Staphylococcus aureus persistence properties associated with bovine mastitis and alternative therapeutic modalities

Staphylococcus aureus is an important agent of contagious bovine intramammary infections in dairy cattle. Its ability to persist inside the udder is based on the presence of important mechanisms such as its ability to form biofilms, polysaccharide capsules small colony variants, and their ability to invade professional and nonprofessional cells, which will protect S. aureus from the innate and adaptive immune response of the cow, and from antibiotics that are no longer considered to be sufficient against S. aureus bovine mastitis. In this review, we present the recent research outlining S. aureus persistence properties inside the mammary gland, including its regulation mechanisms, and we highlight alternative therapeutic strategies that were tested against S. aureus isolated from bovine mastitis such as the use of probiotic bacteria, bacteriocins and bacteriophages. Overall, the persistence of S. aureus inside the mammary gland remains a pressing veterinary problem. A thorough understanding of staphylococcal persistence mechanisms will elucidate novel ways that can help in the identification of novel treatments.     

Expression of Lysostaphin in Milk of Transgenic Mice Affects the Growth of Neonates

As an initial step towards enhancing mastitis resistance in dairy animals, we generated BLG-Lys transgenic mice that secrete lysostaphin, a potent antistaphylococcal protein, in their milk. In the current study, we continue our assessment of lysostaphin as a suitable antimicrobial protein for mastitis resistance and have investigated mammary gland development and function in three lines of transgenic mice. As the lines were propagated, there was a tendency for fewer BLG-Lys litters to survive to weaning (51% as compared to 90% for nontransgenic lines, p = 0.080). Nontransgenic pups fostered on dams from these three lines exhibited diminished growth rates during the first week of lactation. Rates of gain became comparable to pups on nontransgenic dams at later time points. Initial slow growth also resulted in decreased weaning weights for pups nursed by transgenic dams (15.35±0.27 g) when compared to pups delivered and nursed by nontransgenic dams (18.61 ± 0.61 g; p < 0.001), but the effect was temporary, as similar weights were attained by adulthood. Milk yield at peak lactation was not different between BLG-Lys (0.79 ± 0.33 g) and nontransgenic (0.91 ± 0.38 g; p = 0.166) dams. Histological examination of the transgenic mammary glands during gestation revealed no differences when compared to control glands; however, at early lactational stages, the BLG-Lys glands exhibited less alveolar area than control glands and a delay in lobulo-alveolar maturation. The results clearly demonstrate reduced growth of neonates on BLG-Lys dams; whether the poor pup performance can be attributed to delayed mammary development or the gland development merely reflects reduced suckling stimuli from the pups remains to be determined.Authors Abhijit Mitra and Kathleen S. Hruska contributed equally to this work     

Lysostaphin expression in mammary glands confers protection against staphylococcal infection in transgenic mice

Infection of the mammary gland, in addition to causing animal distress, is a major economic burden of the dairy industry. Staphylococcus aureus is the major contagious mastitis pathogen, accounting for approximately 15-30% of infections, and has proved difficult to control using standard management practices. As a first step toward enhancing mastitis resistance of dairy animals, we report the generation of transgenic mice that secrete a potent anti-staphylococcal protein into milk. The protein, lysostaphin, is a peptidoglycan hydrolase normally produced by Staphylococcus simulans. When the native form is secreted by transfected eukaryotic cells it becomes glycosylated and inactive. However, removal of two glycosylation motifs through engineering asparagine to glutamine codon substitutions enables secretion of Gln(125,232)-lysostaphin, a bioactive variant. Three lines of transgenic mice, in which the 5'-flanking region of the ovine beta-lactoglobulin gene directed the secretion of Gln(125,232)-lysostaphin into milk, exhibit substantial resistance to an intramammary challenge of 104 colony-forming units (c.f.u.) of S. aureus, with the highest expressing line being completely resistant. Milk protein content and profiles of transgenic and nontransgenic mice are similar. These results clearly demonstrate the potential of genetic engineering to combat the most prevalent disease of dairy cattle.     

Site-Specific N-Glycosylation of Caprine Lysostaphin Restricts its Bacteriolytic Activity Toward Staphylococcus Aureus

Lysostaphin (LYS) is an anti-staphylococcal prokaryotic polypeptide that has been used to avoid Staphylococcus aureus mastitis through transgenic or viral vector approaches exogenously expressed in dairy animals. However, glycosylation of lysostaphin expressed in mammalian cells results in a loss of bioactivity. Until now, the mechanism of site-specific glycosylation of lysostaphin causing this loss of bioactivity remains unknown. An immortalized caprine mammary epithelial cell line (CMEC-08-D) was used to study recombinant lysostaphin fused with goat β-casein, goat lactoferrin (LF) or prokaryotic signal peptides. These constructs were separately ectopically expressed in CMEC-08-D. Results of site-directed mutagenesis show that Asn¹²⁵ but not Asn²³² is the exact glycosylation site of lysostaphin expressed in CMEC-08-D. In addition, the effect of glycosylation of lysostaphin on its staphylolytic activity was identified through bacterial plate assay. The data indicated that wild type and mutated N232Q-lysostaphin (Asn²³² to Gln²³² substitution) lacked staphylolytic activity. In contrast, mutated N125Q (Asn¹²⁵ to Gln¹²⁵ substitution) and N125Q/N232Q-lysostaphin possessed staphylolytic activity. On the other hand, all mutated lysostaphin showed no change in binding ability to S. aureus. This reveals that N-glycosylation at Asn¹²⁵ of lysostaphin expressed in a eukaryotic system greatly decreases lysostaphin bacteriolytic activity but does not affect its binding ability to S. aureus.     

Adhesion of Staphylococcus aureus to Bovine Mammary Epithelial Cells Induced by Growth in Milk Whey

Staphylococcus aureus strains isolated from bovine intramammary infection (mastitis) were tested for adhesion to bovine mammary epithelial cells after growth in milk whey or TSB. Bacteria grown in milk whey adhered more efficiently to mammary gland epithelial cells in vitro than the corresponding homologous bacteria grown in TSB. Trypsin treatment of milk whey-grown S. aureus had no effect on their adherence. Whereas, pretreatment with periodate significantly decreased bacterial adherence capacity. Periodate treatment of TSB-grown bacteria had no effect on adhesion to the mammary gland epithelial cells.     

Genetically enhanced cows resist intramammary Staphylococcus aureus infection

Mastitis, the most consequential disease in dairy cattle, costs the US dairy industry billions of dollars annually. To test the feasibility of protecting animals through genetic engineering, transgenic cows secreting lysostaphin at concentrations ranging from 0.9 to 14 micrograms/ml [corrected] in their milk were produced. In vitro assays demonstrated the milk's ability to kill Staphylococcus aureus. Intramammary infusions of S. aureus were administered to three transgenic and ten nontransgenic cows. Increases in milk somatic cells, elevated body temperatures and induced acute phase proteins, each indicative of infection, were observed in all of the nontransgenic cows but in none of the transgenic animals. Protection against S. aureus mastitis appears to be achievable with as little as 3 micrograms/ml [corrected] of lysostaphin in milk. Our results indicate that genetic engineering can provide a viable tool for enhancing resistance to disease and improve the well-being of livestock.     

Peptidoglycan Hydrolase Fusions Maintain Their Parental Specificities

The increased incidence of bacterial antibiotic resistance has led to a renewed search for novel antimicrobials. Avoiding the use of broad-range antimicrobials through the use of specific peptidoglycan hydrolases (endolysins) might reduce the incidence of antibiotic-resistant pathogens worldwide. Staphylococcus aureus and Streptococcus agalactiae are human pathogens and also cause mastitis in dairy cattle. The ultimate goal of this work is to create transgenic cattle that are resistant to mastitis through the expression of an antimicrobial protein(s) in their milk. Toward this end, two novel antimicrobials were produced. The (i) full-length and (ii) 182-amino-acid, C-terminally truncated S. agalactiae bacteriophage B30 endolysins were fused to the mature lysostaphin protein of Staphylococcus simulans. Both fusions display lytic specificity for streptococcal pathogens and S. aureus. The full lytic ability of the truncated B30 protein also suggests that the SH3b domain at the C terminus is dispensable. The fusions are active in a milk-like environment. They are also active against some lactic acid bacteria used to make cheese and yogurt, but their lytic activity is destroyed by pasteurization (63°C for 30 min). Immunohistochemical studies indicated that the fusion proteins can be expressed in cultured mammalian cells with no obvious deleterious effects on the cells, making it a strong candidate for use in future transgenic mice and cattle. Since the fusion peptidoglycan hydrolase also kills multiple human pathogens, it also may prove useful as a highly selective, multipathogen-targeting antimicrobial agent that could potentially reduce the use of broad-range antibiotics in fighting clinical infections.     

Milk microbiome signatures of subclinical mastitis-affected cattle analysed by shotgun sequencing

Aims: Metagenomic analysis of milk samples collected from Kankrej, Gir (Bos indicus) and crossbred (Bos taurus × B. indicus) cattle harbouring subclinical mastitis was carried out by next‐generation sequencing 454 GS‐FLX technology to elucidate the microbial community structure of cattle milk. Methods and Results: Milk samples from Kankrej, Gir and crossbred cattle were subjected to metagenomic profiling by pyrosequencing. The Metagenomic analysis produced 63·07, 11·09 and 7·87 million base pairs (Mb) of sequence data, assembled in 264 798, 56 114 and 36 762 sequences with an average read length of 238, 197 and 214 nucleotides in Kankrej, Gir and crossbred cattle, respectively. Phylogenetic and metabolic profiles by the web‐based tool MG‐RAST revealed that the members of Enterobacteriales were predominant in mastitic milk followed by Pseudomonadales, Bacillales and Lactobacillales. Around 56 different species with varying abundance were detected in the subclinically infected milk. Escherichia coli was found to be the most predominant species in Kankrej and Gir cattle followed by Pseudomonas aeruginosa, Pseudomonas mendocina, Shigella flexneri and Bacillus cereus. In crossbred cattle, Staphylococcus aureus followed by Klebsiella pneumoniae, Staphylococcus epidermidis and E. coli were detected in descending order. Metabolic profiling indicated fluoroquinolones, methicillin, copper, cobalt–zinc–cadmium as the groups of antibiotics and toxic compounds to which the organisms showed resistance. Sequences indicating potential of organisms exhibiting multidrug resistance against antibiotics and resistance to toxic compounds were also present. Interestingly, presence of bacteriophages against Staph. aureus, E. coli, Enterobacter and Yersinia species was also observed. Conclusions: The analysis identified potential infectious organisms in mastitis, resistance of organisms to antibiotics and chemical compounds and the natural resistance potential of dairy cows. Significance and Impact of the Study: The findings of this study may help in formulating strategies for the prevention and treatment of mastitis in dairy animals and consequently in reducing economic losses incurred because of it.     

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.     

Antimicrobial resistance and toxin gene profiles of Staphylococcus aureus strains from Holstein milk

Isolation of Staphylococcus aureus (Staph. aureus) from Holstein milk samples with mastitis and nonmastitis was conducted to estimate its prevalence, antimicrobial resistance and toxin genes. A total of 353 milk samples were collected from three Chinese Holstein herds. Fifty‐three Staph. aureus isolates collected from 29 Staph. aureus‐positive samples were characterized via antimicrobial susceptibility, toxin genes and Pulsed‐field Gel Electrophoresis (PFGE) profiles. The prevalence of Staph. aureus was 4·0–9·5% in mastitic and 7·3–11·5% in nonmastitic samples in the analysed herds. Approximately 61·0% of Staph. aureus strains isolated from mastitis cows were resistant to ≥10 antimicrobials compared with 0% of isolates with nonmastitis. The most frequently observed super antigenic toxin gene was pvl (41·5%) followed by seh + pvl (13·2%). We did not find mecA‐positive methicillin‐resistant Staph. aureus (MRSA) strains, while mecA‐negative MRSA strains were identified in the three herds. PFGE results suggested potential transmission of Staph. aureus strains in different farms. These results open new insights into Staph. aureus transmission and antimicrobial resistance of Holstein dairy cows and into developing strategies for udder health improvement of dairy cattle.     

Identification of splice variants,expression analysis and single nucleotide polymorphisms of the PRMT2 gene in dairy cattle

Protein arginine N-methyltransferase 2 (PRMT2), also named HRMT1L1, belongs to the Bovine Protein arginine N-methyltransferase (PRMT) genes which are involved in the immune response. To explore the variability of the PRMT2 gene and resistance to mastitis in cows, splice variant (SV), and single nucleotide polymorphisms (SNPs) were identified in this study. A SV (PRMT2-SV) lacking exon 7 (98-bp) of the PRMT2 gene was found in healthy and mastitis-infected mammary gland tissues. Two of four SNPs were significantly associated with bovine milk yield and protein content. Further, we estimated the relative expression of PRMT2-SV in the mammary gland tissue of dairy cattle by using quantitative real-time polymerase chain reaction. The result showed that expression of the PRMT2-SV mRNA was significantly upregulated 4.02-fold (p < 0.05) in infected mammary tissues (n = 5) compared to healthy tissues (n = 5). Our findings reveal that PRMT2-SV may play an important role in mastitis resistance in dairy cattle. The SNPs may be used as a possible candidate SNPs for marker-assisted selection and management in Chinese Holstein cattle.     

Activation of nuclear factor kappa B in mammary epithelium promotes milk loss during mammary development and infection

We investigated whether nuclear factor kappa B (NF‐κB), which exhibits a regulated pattern of activity during murine mammary gland development, plays an important role during lactation and involution, when milk production ceases and the gland undergoes apoptosis and re‐modeling. We generated a doxycycline inducible transgenic mouse model to activate NF‐κB specifically in the mammary epithelium through expression of a constitutively active form of IKK2, the upstream kinase in the classical NF‐κB signaling cascade. We found that activation of NF‐κB during involution resulted in a more rapid reduction in milk levels and increased cleavage of caspase‐3, an indicator of apoptosis. We also found that activation of NF‐κB during lactation with no additional involution signals had a similar effect. The observation that NF‐κB is a key regulator of milk production led us to investigate the role of NF‐κB during mastitis, an infection of the mammary gland in which milk loss is observed. Mammary gland injection of E. coli LPS resulted in activation of NF‐κB and milk loss during lactation. This milk loss was decreased by selective inhibition of NF‐κB in mammary epithelium. Together, our data reveal that activation of NF‐κB leads to milk clearance in the lactating mammary gland. Therefore, targeting of NF‐κB signaling may prove therapeutic during mastitis in humans and could be beneficial for the dairy industry, where such infections have a major economic impact. J. Cell. Physiol. 222:73–81, 2010. © 2009 Wiley‐Liss, Inc.     

Lactic Acid Bacteria Isolated from Bovine Mammary Microbiota: Potential Allies against Bovine Mastitis

Bovine mastitis is a costly disease in dairy cattle worldwide. As of yet, the control of bovine mastitis is mostly based on prevention by thorough hygienic procedures during milking. Additional strategies include vaccination and utilization of antibiotics. Despite these measures, mastitis is not fully under control, thus prompting the need for alternative strategies. The goal of this study was to isolate autochthonous lactic acid bacteria (LAB) from bovine mammary microbiota that exhibit beneficial properties that could be used for mastitis prevention and/or treatment. Sampling of the teat canal led to the isolation of 165 isolates, among which a selection of ten non-redundant LAB strains belonging to the genera Lactobacillus and Lactococcus were further characterized with regard to several properties: surface properties (hydrophobicity, autoaggregation); inhibition potential of three main mastitis pathogens, Staphylococcus aureus, Escherichia coli and Streptococcus uberis; colonization capacities of bovine mammary epithelial cells (bMEC); and immunomodulation properties. Three strains, Lactobacillus brevis 1595 and 1597 and Lactobacillus plantarum 1610, showed high colonization capacities and a medium surface hydrophobicity. These strains are good candidates to compete with pathogens for mammary gland colonization. Moreover, nine strains exhibited anti-inflammatory properties, as illustrated by the lower IL-8 secretion by E. coli-stimulated bMEC in the presence of these LAB. Full genome sequencing of five candidate strains allowed to check for undesirable genetic elements such as antibiotic resistance genes and to identify potential bacterial determinants involved in the beneficial properties. This large screening of beneficial properties while checking for undesirable genetic markers allowed the selection of promising candidate LAB strains from bovine mammary microbiota for the prevention and/or treatment of bovine mastitis.     

Therapeutic applications of lysostaphin against Staphylococcus aureus

Staphylococcus aureus, an opportunistic pathogen, causes diverse community and nosocomial-acquired human infections, including folliculitis, impetigo, sepsis, septic arthritis, endocarditis, osteomyelitis, implant-associated biofilm infections and contagious mastitis in cattle. In recent days, both methicillin-sensitive and methicillin-resistant S. aureus infections have increased. Highly effective anti-staphylococcal agents are urgently required. Lysostaphin is a 27 kDa zinc metallo antimicrobial lytic enzyme that is produced by Staphylococcus simulans biovar staphylolyticus and was first discovered in the 1960s. Lysostaphin is highly active against S. aureus strains irrespective of their drug-resistant patterns with a minimum inhibitory concentration of ranges between 0·001 and 0·064 μg ml⁻¹. Lysostaphin has activity against both dividing and non-dividing S. aureus cells; and can seep through the extracellular matrix to kill the biofilm embedded S. aureus. In spite of having excellent anti-staphylococcal activity, its clinical application is hindered because of its immunogenicity and reduced bio-availability. Extensive research with lysostaphin lead to the development of several engineered lysostaphin derivatives with reduced immunogenicity and increased serum half-life. Therapeutic efficacy of both native and engineered lysostaphin derivatives was studied by several research groups. This review provides an overview of the therapeutic applications of native and engineered lysostaphin derivatives developed to eradicate S. aureus infections.     

Database of cattle candidate genes and genetic markers for milk production and mastitis

A cattle database of candidate genes and genetic markers for milk production and mastitis has been developed to provide an integrated research tool incorporating different types of information supporting a genomic approach to study lactation, udder development and health. The database contains 943 genes and genetic markers involved in mammary gland development and function, representing candidates for further functional studies. The candidate loci were drawn on a genetic map to reveal positional overlaps. For identification of candidate loci, data from seven different research approaches were exploited: (i) gene knockouts or transgenes in mice that result in specific phenotypes associated with mammary gland (143 loci); (ii) cattle QTL for milk production (344) and mastitis related traits (71); (iii) loci with sequence variations that show specific allele-phenotype interactions associated with milk production (24) or mastitis (10) in cattle; (iv) genes with expression profiles associated with milk production (207) or mastitis (107) in cattle or mouse; (v) cattle milk protein genes that exist in different genetic variants (9); (vi) miRNAs expressed in bovine mammary gland (32) and (vii) epigenetically regulated cattle genes associated with mammary gland function (1). Fourty-four genes found by multiple independent analyses were suggested as the most promising candidates and were further in silico analysed for expression levels in lactating mammary gland, genetic variability and top biological functions in functional networks. A miRNA target search for mammary gland expressed miRNAs identified 359 putative binding sites in 3'UTRs of candidate genes.     

<Emphasis Type="Italic">BRCA1</Emphasis>: a new candidate gene for bovine mastitis and its association analysis between single nucleotide polymorphisms and milk somatic cell score

Bovine mastitis is a very complex and common disease of dairy cattle and a major source of economic losses to the dairy industry worldwide. In this study, the bovine breast cancer 1, early onset gene (BRCA1) was taken as a candidate gene for mastitis resistance. The main object of this study was to investigate whether the BRCA1 gene was associated with mastitis in cattle. Through DNA sequencing, Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) and Created Restriction Site PCR (CRS-PCR) methods, three SNPs (G22231T, T25025A, and C28300A) were detected and twenty-four combinations of these SNPs were observed. The single SNP and their genetic effects on somatic cell score (SCS) were evaluated and a significant association with SCS was found in C28300A. The mean of genotype EE was significantly lower than those of genotypes EF and FF. The results of combined genotypes analysis of three SNPs showed that BBDDFF genotype with the highest SCS were easily for the mastitis susceptibility, whereas AACCEE genotype with the lowest SCS were favorable for the mastitis resistance. The information provided in the present study will be very useful for improving mastitis resistance in dairy cattle by marker-assisted selection.     

Investigation of the lytic ability of South African bacteriophages specific for Staphylococcus aureus,associated with bovine mastitis

Bovine mastitis is an infectious disease of the mammary glands of dairy cattle primarily causaled by the bacterium, Staphylococcus aureus subsp. aureus Rosenbach1884. Traditional control of this organism was through the use of antibiotics. However, S. aureus is developing resistance towards these chemotherapeutic agents faster than they are being developed. Bacteriophages can serve as an alternative control measure for the disease. This study investigated the prevalence of phages and S. aureus within the South African dairy environment, as well as infectivity of phage isolates against antibiotic-resistant S. aureus. The four S. aureus strains used in the study displayed resistance to representative antibiotics from both the β-lactamases and non-β-lactamases, macrolides, aminoglycosides and glycopeptides. Susceptibility was only noted towards the tetracycline antibiotics. Twenty-eight phages were isolated and screened against four strains of S. aureus. Only six phages showed biocontrol potential based on their wide host range, high titres and common growth requirements. Morphological and preliminary genomic analysis was carried out on the three best performing phages. At an optimal titre of between 6.2 × 10⁷ and 2.9 × 10⁸ pfu.ml^?1, the phages were able to reduce live bacterial cell counts between 64% and 95%. In addition, these six phages showed further infectivity towards S. aureus strains that were isolated from different milk-producing regions during a farm survey. The phages isolated in this study show reasonable potential for in vivo applications.