Staphylococcus aureus in Dairy Industry: Enterotoxin Production, Biofilm Formation, and Use of Lactic Acid Bacteria for Its Biocontrol.

Staphylococcus aureus is a well-known pathogen capable of producing enterotoxins during bacterial growth in contaminated food, and the ingestion of such preformed toxins is one of the major causes of food poisoning around the world. Nowadays 33 staphylococcal enterotoxins (SEs) and SE-like toxins have been described, but nearly 95% of confirmed foodborne outbreaks are attributed to classical enterotoxins SEA, SEB, SEC, SED, and SEE. The natural habitat of S. aureus includes the skin and mucous membranes of both humans and animals, allowing the contamination of milk, its derivatives, and the processing facilities. S. aureus is well known for the ability to form biofilms in food processing environments, which contributes to its persistence and cross-contamination in food. The biocontrol of S. aureus in foods by lactic acid bacteria (LAB) and their bacteriocins has been studied for many years. Recently, LAB and their metabolites have also been explored for controlling S. aureus biofilms. LAB are used in fermented foods since in ancient times and nowadays characterized strains (or their purified bacteriocin) can be intentionally added to prolong food shelf-life and to control the growth of potentially pathogenic bacteria. Regarding the use of these microorganism and their metabolites (such as organic acids and bacteriocins) to prevent biofilm development or for biofilm removal, it is possible to conclude that a complex network behind the antagonistic activity remains poorly understood at the molecular level. The use of approaches that allow the characterization of these interactions is necessary to enhance our understanding of the mechanisms that govern the inhibitory activity of LAB against S. aureus biofilms in food processing environments.

Multi-locus sequence typing, antimicrobials resistance and virulence profiles of Salmonella enterica isolated from bovine carcasses in Minas Gerais state, Brazil.

The aim of this study was to identify virulence and antimicrobial resistance profiles and determine the sequence type (ST) by multilocus sequence typing (MLST) of Salmonella enterica isolates from bovine carcasses from slaughterhouse located in Minas Gerais state, Brazil, and its relationship with bovine isolates obtained on the American continent based on sequence type profile. The MLST results were compared with all Salmonella STs associated with cattle on American continent, and a multi-locus sequence tree (MS tree) was built. Among the 17 S. enterica isolates, five ST profiles identified, and ST10 were the most frequent, grouping seven (41.2%) isolates. The isolates presented 11 different profiles of virulence genes, and six different antibiotics resistance profiles. The survey on Enterobase platform showed 333 Salmonella STs from American continent, grouped into four different clusters. Most of the isolates in the present study (13/17), were concentrated in a single cluster (L4) composed by 74 STs. As a conclusion, five different STs were identified, with ST10 being the most common. The isolates showed great diversity of virulence genes and antibiotics resistance profiles. Most of the isolates of this study were grouped into a single cluster composed by 74 STs formed by bovine isolates obtained on the American continent.

Bacterial microbiota shifts in vacuum-packed beef during storage at different temperatures: Impacts on blown pack spoilage.

We aimed to evaluate the bacterial growth and diversity in vacuum-packed beef bags stored at different temperatures and to monitor blown-pack spoilage. We used culture-based methods and high-throughput sequencing to study the development of the main bacterial groups naturally present in beef stored at 4 and 15 °C for 28 days. The growth of sulfite-reducing clostridium (SRC) was impaired in beef bags stored at 4 °C; significant differences among SRC counts were observed in beef bags stored at 4 and 15 °C on days 14, 21, and 28 (P = 0.001). Blown pack was observed in most beef bags stored at 15 °C, from day 14 to day 28, but not in beef bags stored at 4 °C. A storage temperature of 4 °C was able to maintain a stable bacterial microbiota (most prevalent: Photobacterium, Hafnia-Obesumbacterium, and Lactococcus). Remarkable changes in microbial abundance occurred at 15 °C from day 14 to day 28, with a predominance of strict anaerobes (Bacteroides) and the presence of Clostridium spp. The relative frequencies of strict anaerobes and Clostridium were statistically higher in the beef bags stored at 15 °C (P < 0.001 and P = 0.004, respectively). The temperature influenced the microbial counts and relative abundance of spoilage bacteria, leading to blown pack spoilage.

Listeria monocytogenes and Pseudomonas spp. Distribution in a Processing Facility of Spotted Sorubim (Pseudoplatystoma corruscans) in Brazil.

ABSTRACT: The ecology of Listeria monocytogenes and Pseudomonas spp. during the slaughtering of spotted sorubim (Pseudoplatystoma corruscans) in a fish processing facility was assessed. Fish samples (n = 28) were obtained in different points of slaughtering (A, arrival; B, washing; C, gutting; and D, cooling) and subjected to detection of L. monocytogenes and enumeration of Pseudomonas spp. High frequencies of Listeria spp. (17 of 28 to 22 of 28) and L. monocytogenes (6 of 28 to 9 of 28) were identified in all slaughtering points but were not significantly different (P ≥ 0.05). All L. monocytogenes isolates (n = 33) were identified as belonging to serogroup IVb (serotype 4b) and subjected to macrorestriction with ApaI and AscI. The results indicated a continuous entry of L. monocytogenes in the facility, as well as a temporary persistence of a specific pulsotype. Pseudomonas spp. counts significantly decreased between points A and D (P < 0.05), but the mean counts in the end products (D) remained higher than 3 log CFU/g, suggesting the potential for fast spoilage. The obtained results show that L. monocytogenes and Pseudomonas spp. are widely distributed during spotted sorubim slaughtering, indicating the need for proper hygienic procedures to control these bacteria in the processing facility.

Further culture-independent characterization of the lactic microbiota of Serro artisanal cheese.

This study aimed to provide a further characterization of the lactic microbiota present in Minas artisanal cheese (MAC) from the Serro region by using culture-independent methods, as a complementary analysis of a previous study. The total DNA extracted from MAC samples (n = 55) was subjected to repetitive extragenic palindromic-PCR (rep-PCR) and PCR-denaturing gradient gel electrophoresis (PCR-DGGE). Rep-PCR analysis showed that core microbiota of Serro MAC was closely related, independent of the production town, farm size, or time of production. The sequencing of PCR-DGGE bands identified the prevalence of Lactococcus lactis in all samples, and Streptococcus salivarius was also identified. Thus, we conclude that when more accurate methods are unavailable, rep-PCR can be used as a culture-independent method to demonstrate if the microbiota is closely related or not among the samples. PCR-DGGE results also matched to the main findings of high-throughput sequencing, previously presented, confirming its confidence to detect the main microbial groups present in the raw milk cheeses.

Biofilm growth by Listeria monocytogenes on stainless steel and expression of biofilm-related genes under stressing conditions.

This research was carried out to investigate the differences in adhesion and growth during biofilm formation of L. monocytogenes from different sources and clonal complexes. Biofilm by L. monocytogenes (isolates CLIST 441 and 7: both lineage I, serotype 1/2b, CC3; isolates 19 and 508: both lineage II, serotype 1/2c, CC9) was grown on stainless steel coupons under different stressing conditions (NaCl, curing salts and quaternary ammonium compounds-QAC), to determine the expression of different genes involved in biofilm formation and stress response. CLIST 441, which carries a premature stop codon (PMSC) in agrC, formed high-density biofilms in the presence of QAC (7.5% w/v) or curing salts (10% w/v). Reverse Transcriptase-qPCR results revealed that L. monocytogenes isolates presented differences in transcriptional profile of genes related to biofilm formation and adaptation to environmental conditions. Our results demonstrated how L. monocytogenes can survive, multiply and form biofilm under adverse conditions related to food processing environments. Differences in transcriptional expression were observed, highlighting the role of regulatory gene networks for particular serotypes under different stress responses.

Microbial shifts through the ripening of the "Entre Serras" Minas artisanal cheese monitored by high-throughput sequencing.

Minas Gerais is a Brazilian state known as the largest cheese producer in Brazil. Minas Artisanal Cheese (MAC) is produced in different regions of this Brazilian state using raw cow milk to which a natural starter culture ("pingo") is added. "Entre Serras" is one of these regions, in which the MAC production had decreased (even stopped) for decades until recently, when artisanal cheeses production has been resurrected. Here, we aimed to gain insights on the bacterial diversity of "Entre Serras" MAC. 16S rRNA gene amplicon sequencing was used to assess the bacterial community in cheeses produced by four farms (A, B, C, and D) over 60 days of ripening. Overall, Lactococcus lactis was the predominant species found, regardless of the producer/farm. Enterococcus, Streptococcus, Lactobacillus and Leuconostoc genera were also prevalent in the samples microbiota and their levels varied according to the producer/farm. Cheeses produced by Farms A and B presented high contaminant levels (mainly Enterobacteriaceae and S. aureus), which may be attributed to poor hygiene during cheese production and/or herd health management. Chao1 indices varied significantly when the estimated species richness values of the producers/farms were compared (p < 0.05). A principal coordinate analysis also revealed distinct microbial communities for some farms (p < 0.001). However, no statistical significance was identified when samples were grouped by ripening time. Core microbiota analysis indicated that "Entre Serras" MAC microbiota includes not only LAB, but also spoilage and potentially pathogenic bacteria. We provide the first insights on the bacterial diversity of "Entre Serras" MAC, helping the understanding of the inter-regional microbiological diversity of the samples.

Microbiological quality and safety of Brazilian artisanal cheeses.

The establishment of norms that regulates the production and trade of Brazilian Artisanal Cheeses (BAC) has been stimulating many small farmers for this activity. The predominance of lactic acid bacteria (LAB) is a typical characteristic of BAC, which confers desirable attributes to artisanal cheeses. However, these products can be contaminated by other microbial groups, including those that indicate hygienic failures during production and may cause spoilage, or even microorganisms that pose risks to consumers' health. A systematic review of the literature published from January 1996 to November 2020 was carried out to identify scientific data about production characteristics and microbiological aspects of BAC, with a major focus on quality and safety status of these traditional products. Studies that fulfilled the inclusion criteria indicated that artisanal chesses produced in Brazil still do not satisfactorily meet the microbiological criteria established by the national laws, mainly due to the high counts of coagulase-positive Staphylococcus and coliforms. Despite low prevalence, pathogens such as Salmonella and Listeria monocytogenes were isolated in some BAC. This review contributed to better understanding microbiological aspects of BAC, the data compiled by the authors highlight the need to improve hygiene practices along the production chain of these traditional cheeses.

Interference of the acid stress on the expression of llsX by Listeria monocytogenes pathogenic island 3 (LIPI-3) variants.

Listeria monocytogenes harbor different virulence factors, with a highly heterogeneous distribution between distinct lineages and serotypes. The Listeria Pathogenicity Island 3 (LIPI-3), mainly described in lineage I, encodes for Listeriolysin S (LLS), a virulence factor expressed by L. monocytogenes in the gastrointestinal tract during in vivo infections. The aim of this study was to carry out a comparative genotypic analysis of LIPI-3 identified in L. monocytogenes isolates obtained in Brazil and subjected to whole genomic sequencing (WGS). In addition, transcription of llsX expression under different acid stress conditions was evaluated by RT-PCR. Homologues of the eight LIPI-3 genes (llsAGHXBYDP) were identified in 15 isolates (all from lineage I) representative of different sequence types: ST1 (n = 3), ST3 (n = 6), ST218 (n = 5) and ST288 (n = 1). Single nucleotide polymorphism (SNP) analysis revealed that genetic variation resulted in modification of the final peptide LLS for ST218 (serogroup IVb-v1) and ST288 (serogroup IIb). Selected strains from ST3 and ST288 were subjected to acid stress conditions and the expression of llsX, a LIPI-3 gene, was observed: only F2365 (4b/ST1) presented llsX expression after six hours of acid stress, indicating relevant differences when compared to isolates IIb (ST3 and 288). The results highlight the presence of genomic variations on LIPI-3 and llsX expression under acid stress conditions, demanding further studies to evaluate if these mutations have an impact on L. monocytogenes virulence in vivo.

Prevalence, Antimicrobial Resistance, and Diversity of Salmonella along the Pig Production Chain in Southern Brazil.

Control of Salmonella spp. in food production chains is very important to ensure safe foods and minimize the risks of foodborne disease occurrence. This study aimed to identify the prevalence and main contamination sources of Salmonella spp. in a pig production chain in southern Brazil. Six lots of piglets produced at different farms were tracked until their slaughter, and samples were subjected to Salmonella spp. detection. The obtained isolates were serotyped, subjected to antimicrobial resistance testing, and pulsed field gel electrophoresis (PFGE). Salmonella spp. was detected in 160 (10.2%) samples, and not detected in pig carcasses after final washing or chilling. Among the 210 Salmonella spp. isolates, S. Typhimurium was the most prevalent (n = 101) and resistant to at least one antimicrobial. High resistance rates were detected against tetracycline (83.8%), chloramphenicol (54.3%), and trimethoprim-sulfamethoxazole (33.3%). The isolates that were non-susceptible to three or more classes of antimicrobials (n = 60) were considered multidrug-resistant (MDR), and isolates resistant to up to six of the tested antimicrobials were found. PFGE allowed the identification of genetic diversity and demonstrated that farm environment and feed supply may be sources for the dissemination of Salmonella spp. along the production chain. The results revealed the sources of Salmonella contamination in the pig production chain and highlighted the risks of antimicrobial resistance spread.

Microbiological Testing for the Proper Assessment of the Hygiene Status of Beef Carcasses.

Microbiological testing is an important quality management tool in the food industry. In this study, the hygiene status of beef carcasses sampled in eight Brazilian slaughterhouses was assessed by enumeration of different hygiene indicator microorganisms, and a model to establish potential associations among these counts was proposed. The carcasses (n = 464) were surface sampled at four slaughtering steps (step 1: Hide after bleeding; step 2: Carcass after hide removal; step 3: Carcass after evisceration; step 4: Carcass after end washing) and subjected to a counting of mesophilic aerobes (MA), Enterobacteriaceae (EB), total coliforms (TC), and Escherichia coli (EC) using Petrifilm™ plates. Among the sampled beef carcasses (step 4), 32 (6.9%) and 71 (15.3%) presented counts above the microbiological criteria established by (EC) No. 1441/2007 for MA and EB, respectively. Thus, indicating that improvements in slaughter hygiene and a review of process controls are demanded in some of the studied slaughterhouses. The log count differences of EC, TC, and EB from MA were considered as response variables as a function of the slaughtering steps. Differential log counts changed consistently with the steps. The measurements, including the patterns in their inherently random variability, were fairly predictable from steps 1 and 4. The results indicated that differential log counts for TC and EC are not relevant, as their concentrations and random pattern can be inferred from counts of MA and EB. The proposed model can be used as a valuable tool for the design and adoption of feasible quality control programs in beef industries. The adoption of such a tool should have a positive contribution on consumers' health and enhance product quality.

Potential Control of Listeria monocytogenes by Bacteriocinogenic Enterococcus hirae ST57ACC and Pediococcus pentosaceus ST65ACC Strains Isolated From Artisanal Cheese.

Bacteriocinogenic Enterococcus hirae ST57ACC and Pediococcus pentosaceus ST65ACC strains, previously isolated from artisanal cheese, were evaluated for their safety with the aim to determine whether they could be used as beneficial strains, especially in the control of Listeria monocytogenes. Both isolates survived simulated gastrointestinal conditions and showed high levels of auto- and co-aggregation with L. monocytogenes, although the hydrophobicity of cells varied. Using the agar-spot test with 33 commercial drugs from different groups, only anti-inflammatory drugs and drugs containing loratadine and propranolol hydrochloride were able to affect the growth of the tested strains. Both strains were resistant to 3 out of 11 antibiotics tested by the disc diffusion method, and low frequencies of antibiotic resistance-encoding genes were observed by PCR analysis. Tested strains neither presented biogenic amine-related genes nor produced these substances. Aside from some antibiotic resistance characteristics, the tested strains were considered safe as they lack other virulence-related genes. E. hirae ST57ACC and P. pentosaceus ST65ACC both presented beneficial properties, particularly their ability to survive gastrointestinal conditions and to aggregate with L. monocytogenes, which can facilitate the elimination of this pathogen. Further studies should be conducted to better understand these interactions.

Listeria monocytogenes in Food-Processing Facilities, Food Contamination, and Human Listeriosis: The Brazilian Scenario.

Listeria monocytogenes is a foodborne pathogen that contaminates food-processing environments and persists within biofilms on equipment, utensils, floors, and drains, ultimately reaching final products by cross-contamination. This pathogen grows even under high salt conditions or refrigeration temperatures, remaining viable in various food products until the end of their shelf life. While the estimated incidence of listeriosis is lower than other enteric illnesses, infections caused by L. monocytogenes are more likely to lead to hospitalizations and fatalities. Despite the description of L. monocytogenes occurrence in Brazilian food-processing facilities and foods, there is a lack of consistent data regarding listeriosis cases and outbreaks directly associated with food consumption. Listeriosis requires rapid treatment with antibiotics and most drugs suitable for Gram-positive bacteria are effective against L. monocytogenes. Only a minority of clinical antibiotic-resistant L. monocytogenes strains have been described so far; whereas many strains recovered from food-processing facilities and foods exhibited resistance to antimicrobials not suitable against listeriosis. L. monocytogenes control in food industries is a challenge, demanding proper cleaning and application of sanitization procedures to eliminate this foodborne pathogen from the food-processing environment and ensure food safety. This review focuses on presenting the L. monocytogenes distribution in food-processing environment, food contamination, and control in the food industry, as well as the consequences of listeriosis to human health, providing a comparison of the current Brazilian situation with the international scenario.

Molecular tracking of Salmonella spp. in chicken meat chain: from slaughterhouse reception to end cuts.

Due to the importance of Salmonella spp. in poultry products, this study aimed to track its main contamination routes since slaughtering reception to processing of chicken end cuts. Samples from different steps of slaughtering and processing (n = 277) were collected from two chicken slaughterhouses (Sl1 and Sl2) located in Minas Gerais state, Brazil, and subjected to Salmonella spp. detection. The obtained isolates were subjected to serological identification and tested by PCR for specific Salmonella spp. genes (ompC and sifB). Also, Salmonella spp. isolates were subjected to XbaI macrorestriction and pulsed-field gel electrophoresis (PFGE). Sixty-eight samples were positive for Salmonella spp. and 172 isolates were obtained. Sl1 and Sl2 presented similar frequencies of Salmonella spp. positive samples during reception, slaughtering and processing (p > 0.05), except for higher frequencies in Sl1 for chicken carcasses after de-feathering and evisceration (p < 0.05). PFGE allowed the identification of cross contamination and persistence of Salmonella spp. strains in Sl1. The results highlighted the relevance of the initial steps of chicken slaughtering for Salmonella spp. contamination, and the pre-chilling of carcasses as an important controlling tool. In addition, the presence of Salmonella spp. in chicken end cuts samples represents a public health concern.

Hygiene and Safety in the Meat Processing Environment from Butcher Shops: Microbiological Contamination and Listeria monocytogenes.

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.

The Continuous Challenge of Characterizing the Foodborne Pathogen Listeria monocytogenes.

Listeria monocytogenes is an important foodborne pathogen commonly isolated from food processing environments and food products. This organism can multiply at refrigeration temperatures, form biofilms on different materials and under various conditions, resist a range of environmental stresses, and contaminate food products by cross-contamination. L. monocytogenes is recognized as the causative agent of listeriosis, a serious disease that affects mainly individuals from high-risk groups, such as pregnant women, newborns, the elderly, and immunocompromised individuals. Listeriosis can be considered a disease that has emerged along with changing eating habits and large-scale industrial food processing. This disease causes losses of billions of dollars every year with recalls of contaminated foods and patient medical treatment expenses. In addition to the immune status of the host and the infecting dose, the virulence potential of each strain is crucial for the development of disease symptoms. While many isolates are naturally virulent, other isolates are avirulent and unable to cause disease; this may vary according to the presence of molecular determinants associated with virulence. In the last decade, the characterization of genetic profiles through the use of molecular methods has helped track and demonstrate the genetic diversity among L. monocytogenes isolates obtained from various sources. The purposes of this review were to summarize the main methods used for isolation, identification, and typing of L. monocytogenes and also describe its most relevant virulence characteristics.

Molecular Serogrouping of Listeria monocytogenes from Brazil Using PCR.

We assessed the serotype distribution of Listeria monocytogenes isolates from clinical, beef, and environment samples using two PCR-based protocols for serogrouping. A panel of 134 isolates (22 clinical samples, 79 samples of beef cuts, and 33 samples from the beef processing environment) were subjected to conventional serology and identified as serotypes 1/2a (n = 12), 1/2b (n = 21), 1/2c (n = 71), and 4b (n = 30). Isolates from clinical samples were predominantly serotype 4b, and the most prevalent serotype among the beef cut and environment samples was 1/2c. The protocol described by M. Doumith, C. Buchrieser, P. Glaser, C. Jacquet, and P. Martin (J. Clin. Microbiol. 42:3819-3822, 2004) produced contradictory results for seven 1/2a isolates, which were positive for lmo1118 and had the profile IIc (serotypes 1/2c and 3c). Fifteen serotype 4b isolates amplified the target lmo0737, with the atypical profile IVb variant 1. The results obtained with the protocol described by M. K. Borucki and D. R. Call (J. Clin. Microbiol. 41:5537-5540, 2003) were in full agreement with those of the conventional serology. We recommend using this multiplex PCR approach by adding one pair of the reported primers to the panel to reduce total effort by one PCR while maintaining specificity. We present additional recommendations to improve the efficiency and reproducibility of this serogrouping assay.

In Vitro Evaluation of Bacteriocins Activity Against Listeria monocytogenes Biofilm Formation.

The present study aimed to assess the activity of cell-free supernatant (CFS) containing bacteriocins on the formation and maintenance of biofilms developed by Listeria monocytogenes, and the associated effect of bacteriocins and ethylene-diamine-tetra-acetic acid (EDTA) on the formed biofilm. CFS from 9 lactic acid bacteria (LAB) strains was tested for inhibitory activity against 85 L. monocytogenes isolates and 21 LAB strains. Then, 12 L. monocytogenes strains were selected based on genetic profiles and sensitivity to CFS and were subjected to an in vitro assay to assess biofilm formation in microtiter plates, considering different culture media and incubation conditions. Based on these results, 6 L. monocytogenes strains were subjected to the same in vitro procedure to assess biofilm formation, being co-inoculated with CFS. In addition, these strains were subjected to the same in vitro procedure, modified by adding the CFS after biofilm formation. Relevant decrease in biofilm formation was observed in the first experiment, but CFS added after biofilm formation did not eliminate them. CFS from Lactobacillus curvatus ET31 were selected due to its anti-biofilm activity, being associated to EDTA at different concentrations and tested for biofilm control of three strains of L. monocytogenes, using the same in vitro procedure described previously. Concentrated bacteriocin presented poor performance in eliminating formed biofilms, and EDTA concentration presented no evident interference on biofilm elimination. Twelve selected L. monocytogenes strains were positive for investigated virulence makers and negative for luxS gene, recognized as being involved in biofilm formation. Selected L. monocytogenes strains were able to produce biofilms under different conditions. CFSs have the potential to prevent biofilm formation, but they were not able to destroy already formed biofilms. Nevertheless, low concentrations of CFS combined with EDTA caused a relevant reduction in already formed biofilms, but this association was not able to eliminate them. The activity of selected CFS was demonstrated against L. monocytogenes-formed biofilms, being more effective when associated to EDTA at different concentrations.

Adequacy of Petrifilm™ Aerobic Count plates supplemented with de Man, Rogosa & Sharpe broth and chlorophenol red for enumeration of lactic acid bacteria in salami.

The present study aimed to assess the performance of alternative protocols to enumerate lactic acid bacteria (LAB) in salami. Fourteen cultures and two mixed starter cultures were plated using six protocols: 1) Petrifilm™ Aerobic Count (AC) with MRS broth and chlorophenol red (CR), incubated under aerobiosis or 2) under anaerobiosis, 3) MRS agar with CR, 4) MRS agar with bromocresol purple, 5) MRS agar at pH5.7, and 6) All Purpose Tween agar. Samples of salami were obtained and the LAB microbiota was enumerated by plating according protocols 1, 2, 3 and 5. Regression analysis showed a significant correlation between the tested protocols, based on culture counts (p<0.05). Similar results were observed for salami, and no significant differences of mean LAB counts between selected protocols (ANOVA, p>0.05). Colonies were confirmed as LAB, indicating proper selectivity of the protocols. The results showed the adequacy of Petrifilm™ AC supplemented with CR for the enumeration of LAB in salami.

Serotypes and Pulsotypes diversity of Listeria monocytogenes in a beef-processing environment.

Utensils and equipment from meat-processing facilities are considered relevant cross-contamination points of Listeria monocytogenes to foods, demanding tracking studies to identify their specific origins, and predict proper control. The present study aimed to detect L. monocytogenes in a beef-processing facility, investigating the diversity of serotypes and pulsotypes in order to identify the possible contamination routes. Surface samples from knives (n=26), tables (n=78), and employees hands (n=74) were collected before and during the procedures from a beef-processing facility, in addition to surface samples of end cuts: round (n=32), loin (n=30), and chuck (n=32). All samples were subjected to L. monocytogenes screening according ISO 11.290-1, and the obtained isolates were subjected to serotyping and pulsed-field gel electrophoresis. Listeria spp. were identified in all processing steps, in 61 samples, and L. monocytogenes was detected in 17 samples, not being found only in knives. Eighty-five isolates were identified as L. monocytogenes, from serotypes 1/2c (n=65), 4b (n=13), and 1/2b (n=7), being grouped in 19 pulsotypes. Considering these results, cross-contamination among hands, tables, and beef cuts could be identified. The obtained data indicated the relevance of cross-contamination in the beef-processing facility, and the occurrence of serotypes 1/2b and 4b in beef cuts distributed for retail sale is a public health concern.