The quality and safety of meat products can be estimated by assessing their contamination by hygiene indicator microorganisms and some foodborne pathogens, with Listeria monocytogenes as a major concern. To identify the main sources of microbiological contamination in the processing environment of three butcher shops, surface samples were obtained from the hands of employees, tables, knives, inside butcher displays, grinders, and meat tenderizers (24 samples per point). All samples were subjected to enumeration of hygiene indicator microorganisms and detection of L. monocytogenes, and the obtained isolates were characterized by their serogroups and virulence genes. The results demonstrated the absence of relevant differences in the levels of microbiological contamination among butcher shops; samples with counts higher than reference values indicated inefficiency in adopted hygiene procedures. A total of 87 samples were positive for Listeria spp. (60.4%): 22 from tables, 20 from grinders, 16 from knives, 13 from hands, 9 from meat tenderizers, and 7 from butcher shop displays. Thirty-one samples (21.5%) were positive for L. monocytogenes, indicating the presence of the pathogen in meat processing environments. Seventy-four L. monocytogenes isolates were identified, with 52 from serogroups 1/2c or 3c and 22 from serogroups 4b, 4d, 4a, or 4c. All 74 isolates were positive for hlyA, iap, plcA, actA, and internalins (inlA, inlB, inlC, and inlJ). The establishment of appropriate procedures to reduce microbial counts and control the spread of L. monocytogenes in the final steps of the meat production chain is of utmost importance, with obvious effects on the quality and safety of meat products for human consumption.
Meat and meat products are routinely associated with food poisoning outbreaks. During production, processing, and storage, these products are subjected to contamination by pathogenic bacteria, including Listeria monocytogenes (8, 27). For meat products to comply with international standards of quality and safety, one must constantly monitor the hygiene and quality standards in the handling and processing (23) environments of meat products.
Monitoring quality systems are often done by the food industry, and they are applied for the systematic research of indicators on specific points of the processing line and handling of meat products (16). The efficiency of these systems is proven, and it is possible to take corrective measures from these indicators to minimize or avoid possible contamination; the safety and quality of meat products can be estimated through research on various microorganism indicators, such as aerobic mesophilic enumeration and coliforms (14). The aerobic mesophilic enumeration provides an estimate of the overall population of microorganisms present in the environment and items used in the processing of meat products, where high contamination levels are associated with poor hygienic conditions (15). Coliforms are indicators of inadequate sanitary hygienic conditions in the processing, production, and storage of food: Escherichia coli is the classic indicator of the probable presence of enteric pathogenic microorganisms and a good indicator of the sanitary quality of processed foods (12). In addition, direct testing for pathogens is critical to ensure the safety of meat products; therefore, pathogens often associated with meat and meat products, such as L. monocytogenes, must be investigated and controlled in the processing environment (8, 13, 23).
The control of L. monocytogenes in the handling and processing environment of meat products is challenging, because this pathogen survives and multiplies at refrigeration temperatures, tolerates harsh environmental conditions, and has a great ability to form biofilms on processing equipment surfaces (13). Studies show that postprocessing contamination by L. monocytogenes is extremely relevant. Often, the sources of contamination of the final product are the processing equipment, particularly during the cutting and packaging of meat products, and the employees' hands and clothing (24). L. monocytogenes can persist in meat processing plants for long periods, even years, indicating that routine cleaning can be ineffective in removing this pathogen (32).
This study evaluated the level of hygiene and safety of utensils and butcher equipment by measuring hygiene indicator microorganisms and L. monocytogenes, including its potential virulence, and characterized the main sources of contamination.
MATERIALS AND METHODS
Butcher shops and sampling.
Three butcher shops located in Viçosa, Minas Gerais, Brazil, were included in this study with the agreement of the owners and under the official control of the municipality's health surveillance. In these properties, the handling and sale of meat from different animal species and fabrication of meat products and ready-to-eat meat products, especially sausages, occurred. The butcher shops were designated as A, B, and C. Each establishment was visited on eight occasions, when surface samples of manipulation environments (inside the butcher shop display, the hands of employees, and cutting surfaces of the tables), equipment (grinder and meat tenderizer), and utensils (knife) were sampled. Samples were obtained during the usual procedures conducted by the employees in the butcher shops. Table 1 shows the samples and sampling procedure details. Selected points were sampled by surface swabbing two areas of 50 cm2 (a limited area with a sterile template 5 by 10 cm), using two sterile sponges (3M Microbiology, St. Paul, MN) previously moistened in 10 ml of NaCl 0.85% (wt/vol) with buffered peptone solution (0.1% wt/vol; Oxoid Ltd., Basingstoke, UK) and kept under refrigeration until analysis. Each set of two sponges per sample was transferred to a sterile bag, and 80 ml of buffered peptone solution was added (final volume of 100 ml per sample) homogenized for 60 s (Stomacher 400 Circulator, Seward Ltd., Worthing, UK) and subjected to microbiological analysis. Based on this procedure, 1 ml of the obtained homogenate corresponded to 1 cm2 for each sample (9).
Enumeration of hygiene indicator microorganisms.
An aliquot of each collected sample was subjected to 10-fold dilution by using NaCl 0.85% (wt/vol). Two dilutions of each sample were selected considering their likely level of microbiological contamination and plated on Petrifilm Aerobic Count (3M Microbiology) for the enumeration of mesophilic aerobes (MA) bacteria, Petrifilm Enterobacteriaceae (3M) for enumeration of Enterobacteriaceae, and E. coli Petrifilm (3M) to enumerate total coliforms (TC) and E. coli. All plates were incubated at 35°C for 24 to 48 h when the colonies were counted considering the phenotypic characteristics of each group (MA: red colonies formed after 24 h; Enterobacteriaceae: yellow colonies associated or not with gas, associated gas, and colonies formed after 24 h; TC: red and blue colonies associated with gas, formed after 24 h; E. coli: blue colonies associated with gas, formed after 48 h). All results were expressed as CFU per square centimeter. MA, Enterobacteriaceae, TC, and E. coli counts were converted to log, evaluated for normal distribution and homogeneity, and subjected to analysis of variance (ANOVA) and Tukey tests to identify significant differences between butcher shops (P < 0.05). In addition, samples were categorized according to their levels of contamination, based on reference values of good hygienic conditions: 50 CFU/cm2 for MA and Enterobacteriaceae and 10 CFU/cm2 (technique detection limit) for CT and E. coli (19). These frequencies were compared by using chi-square tests and the Marascuilo procedure to identify significant differences between the butcher shops (P < 0.05). All analyses were performed by using Statistica 7.0 (StatSoft Inc., Tulsa, OK) and XLSTAT 2010.2.03 (AddinSoft, New York, NY).
Isolation of Listeria spp. and L. monocytogenes.
Surface samples were checked for the presence of Listeria spp. and L. monocytogenes, according to International Organization for Standardization 11290-1 (20, 21). An aliquot of 40 ml of the suspension obtained as described previously was centrifuged, and the pellet was resuspended in half Fraser broth (Oxoid Ltd.) and incubated at 30°C for 24 h. Then, obtained cultures were streaked onto Listeria agar chromogenic agar (Oxoid Ltd.) and Listeria selective base (Oxford, Oxoid Ltd.) and incubated at 37°C for 48 h. Simultaneously, a 0.1-ml aliquot was transferred to a tube containing 10 ml of Fraser broth (Oxoid Ltd.) and incubated at 35°C for 24 h. Then, the obtained cultures were streaked onto plates containing ALOA and Oxford agar and incubated at 37°C for 48 h. The obtained colonies were subjected to biochemical tests for the production of catalase, motility at 25°C, fermentation of carbohydrates (dextrose, xylose, rhamnose, and mannitol), and production of β-hemolysis on horse blood agar and accordingly identified. The presence or absence of Listeria spp. and L. monocytogenes was expressed per 40 cm2, corresponding to the analyzed aliquot. The frequency of positive samples for Listeria spp. and L. monocytogenes were compared using chi-square test and the Marascuilo procedure for checking significant differences between the butcher shops (P < 0.05). All analyses were performed by using Statistica 7.0 (StatSoft Inc.) and XLSTAT 2010.2.03 (AddinSoft).
Characterization of L. monocytogenes serogroups.
Isolates identified as L. monocytogenes were subjected to DNA extraction and purification by using the Wizard Genomic DNA Purification Kit (Promega Corp., Madison, WI). The extracted DNA from each isolate was subjected to serogrouping by PCR, for categorization of the main phylogenetic divisions by using primers D1 (division I and III, including serogroups 1/2b, 3b, 4b, 4d, 4e, 4a, and 4c) and D2 (division II, including serotypes 1/2a, 1/2c, 3a, and 3c), as described by Borucki and Call (4). The amplification mix was composed of 12.5 μl of GoTaq Green Master Mix (Promega Corp.), 2 μl of DNA, 4 μl of primers D1 and D2, and ultrapure water (Promega Corp.) to bring the total to 25 μl. Two additional PCR reactions were conducted as described, using the primers FlaA and GLT. PCR conditions were 95°C for 3 min, 25 cycles at 95°C for 30 s, annealing temperature for each reaction for 30 s (D1 and D2: 49°C; flA: 54°C; GLT: 45°C), 72°C for 1 min, and 72°C for 10 min. The sequences of each primer used are shown in Table 2. Aliquots of 5 μl of the PCR products were subjected to electrophoresis on 2.0% (mass/vol) agarose gels with 0.5 M Tris-borate-EDTA, stained with GelRed (Biotium Inc., Hayward, CA), and visualized on a transilluminator. For each target DNA region, the following PCR product sizes were observed: 214 bp for D1, 140 bp for D2, 538 bp for flaA, and 483 bp for GLT. In all molecular assays, L. monocytogenes Scott A and ATCC 7644 were used as positive controls. Isolates that were positive for D1 and negative for the GLT gene were classified as serogroups 4b and 4d (division I) or 4a and 4c (division III, rarely found). The isolates that were positive for D1 and GLT were classified as belonging to serogroup 1/2b or 3b. The isolates positive for D2 and negative for the flaA gene were classified as belonging to serogroup 1/2c or 3c (division II), and positive results for flaA characterize serotype 1/2a or 3a (division II) (4).
Virulence potential of L. monocytogenes.
Finally, L. monocytogenes isolates were subjected to molecular analysis to verify the presence of virulence genes, according to the protocols described by Liu et al. (25) and Rawool et al. (29). Multiplex and simplex PCR reactions were used for detection of the following virulence genes: inlA, inlB, inlC, inlJ, plcA, hlyA, actA, and iap. As described by Liu et al. (25), the reaction conditions for multiplex PCR (inlA, inlC, and inlJ) were 94°C for 2 min, 30 cycles of 94°C for 20 s, 55°C for 20 s, 72°C for 50 s, and 72°C for 2 min. The conditions for the simplex PCR (inlB) were 94°C for 2 min, 30 cycles of 94°C for 20 s, 55°C for 20 s, 72°C for 50 s, and 72°C for 2 min. The conditions for the multiplex PCR for hlyA, actA, and iap were described by Rawool et al. (29): 95°C for 2 min, 35 cycles of 95°C for 15 s, 60°C for 30 s, 72°C for 1.5 min, and 72°C for 10 min. For simplex PCR (plcA), the conditions used were 95°C for 2 min, 35 cycles of 95°C for 15 s, 60°C for 30 s, 72°C for 1.5 min, and 72°C for 10 min. The reactions included 12.5 μl of GoTaq Green Master Mix (Promega Corp.), 2.0 μl of extracted DNA, 1.0 μl of each primer, and PCR ultrapure water (Promega Corp.) to bring the total volume to 25 μl. PCR products were separated and visualized, as described in the characterization of serogroups. L. monocytogenes ATCC 7644 was considered as a positive control for the tested virulence genes.
RESULTS AND DISCUSSION
The results from the enumeration of hygiene indicator microorganisms for each butcher shop are shown in Table 3. Overall, the results showed that there were no significant differences in the levels of contamination by hygiene indicator microorganisms enumerated among the three butcher shops (P > 0.05).
All sources in the three butcher shops showed some count for both the MA and Enterobacteriaceae (Table 3). The presence of these microorganisms is indicative that all surfaces in the butcher shops are being used without proper hygiene and that food produced on these surfaces has some risk of spoilage. In particular, MA are able to multiply at temperatures of 35 to 37°C under aerobic conditions, and high counts indicate unsanitary conditions. Enterobacteriaceae enumeration has been used as an important tool for quality systems, indicating when deficiencies in hygiene are present; this group harbors pathogenic microorganisms, such as Salmonella spp., posing a hazard to consumers (19).
Butcher shop B showed a contamination level for TC in its grinder that was significantly higher than the other butcher shops (Table 3; P < 0.05). Coliforms are a group of enterobacteria present in the stool, the environment, soil, and varied surfaces, such as equipment and utensils. This group includes different genera of microorganisms, such as Escherichia, Enterobacter, Citrobacter, and Klebsiella. Because their primary habitat is the animal's intestinal tract, the microorganisms suggest fecal contamination when present (19). In addition, this group indicates inadequate sanitary conditions during processing, production, or storage, and high counts can mean postprocessing contamination, poor cleaning, and disabled sanitization.
Table 4 shows the frequencies of samples with counts above reference values of hygiene indicator microorganisms. There were no statistical differences between the three butcher shops, indicating that the status of contamination between them was equivalent. Samples with counts above reference values indicate inefficiency in cleaning techniques adopted in the three butcher shops (Table 4). From 144 samples analyzed, 54 (37.5%) presented MA counts above the reference value, while 24 samples (16.7%) presented Enterobacteriaceae counts above the reference value. For TC and E. coli, the number of samples with counts above the reference values were seven (4.9%) and three (2.1%), respectively (Table 4).
Samples containing counts above MA reference values were more frequent in grinders (11 samples), tables, and inside butcher shop displays (10 samples), and tenderizers and employees' hands (9 samples). As recommended by the standards of the U.S. Food and Drug Administration and the American Public Health Association, a utensil having less than 100 CFU is considered clean, while for equipment, a score of 2 CFU/cm2 is tolerated (19).
For Enterobacteriaceae, the grinders had the highest frequency of samples with counts above the reference value (Table 4). Because they are difficult to sanitize, tenderizers and grinders can accumulate organic matter, encouraging microbial growth and reducing the efficiency of sanitization procedures. In identifying the main points of microbiological contamination in a meat processing line in Paraná state, Brazil, Barros et al. (2) considered tenderizers and grinders as the main points of contamination by hygiene indicator microorganisms.
Regarding the TC and E. coli counts, tenderizers and grinders, as well as knives and the hands of employees, showed higher frequencies of samples with counts above the reference values (Table 4). The International Commission on Microbiological Specifications for Foods (19) emphasized mishandling as the main source of contamination between raw and processed foods and stressed the importance of cross-contamination for the transmission of pathogenic and spoilage microorganisms. Also, according to the International Commission on Microbiological Specifications for Foods (19), a satisfactory pattern for E. coli and TC is the absence of microorganisms (no count) so that counts above the reference value show inadequate hygiene practices in butcher shops and the potential of harboring enteropathogens.
Among the 144 samples analyzed, 87 (60.4%) were positive for Listeria spp., and 31 (21.5%) were positive for L. monocytogenes (Table 5). All sampled sources tested positive for Listeria spp. in all establishments analyzed. Because 60.4% of samples were positive for Listeria spp. indicates the spread of this genus in the meat processing environment, emphasizing the importance of the points examined as possible sources of contamination of the final products.
According to Table 5, the frequency of positive samples for L. monocytogenes (21.5%) showed the presence of the pathogen on the surfaces with direct and constant contact with meat products, demonstrating how final products could become contaminated. Samples with higher frequencies of Listeria spp. included tables (91.6%) and grinders (83.3%), and the frequencies of L. monocytogenes in the same samples were 20.8 and 29.1%, respectively. These utensils presented the higher frequencies of L. monocytogenes, as well as tenderizers (Table 5). Barros et al. (2) also assessed the frequency of Listeria spp. in the facilities and equipment of establishments that process meat products, describing frequencies of 51.4% (74 samples) in equipment and 35.4% (23 samples) in plants for Listeria spp. The frequencies obtained for L. monocytogenes in the same samples were 9.2% (seven samples) and 10.5% (two samples), respectively.
Table 5 also shows that butcher shop A showed a higher level of contamination by Listeria spp. and L. monocytogenes in tenderizers when compared with butcher shop B and a lower contamination level than butcher shop C (P < 0.05). The presence of these pathogens in meat product processing environments is a microbiological hazard to the final products. Listeria spp. may remain in this environment for months or even years due to their ability to form biofilms, leading to a possible contamination of the final product and potential exposure to pathogenic species (1, 3, 17, 18, 26).
Of the total 31 positive samples for L. monocyotogenes, a collection of 74 isolates was obtained. Fifty-two isolates were characterized as belonging to serogroup 1/2c or 3c, and 22 isolates belonged to serogroups 4b, 4d, 4a, or 4c. Serogroups 4a and 4c belong to division III and can be distinguished by PCR with the primer MAMA-C (22). In this study, isolates were not subjected to this PCR because these serogroups are rarely related to contamination of food products and clinical cases (4, 28). Table 6 shows the distribution of identified serogroups according to the sampled points. The obtained results were similar to studies that focused on identifying L. monocytogenes isolates from food processing environments (10, 11, 28). A frequency of 29.3% of isolates belonged to serogroups 4b, 4d, 4a, or 4c, as observed by Camargo et al. (6) who described the same serogroup in the meat processing environment.
After the characterization of virulence, all 74 isolates were positive for the hlyA, plcA, actA, and iap genes and also for the internalin group (inlA, inlB, inlC, and inlJ), as observed by Camargo et al. (7) in isolates from bovine hides and beef carcass in the state of Minas Gerais, Brazil. These genes play an important role in the virulence mechanisms of this pathogen, and they are involved in different stages of its pathogenesis (5). After invasion of the host cell, additional virulence genes are expressed, including hlyA, which is involved with listeriolysin O. The plcA gene is associated with the production of phospholipase C. The actA gene is related to the expression of the actA protein. The iap gene acts on the protein expression of p60, a protein related to invasion and survival in intestinal cells (31). In another study that evaluated isolates from a food processing environment, meat products, and clinical cases from 11 Brazilian states between the years 1978 and 2013, Camargo et al. (6) found that all L. monocytogenes strains (serotypes 4b, 1/2a, 1/2b, and 1/2c) were positive for the inlA, inlC, and inlJ genes.
Liu et al. (25) found the same genes from the group of internalins (inlA, inlB, inLC, and inlJ) in isolates of divisions I and II that belonged to serotypes recognized as more pathogenic (4b, 1/2a, 1/2b, 1/2c). In another study, Shen et al. (30) detected the presence of inlC and inlJ genes in all isolates (97) of division I and II obtained from meat products. Internalins are proteins that induce internalization of L. monocytogenes in epithelial cells, encoded by a multigene family. Two of these genes (inlA and inlB) encode two internalins particularly important for pathogenicity of L. monocytogenes. The inlC gene, which is not involved in the process of entering the epithelial cell, appears to be involved in virulence and participates in the spread of infection. The inlJ gene is directly related to the passage of L. monocytogenes through the intestinal barrier (25, 31).
According to the results obtained in the enumeration of hygiene indicator microorganisms, the sanitary conditions of meat product processing environments of the three butcher shops analyzed were found to be inadequate, showing a risk of microbiological contamination in the final product. The results showed that no significant differences were observed in the levels of contamination between the three butcher shops analyzed and the frequency of samples with counts above the reference range for each indicator showed poor hygienic practices by the facilities. Additionally, the detection of Listeria spp. indicates the spread of this genus in the butcher shop environment. The isolation of L. monocytogenes in meat product processing environments is indicative of possible contamination of the final products intended for consumers, which, in turn, may lead to cross-contamination of other foods. This study identified the presence of L. monocytogenes isolates belonging to recognized pathogenic serogroups in a butcher shop environment, as well as virulence genes in such isolates, demonstrating the potential exposure to consumers by pathogenic strains. Tables, grinders, and tenderizers have been identified as the main sources of contamination for the pathogenic species studied here, showing that more attention needs to be dedicated to cleaning care in these areas.
The authors thank Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), and Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG).