Enterotoxigenic Staphylococcus aureus in Brazilian artisanal cheeses: Occurrence, counts, phenotypic and genotypic profiles.

The present study aimed to assess the occurrence and counts of Staphylococcus aureus in Brazilian artisanal cheeses (BAC) produced in five regions of Brazil: Coalho and Manteiga (Northeast region); Colonial and Serrano (South); Caipira (Central-West); Marajó (North); and Minas Artisanal cheeses, from Araxá, Campos das Vertentes, Cerrado, Serro and Canastra microregions (Southeast). The resistance to chlorine-based sanitizers, ability to attach to stainless steel surfaces, and antibiogram profile of a large set of S. aureus strains (n = 585) were assessed. Further, a total of 42 isolates were evaluated for the presence of enterotoxigenic genes (sea, seb, sec, sed, see, seg, sei, sej, and ser) and submitted to typing using pulsed-field gel electrophoresis (PFGE). BAC presented high counts of S. aureus (3.4-6.4 log CFU/g), varying from 25 to 62.5%. From the S. aureus strains (n = 585) assessed, 16% could resist 200 ppm of sodium hypochlorite, whereas 87.6% produced strong ability to attach to stainless steel surfaces, corroborating with S. aureus ability to persist and spread in the environment. Furthermore, the relatively high frequency (80.5%) of multidrug-resistant S. aureus and the presence of enterotoxin genes in 92.6% of the strains is of utmost attention. It reveals the lurking threat of SFP that can survive when conditions are favorable. The presence of enterotoxigenic and antimicrobial-resistant strains of S. aureus in cheese constitutes a potential risk to public health. This result calls for better control of cheese contamination sources, and taking hygienic measures is necessary for food safety. More attention should be paid to animal welfare and hygiene practices in some dairy farms during manufacturing to enhance the microbiological quality of traditional cheese products.

Molecular characterization of persistent subclinical mastitis-causing Staphylococcus aureus from dairy farms.

The study aimed to evaluate the genetic diversity of Staphylococcus aureus causing subclinical mastitis (SM) isolated from dairy cows and to assess the effect of the infection status (transient vs. persistent) on the milk and component yield. A total of six dairy farms in São Paulo state were used for the selection of cows with SM caused by S. aureus. S. aureus strains (n = 56) obtained from three biweekly aseptic mammary quarter milk samplings (n = 1140 from 95 cows) were subjected to MALDI-TOF MS analysis for species confirmation and further PFGE analysis. Intramammary infections (IMI) caused by S. aureus were categorized as transient (T: when only one out of 3 milk samplings had positive isolation of any pulsotype) or persistent (P: when two (P2) or three (P3) milk samplings had positive isolation of identical pulsotype over the consecutive episodes of SM. The SmaI macrorestriction fragment profiles of 56 S. aureus isolates showed a dominant S. aureus clonal pattern (PFGE type A; n = 50; 89.3%) within and among the herds. The SM-causing S. aureus represented a reduction of quarter milk yield of 26.2% in transient and 54.8% in persistent cases as well as a reduction of total solid yield of 38.1% and 49.4%, respectively, when compared with the healthy control quarters. Overall, the greater chance of S. aureus to be persistent is when a dominant clonal pattern is present in the herd which consequently may be associated with the cause of accentuated milk loss.

Mycotoxin occurrence in breast milk and exposure estimation of lactating mothers using urinary biomarkers in São Paulo, Brazil.

In this study, the occurrence of aflatoxins (AFs), fumonisins (FBs), ochratoxin A (OTA), deoxynivalenol (DON), zearalenone (ZEN) and some of their metabolites were assessed in breast milk and urine of lactating women (N = 74) from Pirassununga, São Paulo, Brazil. Exposure estimations through urinary mycotoxin biomarkers was also performed. Samples were collected in four sampling times (May and August 2018, February and July 2019) and analyzed by liquid chromatography coupled to tandem mass spectrometry. Aflatoxin M1 (AFM1) was not detected in breast milk. However, two samples (3%) presented FB1 at 2200 and 3400 ng/L, while 4 samples (5%) had OTA at the median level of 360 ng/L. In urine, AFM1 and aflatoxin P1 (AFP1) were found in 51 and 11% of samples, respectively (median levels: 0.16 and 0.07 ng/mg creatinine, respectively). Urinary DON (median level: 38.59 ng/mg creatinine), OTA (median level: 2.38 ng/mg creatinine) and ZEN (median level: 0.02 ng/mg of creatinine) were quantified in 18, 8 and 10% of the samples, respectively. Mean probable daily intake (PDI) values based on urinary biomarkers were 1.58, 1.09, 5.07, and 0.05 µg/kg body weight/day for AFM1, DON, OTA, and ZEN, respectively. Although a low mycotoxin occurrence was detected in breast milk, the PDI for the genotoxic AFs was much higher than those reported previously in Brazil, while PDI values obtained for OTA and DON were higher than recommended tolerable daily intakes. These outcomes warrant concern on the exposure of lactating women to these mycotoxins in the studied area.

Effect of Lactobacillus rhamnosus on growth of Listeria monocytogenes and Staphylococcus aureus in a probiotic Minas Frescal cheese.

This study aimed to evaluate the effect of Lactobacillus rhamnosus GG on growth of Staphylococcus aureus and Listeria monocytogenes, inoculated alone or in combination on surface of Minas Frescal cheeses, during storage for 21 days at 7 °C. Survival percentages of each individual bacterial species after exposure to in vitro simulated gastrointestinal conditions (SGC) were also determined. The addition of L. rhamnosus did not affect (P > 0.05) pH, moisture, fat, protein and texture profile of Minas Frescal cheeses. L. rhamnosus was able to survive in suitable counts (>6 Log CFU/g) in cheeses from the 7th day of storage, with high survival (>74.6-86.4%) after SGC. An inhibitory effect of L. rhamnosus on L. monocytogenes was observed in cheeses (decrease of 1.1-1.6 Log CFU/g) and after SGC (20% reduction in the survival). No inhibitory effect of L. rhamnosus was observed on S. aureus counts (P > 0.05), and this microorganism did not survive the exposure to SGC. In conclusion, the addition of L. rhamnosus in Minas Frescal cheese has a potential for L. monocytogenes inhibition. Further studies are necessary to elucidate the mechanisms involved in the inhibition process and determine the survival ability of the bacterial species evaluated in in vivo experiments.

Behavior of Foodborne Pathogens Listeria monocytogenes and Staphylococcus aureus in Mixed-Species Biofilms Exposed to Biocides.

In nature and man-made environments, microorganisms reside in mixed-species biofilms, in which the growth and metabolism of an organism are different from these behaviors in single-species biofilms. Pathogenic microorganisms may be protected against adverse treatments in mixed-species biofilms, leading to health risk for humans. Here, we developed two mixed five-species biofilms that included one or the other of the foodborne pathogens Listeria monocytogenes and Staphylococcus aureus The five species, including the pathogen, were isolated from a single food-processing environmental sample, thus mimicking the environmental community. In mature mixed five-species biofilms on stainless steel, the two pathogens remained at a constant level of ∼105 CFU/cm2 The mixed five-species biofilms as well as the pathogens in monospecies biofilms were exposed to biocides to determine any pathogen-protective effect of the mixed biofilm. Both pathogens and their associate microbial communities were reduced by peracetic acid treatments. S. aureus decreased by 4.6 log cycles in monospecies biofilms, but the pathogen was protected in the five-species biofilm and decreased by only 1.1 log cycles. Sessile cells of L. monocytogenes were affected to the same extent when in a monobiofilm or as a member of the mixed-species biofilm, decreasing by 3 log cycles when exposed to 0.0375% peracetic acid. When the pathogen was exchanged in each associated microbial community, S. aureus was eradicated, while there was no significant effect of the biocide on L. monocytogenes or the mixed community. This indicates that particular members or associations in the community offered the protective effect. Further studies are needed to clarify the mechanisms of biocide protection and to identify the species playing the protective role in microbial communities of biofilms.IMPORTANCE This study demonstrates that foodborne pathogens can be established in mixed-species biofilms and that this can protect them from biocide action. The protection is not due to specific characteristics of the pathogen, here S. aureus and L. monocytogenes, but likely caused by specific members or associations in the mixed-species biofilm. Biocide treatment and resistance are a challenge for many industries, and biocide efficacy should be tested on microorganisms growing in biofilms, preferably mixed systems, mimicking the application environment.

Staphylococcus aureus in Some Brazilian Dairy Industries: Changes of Contamination and Diversity.

Staphylococcus aureus, a major food-poisoning pathogen, is a common contaminant in dairy industries worldwide, including in Brazil. We determined the occurrence of S. aureus in five dairies in Brazil over 8 months. Of 421 samples, 31 (7.4%) were positive for S. aureus and prevalence varied from 0 to 63.3% between dairies. Sixty-six isolates from the 31 samples were typed by Multi-Locus Sequence Typing to determine if these isolates were persistent or continuously reintroduced. Seven known sequence types (STs), ST1, ST5, ST30, ST97, ST126, ST188 and ST398, and four new ST were identified, ST3531, ST3540, ST3562 and ST3534. Clonal complex (CC) 1 (including the four new ST), known as an epidemic clone, was the dominant CC. However, there were no indications of persistence of particular ST. The resistance toward 11 antibiotic compounds was assessed. Twelve profiles were generated with 75.8% of strains being sensitive to all antibiotic classes and no Methicillin-resistant S. aureus (MRSA) strains were found. The enterotoxin-encoding genes involved in food-poisoning, e.g., sea, sed, see, and seg were targeted by PCR. The two toxin-encoding genes, sed and see, were not detected. Only three strains (4.5%) harbored seg and two of these also harbored sea. Despite the isolates being Methicillin-sensitive S. aureus (MSSA), the presence of CC1 clones in the processing environment, including some harboring enterotoxin encoding genes, is of concern and hygiene must have high priority to reduce contamination.