SummaryThe 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 DagrD 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 DagrD mutant while phagocytosis by THP-1 macrophages was unaffected. Additionally, the level of internalin A (InlA) in the cell wall was decreased in the DagrD 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 DagrD 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 DagrD 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.

ABSTRACTPseudomonas aeruginosais a common cause of infection in the lungs of patients with cystic fibrosis (CF). In addition, biofilm formation and antibiotic resistance ofPseudomonasare 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 ofP. 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 killingP. aeruginosaNH57388A (mucoid) andP. aeruginosaMR299 (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 × 107to 2 × 107P. aeruginosa.Pseudomonaswas 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 clinicalPseudomonasisolates 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-resistantPseudomonaslung infections in CF patients.IMPORTANCEGiven the rise in antibiotic resistance, nonantibiotic therapies are required for the treatment of infection. This is particularly true for the treatment ofPseudomonasinfection in patients with cystic fibrosis. We have identified two bacterial viruses (bacteriophages) that can killPseudomonasgrowing 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.

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.

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.

Five porcine-derived Lactobacillus or Pediococcus isolates administered to pigs (n ‫؍‬ 4), either singly or as a combination at ϳ10 10 CFU per day varied with respect to intestinal survival and persistence. Two Lactobacillus murinus strains survived best and were excreted at ϳ10 7 to 10 8 CFU/g of feces. In contrast, Pediococcus pentosaceus DPC6006 had the lowest fecal count at ϳ10 5 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 (ϳ10 6 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 ϳ10 7 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 ϳ87 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.