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.