Raw milk cheese as a potential infection source of pathogenic and toxigenic food born pathogens

Abstract

The consumption of raw milk cheese has been growing worldwide with S. aureus and E. coli been the leading agents in food poisoning. The present work aims to evaluate the microbiological quality of raw milk cheeses commercialized in Brazil, regarding microbiology safety and enterotoxin gene presence. Forty-three raw milk cheeses samples from five different suppliers were analyzed. Counting and identification of S. aureus, E. coli and Salmonella spp were performed according to BAM from the FDA. Further S. aureus identification was performed by the cydB and Salmonella spp by the invA gene. S. aureus toxin genes (sea, seb, sec, see, ses, seh and sei) and E. coli gene LT, STa, Stb, stx1, stx2, eae, rmpA, wabG, mrkD, kfu, mcgA, fimH and uge were analysed. From the 43 samples analyzed, 18 presented S. aureus with two isolates positive for the tst gene, two for the sec gene, two for the seh gene and four for the sei gene. Thirty-five E. coli and seven. Salmonella spp isolates were obtained. E. coli isolates harbored sta and stx2 genes. The results revealed that raw milk cheeses sold can cause harm consumer's health and highlights the importance of adoption good hygienic-sanitary practices and consumers awareness.

Keywords:
epidemiology; virulence genes; food safety; pathogens

1 Introduction

Due the recent worldwide trend of consumption of unpasteurized (raw) milk and milk products such as raw milk cheese, current information regarding their risks and benefits come into discussion, especially regarding food safety policy. In this regard, Staphylococcal food poisoning is the result of consuming foods containing sufficient amounts of its enterotoxins, with food borne outbreaks of S. aureus intoxication's been associated with the consumption of contaminated milk and dairy products in many countries ((Saka & Terzi Gulel, 2018)⁠⁠. In addition, microbiological analysis of raw milk cheese revealed varying degrees of quality and safety, with Staphylococcus aureus considered a potential food enterotoxin-producing species (Samelis et al., 2019)⁠. Also, it has been shown that coagulase negative staphylococcus (CNS) may be involved in food poisoning because they can also carry enterotoxin encoding genes (se genes) in their genomes that they are thermoresistant (Samelis et al., 2019)⁠. The Staphylococcus spp enterotoxin (SE) genes are encoded in mobile genetic elements, such as plasmids, prophages, and pathogenic islands that are able to be transmitted though S. aureus strains even during food processing (Cardoso & Marin, 2017)⁠⁠. The Shiga toxin–producing E. coli (STEC) is characterized as a group of highly pathogenic strains that produce one or more Shiga toxins that can cause infections ranging from/uncomplicated diarrhea to severe diseases as hemorrhagic colitis and life-threatening hemolytic uremic syndrome (HUS). Ruminants have been identified as a major reservoir of STEC and as reported in the literature, contaminated raw milk and raw milk products are among the main risk factors considered STEC vectors (Nobili et al., 2016)⁠⁠ . The influx of multidrug and/or virulent enterococcal strains from cheese to the human digestive tract has been assessed in some countries all over the world as a serious public health concern when associated with the increasing production and consumption of raw milk cheeses (Cunha-Neto et al., 2020)⁠⁠⁠. Raw milk cheeses have a good presence in the total cheese production of the EU, Canada and especially Switzerland which it correspond of 59.5% of the total cheese produced (Hammad et al., 2015)⁠.⁠, also it is present in Brazil, as it is considered one of the most popular and recognized as Minas frescal cheese (Kamimura et al., 2019)⁠⁠.

Nevertheless, the safety of raw milk cheese has been questioned due to its relation to large outbreaks of foodborne been reported (Cardoso & Marin, 2017)⁠⁠, especially when considered that Shiga toxin–producing E. coli strains have been detected before Cardoso & Marin, 2017)⁠. To better determinate contamination and safety of raw milk cheese a characterization of E. coli strains and of S. aureus were evaluated in Raw milk cheese from various market to at Jaboticabal, Sao Paulo state and at Aracaju, Sergipe, Brazil.

2 Materials and methods

2.1 Sample collection

Cheese samples were collected, from locally artisan producers and farmers’ markets in Jaboticabal, Sao Paulo State and from Aracaju, Sergipe, Brazil. All samples were labeled as having been made from raw milk. Samples were stored at refrigeration temperature (2 to 8 °C) from the time of purchase to delivery to the lab. Once the samples were received, the cheese type, source of milk and purchase location were recorded, and each sample was coded and then repackaged using a clean washable cutting board, sterile knife, and plastic wrap.

2.2 Staphylococcus aureus count, DNA extraction and virulence gene detection

S. aureus count were performed as (Bennett & Lancette, 2017)⁠. After the addition of 25 g of sample to 225 mL of sterilized peptone water, 1 ml of the homogenized samples were serial diluted in sterilized peptone water until it reached 10-6 dilution and were further sown on Braid Parker Agar. Only typical S. aureus colonies were counted. After, isolates were tested for catalase and coagulase production and DNA were extracted for future virulence gene detection. For DNA extraction, well-isolated colonies were inoculated into BHI broth, incubated at 37 °C for 18h and extracted according to the method of Kuramae-Izioka (1997) ⁠with minor modifications as described previously (Almeida et al., 2018)⁠⁠. Virulence gene detection was performed for the Staphylococcus spp. enterotoxin producing genes sea, seb, sec, see, ses, seh and sei genes according to (Paniagua-Contreras et al., 2012)⁠⁠.

2.3 Enumeration of coliform and E. coli, DNA extraction and virulence genes detection

Isolation and enumeration were performed according to (Feng et al., 2018)⁠⁠. Presumptive test for coliforms, fecal coliforms and E. coli were done by the most probable number technique with EC Broth as suggested by (Feng et al., 2018)⁠. 2018. Samples that were positive for E. coli were aliquoted for further virulence and toxin test. Bacterial DNA extraction was done according to (Keskimäki et al., 2001)⁠, and the virulence gene detection by PCR was performed as described by (China et al., 1996)⁠⁠. After the amplification, products were visualized by exposure of the gel to UV light and subsequently photographed. The following virulence genes were analyzed LT, STa, STb, stx1, stx2, eae, rmpA, wabG, mrkD, kfu, mcgA, fimH and uge

2.4 Salmonella spp detection and DNA extraction

Salmonella spp detection was performed according to (Andrews et al., 2018)⁠⁠ with modifications, sample preparation and dilution followed the same as before, after 1 mL of the diluted samples were added to Selenite cystine and Rappaport Vassiliadis for a selective step. After 24 hours incubation period at 37 ^oC, samples were streaked into XLD selective medium, and samples with characteristic black colonies were considered positives. Further, Salmonella isolates were identified by the invA gene and performed as (Fratamico, 2003)⁠⁠, DNA extraction were performed a according to (Keskimäki et al., 2001)⁠⁠.

3 Results and discussion

Among the 43 samples analyzed, coagulase positive Staphylococcus were isolated in 21 (48.83%) with 18 (41.86%) further identified as S. aureus by cydB PCR. Of the 18 isolates of S. aureus obtained, two isolates (11.1%) were positive for the tst gene, two (11.1%) for the sec gene, two (11.1%) for the seh gene and four (22.2%) for the sei gene. No positive isolates were observed for the seb, see and sea genes. Although 18 raw milk cheese evaluated had a S. aureus count within the standard (1x10³CFU/g or ml) allowed by the Brazilian ANVISA RDC No. 12, 2001 (Brasil, 2001)⁠⁠, it was observed that the tst positive and the sec positive isolates were obtained from products that presented S. aureus count within the accepted level (1 x 10³ CFU/g). Also, it is important to highlight that in two samples, S. aureus count were above the 1 x 10⁵ CFU/g that is considered the limit for toxin production (U. S. Department of Health and Human Services, 2011)⁠⁠ and isolates from this samples were sec and seh positive. A high prevalence of S. aureus in Buffalo raw milk cheese have been reported (Saka & Terzi Gulel, 2018)⁠ 17/54 (34%). An author (André et al., 2008)⁠⁠ have reported a prevalence of 70.8% of Staphylococcus spp. in raw milk cheese from Brazil and, (Basanisi et al., 2016)⁠⁠ found a 41.1% S. aureus in goat and sheep cheese samples in Italy. One reason for these differences may be due to the quality of the raw or unpasteurized milk in the dairy production, poor handler personnel hygiene, or cross contamination at the supermarket or at manufacture unit, it can also be related to the use of milk from unhealthy animals, inadequate pasteurization during homemade traditional cheese production, contaminated cheese-making equipment, and improper food handling (Saka & Terzi Gulel, 2018)⁠. The enterotoxigenic profile observed in this study (tst, sec seh sei), differs from those observed by (Saka & Terzi Gulel, 2018)⁠ which had isolates caring the sea, see and sed genes, these authors also had sec positive isolates from Buffalo raw milk cheese in Turkey. This seems to be in agreement with the fact that SEA, either alone or together with other SEs/SEls, is the most common cause of staphylococcal food poisoning worldwide and the SEA, SEC, and SED type toxins seems to be the the most commonly produced toxins in milk and dairy products (Neder et al., 2011)⁠⁠. It is important to notice that the prevalence of enterotoxin-producing S. aureus isolates in milk and dairy products samples may differs among studies due to differences in the source of sampling, and geographical differences.

From the 43 samples analyzed E. coli was observed in 35 (81.3%) and Salmonella spp in 7 (16.2%). When compared with the safety parameters of (Brasil, 2001)⁠, E. coli contamination were above limit (5 x 103 CFU / g) in 19 (44.1%) samples and the presence of Salmonella spp in six (13,9%) samples makes them unsuitable for consumption according to this resolution. Also, sta positive E. coli were detected in two samples and stx2 in one sample. Cheese contamination can occur in various parts of the production chain and, although most cheeses (76.0%) comply with the legislation, detection of E. coli is a large indicator of fecal contamination associated with the presence of S. aureus, may indicate that these products have inadequate hygiene conditions during their manufacture. An aggravating fact is the detection of Salmonella spp in products that are being offered in the market and that can pose a great risk to public health, which could be the result of cross contamination in the establishment. The presence of non pathogenic E. coli, withing the limit, in dairy products are, in general, not harmful (Ombarak et al., 2016)⁠⁠. However, the presence of pathogenic E. coli is problematic and among these virulence genes, the Stx2 is one of the most important virulence factors because it may cause a life-threatening outcomes such as hemolytic uremic syndrome or HUS and neurological disorder (Ombarak et al., 2016)⁠. The detection of STa positives isolates in raw milk cheese could be a problem due to the production of thermostable toxins that could still be viable even if the cheese was heat treated and, according to (Basanisi et al., 2016)⁠⁠ STEC contaminating raw milk persisted through the processes of both the curd and the mozzarella cheese production.

4 Conclusion

The results reveal the cheeses sold can cause harm consumer's health and highlights the importance of adoption good hygienic-sanitary practices in the production and commercialization of cheeses, as well the consumer awareness about the health risks and more effective surveillance. Therefore, a reevaluation of the cheese production, a properly inspection, distribution and marketing conditions is necessary and urgent.

Acknowledgements

To coordenação de aperfeiçoamento de pessoal de nível superior (CAPES) and to the Programa de Microbiologia Agropecuária of the Universidade Estadual Paulista (UNESP), Faculdade de Ciências Agrárias e Veterinárias, Jaboticabal, SP, Brasil for the financia support.

• Practical Application: In this study, potential pathogenic and antibiotic resistant isolates of S. aureus and E. coli were isolated from raw milk cheese from markets in Jaboticabal, Sao Paolo and Aracaju, Sergipe. Although most samples could not be considered suitable for consumption, isolates harboring virulence and resistance genes were also obtained from samples that were within the accepted level of detection in food. Thus, revealing that raw milk cheeses sold can be of harm to consumer's health, especially if they are not consistent evaluated.

References

• Almeida, C. C., Pizauro, L. J. L., Soltes, G. A., Slavic, D., De Ávila, F. A., Pizauro, J. M., & MacInnes, J. I. (2018). Some coagulase negative Staphylococcus spp. isolated from buffalo can be misidentified as Staphylococcus aureus by phenotypic and Sa442 PCR methods. BMC Research Notes, 11(1), 4-9. http://dx.doi.org/10.1186/s13104-018-3449-8 PMid:29848377.
  » http://dx.doi.org/10.1186/s13104-018-3449-8
• André, M. C. D. P. B., Campos, M. R. H., Borges, L. J., Kipnis, A., Pimenta, F. C., & Serafini, Á. B. (2008). Comparison of Staphylococcus aureus isolates from food handlers, raw bovine milk and Minas Frescal cheese by antibiogram and pulsed-field gel electrophoresis following SmaI digestion. Food Control, 19(2), 200-207. http://dx.doi.org/10.1016/j.foodcont.2007.03.010
  » http://dx.doi.org/10.1016/j.foodcont.2007.03.010
• Andrews, W. H., Wang, H., Jacobson, A., Ge, B., Zhang, G., & Hammack, T. (2018). Bacteriological Analytical Manual (BAM). BAM Chapter 5: Salmonella USA: FDA. Retrieved from https://www.fda.gov/food/laboratory-methods-food/bam-chapter-5-salmonella
  » https://www.fda.gov/food/laboratory-methods-food/bam-chapter-5-salmonella
• Basanisi, M. G., Nobili, G., La Bella, G., Russo, R., Spano, G., Normanno, G., & La Salandra, G. (2016). Molecular characterization of Staphylococcus aureus isolated from sheep and goat cheeses in southern Italy. Small Ruminant Research, 135, 17-19. http://dx.doi.org/10.1016/j.smallrumres.2015.12.024
  » http://dx.doi.org/10.1016/j.smallrumres.2015.12.024
• Bennett, R. W., & Lancette, G. A. (2017). Laboratory Methods - BAM: Staphylococcus aureus USA: FDA. Retrieved from https://www.fda.gov/food/foodscienceresearch/laboratorymethods/ucm071429.htm
  » https://www.fda.gov/food/foodscienceresearch/laboratorymethods/ucm071429.htm
• Brasil. Agência Nacional de Vigilância Sanitária – ANVISA. (2001). Aprova o Regulamento Técnico sobre padrões microbiológicos para alimentos (Resolução-RDC nº 12, de 02 de Janeiro de 2001). Diário Oficial [da] República Federativa do Brasil
• Cardoso, P., & Marin, J. M. (2017). Occurrence of non-O157 shiga toxin-encoding Escherichia coli in artisanal mozzarella cheese in Brazil: risk factor associated with food workers. Food Science and Technology (Campinas), 37(1), 41-44. http://dx.doi.org/10.1590/1678-457x.06316
  » http://dx.doi.org/10.1590/1678-457x.06316
• China, B., Pirson, V., & Mainil, J. (1996). Typing of bovine attaching and effacing Escherichia coli by multiplex in vitro amplification of virulence-associated genes. Applied and Environmental Microbiology, 62(9), 3462-3465. http://dx.doi.org/10.1128/AEM.62.9.3462-3465.1996 PMid:8795238.
  » http://dx.doi.org/10.1128/AEM.62.9.3462-3465.1996
• Cunha-Neto, A., Carvalho, L. A., Castro, V. S., Barcelos, F. G., Carvalho, R. C. T., Rodrigues, D. P., Conte-Junior, C. A., & Figueiredo, E. E. S. (2020). Salmonella Anatum, S. Infantis and S. Schwarzengrund in Brazilian Cheeses: occurrence and antibiotic resistance profiles. International Journal of Dairy Technology, 73(1), 296-300. http://dx.doi.org/10.1111/1471-0307.12636
  » http://dx.doi.org/10.1111/1471-0307.12636
• Feng, P., Weagant, S. D., Grant, M. A., & Burkhardt, W. (2018). Bacteriological Analytical Manual (BAM). BAM Chapter 4: enumeration of Escherichia coli and the Coliform bacteria USA: FDA. Retrieved from https://www.fda.gov/food/foodscienceresearch/laboratorymethods/ucm064948.htm
  » https://www.fda.gov/food/foodscienceresearch/laboratorymethods/ucm064948.htm
• Fratamico, P. M. (2003). Comparison of culture, polymerase chain reaction (PCR), TaqMan Salmonella, and Transia Card Salmonella assays for detection of Salmonella spp. in naturally-contaminated ground chicken, ground turkey, and ground beef. Molecular and Cellular Probes, 17(5), 215-221. http://dx.doi.org/10.1016/S0890-8508(03)00056-2 PMid:14580395.
  » http://dx.doi.org/10.1016/S0890-8508(03)00056-2
• Hammad, A. M., Hassan, H. A., & Shimamoto, T. (2015). Prevalence, antibiotic resistance and virulence of Enterococcus spp. in Egyptian fresh raw milk cheese. Food Control, 50, 815-820. http://dx.doi.org/10.1016/j.foodcont.2014.10.020
  » http://dx.doi.org/10.1016/j.foodcont.2014.10.020
• Kamimura, B. A., Magnani, M., Luciano, W. A., Campagnollo, F. B., Pimentel, T. C., Alvarenga, V. O., Pelegrino, B. O., Cruz, A. G., & Sant’Ana, A. S. (2019). Brazilian artisanal cheeses: an overview of their characteristics, main types and regulatory aspects. Comprehensive Reviews in Food Science and Food Safety, 18(5), 1636-1657. http://dx.doi.org/10.1111/1541-4337.12486
  » http://dx.doi.org/10.1111/1541-4337.12486
• Keskimäki, M., Eklund, M., Pesonen, H., Heiskanen, T., & Siitonen, A., & Study Group. (2001). EPEC, EAEC and STEC in stool specimens: prevalence and molecular epidemiology of isolates. Diagnostic Microbiology and Infectious Disease, 40(4), 151-156. http://dx.doi.org/10.1016/S0732-8893(01)00265-6 PMid:11576786.
  » http://dx.doi.org/10.1016/S0732-8893(01)00265-6
• Kuramae-Izioka, E. E. (1997). A rapid, easy and high yield protocol for total genomic DNA isolation of Colletotrichum gloeosporioides and Fusarium oxysporum. Unimar, 19, 683-689.
• Neder, V. E., Canavesio, V. R., & Calvinho, L. F. (2011). Presence of enterotoxigenic staphylococcus aureus in bulk tank milk from Argentine dairy farms. Revista Argentina de Microbiologia, 43(2), 104-106. http://dx.doi.org/10.1590/S0325-75412011000200006 PMid:21731971.
  » http://dx.doi.org/10.1590/S0325-75412011000200006
• Nobili, G., Franconieri, I., Basanisi, M. G., La Bella, G., Tozzoli, R., Caprioli, A., & La Salandra, G. (2016). Short communication: isolation of Shiga toxin-producing Escherichia coli in raw milk and mozzarella cheese in southern Italy. Journal of Dairy Science, 99(10), 7877-7880. http://dx.doi.org/10.3168/jds.2016-11613 PMid:27522413.
  » http://dx.doi.org/10.3168/jds.2016-11613
• Ombarak, R. A., Hinenoya, A., Awasthi, S. P., Iguchi, A., Shima, A., Elbagory, A. R. M., & Yamasaki, S. (2016). Prevalence and pathogenic potential of Escherichia coli isolates from raw milk and raw milk cheese in Egypt. International Journal of Food Microbiology, 221, 69-76. http://dx.doi.org/10.1016/j.ijfoodmicro.2016.01.009 PMid:26824810.
  » http://dx.doi.org/10.1016/j.ijfoodmicro.2016.01.009
• Paniagua-Contreras, G., Sáinz-Espuñes, T., Monroy-Pérez, E., Raymundo Rodríguez-Moctezuma, J., Arenas-Aranda, D., Negrete-Abascal, E., & Vaca, S. (2012). Virulence markers in Staphylococcus aureus strains isolated from hemodialysis catheters of mexican patients. Advances in Microbiology, 2(4), 476-487. http://dx.doi.org/10.4236/aim.2012.24061
  » http://dx.doi.org/10.4236/aim.2012.24061
• Saka, E., & Terzi Gulel, G. (2018). Detection of enterotoxin genes and methicillin-resistance in staphylococcus aureus isolated from water buffalo milk and dairy products. Journal of Food Science, 83(6), 1716-1722. http://dx.doi.org/10.1111/1750-3841.14172 PMid:29802728.
  » http://dx.doi.org/10.1111/1750-3841.14172
• Samelis, J., Kakouri, A., Kondyli, E., & Pappa, E. C. (2019). Effects of curd heating with or without previous milk pasteurisation on the microbiological quality and safety of craft-made ‘Pasta Filata’ Kashkaval cheese curds. International Journal of Dairy Technology, 72(3), 447-455. http://dx.doi.org/10.1111/1471-0307.12601
  » http://dx.doi.org/10.1111/1471-0307.12601
• U. S. Department of Health and Human Services. (2011). Fish and fishery products hazards and controls guidance. In U.S. Department of Health and Human Services. Fish and fishery products hazard and control guidance (4th ed). USA: FDA.

Publication Dates

• Publication in this collection
  28 Sept 2020
• Date of issue
  Apr-Jun 2021

History

• Received
  27 Jan 2020
• Accepted
  05 Mar 2020