Introduction

The prevention of meat product contamination is a challenge faced by the meat industry. The presence of pathogenic microorganisms in these food products may result in foodborne diseases and economic losses to their producers, such as the need to dispose of the contaminated batch and lawsuits by consumers who become ill due to the ingestion of the contaminated product.

Many studies have reported the isolation of diarrheagenic E. coli and Salmonella spp. in pork meat and its derivates (MÜRMANN et al., 2007; LIMA et al., 2011; CHARIMBA et al., 2012; RANTSIOU et al., 2012; CABRAL et al., 2014). According to the Brazilian Health Ministry, 6,848 outbreaks of foodborne diseases were reported between 2007 and 2016 that resulted in 121,283 cases of illness, 17,517 hospitalizations (14.5%), and 111 deaths (0.09%). The main etiologic agents responsible for the outbreaks were Salmonella spp. (7.3%), Escherichia coli (7.3%), and Staphylococcus aureus (5.7%). The contaminated food was not identified in majority of the reported outbreaks (66.9%). Mixed foods were the main cause of the outbreaks where the contaminated food had been identified (2,266, 33.1%). Raw pork meat and derivatives and pork offal were responsible for 103 (4.5%) of the reported outbreaks that had known etiology (BRASIL, 2017).

Eight diarrheagenic E. coli (DEC) groups are known: shiga toxin-producing E. coli (STEC), which includes the subgroup enterohemorrhagic E. coli (EHEC); typical and atypical enteropathogenic E. coli (EPECt and EPECa); enterotoxigenic E. coli (ETEC); enteroinvasive E. coli (EIEC); enteroaggregative E. coli (EAEC); diffusely adherent E. coli (DAEC); and adherent invasive E. coli (AIEC). Enteroaggregative hemorrhagic E. coli (EAHEC) O104:H4 is an emerging E. coli pathotype associated with an outbreak that occurred in Germany in May 2011. The genes used for genetic characterization of these groups are stx1 and stx2 (STEC); stx1, stx2, and eae (EHEC); eae (EPECa); eae and bfp (EPECt); enterotoxin genes LT and ST and colonization factors (ETEC); ipaH and ial (EIEC); and aggR, aap, and AA probe (EAEC) (CROXEN et al., 2013). Genes for the genetic characterization of AIEC have not yet been characterized and there is no agreement among authors for the genetic characterization of DAEC (NATARO; KAPER, 1998; CROXEN et al., 2013). EAHEC O104:H4 is an enteroaggregative E. coli with the stx2a encoding gene (BEUTIN; MARTIN, 2012).

The incidence of gastroenteritis caused by EAEC and EPEC, specially EPECa, has increased in Brazil (REGUA-MANGIA et al., 2004; FRANZOLIN et al., 2005; MORENO et al., 2010; LOZER et al., 2013; DIAS et al., 2016). In the last few years, EPECa was isolated as the cause of gastroenteritis in Brazil in more cases than EPECt, and as a shiga toxin-producing E. coli (STEC), it is an emerging food pathogen (SCHMIDT, 2010).

EPECa serotypes have already been reported in the feces of pig, and pork meat may be a vehicle of transmission of this bacterium to humans. The infection of pig herds by Salmonella tends to be a persistent problem in food production systems. Submandibular lymph nodes and tonsils of pigs contaminated by Salmonella spp. may remain in the carcasses after slaughter, and combined with head region muscles, be used in processed meat manufacturing.

Brazil is the fourth largest producer and exporter of pork meat. In 2016, Brazil produced about 3.7 million tons and 19.6% of this was exported. In Brazil, the per capita consumption of pork meat in 2016 was 14.4 kg. Paraná is the second largest producer of pork meat and produced 13.2% of the total of the pork meat exported by Brazil in 2016. In this year, 13.2% of swine slaughtered in Brazil was in Paraná (ABPA, 2017).

In Londrina, Paraná, Brazil and the surrounding areas there are some meat processing industries that commercialize their products in free fairs, small markets, bars, and restaurants of the city. The Municipal Inspection Service of Londrina is responsible for the inspection of these industries. However, there are no data about the pathogenic microorganisms present in these products.

The aims of this study were to evaluate the presence of Salmonella spp. in sausage samples produced and commercialized in Londrina, and identify eae, bfp, stx1, stx2, hlyA, ipaH, elt, aggR, aap, and AA probe genes in E. coli strains isolated from the sausage samples.

Material and Methods

Sampling

Forty-six samples of three types of fresh sausages (22 fresh pork, 17 Tuscan, and 7 Calabresa) produced by four different producers of Londrina, Paraná, Brazil and the surrounding areas were purchased weekly from March to July 2015 in four supermarkets of Londrina. The brands were nominated as A, B, C, and D. The number of samples from brands C and D were smaller because these brands were not always available for purchase.

Salmonella spp. detection

Salmonella spp. detection was performed according to Silva et al. (2017). Twenty-five grams of each sample was homogenized in 225 mL of buffered peptone water in a Stomacher blender (Stomacher 400, Seward, Worthing, England). After incubation at 35°C for 24 h, 1.0 mL and 0.1 mL aliquots of the non-selective enrichment were inoculated, respectively, in 10 mL of Selenite Cystine Broth (SC) (Himedia Laboratories Pvt. Ltd., Mumbai, India) and in 10 ml of Rappaport-Vassiliadis (RV) broth (Himedia Laboratories). The SC broth was incubated at 35°C and RV broth at 42°C for 24 h. Hektoen agar (Himedia Laboratories) and Xylose Lysine Deoxycholate (XLD) (Himedia Laboratories) used as selective and differential media, were incubated at 35°C for 24 h. Biochemical screening using motility, production of indole, decarboxylation of lysine, and hydrolysis of urea was carried out with the characteristic colonies of Salmonella spp. The confirmation of Salmonella genus was performed using the agglutination technique with polyvalent antiserum anti-Salmonella spp. according to the manufacturer's recommendation (Probac do Brasil, São Paulo, Brasil).

Isolation of E. coli

E. coli isolation was carried out using traditional culture media according to Silva et al. (2017). Twenty-five grams of each sample was homogenized in 225 mL of buffered peptone water in a Stomacher blender (Stomacher 400). Aliquots of 1 mL were inoculated in tubes containing 10 mL of sodium lauryl sulfate (SLS) (Himedia Laboratories) and incubated at 35°C for 24 h. After incubation, SLS tubes were inoculated in eosin methylene blue (EMB) (Himedia Laboratories) and xylose lysine deoxycholate agar (XLD) (Himedia Laboratories) and incubated at 35°C for 24 h. The colonies that were lactose positive and non-H₂S producing were submitted to the following biochemical screening: production of indole, lactose fermentation with gas production, and citrate as the sole source of carbon. The E. coli strains were stored in nutrient agar (Himedia Laboratories).

DNA extraction of E. coli strains for use in PCR

E. coli strains stored in nutrient agar were resuspended in sterile saline solution and cultivated on MacConkey agar (Himedia Laboratories Pvt. Ltd., Mumbai, India). After incubation at 35°C for 24 h, colonies were resuspended in 200 µL of sterile water, boiled in a water bath for 10 min, and centrifuged at 10,000 ×g for 5 min. The supernatant was used as DNA target in the amplification reactions.

Positive and negative controls used in the PCR assays

The diarrheagenic E. coli strains used as positive controls in PCR assays were EHEC ATCC® 43889(tm) (stx2+, eae+, and hlyA+), EPEC 2348/69 (eae+ and bfp+), EHEC H30 (stx1+), EIEC O152 (ipaH+), ETEC H10407 (elt+), ETEC B41 (est+), and EAEC O42 (aggR+). E. coli K12, Hb101, and DH5α were used as negative controls.

PCR conditions for the detection of the virulence genes of diarrheagenic E. coli

The virulence genes of diarrheagenic E. coli were detected using the following virulence markers: eae (a gene that encodes intimin present in EPEC and EHEC), bfp (a gene that encodes bfp present in typical EPEC), aggR (a transcriptional activator for AAFs in EAEC), elt and est (genes that encoded LT and ST toxins in ETEC), ipaH (a gene present in the invasion plasmid that encodes the H antigen in EIEC), stx1 and stx2 (genes that encode the shiga toxin in STEC), and hlyA (a gene that encodes enterohemolysin in EHEC). The primer sequences and sizes of the amplified DNA are described in Table 1. The conditions used in the PCR assays are presented in Table 2.

All amplification reactions were performed in Veriti^® thermocycler (Applied Biosystems®, Foster City, CA, USA). Aliquots of 10 µL of each reaction were homogenized with 1 µL 20x Red Gel (Biotium^®, Hayward, CA, USA). The electrophoresis was carried out on 2% agarose in 1x Tris-Borate-EDTA buffer [Tris (Invitrogen^®) 90 mM; boric acid (Nuclear, Diadema, São Paulo) 90 mM, EDTA (Nuclear) 2 mM] at 70 V for 100 min. A DNA fragment of 1 Kb (Invitrogen^®) was used as molecular size marker. Gels were viewed under ultraviolet light (UV) (Loccus Biotecnologia Molecular, Cotia, São Paulo, Brasil) and photo-documented (L-Pix Image Versão 1.21, Loccus Biotecnologia Molecular). Samples showing bands corresponding to the hlyA gene in the multiplex PCR were confirmed by uniplex PCR using only the hlyA primers.

Serotyping of diarrheagenic E. coli

After detection of the virulence genes of diarrheagenic E. coli by PCR, one of the E. coli strains that harbored the eae and hlyA genes was serotyped at the Health Public Department, Medicine Faculty, National University of Mexico, Mexico City.

Results and Discussion

Salmonella spp. were isolated in 13 (28.3%) and E. coli in 33 (71.3%) of the 46 pork sausage samples analyzed in this study. The contamination by Salmonella spp. was higher in Tuscan sausage samples (8/17, 41.7%) than in fresh pork sausage samples (4/22, 18.2%). E. coli were isolated in 12 of 13 samples positive for Salmonella spp. (Table 3). Seven (53.8%) of 13 samples contaminated with Salmonella spp. were from brand A. Additionally, five from the eight samples of Tuscan sausages positive for Salmonella spp. (62.5%) were also from this brand. All samples of fresh pork and Tuscan sausage from brand A were contaminated by E. coli. A high percentage of contamination by E. coli also was found in fresh pork and Tuscan sausages from brand B (12/15, 80.0%).

The number of samples from brands C (n=5) and D (n=5) were smaller than those from brands A (n=18) and B (n=18). However, E. coli and Salmonella were also isolated in samples from these two brands. Two out of seven samples of Calabresa sausage from brand D were contaminated with Salmonella spp. One of the samples was also contaminated with E. coli serotype O108:H9 that harbored the genes eae and hlyA.

Other studies conducted in the South of Brazil have reported percentages of isolation of Salmonella spp. in sausages similar to that found in this work (13/46, 28.3%). Mürmann et al. (2009) and Souza et al. (2014) detected Salmonella spp. in 24.4% (84/336) and 30.0% (6/20) of the fresh sausage samples analyzed in Porto Alegre, RS and in Cascavel, PR, respectively. In Niteroi, Rio de Janeiro, the percentage of fresh sausages contaminated with Salmonella spp. was higher than that found in the present study and ranged from 37.0% (10/27) to 53.0% (58/91) (LIMA et al., 2011; CABRAL, et al., 2014). However, in other studies conducted in Brazil, the contamination of pork meat and its derivatives was much lower, with percentages ranging from 5.0 to 17.9% (CHAVES et al., 2000; LOBO et al., 2001; SPRICIGO et al., 2008a; BEZERRA et al., 2012).

The prevalence of Salmonella spp. in the brand B samples may be related to the contamination of the pork meat or other ingredients used in the production of some batches. Two fresh pork and one Tuscan sausage sample was collected at an interval of four months. Salmonella spp. was not isolated from samples collected in the intermediate period. However, it should be noted that E. coli was isolated in 12 (80.0%) of the 15-fresh pork and Tuscan sausage samples of this brand, which indicated inadequate hygienic-sanitary conditions of processing, or the contamination of raw pork meat.

Lima et al. (2011) in Niterói, Rio de Janeiro analyzed the surface of sausages using swabs and subsequently the meat from these same samples. The meat of 33.0% of the samples was contaminated with Salmonella spp. while contamination was only present on the surface in 11.0% of the samples. These results indicated that the contamination probably occurred due to the use of pork raw meat contaminated with Salmonella. In another study conducted by Cabral et al. (2014), there was no statistical difference in the Salmonella contamination of sausages analyzed in their original packaging compared to those repackaged by the market itself or sold in bulk to the consumer. These authors have suggested that food handling and storage in retail outlets appear to play a minor role in the contamination of sausages and that the main cause of contamination of the product is the contaminated raw pork meat used in the manufacture of sausages.

Infection of swine herds by Salmonella spp. tends to be a persistent problem in production systems. Castagna et al. (2004b) reported a high percentage of pigs carrying Salmonella spp. in submandibular lymph nodes and tonsils, which may remain in the carcass after slaughter. These parts can be used along with the head muscles in the manufacture of sausages and mechanically separated meat. These ingredients, when contaminated, may be responsible for the presence of Salmonella in those products. The removal of the tonsils from carcass after slaughter does not eliminate the risk of contamination, but it can be reduced in about 30.0%. The removal of the submandibular lymph nodes during processing is difficult because they are firmly attached to the musculature of the head (CASTAGNA et al., 2004a).

It is almost impossible to avoid cross-contamination of carcasses during the slaughter of swine. The use of only Good Manufacturing Practices in the slaughter process does not seem to be effective in reducing the meat contamination by Salmonella spp. More effective measures are related to the production of Salmonella spp. free swine and decontamination of the carcasses (BERENDS, 1998).

The percentage of contamination, 12.6%, found in pork meat samples produced in Portugal is much higher than the percentage reported in the European Union (0.7%). This difference in contamination can be explained by the fact that Portugal does not have a national program for the control of Salmonella in primary production, although it has a monitoring system for the contamination of pork carcasses after slaughter. Control strategies should be implemented on farms to reduce carcass contamination because cross-contamination during slaughter is the most common form of contamination of pork meat and its derivatives (XAVIER et al., 2014).

The highest frequency of Salmonella isolation in the present study was in the Tuscan sausage samples from brand A (71.4%). Samples were collected weekly over a period of three months, suggesting possible contamination of the processing plant and not contamination of a single batch of the product. Two of the nine fresh pork sausage samples from the same brand were also positive for Salmonella.

Alban et al. (2002) reported contamination of <50 to 400 CFU of Salmonella spp./cm² on the surface of the swine carcass, with a reduction of 2.0 to 3.0 log during the sausage manufacture. Mataragas et al. (2015) found that Salmonella enterica contamination can be reduced by 1.0 log or more during the sausage making process, in which changes in pH and water activity occur. Inhibition of multiplication occurs at pH values below 5.3 and therefore, the time and temperature at which raw meat remains at a pH above 5.3 are critical (LUCKE, 2000; ICMSF, 2005).

Although the reduction of contamination during sausage production may occur in Brazil, Mürmann et al. (2007) reported a count ranging from 0.03 NMP g^-1 to 460 NMP g^-1 and Spricigo et al. (2008b) found counts ranging from <3 NMP g^-1 to 1100 NMP g-1 in the 54 positive samples analyzed. The Brazilian legislation defines that Salmonella spp. must be absent in 25 g of the analyzed sample (BRASIL, 2001).

None of the E. coli isolates analyzed in this study presented virulence genes used in the genetic characterization of enteroaggregative E. coli (EAEC), enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIE), or shiga toxin-producing E. coli (STEC). A DEC strain was detected in a Calabresa sausage sample from brand D. This isolate harbored the genes eae and hlyA and was identified as EPECa O108: H9. The O108 serogroup was already associated with an outbreak of human gastroenteritis in the city of Mairiporã, SP, Brazil (VIEIRA et al., 2016) and the serotype O108: H9 has already been isolated from swine feces (FROHLICHER el al., 2008; MALIK et al., 2006). Although there is no evidence of transmission from animals to humans, it is believed that some isolates of EPECa could be zoonotic pathogens (GOMES et al., 2016).

The prevalence of infections caused by EPEC varies considerably according to age, socioeconomic status, and geographic region of the populations studied. EPECt isolates have always been strongly associated with childhood diarrhea; however, recent studies have shown a higher frequency of EPECa as a cause of diarrhea in Brazil and other South American countries (GOMES et al., 2004; ARAUJO et al., 2007; OCHOA et al., 2009; HANNAOUI et al., 2010; LOZER et al., 2013; RUGÉLES et al., 2010; SCHMIDT, 2010; ASSIS et al., 2014; GOMES et al., 2016).

Isolates of EPEC are classified as typical when they have the adhesion factor plasmid (EAF) and the bfp gene, in addition to the eae gene. The serogroups of EPECa differ from those of EPECt, indicating that few EPECa isolates are EPEC isolates that have lost the EAF plasmid (SCHMIDT, 2010). Unlike EPECt, many EPECa isolates are found in several sick or healthy animal species. Recently, Borges et al. (2017) isolated EPEC (typical and atypical) and STEC in wild birds and feral pigeons in the city of São Paulo.

E. coli O108: H9 strain isolated in this study from Calabresa sausage did not present the stx gene. Enterohemorrhagic E. coli (EHEC) is a subtype of STEC that encodes, besides the stx gene, hlyA and eae genes. The eae gene is responsible for the attaching and effacing (AE) lesion (CROXEN et al., 2013). According to Bielaszewska et al. (2007), EHEC isolates may lose stx genes during infection, isolation, or subculture. We do not believe that the serotype O108: H9 isolated in this study had lost the stx gene because it is not one of the classic serotypes of shiga toxin-producing E. coli (O157:H7, O26, O45, O103, O111, O121, and O145) (CROXEN et al., 2013).

Some brands of Calabresa sausages are subject to a cooking process during their production, allowing their consumption without previous cooking. Tuscan and pork sausages, however, are raw and prepared in barbecue or oven grills, baked in conventional ovens, or deep fried. Therefore, the risk for human health that this product may present is more related to the cross contamination to other foods, especially those ingested raw, or the ingestion of the sausage after inadequate thermal process (ESCARTIN et al., 2000; GORMAN et al., 2002).

The standardization and control of all stages of the sausage process are crucial to obtain a product free of contamination. Excessive handling, inappropriate sanitation of instruments and equipment, the absence or deficiency of Good Manufacturing Practices and Hazard Analysis Critical Control Points (HACCP) system affect the sanitary quality of this type of food. In addition, the lack of proper preparation instructions can lead to cross-contamination and consumption of contaminated products.

Conclusion

Salmonella spp. and E. coli were isolated in 28.3% and 71.3% of the analyzed samples, respectively. A sample of Calabresa sausage was contaminated with atypical enteropathogenic E. coli from serotype O108: H9, which harbored the eae and hlyA genes. The results suggest possible contamination of the processing plant and/or raw meat used in the manufacture of sausages. A better inspection of the industries is required to ensure that good manufacturing practices are followed and thus the contamination of the product by pathogenic bacteria be prevented.

Acknowledgment

The authors express their gratitude to the CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) for the Master's scholarship of Paola Bianca Barbosa Cavalin and to the CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) for the financial support. The serotyping of E. coli was performed with the financial support of the Dirección General de Asuntos del Personal Académico (DGAPA), Programa de Apoyo a Proyectos de Investigación e Innovación Tecnológica (PAPIIT), project IN216417, UNAM, Mexico.

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Author notes

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