Genetic barriers more than environmental associations explain Serratia marcescens population structure.

Bacterial species often comprise well-separated lineages, likely emerged and maintained by genetic isolation and/or ecological divergence. How these two evolutionary actors interact in the shaping of bacterial population structure is currently not fully understood. In this study, we investigate the genetic and ecological drivers underlying the evolution of Serratia marcescens, an opportunistic pathogen with high genomic flexibility and able to colonise diverse environments. Comparative genomic analyses reveal a population structure composed of five deeply-demarcated genetic clusters with open pan-genome but limited inter-cluster gene flow, partially explained by Restriction-Modification (R-M) systems incompatibility. Furthermore, a large-scale research on hundred-thousands metagenomic datasets reveals only a partial habitat separation of the clusters. Globally, two clusters only show a separate gene composition coherent with ecological adaptations. These results suggest that genetic isolation has preceded ecological adaptations in the shaping of the species diversity, an evolutionary scenario coherent with the Evolutionary Extended Synthesis.

Cultivation and sequencing-free protocol for Serratia marcescens detection and typing.

Serratia marcescens is an opportunistic pathogen that survives in inhospitable environments causing large outbreaks, particularly in neonatal intensive care units (NICUs). Genomic studies revealed that most S. marcescens nosocomial infections are caused by a specific clone (here "Infectious clone"). Whole genome sequencing (WGS) is the only portable method able to identify this clone, but it requires days to obtain results. We present a cultivation-free hypervariable-locus melting typing (HLMT) protocol for the fast detection and typing of S. marcescens, with 100% detection capability on mixed samples and a limit of detection that can reach the 10 genome copies. The protocol was able to identify the S. marcescens infectious clone with 97% specificity and 96% sensitivity when compared to WGS, yielding typing results portable among laboratories. The protocol is a cost and time saving method for S. marcescens detection and typing for large environmental/clinical surveillance screenings, also in low-middle income countries.

The emergence and diversification of a zoonotic pathogen from within the microbiota of intensively farmed pigs.

The expansion and intensification of livestock production is predicted to promote the emergence of pathogens. As pathogens sometimes jump between species, this can affect the health of humans as well as livestock. Here, we investigate how livestock microbiota can act as a source of these emerging pathogens through analysis of Streptococcus suis, a ubiquitous component of the respiratory microbiota of pigs that is also a major cause of disease on pig farms and an important zoonotic pathogen. Combining molecular dating, phylogeography, and comparative genomic analyses of a large collection of isolates, we find that several pathogenic lineages of S. suis emerged in the 19th and 20th centuries, during an early period of growth in pig farming. These lineages have since spread between countries and continents, mirroring trade in live pigs. They are distinguished by the presence of three genomic islands with putative roles in metabolism and cell adhesion, and an ongoing reduction in genome size, which may reflect their recent shift to a more pathogenic ecology. Reconstructions of the evolutionary histories of these islands reveal constraints on pathogen emergence that could inform control strategies, with pathogenic lineages consistently emerging from one subpopulation of S. suis and acquiring genes through horizontal transfer from other pathogenic lineages. These results shed light on the capacity of the microbiota to rapidly evolve to exploit changes in their host population and suggest that the impact of changes in farming on the pathogenicity and zoonotic potential of S. suis is yet to be fully realized.

Precision Genome Engineering in Streptococcus suis Based on a Broad-Host-Range Vector and CRISPR-Cas9 Technology.

Streptococcussuis is an important zoonotic pathogen that causes severe invasive disease in pigs and humans. Current methods for genome engineering of S. suis rely on the insertion of antibiotic resistance markers, which is time-consuming and labor-intensive and does not allow the precise introduction of small genomic mutations. Here we developed a system for CRISPR-based genome editing in S. suis, utilizing linear DNA fragments for homologous recombination (HR) and a plasmid-based negative selection system for bacteria not edited by HR. To enable the use of this system in other bacteria, we engineered a broad-host-range replicon in the CRISPR plasmid. We demonstrated the utility of this system to rapidly introduce multiple gene deletions in successive rounds of genome editing and to make precise nucleotide changes in essential genes. Furthermore, we characterized a mechanism by which S. suis can escape killing by a targeted Cas9-sgRNA complex in the absence of HR. A characteristic of this new mechanism is the presence of very slow-growing colonies in a persister-like state that may allow for DNA repair or the introduction of mutations, alleviating Cas9 pressure. This does not impact the utility of CRISPR-based genome editing because the escape colonies are easily distinguished from genetically edited clones due to their small colony size. Our CRISPR-based editing system is a valuable addition to the genetic toolbox for engineering of S. suis, as it accelerates the process of mutant construction and simplifies the removal of antibiotic markers between successive rounds of genome editing.

Changes in the oral status and periodontal pathogens in a Sardinian rural community from pre-industrial to modern time.

The oral microbial profile in humans has evolved in response to lifestyle changes over the course of different eras. Here, we investigated tooth lesions and the microbial profile of periodontal bacteria (PB) in dental calculus of a Sardinian pre-industrial rural community. In total, 51 teeth belonging to 12 historical individuals buried in an ossuary in the early 1800s and 26 modern teeth extracted from 26 individuals from the same geographical area were compared to determine the oral health status, bacterial load and amount of most relevant PB. Total caries and bacterial genomes count appeared to be sex-related in historical samples. Historical females presented a higher incidence of caries, PB pathogens and a higher bacterial load than historical males. Furthermore, we compared the PB profile of the historical individuals with the modern ones, revealing a notable increase in modern individuals of PB belonging to "Red complex bacteria" often associated with periodontitis and other chronic diseases of modern life. Our findings could be explained through an analysis of environmental factors such as socioeconomic, hygienic and healthy conditions that can have a great impact on oral health and bacterial composition among individuals of the same and different eras.

Evaluation of "Caterina assay": An Alternative Tool to the Commercialized Kits Used for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Identification.

Here we describe the first molecular test developed in the early stage of the pandemic to diagnose the first cases of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in Sardinian patients in February-March 2020, when diagnostic certified methodology had not yet been adopted by clinical microbiology laboratories. The "Caterina assay" is a SYBR®Green real-time reverse-transcription polymerase chain reaction (rRT-PCR), designed to detect the nucleocapsid phosphoprotein (N) gene that exhibits high discriminative variation RNA sequence among bat and human coronaviruses. The molecular method was applied to detect SARS-CoV-2 in nasal swabs collected from 2110 suspected cases. The study article describes the first molecular test developed in the early stage of the declared pandemic to identify the coronavirus disease 2019 (COVID-19) in Sardinian patients in February-March 2020, when a diagnostic certified methodology had not yet been adopted by clinical microbiology laboratories. The assay presented high specificity and sensitivity (with a detection limit ≥50 viral genomes/µL). No false-positives were detected, as confirmed by the comparison with two certified commercial kits. Although other validated molecular methods are currently in use, the Caterina assay still represents a valid and low-cost detection procedure that could be applied in countries with limited economic resources.

Active Human and Porcine Serum Induce Competence for Genetic Transformation in the Emerging Zoonotic Pathogen Streptococcus suis.

The acquisition of novel genetic traits through natural competence is a strategy used by bacteria in microbe-rich environments where microbial competition, antibiotics, and host immune defenses threaten their survival. Here, we show that virulent strains of Streptococcus suis, an important zoonotic agent and porcine pathogen, become competent for genetic transformation with plasmid or linear DNA when cultured in active porcine and human serum. Competence was not induced in active fetal bovine serum, which contains less complement factors and immunoglobulins than adult serum and was strongly reduced in heat-treated or low-molecular weight fractions of active porcine serum. Late competence genes, encoding the uptake machinery for environmental DNA, were upregulated in the active serum. Competence development was independent of the early competence regulatory switch involving XIP and ComR, as well as sigma factor ComX, suggesting the presence of an alternative stress-induced pathway for regulation of the late competence genes required for DNA uptake.

In vitro Interactions between Streptococcus intermedius and Streptococcus salivarius K12 on a Titanium Cylindrical Surface.

Peri-implantitis is a steadily rising disease and is caused by oral bacterial pathogens able to form biofilm on implant surfaces and peri-implant tissues, making antibiotics treatment less effective. The use of commercial probiotics against oral pathogens could serve as an alternative to prevent biofilm formation. Streptococcus intermedius is one of the early colonizers of biofilm formation in dental implants. The aim of this study was to model the interaction between S. intermedius and Streptococcus salivarius strain K12, a probiotic bacterium producing bacteriocins. S. intermedius was co-cultured with S. salivarius K12 in an in vitro model simulating the biofilm formation in a dental implant composed by a titanium cylinder system. Biofilm formation rate was assessed by Real-Time PCR quantification of bacterial count and expression levels of luxS gene, used in response to cell density in the biofilm. Biofilm formation, bacteriocin production, luxS expression patterns were found to be already expressed within the first 12 h. More importantly, S. salivarius K12 was able to counter the biofilm formation in a titanium cylinder under the tested condition. In conclusion, our dental implant model may be useful for exploring probiotic-pathogen interaction to find an alternative to antibiotics for peri-implantitis treatment.

Human salivary peptide histatin-1 stimulates epithelial and endothelial cell adhesion and barrier function.

Histatins are multifunctional histidine-rich peptides secreted by the salivary glands and exclusively present in the saliva of higher primates, where they play a fundamental role in the protection of the oral cavity. Our previously published results demonstrated that histatin-1 (Hst1) promotes cell-substrate adhesion in various cell types and hinted that it could also be involved in cell-cell adhesion, a process of fundamental importance to epithelial and endothelial barriers. Here we explore the effects of Hst1 on cellular barrier function. We show that Hst1 improved endothelial barrier integrity, decreased its permeability for large molecules, and prevented translocation of bacteria across epithelial cell layers. These effects are mediated by the adherens junction protein E-cadherin (E-cad) and by the tight junction protein zonula occludens 1, as Hst1 increases the levels of zonula occludens 1 and of active E-cad. Hst1 may also promote epithelial differentiation as Hst1 induced transcription of the epithelial cell differentiation marker apolipoprotein A-IV (a downstream E-cad target). In addition, Hst1 counteracted the effects of epithelial-mesenchymal transition inducers on the outgrowth of oral cancer cell spheroids, suggesting that Hst1 affects processes that are implicated in cancer progression.-Van Dijk, I. A., Ferrando, M. L., van der Wijk, A.-E., Hoebe, R. A., Nazmi, K., de Jonge, W. J., Krawczyk, P. M., Bolscher, J. G. M., Veerman, E. C. I., Stap, J. Human salivary peptide histatin-1 stimulates epithelial and endothelial cell adhesion and barrier function.

Streptococcal Adhesin P (SadP) contributes to Streptococcus suis adhesion to the human intestinal epithelium.

BACKGROUND: Streptococcus suis is a zoonotic pathogen, causing meningitis and septicemia. We previously demonstrated that the gastrointestinal tract (GIT) is an entry site for zoonotic S. suis infection. Here we studied the contribution of Streptococcal adhesin Protein (SadP) to host-pathogen interaction at GIT level. METHODS: SadP expression in presence of Intestinal Epithelial Cells (IEC) was compared with expression of other virulence factors by measuring transcript levels using quantitative Real Time PCR (qRT-PCR). SadP variants were identified by phylogenetic analysis of complete DNA sequences. The interaction of SadP knockout and complementation mutants with IEC was tested in vitro. RESULTS: Expression of sadP was significantly increased in presence of IEC. Sequence analysis of 116 invasive strains revealed five SadP sequence variants, correlating with genotype. SadP1, present in zoonotic isolates of clonal complex 1, contributed to binding to both human and porcine IEC and translocation across human IEC. Antibodies against the globotriaosylceramide Gb3/CD77 receptor significantly inhibited adhesion to human IEC. CONCLUSION: SadP is involved in the host-pathogen interaction in the GIT. Differences between SadP variants may determine different affinities to the Gb3/CD77 host-receptor, contributing to variation in adhesion capacity to host IEC and thus to S. suis zoonotic potential.

A hypothetical model of host-pathogen interaction of Streptococcus suis in the gastro-intestinal tract.

Streptococcus suis (SS) is a zoonotic pathogen that can cause systemic infection in pigs and humans. The ingestion of contaminated pig meat is a well-established risk factor for zoonotic S. suis disease. In our studies, we provide experimental evidence that S. suis is capable to translocate across the host gastro-intestinal tract (GIT) using in vivo and in vitro models. Hence, S. suis should be considered an emerging foodborne pathogen. In this addendum, we give an overview of the complex interactions between S. suis and host-intestinal mucosa which depends on the host origin, the serotype and genotype of S. suis, as well as the presence and expression of virulence factors involved in host-pathogen interaction. Finally, we propose a hypothetical model of S. suis interaction with the host-GIT taking in account differences in conditions between the porcine and human host.

Host-pathogen Interaction at the Intestinal Mucosa Correlates With Zoonotic Potential of Streptococcus suis.

BACKGROUND: Streptococcus suis has emerged as an important cause of bacterial meningitis in adults. The ingestion of undercooked pork is a risk factor for human S. suis serotype 2 (SS2) infection. Here we provide experimental evidence indicating that the gastrointestinal tract is an entry site of SS2 infection. METHODS: We developed a noninvasive in vivo model to study oral SS2 infection in piglets. We compared in vitro interaction of S. suis with human and porcine intestinal epithelial cells (IEC). RESULTS: Two out of 15 piglets showed clinical symptoms compatible with S. suis infection 24-48 hours after ingestion of SS2. SS2 was detected in mesenteric lymph nodes of 40% of challenged piglets. SS2 strains isolated from patients showed significantly higher adhesion to human IEC compared to invasive strains isolated from pigs. In contrast, invasive SS9 strains showed significantly higher adhesion to porcine IEC. Translocation across human IEC, which occurred predominately via a paracellular route, was significantly associated with clonal complex 1, the predominant zoonotic genotype. Adhesion and translocation were dependent on capsular polysaccharide production. CONCLUSIONS: SS2 should be considered a food-borne pathogen. S. suis interaction with human and pig IEC correlates with S. suis serotype and genotype, which can explain the zoonotic potential of SS2.

Immunomodulatory effects of Streptococcus suis capsule type on human dendritic cell responses, phagocytosis and intracellular survival.

Streptococcus suis is a major porcine pathogen of significant commercial importance worldwide and an emerging zoonotic pathogen of humans. Given the important sentinel role of mucosal dendritic cells and their importance in induction of T cell responses we investigated the effect of different S. suis serotype strains and an isogenic capsule mutant of serotype 2 on the maturation, activation and expression of IL-10, IL-12p70 and TNF-α in human monocyte-derived dendritic cells. Additionally, we compared phagocytosis levels and bacterial survival after internalization. The capsule of serotype 2, the most common serotype associated with infection in humans and pigs, was highly anti-phagocytic and modulated the IL-10/IL-12 and IL-10/TNF-α cytokine production in favor of a more anti-inflammatory profile compared to other serotypes. This may have consequences for the induction of effective immunity to S. suis serotype 2 in humans. A shielding effect of the capsule on innate Toll-like receptor signaling was also demonstrated. Furthermore, we showed that 24 h after phagocytosis, significant numbers of viable intracellular S. suis were still present intracellularly. This may contribute to the dissemination of S. suis in the body.

ApuA, a multifunctional alpha-glucan-degrading enzyme of Streptococcus suis, mediates adhesion to porcine epithelium and mucus.

We have identified apuA in Streptococcus suis, which encodes a bifunctional amylopullulanase with conserved alpha-amylase and pullulanase substrate-binding domains and catalytic motifs. ApuA exhibited properties typical of a Gram-positive surface protein, with a putative signal sequence and LPKTGE cell-wall-anchoring motif. A recombinant protein containing the predicted N-terminal alpha-amylase domain of ApuA was shown to have alpha-(1,4) glycosidic activity. Additionally, an apuA mutant of S. suis lacked the pullulanase alpha-(1,6) glycosidic activity detected in a cell-surface protein extract of wild-type S. suis. ApuA was required for normal growth in complex medium containing pullulan as the major carbon source, suggesting that this enzyme plays a role in nutrient acquisition in vivo via the degradation of glycogen and food-derived starch in the nasopharyngeal and oral cavities. ApuA was shown to promote adhesion to porcine epithelium and mucus in vitro, highlighting a link between carbohydrate utilization and the ability of S. suis to colonize and infect the host.

Rapid detection and quantitation of Bluetongue virus (BTV) using a Molecular Beacon fluorescent probe assay.

Bluetongue virus (BTV) is the causative agent of Bluetongue (BT) disease in ruminant livestock and occurs almost worldwide between latitudes 35 degrees S and 50 degrees N; 24 serotypes of BTV are known of which 8 circulate periodically within parts of the Mediterranean Region. A fast (about 3.5 h) and versatile diagnostic procedure able to detect and quantify BTV-RNA, has been developed using a Molecular Beacon (MB) fluorescent probe; PCR primers were designed to target 91 bp within the NS3 conserved region of the viral RNA segment 10 (S10) and bracketed the MB fluorescence probe hybridisation site. The MB fluorescent probe was used to develop two Bluetongue serogroup-specific assays: a quantitative real time reverse transcriptase polymerase chain reaction (RT-PCR) and a traditional RT-PCR. These were tested using BTV-RNAs extracted from the blood and organs of BT-affected animals, and from virus isolate suspensions. The samples included ten serotypes (BTV-1-BTV-9 and BTV-16); of these, BTV serotypes -1, -2, -4, -9 and -16 have since 1998 been involved in the extensive outbreaks of BT across the Mediterranean Region. To evaluate the specificity and sensitivity of the MB probe, all positive samples (and negative controls) were tested using the developed quantitative real time RT-PCR and traditional RT-PCR assays. The former test had a detection limit of 10(3) cDNA molecules per reaction with a log-linear quantification range of up to 10(11) (R2 = 0.98), while the latter test was able to detect 500 cDNA-BTV molecules/PCR. The results show that the MB fluorescent probe is both rapid and versatile for the laboratory diagnosis of Bluetongue and for quantifying levels of viraemia in BTV-affected animals. An "in silico" comparison of the primers and MB fluorescent probe used in this study showed that it is possible to detect all 24 serotypes of BTV.