Toxigenic characterization, spoilage potential, and antimicrobial susceptibility of coagulase-positive Staphylococcus species isolated from Minas Frescal cheese.

This study aimed to identify coagulase-positive staphylococci (CPS) species from 21 samples of clandestine Minas Frescal cheese, investigate the potential for deterioration in psychrotrophic and mesophilic conditions, verify the toxigenic potential of Staphylococcus aureus, and determine the antimicrobial susceptibility profile of toxigenic S. aureus. Species determination was performed based on the detection of ß-hemolysis in 5% ovine blood agar; fermentation of mannitol, maltose, and trehalose sugars; and production of acetoin. After species determination, DNA extraction and analysis was performed for S. aureus colonies for genes encoding staphylococcal toxins (eta, etb, tst, sea, seb, sec, sed, and see) using 2 multiplex PCR assays. Isolates identified as toxigenic S. aureus were tested for antimicrobial susceptibility to tetracycline, erythromycin, clindamycin, gentamicin, ciprofloxacin, sulfazotrim, trimethoprim, streptomycin, cefoxitin, vancomycin and enrofloxacin. Elevated CPS counts were observed with an average of >6 log cfu/g. Of the 355 isolates, 177 (49.86%) were identified as S. aureus. Staphylococcus hyicus, Staphylococcus intermedius, Staphylococcus delphini, and Staphylococcus coagulans were identified in 3 (0.84%), 2 (0.56%), 2 (0.56%), and 1 (0.28%) isolates, respectively. Of the total number of S. aureus, 25 (52.08%) were positive for the gene that encodes for toxic shock toxin (TSST-1). Another 16 (33.33%) were positive for the sea gene, and 4 isolates (8.33%) were positive for see and one isolate each was positive for seb (2.08%), sec (2.08%), and etb (2.08%) genes. All isolates demonstrated lipolytic activity under mesophilic and psychrotrophic conditions. S. intermedius and S. hyicus had the most prominent proteolytic potential. Multidrug resistance was observed in most of the potentially toxigenic isolates, with clindamycin having the lowest efficiency (40%), whereas the aminoglycosides (gentamicin and streptomycin) had the highest effectiveness demonstrating inhibition in all evaluated isolates. Methicillin-resistant S. aureus (MRSA) was detected. Minas Frescal cheeses, marketed in the north of Tocantins in the Brazilian Amazon region, do not comply with legal quality standards and pose a public health risk due to the enterotoxigenic potential of multiresistant isolates, in addition to low shelf life of the samples given the high spoilage potential of this microbiota.

Effect of milk bactofugation on the counts and diversity of thermoduric bacteria.

The objective of this work was to determine the effect of milk bactofugation on the counts and microbial diversity of mesophilic (MT), psychrotrophic (PT), and thermophilic (TT) thermoduric bacteria and its potential as a technological method to remove spoilage microorganisms resistant to pasteurization. Different batches of raw milk from 69 dairy farms divided into sets in 3 bulk tanks (A, B, C) were evaluated at different times during the technological process. As the raw milk was preheated (∼55°C) immediately before bactofugation (10,000 × g), the effect of bactofugation was estimated by comparing the counts in raw, preheated, and bactofuged milk. This centrifugation was sufficient to reduce the isolation of 88% of the MT in preheated milk. For PT, it was possible to verify a reduction of 72.5% in batch C. The TT were not recovered at higher detection limits (<5 cfu/mL). For diversity, 310 isolates were identified using a molecular approach; 15 species of contaminating thermoduric bacteria were identified from raw and preheated milk, and only 6 species were recovered in bactofuged milk. Only MT were recovered from the bactofuged milk, mainly the species Lysinibacillus fusiformis (61.7%) and Bacillus licheniformis (12.3%). Both species are known to be endospore-forming psychrotrophs and have proteolytic or lipolytic activity. The bactofugation of raw milk reduced the number of isolates of B. licheniformis, Bacillus toyonensis, Micrococcus aloeverae, and Aestuariimicrobium kwangyangense by 33, 43, 86, and 92%, respectively, and reduced the isolates of Macrococcus caseolyticus, Lysinibacillus varians, Carnobacterium divergens, Microbacterium hominis, Kocuria indica, Micrococcus yunnanensis, Gordonia paraffinivorans, Bacillus invictae, and Kocuria kristinae to undetectable levels. The results of this study indicate that bactofugation can be applied by the dairy industry to reduce pasteurization-resistant microorganisms in combination with prophylactic measures to prevent the contamination of raw milk by spores and vegetative forms of bacteria.

Short communication: Molecular characterization and antimicrobial resistance of pathogenic Escherichia coli isolated from raw milk and Minas Frescal cheeses in Brazil.

The aim of this study was to quantify, identify, evaluate antimicrobial resistance, and characterize the virulence factors of enteropathogenic (EPEC), Shiga-toxigenic (STEC), and enterohemorrhagic (EHEC) Escherichia coli in raw milk (RM) and legal (LMFC) and illegal (IMFC) Minas Frescal cheeses in southern and northeast Brazil. Illegal cheeses are those made without official inspection service or sanitary surveillance. We evaluated samples of RM produced in Paraná (southern) and Maranhão (northeast) States, LMFC produced using pasteurized milk in inspected industries, and IMFC potentially produced with raw milk. Mean total coliform counts were 8.4 × 104 cfu/mL for RM, 1.4 × 107 cfu/mL for LMFC, and 2.9 × 107 cfu/mL for IMFC. Mean E. coli counts were 2.4 × 103 cfu/mL for RM, 1.9 × 102 cfu/mL for LMFC, and 1.1 × 105 cfu/mL for IMFC. Among the 205 E. coli isolates from RM, 9.75% were identified as EPEC, mainly (90%) in samples from Paraná. Of the total isolates from the cheese samples, 97.4% (n = 111) came from IMFC, of which 1.8 and 2.7% were identified as EPEC and STEC, respectively; no EHEC was detected. The phylogenetic group A (60%) and typical EPEC (68%) predominated, which confirms the possible human origin of pathogenic isolates in RM and IMFC. Of these, 50% were resistant to at least one antibiotic, and streptomycin was the antimicrobial with the highest number (8) of EPEC and STEC resistant isolates. This study reports the first isolation of serogroup O28ac in Brazilian milk. We found no predominance of a specific serogroup of EPEC or STEC in milk or cheese or clonal isolates in the same sample, indicating different origins of the contamination in these products, presumably mostly related to poor hygienic handling.

Genetic diversity of thermoduric spoilage microorganisms of milk from Brazilian dairy farms.

When correctly pasteurized, packaged, and stored, milk with low total bacterial counts (TBC) has a longer shelf life. Therefore, microorganisms that resist heat treatments are especially important in the deterioration of pasteurized milk and in its shelf life. The aim of this work was to quantify the thermoduric microorganisms after the pasteurization of refrigerated raw milk samples with low TBC and to identify the diversity of these isolates with proteolytic or lipolytic potential by RFLP analysis. Twenty samples of raw milk were collected in bulk milk tanks shortly after milking in different Brazilian dairy farms and pasteurized. The mean thermoduric count was 3.2 (±4.7) × 102 cfu/mL (2.1% of the TBC). Of the 310 colonies obtained, 44.2% showed milk spoilage potential, 32.6% were proteolytic and lipolytic simultaneously, 31% were exclusively proteolytic, and 48 (36.4%) were only lipolytic. Regarding the diversity, 8 genera were observed (Bacillus, Brachybacterium, Enterococcus, Streptococcus, Micrococcus, Kocuria, Paenibacillus, and Macrococcus); there was a predominance of endospore-forming bacteria (50%), and Bacillus licheniformis was the most common (34.1%) species. Considering the RFLP types, it was observed that the possible clonal populations make up the microbiota of different milk samples, but the same milk samples contain microorganisms of a single species with different RFLP types. Thus, even in milk with a high microbiological quality, it is necessary to control the potential milk-deteriorating thermoduric microorganisms to avoid the risk of compromising the shelf life and technological potential of pasteurized milk.

The main spoilage-related psychrotrophic bacteria in refrigerated raw milk.

Refrigerated raw milk may contain psychrotrophic microorganisms that produce thermoresistant exoproteases and lipases, which may compromise the quality of processed fluid milk and dairy products during storage. The aim of this work was to quantify and identify the deteriorating psychrotrophic microbiota in Brazilian refrigerated raw milk using genetic diversity analysis. The mean psychrotrophic count was 1.1 × 104 cfu/mL. Of the total isolates, 47.8 and 29.8% showed deteriorating activity at 35°C within 48 h and 7°C within 10 d, respectively. Among the proteolytic species, more isolated by this study were Lactococcus lactis (27.3%), Enterobacter kobei (14.8%), Serratia ureilytica (8%), Aerococcus urinaeequi (6.8%), and Bacillus licheniformis (6.8%). Observed among lipolytics were E. kobei (17.7%), L. lactis (15.6%), A. urinaeequi (12.5%), and Acinetobacter lwoffii (9.4%). The isolates S. ureilytica, E. kobei, Pseudomonas spp., and Yersinia enterocolitica potentially produced alkaline metalloprotease (aprX). Despite the low counts, a considerable portion of the psychrotrophic microbiota presented spoilage potential, which reaffirms the need for rigor in the control of contamination and the importance of rapid processing as factors that maintain the quality of milk and dairy products.