Extensive horizontal gene transfer during Staphylococcus aureus co-colonization in vivo.

Staphylococcus aureus is a commensal and major pathogen of humans and animals. Comparative genomics of S. aureus populations suggests that colonization of different host species is associated with carriage of mobile genetic elements (MGE), particularly bacteriophages and plasmids capable of encoding virulence, resistance, and immune evasion pathways. Antimicrobial-resistant S. aureus of livestock are a potential zoonotic threat to human health if they adapt to colonize humans efficiently. We utilized the technique of experimental evolution and co-colonized gnotobiotic piglets with both human- and pig-associated variants of the lineage clonal complex 398, and investigated growth and genetic changes over 16 days using whole genome sequencing. The human isolate survived co-colonization on piglets more efficiently than in vitro. During co-colonization, transfer of MGE from the pig to the human isolate was detected within 4 h. Extensive and repeated transfer of two bacteriophages and three plasmids resulted in colonization with isolates carrying a wide variety of mobilomes. Whole genome sequencing of progeny bacteria revealed no acquisition of core genome polymorphisms, highlighting the importance of MGE. Staphylococcus aureus bacteriophage recombination and integration into novel sites was detected experimentally for the first time. During colonization, clones coexisted and diversified rather than a single variant dominating. Unexpectedly, each piglet carried unique populations of bacterial variants, suggesting limited transmission of bacteria between piglets once colonized. Our data show that horizontal gene transfer occurs at very high frequency in vivo and significantly higher than that detectable in vitro

Functional traits of expanding, thicket-forming shrubs: contrasting strategies between exotic and native species

Woody expansion has been documented for decades in many different systems globally, often yielding vast changes in ecosystem functioning. While causes and consequences of woody expansion have been well documented, few studies have addressed plant functional traits that promote dramatic and rapid expansion in range. Our objectives were to investigate plant functional traits that contribute to the colonization, rapid expansion, and thicket formation of an invasive, N-fixing shrub, Elaeagnus umbellata Thunb. (Elaeagnaceae), and a native, N-fixing shrub Morella cerifera (L.) Small (Myricaceae) and compare to native, sympatric, non-expanding shrub species. Quantified functional traits included morphological (e.g., specific leaf area, leaf area) and physiological characteristics (e.g., electron transport rate, hydraulic conductivity) and were linked to two primary resources: light and water, which directly influence plant growth. Elaeagnus umbellata and M. cerifera rely on different strategies to maximize carbon gain, yet resulting physiological efficiency is similar. Elaeagnus umbellata invests a substantial amount of energy into growth during a short amount of time (i.e., deciduous growing season), using an acquisitive trait strategy to outcompete co-occurring woody species, while M. cerifera is productive year-round and uses a combination of conservative and acquisitive traits to outcompete co-occurring woody species. The majority of quantified functional traits of E. umbellata and several of M. cerifera are indicative of efficient light capture, utilization, and internal water movement. These factors contribute to rapid range expansion and thicket formation by promoting enhanced productivity while simultaneously inhibiting colonization and expansion of co-occurring species. Suites of functional traits are important for expansive success and thicket formation, yet differences in functional traits represent alternative strategies for colonization, rapid expansion, and thicketization

Evaluation of differences in S. pneumoniae colonization among children with and without clinically diagnosed asthma/wheeze

Streptococcus pneumoniae colonization is the nasopharynx is common in young children. Colonization of S. pneumoniae is a necessary precursor for invasive pneumococcal disease (IPD), which is a major cause of morbidity and mortality among children less than five years of age globally. Co-morbidities such as asthma have been identified as risk factors for IPD but little is known about why. Children with co-morbidities have a higher likelihood of progressing to IPD because they are colonized at higher rates or because their immune systems respond differently than children without co-morbidities. In addition, vaccination was introduced in 2010 to help decrease pneumococcal colonization rates from the 13 most common serotypes. We used data from the pediatric primary care clinic at Boston Medical Center to study the relationship between asthma/wheeze and S. pneumoniae colonization among children under the age of five years. Information about colonization serotype distribution was also assessed in this study. Data was accessed from 3098 children from 4–59 months old visiting the pediatric primary care clinic at Boston Medical Center from July 2010 to March 2014. In multivariable logistic regression models, the odds of colonization increased 80% (OR 1.80, 95% CI 1.2, 2.6) in children with asthma/wheeze under 24 months old. Adjustment for presence of URTI or recent exposure to antibiotics slightly mitigates this relationship. Children with clinically diagnosed asthma/wheeze have 80% increased odds of being colonized than children without asthma/wheeze

Nod1 signaling overcomes resistance of S. pneumoniae to opsonophagocytic killing

Airway infection by the Gram-positive pathogen Streptococcus pneumoniae (Sp) leads to recruitment of neutrophils but limited bacterial killing by these cells. Co-colonization by Sp and a Gram-negative species, Haemophilus influenzae (Hi), provides sufficient stimulus to induce neutrophil and complement-mediated clearance of Sp from the mucosal surface in a murine model. Products from Hi, but not Sp, also promote killing of Sp by ex vivo neutrophil-enriched peritoneal exudate cells. Here we identify the stimulus from Hi as its peptidoglycan. Enhancement of opsonophagocytic killing was facilitated by signaling through nucleotide-binding oligomerization domain-1 (Nod1), which is involved in recognition of γ-D-glutamyl-meso-diaminopimelic acid (meso-DAP) contained in cell walls of Hi but not Sp. Neutrophils from mice treated with Hi or compounds containing meso-DAP, including synthetic peptidoglycan fragments, showed increased Sp killing in a Nod1-dependent manner. Moreover, Nod1-/- mice showed reduced Hi-induced clearance of Sp during co-colonization. These observations offer insight into mechanisms of microbial competition and demonstrate the importance of Nod1 in neutrophil-mediated clearance of bacteria in vivo

The blp locus of Streptococcus pneumoniae plays a limited role in the selection of which strains can co-colonize the human nasopharynx.

Nasopharyngeal colonization is important for Streptococcus pneumoniae evolution, providing the opportunity for horizontal gene transfer when multiple strains co-occur. Although colonization with more than one strain of pneumococcus is common, the factors that influence the ability of strains to co-exist are not known. A highly variable blp (bacteriocin-like peptide) locus has been identified in all sequenced strains of S. pneumoniae This locus controls the regulation and secretion of bacteriocins, small peptides that target other bacteria. In this study, we analyzed a series of co-colonizing isolates to evaluate the impact of the blp locus on human colonization to determine whether competitive phenotypes of bacteriocin secretion restrict co-colonization.We identified a collection of 135 nasopharyngeal samples with two or more strains totaling 285 isolates. The blp locus of all strains was characterized genetically with regards to pheromone type, bacteriocin/immunity content and potential for locus functionality. Inhibitory phenotypes of bacteriocin secretion and locus activity were assessed through overlay assays. Isolates from single colonization (n=298) were characterized for comparison.Co-colonizing strains had a high diversity of blp cassettes; approximately one third displayed an inhibitory phenotype in vitro Despite in vitro evidence of competition, pneumococci co-colonized individuals independently of their blp pheromone type (p=0.577), bacteriocin/immunity content, blp locus activity (p=0.798) and inhibitory phenotype (p=0.716). In addition, no significant differences were observed when single and co-colonizing strains were compared.Despite clear evidence of blp-mediated competition in experimental models, our study suggests that the blp locus plays a limited role in restricting pneumococcal co-colonization in humans. IMPORTANCE: Nasopharyngeal colonization with Streptococcus pneumoniae (pneumococcus) is important for pneumococcal evolution as it represents the major site for horizontal gene transfer when multiple strains co-occur, a phenomenon known as co-colonization. Understanding how pneumococcal strains interact within the competitive environment of the nasopharynx is of chief importance in the context of pneumococcal ecology. In this study we used an unbiased collection of naturally co-occurring pneumococcal strains and showed that a biological process frequently used by bacteria for competition - bacteriocin production - is not decisive in the co-existence of pneumococci in the host, contrary to what has been shown in experimental models

A host KH RNA-binding protein is a susceptibility factor targeted by an RXLR effector to promote late blight disease

Plant pathogens deliver effector proteins that alter host processes to create an environment conducive to colonization. Attention has focused on identifying the targets of effectors and how their manipulation facilitates disease. RXLR effector Pi04089 from the potato blight pathogen Phytophthora infestans accumulates in the host nucleus and enhances colonization when transiently expressed in planta. Its nuclear localization is required for enhanced P. infestans colonization. Pi04089 interacts in yeast and in planta with a putative potato K-homology (KH) RNA-binding protein, StKRBP1. Co-localization of Pi04089 and StKRBP1, and bimolecular fluorescence complementation between them, indicate they associate at nuclear speckles. StKRBP1 protein levels increased when it was co-expressed with Pi04089. Indeed, such accumulation of StKRBP1 was observed also on the first day of leaf colonization by the pathogen. Remarkably, overexpression of StKRBP1 significantly enhances P. infestans infection. Mutation of the nucleotide-binding motif GxxG to GDDG in all three KH domains of StKRBP1 abolishes its interaction with Pi04089, its localization to nuclear speckles, and its increased accumulation when co-expressed with the effector. Moreover, the mutant StKRBP1 protein no longer enhances leaf colonization by P. infestans, implying that nucleotide binding is likely required for this activity. We thus argue that StKRBP1 can be regarded as a susceptibility factor, as its activity is beneficial to the pathogen

Effect of hydrolyzed milk on the adhesion of Lactobacilli to intestinal cells

Milk is an essential part of the human diet and is undoubtedly a major calcium source in human nutrition, accepted well by most individuals. Knowledge on how the components from dairy products support or reduce the adherence of probiotics to the intestinal epithelium is limited. The purpose of this study was to investigate the effect of acid-hydrolyzed milk on the adhesion ability of two potentially probiotic strains (Lactobacillus plantarum S2, Lactobacillus gasseri R) to in vitro human intestinal epithelial model consisting of Caco-2 and mucus-secreting HT29-MTX co-culture. The adhesion of our tested strains L. gasseri and L. plantarum was 4.74 and 7.16%, respectively, when using inoculum of 2 × 108 CFU ml–1. Addition of acid-hydrolyzed milk to co-culture decreased the adherence by 53.7% for L. gasseri R and by 62.2% for L. plantarum S2. The results of this study evidently indicate the potential importance of the food matrix as a factor influencing probiotic colonization of the gut

Three-dimensional organotypic co-culture model of intestinal epithelial cells and macrophages to study Salmonella enterica colonization patterns

Three-dimensional models of human intestinal epithelium mimic the differentiated form and function of parental tissues often not exhibited by two-dimensional monolayers and respond to Salmonella in key ways that reflect in vivo infections. To further enhance the physiological relevance of three-dimensional models to more closely approximate in vivo intestinal microenvironments encountered by Salmonella, we developed and validated a novel three-dimensional co-culture infection model of colonic epithelial cells and macrophages using the NASA Rotating Wall Vessel bioreactor. First, U937 cells were activated upon collagen-coated scaffolds. HT-29 epithelial cells were then added and the three-dimensional model was cultured in the bioreactor until optimal differentiation was reached, as assessed by immunohistochemical profiling and bead uptake assays. The new co-culture model exhibited in vivo-like structural and phenotypic characteristics, including three-dimensional architecture, apical-basolateral polarity, well-formed tight/adherens junctions, mucin, multiple epithelial cell types, and functional macrophages. Phagocytic activity of macrophages was confirmed by uptake of inert, bacteria-sized beads. Contribution of macrophages to infection was assessed by colonization studies of Salmonella pathovars with different host adaptations and disease phenotypes (Typhimurium ST19 strain SL1344 and ST313 strain D23580; Typhi Ty2). In addition, Salmonella were cultured aerobically or microaerobically, recapitulating environments encountered prior to and during intestinal infection, respectively. All Salmonella strains exhibited decreased colonization in co-culture (HT-29-U937) relative to epithelial (HT-29) models, indicating antimicrobial function of macrophages. Interestingly, D23580 exhibited enhanced replication/survival in both models following invasion. Pathovar-specific differences in colonization and intracellular co-localization patterns were observed. These findings emphasize the power of incorporating a series of related three-dimensional models within a study to identify microenvironmental factors important for regulating infection

The multiplicity of cellular infection changes between primary and secondary infected cells during systemic infection by a plant virus. [P.45]

The multiplicity of cellular infection (MOI) is the number of virus genomes of a given virus species that infect individual cells. This parameter chiefly impacts the severity of within-host population bottlenecks, the intensity of genetic exchange, as well as the competition and complementation among viral genotypes. Only a fistful of formal estimations of MOI is currently available, and most reports have considered the MOI as a constant within the infected host. Nevertheless, the colonization of a multicellular host is a complex process during which the MOI may dramatically change in different organs and at different stage of the infection. We have used both qualitative and quantitative approaches to analyze the MOI during the colonization of turnip plants by the Turnip mosaic virus. Remarkably, different MOI values were observed at two phases of the systemic infection of a leaf. The MOI was low in cells primarily infected from virus circulating within the vasculature. Then, the founded populations moved from cell to cell at a very high MOI. Despite this elevated MOI during cell-to-cell progression, the viral lines displayed a territorial behavior and severely limited co-infection of cells by lineages originated in different primary sites. Our results thus unveil an intriguing colonization pattern where individual viral genomes initiate distinct lineages within a leaf. Kin genomes massively co-infect cells within a lineage, but co-infection by two distinct lineages is precluded. This pattern explains in an unforeseen way the common but uncharacterized phenomenon of spatial segregation of virus genotypes in infected plants. (Résumé d'auteur

Assessing hepatic metabolic changes during progressive colonization of germ-free mouse by 1H NMR spectroscopy

It is well known that gut bacteria contribute significantly to the host homeostasis, providing a range of benefits such as immune protection and vitamin synthesis. They also supply the host with a considerable amount of nutrients, making this ecosystem an essential metabolic organ. In the context of increasing evidence of the link between the gut flora and the metabolic syndrome, understanding the metabolic interaction between the host and its gut microbiota is becoming an important challenge of modern biology.1-4 Colonization (also referred to as normalization process) designates the establishment of micro-organisms in a former germ-free animal. While it is a natural process occurring at birth, it is also used in adult germ-free animals to control the gut floral ecosystem and further determine its impact on the host metabolism. A common procedure to control the colonization process is to use the gavage method with a single or a mixture of micro-organisms. This method results in a very quick colonization and presents the disadvantage of being extremely stressful5. It is therefore useful to minimize the stress and to obtain a slower colonization process to observe gradually the impact of bacterial establishment on the host metabolism. In this manuscript, we describe a procedure to assess the modification of hepatic metabolism during a gradual colonization process using a non-destructive metabolic profiling technique. We propose to monitor gut microbial colonization by assessing the gut microbial metabolic activity reflected by the urinary excretion of microbial co-metabolites by 1H NMR-based metabolic profiling. This allows an appreciation of the stability of gut microbial activity beyond the stable establishment of the gut microbial ecosystem usually assessed by monitoring fecal bacteria by DGGE (denaturing gradient gel electrophoresis).6 The colonization takes place in a conventional open environment and is initiated by a dirty litter soiled by conventional animals, which will serve as controls. Rodents being coprophagous animals, this ensures a homogenous colonization as previously described.7 Hepatic metabolic profiling is measured directly from an intact liver biopsy using 1H High Resolution Magic Angle Spinning NMR spectroscopy. This semi-quantitative technique offers a quick way to assess, without damaging the cell structure, the major metabolites such as triglycerides, glucose and glycogen in order to further estimate the complex interaction between the colonization process and the hepatic metabolism7-10. This method can also be applied to any tissue biopsy11,12