Contamination Of Raw Milk Research Articles

The First Pseudomonas Phage vB_PseuGesM_254 Active against Proteolytic Pseudomonas gessardii Strains

Bacteria of the Pseudomonas genus, including the Pseudomonas gessardii subgroup, play an important role in the environmental microbial communities. Psychrotolerant isolates of P. gessardii can produce thermostable proteases and lipases. When contaminating refrigerated raw milk, these bacteria spoil it by producing enzymes resistant to pasteurization. One possible way to prevent spoilage of raw milk is to use Pseudomonas lytic phages specific to undesirable P. gessardii isolates. The first phage, Pseudomonas vB_PseuGesM_254, was isolated and characterized, which is active against several proteolytic P. gessardii strains. This lytic myophage can infect and lyse its host strain at 24 °C and at low temperature (8 °C); so, it has the potential to prevent contamination of raw milk. The vB_PseuGesM_254 genome, 95,072 bp, shows a low level of intergenomic similarity with the genomes of known phages. Comparative proteomic ViPTree analysis indicated that vB_PseuGesM_254 is associated with a large group of Pseudomonas phages that are members of the Skurskavirinae and Gorskivirinae subfamilies and the Nankokuvirus genus. The alignment constructed using ViPTree shows that the vB_PseuGesM_254 genome has a large inversion between ~53,100 and ~70,700 bp, which is possibly a distinctive feature of a new taxonomic unit within this large group of Pseudomonas phages.

Evolution of bacterial flora in raw yak milk and its effect on quality characteristics during frozen storage

Due to the scarcity of yak milk in Tibetan areas, local herders primarily use traditional freezing methods for preservation. However, the extent of microbial contamination in raw yak milk during frozen storage and its impact on milk quality remains largely unexplored. This study analyzed the changes in bacterial populations and quality of raw yak milk stored at −18 °C for 6 months. The results indicated that, compared to fresh milk, the freshness of yak milk showed no significant change after 2 months of frozen storage (P > 0.05). After 4 months, the bacterial diversity of flora in yak milk increased significantly, with Pseudomonas and Acinetobacter emerging as the dominant psychrophilic bacteria. Correspondingly, the expression of metabolic pathways related to quality deterioration increased, with a significant increase in physicochemical indicators such as acidity, proteolysis degree, acid value and fat oxidation degree (P < 0.05). Extending the frozen storage to 6 months resulted in a slight but no significant decrease in bacterial diversity, with no significant difference in quality deterioration compared to the 4-month group (P > 0.05). Correlation analysis further revealed that Acinetobacter and Psychrobacter were significantly positively correlated with quality deterioration of frozen milk (P < 0.05). In summary, when frozen for more than 4 months, raw yak milk exhibited the highest bacterial diversity and predicted the most abundant metabolic pathways, with dominant psychrophilic bacteria massive proliferation leading to a decline in quality and safety.

Микробиологические риски как причина пороков внешнего вида сыра

Aerobic Bacillus bacteria, yeasts, and micrococci are pigment-forming microorganisms responsible for color defects in cheese production. The article describes their environment, industry-related characteristics, types of defects they cause, and various color violations in commercial cheeses. The research featured domestic cheeses with yellow and red surface spots that appeared during industrial ripening and storage. Pigment-forming microorganisms developed on cheese surface at a low ripening temperature, high concentration of table salt, and critically low oxygen. Pigment-forming microflora proved to enter cheese from milk, brine, or production environment, e.g., water, air, and processing equipment, dry and succulent feeds being the main source of milk contamination with spore-forming aerobic bacteria, yeast, mold fungi, etc. Contamination of raw milk, milking equipment, and air environment with micrococci was primarily associated with cows’ udder and skin. Cheese production presupposes low-temperature heat treatment of raw milk, which means possible residual microflora in pasteurized milk. As a result, the total count of mesophilic aerobic and optionally anaerobic microorganisms, i.e., the potential pigment formers, was to stay below 104 CFU/cm3. Yeast and spore-forming Bacillus microorganisms demonstrated the acceptable level of ≤102 CFU/cm3 in milk intended for cheesemaking. If their total count exceeded 103 CFU/cm3, it indicated high microbiological risks of cheese appearance defects. The microflora responsible for discoloration of cheese surface was represented by yeast, micrococci, aerobic spores, or their combinations.

Epidemiology, Virulence and Antimicrobial Resistance of Escherichia coli Isolated from Small Brazilian Farms Producers of Raw Milk Fresh Cheese.

This study aimed to identify contamination sources in raw milk and cheese on small farms in Brazil by isolating Escherichia coli at various stages of milk production and cheese manufacturing. The study targeted EAEC, EIEC, ETEC, EPEC, STEC, and ExPEC pathotypes, characterizing isolates for the presence of virulence genes, phylogroups, antimicrobial susceptibility, and phylogenetic relationships using PFGE and MLST. The presence of antimicrobial resistance genes and serogroups was also determined. Three categories of E. coli were identified: pathogenic, commensal, and ceftriaxone-resistant (ESBL) strains. Pathogenic EPEC, STEC, and ExPEC isolates were detected in milk and cheese samples. Most isolates belonged to phylogroups A and B1 and were resistant to antimicrobials such as nalidixic acid, ampicillin, kanamycin, streptomycin, sulfisoxazole, and tetracycline. Genetic analysis revealed that E. coli with identical virulence genes were present at different stages within the same farm. The most frequently identified serogroup was O18, and MLST identified ST131 associated with pathogenic isolates. The study concluded that E. coli was present at multiple points in milk collection and cheese production, with significant phylogroups and high antimicrobial resistance. These findings highlight the public health risk posed by contamination in raw milk and fresh cheese, emphasizing the need to adopt hygienic practices to control these microorganisms.

Comparative analysis of nutritional quality and microbial contamination in camel milk: Evaluating safety and public health risks between camel milk obtained from street vendors and farms in Saudi Arabia.

To evaluate the nutritional quality and microbial contamination of raw camel milk sourced from street vendors and compare it with milk obtained from farms, with a particular emphasis on pathogenic organisms. Twenty samples were systematically collected from street vendors and farms between July 2022 and February 2023 and analyzed at King Fahad Medical Research Centre and the Pharmacy College, King Abdulaziz University, Jeddah, Saudi Arabia. The microbial analysis employed culture-dependent techniques for colony-forming unit analysis and isolation of microbial colonies from milk samples. Microbial identification utilized advanced methods, including VITEK-MS equipment and the MALDI-TOF technique. The chemical composition was analysed through enzyme-linked immunosorbent assays. The findings revealed significant differences in microbial loads, with milk sourced from street vendors exhibiting considerably higher microbial counts than farm-sourced milk, including pathogenic species like Staphylococcus aureus and Escherichia coli. The results indicated that camel milk from street vendors possessed a higher level of microbial contamination, suggesting potential health risks associated with its purchase and consumption from these sources. This study highlights the urgent need for stringent food safety practices in handling, selling, and distributing camel milk to reduce microbial risks to safe levels, thereby mitigating potential health hazards.

Assessment of elements (toxic and essential) concentrations and methicillin resistance in Staphylococcus aureus strains isolated from raw milk in the Kurdistan province

The presence of residual antibiotics and the excessive accumulation of metals beyond permissible limits in milk represent significant health challenges. This study aimed to analyse the levels of heavy metals and trace elements in raw milk from Kurdistan province using inductively coupled plasma mass spectrometry (ICP‐MS) following digestion via thermal block. Subsequently, the samples were evaluated for contamination with MRSA (methicillin‐resistant Staphylococcus aureus), taking into account the potential correlation between methicillin‐resistant strains and metal accumulation. Finally, milk safety was assessed based on the presence of toxic metals. A total of 180 milk samples were examined. Among them, 13.34% were found to be contaminated with S. aureus, and of those, 62.48% tested positive for MRSA. The metal concentrations (mg/kg) were measured as follows: arsenic 7 × 10−2 ± 6 × 10−3, cadmium 4.70 × 10−1 ± 9.70 × 10−2, lead 7 × 10−2 ± 5 × 10−3, mercury 3 × 10−2 ± 9 × 10−3 and iron 4 × 10−2 ± 9 × 10−3. Interestingly, arsenic, cadmium and iron levels were found to be correlated with the presence of MRSA. Additionally, there was an inverse relationship between toxic metals and essential ones in samples sensitive to methicillin (P &lt; 0.001). The Target Hazard Quotient (THQ) for cadmium is indicated at 9.94. Furthermore, the calculated Carcinogenic Risk (CR) parameters for cadmium and arsenic were 6.06 × 10−3 and 2.22 × 10−4, respectively, suggesting that their concentrations fall within the carcinogenic range. The coexistence of MRSA and metal contamination in raw milk highlights the potential risks linked to its consumption. Consequently, the adoption of rigorous hygiene protocols is imperative to effectively address these concerns.

Multipathogen quantitative risk assessment in raw milk soft cheese

We propose a multipathogen Quantitative Microbiological Risk Assessment (QMRA) model to estimate the risk of foodborne illness from bacterial pathogens in raw milk soft cheese. Our work extends an existing QMRA model for pathogenic Shiga toxin-producing Escherichia coli (STEC) (Basak et al.,under review; Perrin et al., 2014) by incorporating the effects of Salmonella and Listeria monocytogenes. This multipathogen model integrates microbial contamination of raw milk at the farm level, as well as the growth and survival of these bacteria during cheese fabrication, ripening, and storage. The public health impact of multipathogen risk associated with raw milk cheese consumption is assessed using Disability-Adjusted Life Years (DALYs). The model evaluates intervention strategies at both pre- and post-harvest stages to estimate intervention costs. Furthermore, it tests various scenarios of these strategies and optimizes intervention parameters to minimize multipathogen risk and associated costs. This article discusses challenges in QMRA model validation, emphasizes model limitations, and explores future perspectives for improvement.

Determination of microbial contamination in raw milk, processed milk and yoghurt consumed in Mbarara city, western part of Uganda

he microbial composition of milk serves as a key indicator of its quality. While its nutritional value is significant, the inherent nature of milk also makes it an ideal environment for the proliferation of microorganisms, some of which lead to human illnesses, while others contribute to the spoilage of milk, making it unsafe for consumption. This research therefore aimed to assess the bacterial contamination content in raw milk, UHT and yoghurt, a total of 36 samples were gathered from various retailers, markets and supermarkets across Mbarara city. Contaminated milk and its products are accountable for human dairy related illnesses. This cross-sectional study was conducted to assess micro bacterial contamination and the presence of selected milk-borne zoonotic pathogens. The study involved 36 samples of milk and milk products mainly raw milk, UHT, or processed milk and yoghurt. Laboratory analysis conducted included Total Plate Count (TPC), Yeasts and molds (YM), staphylococcus, salmonella and coliform, as well as detection of Escherichia coli (E coli), biochemical tests including gram staining were employed to isolate and identify bacteria in these samples. The findings indicated the presence of these types of bacteria in raw milk, Staphylococcus spp (25%), Salmonella spp (33.3%), YM (41.6%), TPC (50%), coliforms (33.3%) and E.coli (25%), for UHT milk the percentage was Staphylococcus spp (0%), Salmonella spp (16.6%), YM (8.3%), TPC(16.6%), coliforms (16.6%) and E.coli (8.3%), finally for yoghurt the results were Staphylococcus spp (8.3%), Salmonella spp (8.3%), YM (16.6%), TPC (25%), coliforms (16.6%) and E.coli (0%). In conclusion, the detection of microbial contamination in raw milk and milk products suggests inadequate sanitary practices and poor storage practices. Products that are contaminated by these pathogenic organisms when consumed may result in food poisoning and a proper awareness among the stakeholders and consumers regarding possible outcomes of consuming contaminated food items is necessary. keywords: Milk, Yoghurt, Food poisoning, Microorganisms, Contamination

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 dPAB have also 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) and 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.

One-Year Monitoring of Prevalence and Diversity of Dairy Propionic Acid Bacteria in Raw Milk by Means of Culture-Dependent and Culture-Independent Methods.

Even low levels of dairy propionic acid bacteria (dPAB) can cause cheese defects, resulting in severe economic losses for the producers of selected raw milk cheeses. Therefore, routine quality control of raw cheese milk for dPAB contamination is essential if propionic acid fermentation is undesired. Although knowledge of dPAB contamination of raw milk is important to understand cheese spoilage, long-term dPAB screening data are outdated, and studies taking into account different farm management parameters and their potential influence on dPAB levels are scarce. This study aims to provide insight into the dPAB levels of raw milk over time, to identify farm management factors that potentially influence dPAB levels, and to compare a cultural yeast extract lactate agar (YELA) and lithium glycerol agar (LGA) and a culture-independent method (qPCR) for dPAB quantification with respect to their applicability in routine quality control for the dairy industry. For this purpose, bulk tank milk from 25 dairy farms was screened for dPAB contamination over a one-year period. We were able to identify significant differences in the dPAB contamination levels in raw milk depending on selected farm-specific factors and observed relationships between the different types of milking systems and dPAB contamination levels in raw milk. When dPAB were quantified by cultivation on YELA, strong overgrowth of commensal microbiota impeded counting. Therefore, we conclude that quantification on LGA or by qPCR is preferable. Both methods, colony counting on LGA as well as quantification of dPAB using qPCR, have advantages for the application in (routine) quality control of raw milk, one being low-tech and inexpensive, the other being fast and highly specific, but the detection of (low level) dPAB contamination in raw milk remains a challenge.

Extended-SpectrumBeta-LactamaseProducingEscherichia coli in Raw Cow Milk At Selling Points and Determinants of Contamination in and Around Chencha, Southern Ethiopia.

Bacterial foodborne infections are a major public health concern globally, and the emergence of antimicrobial-resistant bacteria in food worsens the associated problems which are alarming issues. The current study aimed to isolate E. coli, determine antimicrobial resistance patterns, estimate the prevalence of extended-spectrum beta-lactamase-producing E. coli, and determinants of raw cow milk contamination at selling points in and around Chencha, Southern Ethiopia from January 2021 to April, 2022. A total of 384 milk samples were collected randomly using a cross-sectional study and processed in the laboratory for evaluation of microbial load, bacterial isolation, and antimicrobial susceptibility patterns. The determinants were assessed through a prepared questionnaire, and descriptive statistics and multivariable logistic regression analysis were performed using SPSS version 21. Out of 384 milk samples analyzed for microbial loads, 5.3±1.68 log and 2.17±1.51 log CFU/mL respectively were the mean values of total viable count and total coliform count. A total of 118 (30.7%) samples were contaminated with E. coli of about 11.8% extended-spectrum beta-lactamase producers. Notably, 91 (77.1%) of isolates were multiple drug-resistant isolates. The multivariable logistic regression showed that variables of educational status of milk handlers, hand washing activities, nose picking habit of milk handlers, milk container cleaning activity, milk container type, physical abnormal milk checking status, and attended training on hygienic food handling practices type were significantly associated with the milk contamination in the study area. The results implied that the raw milk samples served in the study area contained bacterial pathogens and a higher microbial load than recommended standards. Our data also confirmed the presence of multiple drug resistant and extended-spectrum beta-lactamase producing E. coli. Therefore, a close follow-up and training of milk handlers is needed. Besides, consumers should be made aware of the risks of consuming raw milk.

Molecular diagnosis for camel raw milk microbiota.

The existence of diverse microbes in unprocessed camel milk poses a significant threat to the well-being of a large population, especially infants and toddlers. The objective of this study was to ascertain the existence of microorganisms in unprocessed raw camel milk by employing a molecular-based technique in combination with a histological examination of bacteria. The identification of microbial species was achieved by employing PCR amplification and sequencing of 16s rRNA gene fragments. Various micorganisms found includes the probiotic Lactobacillus species, Staphylococcus succinic, Macrococcus casealyticus, Bacillus cohnii, and Salinicoccus kunmingensis. To prevent microbial contamination in raw milk, it is necessary to adequately heat or pasteurise the milk and to wash and sterilise the udder before milking the camel. This is because raw milk contains microbes that cause multiple diseases. Moreover, in the current era of the COVID-19 pandemics, ensuring proper sanitary conditions in milk and its derivatives might potentially mitigate the transmission of various diseases among consumers shortly. Keywords: camel, microbiota, 16s rRNA gene, PCR.

An automated fluorescent immunoassay for on-site screening of AFM1 in raw milk at the ppt level

In the dairy industry, the quality of raw milk as it is collected from dairy farmers must be strictly ensured. Therefore, when on-site screening of typical carcinogens in raw milk, this technology must simultaneously be convenient to operate and highly sensitive. Here, an automated and fluorescent immunoassay system for screening trace amounts of aflatoxin M1 (AFM1) in raw milk was developed. The whole immunoassay procedure can be processed in a true “sample-to-results” paradigm, avoiding the tedious manual operation of the traditional indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) method. In addition, we designed an integrated fluorescent spectrometer that can quantitatively measure fluorescent signals with high sensitivity. The automated and fluorescent immunoassay system can screen AFM1 in raw milk samples with an ultra low limit of detection (4.7 pg/mL in raw milk). The half maximal inhibitory concentration (IC50) was 8.3 pg/mL, which is approximately 4-fold lower than that of traditional ic-ELISA. When the system was applied to screen actual raw milk samples, the recovery rates ranged from 79.4% to 104.6%, with a coefficient of variation ranging from 8.9% to 15.2%. Overall, we believe that the automated and fluorescent immunoassay system can provide a one-stop solution that is user-friendly and highly sensitive for screening trace AFM1 contaminants in raw milk.

Hygienic Practices of Vendors and Their Contribution to Coliform, Salmonella, and Shigella Bacteria of Raw Milk at Asella Town, Oromia, Ethiopia.

Coliform, Salmonella, and Shigella are among the most encountered bacteria in raw milk. This study is aimed at determining the extent of coliform, Salmonella, and Shigella bacteria in raw milk and vendor hygiene practices at Asella town, Oromia Regional State, Ethiopia, from March 1 to 30, 2022. In this study, 210 milk vendors were included; each vendor provided a 50 ml sample of raw milk. Bacteria were isolated and identified using standard bacteriological techniques. Data were entered and analyzed using EPI info version 7 and SPSS version 22, respectively. A binary logistic regression model was applied to determine the factors associated with bacterial contamination of raw milk. The total contamination percentage of raw milk was 50 (23.8%) (95% CI: 18.1-29.5%). The predominant bacteria identified were coliform 43 (20.5%) followed by Salmonella species 7 (3.3%). Among coliforms, the predominant bacteria were Citrobacter species 15 (34.9%) followed by Enterobacter species 11 (25.6%), Escherichia coli and Serratia species each 6 (14%), and Klebsiella species 5 (11.6%). However, no Shigella was isolated in this study. Not having the habit of washing cow teats (p < 0.0001), the habit of washing teats with tap water (p < 0.0001), not having separate cloth during milking (p < 0.0001), not having a practice of testing milk for bacterial contamination (p = 0.027), and not having separate vending environment (p = 0.039) were significantly associated with bacterial contamination of raw milk. The percentage of bacterial contamination of milk was found to be high. Participants without a habit of washing cow teats, a habit of washing milk utensils with only tap water, and not having separate vending environments were associated factors for bacterial contamination of raw milk. Milk vendors are advised to develop the habit of washing teats before milking, avoid washing teat/milk utensils only with tap water, and have a separate vending environment.

Listeria Contamination in Milk-Processing Chain and Proficiency in Listeria monocytogenes Decontamination of Small-Scale Milk Retailers

Listeria spp. is an important foodborne bacterium. This microorganism can be discarded from milk using high temperatures such as pasteurization. The milk-processing methods of many small-scale retailers lack quality control. This study was to survey Listeria contamination at the farm and retailer levels. The retailers were to be interviewed for knowledge, attitude, and practice as well. Finally, we were to determine the heating processes employed to decontaminate microorganisms by the retailers using a reference strain of L. monocytogenes. Milk samples were collected from milk-collecting centers and small-scale retailers. In clinical trial, the processing measures were proved for the proficiency in L. monocytogenes decontamination. One out of 99 farms presented Listeria contamination, confirmed to L. marthii. Fifty small-scale retailers participated in the second part, including 13 males and 37 females. No Listeria spp. but Staphylococcus spp. and Bacillus spp. were identified in the processed milk. Data analyses revealed that the location of the retailer was significantly associated with the volume they routinely ordered per lot and the milk-processing time the retailers used to treat milk. Knowledge on raw milk contamination is significantly associated with the stocking or processing of the whole milk lot. Processing measures presale were significantly influenced by the gender of the retailer. The male retailer reportedly spent less time treating milk compared to their female counterparts. To assess the efficacy of the processing methods, a trial using L. monocytogenes as a reference strain was conducted. Interestingly, no L. monocytogenes was detected after sample treatment, but other microorganisms such as S. epidermidis, S. warneri, and Escherichia coli were found, suggesting potential issues with cross-contamination. In conclusion, while the trial implied that the retailers’ processes were effective in L. monocytogenes decontamination, the study highlighted inappropriate practices and the risk of cross-contamination. Continuous monitoring of product safety in small-scale milk retailers is imperative to ensure consumer well-being.

Identification and Characterisation of Spore-Forming Bacteria in Bovine Raw Milk Collected from Four Dairy Farms in New Zealand

Contamination of milk and dairy products with pathogenic and spoilage bacteria may result in huge economic loss due to recalls of products. This study aimed to identify spore-forming bacteria from raw milk and characterise those for toxin production and their spoilage ability. Bovine raw milk collected from dairy farms in the Manawatu region of the North Island of New Zealand was tested for the presence of both aerobic and anaerobic spore-forming bacteria using standard culture-based techniques, as well as genomic analysis. The spore-forming bacteria were investigated for the presence of toxin genes and their spoilage potential. A low number of aerobic spore-forming bacteria were detected in raw-milk samples collected from the four farms in summer and winter. The 16S rRNA sequence types similar to important food spoilage bacteria like C. beijerinckii, C. sporogenes, B. licheniformis and members of the Paenibacillus genus, as well as potentially toxigenic bacteria such as B. cereus and C. perfringens were isolated. Genes responsible for important toxin production were present in some of the tested spore-forming bacteria. This pilot study highlights the presence of various spoilage and pathogenic spore-forming bacteria in raw milk from these farms. A low number of spore-forming bacteria indicates the implementation of good hygienic farm practices and management to reduce the contamination of raw milk with spore-forming bacteria.

Phylogenetic variation in raw cow milk microbiota and the impact of forage combinations and use of silage inoculants.

The microbiota of bulk tank raw milk is known to be closely related to that of microbial niches of the on-farm environment. Preserved forage types are partof this ecosystem and previous studies have shown variations in their microbial ecology. However, little is known of the microbiota of forage ration combinations and the transfer rates of associated species to milk. We identified raw milk bacteria that may originate from forage rations encompassing either hay (H) or grass/legume silage uninoculated (GL) as the only forage type, or a combination of GL and corn silage uninoculated (GLC), or grass/legume and corn silage both inoculated (GLICI). Forage and milk samples collected in the fall and spring from 24 dairy farms were analyzed using 16S rRNA gene high-throughput sequencing following a treatment with propidium monoazide to account for viable cells. Three community types separating H, GL, and GLICI forage were identified. While the H community was co-dominated by Enterobacteriaceae, Microbacteriaceae, Beijerinckiaceae, and Sphingomonadaceae, the GL and GLICI communities showed high proportions of Leuconostocaceae and Acetobacteraceae, respectively. Most of the GLC and GLICI rations were similar, suggesting that in the mixed forage rations involving grass/legume and corn silage, the addition of inoculant in one or both types of feed does not considerably change the microbiota. Raw milk samples were not grouped in the same way, as the GLC milk was phylogenetically different from that of GLICI across sampling periods. Raw milk communities, including the GLICI group for which cows were fed inoculated forage, were differentiated by Enterobacteriaceae and other Proteobacteria, instead of by lactic acid bacteria. Of the 113 amplicon sequence variants (ASVs) shared between forage rations and corresponding raw milk, bacterial transfer rates were estimated at 18 to 31%. Silage-based forage rations, particularly those including corn, share more ASVs with raw milk produced on corresponding farms compared to that observed in the milk from cows fed hay. These results show the relevance of cow forage rations as sources of bacteria that contaminate milk and serve to advance our knowledge of on-farm raw milk contamination.

Seasonal effect on the prevalence of virulence genes of non-O157 Verotoxic E.coli serogroups in faeces of cattle calves

Calves faecal samples (n=216) were investigated to observe the effect of seasons on the prevalence of serogroups and virulence genes of non-O157 VTEC. A total of 177 (81.94%) E. coli were isolated and 32 (14.81%) were identified as VTEC and serotyping resulted in 13 different non-O157 ‘O’ serogroups. The prevalence of serogroups and their virulence genes was found to be influenced by seasons and highest number were shed in summer (22.22%) followed by rainy (13.88%) and winter season (8.33%), respectively. A higher prevalence of O9 and O11 serogroups (25% each) was observed in summers. Molecular detection of virulence genes revealed the overall prevalence of vt1 to be 37.5%, vt2 43.8%, (vt1+vt2) 18.8%, eaeA 21.9% and hlyA 34.4% genes. Dominance of hlyA 50% was observed in summers, whereas vt1 and vt2 were more prevalent during rain (50% each). The study revealed the link between the occurrence of hlyA gene and O9, O11 serogroups in summers as both the serogroups were hlyA gene bearer. This association might be responsible for more VTEC outbreaks in summers. So, faecal contamination of raw milk seems to pose greater threat of non-O157 VTEC outbreak during hotter and humid months.

Presence of methicillin-resistant Staphylococcus aureus in raw cow’s milk: adhesive capacities and extracellular enzymes characterization

Raw milk contamination by Methicillin-resistant S. aureus (MRSA) threatens food safety and leads to public health problems. Our study evaluates biofilm formation and virulence factors among 21 MRSA in raw cow’s milk. Methicillin resistance was confirmed by cefoxitin screening using the automated VITEK2 system, with a minimum inhibitory concentration greater than 8 mg/l. Qualitative characterization of biofilm-producing strains was performed on Congo red agar (CRA), with a semi-quantitative adhesion test on 96-well tissue culture plates (TCP). The ability to produce different enzymes was evaluated, such as caseinase, lipase, and phospholipase (lecithinase). The surface hydrophobicity of the bacteria was determined, and the autoaggregation test was used to predict the interactions between bacterial cells. Among the tested strains, 61.9% were biofilm producers in the CRA, developing a positive and variable phenotype. Furthermore, 19.05% and 80.95% of isolates were high and low biofilm formation on TCP. The enzymatic activity showed that lécithinase, caseinase, and lipase activities were detected in 100%, 80.95%, and 80.95% of cases. Highly hydrophilic (85.71%) and weakly hydrophobic (14.29%) were detected in MRSA isolates. The strains showed that 71.43% were moderate autoaggregation and 28.57% were low autoaggregation. No significant difference was found between the CRA method and TCP (P&gt; 0.05). A significant association was found between adhesion capacity and bacterial autoaggregation in S. aureus strains (p&gt;0.05). On the other hand, no statistical association between the hydrophobicity of microbial strains and adhesion capacity (p&lt;0.05) was found. The same result was for the hydrophobicity of microbial strains and autoaggregation (p&lt;0.05). This investigation could be beneficial for developing new control measures, prevention, and effective treatment against staphylococcal bacterial resistance.

Development of Polymerase Chain Reaction assay protocol for assessment Salmonella sp. in cow raw milk

Salmonella sp. is a pathogenic bacterium that may associated with acute diarrhoea in human. These bacteria may be transmitted in a variety of ways, including consumption of contaminated cow raw milk. Salmonella sp. is troublesome to assessment due to methodological restrictions. The aim of this study was to development the protocol of Polymerase Chain reaction (PCR) assay for Assessment Salmonella sp. in cow raw milk. This method is comprised of (1) biochemical assay and (2) bacterial DNA purification from Selenite Cystine Broth and XLD Xylose Lysine Deoxycholate culture using Wizard® Genomic DNA Purification Kit followed by PCR detection. The biochemical assay is divided into several stages, namely bacterial isolation, Gram staining and conventional biochemical tests. The PCR optimization was done with Salmonella sp. The oligonucleotide primer invasion protein (invA) gene (F: 5’-TCGTCATTCCATTACCTACC-3’; R: 5’-AAACGTTGAAAAACTGAGGA-3’) were used for targeting the diagnosis of Salmonella at the genus level. In biochemical tests, Salmonella sp. results showed posit if result of catalase, oxidase, citrate, TSIA, lactose-sucrose-mannitol fermentation, urea, and methyl-red. Conversely, negative result for Voges-Proskauer. PCR protocol consist of 30 PCR cycles with initial denaturation at 94°C for 45 seconds, denaturation at 94°C for 20 seconds, annealing at 57°C for 15 seconds, extension at 72°C for 15 seconds and finally at 72°C for 2 minutes. The conventional method detection results obtained as many as 9 positive samples Salmonella sp. and the PCR method obtained 7 positive samples Salmonella sp. In conclusion, our results indicate that the developed protocol would be utilized as a routine analysis for monitoring cow raw milk contamination and the protocol of the PCR technique provides results at a handful of time required by the biochemical assay (24 hours compared to 2–3 days).