Identification and antimicrobial susceptibility profiles of Staphylococcus species isolated from raw cow milk, and swabs in smallholder dairy farms in Meta district, Eastern Ethiopia.

BACKGROUND: The safety of milk production in terms of foodborne infections is a worldwide issue, particularly in developing countries where production is often unhygienic. A cross-sectional study was conducted from December 2018 to August 2019 in the Meta District of Eastern Hararghe Zone, Oromia Regional State, Ethiopia. We aim to assess milk hygiene practices among smallholder dairy farmers, estimate the prevalence of Staphylococcus aureus in raw cow milk and swabs, assess associated risk factors, and the antimicrobial susceptibility test of S. aureus isolates. Face-to-face interviews with 30 respondents randomly selected from smallholder dairy farmers were used to assess the potential risk factors for S. aureus contaminations in milk. A total of 177 samples were examined using standard microbiological testing. The disc diffusion technique was also employed to assess the antibiotic susceptibility of the isolates. The data was analyzed using STATA® version 14.0 statistical software. RESULTS: According to the milk hygiene assessment, 80% of respondents did not wash cow udder before milking, did not use detergent to clean milk containers, and did not keep milk refrigerated before consumption or sale, while 63.3% of milk consumers ingested raw milk. They had never heard of staphylococci foodborne disease. Likewise, the overall prevalence of S. aureus was 12.42% (95%CI: 8.32-18.98). The prevalence of S. aureus in udder milk, equipment swabs, and milkers' hands was 18.8%, 26.7%, and 30%, respectively. The prevalence of S. aureus in milk is significantly associated with age, and mastitis history (p < 0.05). Moreover, old and mastitis positive animals were eight (OR: 8.40; 95%CI: 1.68-41.89) and four (OR: 4.33; 95%CI: 1.37-13.66) times more likely to be infected by S. aureus than adult, and mastitis negative animal. The isolates were resistant to penicillin G (97.4%) and tetracycline (69.2%) whereas susceptible to kanamycin, streptomycin, vancomycin, and cefotaxime, at 84.6%, 71.8%, 64%, and 58.8%, respectively. CONCLUSION: This study revealed the presence of antimicrobial-resistant patterns of S. aureus on commonly used antibiotics, as well as inadequate milk handling practices in the study area. Thus, awareness should be created on proper milk handling and hygiene as well as appropriate uses of antibiotics should be encouraged.

Milk-derived exosome nanovesicles: recent progress and daunting hurdles.

Raw milk is the foundation of quality and safety in the dairy industry, and improving milk source management is the fundamental guarantee. Milk-derived exosomes (MDEs) are nanoscale information transfer molecules secreted by mammary cells with unique content and high stability, which can be used not only as potential markers to analyze key traits of lactation, reproduction, nutrition and health of animals, but also help farm managers to take timely interventions to improve animal welfare, milk quality, and functional traits. Our review first outlines the latest advances in MDEs isolation and purification, compositional analysis and characterization tools. We then provide a comprehensive summary of recent applications of MDEs liquid biopsy in breed selection, disease prevention and control, and feeding management. Finally, we evaluate the impact of processing on the stability of MDEs to offer guidance for dairy production and storage. The limitations and challenges in the development and use of MDEs markers are also discussed. As a noninvasive marker with high sensitivity and specificity, the MDEs-mediated assay technology is expected to be a powerful tool for measuring cow health and raw milk quality, enabling dynamic and precise regulation of dairy cows and full traceability of raw milk.

Serial cultures in invert emulsion and monophase systems for microbial community shaping and propagation.

BACKGROUND: Microbial communities harbor important biotechnological potential in diverse domains, however, the engineering and propagation of such communities still face both knowledge and know-how gaps. More specifically, culturing tools are needed to propagate and shape microbial communities, to obtain desired properties, and to exploit them. Previous work suggested that micro-confinement and segregation of microorganisms using invert (water-in-oil, w/o) emulsion broth can shape communities during propagation, by alleviating biotic interactions and inducing physiological changes in cultured bacteria. The present work aimed at evaluating invert emulsion and simple broth monophasic cultures for the propagation and shaping of bacterial communities derived from raw milk in a serial propagation design. RESULTS: The monophasic setup resulted in stable community structures during serial propagation, whereas the invert emulsion system resulted in only transiently stable structures. In addition, different communities with different taxonomic compositions could be obtained from a single inoculum. Furthermore, the implementation of invert emulsion systems has allowed for the enrichment of less abundant microorganisms and consequently facilitated their isolation on culture agar plates. CONCLUSIONS: The monophasic system enables communities to be propagated in a stable manner, whereas the invert emulsion system allowed for the isolation of less abundant microorganisms and the generation of diverse taxonomic compositions from a single inoculum.

Growth of methicillin-resistant Staphylococcus aureus during raw milk soft cheese-production and the inhibitory effect of starter cultures.

The consumption of raw milk or raw milk products might be a potential risk factor for the transmission of methicillin-resistant Staphylococcus aureus (MRSA). Therefore, we studied MRSA growth during raw milk soft cheese-production. Furthermore, we investigated the inhibitory effect of four starter cultures (Lactococcus lactis, Lacticaseibacillus rhamnosus, Lactiplantibacillus plantarum, Lactobacillus helveticus) on the growth of MRSA in a spot-agar-assay and in raw milk co-culture following a cheesemaking temperature profile. During the initial phases of raw milk cheese-production, MRSA counts increased by 2 log units. In the ripening phase, MRSA counts only dropped slightly and remained high up to the end of the storage. Comparable MRSA counts were found in the rind and core and strain-specific differences in survival were observed. In the spot-agar-assay, all four starter cultures showed strong or intermediate inhibition of MRSA growth. In contrast, in raw milk, only Lactococcus lactis strongly inhibited MRSA, whereas all other starter cultures only had minor inhibitory effects on MRSA growth. Our results indicate that MRSA follow a similar growth pattern as described for other S. aureus during raw milk soft cheese-production and illustrate the potential use of appropriate starter cultures to inhibit MRSA growth during the production of raw milk cheese.

Comparative study of the bacterial community of organic and conventional cow's milk.

Agricultural practises such as conventional and organic farming can potentially affect the microbial communities in milk. In the present study, the bacterial diversity of milk was investigated using high-throughput sequencing on ten organic and ten conventional farms in the Azores, a region where milk production is largely based on year-round grazing systems. The microbiota of milk from both production systems was dominated by Bacillota, Pseudomonadota, Actinomycetota and Bacteroidota. The organic milk showed greater heterogeneity between farms, as reflected in the dispersion of diversity indices and the large variation in the relative abundances of the dominant genera. In contrast, conventionally produced milk showed a high degree of similarity within each season. In the conventional production system, the season also had a strong influence on the bacterial community, but this effect was not observed in the organic milk. The LEfSe analysis identified the genus Iamia as significantly (p < 0.05) more abundant in organic milk, but depending on the season, several other genera were identified that distinguished organic milk from conventionally produced milk. Of these, Bacillus, Iamia and Nocardioides were associated with the soil microbiota in organic farming.

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.

Sources of propionic acid bacteria contamination in the milking parlor environment on Alpine Dairy Farms.

High quality raw milk is an important prerequisite for the production of long ripened raw milk cheeses. This implies not only the absence of pathogenic microorganisms in raw milk, but also low levels of spoilage bacteria, including dairy propionic acid bacteria (dPAB), that can cause blowing and sensory defects in cheese, resulting in severe economic losses for producers. Raw milk contamination with dPAB has been primarily associated with improperly cleaned milking systems, but they have been detected in feed, soil, feces and on the teat skin. The objective of this study was to identify potential sources of raw milk contamination with dPAB in the barn and milking parlor environments. We also wanted to know more about the prevalence of the dPAB species in these environments and the levels of contamination. For this purpose, 16 small scale Alpine dairy farms were visited in August 2022: samples were taken from the barn environment (e.g., swab samples, air, feed, bedding), the milking system (swab samples, residual cleaning water, cleaning sponges, milk filters) and milk samples were collected at various sampling points along the milking system. Samples were analyzed for dPAB contamination, and results showed contamination at multiple sampling locations. We observed potential adverse effects of improperly set cleaning parameters of the milking system, as well as of farm specific practices. In addition, we identified cleaning water residues as an important source of contamination. Based on these findings, we propose potential mitigation strategies to reduce the risk of raw milk contamination with cheese spoilage bacteria, thereby contributing to a more sustainable food production.

Troubleshooting high laboratory pasteurization counts in organic raw milk requires characterization of dominant thermoduric bacteria, which includes nonsporeformers as well as sporeformers.

Laboratory pasteurization count (LPC) enumerates thermoduric bacteria and is one parameter used to assess raw milk quality. No regulatory limit has presently been set for LPC, but LPC data are used by some dairy processors and cooperatives to designate raw milk quality premiums paid to farmers and may also be used for troubleshooting bacterial contamination issues. Although it is occasionally used as a proxy for levels of bacterial spores in raw milk, limited knowledge is available on the types of organisms that are enumerated by LPC in contemporary raw milk supplies. Although historical studies have reported that thermoduric bacteria quantified by LPC may predominantly represent gram-positive cocci, updated knowledge on microbial populations enumerated by LPC in contemporary organic raw milk supplies is needed. To address this gap, organic raw milk samples from across the United States (n = 94) were assessed using LPC, and bacterial isolates were characterized. LPC ranged from below detection (<0.70 log cfu/mL) to 4.07 log cfu/mL, with a geometric mean of 1.48 log cfu/mL. Among 380 isolates characterized by 16S rDNA sequencing, 52.6%, 44.5%, and 2.4% were identified as gram-positive sporeformers, gram-positive nonsporeformers, and gram-negatives, respectively; 0.5% could not be categorized into those groups because they could only be assigned a higher level of taxonomy. Isolates identified as gram-positive sporeformers were predominantly Bacillus (168/200), and gram-positive nonsporeformers were predominantly Brachybacterium (56/169) and Kocuria (47/169). To elucidate if the LPC level can be an indicator of the type of thermoduric (e.g., sporeforming bacteria) present in raw milk, we evaluated the proportion of sporeformers in raw milk samples with LPC of ≤100 cfu/mL, 100 to 200 cfu/mL, and ≥200 cfu/mL (51%, 67%, and 35%), showing a trend for sporeformers to represent a smaller proportion of the total thermoduric population when LPC increases, although overall linear regression showed no significant association between the proportion of sporeformers and the LPC concentration. Hence, LPC level alone provides no insight into the makeup of the thermoduric population in raw milk, and further characterization is needed to elucidate the bacterial drivers of elevated LPC in raw milk. We therefore further characterized the isolates from this study using MALDI-TOF mass spectrometry (MALDI-TOF MS), a rapid microbial identification tool that is more readily available to dairy producers than 16S rDNA PCR and sequencing. Although our data indicated agreement between 16S rDNA sequencing and MALDI-TOF MS for 66.6% of isolates at the genus level, 24.2% and 9.2% could not be reliably identified or were mischaracterized using MALDI-TOF MS, respectively. This suggests that further optimization of this method is needed to allow for accurate characterization of thermoduric organisms commonly found in raw milk. Ultimately, our study provides a contemporary perspective on thermoduric bacteria selected by the LPC method and establishes that the LPC alone is not sufficient for identifying the bacterial drivers of LPC levels. Further development of rapid characterization methods that are accessible to producers, cooperatives, and processors will support milk quality troubleshooting efforts and ultimately improve outcomes for dairy industry community members.

Deciphering the Mechanism by Which Carbon Dioxide Extends the Shelf Life of Raw Milk: A Microbiomics- and Metabolomics-Based Approach.

Microbial community succession in raw milk determines its quality and storage period. In this study, carbon dioxide (CO2) at 2000 ppm was used to treat raw milk to investigate the mechanism of extending the shelf life of raw milk by CO2 treatment from the viewpoint of microbial colonies and metabolites. The results showed that the shelf life of CO2-treated raw milk was extended to 16 days at 4 °C, while that of the control raw milk was only 6 days. Microbiomics analysis identified 221 amplicon sequence variants (ASVs) in raw milk, and the alpha diversity of microbial communities increased (p < 0.05) with the extension of storage time. Among them, Pseudomonas, Actinobacteria and Serratia were the major microbial genera responsible for the deterioration of raw milk, with a percentage of 85.7%. A combined metagenomics and metabolomics analysis revealed that microorganisms altered the levels of metabolites, such as pyruvic acid, glutamic acid, 5'-cmp, arginine, 2-propenoic acid and phenylalanine, in the raw milk through metabolic activities, such as ABC transporters, pyrimidine metabolism, arginine and proline metabolism and phenylalanine metabolism, and reduced the shelf life of raw milk. CO2 treatment prolonged the shelf life of raw milk by inhibiting the growth of Gram-negative aerobic bacteria, such as Acinetobacter guillouiae, Pseudomonas fluorescens, Serratia liquefaciens and Pseudomonas simiae.

Potentially toxic elements in soil-plant-water-animal continuum in a mining area from Northwestern Mexico: animal exposure pathways and health risks for children.

Mining increases environmental concentrations of potentially toxic elements (PTEs) accumulating in organisms and spreading in the human food chain-their presence in milk is of great human health concern. Pathways were identified by which these elements reach raw milk from farms within a mining area in Northwestern Mexico; health risks for dairy cattle and children were also evaluated. Water from river and cattle waterers, as well as, soils showed that PTE concentrations generally below the Mexican and international limits; cattle forage concentrations were above the World Health Organization limits. Al, Mg, Mo, Ni and Zn were recorded in raw milk samples from the mining area, showing that Cd, Co, Cr, Cu, Pb and V are transferred from soil to plants but not accumulated in raw milk. Zn concentrations in raw milk exceeded the permissible limit; milk from farms without mining operations (comparison site) showed the presence of Al, Cr and Cu. In cattle tail hair, PTE did not correlate with raw milk concentrations. Metal accumulation in milk was higher through water consumption than that accumulated through forage consumption. Daily intakes (DI) of Al, Mg and Zn in cows could represent a risk for their health. The observed biotransference was higher than in other parts of Mexico, and the calculated DI and hazard quotients indicate no adverse health effects for children. However, the hazard Index values indicate that exposure to multiple PTE represents a risk for children. Management measures should be performed to control the cumulative risks to protect young children's health.

Severe Streptococcus equi Subspecies zooepidemicus Outbreak from Unpasteurized Dairy Product Consumption, Italy.

During November 2021-May 2022, we identified 37 clinical cases of Streptococcus equi subspecies zooepidemicus infections in central Italy. Epidemiologic investigations and whole-genome sequencing showed unpasteurized fresh dairy products were the outbreak source. Early diagnosis by using sequencing technology prevented the spread of life-threatening S. equi subsp. zooepidemicus infections.

Corynebacterium suedekumii sp. nov and Corynebacterium breve sp. nov., isolated from raw cow's milk.

Four Gram-positive, rod-shaped, none-sporeforming, non-motile isolates were obtained from various raw milk samples taken from the cooling tank on a research farm in Königswinter, Germany. Based on phylogenetic analysis of the 16S rRNA genes and whole genome sequences, all isolates were assigned to the genus Corynebacterium, but were divided in two different groups. All isolates contained C18 : 1 cis 9 and C16 : 0 as predominant fatty acids, as well as traces of C18 : 0. They all contained menaquinones MK-8 (H2) and MK-9 (H2) and produced mycolic acids characteristic for the majority of species belonging to the genus Corynebacterium. 16S rRNA gene sequence similarity values to the closest related type strains Corynebacterium humireducens DSM 45392T and Corynebacterium pilosum DSM 20521T were below 98.7 %, average nucleotide identity values were below 86 % and digital DNA-DNA-hybridization values were below 25 %, indicating that the isolates represent two novel species. The names Corynebacterium suedekumii sp. nov. and Corynebacterium breve sp. nov. are proposed, represented by the type strains LM112T (=DSM 116216T=HAMBI 3782T) and R4T (=DSM 116183T=HAMBI 3785T), respectively.

A systematic literature review of milk consumption and associated bacterial zoonoses in East Africa.

Consumption of unsafe animal-source foods is the major cause of foodborne disease outbreaks in low-income countries. Despite current knowledge of the threat posed by raw milk consumption to human health, people in many countries in East Africa still consume unboiled milk. This literature review explored the association between milk consumption and the occurrence of five milk-borne bacterial zoonoses: brucellosis, salmonellosis, campylobacteriosis, Escherichia coli infections, and tuberculosis. A search for literature published up to 1 October 2021 was conducted through the Web of Science, PubMed, and Scopus databases, using Preferred Reporting Items for Systematic reviews and Meta-Analyses guidelines. The selection process yielded 65 articles describing studies conducted in East Africa 2010-2021, which were carefully scrutinized. The most investigated pathogen was Brucella spp. (54.5%), followed by E. coli (18.2%), Salmonella spp. (12.1%), Mycobacterium spp. (6.1%), and E. coli O157: H7 (6.1%). The most common predisposing factors for potential milk-borne disease outbreaks were consumption of contaminated raw milk, inadequate cold storage along the milk value chain, poor milk handling practices, and lack of awareness of the health risks of consuming unpasteurized milk. Thus, a tailor-made training program is needed for all milk value chain actors to enhance the safety of milk sold in informal markets, and a One Health approach should be applied. Future studies should employ more advanced diagnostic techniques and countries in East Africa should invest in modern diagnostic tools and equipment, both in hospitals and in local rural settings where most cases occur.

Carbapenem resistance and biofilm formation status of Enterobacterales isolated from raw milk via molecular versus phenotypic methods.

Antibiotic resistance genes can easily be transferred between bacteria in the biofilm. In the dairy industry, many bacterial species forming biofilms on the surfaces of equipment are widely reported. The experiments reported in this research paper aimed to investigate the carbapenem resistance and biofilm formation properties of Enterobacterales isolates which are spoilage microorganisms obtained from raw milk. In addition, the study determined that whether there was a relationship between the biofilm formation ability or the protein spectra of these isolates. In this study, ninety-two Enterobacterales isolates collected from 173 raw milk samples were investigated. Initially, the isolates were identified as Citrobacter braakii (n = 18), Citrobacter freundii (n = 12), Enterobacter asburiae (n = 1), Enterobacter cloacae (n = 3), Escherichia coli (n = 10), Hafnia alvei (n = 18), Klebsiella oxytoca (n = 1), Serratia fonticola (n = 24), Serratia liquefaciens (n = 4), and Serratia marcescens (n = 1) using MALDI-TOF MS. As a result, carbapenem resistance was determined in 6.5% of the isolates by CIM test, MHT, and the disk diffusion methods, but none of them had blaOXA-48, blaKPC, blaNDM-1, blaOXA23, blaOXA-58, blaOXA-51, blaVIM, and blaIMP genes. This may be due to the effect of other resistance mechanisms such as porin loss or increased flow pump activity. Furthermore, biofilm formation (weak and moderate) was detected in 97.8% of the Enterobacterales isolates. The mass spectra of the moderate biofilm producer isolate of Serratia spp. and the mass spectra of the weak biofilm producers of E.coli presented similarities.

A novel Staphylococcus phage, vB_Sau-RP15, and its application in contaminated milk.

The vB_Sau-RP15 phage, selected for its potential use as a phage treatment in milk, was isolated from raw milk using Staphylococcusaureus NP01 as the host. The host range test revealed that the phage was able to lyse 12 strains of Staph. aureus from raw milk. This phage was stable at 4-37°C and pH 6-9 for at least 1 h. The adsorption rate was ~78% within the first 3 min. A low frequency of phage-insensitive mutant induction (4.6 × 10-6) was observed. Genomic analyses revealed that the vB_Sau-RP15 represented a novel species in the genus Silviavirus. Even though no virulence or antibiotic resistance genes were detected, the phage genome carried lysogenic-associated genes. Phage treatments (108 PFU per ml) in pasteurized milk contaminated with low (104 CFU per ml) and high (107 CFU per ml) concentrations of Staph. aureus confirmed the proficiency of the phage in the diminishing of the number of bacterial cells at 4°C and ambient temperature. A Staphylococcus phage, vB_Sau-RP15, could be a promising agent for controlling Staph. aureus contamination in milk.

Effects of storage temperature on microbiota shifts in raw milk biofilm developed on stainless steel.

This study aimed to investigate the microbiota in raw milk and the influence of storage temperature on the microbiota shift after biofilm formation. Raw milk stored at 4 °C and biofilms developed in raw milk incubated at 4 °C or 25 °C for 7 days were subjected to microbiota analysis as well as quantitative analyses of aerobic or anaerobic bacteria. The levels of aerobic bacteria increased during biofilm formation, while no significant changes were observed within anaerobic bacteria. In addition, there was a difference between aerobic and anaerobic bacterial counts in raw milk and biofilm stored for 7 days. The pattern of microbial composition differed by temperature. In addition, the genus Pseudomonas (53-71%) occupied a high proportion in raw milk, and the raw milk biofilm developed at 4 °C, while the genus Lactobacillus (75-83%) was predominant in biofilms developed at 25 °C. Intriguingly, bacterial richness was higher in raw milk on day 0 and biofilm developed at 4 °C than raw milk after 7 days of storage at 4 °C. These findings suggest that temperature critically affects the bacterial composition of both raw milk and its associated biofilm.

Prevalence of Campylobacter species and associated risk factors for contamination of dairy products collected in a dry season from major milk sheds in Ethiopia.

A cross-sectional study was conducted to investigate the prevalence and risk factors for contamination of Ethiopian dairy products with Campylobacter. A total of 912 dairy food samples were collected from establishments of 682 study participants that were interviewed. Samples were tested for Campylobacter by following the ISO 10272-1:2017 standard and PCR confirmation. Campylobacter was detected in 11% of tested food samples and all detected Campylobacter were C. jejuni. The highest prevalence of C. jejuni was found in raw milk (16%), followed by pasteurized milk (9%) and cottage cheese (2%) (P < 0.001). Using warm water and soap for cleaning cow udders and teats on farms reduced the likelihood of detecting Campylobacter in milk (AOR = 0.3, P = 0.023). Filtering milk with a cloth, using a plastic filter (AOR = 0.065, P = 0.005), and storing milk in an aluminum container (AOR = 0.23, P = 0.027) reduced the likelihood of detecting Campylobacter in milk at the collection facilities. In contrast, Campylobacter detection was significantly more likely in milk collected at collection centers with concrete floors (AOR = 5.2, P = 0.004). The odds of detecting Campylobacter in milk were 17 times greater (AOR = 17, P = 0.007) in milk processing facilities that did not calibrate a pasteurizer on an annual basis. Finally, having a separate refrigerator for milk storage reduced the odds of detecting Campylobacter in retail (AOR = 0.29, P = 0.021).

Invited review: Redefining raw milk quality-Evaluation of raw milk microbiological parameters to ensure high-quality processed dairy products.

Raw milk typically has little bacterial contamination as it leaves the udder of the animal; however, through a variety of pathways, it can become contaminated with bacteria originating from environmental sources, the cow herself, and contact with contaminated equipment. Although the types of bacteria found in raw milk are very diverse, select groups are particularly important from the perspective of finished product quality. In particular, psychrophilic and psychrotolerant bacteria that grow quickly at low temperatures (e.g., species in the genus Pseudomonas and the family Enterobacteriaceae) and produce heat-stable enzymes, and sporeforming bacteria that survive processing hurdles in spore form, are the 2 primary groups of bacteria related to effects on processed dairy products. Understanding factors leading to the presence of these important bacterial groups in raw milk is key to reducing their influence on processed dairy product quality. Here we examine the raw milk microbiological parameters used in the contemporary dairy industry for their utility in identifying raw milk supplies that will perform well in processed dairy products. We further recommend the use of a single microbiological indicator of raw milk quality, namely the total bacteria count, and call for the development of a whole-farm approach to raw milk quality that will use data-driven, risk-based tools integrated across the continuum from production to processing and shelf-life to ensure continuous improvement in dairy product quality.

Effect of cooking temperature on alkaline phosphatase in the production of raw-milk Pecorino cheese.

Alkaline phosphatase (ALP) is a native raw-milk enzyme used in many countries as the standard assay for rapidly validating the milk pasteurization process. Due to the increased restrictions on the production or import of cheeses produced from unpasteurized milk, ALP activity (<10 mU/g) in cheese was measured as a simple and reliable method to check proper milk pasteurization in cheese for both safety inspection and trading controls. In Sicily, the artisanal cheesemaking of the Protected Denomination of Origin (PDO) semi-hard cheeses made with raw sheep milk, includes the cooking of the curd, after whey separation, in a wooden vat under hot Scotta whey (≥80°C), for 3 to 4 h, and finally is left to cool at ambient temperature. Thus, the temperatures adopted during cheesemaking may inactivate the ALP enzyme. To this purpose, the aim of this study was to demonstrate how different temperatures of Scotta whey (35°C [T35], 60°C [T60], 70°C [T70], 80°C [T80], 90°C [T90], and 100°C [T100]) used during the second cooking of Pecorino cheeses after molding for 3 h, influence the ALP activity in fresh and 3-mo aged cheese, both at core and outside. The results highlight that the rate of reduction of ALP was greater with increasing temperature of the second cooking, in particular for T 80°C curd, indicating that the use of Scotta whey >80°C could be a breakpoint able to reduce the ALP activity to values <10 mU/g. Different effects between the core and the outside portions of the experimental cheeses were found, with a decrease in ALP activity more on the outside than in the core portions, in both fresh and 3-mo aged cheeses, for T80, T90, and T100 treatments. Care must be taken in using ALP to control the use of pasteurized milk in the production of PDO cheeses without considering the cheesemaking processes, such as the second cooking, which could be equal to pasteurization, and an adequate interaction of time and temperature can reduce the ALP activity to values comparable with cheeses produced with pasteurized milk.

Outbreaks of Campylobacteriosis Caused by Drinking Raw Milk in Japan: Evidence of Relationship Between Milk and Patients by Using Whole Genome Sequencing.

Raw milk may contain some infectious bacteria and usually requires pasteurization before drinking. In this study, we report rare outbreaks of campylobacteriosis associated with raw milk in Japan, and the application of whole genome sequencing (WGS) to studies on foodborne diseases. In August 2018, there were three outbreaks of campylobacteriosis, presumably caused by the consumption of unpasteurized raw milk, derived from the same farm; thus, these three outbreaks seemed to be associated with a single contaminant at the farm. Therefore, we analyzed Campylobacter jejuni isolates obtained at the three locations using several genetic methods. The sequence type of each isolate, revealed by multilocus sequence typing, was ST-61, and the profile determined using pulsed-field gel electrophoresis was the same; however, neither method could distinguish these from previously obtained strains. Subsequently, we performed WGS and single nucleotide variant (SNV) analysis that provided evidence of clonality, indicating that C. jejuni contamination was attributed to the farm. As in this study, evidence suggests that SNV analysis provides molecular biological support in cases with sufficient epidemiological information. Hence, similar analytical methods may be used in other sporadic cases to elucidate the relevance of the cases.