The effects of lactate on nitrosylmyoglobin formation from nitrite and metmyoglobin in a cured meat system

Two experiments were conducted to determine the effects of lactate on nitrite during meat curing. In the first experiment, using a model system, eight reaction components including nitrite and lactate, were used to assess the effect of each component on metmyoglobin reducing activity by excluding one component at a time. Excluding lactate, nicotinamide adenine dinucleotide (NAD), l-lactate dehydrogenase (LDH) or phenazine methosulfate (PMS) resulted in no reducing activity. A second experiment, utilising a meat mixture, investigated the effects of lactate (0%, 2%, 4% or 6%), nitrite (0 or 156 ppm), and packaging (oxygen-permeable or vacuum) on residual nitrite, meat colour and pH. Addition of lactate reduced residual nitrite in the meat mixtures. Both experiments support the hypothesis that lactate generates NADH which then reduces metmyoglobin to deoxymyoglobin. The resulting greater concentration of reduced myoglobin subsequently reacted with nitrite to produce more nitric oxide, reducing nitrite concentration and accelerating curing reactions.     

Short communication: Biofilm production characterization of mecA and mecC methicillin-resistant Staphylococcus aureus isolated from bovine milk in Great Britain

Staphylococcus aureus is an important cause of contagious intramammary infection in dairy cattle, and the ability to produce biofilm is considered to be an important virulence property in the pathogenesis of mastitis. The aim of this study was to characterize the biofilm formation capacity of methicillin-resistant Staph. aureus (MRSA), encoding mecA or mecC, isolated from bulk tank milk in Great Britain. For this purpose, 20 MRSA isolates were grown on microtiter plates to determine the biofilm production. Moreover, the spa-typing and the presence of the intercellular adhesion genes icaA and icaD were analyzed by PCR. All MRSA isolates tested belonged to 9 spa-types and were PCR-positive for the ica genes; 10 of them (50%) produced biofilm in the microtiter plate assay. This is also the first demonstration of biofilm production by mecC MRSA.     

Contribution of hydrogen peroxide to the inhibition of Staphylococcus aureus by Lactococcus garvieae in interaction with raw milk microbial community

The response of Staphylococcus aureus growth inhibition by Lactococcus garvieae to catalase and milk lactoperoxidase, and its efficiency in raw milk cheese were evaluated. S. aureus and L. garvieae were co-cultivated in broth buffered at pH 6.8, and in raw, pasteurized and microfiltered milk, in presence and absence of catalase. Although H₂O₂ production by L. garvieae was detected only in agitated broth, the inhibition of S. aureus by L. garvieae was reduced by catalase both in static and shaking cultures by 2.7 log, pasteurized milk (∼0.7 log), microfiltered milk (∼0.6 log) and raw milk (∼0.2 log). The growth of S. aureus alone in microfiltered milk was delayed compared with that in pasteurized milk and inhibition of S. aureus by L. garvieae was stronger in microfiltered milk. The inhibition of coagulase-positive staphylococci (CPS) by L. garvieae in raw milk cheese was similar to that in raw milk (∼0.8 log), but weaker than that in pasteurized and microfiltered milks. L. garvieae also had an early antagonistic effect on the growth of several other microbial groups, which lastingly affected populations levels and balance during cheese ripening.     

Characterization and technological properties of Staphylococcus xylosus strains isolated from a Tunisian traditional salted meat

The technological properties of strains of Staphylococcus xylosus were studied to select the most suitable for use as starter cultures for the production of dried fermented meat products. Strains of S. xylosus were isolated from traditional salted Tunisian meat and were identified by biochemical and molecular methods. Thirty strains of S. xylosus were studied to evaluate their catalase, nitrate reductase, lipolytic, proteolytic and antibacterial activities as well as growth ability at different temperatures, pH's and NaCl concentrations. All strains of S. xylosus had catalase activity and were able to reduce nitrates to nitrites. The nitrate reductase activity increased when the strains were kept under anaerobic conditions. Proteolytic activity on milk and on gelatin agar was demonstrated for 100% and 83.3% of the S. xylosus isolates, respectively. However extracellular proteolytic activity as assessed by the azocasein method was poor in all the strains. Lipolytic activity as assessed by the agar method showed that 76.6% of strains of S. xylosus could hydrolyze Tween 20 against 33.3% that could hydrolyze tributyrin. Tween 80 was hydrolyzed by only 10% of strains. Strains of S. xylosus hydrolyzed pork fat better than beef and lamb fat. The majority of strains had antibacterial activity against Salmonella arizonae, Staphylococcus aureus, Pseudomonas aeuroginosa, Escherichia coli and Enterococcus faecalis.     

Enterotoxin-producing Staphylococcus aureus genotype B as a major contaminant in Swiss raw milk cheese

The objective of this study was to characterize Staphylococcus aureus isolates from Swiss raw milk cheeses that had been found to be contaminated with coagulase-positive staphylococci and to estimate the frequency of the various genotypes, in particular the mastitis-associated Staph. aureus genotype B (GTB). The isolates were also tested for staphylococcal enterotoxin (SE) genes and other virulence factors. From 623 coagulase-positive staphylococci isolated from 78 contaminated raw milk cheeses, 609 were found to be Staphylococcus aureus. Genotyping of all Staph. aureus isolates was performed by PCR amplification of the 16S–23S rRNA intergenic spacer region, as this method was used previously to differentiate between mastitis subtypes associated with their clinical outcome. In total, 20 different genotypes were obtained and the 5 most frequently occurring genotypes were distributed in 6.4% or more of the samples. The enterotoxin-producing Staph. aureus GTB, known for its high contagiousness and increased pathogenicity in Swiss mastitis herds, was found to be the most abundant subtype at the sample level (71.8%) as well as among the isolates (62.0%). A subset of 107 isolates of the different genotypes were analyzed for the presence of SE genes and revealed 9 different SE gene patterns, with sed being most frequently detected and 26% being PCR-negative for SE genes. Almost all isolates of the major contaminant GTB contained the SE gene pattern sed, sej, ser, with half of them additionally carrying sea. Production of SE in vitro was consistent with the SE genes detected in most of the cases; however, some isolated GTB did not produce SEA. Staphylococcus aureus Protein A (spa) typing revealed 30 different subtypes and most GTB isolates belonged to the bovine spa type t2953; GTB/t2953 was linked among other subtypes to SE production in cheese and staphylococcal intoxication cases. Furthermore, 1 of the 623 isolates was a methicillin-resistant Staph. aureus, which was an seh-carrying Staph. aureus spa type tbl 0635 (non-GTB). We conclude that control and reduction of enterotoxigenic Staph. aureus GTB in dairy herds in Switzerland will not only prevent economic losses at the farm level but also improve the safety of raw milk cheeses; distribution of methicillin-resistant Staph. aureus via raw milk cheese is of less concern.     

Prevalence of bacteriophages infecting Staphylococcus aureus in dairy samples and their potential as biocontrol agents

The prevalence of bacteriophages infecting Staphylococcus aureus in dairy samples was assessed. Fourteen Staph. aureus strains were used in enrichment cultures of 75 dairy samples. All samples grew specific Staph. aureus bacteriophages. According to the host range, 8 different phages were isolated. Three of them, phages ΦH5, ΦG7, and ΦA72, were found in 89% of the samples; all the isolated phages were temperate. Phages ΦH5 and ΦA72 were used in preliminary bacterial challenge tests against Staph. aureus in milk. A phage mixture (1:1) was more effective than each single phage, most likely by preventing the survival of lysogenized cells. Phages inhibited Staph. aureus in UHT and pasteurized whole-fat milk. However, the phages were less active in semi-skimmed raw milk and little inhibition was achieved in whole, raw milk. Killing of Staph. aureus was observed at room temperature and at 37°C, but not at refrigeration temperature.     

Identification of potential sources of Staphylococcus aureus in herds with mastitis problems

Staphylococcus aureus is a common udder pathogen of dairy cows that often causes herd problems. Various mastitis control programs have been used to combat the problem but have not always been efficient in preventing new Staph. aureus infections, indicating the presence of possible sources of infection other than those traditionally considered. Therefore, the aim of the study was to identify potential sources of infection relevant for Staph. aureus mastitis within 5 dairy herds with udder health problems caused by Staph. aureus. Samples were collected from milk of lactating cows, from body sites, and from the environment of lactating cows, dry cows, late pregnant heifers, young heifers 4 to 12 mo old, and heifer calves 0 to 3 mo old. Isolates of Staph. aureus were identified and compared using pulsed-field gel electrophoresis. Four to 7 unique Staph. aureus pulsotypes were found within each herd, with one strain predominating in milk in each herd. The milk pulsotypes were also frequently isolated in body samples, especially on hock skin, and in the immediate environment of lactating cows, and were sometimes found in other animal groups, especially in dry cows and heifer calves 0 to 3 mo old. The prevalence of Staph. aureus in milk and other types of samples varied markedly between herds. Staphylococcus aureus isolates with genotypes indistinguishable from those found in milk also dominated in extra-mammary sites within the dairy herds studied, and hock skin was identified as an important reservoir of Staph. aureus. The results contribute new knowledge necessary to improve strategies for udder health control in herds.     

Effect of a bacteriocin produced by Pediococcus acidilactici against Listeria monocytogenes and Clostridium perfringens on Spanish raw meat

The inhibitory effect of a bacteriocin, produced by Pediococcus acidilactici, against Listeria monocytogenes and Clostridium perfringens on Spanish raw meat surface, was evaluated by in situ assays. Samples were incubated with the bacteriocin and then with a culture of the pathogenic bacteria. The treatment with 500, 1000 or 5000 bacteriocin units/ml (BU/ml) reduced the counts of L. monocytogenes after storage at 15°C during 72h by 1, 2 or 3 log cycles and with 1000 or 5000 BU/ml after storage at 4°C during 21 days by 2.5 or 3.5 log cycles, respectively, compared to the control. With C. perfringens a bacteriostatic effect could be observed.     

Mastitis-related subtypes of bovine Staphylococcus aureus are characterized by different clinical properties

Based on a former study from our group, one subtype of Staphylococcus aureus was associated with high within-herd prevalence of mastitis, whereas the other subtypes were associated with a low prevalence (sporadic intramammary infection). To confirm this hypothesis, a prospective study was done in 29 Swiss dairy herds. In particular, milk samples were collected from 10 herds with Staph. aureus herd problems (cases) and compared with samples from 19 herds with only sporadic cases of with Staph. aureus intramammary infection (controls). The isolates were tested for their virulence gene pattern and genotyped by PCR amplification of the 16S-23S rRNA intergenic spacer. The patterns and genotypes were then associated and compared with epidemiological and clinical data. Confirming the hypothesis, one particular subtype (genotype B) was associated with high within-herd and within-cow prevalence of intramammary infection, whereas the other subtypes were associated with low within-herd prevalence and infected single quarters. The gene patterns and genotypes were highly related, demonstrating the genetic diversity of the genotypes. The somatic cell counts were clearly increased in herds with a genotype B problem compared with herds with infections of other genotypes. Based on the different clinical properties and treatment consequences associated with these different genotypes found in Switzerland, we recommend subtyping Staph. aureus in other countries to determine if this finding is universally applicable.     

Multiple cases of methicillin-resistant CC130 Staphylococcus aureus harboring mecC in milk and swab samples from a Bavarian dairy herd

The discovery of a new mecA homolog, mecC, necessitates a modification of diagnostic procedures for the identification of methicillin-resistant Staphylococcus aureus (MRSA), as most assays used for the genotypic and phenotypic mecA detection cannot currently recognize mecC. Although the prevalence, distribution, and importance of mecC are not yet completely understood, an exchange of mecC-MRSA between humans and animals seems possible. All previously reported observations of mecC-positive strains have been sporadic. To the best of our knowledge, this is the first report about multiple cases of mecC-positive Staph. aureus in 1 dairy herd. Clonal complex 130 Staph. aureus harboring mecC were found in milk samples from 16 of 56 lactating cows kept in a herd in Bavaria, Germany. Almost all quarter milk samples positive for mecC-MRSA had the lowest possible California Mastitis Test score; composite somatic cell counts obtained from monthly milk recordings showed a mean of 51,600 cells/mL in mecC-MRSA affected cows. Additionally, mecC-positive clonal complex 130 Staph. aureus were detected in swab samples from the mammary skin and a teat lesion of 1 cow from this herd. This report suggests that mecC-carrying strains are able to spread among livestock, and that they have the ability to cause multiple cases in single herds. Therefore, future studies targeting MRSA in dairy cows need to consider mecC.     

Short communication: Antimicrobial susceptibility profiling and genotyping of Staphylococcus aureus isolates from bovine mastitis in Poland

Staphylococcus aureus is the predominant causative agent of bovine mastitis, a disease that remains a major economic burden for the dairy industry worldwide. In this study, the antimicrobial resistance patterns and the genetic composition of 80 S. aureus mastitis isolates collected from 14 dairy farms in Eastern Poland were determined. Of the 10 antimicrobial agents evaluated, only testing for penicillin G produced drug resistance. As 41% of the S. aureus isolates were penicillin resistant, this drug along with other β-lactamase-sensitive β-lactams, should rather not be considered for the treatment of bovine mastitis caused by S. aureus. Upon genotyping, with a triplex PCR method, a total of 11 distinct PCR types were produced. The population structure of S. aureus isolates was highly clonal, with 1 predominant genotype circulating on each farm. The observed similarities in the genotype composition of S. aureus populations from geographically distant farms underscore the significance of interfarm transmission of S. aureus in Poland. This, in turn, argues for the establishment of a nationwide surveillance program for bovine mastitis due to this pathogen.     

Biofilm-producing ability of Staphylococcus aureus isolates from Brazilian dairy farms

This study aimed to investigate the in silico biofilm production ability of Staphylococcus aureus strains isolated from milking parlor environments on dairy farms from São Paulo, Brazil. The Staph. aureus isolates were obtained from 849 samples collected on dairy farms, as follows: milk from individual cows with subclinical mastitis or history of the disease (n = 220); milk from bulk tank (n = 120); surfaces of milking machines and utensils (n = 389); and milk handlers (n = 120). Thirty-one Staph. aureus isolates were obtained and categorized as pulsotypes by pulsed-field gel electrophoresis and submitted to assays for biofilm formation on polystyrene, stainless steel, rubber, and silicone surfaces. Fourteen (45.2%) pulsotypes were considered producers of biofilm on the polystyrene microplate assay, whereas 13 (41.9%) and 12 (38.7%) pulsotypes were biofilm producers on stainless steel and rubber, respectively. None of the pulsotypes evaluated produced biofilms on silicone. Approximately 45% of Staph. aureus pulsotypes isolated from different sources on dairy farms showed the ability to produce biofilms in at least one assay, indicating possible persistence of this pathogen in the milking environment. The potential involvement of Staph. aureus in subclinical mastitis cases and its occurrence in milk for human consumption emphasize the need to improve hygiene practices to prevent biofilm formation on the farms studied.     

The influence of precultivation parameters on the catabolism of branched-chain amino acids by Staphylococcus xylosus and Staphylococcus carnosus

The influence of precultivation parameters on the ability of Staphylococcus xylosus and Staphylococcus carnosus to convert branched-chain amino acids—leucine, isoleucine and valine—into volatile flavour compounds was investigated using resting cells in a defined reaction medium. The studied precultivation parameters were: growth phase, temperature, NaCl concentration and the concentration of leucine, isoleucine and valine (only for S. xylosus). Flavour compounds were sampled by automatic static headspace collection and separated/quantified using gas chromatography/flame ionization detection (GC/FID).Main catabolic products from degradation of leucine, isoleucine and valine were the flavour intensive branched-chain acids: 2- and 3-methylbutanoic and 2-methylpropanoic acids. The precultivation parameters altered the production of the branched-chain acids significantly, but to various degrees for S. xylosus and S. carnosus.Production of branched-chain acids by S. carnosus was only influenced slightly by the growth phase and not by changing the NaCl concentration between 4.0% and 10.0% (w/w). Lowering the temperature from 28°C to 18°C significantly decreased S. carnosus’ generation of branched-chain acids. In contrast, S. xylosus was significantly influenced by all precultivation parameters, in particular by the growth phase. Cells taken from growing cultures had a much higher production of branched-chain acids compared to cells taken from stationary cultures. Addition of leucine and valine to the precultivation medium enhanced the production of branched-chain acids whereas addition of isoleucine had the opposite effect.     

Short communication: Prevalence and antibiotic resistance of Staphylococcus aureus isolated from bovine clinical mastitis

The aims of this study were to determine the prevalence and antibiotic resistance of Staphylococcus aureus isolated from bovine clinical mastitis in Varamin, Tehran Province, Iran. All of the isolated Staph. aureus were identified by morphology and culture and confirmed using the API Staph identification system (bioMérieux, Marcy-l’Étoile, France). Antibiotic resistance genes were detected by PCR with oligonucleotide primers specific for each gene. Staphylococcus aureus was recovered from 43 of 207 (20.1%) bovine clinical milk samples. Using disk diffusion, methicillin-resistant Staph. aureus was detected in 5 of 43 (11.6%) samples. The pathogen showed high resistance against penicillin G (86%) and tetracycline (76.7%). The blaZ (penicillin) (86%), tetM (tetracycline), and ermC (erythromycin) genes (39.5% each) were the most prevalent antibiotic resistance genes. The findings of this study are useful for designing specific control programs for bovine clinical mastitis caused by Staph. aureus in this region of Iran.     

Short communication: Antimicrobial efficacy of intramammary treatment with a novel biphenomycin compound against Staphylococcus aureus, Streptococcus uberis, and Escherichia coli-induced mouse mastitis

Bovine mastitis undermines udder health, jeopardizes milk production, and entails prohibitive costs, estimated at $2 billion per year in the dairy industry of the United States. Despite intensive research, the dairy industry has not managed to eradicate the 3 major bovine mastitis-inducing pathogens: Staphylococcus aureus, Streptococcus uberis, and Escherichia coli. In this study, the antimicrobial efficacy of a newly formulated biphenomycin compound (AIC102827) was assessed against intramammary Staph. aureus, Strep. uberis, and E. coli infections, using an experimental mouse mastitis model. Based on its effective and protective doses, AIC102827 applied into the mammary gland was most efficient to treat Staph. aureus, but also adequately reduced growth of Strep. uberis or E. coli, indicating its potential as a broad-spectrum candidate to treat staphylococcal, streptococcal, and coliform mastitis in dairy cattle.     

The antioxidative properties of mustard leaf (Brassica juncea) kimchi extracts on refrigerated raw ground pork meat against lipid oxidation

The efficacy of varying concentration of mustard leaf kimchi ethanolic extracts (MK) in retarding oxidative rancidity was tested with raw ground pork. Freshly ground pork meat was assigned to one of the following five treatments: control (no antioxidants); AC-0.02 (0.02% ascorbic acid); MK-0.05, 0.1, and 0.2 (0.05%, 0.1% and 0.2% MK, respectively). The pH of the samples decreased and the TBARS values and free fatty acids (%) increased considerably (P < 0.05) during storage. The total bacterial count was lower in MK-0.1 and MK-0.2 than the control during storage. The internal L∗ value and a∗ value decreased (P < 0.05) with the addition of MK. The internal b∗ value of MK treatments were higher (P < 0.05) than that for the control and increased incrementally with MK concentration. The TBARS values and free fatty acids (%) of MK-0.02 was lowest among the treatments. The peroxide value of the control increased until 7 days and reached the maximum value at a certain storage time and decreased thereafter. In the other treatments it increased. All treatments had lower concentration of conjugated dienes (P < 0.05) compared to the control sample, after the first day. Mustard leaf kimchi ethanolic extracts exhibited a protective effect against lipid oxidation in raw ground pork.     

Satureja horvatii essential oil: In vitro antimicrobial and antiradical properties and in situ control of Listeria monocytogenes in pork meat

The dominant compounds in Satureja horvatii oil were p-cymene (33.14%), thymol (26.11%) and thymol methyl ether (15.08%). The minimum inhibitory concentration (MIC) varied from 0.03 to 0.57 mg/mL for bacteria, and from 0.56 to 2.23 mg/mL for yeast strains, while minimum bactericidal/yeast-cidal concentration (MBC/MYC) varied from 0.07 to 1.15 mg/mL and 1.11 to 5.57 mg/mL for bacteria and yeasts, respectively. The antiradical potential of the essential oil was evaluated using hydroxyl radical (•OH) generated in Fenton reaction. The meat preserving potential of essential oil from Satureja horvatii was investigated against L. monocytogenes. Essential oil successfully inhibited development of L. monocytogenes in pork meat. Sensorial evaluation on flavor and color of meat was performed. The color and flavor of meat treated with essential oil improved after 4 days of storage. S. horvatii essential oil can act as a potent inhibitor of food spoiling microorganisms, in meat products and also can be a useful source of natural antioxidants.     

Staphylococcus aureus and Listeria monocytogenes in Norwegian raw milk cheese production

The aim of this study was to survey the presence of Staphylococcus aureus and Listeria monocytogenes during the cheese making process in small-scale raw milk cheese production in Norway.The prevalence of S. aureus in bovine and caprine raw milk samples was 47.3% and 98.8%, respectively. An increase in contamination during the first 2-3 h resulted in a 73.6% prevalence of contamination in the bovine curd, and 23 out of 38 S. aureus-negative bovine milk samples gave rise to S. aureus-positive curds. The highest contamination levels of S. aureus were reached in both caprine and bovine cheese after 5-6 h (after the first pressing). There was no contamination of L. monocytogenes in caprine cheeses and only one (1.4%) contaminated bovine cheese.This work has increased our knowledge about S. aureus and L. monocytogenes contamination during the process of raw milk cheese production and gives an account of the hygiene status during the manufacture of Norwegian raw milk cheeses.     

A longitudinal study investigating the prevalence of Staphylococcus aureus genotype B in seasonally communal dairy herds

Staphylococcus aureus is a major mastitis-causing pathogen. Various genotypes have been recently identified in Switzerland but Staph. aureus genotype B (GTB) was the only genotype associated with high within-herd prevalence. The risk of introducing this Staph. aureus genotype into a herd may be increased by frequent animal movements. This may also be the case when cows from different herds of origin are commingled and share their milking equipment for a limited period of time. The aim of the present study was to determine the prevalence of Staph. aureus GTB in seasonally communal dairy herds before and after a summer period when dairy farming is characterized by mixing cows from different herds of origin in 1 communal operation. In addition, the environment was investigated to identify potential Staph. aureus GTB reservoirs relevant for transmission of the disease. A total of 829 cows from 110 herds of origin in 9 communal operations were included in the study. Composite milk samples were collected from all cows during the first or second milking after arrival at the communal operation and again shortly before the end of the season. Swab samples from the environment, involved personnel, and herding dogs present were collected before the cows arrived. At the end of the season, sampling of personnel was repeated. All samples were analyzed for the presence of Staph. aureus GTB using an established quantitative PCR. At the beginning of the season, Staph. aureus GTB-positive cows were identified in 7 out of 9 communal operations and the within-communal operation prevalence ranged from 2.2 to 38.9%. At the second sampling, all communal operations were Staph. aureus GTB positive, showing within-communal operation prevalence from 1 to 72.1%. The between-herd of origin prevalence increased from 27.3 to 56.6% and the cow-level prevalence increased from 11.2% at the beginning of the season to 29.6% at the end of the season. On 3 different communal operations, Staph. aureus GTB-positive swabs from seasonally employed personnel were identified at the end of the season. The results indicate that Staph. aureus GTB can easily spread in communal operations when cows from different herds of origin are mixed during the summer season. Effective management measures need to be designed to prevent the spread of Staph. aureus GTB in seasonally communal herds.