Survey of Campylobacter in foods implicated in foodborne diseases in Southern Brazil

Marjo Cadó Bessa Simone Haas Rosane Campanher Ramos Raquel Rocha Daiane Voss-Rech Raquel Rebelatto Sabrina Castilho Duarte Arlei Coldebella Clarissa Silveira Luiz Vaz About the authors

ABSTRACT

Campylobacter is not routinely tested in foodborne disease investigations in Brazil. Here, we studied the occurrence of Campylobacter among other food-related bacteria commonly found in foodborne disease outbreaks reported in Rio Grande do Sul State, Southern Brazil. Seventy-two food samples were analyzed by using culture-based detection methods during the 18-month investigation of 36 foodborne disease outbreaks. The sampled foods from the foodborne disease outbreaks were all negative for Campylobacter . However, at least one of other routinely investigated foodborne-related bacteria was detected in 29.17% (21/72) of the samples. Taken together, these results suggest the need to monitor Campylobacter in foodborne diseases to detect sporadic cases caused by Campylobacter that might go unnoticed in Rio Grande do Sul.

KEYWORDS:
Campylobacter; Food safety; Outbreak

INTRODUCTION

Campylobacter is the most prevalent pathogen in foodborne gastroenteritis infections in developed countries 11. European Food Safety Authority, European Centre for Disease Prevention and Control. The European Union summary report on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks in 2017. EFSA J. 2018;16:5500.,22. Tack DM, Ray L, Griffin PM, Cieslak PR, Dunn J, Rissman T, et al. Preliminary incidence and trends of infections with pathogens transmitted commonly through food — Foodborne Diseases Active Surveillance Network, 10 U.S. sites, 2016—2019. MMWR Morb Mortal Wkly Rep. 2020;69:509-14. . Campylobacter jejuni is mostly associated with human infections and is more frequent in laboratory-confirmed cases, followed by C. coli and C. lari11. European Food Safety Authority, European Centre for Disease Prevention and Control. The European Union summary report on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks in 2017. EFSA J. 2018;16:5500. . Epidemiological studies have demonstrated that campylobacteriosis primarily results from the consumption or mishandling of contaminated food of animal origin, especially from chicken 11. European Food Safety Authority, European Centre for Disease Prevention and Control. The European Union summary report on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks in 2017. EFSA J. 2018;16:5500.,33. Silva WC, Targino BN, Mendonça RS, Sant'Ana AS, Hungaro HM. Campylobacter: an overview of cases, occurrence in food, contamination sources and antimicrobial resistance in Brazil. Food Rev Int. 2018;34:364-89. .

In Brazil, Campylobacter has been reported in chicken meat and other foods of animal origin 33. Silva WC, Targino BN, Mendonça RS, Sant'Ana AS, Hungaro HM. Campylobacter: an overview of cases, occurrence in food, contamination sources and antimicrobial resistance in Brazil. Food Rev Int. 2018;34:364-89.,44. Silva DT, Tejada TS, Blum-Menezes D, Dias PA, Timm CD. Campylobacter species isolated from poultry and humans, and their analysis using PFGE in southern Brazil. Int J Food Microbiol. 2016;217:189-94. , as well as in human feces 44. Silva DT, Tejada TS, Blum-Menezes D, Dias PA, Timm CD. Campylobacter species isolated from poultry and humans, and their analysis using PFGE in southern Brazil. Int J Food Microbiol. 2016;217:189-94. . However, only eight foodborne disease outbreaks involving Campylobacter spp. were reported to the Brazilian Ministry of Health among the approximately 14,000 outbreaks occurring in the period 2000 to 2019 55. Brasil. Ministério da Saúde. Doenças transmitidas por alimentos: causas, sintomas, tratamento e prevenção. [cited 2020 Oct 29]. Available from: https://antigo.saude.gov.br/saude-de-a-z/doencas-transmitidas-por-alimentos
https://antigo.saude.gov.br/saude-de-a-z...
. From 2009 to 2018, 6,903 foodborne disease outbreaks were reported in Brazil, and Escherichia coli (24.0%), Salmonella spp. (11.2%), and Staphylococcus aureus (9.5%) were the most frequently identified microorganisms in laboratory-confirmed cases 66. Brasil. Ministério da Saúde. Secretaria de Vigilância em Saúde. Departamento de Vigilância das Doenças Transmissíveis. Coordenação Geral de Doenças Transmissíveis. Surtos de doenças transmitidas por alimentos no Brasil: informe 2018. Brasília: Ministério da Saúde; 2019. [cited 2020 Oct 29]. Available from: https://portalarquivos2.saude.gov.br/images/pdf/2019/fevereiro/15/Apresenta––––o-Surtos-DTA–––Fevereiro-2019.pdf
https://portalarquivos2.saude.gov.br/ima...
. The Central Public Health Laboratories (LACENs) network is structured to provide, among other attributes, the laboratory analyses to support the Brazilian epidemiological surveillance system for foodborne diseases. However, Campylobacter is not routinely tested in foodborne disease investigations. Hence, the incidence of foodborne campylobacteriosis may be underestimated in Brazil. This study aimed to analyze the occurrence of Campylobacter in foods implicated in foodborne diseases reported in Rio Grande do Sul State, Southern Brazil.

MATERIALS AND METHODS

Food samples

In the period from March 2015 to September 2016, the LACEN-RS (Porto Alegre, Rio Grande do Sul State) provided 72 subsamples of foodborne disease-associated foods for Campylobacter analysis. Samples were of dairy products; sausages, eggs, meals prepared with eggs, chicken, pork, beef and fish, and foods of other origins. Samples were collected by the municipal health surveillance services as part of the investigation of 36 foodborne disease outbreaks to determine their etiological agents. These outbreaks comprised cases of a similar disease with similar incubation period distribution, deriving from common foods sources, and patients presented with at least one of the following symptoms: nausea; vomiting; stomach cramps; diarrhea and headache. The foods came from nine of the 19 Regional Health Coordinating Units (CRS) in Rio Grande do Sul, covering different municipalities ( Figure 1 ). Samples were transported chilled to the laboratory and analyzed within 24 h. A descriptive analysis was carried out to determine the relationship between the categories of foods analyzed, the bacteria identified and the CRS of origin.

Figure 1
Provenance (number of samples/total analyzed number) of the foods according to the Regional Health Coordinating Unit (CRS) in Rio Grande do Sul, Southern Brazil. 1: 1st CRS Porto Alegre (6/72); 2: 2nd CRS Porto Alegre (27/72); 4: 4th CRS Santa Maria (1/72); 5: 5th CRS Caxias do Sul (23/72); 6: 6th CRS Passo Fundo (7/72); 9: 9th CRS Cruz Alta (1/72); 11: 11th CRS Erechim (4/72); 13: 13th CRS Santa Cruz do Sul (2/72); 16: 16th CRS Lajeado (1/72).

Routine bacteriological analysis

Foods were submitted to conventional bacteriological analyses at LACEN-RS to detect pathogenic ( Salmonella spp., coagulase-positive staphylococci, Listeria monocytogenes , Bacillus cereus and sulphite-reducing Clostridium at 46 °C) and non-pathogenic ( Escherichia coli ) bacteria 77. Downes FP, Ito K, editors. Compendium of methods for the microbiological examination of foods. 4th ed. Washington: APHA; 2001. . Samples were tested according to the clinical presentation and food microbiological criteria established in Brazil 88. Brasil. Ministério da Saúde. Agência Nacional de Vigilância em Saúde. Resolução - RDC n° 12, de 2 de janeiro de 2001. Aprovar o regulamento técnico sobre padrões microbiológicos para alimentos. Diário Oficial da União, Brasília, 10 jan. 2001. [cited 2020 Oct 29]. Available from: http://portal.anvisa.gov.br/documents/33880/2568070/RDC_12_2001.pdf/15ffddf6-3767-4527-bfac-740a0400829b
http://portal.anvisa.gov.br/documents/33...
.

Qualitative analysis of thermotolerant Campylobacter

Subsamples of either cooked or raw foods were subjected to Campylobacter qualitative analysis using a protocol applicable to products for human consumption 99. International Organization for Standardization. ISO 10272-1:2006: microbiology of food and animal feeding stuffs: horizontal method for detection and enumeration of thermotolerant Campylobacter spp.: part 1: detection method. Geneva: ISO; 2006. . Briefly, the samples were homogenized in Bolton broth (Fluka, Darmstadt, Hesse, Germany) and incubated in a microaerobic atmosphere (5% O2, 10% CO2, with balance in N2) at 37 °C for 4 h, followed by incubation at 41.5 °C (± 1 °C) for 44 h (± 4 h). Enriched samples were plated on modified Charcoal Cefoperazone Deoxycholate agar (mCCD, Fluka, Darmstadt, Hesse, Germany) and on Preston agar (Fluka, Darmstadt, Hesse, Germany), and incubated in microaerobic atmosphere at 41.5 °C (± 1 °C) for 44 h (± 4 h). Typical or putative Campylobacter colonies were subcultured on blood agar base no. 2 (Fluka, Darmstadt, Hesse, Germany) to be further analyzed by Gram staining, motility, microaerobic growth at 25 °C, aerobic growth at 41.5 °C and oxidase. C. jejuni (BRM 34342) and C. coli (BRM 34343) from the microbial culture collection (CMISEA) of Embrapa (Empresa Brasileira de Pesquisa Agropecuaria) Swine and Poultry were used as controls strains.

RESULTS AND DISCUSSION

In the period studied, the most common analyzed food samples were from the category of beef meals (14/72, 19.44%), followed by fish meals (11/72, 15.28%) and sausages (11/72, 15.28%). Samples were primarily collected by the 2nd CRS (27/72, 37.50%) and the 5th CRS (23/72, 31.94%). Contrary to the known high rates of Campylobacter in chicken and poultry samples 33. Silva WC, Targino BN, Mendonça RS, Sant'Ana AS, Hungaro HM. Campylobacter: an overview of cases, occurrence in food, contamination sources and antimicrobial resistance in Brazil. Food Rev Int. 2018;34:364-89.,44. Silva DT, Tejada TS, Blum-Menezes D, Dias PA, Timm CD. Campylobacter species isolated from poultry and humans, and their analysis using PFGE in southern Brazil. Int J Food Microbiol. 2016;217:189-94. in Brazil, thermotolerant Campylobacter species were not detected in the foods analyzed in this study. Differences in campylobacteriosis incidence vary among countries or regions within the same country due to the sensitivity of laboratory procedures, population or surveillance bias 1010. Kaakoush NO, Castaño-Rodríguez N, Mitchell HM, Man SM. Global epidemiology of Campylobacter infection. Clin Microbiol Rev. 2015;28:687-720. .

Notably, campylobacteriosis generally occurs as sporadic cases 11. European Food Safety Authority, European Centre for Disease Prevention and Control. The European Union summary report on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks in 2017. EFSA J. 2018;16:5500. . Sporadic campylobacteriosis might be part of the underreported foodborne diseases in Brazil as they are seldom identified. In the USA, Campylobacter accounts for the highest incidence of bacterial foodborne infections per 100,000 inhabitants 22. Tack DM, Ray L, Griffin PM, Cieslak PR, Dunn J, Rissman T, et al. Preliminary incidence and trends of infections with pathogens transmitted commonly through food — Foodborne Diseases Active Surveillance Network, 10 U.S. sites, 2016—2019. MMWR Morb Mortal Wkly Rep. 2020;69:509-14. . However, when considering bacterial foodborne disease outbreaks, it is the third most common (4%), after Salmonella spp. (19%) and C. perfringens (6%) 1111. Centers for Disease Control and Prevention. Surveillance for foodborne disease outbreaks, United States, 2017: annual report. Atlanta: CDC; 2019. . The campylobacteriosis foodborne outbreaks in Brazil occurred in 2001, 2003, 2005, 2014, 2016 and 2019 and involved fruits, desserts, water, mixed foods, milk, dairy products and poultry meat. In total 299 people fell ill 55. Brasil. Ministério da Saúde. Doenças transmitidas por alimentos: causas, sintomas, tratamento e prevenção. [cited 2020 Oct 29]. Available from: https://antigo.saude.gov.br/saude-de-a-z/doencas-transmitidas-por-alimentos
https://antigo.saude.gov.br/saude-de-a-z...
.

On the other hand, the competing microbiota could have hindered the detection of low levels of Campylobacter in the foods, since a relatively low dose (500 CFU) is able to cause intestinal colonization and trigger acute enteritis 1212. Rosenquist H, Nielsen NL, Sommer HM, Nørrung B, Christensen BB. Quantitative risk assessment of human campylobacteriosis associated with thermophilic Campylobacter species in chickens. Int J Food Microbiol. 2003;83:87-103. . Campylobacter -negative samples may reflect the effect of several intrinsic and extrinsic factors to which the foods were exposed to. For example, Campylobacter cells can suffer sublethal injuries caused by oxidative stress 1313. Oh E, McMullen L, Jeon B. Impact of oxidative stress defense on bacterial survival and morphological change in Campylobacter jejuni under aerobic conditions. Front Microbiol. 2015;6:295. , acidic conditions 1414. Chaveerach P, ter Huurne AA, Lipman LJ, van Knapen F. Survival and resuscitation of ten strains of Campylobacter jejuni and Campylobacter coli under acid conditions. Appl Environ Microbiol. 2003;69:711-4. , low temperatures 1515. Chaisowwong W, Kusumoto A, Hashimoto M, Harada T, Maklon K, Kawamoto K. Physiological characterization of Campylobacter jejuni under cold stresses conditions: its potential for public threat. J Vet Med Sci. 2012;74:43-50. or excessive use of disinfectants and preservative additives 1616. Meyer B. Does microbial resistance to biocides create a hazard to food hygiene? Int J Food Microbiol. 2006;112:275-9. , which might trigger the viable but non-culturable (VBNC) state. It has been hypothesized that VBNC Campylobacter cells may proliferate in the human intestine and cause infection 1717. Baffone W, Casaroli A, Citterio B, Pierfelici L, Campana R, Vittoria E, et al. Campylobacter jejuni loss of culturability in aqueous microcosms and ability to resuscitate in a mouse model. Int J Food Microbiol. 2006;107:83-91.1919. Ayrapetyan M, Oliver JD. The viable but non-culturable state and its relevance in food safety. Curr Opin Food Sci. 2016;8:127-33. . Despite the presence of VBNC forms that were not evaluated in this study and the lack of evidence that their presence in foods could trigger campylobacteriosis in consumers, the public health risk should not be ignored.

At least one of the other pathogenic and nonpathogenic bacteria tested was detected in 29.17% (21/72) of the analyzed samples ( Figure 2 ), involved in 17 of the 36 investigated outbreaks. Five samples presented contamination by more than one bacterial agent (data not shown). Among the microorganisms that are not normally harmful but may be used as an indirect indicator of health hazard in food, non-pathogenic E . coli was detected in 20.83% of the analyzed samples (15/72, Figure 2). E . coli was the second most frequently reported microorganism in foodborne disease outbreak investigations in Brazil from 2000 to 2019 55. Brasil. Ministério da Saúde. Doenças transmitidas por alimentos: causas, sintomas, tratamento e prevenção. [cited 2020 Oct 29]. Available from: https://antigo.saude.gov.br/saude-de-a-z/doencas-transmitidas-por-alimentos
https://antigo.saude.gov.br/saude-de-a-z...
. Such a number includes non-pathogenic E . coli and Shiga toxin-producing E. coli55. Brasil. Ministério da Saúde. Doenças transmitidas por alimentos: causas, sintomas, tratamento e prevenção. [cited 2020 Oct 29]. Available from: https://antigo.saude.gov.br/saude-de-a-z/doencas-transmitidas-por-alimentos
https://antigo.saude.gov.br/saude-de-a-z...
. Most of the samples evaluated in this study (51/72, 70.83%) were negative for the routinely tested bacteria. Other foodborne microorganisms, such as zoonotic viruses, may be related to outbreak-associated foodborne diseases, which, however, were not focused on in this study. Similar frequency of food-related bacteria has been reported in foodborne disease outbreaks between 2009 and 2018 in Brazil, whose etiological agent was laboratory-confirmed in approximately 30% of the cases 55. Brasil. Ministério da Saúde. Doenças transmitidas por alimentos: causas, sintomas, tratamento e prevenção. [cited 2020 Oct 29]. Available from: https://antigo.saude.gov.br/saude-de-a-z/doencas-transmitidas-por-alimentos
https://antigo.saude.gov.br/saude-de-a-z...
. Despite the number of cases in which the involved food was not identified, the closure of foodborne disease outbreaks investigations has been mostly achieved based on epidemiological criteria without considering the laboratory result 2020. Finger JA, Baroni WS, Maffei DF, Bastos DH, Pinto UM. Overview of foodborne disease outbreaks in Brazil from 2000 to 2018. Foods 2019;8:434. . Moreover, the attribution of outbreaks to any of the agents detected during this study depends on the establishment of a clear epidemiological relationship.

Figure 2
Distribution of the pathogenic and non-pathogenic bacteria identified in the foods involved in foodborne disease outbreaks in Rio Grande do Sul, Southern Brazil, in the period 2015-2016.

In conclusion, the foods analyzed in the studied period tested negative for thermotolerant Campylobacter . Nevertheless, other bacteria were laboratory-confirmed in 29.17% of the samples. Taken together, the results suggest the need to monitor Campylobacter in foodborne diseases to detect sporadic Campylobacter-associated cases that might go unnoticed in Rio Grande do Sul.

ACKNOWLEDGMENTS

We thank LACEN/CEVS/SES-RS and its technical team who has assisted this study, and PUCRS for the research grant to MCB. This work was supported by the National Council for Scientific and Technological Development — CNPq (grant N° 477431/2013-5).

REFERENCES

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    European Food Safety Authority, European Centre for Disease Prevention and Control. The European Union summary report on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks in 2017. EFSA J. 2018;16:5500.
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    Tack DM, Ray L, Griffin PM, Cieslak PR, Dunn J, Rissman T, et al. Preliminary incidence and trends of infections with pathogens transmitted commonly through food — Foodborne Diseases Active Surveillance Network, 10 U.S. sites, 2016—2019. MMWR Morb Mortal Wkly Rep. 2020;69:509-14.
  • 3
    Silva WC, Targino BN, Mendonça RS, Sant'Ana AS, Hungaro HM. Campylobacter: an overview of cases, occurrence in food, contamination sources and antimicrobial resistance in Brazil. Food Rev Int. 2018;34:364-89.
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    Silva DT, Tejada TS, Blum-Menezes D, Dias PA, Timm CD. Campylobacter species isolated from poultry and humans, and their analysis using PFGE in southern Brazil. Int J Food Microbiol. 2016;217:189-94.
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    » https://portalarquivos2.saude.gov.br/images/pdf/2019/fevereiro/15/Apresenta––––o-Surtos-DTA–––Fevereiro-2019.pdf
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    Downes FP, Ito K, editors. Compendium of methods for the microbiological examination of foods. 4th ed. Washington: APHA; 2001.
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    Brasil. Ministério da Saúde. Agência Nacional de Vigilância em Saúde. Resolução - RDC n° 12, de 2 de janeiro de 2001. Aprovar o regulamento técnico sobre padrões microbiológicos para alimentos. Diário Oficial da União, Brasília, 10 jan. 2001. [cited 2020 Oct 29]. Available from: http://portal.anvisa.gov.br/documents/33880/2568070/RDC_12_2001.pdf/15ffddf6-3767-4527-bfac-740a0400829b
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    Kaakoush NO, Castaño-Rodríguez N, Mitchell HM, Man SM. Global epidemiology of Campylobacter infection. Clin Microbiol Rev. 2015;28:687-720.
  • 11
    Centers for Disease Control and Prevention. Surveillance for foodborne disease outbreaks, United States, 2017: annual report. Atlanta: CDC; 2019.
  • 12
    Rosenquist H, Nielsen NL, Sommer HM, Nørrung B, Christensen BB. Quantitative risk assessment of human campylobacteriosis associated with thermophilic Campylobacter species in chickens. Int J Food Microbiol. 2003;83:87-103.
  • 13
    Oh E, McMullen L, Jeon B. Impact of oxidative stress defense on bacterial survival and morphological change in Campylobacter jejuni under aerobic conditions. Front Microbiol. 2015;6:295.
  • 14
    Chaveerach P, ter Huurne AA, Lipman LJ, van Knapen F. Survival and resuscitation of ten strains of Campylobacter jejuni and Campylobacter coli under acid conditions. Appl Environ Microbiol. 2003;69:711-4.
  • 15
    Chaisowwong W, Kusumoto A, Hashimoto M, Harada T, Maklon K, Kawamoto K. Physiological characterization of Campylobacter jejuni under cold stresses conditions: its potential for public threat. J Vet Med Sci. 2012;74:43-50.
  • 16
    Meyer B. Does microbial resistance to biocides create a hazard to food hygiene? Int J Food Microbiol. 2006;112:275-9.
  • 17
    Baffone W, Casaroli A, Citterio B, Pierfelici L, Campana R, Vittoria E, et al. Campylobacter jejuni loss of culturability in aqueous microcosms and ability to resuscitate in a mouse model. Int J Food Microbiol. 2006;107:83-91.
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    Li L, Mendis N, Trigui H, Oliver JD, Faucher SP. The importance of the viable but non-culturable state in human bacterial pathogens. Front Microbiol. 2014;5:258.
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    Ayrapetyan M, Oliver JD. The viable but non-culturable state and its relevance in food safety. Curr Opin Food Sci. 2016;8:127-33.
  • 20
    Finger JA, Baroni WS, Maffei DF, Bastos DH, Pinto UM. Overview of foodborne disease outbreaks in Brazil from 2000 to 2018. Foods 2019;8:434.

Publication Dates

  • Publication in this collection
    13 Nov 2020
  • Date of issue
    2020

History

  • Received
    03 Aug 2020
  • Accepted
    28 Oct 2020
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