Pat G. Casey

Functional genome analysis of Bifidobacterium breve UCC2003 reveals type IVb tight adherence (Tad) pili as an essential and conserved host-colonization factor

Development of the human gut microbiota commences at birth, with bifidobacteria being among the first colonizers of the sterile newborn gastrointestinal tract. To date, the genetic basis of Bifidobacterium colonization and persistence remains poorly understood. Transcriptome analysis of the Bifidobacterium breve UCC2003 2.42-Mb genome in a murine colonization model revealed differential expression of a type IVb tight adherence (Tad) pilus-encoding gene cluster designated “tad2003.” Mutational analysis demonstrated that the tad2003 gene cluster is essential for efficient in vivo murine gut colonization, and immunogold transmission electron microscopy confirmed the presence of Tad pili at the poles of B. breve UCC2003 cells. Conservation of the Tad pilus-encoding locus among other B. breve strains and among sequenced Bifidobacterium genomes supports the notion of a ubiquitous pili-mediated host colonization and persistence mechanism for bifidobacteria.

A Five-Strain Probiotic Combination Reduces Pathogen Shedding and Alleviates Disease Signs in Pigs Challenged with Salmonella enterica Serovar Typhimurium

ABSTRACT Salmonella spp. infection is a major cause of gastroenteritis, with many thousands of cases reported in the European Union every year. The use of probiotics offers the potential to improve this situation. Here, we investigate the effects of oral treatment of pigs with a defined lactic acid bacteria culture mixture on both clinical and microbiological signs of Salmonella enterica serovar Typhimurium infection. Fifteen weaned pigs blocked by sex and weight were administered control milk or a mixture of five probiotic strains as either a milk fermentate or milk suspension for a total of 30 days. The mixture consisted of two strains of Lactobacillus murinus and one strain each of Lactobacillus salivarius subsp. salivarius, Lactobacillus pentosus, and Pediococcus pentosaceous. Following probiotic administration for 6 days, animals were challenged orally with serovar Typhimurium; the health of the animals and the microbiological composition of their feces were monitored for 23 days postinfection. Animals treated with probiotic showed reduced incidence, severity, and duration of diarrhea. These animals also gained weight at a greater rate than control pigs administered skim milk. Mean fecal numbers of Salmonella were significantly reduced in probiotic-treated animals at 15 days postinfection (P = 0.01). The administered probiotic bacteria improved both the clinical and microbiological outcome of Salmonella infection. These strains offer significant benefit for use in the food industry and may have potential in human applications.

Regulation of host weight gain and lipid metabolism by bacterial bile acid modification in the gut.

Significance It is known that the gastrointestinal microbiota influences adiposity and weight gain in the host. However the mechanisms by which gut microorganisms coordinate host physiological processes are currently unclear. We demonstrate that a single, widely distributed function of the gut microbiota, bile salt hydrolase (BSH) activity, significantly influences lipid metabolism, weight gain, and cholesterol levels in the host. In our study microbial BSH activity was shown to direct expression of host signalling pathways with known roles in lipid metabolism, circadian rhythm, and epithelial cell function. The work defines the significant impact of in situ bile hydrolysis on host metabolism and indicates how this finding may be exploited as a potential intervention strategy for the control of obesity and metabolic syndrome. Alterations in the gastrointestinal microbiota have been implicated in obesity in mice and humans, but the key microbial functions influencing host energy metabolism and adiposity remain to be determined. Despite an increased understanding of the genetic content of the gastrointestinal microbiome, functional analyses of common microbial gene sets are required. We established a controlled expression system for the parallel functional analysis of microbial alleles in the murine gut. Using this approach we show that bacterial bile salt hydrolase (BSH) mediates a microbe–host dialogue that functionally regulates host lipid metabolism and plays a profound role in cholesterol metabolism and weight gain in the host. Expression of cloned BSH enzymes in the gastrointestinal tract of gnotobiotic or conventionally raised mice significantly altered plasma bile acid signatures and regulated transcription of key genes involved in lipid metabolism (Pparγ, Angptl4), cholesterol metabolism (Abcg5/8), gastrointestinal homeostasis (RegIIIγ), and circadian rhythm (Dbp, Per1/2) in the liver or small intestine. High-level expression of BSH in conventionally raised mice resulted in a significant reduction in host weight gain, plasma cholesterol, and liver triglycerides, demonstrating the overall impact of elevated BSH activity on host physiology. In addition, BSH activity in vivo varied according to BSH allele group, indicating that subtle differences in activity can have significant effects on the host. In summary, we demonstrate that bacterial BSH activity significantly impacts the systemic metabolic processes and adiposity in the host and represents a key mechanistic target for the control of obesity and hypercholesterolemia.

Isolation and characterization of anti-Salmonella lactic acid bacteria from the porcine gastrointestinal tract

Aims:  To identify lactic acid bacteria (LAB) of porcine intestinal origin with anti‐Salmonella activity.

Bacteriophages MR299-2 and NH-4 Can Eliminate Pseudomonas aeruginosa in the Murine Lung and on Cystic Fibrosis Lung Airway Cells

ABSTRACT Pseudomonas aeruginosa is a common cause of infection in the lungs of patients with cystic fibrosis (CF). In addition, biofilm formation and antibiotic resistance of Pseudomonas are major problems that can complicate antibiotic therapy. We evaluated the efficacy of using bacteriophages to kill the pathogen in both biofilms and in the murine lung. We isolated and characterized two phages from a local wastewater treatment plant, a myovirus (ϕNH-4) and a podovirus (ϕMR299-2). Both phages were active against clinical isolates of P. aeruginosa. Together, the two phages killed all 9 clinical isolate strains tested, including both mucoid and nonmucoid strains. An equal mixture of the two phages was effective in killing P. aeruginosa NH57388A (mucoid) and P. aeruginosa MR299 (nonmucoid) strains when growing as a biofilm on a cystic fibrosis bronchial epithelial CFBE41o- cell line. Phage titers increased almost 100-fold over a 24-h period, confirming replication of the phage. Furthermore, the phage mix was also effective in killing the pathogen in murine lungs containing 1 × 107 to 2 × 107 P. aeruginosa. Pseudomonas was effectively cleared (reduced by a magnitude of at least 3 to 4 log units) from murine lungs in 6 h. Our study demonstrates the efficacy of these two phages in killing clinical Pseudomonas isolates in the murine lung or as a biofilm on a pulmonary cell line and supports the growing interest in using phage therapy for the control and treatment of multidrug-resistant Pseudomonas lung infections in CF patients. IMPORTANCE Given the rise in antibiotic resistance, nonantibiotic therapies are required for the treatment of infection. This is particularly true for the treatment of Pseudomonas infection in patients with cystic fibrosis. We have identified two bacterial viruses (bacteriophages) that can kill Pseudomonas growing on human lung cells and in an animal model of lung infection. The use of bacteriophages is particularly appropriate because the killing agent can replicate on the target cell, generating fresh copies of the bacteriophage. Thus, in the presence of a target, the killing agent multiplies. By using two bacteriophages we can reduce the risk of resistant colonies developing at the site of infection. Bacteriophage therapy is an exciting field, and this study represents an important demonstration of efficacy in validated infection models. Given the rise in antibiotic resistance, nonantibiotic therapies are required for the treatment of infection. This is particularly true for the treatment of Pseudomonas infection in patients with cystic fibrosis. We have identified two bacterial viruses (bacteriophages) that can kill Pseudomonas growing on human lung cells and in an animal model of lung infection. The use of bacteriophages is particularly appropriate because the killing agent can replicate on the target cell, generating fresh copies of the bacteriophage. Thus, in the presence of a target, the killing agent multiplies. By using two bacteriophages we can reduce the risk of resistant colonies developing at the site of infection. Bacteriophage therapy is an exciting field, and this study represents an important demonstration of efficacy in validated infection models.

Relative Ability of Orally Administered Lactobacillus murinus To Predominate and Persist in the Porcine Gastrointestinal Tract

ABSTRACT Five porcine-derived Lactobacillus or Pediococcus isolates administered to pigs (n = 4), either singly or as a combination at ∼1010 CFU per day varied with respect to intestinal survival and persistence. Two Lactobacillus murinus strains survived best and were excreted at ∼107 to 108 CFU/g of feces. In contrast, Pediococcus pentosaceus DPC6006 had the lowest fecal count at ∼105 CFU/g and was excreted at a significantly lower level than both L. murinus strains. Fecal L. murinus DPC6003 counts were also significantly higher than both Lactobacillus salivarius DPC6005 and Lactobacillus pentosus DPC6004 (∼106 CFU/g). The L. murinus strains persisted for at least 9 days postadministration in both the feces and the cecum. Animals fed a combination of all five strains excreted ∼107 CFU of the administered strains/g, with L. murinus predominating, as determined by randomly amplified polymorphic DNA PCR. Postadministration, variation was observed between animals fed the strain combination, but in general, L. murinus DPC6002 and DPC6003 and L. pentosus DPC6004 predominated in the feces and the cecum while P. pentosaceus DPC6006 was detected only in the cecum. Fifteen days after the start of culture administration, mean fecal Enterobacteriaceae counts were significantly lower in some of the treatment groups. In addition, when mean preadministration counts were compared with those obtained after 21 days of culture administration, Enterobacteriaceae counts were reduced by ∼87 to 98% in pigs fed L. salivarius DPC6005, P. pentosaceus DPC6006, L. pentosus DPC6004, and the culture mix. In conclusion, the porcine intestinal isolates have potential as probiotic feed additives for pigs, with differences in strain performance highlighting the advantages of using culture combinations.

AgrD‐dependent quorum sensing affects biofilm formation, invasion, virulence and global gene expression profiles in Listeria monocytogenes

The Listeria monocytogenes Agr peptide‐sensing system has been analysed by creating a deletion mutant in agrD, the structural gene for the putative quorum‐sensing peptide. The ΔagrD mutant displayed significantly reduced biofilm formation, a defect which could be restored by genetic or physical complementation. A reduced invasion of Caco‐2 intestinal epithelial cells was observed for the ΔagrD mutant while phagocytosis by THP‐1 macrophages was unaffected. Additionally, the level of internalin A (InlA) in the cell wall was decreased in the ΔagrD mutant. Expression profiling of virulence genes (hlyA, actA, plcA, prfA and inlA) identified a finely tuned regulation which resulted in an impaired virulence response in the ΔagrD mutant. The mutant is also significantly attenuated for virulence in mice, as revealed by bioluminescent in vivo imaging. On day 3 post infection, systemic dissemination to livers and spleens had occurred for the wild type, whereas the ΔagrD mutant remained localized to the liver. Microarray analysis identified 126 and 670 genes as significantly regulated in exponential and stationary phase respectively. The results presented here suggest that peptide sensing plays an important role in the biology of L. monocytogenes, with relevant phenotypes in both the saprophytic and parasitic lifecycles.

Listeriolysin S, a Novel Peptide Haemolysin Associated with a Subset of Lineage I Listeria monocytogenes

Streptolysin S (SLS) is a bacteriocin-like haemolytic and cytotoxic virulence factor that plays a key role in the virulence of Group A Streptococcus (GAS), the causative agent of pharyngitis, impetigo, necrotizing fasciitis and streptococcal toxic shock syndrome. Although it has long been thought that SLS and related peptides are produced by GAS and related streptococci only, there is evidence to suggest that a number of the most notorious Gram-positive pathogenic bacteria, including Listeria monocytogenes, Clostridium botulinum and Staphylococcus aureus, produce related peptides. The distribution of the L. monocytogenes cluster is particularly noteworthy in that it is found exclusively among a subset of lineage I strains; i.e., those responsible for the majority of outbreaks of listeriosis. Expression of these genes results in the production of a haemolytic and cytotoxic factor, designated Listeriolysin S, which contributes to virulence of the pathogen as assessed by murine- and human polymorphonuclear neutrophil–based studies. Thus, in the process of establishing the existence of an extended family of SLS-like modified virulence peptides (MVPs), the genetic basis for the enhanced virulence of a proportion of lineage I L. monocytogenes may have been revealed.

Improved Luciferase Tagging System for Listeria monocytogenes Allows Real-Time Monitoring In Vivo and In Vitro

ABSTRACT An improved system for luciferase tagging Listeria monocytogenes was developed by constructing a highly active, constitutive promoter. This construct gave 100-fold-higher activity in broth than any native promoter tested and allowed for imaging of lux-tagged L. monocytogenes in food products, during murine infections, and in tumor targeting studies.

Effect of Lactobacillus salivarius Bacteriocin Abp118 on the Mouse and Pig Intestinal Microbiota

Lactobacilli are Gram-positive bacteria that are a subdominant element in the human gastrointestinal microbiota, and which are commonly used in the food industry. Some lactobacilli are considered probiotic, and have been associated with health benefits. However, there is very little culture-independent information on how consumed probiotic microorganisms might affect the entire intestinal microbiota. We therefore studied the impact of the administration of Lactobacillus salivarius UCC118, a microorganism well characterized for its probiotic properties, on the composition of the intestinal microbiota in two model animals. UCC118 has anti-infective activity due to production of the bacteriocin Abp118, a broad-spectrum class IIb bacteriocin, which we hypothesized could impact the microbiota. Mice and pigs were administered wild-type (WT) L. salivarius UCC118 cells, or a mutant lacking bacteriocin production. The microbiota composition was determined by pyrosequencing of 16S rRNA gene amplicons from faeces. The data show that L. salivarius UCC118 administration had no significant effect on proportions of major phyla comprising the mouse microbiota, whether the strain was producing bacteriocin or not. However, L. salivarius UCC118 WT administration led to a significant decrease in Spirochaetes levels, the third major phylum in the untreated pig microbiota. In both pigs and mice, L. salivarius UCC118 administration had an effect on Firmicutes genus members. This effect was not observed when the mutant strain was administered, and was thus associated with bacteriocin production. Surprisingly, in both models, L. salivarius UCC118 administration and production of Abp118 had an effect on Gram-negative microorganisms, even though Abp118 is normally not active in vitro against this group of microorganisms. Thus L. salivarius UCC118 administration has a significant but subtle impact on mouse and pig microbiota, by a mechanism that seems at least partially bacteriocin-dependent.