Paula Mendonça Moraes

Bacteriocinogenic and virulence potential of Enterococcus isolates obtained from raw milk and cheese.

To provide molecular and phenotypical characterization of Enterococcus isolates obtained from raw milk and cheese, regarding their bacteriocinogenic and virulence activity.

Microbiological Quality and Safety of Raw Milk and Soft Cheese and Detection of Autochthonous Lactic Acid Bacteria with Antagonistic Activity Against Listeria monocytogenes, Salmonella Spp., and Staphylococcus aureus

This study aimed to characterize the microbiological quality and safety of raw milk and soft cheese, verifying possible associations between microbial populations, and the detection of lactic acid bacteria (LAB) with antagonistic activity against foodborne pathogens. Raw milk (n = 36) and soft cheese (n = 18) samples were collected and submitted for the analysis of mesophilic aerobes, total coliforms, Escherichia coli, LAB, coagulase-positive Staphylococcus (CPS), Listeria monocytogenes, and Salmonella spp. In all, 389 LAB isolates were randomly selected and submitted for antagonistic tests against L. monocytogenes, St. aureus, Salmonella Typhimurium, and Lactobacillus sakei. The samples presented high counts of mesophilic aerobes, total coliforms, and LAB, and also high and significant correlation indices between these populations. Low levels of CPS and E. coli were observed, as well as an absence of Salmonella spp. and L. monocytogenes. A substantial portion of the analyzed samples presented LAB cultures with antagonistic activity, but not against Salmonella Typhimurium. The obtained results indicate the antimicrobial potential of the autochthonous microbiota of raw milk and soft cheese. Despite the spoilage potential, the LAB present in the studied food products can be isolated and properly characterized as antagonistic cultures, to be used in bioconservation studies for pathogen control in foods.

Molecular identification of naturally occurring bacteriocinogenic and bacteriocinogenic-like lactic acid bacteria in raw milk and soft cheese.

Lactic acid bacteria (LAB) are currently used by food industries because of their ability to produce metabolites with antimicrobial activity against gram-positive pathogens and spoilage microorganisms. The objectives of this study were to identify naturally occurring bacteriocinogenic or bacteriocinogenic-like LAB in raw milk and soft cheese and to detect the presence of nisin-coding genes in cultures identified as Lactococcus lactis. Lactic acid bacteria cultures were isolated from 389 raw milk and soft cheese samples and were later characterized for the production of antimicrobial substances against Listeria monocytogenes. Of these, 58 (14.9%) LAB cultures were identified as antagonistic; the nature of this antagonistic activity was then characterized via enzymatic tests to confirm the proteinaceous nature of the antimicrobial substances. In addition, 20 of these antagonistic cultures were selected and submitted to genetic sequencing; they were identified as Lactobacillus plantarum (n=2) and Lactococcus lactis ssp. lactis (n=18). Nisin genes were identified by polymerase chain reaction in 7 of these cultures. The identified bacteriocinogenic and bacteriocinogenic-like cultures were highly variable concerning the production and activity of antimicrobial substances, even when they were genetically similar. The obtained results indicated the need for molecular and phenotypic methodologies to properly characterize bacteriocinogenic LAB, as well as the potential use of these cultures as tools to provide food safety.

Foodborne Pathogens and Microbiological Characteristics of Raw Milk Soft Cheese Produced and on Retail Sale in Brazil

The consumption of raw milk soft cheeses (RMSC), which are typically manufactured in small dairy farms under unsatisfactory hygiene conditions, is common in Brazil. Due to these production characteristics, this type of cheese is a potential carrier of pathogenic microorganisms, such as Listeria monocytogenes, Salmonella, and enterotoxin-producing Staphylococcus spp. Considering these characteristics, in this work, we aimed to detect the presence of these pathogenic microorganisms in RMC and to evaluate their microbiological quality. Fifty-five samples of this product were collected from different noninspected commercial establishments and submitted to the enumeration of mesophilic aerobes (MA), total coliforms (TC), Escherichia coli, and coagulase-positive staphylococci (CPS), and detection of L. monocytogenes and Salmonella spp. All analyzed samples were negative for Salmonella spp. and L. monocytogenes. All samples presented counts of MA higher than 10(6) colony forming units/g (CFU/g; range, 3.0x10(6) to 4.0x10(9)). TC were present at levels between 1.0x10(3) and 1.8x10(8) CFU/g, and E. coli between 1.0x10(2) and 3.5x10(6) CFU/g. CPS were detected in 17 (30.9%) samples at levels higher than 10(4) CFU/g. These results confirm the poor microbiological quality of raw milk used in the manufacturing of RMC samples, and also the inadequate production conditions. Therefore, the evaluation of microbiological safety and quality of these products must be constantly reported to alert the official agencies about the significance of proper inspection.

Enumeration of coagulase and thermonuclease-positive Staphylococcus spp. in raw milk and fresh soft cheese: an evaluation of Baird-Parker agar, Rabbit Plasma Fibrinogen agar and the Petrifilm Staph Express count system.

Staphylococcus spp. are microorganisms that are naturally present in milk and dairy products and are often associated with food-borne diseases outbreaks due to the ability of some strains to produce thermostable enterotoxins. This ability is usually associated with coagulase and thermonuclease production, characteristics that are considered in the microbiological analyses for the control of such microorganisms. The objective of this study was to evaluate the culture media and the methodologies used for the enumeration of coagulase and thermonuclease-positive Staphylococcus spp. in raw milk and fresh soft cheese. Samples of artificially contaminated milk (with coagulase-positive Staphylococcus reference strains) and samples of naturally contaminated raw milk and cheese were submitted for enumeration in Baird-Parker agar (BP), Rabbit Plasma Fibrinogen agar (RPFA) and in the Petrifilm Staph Express count system (STX). No significant differences (P > 0.05) were observed between the mean counts obtained in all of the evaluated culture media. RPFA and STX had good correlation indices between the total and typical colony counts as well as with coagulase and the thermonuclease-positive colony counts. Thus, there is a better association between coagulase and thermonuclease production to typical colony morphology developed on these culture media, leading to more accurate and reliable results than with BP, which demonstrated lower correlation indices between these counts.

Comparison of phenotypic and molecular tests to identify lactic acid bacteria

Twenty-nine lactic acid bacteria (LAB) isolates were submitted for identification using Biolog, API50CHL, 16S rDNA sequencing, and species-specific PCR reactions. The identification results were compared, and it was concluded that a polyphasic approach is necessary for proper LAB identification, being the molecular analyzes the most reliable.

Glucuronidase activity of Escherichia coli isolated from chicken carcasses

To identify Escherichia coli through the production of β-D-glucuronidase (GUD), 622 suspect cultures were isolated from chicken carcasses and plated in Petrifilm™ EC. Of these cultures, only 44 (7.1%) failed to produce GUD. This result indicates the usefulness of GUD production for estimating E. coli populations in chicken.To identify Escherichia coli through the production of β-D-glucuronidase (GUD), 622 suspect cultures were isolated from chicken carcasses and plated in PetrifilmTM EC. Of these cultures, only 44 (7.1%) failed to produce GUD. This result indicates the usefulness of GUD production for estimating E. coli populations in chicken.