended juices in Bangladesh. Foodborne Path Dis 7: 1381–85. Ling OW, Radu S, Rusul G, Karim MI, Purwati E, Lihan S. 2000. Enterobacterial Repetitive Intragenic Consensus (ERIC) genotyping of Escherichia coli O157:H7. Pakistan J Biol Sci 3:35–7. Manna SK, Brahmane MP, Das R, Manna C, Batabyal S. 2006. Detection of Escherichia coli O157 in foods of animal origin by culture and multiplex polymerase chain reaction. J Food Sci Technol 46:77–9. Martins RP, Nakazato L, Dutra V, Leite DS. 2011. Analysis of virulence genes in Escherichia coli isolated from grated cheese. Ciˆenc Tecnol Aliment 31:106–8. MilkPoint. 2012. Consumo de l´acteos tem expans˜ao. Available from: http://www.milkpoint. com.br/cadeia-do-leite/giro-lacteo/consumo-de-lacteos-tem-expansao-78049n.aspx. Accessed 2012 February 12. Oliveira MG, Brito JRF, Gomes TAT, Guth BECG, Vieira MAM, Naves ZVF, Vaz TMI, Irino K. 2008. Diversity of virulence profiles of Shiga toxin-producing Escherichia coli serotypes in food-producing animals in Brazil. Int J Food Microbiol 127: 139–46. Paneto BR, Schocken-Iturrino RP, Macedo C, Santo E, Marin JM. 2007. Occurrence of toxigenic Escherichia coli in raw milk cheese in Brazil. Arq Bras Med Vet Zootec 59: 508–12. Paton AW, Paton JC. 1998. Detection and characterization of Shiga toxigenic Escherichia coli by using multiplex PCR assays for stx1, stx2, eaeA, Enterohemorrhagic E. coli hlyA, rfbO111, and rfbO157. J Clinical Microbiol 11:598–602
M: Food Microbiology & Safety M1180 Journal of Food Science r Vol. 79, Nr. 6, 2014
Rahimi E, Chaleshtori SS, Parsaei P. 2011. Prevalence and antimicrobial resistance of Escherichia coli O157 isolated from traditional cheese, ice cream and yoghurt in Iran. African J Microbiol 5:3706–10. Rantsiou K, Alessandria V, Cocolin L. 2012. Prevalence of Shiga toxin-producing Escherichia coli in food products of animal origin as determined by molecular methods. Int J Food Microbiol 154:37–43. Solomakos N, Govaris A, Angelidis AS, Pournaras S, Burriel AR, Kritas SK, Papageorgiou DK. 2009. Occurrence, virulence genes and antibiotic resistance of Escherichia coli O157 isolated from raw bovine, caprine and ovine milk in Greece. Food Microbiol 26: 865–71. Swanenburg M, Urlings HAP, Keuzenkamp DA, Snijders JMA. 1998. Validation of ERIC-PCR as a tool in epidemiology research of Salmonella in slaughter pigs. J Ind Microbiol Biotechn 21:141–4. Timm CD, Conceic¸a˜ o FR, Menin A, Conceic¸a˜ o RCS, Dellagostin OA, Aleixo JAG. 2009. Prevalence of Shiga toxin-producing Escherichia coli in southern Brazil isolated from ground beef and raw milk. Ciˆenc Anim Brasi 10:641–9. Versalovic J, Koeuth T, Lupski JR. 1991. Distribution of repetitive DNA sequences in eubacteria and application to fingerprinting of bacterial genomes. Nucl Acids Res 19:6823–31. Vicente HIG, Amaral LA, Cerqueira AMF. 2005. Shigatoxigenic Escherichia coli serogroups O157, O111 and O113 in feces, water and milk samples from dairy farms. Brazilian J Microbiol 36:217–22. Zanella GN, Mikcha JMG, Bando E, Siqueira VLD, Machinski Jr. M. 2010. Occurrence, antibiotic resistance of coliforms and antimicrobial residues from cow’s milk in Brazil. J Food Prot 73 1684–7. Wenz JR, Barrington GM, Garry FB, Ellis RP, Magnuson RJ. 2006. Escherichia coli isolates’ serotypes, genotypes, and virulence genes and clinical coliform mastitis severity. J Dairy Sci 89: 3408–12.
