Characterization of Staphylococcus aureus isolates recovered from dairy sheep farms (agr group,adherence, slime,resistance to antibiotics)

Staphylococcus aureus is one of the major causes of dairy sheep mastitis. The S. aureus agr locus (accessory gene regulator) regulates the production of most staphylococcal exoproteins, including exoenzymes, toxins, surface proteins, and other virulence factors. S. aureus have four agr groups (alleles) determined by PCR. In this study, 46 S. aureus isolates, recovered in south-east of France, were also characterized by their properties of adherence to smooth surfaces, slime production and resistance to 10 antibiotics. For 46 S. aureus associated with dairy sheep mastitis (subclinical mastitis, clinical mastitis, environment of dairy sheep farm), 80% (37/46) belonged to agr group 3, 39% (18/46) were adherent (adherent, strongly adherent or with maximal adherence). For the same isolates, 26% (12/46) were slime producers (moderate or strong producers). All the 46 isolates were susceptible to oxacillin, except for two isolates including two sheep subclinical mastitis isolates. The dairy sheep subclinical mastitis isolates were for 79% (22/28), susceptible to nine other antibiotics tested.     

Dual Role of the Oligopeptide Permease Opp3 during Growth of Staphylococcus aureus in Milk

Staphylococcus aureus RN6390 presents a diauxic growth in milk, due to amino acid limitation. Inactivation of the oligopeptide permease Opp3 (dedicated to the nitrogen nutrition of the strain) not only affects the growth of the strain but also results in reduced expression levels of three major extracellular proteases.Staphylococcus aureus is a ubiquitous gram-positive pathogen commonly found in the environment. Some S. aureus strains are able to produce enterotoxins that cause food poisoning (6). In France, contaminated dairy products are the main source of staphylococcal food-borne disease outbreaks (3). Thus, the development of this bacterium in milk is a major concern for the safety of dairy products. Our objective was to evaluate the growth of S. aureus in milk, with special attention focused on the role of the peptide transport systems.     

Subinhibitory concentrations of perilla oil affect the expression of secreted virulence factor genes in Staphylococcus aureus

Background

The pathogenicity of staphylococcus aureus is dependent largely upon its ability to secrete a number of virulence factors, therefore, anti-virulence strategy to combat S. aureus-mediated infections is now gaining great interest. It is widely recognized that some plant essential oils could affect the production of staphylococcal exotoxins when used at subinhibitory concentrations. Perilla [Perilla frutescens (L.) Britton], a natural medicine found in eastern Asia, is primarily used as both a medicinal and culinary herb. Its essential oil (perilla oil) has been previously demonstrated to be active against S. aureus. However, there are no data on the influence of perilla oil on the production of S. aureus exotoxins.

Methodology/Principal Findings

A broth microdilution method was used to determine the minimum inhibitory concentrations (MICs) of perilla oil against S. aureus strains. Hemolysis, tumour necrosis factor (TNF) release, Western blot, and real-time RT-PCR assays were performed to evaluate the effects of subinhibitory concentrations of perilla oil on exotoxins production in S. aureus. The data presented here show that perilla oil dose-dependently decreased the production of α-toxin, enterotoxins A and B (the major staphylococcal enterotoxins), and toxic shock syndrome toxin 1 (TSST-1) in both methicillin-sensitive S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA).

Conclusions/Significance

The production of α-toxin, SEA, SEB, and TSST-1 in S. aureus was decreased by perilla oil. These data suggest that perilla oil may be useful for the treatment of S. aureus infections when used in combination with β-lactam antibiotics, which can increase exotoxins production by S. aureus at subinhibitory concentrations. Furthermore, perilla oil could be rationally applied in food systems as a novel food preservative both to inhibit the growth of S. aureus and to repress the production of exotoxins, particularly staphylococcal enterotoxins.     

Prevalence of Clonal Complexes and Virulence Genes among Commensal and Invasive Staphylococcus aureus Isolates in Sweden

Staphylococcus aureus encodes a remarkable number of virulence factors which may contribute to its pathogenicity and ability to cause invasive disease. The main objective of this study was to evaluate the association between S. aureus invasiveness and bacterial genotype, in terms of the presence of virulence genes and affiliation to clonal complexes. Also, the significance of different virulence genes, mainly adhesins, for the development of infective endocarditis was investigated.DNA microarray technology was used to analyze 134 S. aureus isolates, all methicillin-susceptible, derived from three groups of clinically well-characterized patients: nasal carriers (n=46), bacteremia (n=55), and bacteremia with infective endocarditis (n=33).Invasive isolates were dominant in four of the major clonal complexes: 5, 8, 15, and 25. Of the 170 virulence genes examined, those encoding accessory gene regulator group II (agr II), capsule polysaccharide serotype 5 (cap5), and adhesins such as S. aureus surface protein G (sasG) and fibronectin-binding protein B (fnbB) were found to be associated with invasive disease. The same was shown for the leukocidin genes lukD/lukE, as well as the genes encoding serine protease A and B (splA/splB), staphylococcal complement inhibitor (scn) and the staphylococcal exotoxin-like protein (setC or selX). In addition, there was a trend of higher prevalence of certain genes or gene clusters (sasG, agr II, cap5) among isolates causing infective endocarditis compared to other invasive isolates. In most cases, the presence of virulence genes was linked to clonal complex affiliation.In conclusion, certain S. aureus clonal lineages harboring specific sets of virulence genes seem to be more successful in causing invasive disease.     

Production and Targeting of the Brucella abortus Antigen L7/L12 in Lactococcus lactis: a First Step towards Food-Grade Live Vaccines against Brucellosis

Brucella abortus is a facultative intracellular gram-negative bacterial pathogen that infects humans and animals by entry mainly through the digestive tract. B. abortus causes abortion in pregnant cattle and undulant fever in humans. The immunogenic B. abortus ribosomal protein L7/L12 is a promising candidate antigen for the development of oral live vaccines against brucellosis, using food-grade lactic acid bacteria (LAB) as a carrier. The L7/L12 gene was expressed in Lactococcus lactis, the model LAB, under the nisin-inducible promoter. Using different signals, L7/L12 was produced in cytoplasmic, cell-wall-anchored, and secreted forms. Cytoplasmic production of L7/L12 gave a low yield, estimated at 0.5 mg/liter. Interestingly, a secretable form of this normally cytoplasmic protein via fusion with a signal peptide resulted in increased yield of L7/L12 to 3 mg/liter; secretion efficiency (SE) was 35%. A fusion between the mature moiety of the staphylococcal nuclease (Nuc) and L7/L12 further increased yield to 8 mg/liter. Fusion with a synthetic propeptide (LEISSTCDA) previously described as an enhancer for heterologous protein secretion in L. lactis (Y. Le Loir, A. Gruss, S. D. Ehrlich, and P. Langella, J. Bacteriol. 180:1895-1903, 1998) raised the yield to 8 mg/liter and SE to 50%. A surface-anchored L7/L12 form in L. lactis was obtained by fusing the cell wall anchor of Streptococcus pyogenes M6 protein to the C-terminal end of L7/L12. The fusions described allow the production and targeting of L7/L12 in three different locations in L. lactis. This is the first example of a B. abortus antigen produced in a food-grade bacterium and opens new perspectives for alternative vaccine strategies against brucellosis.     

Population Diversification in Staphylococcus aureus Biofilms May Promote Dissemination and Persistence

The biofilm mode of growth can lead to diversification of the bacterial population by promoting the emergence of variants. Here we report the identification and characterization of two major subpopulations of morphological variants arising in biofilms of S. aureus. One of these lacked pigmentation (termed white variants; WVs), whilst the other formed colonies on agar that were larger and paler than the parental strain (termed large pale variants; LPVs). WVs were unable to form biofilms, and exhibited increased proteolysis and haemolysis; all phenotypes attributable to loss-of-function mutations identified in the gene encoding the alternative sigma factor, sigB. For LPVs, no differences in biofilm forming capacity or proteolysis were observed compared with the parental strain. Genetic analysis of LPVs revealed that they had undergone mutation in the accessory gene regulator system (agrA), and deficiency in agr was confirmed by demonstrating loss of both colony spreading and haemolytic activity. The observation that S. aureus biofilms elaborate large subpopulations of sigB and agr mutants, both genotypes that have independently been shown to be of importance in staphylococcal disease, has implications for our understanding of staphylococcal infections involving a biofilm component.     

Natural populations of lactic acid bacteria associated with silage fermentation as determined by phenotype, 16S ribosomal RNA and recA gene analysis

One hundred and fifty-six strains isolated from corn (Zea mays L.), forage paddy rice (Oryza sativa L.), sorghum (Sorghum bicolor L.) and alfalfa (Medicago sativa L.) silages prepared on dairy farms were screened, of which 110 isolates were considered to be lactic acid bacteria (LAB) according to their Gram-positive and catalase-negative characteristics and, mainly, the lactic acid metabolic products. These isolates were divided into eight groups (A-H) based on the following properties: morphological and biochemical characteristics, γ-aminobutyric acid production capacity, and 16S rRNA gene sequences. They were identified as Weissella cibaria (36.4%), Weissella confusa (9.1%), Leuconostoc citreum (5.3%), Leuconostoc lactis (4.9%), Leuconostoc pseudomesenteroides (8.0%), Lactococcus lactis subsp. lactis (4.5%), Lactobacillus paraplantarum (4.5%) and Lactobacillus plantarum (27.3%). W. cibaria and W. confusa were mainly present in corn silages, and L. plantarum was dominant on sorghum and forage paddy rice silages, while L. pseudomesenteroides, L. plantarum and L. paraplantarum were the dominant species in alfalfa silage. The corn, sorghum and forage paddy rice silages were well preserved with lower pH values and ammonia-N concentrations, but had higher lactic acid content, while the alfalfa silage had relatively poor quality with higher pH values and ammonia-N concentrations, and lower lactic acid content. The present study confirmed the diversity of LAB species inhabiting silages. It showed that the differing natural populations of LAB on these silages might influence fermentation quality. These results will enable future research on the relationship between LAB species and silage fermentation quality, and will enhance the screening of appropriate inoculants aimed at improving such quality.     

Expression of virulence factors by Staphylococcus aureus grown in serum

Staphylococcus aureus produces many virulence factors, including toxins, immune-modulatory factors, and exoenzymes. Previous studies involving the analysis of virulence expression were mainly performed by in vitro experiments using bacterial medium. However, when S. aureus infects a host, the bacterial growth conditions are quite different from those in a medium, which may be related to the different expression of virulence factors in the host. In this study, we investigated the expression of virulence factors in S. aureus grown in calf serum. The expression of many virulence factors, including hemolysins, enterotoxins, proteases, and iron acquisition factors, was significantly increased compared with that in bacterial medium. In addition, the expression of RNA III, a global regulon for virulence expression, was significantly increased. This effect was partially restored by the addition of 300 μM FeCl₃ into serum, suggesting that iron depletion is associated with the increased expression of virulence factors in serum. In chemically defined medium without iron, a similar effect was observed. In a mutant with agr inactivated grown in serum, the expression of RNA III, psm, and sec4 was not increased, while other factors were still induced in the mutant, suggesting that another regulatory factor(s) is involved. In addition, we found that serum albumin is a major factor for the capture of free iron to prevent the supply of iron to bacteria grown in serum. These results indicate that S. aureus expresses virulence factors in adaptation to the host environment.     

Mobile CRISPR/Cas-Mediated Bacteriophage Resistance in Lactococcus lactis

Lactococcus lactis is a biotechnological workhorse for food fermentations and potentially therapeutic products and is therefore widely consumed by humans. It is predominantly used as a starter microbe for fermented dairy products, and specialized strains have adapted from a plant environment through reductive evolution and horizontal gene transfer as evidenced by the association of adventitious traits with mobile elements. Specifically, L. lactis has armed itself with a myriad of plasmid-encoded bacteriophage defensive systems to protect against viral predation. This known arsenal had not included CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins), which forms a remarkable microbial immunity system against invading DNA. Although CRISPR/Cas systems are common in the genomes of closely related lactic acid bacteria (LAB), none was identified within the eight published lactococcal genomes. Furthermore, a PCR-based search of the common LAB CRISPR/Cas systems (Types I and II) in 383 industrial L. lactis strains proved unsuccessful. Here we describe a novel, Type III, self-transmissible, plasmid-encoded, phage-interfering CRISPR/Cas discovered in L. lactis. The native CRISPR spacers confer resistance based on sequence identity to corresponding lactococcal phage. The interference is directed at phages problematic to the dairy industry, indicative of a responsive system. Moreover, targeting could be modified by engineering the spacer content. The 62.8-kb plasmid was shown to be conjugally transferrable to various strains. Its mobility should facilitate dissemination within microbial communities and provide a readily applicable system to naturally introduce CRISPR/Cas to industrially relevant strains for enhanced phage resistance and prevention against acquisition of undesirable genes.     

Staphylococcus aureus Interaction with Phospholipid Vesicles – A New Method to Accurately Determine Accessory Gene Regulator (agr) Activity

The staphylococcal accessory gene regulatory (agr) operon is a well-characterised global regulatory element that is important in the control of virulence gene expression for Staphylococcus aureus, a major human pathogen. Hence, accurate and sensitive measurement of Agr activity is central in understanding the virulence potential of Staphylococcus aureus, especially in the context of Agr dysfunction, which has been linked with persistent bacteraemia and reduced susceptibility to glycopeptide antibiotics. Agr function is typically measured using a synergistic haemolysis CAMP assay, which is believe to report on the level of expression of one of the translated products of the agr locus, delta toxin. In this study we develop a vesicle lysis test (VLT) that is specific to small amphipathic peptides, most notably delta and Phenol Soluble Modulin (PSM) toxins. To determine the accuracy of this VLT method in assaying Agr activity, we compared it to the CAMP assay using 89 clinical Staphylococcus aureus isolates. Of the 89 isolates, 16 were designated as having dysfunctional Agr systems by the CAMP assay, whereas only three were designated as such by VLT. Molecular analysis demonstrated that of these 16 isolates, the 13 designated as having a functional Agr system by VLT transcribed rnaIII and secreted delta toxin, demonstrating they have a functional Agr system despite the results of the CAMP assay. The agr locus of all 16 isolates was sequenced, and only the 3 designated as having a dysfunctional Agr system contained mutations, explaining their Agr dysfunction. Given the potentially important link between Agr dysfunction and clinical outcome, we have developed an assay that determines this more accurately than the conventional CAMP assay.     

Bacterial Competition for Human Nasal Cavity Colonization: Role of Staphylococcal agr Alleles

We examined the bacterial aerobic nasal flora of 216 healthy volunteers to identify potential competitive interactions among different species, with special emphasis on the influence of staphylococcal agr alleles. The Staphylococcus aureus colonization rate correlated negatively with the rate of colonization by Corynebacterium spp. and non-aureus staphylococci, especially S. epidermidis, suggesting that both Corynebacterium spp. and S. epidermidis antagonize S. aureus colonization. Most of the S. aureus and S. epidermidis isolates were agr typed by a PCR method. Only one S. aureus agr (agr_Sa) allele was detected in each carrier. Multiple logistic regression of the two most prevalent agr_Sa alleles (agr-1_Sa and agr-2_Sa) and the three S. epidermidis agr (agr_Se) alleles showed a specific influence of the agr system. The results of this model did not support conclusions drawn from previous in vitro agr-specific cross-inhibition experiments. Our findings suggest that the agr alleles, which are strongly linked to the bacterial genetic background, may simply be associated with common biological properties—including mediators of bacterial interference—in the strains that bear them.     

Genome sequence of Staphylococcus aureus strain Newman and comparative analysis of staphylococcal genomes: polymorphism and evolution of two major pathogenicity islands

Strains of Staphylococcus aureus, an important human pathogen, display up to 20% variability in their genome sequence, and most sequence information is available for human clinical isolates that have not been subjected to genetic analysis of virulence attributes. S. aureus strain Newman, which was also isolated from a human infection, displays robust virulence properties in animal models of disease and has already been extensively analyzed for its molecular traits of staphylococcal pathogenesis. We report here the complete genome sequence of S. aureus Newman, which carries four integrated prophages, as well as two large pathogenicity islands. In agreement with the view that S. aureus Newman prophages contribute important properties to pathogenesis, fewer virulence factors are found outside of the prophages than for the highly virulent strain MW2. The absence of drug resistance genes reflects the general antibiotic-susceptible phenotype of S. aureus Newman. Phylogenetic analyses reveal clonal relationships between the staphylococcal strains Newman, COL, NCTC8325, and USA300 and a greater evolutionary distance to strains MRSA252, MW2, MSSA476, N315, Mu50, JH1, JH9, and RF122. However, polymorphism analysis of two large pathogenicity islands distributed among these strains shows that the two islands were acquired independently from the evolutionary pathway of the chromosomal backbones of staphylococcal genomes. Prophages and pathogenicity islands play central roles in S. aureus virulence and evolution.     

Antibacterial metabolites of lactic acid bacteria: Their diversity and properties

The review is devoted to literature data on antimicrobial metabolites produced by lactic acid bacteria (LAB), which have long been used for the preparation of cultured dairy products. This paper summarizes data on low-molecular-weight antimicrobial substances, which are primary products or by-products of lactic fermentation. Individual sections are devoted to a variety of antifungal agents and bacteriocins produced by LAB; their potential use as food preservatives has been discussed. The characteristics and classification of bacteriocins are presented in a greater detail; their synthesis and mechanism of action are described using the example of nisin A, which belongs to class I lantibiotics synthesized by the bacterium Lactococcus lactis subsp. lactis. The mechanism of action of class II bacteriocins has been demonstrated with lacticin. Prospective directions for using LAB antimicrobial metabolites in industry and medicine are discussed in the Conclusion.     

Characterisation of the Virulence Factors and Genetic Types of Methicillin Susceptible Staphylococcus aureus from Patients and Healthy Individuals

Methicillin sensitive Staphylococcus aureus is an important bacterial pathogen associated with hospital- and community-acquired infections leading to endocarditis, skin tissue infection and pneumonia. The objective of this study was to determine both the genetic characteristics of methicillin-sensitive S. aureus (MSSA) strains, and the occurrence of virulence factors produced by S. aureus strains isolated from UMMC and healthy students in the University from year 2009. Out of 429 nasal swab samples, 67 were MSSA. The prevalence of 21 different virulence genes among 67 Malaysian clinical and community MSSA strains was determined by PCR, and their genetic features were assessed by PCR-RFLP of coa gene, agr types, spa typing and PFGE. The five predominant virulence genes were ica (79 %), efb and fnbA (61 % each), sdrE (57 %) and hlg (45 %). Toxin genes (enterotoxin, etd and pvl) were significantly more common (P < 0.05) in clinical strains compared to community strains. Three agr genotypes were observed: agr type I (45 %), agr type III (25 %) and agr type II (19 %). All 67 MSSA strains were distinguished into 26 profiles by PCR-RFLP of coa, 55 pulsotypes and 21 spa types. Four novel spa types (t7312, t7581, t7582 and t7583) were observed. In conclusion, different virulence profiles were observed in MSSA strains in Malaysia where toxin genes were more prevalent among clinical strains. No correlation between DNA profiles (coa-RFLP, PFGE and spa) and virulotypes was observed. The Malaysian MSSA strains from clinical and community sources were genetically diverse and heterogeneous.     

Safety and probiotic functionality of isolated goat milk lactic acid bacteria

Purpose

Lactic acid bacteria (LAB) are traditionally employed in the food industry. LAB strains from goat milk may also present probiotic potential, and it is fundamental to study the safety and functionality aspects which are desirable for their use in food. The objective of this study was to verify the probiotic potential of lactic bacteria isolated from goat milk.

Methods

The presence of safety-related virulence factors (hemolytic activity, gelatinase production, coagulase, and sensitivity to antibiotics) as well as functionality (exopolysaccharide (EPS) production, proteolytic activity, autoaggregation, gas production, survival in the gastrointestinal tract, and antimicrobial activity against bacteria that impair oral health) were determined.

Result

The selected LAB strains are safe against the evaluated parameters and have characteristics of possible probiotic candidates. Especially L. plantarum (DF60Mi) and Lactococcus lactis (DF04Mi) have potential to be added to foods because they have better resistance to simulated gastrointestinal conditions. In addition, they are isolated with already proven antimicrobial activity against Listeria monocytogenes, an important food-borne pathogen. DF60Mi was able to produce EPS (exopolysaccharides). LS2 and DF4Mi strains, both Lactococcus lactis subsp. lactis, demonstrated antimicrobial activity against S. mutans ATCC 25175, a recurrent microorganism in oral pathologies, mainly caries.

Conclusion

This study provides subsidies for future exploration of the potentialities of these LAB strains for both the development of new functional foods and for application in oral health.