Biodiversity and antibiotic resistance profile provide new evidence for a different origin of enterococci in bovine raw milk and feces.

Enterococci are widely distributed in dairy sector. They are commensals of the gastrointestinal tract of animals, thus, via fecal contamination, could reach raw milk and dairy products. The aims of this study were: 1) to investigate the enterococcal diversity in cow feces and milk samples and 2) to evaluate the antibiotic resistance (AR) of dairy-related enterococci and their ability to transfer resistance genes. E. faecalis (59.9%), E. faecium (18.6%) and E. lactis (12.4%) were prevalent in milk, while E. faecium (84.2%) and E. hirae (15.0%) were dominant in bovine feces. RAPD-PCR highlighted a high number of Enterococcus biotypes (45 from milk and 37 from feces) and none of the milk strains exhibited genetic profiles similar to those of feces biotypes. A high percentage of enterococci isolated from milk (71%) were identified as multidrug resistant and resistance against streptomycin and tetracycline were widespread among milk strains while enterococci from feces were commonly resistant to linezolid and quinupristin/dalfopristin. Only E. faecalis strains were able to transfer horizontally the tetM gene to Lb. delbrueckii subsp. lactis. Our results indicated that Enterococcus biotypes from milk and bovine feces belong to different community and the ability of these microorganisms to transfer AR genes is strain-dependent.

Air Quality, Management Practices and Calf Health in Italian Dairy Cattle Farms.

Among factors that can affect calf health, microbial quality of the pen air is poorly studied. In 25 Italian dairy farms, data concerning air quality in the calf pens, hygiene of pens and equipment, microclimatic conditions, calf health and management were collected during the winter season (January-March 2020 and December-March 2021). The average air Standard Plate Count (SPC) of 85 pens was 4.51 (SD = 0.52) log10 cfu/m3 whereas the average air ammonia concentration was 0.66 (SD = 0.53) ppm. Positive correlations were found between average Temperature Humidity Index (THI) in the pen and air SPC, night maximum THI and air SPC and between SPC and yeast, mould and ammonia concentration in the pen air. The concentrations of E. coli, Enterobacteriaceae and yeasts in the pen air were higher and calf cough increased as the renewal interval of bedding material became longer. High bedding dry matter and low THI were associated with low air SPC, good calf health scores and low mortality. Maintaining low bedding humidity and controlling microclimatic conditions can contribute to enhancing air microbiological quality in the pen and reduce calf diseases and mortality.

Antibiotic resistance in wild and commercial non-enterococcal Lactic Acid Bacteria and Bifidobacteria strains of dairy origin: An update.

Antibiotic Resistance is a growing concern for public health and global economy. Lactic acid bacteria (LAB) involved in the production of dairy products and commonly present in the agro-zootechnical environment can act as reservoirs of antibiotic resistance genes, acquiring or transferring them to other microorganisms. The review focuses on LAB group of dairy origin (Lactobacillus, Lactococcus, Streptococcus, Leuconostoc, Pediococcus and Weissella) and Bifidobacterium genus, considering its large use in dairy industry. We have analyzed data in the last 25 years, highlighting atypical resistance, genetic traits correlated to antibiotic resistance and their ability to be transmitted to other microorganisms; comparative analysis of resistomes was also considered. Differences were observed among wild strains isolated from different regions because of authorized antibiotic use. Commercial strains belonging to Lactobacillus, Streptococcus and Bifidobacterium currently used for industrial dairy products are frequently resistant to gentamycin, kanamycin, chloramphenicol together with tetracycline. The presence of resistant wild LAB in raw milk products has been significantly reduced as a result of worldwide restrictions on the use of antibiotics in animal husbandry. Transmissible resistances are still present in industrial cultures, despite the great effort of starter industries in the process control and the safety screening of commercial cultures.

Bacterial Communities in the Embryo of Maize Landraces: Relation with Susceptibility to Fusarium Ear Rot.

Locally adapted maize accessions (landraces) represent an untapped resource of nutritional and resistance traits for breeding, including the shaping of distinct microbiota. Our study focused on five different maize landraces and a reference commercial hybrid, showing different susceptibility to fusarium ear rot, and whether this trait could be related to particular compositions of the bacterial microbiota in the embryo, using different approaches. Our cultivation-independent approach utilized the metabarcoding of a portion of the 16S rRNA gene to study bacterial populations in these samples. Multivariate statistical analyses indicated that the microbiota of the embryos of the accessions grouped in two different clusters: one comprising three landraces and the hybrid, one including the remaining two landraces, which showed a lower susceptibility to fusarium ear rot in field. The main discriminant between these clusters was the frequency of Firmicutes, higher in the second cluster, and this abundance was confirmed by quantification through digital PCR. The cultivation-dependent approach allowed the isolation of 70 bacterial strains, mostly Firmicutes. In vivo assays allowed the identification of five candidate biocontrol strains against fusarium ear rot. Our data revealed novel insights into the role of the maize embryo microbiota and set the stage for further studies aimed at integrating this knowledge into plant breeding programs.

Genotyping and Antimicrobial Susceptibility Profiling of Streptococcus uberis Isolated from a Clinical Bovine Mastitis Outbreak in a Dairy Farm.

Streptococcus uberis, an environmental pathogen responsible also for contagious transmission, has been increasingly implicated in clinical mastitis (CM) cases in Europe. We described a 4-month epidemiological investigation of Strep. uberis CM cases in an Italian dairy farm. We determined molecular characteristics and phenotypic antimicrobial resistance of 71 Strep. uberis isolates from dairy cows with CM. Genotypic variability was investigated via multiplex PCR of housekeeping and virulence genes, and by RAPD-PCR typing. Antimicrobial susceptibility was assessed for 14 antimicrobials by MIC assay. All the isolates carried the 11 genes investigated. At 90% similarity, two distinct clusters, grouping 69 of the 71 isolates, were detected in the dendrogram derived from the primer ERIC1. The predominant cluster I could be separated into two subclusters, containing 38 and 14 isolates, respectively. Strep. uberis strains belonging to the same RAPD pattern differed in their resistance profiles. Most (97.2%) of them were resistant to at least one of the drugs tested, but only 25.4% showed a multidrug resistance phenotype. The highest resistance rate was observed for lincomycin (93%), followed by tetracycline (85.9%). This study confirmed a low prevalence of ß-lactam resistance in Strep. uberis, with only one isolate showing resistance to six antimicrobial classes, including cephalosporins.

Proteolytic Traits of Psychrotrophic Bacteria Potentially Causative of Sterilized Milk Instability: Genotypic, Phenotypic and Peptidomic Insight.

The proteolytic traits of the psychrotrophic strains Pseudomonas poae LP5, Pseudomonas fluorescens LPF3, Chryseobacterium joostei LPR1, Pseudomonas fulva PS1, Citrobacter freundii PS37, Hafnia alvei PS46, and Serratia marcescens PS92 were initially investigated by phenotypic and genotypic approaches. Six strains elicited extracellular proteolytic activity, and five expressed the thermostable AprX or (likely) Ser1 enzymes. Then, the strains were inoculated (104 CFU/mL) in microfiltered pasteurized milk and kept at 4 °C for five days. All of the strains reached 108 CFU/mL at the end of storage and five produced thermostable extracellular proteolytic enzymes. The freshly inoculated samples and the corresponding samples at 108 CFU/mL were batch-sterilized (131 °C, 30 s) and kept at 45 °C up to 100 days. The former samples did not gel until the end of incubation, whereas the latter, containing P. poae, P. fluorescens, C. joostei, C. freundii, and S. marcescens, gelled within a few days of incubation. The thermostable proteolytic activity of strains affected the peptidomic profile, and specific proteolyzed zones of ß-CN were recognized in the gelled samples. Overall, the results confirm some proteolytic traits of psychrotrophic Pseudomonas spp. strains and provide additional insights on the proteolytic activity of psychrotrophic bacteria potentially responsible for sterilized milk destabilization.

Paraprobiotics: A New Perspective for Functional Foods and Nutraceuticals.

Probiotics are live microorganisms that confer health benefits on the host. However, in recent years, several concerns on their use have been raised. In particular, industrial processing and storage of probiotic products are still technological challenges as these could severely impair cell viability. On the other hand, safety of live microorganisms should be taken into account, especially when administered to vulnerable people, such as the elderly and immunodeficient individuals. These drawbacks have enhanced the interest toward new products based on non-viable probiotics such as paraprobiotics and postbiotics. In particular, paraprobiotics, defined as "inactivated microbial cells (non-viable) that confer a health benefit to the consumer," hold the ability to regulate the adaptive and innate immune systems, exhibit anti-inflammatory, antiproliferative and antioxidant properties and exert antagonistic effect against pathogens. Moreover, paraprobiotics can exhibit enhanced safety, assure technological and practical benefits and can also be used in products suitable for people with weak immunity and the elderly. These features offer an important opportunity to prompt the market with novel functional foods or nutraceuticals that are safer and more stable. This review provides an overview of central issues on paraprobiotics and highlights the urgent need for further studies aimed at assessing safety and efficacy of these products and their mechanisms of action in order to support decisions of regulatory authorities. Finally, a definition is proposed that unambiguously distinguishes paraprobiotics from postbiotics.

Assessment of Possible Application of an Atmospheric Pressure Plasma Jet for Shelf Life Extension of Fresh-Cut Salad.

Ready-to-eat salads are very perishable with quality losses within 6-7 days, and the extension of their shelf life is still a challenge. In this work, an atmospheric pressure plasma jet (APPJ) was applied for the surface decontamination of fresh-cut lettuce baby leaves. The APPJ antimicrobial efficiency on the natural microbiota and its impact on some physicochemical attributes of lettuce were evaluated as a function of the treatment duration (0-30 s). Then, the influence of plasma treatment on the salad shelf life was studied, following the growth of aerobic mesophilic bacteria in both untreated and plasma-treated samples during 9 days of storage at 4 °C, together with the plasma-induced changes in physicochemical parameters of lettuce leaves. The APPJ induced a fast (15 s) microbial decontamination (1.3 log10 CFU/g) of the salad surface. Exposure time and salad-plasma plume distance were the parameters that substantially affected the microbial inactivation. APPJ treatment retarded bacterial growth during the refrigerated storage, as plasma-treated samples were noticeably less contaminated than the non-treated ones in the first 3-4 days. No significant effect were observed on electrolyte leakage, pH, and dry matter content in both the set up phase and the shelf life study.

Effect of Different Farming Practices on Lactic Acid Bacteria Content in Cow Milk.

The natural load of lactic acid bacteria (LAB) in milk is the basis of the production of raw milk cheeses, such as Grana Padano PDO. In the last decades, improvements in livestock hygiene management resulted in bulk cow milk with less than 20,000 colony forming units (CFU) of bacterial count, unable to ensure a sufficient supply of LAB, with a negative impact on cheese quality. This study investigated the relations between farm management practices and prevalence of different groups of bacteria in cow milk. Sixty-two intensive dairy farms located in Lombardy (Italy) where involved, most of them destined as milk for the production of Grana Padano. Season had no significant effect on the content of most of the bacterial groups, except for coliforms. A strong relation among standard plate count (SPC) and other bacterial groups was evidenced. Cluster analysis showed that the most productive farms applied a complete milking routine and produced milk with the lowest value of SPC, the lowest count of the other bacteria, including LAB, but the highest LAB/SPC. The study suggests that complexity of farming practices can affect the microbial population of milk.

Raw Milk Microbiota Modifications as Affected by Chlorine Usage for Cleaning Procedures: The Trentingrana PDO Case.

Milk microbiota represents a key point in raw milk cheese production and contributes to the development of typical flavor and texture for each type of cheese. The aim of the present study was to evaluate the influence of chlorine products usage for cleaning and sanitizing the milking equipment on (i) raw milk microbiota; (ii) the deriving whey-starter microbiota; and (iii) Trentingrana Protected Designation of Origin (PDO) cheese microbiota and volatilome. Milk samples from three farms affiliated to a Trentingrana PDO cheese factory were collected three times per week during a 6-weeks period in which a sodium hypochlorite detergent (period C) was used and during a subsequent 6-weeks period of non-chlorine detergent usage (period NC). Samples were subjected to microbiological [Standard Plate Count; coliforms; coagulase-positive staphylococci; and lactic acid bacteria (LAB)] and metagenomic analysis (amplification of V3-V4 regions of 16S rRNA gene performed on Illumina MiSeq platform). In addition, cheese volatilome was determined by SPME-GC-MS. In the transition from period C to period NC, higher SPC and LAB counts in milk were recorded. Milk metagenomic analysis showed a peculiar distinctive microbiota composition for the three farms during the whole experimental period. Moreover, differences were highlighted comparing C and NC periods in each farm. A difference in microbial population related to chlorine usage in bulk milk and vat samples was evidenced. Moreover, chlorine utilization at farm level was found to affect the whey-starter population: the usually predominant Lactobacillus helveticus was significantly reduced during NC period, whereas Lactobacillus delbrueckii had the exact opposite trend. Alpha- and beta-diversity revealed a separation between the two treatment periods with a higher presence of L. helveticus, L. delbrueckii, and Streptococcus thermophilus in cheese samples after NC detergent period. Cheese volatilome analysis showed a slight decrease in lipolysis during C period in the inner part of the cheese wheel. Although preliminary, these results suggest a profound influence on milk and cheese microbiota, as well as on raw milk cheese production and quality, due to the use of chlorine. However, further studies will be needed to better understand the complex relationship between chlorine and microbiota along all the cheese production steps.

How we can improve the antimicrobial performances of lactic acid bacteria? A new strategy to control Listeria monocytogenes in Gorgonzola cheese.

This study was aimed to evaluate the effectiveness of two lactic acid bacteria (LAB) cultures (Lactococcus lactis FT27 and Carnobacteroim divergens SCA), lactic acid/sodium lactate (LASL - l-lactic acid 61% (w/w) and L-sodium lactate 21% (w/w)) and their combination against four Listeria monocytogenes biotypes isolated from Gorgonzola cheese. In vitro antilisterial activity showed that the sensitivity to antimicrobials was strain-dependent. Antimicrobial challenge testing was performed on Gorgonzola rinds simulating contamination occurring at the beginning (6 days) and at the end (55 days) of the ripening period, to assess the antilisterial activity of LAB strains and LASL during the subsequent 60 days at 4 °C. LASL showed a higher antilisterial activity than LAB, maintaining the pathogen content below the EC limit (<2.0 log10 CFU/g) for 60 days. A strong listericidal effect was observed combining LAB with LASL (2,8 µL/cm2) Lc. lactis in combination with LASL completely inhibited the two L. monocytogenes strains in the first 50 days, while LASL with C. divergens was more effective in the second part of ripening when the pH raised. Data obtained encourage the use of LASL along with antimicrobial LAB rotation schemes during cheese ripening for the prevention and/or control of the L. monocytogenes on cheese surface.

Preliminary Results, Perspectives, and Proposal for a Screening Method of In Vitro Susceptibility of Prototheca Species to Antimicrotubular Agents.

Microorganisms belonging to the genus Prototheca are achlorophyllous microalgae, occasionally behaving as environmental pathogens that cause severe mastitis in milk cows, as well as localized or systemic infections in humans and animals. Among the different species belonging to the genus, Prototheca zopfii genotype 2 (recently reclassified as P. bovis) and P. blaschkeae are most commonly associated with bovine mastitis. To date, no pharmacological treatment is available to cure protothecal mastitis, and infected animals must be quarantined to avoid spreading the infection. The few antibiotic and antifungal drugs effective in vitro against Prototheca give poor results in vivo This failure is likely due to the lack of specificity of such drugs. As microalgae are more closely related to plants than to bacteria or fungi, an alternative possibility is to test molecules with herbicidal properties, in particular, antimicrotubular herbicides, for which plant rather than animal tubulin is the selective target. Once a suitable test protocol was set up, a panel of 11 antimicrotubular agents belonging to different chemical classes and selective for plant tubulin were tested for the ability to inhibit growth of Prototheca cells in vitro Two dinitroanilines, dinitramine and chloralin, showed strong inhibitory effects on P. blaschkeae at low micromolar concentrations, with half-maximal inhibitory concentrations (IC50) of 4.5 and 3 µM, respectively, while both P. zopfii genotype 1 (now reclassified as P. ciferrii) and P. bovis showed susceptibility to dinitramine only, to different degrees. Suitable screening protocols for antimitotic agents are suggested.

Bacterial proteolysis of casein leading to UHT milk gelation: An applicative study.

Heat-stable peptidases released in refrigerated raw milk by psychrotrophic bacteria are responsible for UHT milk gelation. K-casein-derived caseinomacropeptides, identified by mass spectrometry, were constantly detected in gelled milk by capillary electrophoresis. Strains of Pseudomonas fluorescens, Ps. poae and Chryseobacterium joostei, selected among aprX-positive strains from raw milk, were incubated in milk up to 6 days at 4 °C before sterilization (98 °C/4 min). Samples were then stored at 25 or 40 °C, visually observed for gelation, and analysed for presence of caseinomacropeptides throughout 90 days of storage. Depending on cold pre-incubation time, caseinomacropeptides accumulated well before gelation onset in milk stored at 25 °C. Caseinomacropeptides were successively degraded, especially in milk stored at 40 °C, due to extensive proteolysis, and an abundant sediment developed instead of a gel. The caseinomacropeptides are here presented as an early indicator of UHT milk gelation and a mechanism explaining this phenomenon is proposed.

Does Enterococcus faecalis from Traditional Raw Milk Cheeses Serve as a Reservoir of Antibiotic Resistance and Pathogenic Traits?

Enterococcus faecalis is not only a prevalent species among dairy microbial community but also a well-documented opportunistic pathogen. Food safety should exclude the possibility of consumer exposure to its virulence traits through consumption of dairy products. In this study, an integrated approach based on both phenotypic and genotypic methods was applied to investigate the incidence of antibiotic resistance and pathogenicity potential in 40 E. faecalis isolated from 10 Italian raw milk cheeses over a 13-year period (1997-2009). Among the 14 tested antibiotics, resistance to tetracycline, rifampicin, chloramphenicol, and erythromycin was observed, whereas vancomycin-resistant enterococci were not found. A high incidence (90% of strains) of the tet(M) gene emerged, whereas tet(K), tet(S), tet(L), int, and ermB genes were occasionally amplified (12.5%, 10%, 7.5%, 2.5% and 30%, respectively). No strain was positive for vancomycin-resistant determinants. Among the seven virulence determinants considered, the asa1, gelE, esp, and efaA genes were harbored. No other gene encoding for either different virulence factors (cylA, hyl, and ace) or amino acid decarboxylase activity (hdc, tdc, and odc) was detected. Consequently, E. faecalis isolated from raw milk cheeses does not represent a substantial reservoir of antimicrobial resistance and virulence factors if compared with clinical strains. However, this species occasionally harbors detrimental traits; thus, the possibility that it could be a route for transmission of pathogenic genes through dairy products should never be disregarded.

Bio-guided Purification and Mass Spectrometry Characterisation Exploring the Lysozyme-like Protein from Enterococcus lactis Q1, an Unusual Marine Bacterial Strain.

Lactic acid bacteria produce various antibacterial peptides such as bacteriocins that are active against pathogenic and spoilage microorganisms. Very little attention has been paid to the production of lysozyme as an antimicrobial enzyme. The present work represents one of the few studies reporting lysozyme production by enterococci. Indeed, this study was first conducted to evaluate the antimicrobial activity of Enterococcus lactis Q1, an enterocin P-producing strain previously isolated from fresh shrimp (Penaeus vannamei), against multidrug-resistant clinical isolates. Results showed significant inhibitory activity (P < 0.05) towards diverse pathogens. The purification of the antimicrobial substances produced by Q1 strain leads to the isolation of two active fractions. The SDS-PAGE and mass spectrometry analyses of fraction number 2 (fraction 2) revealed the presence of a protein with molecular mass of 14.3 kDa. Additionally, the experimental results are consistent with mass spectra of industrial lysozyme (Fluka ref. 62970). The lysozyme produced by Enterococcus lactis Q1 strain was confirmed by a plate method against Micrococcus luteus ATCC 4698. Also, sensitivity of the Q1 strain to different concentrations of lysozyme was investigated. For the first time, this study shows that E. lactis Q1 produces lysozyme which could be an excellent candidate in food biopreservation or production of functional foods to promote health benefits.

Safety characterisation and inhibition of fungi and bacteria by a novel multiple enterocin-producing Enterococcus lactis 4CP3 strain.

This study aims to characterise a potential bacteriocinogenic lactic acid bacterial strain isolated from a raw pink shrimp (Palaemon serratus) and evaluate its safety aspect. The strain designated as 4CP3 was noted to display antibacterial activities (P < 0.05) against Gram-positive and Gram-negative foodborne pathogens (Listeria monocytogenes and Pseudomonas aeruginosa) and some filamentous fungi (e.g. Aspergillus niger A79). Phenotypic and molecular techniques as well as phylogenetic analysis identified the isolate 4CP3 as Enterococcus lactis. Its produced antimicrobial substance was determined as a bacteriocin that was stable over a wide range of pH (2-10) and after heating at 100 °C for 15 min. The maximum bacteriocin production was 1400 AU/ml recorded after 12 h of incubation in de Man, Rogosa and Sharpe (MRS) broth medium at 30 °C. The mode of action of the bacteriocin produced by 4CP3 strain was identified as bactericidal against L. monocytogenes EGDe 107776 and P. aeruginosa ATCC 27853. By specific PCR amplifications, E. lactis 4CP3 was shown to produce the enterocins A, B and P. To our knowledge, this feature is newly described for E. lactis strain isolated from raw shrimps. Regarding safety aspect of E. lactis 4CP3, it has been demonstrated that this strain was not haemolytic, gelatinase negative, sensitive to vancomycin, and free of common antibiotic resistance genes and virulence factors. Therefore, it may be useful as a safe natural agent in preservation of foods or as a new probiotic strain in food and feed.

Safety, potential biotechnological and probiotic properties of bacteriocinogenic Enterococcus lactis strains isolated from raw shrimps.

The aims of this study are to isolate new bacteriocinogenic lactic acid bacterial strains from white (Penaeus vannamei) and pink (Palaemon serratus) raw shrimps and evaluate their technological and probiotic potentialities. Seven strains were selected, among fifty active isolates, as producing interesting antimicrobial activity. Identified as Enterococcus lactis, these isolates were able to produce enterocins A, B and/or P. The safety aspect, assessed by microbiological and molecular tests, demonstrated that the strains were susceptible to relevant antibiotics such as vancomycin, negative for haemolysin and gelatinase activities, and did not harbour virulence and antibiotic resistance genes. The assessment of potential probiotic and technological properties showed a low or no lipolytic activity, moderate milk-acidifying ability, high reducing power, proteolytic activity and tolerance to bile (P < 0.05) and good autoaggregation and coaggregation capacities. Two strains designated as CQ and C43 exhibiting high enzymatic activities and bile salt hydrolase activity were found to display high survival under simulated in vitro oral cavity and gastrointestinal tract conditions caused by presence of lysozyme, pepsin, pancreatin, bile salts and acidic pH. This study highlights safe Enterococcus lactis strains with great technological and probiotic potentials for future application as new starter, adjunct, protective or probiotic cultures in food industry.

Prototheca blaschkeae subsp. brasiliensis subsp. nov., isolated from cow milk.

A strain of an achlorophyllic alga, named PR24T, was isolated from cow milk samples from the state of Minas Gerais, Brazil. Based on 18S rDNA, 28S rRNA, D1/D2 region of the LSU rDNA and SSU rRNA gene sequence similarities, this strain was found to be a member of the genus Prototheca and closely related to Protothecablaschkeae SAG2064T. However, the novel strain could easily be distinguished from recognized Prototheca species by internal transcribed spacer, species-specific PCR, single-strand conformation polymorphism-PCR analysis and phenotypic characteristics. The inability to grow in Sabouraud broth at pH 4.0 and the different cellular fatty acid composition clearly distinguished PR24T from the reference strain of P. blaschkeae. The combination of genotypic and phenotypic data indicates that strain PR24T represents a subspecies of P. blaschkeae, for which the name Prototheca blaschkeae subsp. brasiliensis subsp. nov. is proposed. The respective type strain is PR24T (=DSM 103592T=IHEM 26958T).

Efficient milking hygiene reduces bacterial spore contamination in milk.

Clostridia in the milk can lead to late blowing, a cheese defect. Clostridia are ubiquitous, deriving from both the farm environment and the feed ingested by the cows, and are transferred into the milk through faecal contamination. Our aim was to investigate the effect of different in-parlour practices on the content of anaerobic spore-forming bacteria in milk, and to monitor the variation in spore content in the feed and environment. The experiment, conducted in an experimental dairy during autumn, was repeated in exactly the same way for two consecutive years. The experimental design applied three different milking routines in three consecutive 7-d periods: forestripping alone (F); forestripping and post-dipping (F+Post); pre-dipping, wiping, forestripping and post-dipping (Pre+F+Post). Teat skin swabs and samples of feed, faeces, bedding materials and milk were collected for microbiological analyses. The dietary forage of the lactating cows included maize silage, which, in both years, was found to have the highest level of clostridial spore contamination. Pre-dipping with a detergent/emollient solution, and drying with a disposable paper towel, proved much more efficient in reducing spore contamination than forestripping alone, both on the teats (1·30 vs. 2·20 log10 MPN/swab; P < 0·001) and in the milk (1·82 vs. 2·47 log10 MPN/L, P < 0·02), while post-dipping had little influence on spore count. The standard plate count in milk was significantly lower with Pre+F+Post treatment than with F (3·80 vs. 4·51 log10 CFU/mL, P < 0·01). The teat preparation procedure did not influence the lactic acid bacterial levels in the milk, which is very positive in that decreased lactic acid bacterial content can lessen raw milk cheese quality.

Isolation and characterisation of an enterocin P-producing Enterococcus lactis strain from a fresh shrimp (Penaeus vannamei).

Screening for lactic acid bacteria (LAB) from fresh shrimp samples (Penaeus vannamei) collected from retail seafood markets in the Tunisian's coast, resulted in the isolation of an Enterococcus strain termed Q1. This strain was selected for its antagonistic activity against pathogenic bacteria such as Listeria monocytogenes, Pseudomonas aeruginosa, Lactococcus garvieae and against fungi (Aspergillus niger and Fusarium equiseti). The Q1 strain was characterised using standard morphological and biochemical tests, growth assays at different temperatures, pH and salinity. 16S rRNA, rpoA and pheS gene sequencing, as well as the 16S-23S rRNA intergenic spacer analyses, were combined to identify strain Q1 as a strain of Enterococcus lactis. The bacteriocin produced by E. lactis Q1 is thermostable, active in the pH range from 4.0 to 9.0 and has a bactericidal mode of action. The enterocin P structural gene was detected by specific PCR in strain E. lactis Q1, which is in good agreement with SDS-PAGE data of the purified bacteriocin. A lack of significant antibiotic resistance genes and virulence determinants was confirmed by specific PCRs. This work provides the first description of an enterocin P producer E. lactis strain isolated from a fresh shrimp. Based on its safety properties (absence of haemolytic activity, virulence factors and antibiotic resistance genes), this strain has the potential to be used as a natural additive or adjunct protective culture in food biopreservation and/or probiotic culture.