Emergence of Capnocytophaga canimorsus and Capnocytophaga cynodegmi in oral cavities of newborn puppies, a pilot study.

Capnocytophaga canimorsus and Capnocytophaga cynodegmi are commensal bacteria in the oral cavities of dogs. Both are zoonotic pathogens that could infect humans via dog bites. C. canimorsus may cause life-threatening infections in humans, whereas C. cynodegmi infections tend to be milder and more localized. Capsular serovars A-C of C. canimorsus seem to be virulence-associated. Some of the C. canimorsus serovars described to date can also be detected in other Capnocytophaga species, including C. cynodegmi. The objective of this pilot study was to investigate the emergence of C. canimorsus and C. cynodegmi after birth in oral cavities of puppies and to evaluate the impact of the dam's Capnocytophaga spp. carrier status on the emergence. Ten litters, altogether 59 puppies, were included in the study. The puppies and their dams were sampled at five time points over seven weeks after whelping. Oral swab samples taken were investigated for the presence of C. canimorsus and C. cynodegmi by species-specific polymerase chain reaction (PCR), the specificity of which was verified by sequencing a selection of the PCR products. Samples that were positive in Capnocytophaga PCR reactions were also capsular-typed by PCR to gain more knowledge about the Capnocytophaga spp. present in the samples. Altogether 10.2% and 11.9% of puppies, or 20.0% and 30.0% of litters tested PCR-positive for C. canimorsus and C. cynodegmi, respectively. Capnocytophaga PCR-positive puppy samples were always positive for only C. cynodegmi or C. canimorsus, not both. Most Capnocytophaga PCR-positive puppies became positive at the age of 5 to 7 weeks. Only a minority (5/16) of the C. cynodegmi PCR-positive dog samples were positive in capsular typing PCR, whereas all C. canimorsus PCR-positive dog samples were negative in capsular typing PCR. For all Capnocytophaga PCR-positive puppies, their dam was positive for the same Capnocytophaga species. These results suggest that puppies become colonized by C. cynodegmi or C. canimorsus from their dams at the time of deciduous teeth eruption.

Redesigning and teaching veterinary microbiology laboratory exercises with combined on-site and online participation during the COVID-19 pandemic.

The COVID-19 (coronavirus disease 2019) pandemic has forced universities to find new ways to conduct learning and teaching, as traditional face-to-face teaching has been prevented or restricted to an absolute minimum in many instances. Therefore, we redesigned and taught second-year veterinary student microbiology laboratory exercises (labs) with a hybrid learning approach. For this, a novel 'remote partner' model was implemented in which students present on-site in the laboratory worked synchronously pairwise with their remote partner present online. A student feedback survey revealed that in this remote partner model, both on-site and online participation in the labs were experienced as being useful in improving their laboratory skills. The students' overall performance in hands-on microbiological laboratory skills and safe working practices was similar in the hybrid learning approach (the 2021 class) and in the traditional on-site participation approach (the 2018-20 classes). This study suggests that the remote partner model is an effective way to acquire microbiological laboratory skills. This learning approach can be used in the non-pandemic future and/or also be applied to other fields.

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.

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.

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-κB 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.

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.

Genome Sequences of Four Staphylococcus aureus Strains Isolated from Bovine Mastitis.

Staphylococcus aureus is a major causative agent of mastitis in dairy cows. The pathogenicity of S. aureus may vary; it is able to cause severe clinical mastitis, but most often it is associated with chronic subclinical mastitis. Here, we present the genome assemblies of four S. aureus strains from bovine mastitis.

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

BACKGROUND: Adhesiveness to intestinal epithelium, beneficial immunomodulating effects and the production of pathogen-inhibitory compounds are generally considered as beneficial characteristics of probiotic organisms. We showed the potential health-promoting properties and the mechanisms of probiotic action of seven swine intestinal Lactobacillus amylovorus isolates plus the type strain (DSM 20531T) by investigating their adherence to porcine intestinal epithelial cells (IPEC-1) and mucus as well as the capacities of the strains to i) inhibit the adherence of Escherichia coli to IPEC-1 cells, ii) to produce soluble inhibitors against intestinal pathogens and iii) to induce immune signaling in dendritic cells (DCs). Moreover, the role of the L. amylovorus surface (S) -layers - symmetric, porous arrays of identical protein subunits present as the outermost layer of the cell envelope - in adherence to IPEC-1 cells was assessed using a novel approach which utilized purified cell wall fragments of the strains as carriers for the recombinantly produced S-layer proteins. RESULTS: Three of the L. amylovorus strains studied adhered to IPEC-1 cells, while four strains inhibited the adherence of E. coli, indicating additional mechanisms other than competition for binding sites being involved in the inhibition. None of the strains bound to porcine mucus. The culture supernatants of all of the strains exerted inhibitory effects on the growth of E. coli, Salmonella, Listeria and Yersinia, and a variable, strain-dependent induction was observed of both pro- and anti-inflammatory cytokines in human DCs. L. amylovorus DSM 16698 was shown to carry two S-layer-like proteins on its surface in addition to the major S-layer protein SlpA. In contrast to expectations, none of the major S-layer proteins of the IPEC-1 -adhering strains mediated bacterial adherence. CONCLUSIONS: We demonstrated adhesive and significant pathogen inhibitory efficacies among the swine intestinal L. amylovorus strains studied, pointing to their potential use as probiotic feed supplements, but no independent role could be demonstrated for the major S-layer proteins in adherence to epithelial cells. The results indicate that many intestinal bacteria may coexist with and confer benefits to the host by mechanisms not attributable to adhesion to epithelial cells or mucus.

Bovine-associated CNS species resist phagocytosis differently.

BACKGROUND: Coagulase-negative staphylococci (CNS) cause usually subclinical or mild clinical bovine mastitis, which often remains persistent. Symptoms are usually mild, mostly only comprising slight changes in the appearance of milk and possibly slight swelling. However, clinical mastitis with severe signs has also been reported. The reasons for the differences in clinical expression are largely unknown. Macrophages play an important role in the innate immunity of the udder. This study examined phagocytosis and killing by mouse macrophage cells of three CNS species: Staphylococcus chromogenes (15 isolates), Staphylococcus agnetis (6 isolates) and Staphylococcus simulans (15 isolates). Staphylococcus aureus (7 isolates) was also included as a control. RESULTS: All the studied CNS species were phagocytosed by macrophages, but S. simulans resisted phagocytosis more effectively than the other CNS species. Only S. chromogenes was substantially killed by macrophages. Significant variations between isolates were seen in both phagocytosis and killing by macrophages and were more common in the killing assays. Significant differences between single CNS species and S. aureus were observed in both assays. CONCLUSION: This study demonstrated that differences in the phagocytosis and killing of mastitis-causing staphylococci by macrophages exist at both the species and isolate level.

Characterization and separate activities of the two promoters of the Lactobacillus brevis S-layer protein gene.

Lactobacillus brevis ATCC 8287 possesses a surface (S)-layer protein SlpA, the gene of which is very efficiently expressed. To study the expression signals of the slpA gene, several different reporter plasmids, based on the low-copy-number vector pKTH2121 derived from pGK12, were constructed. In the reporter plasmids, only one of the two consecutive slpA promoters (P1, P2) was placed upstream of the Lactobacillus helveticus proline iminopeptidase (pepI) gene, and defined parts of the sequences upstream of the promoter were deleted. As indicated by reporter enzyme activities, both promoters were efficiently recognized at different growth stages in L. brevis. An upstream region important for the full activity of P1 was identified. The quantification of pepI-specific mRNA in L. brevis and SDS-PAGE indicated that slpA expression is not regulated at the post-transcriptional level and revealed no regulation of slpA promoters under the conditions tested. The high expression levels of both slpA and the reporter gene in L. brevis were found to remain at a high level after the addition of bile or pancreatin in the growth medium or after a change of the carbon source, which is advantageous for the potential use of SlpA as a carrier in live oral vaccines.

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 179-435 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.

Secretion of biologically active porcine interleukin-2 by Lactococcus lactis.

In this study, secretion of two functional recombinant porcine interleukin-2 (rIL-2) proteins by Lactococcus lactis was studied. Two secretion cassettes were constructed in which the secretion was achieved by gene fusion between the lactococcal usp45 secretion signal, a synthetic propeptide and the sequence encoding the mature IL-2. In addition, one of the two secretion cassettes contained the H-domains of L. lactis PrtP. Both of the constructed recombinant IL-2 proteins were found to be secreted in the same quantities, approximately 0.5mg/l. According to a cell proliferative assay using CTLL-2 cell line the specific biological activities of both purified rIL-2 proteins were found to be of similar levels.

Lactobacillus surface layers and their applications.

Surface (S-) layers are crystalline arrays of proteinaceous subunits present as the outermost component of cell wall in several species of the genus Lactobacillus, as well as in many other bacteria and Archaea. Despite the high similarity of the amino acid composition of all known S-layer proteins, the overall sequence similarity is, however, surprisingly small even between the Lactobacillus S-layer proteins. In addition, the typical characteristics of Lactobacillus S-layer proteins, distinguishing them from other S-layer proteins, are small size and high-predicted pI value. Several lactobacilli possess multiple S-layer protein genes, which can be differentially or simultaneously expressed. To date, the characterized functions of Lactobacillus S-layers are involved in mediating adhesion to different host tissues. A few applications for the S-layer proteins of lactobacilli already exist, including their use as antigen delivery vehicles.

Surface display of the receptor-binding region of the Lactobacillus brevis S-layer protein in Lactococcus lactis provides nonadhesive lactococci with the ability to adhere to intestinal epithelial cells.

Lactobacillus brevis is a promising lactic acid bacterium for use as a probiotic dietary adjunct and a vaccine vector. The N-terminal region of the S-layer protein (SlpA) of L. brevis ATCC 8287 was recently shown to mediate adhesion to various human cell lines in vitro. In this study, a surface display cassette was constructed on the basis of this SlpA receptor-binding domain, a proteinase spacer, and an autolysin anchor. The cassette was expressed under control of the nisA promoter in Lactococcus lactis NZ9000. Western blot assay of lactococcal cell wall extracts with anti-SlpA antibodies confirmed that the SlpA adhesion domain of the fusion protein was expressed and located within the cell wall layer. Whole-cell enzyme-linked immunosorbent assay and immunofluorescence microscopy verified that the SlpA adhesion-mediating region was accessible on the lactococcal cell surface. In vitro adhesion assays with the human intestinal epithelial cell line Intestine 407 indicated that the recombinant lactococcal cells had gained an ability to adhere to Intestine 407 cells significantly greater than that of wild-type L. lactis NZ9000. Serum inhibition assay further confirmed that adhesion of recombinant lactococci to Intestine 407 cells was indeed mediated by the N terminus-encoding part of the slpA gene. The ability of the receptor-binding region of SlpA to adhere to fibronectin was also confirmed with this lactococcal surface display system. These results show that, with the aid of the receptor-binding region of the L. brevis SlpA protein, the ability to adhere to gut epithelial cells can indeed be transferred to another, nonadhesive, lactic acid bacterium.

Isolation of three new surface layer protein genes (slp) from Lactobacillus brevis ATCC 14869 and characterization of the change in their expression under aerated and anaerobic conditions.

Two new surface layer (S-layer) proteins (SlpB and SlpD) were characterized, and three slp genes (slpB, slpC, and slpD) were isolated, sequenced, and studied for their expression in Lactobacillus brevis neotype strain ATCC 14869. Under different growth conditions, L. brevis strain 14869 was found to form two colony types, smooth (S) and rough (R), and to express the S-layer proteins differently. Under aerobic conditions R-colony type cells produced SlpB and SlpD proteins, whereas under anaerobic conditions S-colony type cells synthesized essentially only SlpB. Anaerobic and aerated cultivations of ATCC 14869 cells in rich medium also resulted in S-layer protein patterns similar to those of the S- and R-colony type cells, respectively. Electron microscopy suggested the presence of only a single S-layer with an oblique structure on the cells of both colony forms. The slpB and slpC genes were located adjacent to each other, whereas the slpD gene was not closely linked to the slpB-slpC gene region. Northern analyses confirmed that both slpB and slpD formed a monocistronic transcription unit and were effectively expressed, but slpD expression was induced under aerated conditions. slpC was a silent gene under the growth conditions tested. The amino acid contents of all the L. brevis ATCC 14869 S-layer proteins were typical of S-layer proteins, whereas their sequence similarities with other S-layer proteins were negligible. The interspecies identity of the L. brevis S-layer proteins was mainly restricted to the N-terminal regions of those proteins. Furthermore, Northern analyses, expression of a PepI reporter protein under the control of the slpD promoter, and quantitative real-time PCR analysis of slpD expression under aerated and anaerobic conditions suggested that, in L. brevis ATCC 14869, the variation of S-layer protein content involves activation of transcription by a soluble factor rather than DNA rearrangements that are typical for most of the S-layer phase variation mechanisms known.

Surface display of foreign epitopes on the Lactobacillus brevis S-layer.

So far, the inability to establish viable Lactobacillus surface layer (S-layer) null mutants has hampered the biotechnological applications of Lactobacillus S-layers. In this study, we demonstrate the utilization of Lactobacillus brevis S-layer subunits (SlpA) for the surface display of foreign antigenic epitopes. With an inducible expression system, L. brevis strains producing chimeric S-layers were obtained after testing of four insertion sites in the slpA gene for poliovirus epitope VP1, that comprises 10 amino acids. The epitope insertion site allowing the best surface expression was used for the construction of an integration vector carrying the gene region encoding the c-Myc epitopes from the human c-myc proto-oncogene, which is composed of 11 amino acids. A gene replacement system was optimized for L. brevis and used for the replacement of the wild-type slpA gene with the slpA-c-myc construct. A uniform S-layer, displaying on its surface the desired antigen in all of the S-layer protein subunits, was obtained. The success of the gene replacement and expression of the uniform SlpA-c-Myc recombinant S-layer was confirmed by PCR, Southern blotting MALDI-TOF mass spectrometry, whole-cell enzyme-linked immunosorbent assay, and immunofluorescence microscopy. Furthermore, the integrity of the recombinant S-layer was studied by electron microscopy, which indicated that the S-layer lattice structure was not affected by the presence of c-Myc epitopes. To our knowledge, this is the first successful expression of foreign epitopes in every S-layer subunit of a Lactobacillus S-layer while still maintaining the S-layer lattice structure.