Rita de Cássia dos Santos da Conceição

Condições higiênico-sanitárias no comércio ambulante de alimentos em Pelotas-RS

Street-vending of ready-to-eat (RTE) foods can be a risk to the consumers health, since people usually involved in this activity does not have proper knowledge in safe handling of foods. Despite this, only a few studies have been made on the microbiological quality of these foods and places where they are prepared. In this paper we report on the higienic-sanitary quality of hot-dogs sold by street-vendors from Pelotas,RS. Samples of hot-dogs, water and work surfaces, were collected from 60 street-vending places and taken to the laboratory for analysis. Counts of aerobic mesophilic bacteria (TPC), Staphylococcus coagulase positive (SCP), total coliforms (TC) and coliforms at 45oC (FC) were made on samples of hot-dogs. SPC, TC and FC counts were made on the water and surface samples. Among the 60 samples of hot-dogs 53%, 48%, 37% and 25% were found unsatisfactory for TC, TPC, STA and FC, respectively. Only 3 (5%) water samples were found unsatisfactory according to the TPC standard used, and 27% and 23% did not read the standards for TC and FC. Regarding the surfaces, 70% were found unsatisfactory for TPC, 68% for TC and 67% for FC. Salmonella sp was not detected in any of the samples tested. These results suggest that the hygiene practices of many food street-vending places are not adequate, resulting in a high proportion of read-to-eat RTE) foods with microbiological quality unsatisfactory for consumption.

Detection of Salmonella Typhimurium in Raw Meats using In‐House Prepared Monoclonal Antibody Coated Magnetic Beads and PCR Assay of the fimA Gene

Abstract A method for detection of Salmonella Typhimurium in meat samples that uses in‐house monoclonal antibody (MAb) coated magnetic beads for immunomagnetic separation (IMS) associated with PCR amplification of the gene fimA was developed. An internal amplification control (IAC) of the PCR reaction was constructed. The fimA PCR has shown 100% sensitivity and specificity when tested with various bacteria. The detection limit of the IMS‐PCR method, using a post‐enrichment in BHI broth for 6 h between IMS and PCR, was 1–10 CFU/mL. The method proved to be rapid (27 hrs), highly sensitive (1–10 CFU/25 g), and specific for detection of S. Typhimurium from experimentally contaminated pork and chicken meat samples.

The role of quorum sensing in Escherichia coli (ETEC) virulence factors.

Quorum sensing (QS) is a signaling system among bacteria mediated by auto-inducer substances (AI). Whenever the concentration of these molecules reaches a threshold corresponding to a high cell density or quorum, the whole population starts a coordinated expression of specific genes. Studies have shown that epinephrine is also responsible for activating specific bacterial genes. This work aimed to investigate the role of conditioned medium (containing AI), epinephrine and their association on growth, motility, F4 fimbriae and heat-labile toxin (LT) expression on enterotoxigenic Escherichia coli (ETEC, E68). A significant increase in motility, F4 and LT expression, was observed in the ETEC culture supplemented with conditioned medium and epinephrine. These findings suggest that ETEC uses some components of conditioned medium (e.g., AI molecules), host molecules (epinephrine), and their association to modulate the expression of important virulence genes.

Detection of salmonella sp in chicken cuts using immunomagnetic separation

The immunomagnetic separation (IMS) is a technique that has been used to increase sensitivity and specificity and to decrease the time required for detection of Salmonella in foods through different methodologies. In this work we report on the development of a method for detection of Salmonella in chicken cuts using in house antibody-sensitized microspheres associated to conventional plating in selective agar (IMS-plating). First, protein A-coated microspheres were sensitized with polyclonal antibodies against lipopolysacharide and flagella from salmonellae and used to standardize a procedure for capturing Salmonella Enteritidis from pure cultures and detection in selective agar. Subsequently, samples of chicken meat experimentally contaminated with S. Enteritidis were analyzed immediately after contamination and after 24h of refrigeration using three enrichment protocols. The detection limit of the IMS-plating procedure after standardization with pure culture was about 2x10 CFU/mL. The protocol using non-selective enrichment for 6-8h, selective enrichment for 16-18h and a post-enrichment for 4h gave the best results of S. Enteritidis detection by IMS-plating in experimentally contaminated meat. IMS-plating using this protocol was compared to the standard culture method for salmonellae detection in naturally contaminated chicken cuts and yielded 100% sensitivity and 94% specificity. The method developed using in house prepared magnetic microespheres for IMS and plating in selective agar was able to diminish by at least one day the time required for detection of Salmonella in chicken products by the conventional culture method.

Monoclonal antibodies against serogroup B salmonellae: production, characterisation and use in a sandwich ELISA

Abstract Ten monoclonal antibodies (MAbs) against serogroup B salmonellae were obtained after immunisation of BALB/c mice with outer membrane proteins (OMPs) from Salmonella enterica serovar Typhimurium. Affinity constants, measured by enzyme-linked immunosorbent assay (ELISA), ranged from 8×05 to 6×07 l mol−1. Additivity ELISA demonstrated that most MAbs recognise different epitopes. ELISA determined the antigen specificity of the MAbs. The MAbs were more reactive with live Salmonella Typhimurium than with heat-treated cells. Two MAbs were used to develop a sandwich ELISA for rapid detection of Salmonella Typhimurium in experimentally contaminated meats. Its detection limit was 105 CFU ml−1 and it was able to detect 1–10 CFU in post-enrichment broth from 25 g of beef and chicken meat samples. The sandwich ELISA developed was shown to be specific and sensitive for the detection of Salmonella Typhimurium in meat, and produced results comparable to the culture methods in 57 h.

Biofilm Formation by Coagulase-Positive Staphylococcus aureus Isolated from Mozzarella Cheese Elaborated with Buffalo Milk and its Effect on Sensitivity to Sanitizers

Background: The buffalo milk mozzarella cheese is a new product in the market, with high consumer acceptance and excellent prospects for trade. The cheese is rich in nutrients, which favors the proliferation of microorganisms that can cause food-borne diseases in the consumer. Staphylococcus aureus can cause gastro-enteritis in humans by the production of enterotoxins in food. One problem that may hinder the elimination of undesirable microorganisms in the food industry is the formation of biofilms. The objective of this study was to determine the effect of biofilm formation by Staphylococcus aureus isolated from buffalo mozzarella cheese on sensitivity to sanitizers. Materials, Methods & Results: Fifty samples of buffalo mozzarella cheese were analyzed to investigate the presence of S. aureus. The isolates were obtained through microbiological analysis and identified by PCR. The similarity of the strains was compared through rep-PCR. The distinct strains were tested for biofilm formation in microtiter plates. Soy Tripticase Broth (TSB) was placed in each well of the microtiter plate and overnight cultures of each strain was added. Wells without bacterial culture were used as controls. A villous cap was then placed on the plate and incubated for 48 h at 37°C. During incubation, the biofilms formed on the surface of the villi of the caps. For quantification of biofilm formation, material that remained attached to the cap was stained with crystal violet, the stained biofilm was extracted and the OD570 of each well was measured. Each strain was classified as non-biofilm forming, weak forming, moderately formed or formative strong. Strong forming and non-biofilm forming strains were tested on high density polyethylene, stainless steel and glass surfaces. Plates of 4 cm² of the different materials were placed in TSB where the culture of each isolate was inoculated separately. At each 48 h incubation the plates were washed to remove unbound cells and re-inserted into TSB without the inoculum. After five replicates of the procedure, sterile swabs were passed over the entire surface of each plate for counting in Baird-Parker agar. They were also tested for sensitivity to sodium hypochlorite and iodine after biofilm formation. The biofilm plates were immersed in flasks containing sanitizers, where they remained for 10 min. At the established contact time, the plates were immersed in neutralizing solution for 30 s. After washing with PBS, a sterile swab was passed on the surface of each plate and counts on Baird-Parker agar were performed. The bands profiles obtained on rep-PCR were identical when compared to isolates from the same sample, indicating that each sample was contaminated with only one S. aureus strain. From the twenty S. aureus strain identified, two isolates were classified as strong biofilm formers, seven as moderate formers, ten weak formers and one as non-biofilm builder. The two strong forming strains produced biofilm on the three surfaces tested. The application of sodium hypochlorite and iodine sanitizers promoted a reduction of approximately 2 log bacterial populations on all surfaces of both the biofilm and non-forming strains. Discussion: Most strains of S. aureus isolated from buffalo milk mozzarella cheese have the ability to form biofilm on the surfaces of equipment and utensils that have stainless steel, glass or high density polyethylene components. Although biofilm forming strains are no longer resistant to sanitizers sodium hypochlorite and iodine than non-forming sanitizers, they reach higher concentrations in the biofilm, resulting in larger bacterial populations remaining after application of the sanitizers. These results support the recommendation that the good hygienic practices adopted by industries processing buffalo milk mozzarella cheese should include specific measures to control the Staphylococcus aureus contamination.

Virulence and antimicrobial resistance of Salmonella spp. and Escherichia coli in the beef jerky production line

Abstract Intense manipulation during beef jerky production increases the possibility of contamination with pathogenic microorganisms. This study evaluated the contamination by thermotolerant coliforms, Escherichia coli and Salmonella spp., on processing surfaces and raw materials during beef jerky production, as well as in the final product. Thermotolerant coliforms were found on all surfaces tested and in the raw material. Escherichia coli was identified in 6.7% of the surface samples, while Salmonella spp. was found in 3.3% of the surface samples and 8.6% of raw material samples. Virulence genes were detected in Salmonella spp. isolates. One Salmonella spp. isolate was resistant to sulfonamide, while one E. coli isolate was multiresistant, including the presence of resistance genes sul2, strA, strB, tetA and tetB. The presence of coliforms demonstrates failings in hygienic‐sanitary procedures. The presence of pathogenic microorganisms causing foodborne diseases in the production line indicates persistent contamination in the production plant. Although the drying process applied to beef jerky should guarantee the safety of the final product, the presence of multiresistant pathogenic microorganisms, presenting virulence genes, should be a matter of concern. Because beef jerky is a ready‐to‐eat product, a failure in the production process may cause such microorganisms to pose a public health risk.

Sanitizer resistance of biofilm-forming Salmonellaisolated from meat products

Este estudo avaliou a capacidade de Salmonella enterica subsp. enterica isolada de produtos carneos formar biofilme e testou sua resistencia a diferentes sanitizantes. Vinte cepas foram avaliadas quanto a capacidade de formar biofilme em placas de microtitulacao. As cepas formadoras de biofilme foram testadas em superficies de polietileno de alta densidade, aco inoxidavel e vidro e tiveram a sensibilidade ao hipoclorito de sodio e ao iodo avaliada. Duas cepas de Salmonella Enteritidis isoladas de produtos de frango apresentaram capacidade de formar biofilme nas superficies testadas. Essas cepas alcancaram maiores populacoes nas superficies do que aquelas nao formadoras de biofilme, e foram mais dificeis de remover ou reduzir. Devido a acao sanitizante ser menos eficiente sobre bacterias formadoras de biofilme, esses micro-organismos podem persistir no biofilme formado sobre as superficies de equipamentos e utensilios e ocasionalmente contaminar os alimentos antes da sua expedicao, aumentando dessa forma o risco de ocorrencia de doencas transmitidas por alimentos.