Mastitis Strains Research Articles

Comparative genomics study of Staphylococcus aureus isolated from cattle and humans reveals virulence patterns exclusively associated with bovine clinical mastitis strains.

Staphylococcus aureus causes nosocomial and intramammary infections in humans and cattle, respectively. A large number of virulence factors are thought to play important roles in the pathogenesis of this bacterium. Currently, genome-wide and data-analysis studies are being used to better understand its epidemiology. In this study, we conducted a genome wide comparison and phylogenomic analyses of S. aureus to find specific virulence patterns associated with clinical and subclinical mastitis strains in cattle and compare them with those of human origin. The presence/absence of key virulence factors such as adhesin, biofilm, antimicrobial resistance, and toxin genes, as well as the phylogeny and sequence type of the isolates were evaluated. A total of 248 genomes (27 clinical mastitis, 43 subclinical mastitis, 21 milk, 53 skin-related abscesses, 49 skin infections, and 55 pus from cellulitis) isolated from 32 countries were evaluated. We found that the cflA, fnbA, ebpS, spa, sdrC, coa, emp, vWF, atl, sasH, sasA, and sasF adhesion genes, as well as the aur, hglA, hglB, and hglC toxin genes were highly associated in clinical mastitis strains. The strains had diverse genetic origins (72 protein A and 48 sequence types with ST97, ST8 and ST152 being frequent in isolates from clinical mastitis, abscess, and skin infection, respectively). Further, our phylogenomic analyses suggested that zoonotic and/or zooanthroponotic transmission may have occurred. These findings contribute to a better understanding of S. aureus epidemiology and the relationships between adhesion mechanisms, biofilm formation, antimicrobial resistance, and toxins and could aid in the development of improved vaccines and strain genotyping methods.

Whole-genome analysis of Klebsiella pneumoniae from bovine mastitis milk in the U.S.

Dairy cattle mastitis has long been one of the most common and costly diseases in the dairy industry worldwide, due to its significant impact on milk production and animal welfare. Among all mastitis causing bacterial pathogens, Klebsiella pneumoniae causes the largest milk loss. To better understand the genomic features of this population, 180 K. pneumoniae strains isolated from dairy cattle mastitis milk in 11 U.S. states were sequenced. The phylogenetic analysis classified all mastitis-causing K. pneumoniae into two major phylogroups, with exclusive predominance in phylogroup KpI. Analysis of more than 61 sequence types, 51 capsular types and 12 lipopolysaccharide O-antigen types revealed great genomic diversity of this K. pneumoniae population. Approximately 100 gene units in accessory genomes were detected with significantly higher prevalence in bovine mastitis strains, compared to human-sourced or dairy environmental strains. The most notable genes were identified associated with ferric citrate uptake, lactose fermentation and resistance to heavy metals. The acquired antimicrobial resistance genes were identified in sporadic mastitis strains. This comprehensive genomic epidemiological study provides insights for a better understanding of the virulence of mastitis-causing K. pneumoniae strains and may lead to the development of novel diagnostic tools and preventive strategies.

Relative virulence of Staphylococcus aureus bovine mastitis strains representing the main Canadian spa types and clonal complexes as determined using in vitro and in vivo mastitis models

Staphylococcus aureus is one of the main pathogens leading to both clinical and subclinical bovine mastitis in dairy cattle. Prediction of disease evolution based on the characteristics of Staph. aureus isolates that cause intramammary infections and understanding the host-pathogen interactions may improve management of mastitis in dairy herds. For this study, several strains were selected from each of the 6 major Canadian spa types associated with mastitis (t267, t359, t529, t605, t2445, and t13401). Adherence to host cells and intracellular persistence of these strains were studied using a bovine mammary gland epithelial cell line (MAC-T). Additionally, relative virulence and host response (cytokines production) were also studied in vivo using a mouse model of mastitis. Whole-genome sequencing was performed on all strains and associations between clonal complex, sequence type, and presence of certain virulence factors were also investigated. Results show that spa type t2445 was correlated with persistence in MAC-T cells. Strains from spa t359 and t529 showed better ability to colonize mouse mammary glands. The exception was strain sa3154 (spa t529), which showed less colonization of glands compared with other t359 and t529 strains but possessed the highest number of superantigen genes including tst. All strains possessed hemolysins, but spa types t529 and t2445 showed the largest diameter of β-hemolysis on blood agar plates. Although several spa types possessed 2 or 3 serine-aspartate rich proteins (Sdr) believed to be involved in many pathogenic processes, most t529 strains expressed only an allelic variant of sdrE. The spa types t605 (positive for the biofilm associated protein gene; bap+) and t13401 (bap-), that produced the largest amounts of biofilm in vitro, were the least virulent in vivo. Finally, strains from spa type t529 (ST151) elicited a cytokine expression profile (TNF-α, IL-1β and IL-12) that suggests a potential for severe inflammation. This study suggests that determination of the spa type may help predict the severity of the disease and the ability of the immune system to eliminate intramammary infections caused by Staph. aureus.

Genome based phylogeny and virulence factor analysis of mastitis causing Escherichia coli isolated from Indian cattle

Mastitis is a highly infectious disease prevalent in dairy cattle and it is majorly caused by Escherichia coli (E. coli). The objective of present study is to investigate the occurrence of virulence genes, antimicrobial susceptibility and comparative analysis of E. coli (IVRI KOL CP4 and CM IVRI KOL-1) isolates from mastitis infected animal. Whole-genome sequencing (WGS) was performed using a PacBio RS II system and de novo assembled using Hierarchical Genome Assembly Process (HGAP3). Bacterial Pan Genome Analysis Pipeline (BPGA) was used for pangenome analysis. A set of 50 E. coli isolates were used for comparative analysis (48 collected from the database and 2 reference sequences). Core genes were further concatenated for phylogenetic analyses. In silico analysis was performed for antibiotic resistance and virulence gene identification. Both of the E. coli isolates carried many resistance genes including, b-lactamase, quinolones, rifampicin, macrolide, aminoglycoside and phenicols resistance. We detected 39 virulence genes in IVRI KOL CP4 and 52 in CM IVRI KOL-1 which include toxins, adhesions, invasins, secretion machineries or iron acquisition system. High prevalence of mastitis strains belongs to phylogroups A, although few isolates were also assigned to phylogenetic groups B1 and B2. In conclusion, the present study reported the presence of genes involved in Adherence, Iron acquisition, secretion system and toxins which shown to be crucial in MPEC pathogenicity. This is the first whole genome analysis of MPEC strains to be carried out in Indian isolate to highlights the spread of resistance and virulence genes in food animals.

Bile Salts Regulate Zinc Uptake and Capsule Synthesis in a Mastitis-Associated Extraintestinal Pathogenic Escherichia coli Strain.

Extraintestinal pathogenic Escherichia coli (ExPEC) strains are major causes of urinary and bloodstream infections. ExPEC reservoirs are not completely understood. Some mastitis-associated E. coli (MAEC) strains carry genes associated with ExPEC virulence, including metal scavenging, immune avoidance, and host attachment functions. In this study, we investigated the role of the high-affinity zinc uptake (znuABC) system in the MAEC strain M12. Elimination of znuABC moderately decreased fitness during mouse mammary gland infections. The ΔznuABC mutant strain exhibited an unexpected growth delay in the presence of bile salts, which was alleviated by the addition of excess zinc. We isolated suppressor mutants with improved growth in bile salts, several of which no longer produced the K96 capsule made by strain M12. The addition of bile salts also reduced capsule production by strain M12 and ExPEC strain CP9, suggesting that capsule synthesis may be detrimental when bile salts are present. To better understand the role of the capsule, we compared the virulence of mastitis strain M12 with that of its unencapsulated ΔkpsCS mutant in two models of ExPEC disease. The wild-type strain successfully colonized mouse bladders and kidneys and was highly virulent in intraperitoneal infections. Conversely, the ΔkpsCS mutant was unable to colonize kidneys and was unable to cause sepsis. These results demonstrate that some MAEC strains may be capable of causing human ExPEC illness. The virulence of strain M12 in these infections is dependent on its capsule. However, capsule may interfere with zinc homeostasis in the presence of bile salts while in the digestive tract.

Insight Into the Virulence Related Secretion Systems, Fimbriae, and Toxins in O2:K1 Escherichia coli Isolated From Bovine Mastitis.

Mastitis remains a major infection of dairy cows and an important issue for the dairy farmers, and Escherichia coli (E. coli) bovine mastitis is a disease of significant economic importance in the dairy industry. Our study identified six isolates belong to phylogroup B2 from 69 bovine mastitis E. coli strains. Except for one serotype O1 strain, all group B2 isolates were identified into serotype O2 and showed significantly higher mortality in the mouse infection than other phylogroups' strains. Genomic analyses and further tests were performed to examine the role of secretion systems, fimbriae, and toxins during the systemic infection of O2:K1 strain BCE049. Two integral T6SS loci and three predicted effectors clusters were found to assemble the functional T6SS complex and deliver diverse toxic effectors to modulate bacterial virulence in the mouse infection model. A total of four T4SS loci were harbored in the BCE049 genome, three of them are encoded in different plasmids, respectively, whereas the last one locates within the bacterial chromosome at FQU84_16715 to FQU84_16760, and was significantly involved in the bacterial pathogenicity. Numerous predicted pilus biosynthesis gene loci were found in the BCE049 genome, whereas most of them lost long fragments encoding key genes for the pili assembly. Unexpectedly, a type IV pilus gene locus locating at FQU84_01405 to FQU84_01335 in the plasmid 2, was found to be required for the full virulence of mastitis strain BCE049. It should be noted that a genetic neighborhood inserted with diverse genes is encoded by the plasmid 1, which harbors three prominent toxins including β-hemolysin, cytotoxic necrotizing factor 2 and cytolethal distending toxin type III. Consequent studies verified that these toxins significantly contributed to the bacterial pathogenicity. These findings provide a molecular blueprint for understanding the underlying mechanisms employed by the bovine mastitis E. coli to colonize in host and cause systemic infection.

Phenolic Plant Extracts Versus Penicillin G: In Vitro Susceptibility of Staphylococcus aureus Isolated from Bovine Mastitis.

Antibiotics are the elective drugs in bovine mastitis (BM) treatment, despite their low rates of efficiency and effectiveness and increasing risk of pathogen resistance. In this sense, it is urgent to discover new and effective antimicrobial agents to apply in BM control and even treatment. Plant extracts have been widely recognized as a rich source of phytochemicals with antimicrobial potential. Thus, the present work aims to compare the bioactivity of Eucalyptus globulus and Juglans regia extracts against Staphylococcus aureus bovine mastitis strains with penicillin G. At non-toxic concentrations, E. globulus exerted a bacteriostatic effect in planktonic cells and J. regia had no antimicrobial activity. Penicillin G, at minimum inhibitory concentration (MIC), demonstrated bactericidal activity, but just for S. aureus 3, 5, 6 and ATCC 25923, while the other strains seem to have acquired resistance. On the other hand, E. globulus and penicillin G in combination demonstrated synergy, being the most effective approach against S. aureus 1, 2 and 4. Thus, penicillin alone and in combination with E. globulus or J. regia seems to be promissory strategies to control bovine mastitis infections.

New Insight into Genotypic and Phenotypic Relatedness of Staphylococcus aureus Strains from Human Infections or Animal Reservoirs.

Staphylococcus aureus is a common human and livestock opportunistic pathogen, and there is evidence of animal to human transmission. This paper aimed to recognize properties of the isolates from collections of human and livestock S. aureus strains and to estimate compatibility of results based on phenotypic tests, microarrays and the spa typing methods. The second goal was to study differences between human and animal isolates in terms of specificity of their hosts and the strain transmission among various hosts. Most strains showed multi-susceptible profiles and produced enzymes on a high level, and they were phenotypically and genetically similar. However, in contrast to the Polish bovine mastitis strains, the Slovakian strains were multi-resistant. In this research, the strains showed significant differences in terms of their phenotypic manifestations and the presence of hemolysins genes; however, other enzyme-encoding genes correlated to a higher extent with the microarrays results. Interestingly, there was a lack of enterotoxin genes in human Poultry-like protein A+ strains in comparison to other human strains. Our study showed that differences between virulence profiles of the human and animal strains correlated with their origin rather than their hosts, and any trait allowed clearly distinguishing between them based on the microarray results.

Escherichia coli mastitis strains: In vitro phenotypes and severity of infection in vivo.

Mastitis remains a major infection of dairy cows and an important issue for dairy farmers and the dairy industry, in particular infections due to Escherichia coli strains. So far, properties specific to E. coli causing mastitis remain ill defined. In an attempt to better understand the properties required for E. coli to trigger mastitis, we used a range of in vitro assays to phenotypically characterize four E. coli strains, including the prototypical E. coli mastitis strain P4, possessing different relative abilities to cause mastitis in a mouse model. Our results indicate that a certain level of serum resistance might be required for colonization of the mammary gland. Resistance to neutrophil killing is also likely to contribute to a slower clearance of bacteria and higher chances to colonize the udder. In addition, we show that the four different strains do induce a pro-inflammatory response by mammary epithelial cells but with different intensities. Interestingly, the prototypical mastitis strain P4 actually induces the less intense response while it is responsible for the most severe infections in vivo. Altogether, our results suggest that different strategies can be used by E. coli strains to colonize the mammary gland and cause mastitis.

Characterization of a vraG Mutant in a Genetically Stable Staphylococcus aureus Small-Colony Variant and Preliminary Assessment for Use as a Live-Attenuated Vaccine against Intrammamary Infections.

Staphylococcus aureus is a leading cause of bovine intramammary infections (IMIs) that can evolve into difficult-to-treat chronic mastitis. To date, no vaccine formulation has shown high protective efficacy against S. aureus IMI, partly because this bacterium can efficiently evade the immune system. For instance, S. aureus small colony variants (SCVs) have intracellular abilities and can persist without producing invasive infections. As a first step towards the development of a live vaccine, this study describes the elaboration of a novel attenuated mutant of S. aureus taking advantage of the SCV phenotype. A genetically stable SCV was created through the deletion of the hemB gene, impairing its ability to adapt and revert to the invasive phenotype. Further attenuation was obtained through inactivation of gene vraG (SACOL0720) which we previously showed to be important for full virulence during bovine IMIs. After infection of bovine mammary epithelial cells (MAC-T), the double mutant (ΔvraGΔhemB) was less internalized and caused less cell destruction than that seen with ΔhemB and ΔvraG, respectively. In a murine IMI model, the ΔvraGΔhemB mutant was strongly attenuated, with a reduction of viable counts of up to 5-log10 CFU/g of mammary gland when compared to the parental strain. A complete clearance of ΔvraGΔhemB from glands was observed whereas mortality rapidly (48h) occurred with the wild-type strain. Immunization of mice using subcutaneous injections of live ΔvraGΔhemB raised a strong immune response as judged by the high total IgG titers measured against bacterial cell extracts and by the high IgG2a/IgG1 ratio observed against the IsdH protein. Also, ΔvraGΔhemB had sufficient common features with bovine mastitis strains so that the antibody response also strongly recognized strains from a variety of mastitis associated spa types. This double mutant could serve as a live-attenuated component in vaccines to improve cell-mediated immune responses against S. aureus IMIs.

In vitro evaluation of a novel bacteriophage cocktail as a preventative for bovine coliform mastitis

The objective of this study was to investigate the potential use of bacteriophage in preventing Escherichia coli mastitis on dairies. A cocktail consisting of 4 distinct bacteriophages was generated by screening against 36 E. coli isolates from dairy cows in Washington State with clinical mastitis. The bacteriophage significantly inhibited growth of 58% of the Washington State isolates and 54% of E. coli mastitis isolates from New York State, suggesting that the cocktail of phages had a relatively broad spectrum of action against relevant strains from 2 distinct geographies. The ability to suppress bacterial growth of these isolates in a liquid growth medium was not affected by the ratio of bacteriophage particles to bacterial cells (multiplicity of infection, MOI). For those E. coli that were completely inhibited by the phage cocktail, an MOI as low as 10had the same effect as 10µg/mL of ceftiofur on the growth rate of E. coli over a 12-h period using optical density measurements. A 3.3- to 5.6-log reduction of growth was achieved when E. coli was co-incubated with our phage cocktail in raw milk over a 12-h period at physiologic temperature. A modified gentamicin protection assay using bovine mammary epithelial cells provided a model to test whether bacteriophage could prevent cell attachment and invasion by chronic coliform mastitis strains. Pretreatment of cell cultures with the phage cocktail significantly reduced adhesion and intracellular survival of E. coli compared with controls. When combined with a bismuth-based teat sealant, the phage cocktail was able to inhibit bacterial growth when challenged with 1.6×103 cfu/mL of a clinical mastitis E. coli strain. In vitro results show bactericidal activity by our phage in raw milk and mammary tissue culture systems. Before a bacteriophage-based dry-cow treatment becomes a potential option for dairies, in vivo studies must be able to demonstrate that a specific dose of bacteriophage can protect cows from experimentally induced E. coli mastitis without inducing an inflammatory reaction.

Composição química e eficácia do óleo essencial e do extrato etanólico de Alpinia zerumbet sobre Staphylococcus aureus

RESUMO: O presente trabalho descreve a atividade antibacteriana do óleo essencial e do extrato etanólico das folhas de Alpinia zerumbet (colônia) sobre cepas de Staphylococcus aureus isoladas de vacas com mastite subclínica e cepas padrão ATCC 29213 e ATCC 25923, por meio do método de difusão em ágar. Foram utilizados 10 tratamentos contendo diferentes concentrações do óleo essencial e do extrato etanólico (100,0; 50,0; 25,0; 12,5 e 6,3 mg.mL-1) e o grupo controle (álcool etílico a 50% e Tween a 1%). Os constituintes majoritários do óleo essencial foram p -cimeno (32,72%), 1,8-cineol (24,05%) e 4-terpineol (20,23%), sendo esses determinados por cromatografia a gás acoplada a espectrometria de massas e cromatografia a gás com detector de ionização de chama (CG-EM/DIC). No extrato etanólico foi detectado o ácido elágico e três flavonoides: rutina, quercetina e campferol, por meio de cromatografia a líquido de alta eficiência acoplada a detector de arranjo de diodo (CLAE-DAD). Todas as cepas apresentaram sensibilidade aos tratamentos com óleo essencial e extrato etanólico. A melhor resposta foi obtida com o óleo essencial de A. zerumbet que, na concentração de 100 mg.mL-1 proporcionou inibição total do crescimento bacteriano. Esses resultados sugerem o potencial antibacteriano do óleo essencial e do extrato etanólico de A. zerumbet no controle da mastite bovina.

Clustering of Staphylococcus aureus bovine mastitis strains from regions of Central-Eastern Poland based on their biochemical and genetic characteristics.

Staphylococcus aureus strains were isolated from mastitic milk of cows with infected mammary glands. The animals were living in 12 different farms near Lublin, in Central-Eastern Poland. A biochemical identification method based on enzymatic assay was performed, followed by haemolytic and proteolytic tests. PCR-RFLP targeted on the gap gene allowed the genetic identification of strains at the species level and verified phenotypic identification results. A molecular typing method using triplex PCR was performed to recognize the genetic similarity of the analyzed strains. DNA microarray hybridization (StaphyType, Alere Technologies) was used for detection of antibiotic resistance and virulence associated markers. The results obtained indicate high genetic similarity in strains isolated from the same sites. High genetic similarities were also detected between strains isolated from cows from different farms of the same region. A slightly lower similarity was noted however, in strains from various regions indicating that the strains are herd specific and that the cow's infections caused by S. aureus were of a clonal character. In 21 representative isolates selected for DNA-microarray testing, only fosfomycin (fosB) and penicillin resistance markers (blaZ, blaI, blaR) were detected. The presence of genes coding for haemolysins (lukF, lukS, hlgA, hla, hld, hlb), proteases (aur, sspA, sspB, sspP), enterotoxins (entA, entD, entG, entI, entJ, entM, entN, entO, entR, entU, egc-cluster), adhesins (icaA, icaC, icaD, bbp, clfA, clfB, fib, fnbA, map, vwb) or immune evasion proteins (scn, chp, sak) was common and, with exceptions, matched triplex PCR-defined clusters.

Molecular characteristics of bap-positive Staphylococcus aureus strains from dairy cow mastitis.

The biofilm-associated protein (Bap) of Staphylococcus aureus is a high molecular weight cell-wall-anchored protein involved in biofilm formation, first described in bovine mastitis strains from Spain. So far, studies regarding Bap were mainly based on the Spanish strain V329 and its mutants, but no information on the genetic variability of bap-positive Staph. aureus strains is yet available in the literature. The present study investigated the molecular characteristics of 8 bap-positive Staph. aureus strains from subclinical bovine mastitis, isolated in 5 herds; somatic cell counts (SCC) of milk samples were also registered. Strains were characterised using MLST, SPA typing and microarray and the results were compared with V329. All isolates from this study and V329 were assigned to ST126, t605, but some molecular differences were observed. Only herd A and B strains harboured the genes for β-lactams resistance; the leukocidin D/E gene, a type I site-specific deoxyribonuclease subunit, 3rd locus gene and serin-protease A and B were carried by all strains, but not by V329, while serin-protease E was absent in V329 and in another isolate. Four isolates and V329 harboured the fibronectin-binding protein B gene. SCC showed the highest value in the milk sample affected by the only strain carrying all the virulence factors considered. Potential large variability of virulence was evidenced among V329 and all bap-positive Staph. aureus strains considered: the carriage of fnb could enhance the accumulation of biofilm, but the lack of lukD/E and splA, B or E might decrease the invasiveness of strain.

Reduction of teat skin mastitis pathogen loads: Differences between strains, dips, and contact times

The purpose of these experiments was to (1) assess differences in mastitis pathogen strain sensitivities to teat disinfectants (teat dips), and (2) determine the optimum time for premilking teat dips to remain in contact with teat skin to reduce pathogen loads on teat skin. Two experiments were conducted using the excised teat model. In experiment 1, the differences in mastitis pathogen strain sensitivities to 4 commercially available dips (dip A: 1% H2O2; dip B: 1% chlorine dioxide; dip C: 1% iodophor; and dip D: 0.5% iodophor) were evaluated. Four strains of 11 common mastitis pathogens (Staphylococcus aureus, Streptococcus agalactiae, Mycoplasma bovis, Streptococcus dysgalactiae, Streptococcus uberis, Escherichia coli, Staphylococcus chromogenes, Staphylococcus epidermidis, Staphylococcus hyicus, Staphylococcus xylosus, and Staphylococcus haemolyticus) were tested. In experiment 2, the percentage log reduction of mastitis pathogens (Escherichia coli, Streptococcus uberis, Streptococcus dysgalactiae, Klebsiella species, Staphylococcus chromogenes, Staphylococcus haemolyticus, Staphylococcus xylosus, and Staphylococcus epidermidis) on teat skin with 3 commercially available teat dips: dip A; dip D; and dip E: 0.25% iodophor, using dip contact times of 15, 30, and 45 s, was evaluated. Experiment 1 results indicated significant differences in strain sensitivities to dips within pathogen species: Staphylococcus aureus, Staphylococcus chromogenes, and Streptococcus uberis. Species differences were also found where Mycoplasma bovis (97.9% log reduction) was the most sensitive to tested teat dips and Staphylococcus haemolyticus (71.4% log reduction) the most resistant. Experiment 2 results indicated that contact times of 30 and 45 s were equally effective in reducing recovered bacteria for dips D and E and were also significantly more effective than a 15-s contact time. No differences were seen in recovered bacteria between tested contact times after treatment with dip A. It can be concluded that different mastitis pathogen species and strains within species may possess different sensitivities to teat dips, which may have implications in selection of teat dips on dairies. Furthermore, a 30-s premilking dip contact time for iodophors and 15 s for H2O2 dips may be optimal in reducing pathogen load in the shortest amount of time. A reduction in premilking teat dip contact time may improve milking parlor efficiency.

Investigation of biofilm production and icaA and icaD genes in Staphylococcus aureus isolated from heifers and cows with mastitis

Biofilm formation and antimicrobial resistance of Staphylococcus aureus are important virulence factors in cases of mastitis in dairy cows. However, few studies have investigated mastitis strains isolated from heifers. Within this context, the objective of the present study was to investigate biofilm formation on Congo red agar, the presence of the icaA and icaD genes by polymerase chain reaction (PCR), and the percentage of in vitro antimicrobial resistance of 110 S. aureus isolates from mammary gland secretions of heifers and cows with mastitis. PCR detected the icaA and icaD genes in 98% and 100% of isolates, respectively. However, only 55.5% of all isolates produced a biofilm on Congo red agar. Antimicrobial susceptibility testing revealed that 47.0% of isolates from heifers and 70.4% of isolates from cows were resistant to at least one of the antimicrobial agents tested. Resistance to penicillin and/or ampicillin was the most frequent (44.5%). These results indicate the need to implement prophylactic and control measures of mastitis for heifers. Heifers and cows can carry resistant strains with the capacity of biofilm production, a fact representing a threat to public health and animal well-being and generating losses to dairy farmers.

Comparative Exoprotein Profiling of Different Staphylococcus epidermidis Strains Reveals Potential Link between Nonclassical Protein Export and Virulence

Staphylococcus epidermidis (SE) includes commensal and pathogenic strains capable of infecting humans and animals. This study reports global exoproteome profiling of bovine mastitis strain PM221 and two human strains, commensal-type ATCC12228 and sepsis-associated RP62A. We identified 451, 395, and 518 proteins from culture supernatants of PM221, ATCC12228, and RP62A, respectively. Comparison of the identified exoproteomes revealed several strain-specific differences related to secreted antigens and adhesins, higher virulence capability for RP62A, and similarities between the PM221 and RP62A exoproteomes. The majority of the identified proteins (∼80%) were predicted to be cytoplasmic, including proteins known to be associated in membrane vesicles (MVs) in Staphylococcus aureus and immunogenic/adhesive moonlighting proteins. Enrichment of MV fractions from culture supernatants and analysis of their protein composition indicated that this nonclassical protein secretion pathway was being exploited under the conditions used and that there are strain-specific differences in nonclassical protein export. In addition, several predicted cell-surface proteins were identified in the culture media. In summary, the present study is the first in-depth exoproteome analysis of SE highlighting strain-specific factors able to contribute to virulence and adaptation.

New properties of wheat bran: anti‐biofilm activity and interference with bacteria quorum‐sensing systems

Some plant extracts, have been demonstrated to interfere with the microbial metabolism of several pathogenic bacteria. Within this antimicrobial properties it has been described the potential to inhibit or destroy biofilms or to interfere in quorum-sensing (QS) systems. However, to our knowledge, no study exploring this potential of wheat-bran (WB) has been published. The purpose of the present study is to evaluate the anti-biofilm activity of WB against a cow mastitis strain of Staphylococcus aureus and also its possible interference with bacterial QS systems. The potential of inhibition and destruction of the biofilm was studied by different in vitro assays. Also, we tested the ability of WB to interfere in bacterial QS by degrading acyl-homoserine lactones (AHL) as one of the most studied QS signal molecules for Gram-negative bacteria. The soluble extract of WB at 0.5% showed anti-biofilm activity, inhibiting biofilm formation and also destroying it. Similarly, the > 300 kDa fraction from WB had significant anti-biofilm activity in both in vitro assays. The WB also showed a potential to interfere with bacterial QS systems, as it was demonstrated to contain certain lactonase activity able to reduce AHL concentration in the medium. The present study reveals two additional beneficial properties of WB extract never explored before, which may be related to the presence of defence compounds in the plant extract able to interfere with microbial biofilms and also QS systems.

Prevalence of mastitis pathogens in milk from clinically healthy cows

A total of 669 individual cow milk samples originating from asymptomatic cows from 16 dairy farms were examined for the presence of microorganisms with the potential to cause mastitis. Coagulase-negative staphylococci clearly predominated (53.5% positive samples) followed by streptococci and enterococci (both occurring in 16.1% samples). Among streptococci, so-called mastitis streptococci (S. uberis, S. dysgalactiae and S. agalactiae) prevailed (11.7% positive samples). Enterobacteriaceae were found in 10.0% samples, most of which (6.6% samples) were positive for Escherichia coli. Yeasts (mainly Candida spp.) were found in 8.2% samples. One of the major mastitis pathogens, Staphylococcus aureus subsp. aureus, was isolated from 9.0% of samples. S. aureus isolates were further characterised in terms of their capability to form biofilm, antimicrobial susceptibility and clonality (PFGE). All S. aureus isolates were capable of biofilm formation and were generally susceptible to the majority of tested antibiotics. The exception was ampicillin, resistance to which was observed in 27.7% isolates. Therefore, the relatively frequent occurrence of S. aureus could be attributed to persistent intramammary infections due to biofilm formation rather than low efficacy of particular antibiotics. PFGE analysis revealed clonal spread of certain S. aureus isolates within and between farms indicating that certain lineages of S. aureus mastitis strains are particularly successful.

Genotyping and virulence factors assessment of bovine mastitis Escherichia coli

Escherichia coli is a major agent of bovine mastitis worldwide. However, specific E. coli virulence factors associated to pathogenicity during intra-mammary infections are yet unknown and this pathotype remains uncharacterized. The objectives of the present work were to assess the presence of a wide range of known virulence factors in a large set of E. coli strains isolated from bovine mastitis (mastitis set) and to study the genotypic distribution of strains in the mastitis set in comparison to a set of strains isolated from cows' environment in dairy farms (environmental set). Virulence factors were assessed by DNA hybridization microarray. The three most prevalent virulence factors found in the mastitis set were lpfA (long polar fimbriae), iss (increased serum resistance) and astA (enteroaggregative E. coli heat-stable enterotoxin 1). None, however, characterized the majority of these strains. Genotyping was assessed by ECOR phylogenetic grouping, multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). Strains in the mastitis and environmental sets were differentially distributed into ECOR phylogenetic groups; groups A and B1 being the most prevalent ones. Multiple MLST strain types were found in the two sets of strains, but only a few were common to both, and diversity was higher in the environmental set. A variety of PFGE patterns were found in the mastitis and environmental sets. Two clusters comprising mostly highly similar mastitis strains were identified. The results confirm that mastitis E. coli strains mostly lack known E. coli virulence factors. In addition, it is shown that the genotypic diversity of mastitis strains does not reflect the diversity found in the environmental E. coli population.