Genomic Analysis of Staphylococcus aureus Isolates Associated With Peracute Non-gangrenous or Gangrenous Mastitis and Comparison With Other Mastitis-Associated Staphylococcus aureus Isolates

Staphylococcus aureus is a highly prevalent cause of mastitis in dairy herds worldwide, capable of causing outcomes that vary from subclinical to peracute gangrenous mastitis. We performed a comparative genomic analysis between 14 isolates of S. aureus, originating from peracute bovine mastitis with very severe signs (9 gangrenous, 5 non-gangrenous) and six isolates originating from subclinical or clinical mastitis with mild to moderate signs, to find differences that could be associated with the clinical outcome of mastitis. Of the 296 virulence factors studied, 219 were detected in all isolates. No difference in the presence of virulence genes was detected between the peracute and control groups. None of the virulence factors were significantly associated with only a single study group. Most of the variation in virulence gene profiles existed between the clonal complexes. Our isolates belonged to five clonal complexes (CC97, CC133, CC151, CC479, and CC522), of which CC522 has previously been detected only in isolates originating from caprine and ovine mastitis, but not from bovine mastitis. For statistical analysis, we sorted the CCs into two groups. The group of CCs including CC133, CC479, and CC522 was associated with gangrenous mastitis, in contrast to the group of CCs including CC97 and CC151. The presence of virulence genes does not explain the clinical outcome of mastitis, but may be affected by allelic variation, and especially different regulation and thus expression in the virulence genes.Peer reviewe

Identification and characterization of domains responsible for self-assembly and cell wall binding of the surface layer protein of Lactobacillus brevis ATCC 8287

Background: Lactobacillus brevis ATCC 8287 is covered by a regular surface (S-) layer consisting of a 435 amino acid protein SlpA. This protein is completely unrelated in sequence to the previously characterized S-layer proteins of Lactobacillus acidophilus group. Results: In this work, the self-assembly and cell wall binding domains of SlpA were characterized. The C-terminal self-assembly domain encompassed residues 179435 of mature SlpA, as demonstrated by the ability of N-terminally truncated recombinant SlpA to form a periodic structure indistinguishable from that formed by full length SlpA. Furthermore, a trypsin degradation analysis indicated the existence of a protease resistant C-terminal domain of 214 amino acids. By producing a set of C-terminally truncated recombinant SlpA (rSlpA) proteins the cell wall binding region was mapped to the N-terminal part of SlpA, where the first 145 amino acids of mature SlpA alone were sufficient for binding to isolated cell wall fragments of L. brevis ATCC 8287. The binding of full length rSlpA to the cell walls was not affected by the treatment of the walls with 5% trichloroacetic acid (TCA), indicating that cell wall structures other than teichoic acids are involved, a feature not shared by the Lactobacillus acidophilus group S-layer proteins characterized so far. Conserved carbohydrate binding motifs were identified in the positively charged N-terminal regions of six Lactobacillus brevis S-layer proteins. Conclusion: This study identifies SlpA as a two-domain protein in which the order of the functional domains is reversed compared to other characterized Lactobacillus S-layer proteins, and emphasizes the diversity of potential cell wall receptors despite similar carbohydrate binding sequence motifs in Lactobacillus S-layer proteins.(VLID)90437

A comparative characterization of different host-sourced Lactobacillus ruminis strains and their adhesive, inhibitory, and immunomodulating functions

Lactobacillus ruminis, an autochthonous member of the gastrointestinal microbiota of humans and many animals, is a less characterized but interesting species for many reasons, including its intestinal prevalence and possible positive roles in host-microbe crosstalk. In this study, we isolated a novel L. ruminis strain (GRL 1172) from porcine feces and analyzed its functional characteristics and niche adaptation factors in parallel with those of three other L. ruminis strains (a human isolate, ATCC 25644, and two bovine isolates, ATCC 27780 and ATCC 27781). All the strains adhered to fibronectin, type I collagen, and human colorectal adenocarcinoma cells (HT-29), but poorly to type IV collagen, porcine intestinal epithelial cells (IPEC-1), and human colon adenocarcinoma cells (Caco-2). In competition assays, all the strains were able to inhibit the adhesion of Yersinia enterocolitica and enterotoxigenic Escherichia coli (ETEC, F4(+)) to fibronectin, type I; collagen, IPEC-1, and Caco-2 cells, and the inhibition rates tended to be higher than in exclusion assays. The culture supernatants of the tested strains inhibited the growth of six selected pathogens to varying extents. The inhibition was solely based on the low pH resulting from acid production during growth. All four L. ruminis strains supported the barrier function maintenance of Caco-2 cells, as shown by the modest increase in trans-epithelial electrical resistance and the prevention of dextran diffusion during co-incubation. However, the strains could not prevent the barrier damage caused by ETEC in the Caco-2 cell model. All the tested strains and their culture supernatants were able to provoke Toll-like receptor (TLR) 2-mediated NF-kappa B activation and IL-8 production in vitro to varying degrees. The induction of TLR5 signaling revealed that flagella were expressed by all the tested strains, but to different extents. Flagella and pili were observed by electron microscopy on the newly isolated strain GRL 1172.Peer reviewe

Genome Sequences of Four Staphylococcus aureus Strains Isolated from Bovine Mastitis

Peer reviewe

Comparative genome analysis of 24 bovine-associated Staphylococcus isolates with special focus on the putative virulence genes

Non-aureus staphylococci (NAS) are most commonly isolated from subclinical mastitis. Different NAS species may, however, have diverse effects on the inflammatory response in the udder. We determined the genome sequences of 20 staphylococcal isolates from clinical or subclinical bovine mastitis, belonging to the NAS species Staphylococcus agnetis S. chromogenes, and S. simulans, and focused on the putative virulence factor genes present in the genomes. For comparison we used our previously published genome sequences of four S. aureus isolates from bovine mastitis. The pan-genome and core genomes of the non-aureus isolates were characterized. After that, putative virulence factor orthologues were searched in silico. We compared the presence of putative virulence factors in the NAS species and S. aureus and evaluated the potential association between bacterial genotype and type of mastitis (clinical vs. subclinical). The NAS isolates had much less virulence gene orthologues than the S. aureus isolates. One third of the virulence genes were detected only in S. aureus. About 100 virulence genes were present in all S. aureus isolates, compared to about 40 to 50 in each NAS isolate. S. simulans differed the most. Several of the virulence genes detected among NAS were harbored only by S. simulans, but it also lacked a number of genes present both in S. agnetis and S. chromogenes. The type of mastitis was not associated with any specific virulence gene profile. It seems that the virulence gene profiles or cumulative number of different virulence genes are not directly associated with the type of mastitis (clinical or)subclinical), indicating that host derived factors such as the immune status play a pivotal role in the manifestation of mastitis.Peer reviewe

Human gut-commensalic Lactobacillus ruminis ATCC 25644 displays sortase-assembled surface piliation: Phenotypic characterization of its fimbrial operon through in silico predictive analysis and recombinant expression in Lactococcus lactis

Sortase-dependent surface pili (or fimbriae) in Gram-positive bacteria are well documented as a key virulence factor for certain harmful opportunistic pathogens. However, it is only recently known that these multi-subunit protein appendages are also belonging to the "friendly" commensals and now, with this new perspective, they have come to be categorized as a niche-adaptation factor as well. In this regard, it was shown earlier that sortase-assembled piliation is a native fixture of two human intestinal commensalics (i.e., Lactobacillus rhamnosus and Bifidobacterium bifidum), and correspondingly where the pili involved have a significant role in cellular adhesion and immunomodulation processes. We now reveal that intestinal indigenous (or autochthonous) Lactobacillus ruminis is another surface-piliated commensal lactobacillar species. Heeding to in silico expectations, the predicted loci for the LrpCBA-called pili are organized tandemly in the L. ruminis genome as a canonical fimbrial operon, which then encodes for three pilin-proteins and a single C-type sortase enzyme. Through electron microscopic means, we showed that these pilus formations are a surface assemblage of tip, basal, and backbone pilin subunits (respectively named LrpC, LrpB, and LrpA) in L. ruminis, and also when expressed recombinantly in Lactococcus lactis. As well, by using the recombinant-piliated lactococci, we could define certain ecologically relevant phenotypic traits, such as the ability to adhere to extracellular matrix proteins and gut epithelial cells, but also to effectuate an induced dampening on Toll-like receptor 2 signaling and interleukin-8 responsiveness in immune-related cells. Within the context of the intestinal microcosm, by wielding such niche-advantageous cell-surface properties the LrpCBA pilus would undoubtedly have a requisite functional role in the colonization dynamics of L. ruminis indigeneity. Our study provides only the second description of a native-piliated Lactobacillus species, but at the same time also involves the structural and functional characterization of a third type of lactobacillar pilus.Peer reviewe

Functional characterization of probiotic surface layer protein-carrying Lactobacillus amylovorus strains.

Peer reviewe