Survival of Streptococcus uberis on bedding substrates.

Streptococcus uberis (S. uberis) is a mastitis pathogen with an environmental reservoir. Management factors related to housing design and bedding are associated with the risk of S. uberis mastitis. This study aimed to investigate the ability of five distinct strains of S. uberis to survive and replicate on three common bedding materials (sand, wheat straw and kiln dried pine sawdust). Sterilized bedding substrates were inoculated with S. uberis and incubated at room temperature. Bacterial recovery from these media over time indicated that S. uberis numbers increased on used bedding materials, suggesting the addition of faeces and urine promoted replication. The bacterium was recovered for at least 35 days on straw and sand bedding, but could not be recovered beyond 7 days on clean or used sawdust. This study demonstrates the importance of bedding type and management on the environmental survival of S. uberis.

Assessment of the prevalence of Streptococcus uberis in dairy cow feces and implications for herd health.

Streptococcus uberis is a major causative agent of bovine mastitis worldwide, negatively affecting both milk production and animal welfare. Mammary infections result from environmental reservoirs, with cattle themselves required to propagate the infection cycle. Two longitudinal studies were performed to investigate the prevalence of Streptococcus uberis within feces and to evaluate factors which may affect gastrointestinal carriage. Bacterial detection was confirmed using a PCR-based method directed against sub0888 that detected S. uberis at an analytical sensitivity of 12 cfu/g of bovine feces. The first study sampled an entire herd at 8-wk intervals, over a 10-mo period and identified that maintenance of S. uberis within the dairy cow environment was due to a high proportion of animals shedding S. uberis and not due to a low number of "super-shedding" cows within the herd. Seasonality influenced detection rates, with detection levels significantly higher for housed cattle compared with those at pasture. Multilevel logistic regression was used to identify significant factors that affected S. uberis detection; these included parity, stage of lactation, and body condition score. An additional study involved screening a smaller cohort of cows housed over a 4-wk period and identified an increased probability of detection if cows were housed in loose straw yards, compared those in straw cubicles. This study highlighted several cow and management related factors that affect both detection of S. uberis and future infection risks.

PCR-Based Direct Detection of Streptococcus uberis from Subclinical and Clinical Dairy Cattle Milk Samples.

Streptococcus uberis is one of the leading causes worldwide of mastitis in the dairy industry, with the most likely sources of infection attributed to environmental reservoirs such as contaminated bedding materials. Early detection of those cases most likely to progress to clinical disease would lead to improved animal welfare, a critical component of overall health and productivity. A multiplex PCR-based diagnostic test was developed for detection of S. uberis directly from milk and targeting two genes previously identified as important for intramammary colonisation and persistence in dairy cattle. Results indicated the threshold for detection directly from milk was 20,000 CFU/ml and this was achieved without the need for preenrichment. In addition, S. uberis could be identified from milk samples collected during intramammary challenge studies, prior to clinical signs of infection and at much lower detection limits. The PCR test developed for confirmation of the presence of S. uberis directly from infected milk has potential value as a diagnostic test to identify early infection and/or to confirm that antibiotic therapy has been successful.

A Paradox in Bacterial Pathogenesis: Activation of the Local Macrophage Inflammasome Is Required for Virulence of Streptococcus uberis.

Streptococcus uberis is a common cause of intramammary infection and mastitis in dairy cattle. Unlike other mammary pathogens, S. uberis evades detection by mammary epithelial cells, and the host-pathogen interactions during early colonisation are poorly understood. Intramammary challenge of dairy cows with S. uberis (strain 0140 J) or isogenic mutants lacking the surface-anchored serine protease, SUB1154, demonstrated that virulence was dependent on the presence and correct location of this protein. Unlike the wild-type strain, the mutant lacking SUB1154 failed to elicit IL-1ß from ex vivo CD14+ cells obtained from milk (bovine mammary macrophages, BMM), but this response was reinstated by complementation with recombinant SUB1154; the protein in isolation elicited no response. Production of IL-1ß was ablated in the presence of various inhibitors, indicating dependency on internalisation and activation of NLRP3 and caspase-1, consistent with inflammasome activation. Similar transcriptomic changes were detected in ex vivo BMM in response to the wild-type or the SUB1154 deletion mutant, consistent with S. uberis priming BMM, enabling the SUB1154 protein to activate inflammasome maturation in a transcriptionally independent manner. These data can be reconciled in a novel model of pathogenesis in which, paradoxically, early colonisation is dependent on the innate response to the initial infection.

Complete Genome Sequence of Streptococcus agalactiae Strain 01173, Isolated from Kuwaiti Wild Fish.

Here, we report the complete genome of piscine Streptococcus agalactiae 01173 serotype Ia, which was generated using long-read sequencing technology. The bacteria were isolated from wild fish displaying signs of streptococcosis, from a fish kill incident in Kuwait.

Macrophage-Derived IL1ß and TNFα Regulate Arginine Metabolism in Neuroblastoma.

Neuroblastoma is the most common childhood solid tumor, yet the prognosis for high-risk disease remains poor. We demonstrate here that arginase 2 (ARG2) drives neuroblastoma cell proliferation via regulation of arginine metabolism. Targeting arginine metabolism, either by blocking cationic amino acid transporter 1 (CAT-1)-dependent arginine uptake in vitro or therapeutic depletion of arginine by pegylated recombinant arginase BCT-100, significantly delayed tumor development and prolonged murine survival. Tumor cells polarized infiltrating monocytes to an M1-macrophage phenotype, which released IL1ß and TNFα in a RAC-alpha serine/threonine-protein kinase (AKT)-dependent manner. IL1ß and TNFα established a feedback loop to upregulate ARG2 expression via p38 and extracellular regulated kinases 1/2 (ERK1/2) signaling in neuroblastoma and neural crest-derived cells. Proteomic analysis revealed that enrichment of IL1ß and TNFα in stage IV human tumor microenvironments was associated with a worse prognosis. These data thus describe an immune-metabolic regulatory loop between tumor cells and infiltrating myeloid cells regulating ARG2, which can be clinically exploited. SIGNIFICANCE: These findings illustrate that cross-talk between myeloid cells and tumor cells creates a metabolic regulatory loop that promotes neuroblastoma progression.

Bovine Neonatal Monocytes Display Phenotypic Differences Compared With Adults After Challenge With the Infectious Abortifacient Agent Neospora caninum.

The neonatal period represents a window of susceptibility for ruminants given the abundance of infectious challenges in their environment. Maternal transfer of immunity does not occur in utero but post-parturition, however this does not compensate for potential deficits in the cellular compartment. Here we present a cellular and transcriptomic study to investigate if there is an age-related difference in the monocyte response in cattle during intra-cellular protozoan infection. We utilized Neospora caninum, an obligate intracellular protozoan parasite that causes abortion and negative economic impacts in cattle worldwide, to study these responses. We found neonatal animals had a significant greater percentage of CD14+ monocytes with higher CD80 cell surface expression. Adult monocytes harbored more parasites compared to neonatal monocytes; additionally greater secretion of IL-1ß was observed in neonates. Microarray analysis revealed neonates have 535 genes significantly upregulated compared to adult with 23 upregulated genes. Biological pathways involved in immune response were evaluated and both age groups showed changes in the upregulation of tyrosine phosphorylation of STAT protein and JAK-STAT cascade pathways. However, the extent to which these pathways were upregulated in neonates was much greater. Our findings suggest that neonates are more resistant to cellular invasion with protozoan parasites and that the magnitude of the responses is related to significant changes in the JAK-STAT network.

PIMMS (Pragmatic Insertional Mutation Mapping System) Laboratory Methodology a Readily Accessible Tool for Identification of Essential Genes in Streptococcus.

The Pragmatic Insertional Mutation Mapping (PIMMS) laboratory protocol was developed alongside various bioinformatics packages (Blanchard et al., 2015) to enable detection of essential and conditionally essential genes in Streptococcus and related bacteria. This extended the methodology commonly used to locate insertional mutations in individual mutants to the analysis of mutations in populations of bacteria. In Streptococcus uberis, a pyogenic Streptococcus associated with intramammary infection and mastitis in ruminants, the mutagen pGhost9:ISS1 was shown to integrate across the entire genome. Analysis of >80,000 mutations revealed 196 coding sequences, which were not be mutated and a further 67 where mutation only occurred beyond the 90th percentile of the coding sequence. These sequences showed good concordance with sequences within the database of essential genes and typically matched sequences known to be associated with basic cellular functions. Due to the broad utility of this mutagen and the simplicity of the methodology it is anticipated that PIMMS will be of value to a wide range of laboratories in functional genomic analysis of a wide range of Gram positive bacteria (Streptococcus, Enterococcus, and Lactococcus) of medical, veterinary, and industrial significance.

Virulence related sequences; insights provided by comparative genomics of Streptococcus uberis of differing virulence.

BACKGROUND: Streptococcus uberis, a Gram-positive, catalase-negative member of the family Streptococcaceae is an important environmental pathogen responsible for a significant proportion of subclinical and clinical bovine intramammary infections. Currently, the genome of only a single reference strain (0140J) has been described. Here we present a comparative analysis of complete draft genome sequences of an additional twelve S. uberis strains. RESULTS: Pan and core genome analysis revealed the core genome common to all strains to be 1,550 genes in 1,509 orthologous clusters, complemented by 115-246 accessory genes present in one or more S. uberis strains but absent in the reference strain 0140J. Most of the previously predicted virulent genes were present in the core genome of all 13 strains but gene gain/loss was observed between the isolates in CDS associated with clustered regularly interspaced short palindromic repeats (CRISPRs), prophage and bacteriocin production. Experimental challenge experiments confirmed strain EF20 as non-virulent; only able to infect in a transient manner that did not result in clinical mastitis. Comparison of the genome sequence of EF20 with the validated virulent strain 0140J identified genes associated with virulence, however these did not relate clearly with clinical/non-clinical status of infection. CONCLUSION: The gain/loss of mobile genetic elements such as CRISPRs and prophage are a potential driving force for evolutionary change. This first "whole-genome" comparison of strains isolated from clinical vs non-clinical intramammary infections including the type virulent vs non-virulent strains did not identify simple gene gain/loss rules that readily explain, or be confidently associated with, differences in virulence. This suggests that a more complex dynamic determines infection potential and clinical outcome not simply gene content.

Transposon insertion mapping with PIMMS - Pragmatic Insertional Mutation Mapping System.

UNLABELLED: The PIMMS (Pragmatic Insertional Mutation Mapping System) pipeline has been developed for simple conditionally essential genome discovery experiments in bacteria. Capable of using raw sequence data files alongside a FASTA sequence of the reference genome and GFF file, PIMMS will generate a tabulated output of each coding sequence with corresponding mapped insertions accompanied with normalized results enabling streamlined analysis. This allows for a quick assay of the genome to identify conditionally essential genes on a standard desktop computer prioritizing results for further investigation. AVAILABILITY: The PIMMS script, manual and accompanying test data is freely available at https://github.com/ADAC-UoN/PIMMS.

Vru (Sub0144) controls expression of proven and putative virulence determinants and alters the ability of Streptococcus uberis to cause disease in dairy cattle.

The regulation and control of gene expression in response to differing environmental stimuli is crucial for successful pathogen adaptation and persistence. The regulatory gene vru of Streptococcus uberis encodes a stand-alone response regulator with similarity to the Mga of group A Streptococcus. Mga controls expression of a number of important virulence determinants. Experimental intramammary challenge of dairy cattle with a mutant of S. uberis carrying an inactivating lesion in vru showed reduced ability to colonize the mammary gland and an inability to induce clinical signs of mastitis compared with the wild-type strain. Analysis of transcriptional differences of gene expression in the mutant, determined by microarray analysis, identified a number of coding sequences with altered expression in the absence of Vru. These consisted of known and putative virulence determinants, including Lbp (Sub0145), SclB (Sub1095), PauA (Sub1785) and hasA (Sub1696).

Sortase anchored proteins of Streptococcus uberis play major roles in the pathogenesis of bovine mastitis in dairy cattle.

Streptococcus uberis, strain 0140J, contains a single copy sortase A (srtA), encoding a transamidase capable of covalently anchoring specific proteins to peptidoglycan. Unlike the wild-type, an isogenic mutant carrying an inactivating ISS1 insertion within srtA was only able to infect the bovine mammary gland in a transient fashion. For the first 24 h post challenge, the srtA mutant colonised at a similar rate and number to the wild type strain, but unlike the wild type did not subsequently colonise in higher numbers. Similar levels of host cell infiltration were detected in response to infection with both strains, but only in those mammary quarters infected with the wild type strain were clinical signs of disease evident. Mutants that failed to express individual sortase substrate proteins (sub0135, sub0145, sub0207, sub0241, sub0826, sub0888, sub1095, sub1154, sub1370, and sub1730) were isolated and their virulence determined in the same challenge model. This revealed that mutants lacking sub0145, sub1095 and sub1154 were attenuated in cattle. These data demonstrate that a number of sortase anchored proteins each play a distinct, non-redundant and important role in pathogenesis of S. uberis infection within the lactating bovine mammary gland.

Identification of sortase A (SrtA) substrates in Streptococcus uberis: evidence for an additional hexapeptide (LPXXXD) sorting motif.

Sortase (a transamidase) has been shown to be responsible for the covalent attachment of proteins to the bacterial cell wall. Anchoring is effected on secreted proteins containing a specific cell wall motif toward their C-terminus; that for sortase A (SrtA) in Gram-positive bacteria often incorporates the sequence LPXTG. Such surface proteins are often characterized as virulence determinants and play important roles during the establishment and persistence of infection. Intramammary infection with Streptococcus uberis is a common cause of bovine mastitis, which impacts on animal health and welfare and the economics of milk production. Comparison of stringently produced cell wall fractions from S. uberis and an isogenic mutant strain lacking SrtA permitted identification of 9 proteins likely to be covalently anchored at the cell surface. Analysis of these sequences implied the presence of two anchoring motifs for S. uberis, the classical LPXTG motif and an additional LPXXXD motif.

Evidence for niche adaptation in the genome of the bovine pathogen Streptococcus uberis.

BACKGROUND: Streptococcus uberis, a Gram positive bacterial pathogen responsible for a significant proportion of bovine mastitis in commercial dairy herds, colonises multiple body sites of the cow including the gut, genital tract and mammary gland. Comparative analysis of the complete genome sequence of S. uberis strain 0140J was undertaken to help elucidate the biology of this effective bovine pathogen. RESULTS: The genome revealed 1,825 predicted coding sequences (CDSs) of which 62 were identified as pseudogenes or gene fragments. Comparisons with related pyogenic streptococci identified a conserved core (40%) of orthologous CDSs. Intriguingly, S. uberis 0140J displayed a lower number of mobile genetic elements when compared with other pyogenic streptococci, however bacteriophage-derived islands and a putative genomic island were identified. Comparative genomics analysis revealed most similarity to the genomes of Streptococcus agalactiae and Streptococcus equi subsp. zooepidemicus. In contrast, streptococcal orthologs were not identified for 11% of the CDSs, indicating either unique retention of ancestral sequence, or acquisition of sequence from alternative sources. Functions including transport, catabolism, regulation and CDSs encoding cell envelope proteins were over-represented in this unique gene set; a limited array of putative virulence CDSs were identified. CONCLUSION: S. uberis utilises nutritional flexibility derived from a diversity of metabolic options to successfully occupy a discrete ecological niche. The features observed in S. uberis are strongly suggestive of an opportunistic pathogen adapted to challenging and changing environmental parameters.