Autochthonous faecal viral transfer (FVT) impacts the murine microbiome after antibiotic perturbation.

BACKGROUND: It has become increasingly accepted that establishing and maintaining a complex and diverse gut microbiota is fundamental to human health. There are growing efforts to identify means of modulating and influencing the microbiota, especially in individuals who have experienced a disruption in their native microbiota. Faecal microbiota transplantation (FMT) is one method that restores diversity to the microbiota of an individual by introducing microbes from a healthy donor. FMT introduces the total microbial load into the recipient, including the bacteria, archaea, yeasts, protists and viruses. In this study, we investigated whether an autochthonous faecal viral transfer (FVT), in the form of a sterile faecal filtrate, could impact the recovery of a bacteriome disrupted by antibiotic treatment. RESULTS: Following antibiotic disruption of the bacteriome, test mice received an FVT harvested prior to antibiotic treatment, while control mice received a heat- and nuclease-treated FVT. In both groups of mice, the perturbed microbiome reverted over time to one more similar to the pre-treatment one. However, the bacteriomes of mice that received an FVT, in which bacteriophages predominate, separated from those of the control mice as determined by principal co-ordinate analysis (PCoA). Moreover, analysis of the differentially abundant taxa indicated a closer resemblance to the pre-treatment bacteriome in the test mice that had received an FVT. Similarly, metagenomic sequencing of the virome confirmed that faecal bacteriophages of FVT and control mice differed over time in both abundance and diversity, with the phages constituting the FVT persisting in mice that received them. CONCLUSIONS: An autochthonous virome transfer reshaped the bacteriomes of mice post-antibiotic treatment such that they more closely resembled the pre-antibiotic microbiota profile compared to mice that received non-viable phages. Thus, FVT may have a role in addressing antibiotic-associated microbiota alterations and potentially prevent the establishment of post-antibiotic infection. Given that bacteriophages are biologically inert in the absence of their host bacteria, they could form a safe and effective alternative to whole microbiota transplants that could be delivered during/following perturbation of the gut flora.

Bioavailability of the anti-clostridial bacteriocin thuricin CD in gastrointestinal tract.

Thuricin CD is a two component narrow spectrum bacteriocin comprising two peptides with targeted activity against Clostridium difficile. This study examined the bioavailability of thuricin with a view to developing it as an effective antimicrobial against intestinal infection. One of the peptides, Trn-ß, was found to be degraded by the gastric enzymes pepsin and α-chymotrypsin both in vitro and in vivo, whereas Trn-α was resistant to digestion by these enzymes and hence was detected in the intestinal porcine digesta following oral ingestion by pigs. In order to determine if spores of the producing organism Bacillus thuringiensis DPC 6431 could be used to deliver the bacteriocin to the gut, spores were fed to 30 mice (approx. 10(8)-2×10(8) per animal) and their germination, growth and production of thuricin in the gastrointestinal tract (GIT) of the animals was monitored. Almost 99 % of the spores delivered to the GIT were excreted in the first 24 h and neither Trn-α nor Trn-ß was detected in the gut or faecal samples of the test mice, indicating that ingestion of B. thuringiensis spores may not be a suitable vehicle for the delivery of thuricin CD. When thuricin CD was delivered rectally to mice (n = 40) and C. difficile shedding monitored at 1, 6, 12 and 24 h post-treatment, there was a >95 % (>1.5 log units) reduction of C. difficile 027 in the colon contents of infected mice (n = 10) 1 h post-treatment compared with the control group (n = 10; P<0.001). Furthermore, 6 h post-treatment there was a further 1.5 log reduction in C. difficile numbers (n = 10) relative to the control group (n = 10; P<0.05). These results would suggest that rectal administration of thuricin may be a promising mode of delivery of thuricin CD to the colon.

Exopolysaccharide-producing probiotic Lactobacilli reduce serum cholesterol and modify enteric microbiota in ApoE-deficient mice.

BACKGROUND: Probiotic bacteria have been associated with a reduction in cardiovascular disease risk, a leading cause of death and disability. OBJECTIVES: The aim of this study was to assess the impact of dietary administration of exopolysaccharide-producing probiotic Lactobacillus cultures on lipid metabolism and gut microbiota in apolipoprotein E (apoE)-deficient mice. METHODS: First, we examined lipid metabolism in response to dietary supplementation with recombinant ß-glucan-producing Lactobacillus paracasei National Food Biotechnology Centre (NFBC) 338 expressing the glycosyltransferase (Gtf) gene from Pediococcus parvulus 2.6 (GTF), and naturally exopolysaccharide-producing Lactobacillus mucosae Dairy Product Culture Collection (DPC) 6426 (DPC 6426) compared with the non-ß-glucan-producing isogenic control strain Lactobacillus paracasei NFBC 338 (PNZ) and placebo (15% wt:vol trehalose). Second, we examined the effects on the gut microbiota of dietary administration of DPC 6426 compared with placebo. Probiotic Lactobacillus strains at 1 × 10(9) colony-forming units/d per animal were administered to apoE(-/-) mice fed a high-fat (60% fat)/high-cholesterol (2% wt:wt) diet for 12 wk. At the end of the study, aortic plaque development and serum, liver, and fecal variables involved in lipid metabolism were analyzed, and culture-independent microbial analyses of cecal content were performed. RESULTS: Total cholesterol was reduced in serum (P < 0.001; ∼33-50%) and liver (P < 0.05; ∼30%) and serum triglyceride concentrations were reduced (P < 0.05; ∼15-25%) in mice supplemented with GTF or DPC 6426 compared with the PNZ or placebo group, respectively. In addition, dietary intervention with GTF led to increased amounts of fecal cholesterol excretion (P < 0.05) compared with all other groups. Compositional sequencing of the gut microbiota revealed a greater prevalence of Porphyromonadaceae (P = 0.001) and Prevotellaceae (P = 0.001) in the DPC 6426 group and lower proportions of Clostridiaceae (P < 0.05), Peptococcaceae (P < 0.001), and Staphylococcaceae (P < 0.01) compared with the placebo group. CONCLUSION: Ingestion of exopolysaccharide-producing lactobacilli resulted in seemingly favorable improvements in lipid metabolism, which were associated with changes in the gut microbiota of mice.

In vivo assessment of growth and virulence gene expression during commensal and pathogenic lifestyles of luxABCDE-tagged Enterococcus faecalis strains in murine gastrointestinal and intravenous infection models.

Cytolysin and gelatinase are prominent pathogenicity determinants associated with highly virulent Enterococcus faecalis strains. In an effort to explore the expression profiles of these virulence traits in vivo, we have employed E. faecalis variants expressing the luxABCDE cassette under the control of either the P16S, cytolysin, or gelatinase promoter for infections of Galleria mellonella caterpillars and mice. Systemic infection of G. mellonella with bioluminescence-tagged E. faecalis MMH594 revealed temporal regulation of both gelatinase and cytolysin promoters and demonstrated that these traits were induced in response to the host environment. Gavage of mice pretreated perorally with antibiotics resulted in efficient colonization of the murine gastrointestinal tract (GIT) in a strain-dependent manner, where the commensal baby isolate EF62 was more persistent than the nosocomial isolate MMH594. A highly significant correlation (R(2) > 0.94) was found between bioluminescence and the CFU counts in mouse fecal samples. Both strains showed similar preferences for growth and persistence in the ileum, cecum, and colon. Cytolysin expression was uniform in these compartments of the intestinal lumen. In spite of high numbers (10(9) CFU/g of intestinal matter) in the ileum, cecum, and colon, no evidence of translocation or systemic infection could be observed. In the murine intravenous infection model, cytolysin expression was readily detected in the liver, kidneys, and bladder. At 72 h postinfection, the highest bacterial loads were found in the liver, kidneys, and spleen, with organ-specific expression levels of cytolysin ~400- and ~900-fold higher in the spleen and heart, respectively, than in the liver and kidneys. Taken together, this system based on the bioluminescence imaging technology is established as a new, powerful method to monitor the differential regulation of E. faecalis virulence determinants and to study the spatiotemporal course of infection in living animals in real time.

In vivo activity of nisin A and nisin V against Listeria monocytogenes in mice.

BACKGROUND: Lantibiotics are post-translationally modified antimicrobial peptides, of which nisin A is the most extensively studied example. Bioengineering of nisin A has resulted in the generation of derivatives with increased in vitro potency against Gram-positive bacteria. Of these, nisin V (containing a Met21Val change) is noteworthy by virtue of exhibiting enhanced antimicrobial efficacy against a wide range of clinical and food-borne pathogens, including Listeria monocytogenes. However, this increased potency has not been tested in vivo. RESULTS: Here we address this issue by assessing the ability of nisin A and nisin V to control a bioluminescent strain of Listeria monocytogenes EGDe in a murine infection model.More specifically, Balb/c mice were infected via the intraperitoneal route at a dose of 1 × 10(5) cfu/animal and subsequently treated intraperitoneally with either nisin V, nisin A or a PBS control. Bioimaging of the mice was carried out on day 3 of the trial. Animals were then sacrificed and levels of infection were quantified in the liver and spleen. CONCLUSION: This analysis revealed that nisin V was more effective than Nisin A with respect to controlling infection and therefore merits further investigation with a view to potential chemotherapeutic applications.

Virulence aspects of Listeria monocytogenes LO28 high pressure-resistant variants.

High pressure treatment is a novel food processing approach for reducing pathogens in foods and food ingredients. However, relatively little is known about the pathogenic potential of organisms that survive the treatment. Twelve previously isolated and characterized variants of Listeria monocytogenes LO28 obtained after a high pressure treatment were assessed for their virulence potential and antibiotic susceptibility. Ten variants showed attenuated virulence while two variants retained full virulence in a mouse model of infection. Seven of the attenuated variants demonstrated a reduction in virulence factor activity. Compared to the wild type, all variants exhibited similar or increased susceptibility to multiple antibiotics commonly used in listeriosis treatment.

A novel host-responsive sensor mediates virulence and type III secretion during Pseudomonas aeruginosa-host cell interactions.

Sensitive sensory mechanisms are instrumental in affording Pseudomonas aeruginosa the capacity to establish diverse yet severe human infections, which can manifest themselves in long-term untreatable disease. The ability of P. aeruginosa to tightly regulate gene expression and virulence factor production, in response to activation of these sensory components, enables the pathogen to sustain infection despite the host immune response and aggressive antibiotic treatment. Although a number of factors are recognized as playing a role in early infection, very little is known regarding the sensors involved in this process. In this study, we identified P. aeruginosa PA3191 as a novel host-responsive sensor that plays a key role during P. aeruginosa-host interactions and is required for optimum colonization and dissemination in a mouse model of infection. We demonstrated that PA3191 contributed to modulation of the type III secretion system (T3SS) in response to host cells and T3SS-inducing conditions in vitro. PA3191 (designated GtrS) acted in concert with the response regulator GltR to regulate the OprB transport system and subsequently carbon metabolism. Through this signal transduction pathway, T3SS activation was mediated via the RsmAYZ regulatory cascade and involved the global anaerobic response regulator Anr.

HmgR, a key enzyme in the mevalonate pathway for isoprenoid biosynthesis, is essential for growth of Listeria monocytogenes EGDe.

Isoprenoids may be synthesized via one of two pathways, the classical mevalonate pathway or the alternative 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. While the majority of bacteria utilize a single pathway for isoprenoid biosynthesis, Listeria monocytogenes is unusual in possessing the complete set of genes for both pathways. Here, we utilized new molecular tools to create precise gene deletions in selected genes encoding enzymes of both pathways, gcpE, lytB (encoding proteins in the MEP pathway) and hmgR (encoding a protein in the mevalonate pathway). We demonstrate that the hmgR gene can only be deleted when the growth medium is supplemented with exogenous mevalonate. Furthermore, full growth of the mutant in the absence of mevalonate was only possible when the intact hmgR gene was supplied in trans using an IPTG-inducible expression system. Murine competitive index assays performed via the oral and intraperitoneal routes of infection revealed that the mevalonate hmgR mutant could not be recovered from livers and spleens 3 days post-infection. We propose that HmgR in L. monocytogenes EGDe is involved in essential metabolic functions and that an intact MEP pathway is not capable of sustaining growth.

Factors affecting survival of Listeria monocytogenes and Listeria innocua in soil samples.

We investigated the ability of several strains of L. monocytogenes and Listeria innocua strains to survive in local soil samples in vitro. Survival of three L. monocytogenes strains, EGDe, CD83, and CD1038, and three L. innocua strains, CLIP, FH2117, FH2152, was monitored in soil samples by direct enumeration of colony-forming units on selective agar. The study did not demonstrate any species-specific difference in soil survival, and all Listeria strains exhibited a marked decline in numbers over time. Bioluminescence imaging approaches to detect lux-tagged strains in soil proved largely ineffective, most likely due to the reduced metabolic activity of strains in this environment. We investigated the influence of specific factors including the presence of a background microbiota, growth temperature, moisture and strain motility upon persistence in this environment. A sequenced L. monocytogenes strain, EGDe, was capable of active growth in sterile soil yet exhibited a decline in the presence of the normal soil microbiota. Furthermore, greater survival was seen at lower incubation temperatures in normal soil. Finally, we demonstrated a direct correlation between motility and survival of L. monocytogenes in soil with highly motile L. monocytogenes strains exhibiting greater soil survival than non-motile mutants.

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

Directed evolution and targeted mutagenesis to murinize Listeria monocytogenes internalin A for enhanced infectivity in the murine oral infection model.

BACKGROUND: Internalin A (InlA) is a critical virulence factor which mediates the initiation of Listeria monocytogenes infection by the oral route in permissive hosts. The interaction of InlA with the host cell ligand E-cadherin efficiently stimulates L. monocytogenes entry into human enterocytes, but has only a limited interaction with murine cells. RESULTS: We have created a surface display library of randomly mutated InlA in a non-invasive heterologous host Lactococcus lactis in order to create and screen novel variants of this invasion factor. After sequential passage through a murine cell line (CT-26), multiple clones with enhanced invasion characteristics were identified. Competitive index experiments were conducted in mice using selected mutations introduced into L. monocytogenes EGD-e background. A novel single amino acid change was identified which enhanced virulence by the oral route in the murine model and will form the basis of further engineering approaches. As a control a previously described EGD-InlA(m) murinized strain was also re-created as part of this study with minor modifications and designated EGD-e InlA(m)*. The strain was created using a procedure that minimizes the likelihood of secondary mutations and incorporates Listeria-optimized codons encoding the altered amino acids. L. monocytogenes EGD-e InlA(m)* yielded consistently higher level murine infections by the oral route when compared to EGD-e, but did not display the two-fold increased invasion into a human cell line that was previously described for the EGD-InlA(m) strain. CONCLUSIONS: We have used both site-directed mutagenesis and directed evolution to create variants of InlA which may inform future structure-function analyses of this protein. During the course of the study we engineered a murinized strain of L. monocytogenes EGD-e which shows reproducibly higher infectivity in the intragastric murine infection model than the wild type, but does not display enhanced entry into human cells as previously observed. This murinized L. monocytogenes strain will provide a useful tool for the analysis of the gastrointestinal phase of listeriosis.

Allelic variation of bile salt hydrolase genes in Lactobacillus salivarius does not determine bile resistance levels.

Commensal lactobacilli frequently produce bile salt hydrolase (Bsh) enzymes whose roles in intestinal survival are unclear. Twenty-six Lactobacillus salivarius strains from different sources all harbored a bsh1 allele on their respective megaplasmids. This allele was related to the plasmid-borne bsh1 gene of the probiotic strain UCC118. A second locus (bsh2) was found in the chromosomes of two strains that had higher bile resistance levels. Four Bsh1-encoding allele groups were identified, defined by truncations or deletions involving a conserved residue. In vitro analyses showed that this allelic variation was correlated with widely varying bile deconjugation phenotypes. Despite very low activity of the UCC118 Bsh1 enzyme, a mutant lacking this protein had significantly lower bile resistance, both in vitro and during intestinal transit in mice. However, the overall bile resistance phenotype of this and other strains was independent of the bsh1 allele type. Analysis of the L. salivarius transcriptome upon exposure to bile and cholate identified a multiplicity of stress response proteins and putative efflux proteins that appear to broadly compensate for, or mask, the effects of allelic variation of bsh genes. Bsh enzymes with different bile-degrading kinetics, though apparently not the primary determinants of bile resistance in L. salivarius, may have additional biological importance because of varying effects upon bile as a signaling molecule in the host.

Specific osmolyte transporters mediate bile tolerance in Listeria monocytogenes.

The food-borne pathogenic bacterium Listeria monocytogenes has the potential to adapt to an array of suboptimal growth environments encountered within the host. The pathogen is relatively bile tolerant and has the capacity to survive and grow within both the small intestine and the gallbladder in murine models of oral infection. We have previously demonstrated a role for the principal carnitine transport system of L. monocytogenes (OpuC) in gastrointestinal survival of the pathogen (R. Sleator, J. Wouters, C. G. M. Gahan, T. Abee, and C. Hill, Appl. Environ. Microbiol. 67:2692-2698, 2001). However, the mechanisms by which OpuC, or indeed carnitine, protects the pathogen in this environment are unclear. In the current study, systematic analysis of strains with mutations in osmolyte transporters revealed a role for OpuC in resisting the acute toxicity of bile, with a minor role also played by BetL, a secondary betaine uptake system which also exhibits a low affinity for carnitine. In addition, the toxic effects of bile on wild-type L. monocytogenes cells were ameliorated when carnitine (but not betaine) was added to the medium. lux-promoter fusions to the promoters of the genes encoding the principal osmolyte uptake systems Gbu, BetL, and OpuC and the known bile tolerance system BilE were constructed. Promoter activity for all systems was significantly induced in the presence of bile, with the opuC and bilE promoters exhibiting the highest levels of bile-dependent expression in vitro and the betL and bilE promoters showing the highest expression levels in the intestines of orally inoculated mice. A direct comparison of all osmolyte transporter mutants in a murine oral infection model confirmed a major role for OpuC in intestinal persistence and systemic invasion and a minor role for the BetL transporter in fecal carriage. This study therefore demonstrates a previously unrecognized function for osmolyte uptake systems in bile tolerance in L. monocytogenes.

Short-term consumption of a high-fat diet increases host susceptibility to Listeria monocytogenes infection.

BACKGROUND: A westernized diet comprising a high caloric intake from animal fats is known to influence the development of pathological inflammatory conditions. However, there has been relatively little focus upon the implications of such diets for the progression of infectious disease. Here, we investigated the influence of a high-fat (HF) diet upon parameters that influence Listeria monocytogenes infection in mice. RESULTS: We determined that short-term administration of a HF diet increases the number of goblet cells, a known binding site for the pathogen, in the gut and also induces profound changes to the microbiota and promotes a pro-inflammatory gene expression profile in the host. Host physiological changes were concordant with significantly increased susceptibility to oral L. monocytogenes infection in mice fed a HF diet relative to low fat (LF)- or chow-fed animals. Prior to Listeria infection, short-term consumption of HF diet elevated levels of Firmicutes including Coprococcus, Butyricicoccus, Turicibacter and Clostridium XIVa species. During active infection with L. monocytogenes, microbiota changes were further exaggerated but host inflammatory responses were significantly downregulated relative to Listeria-infected LF- or chow-fed groups, suggestive of a profound tempering of the host response influenced by infection in the context of a HF diet. The effects of diet were seen beyond the gut, as a HF diet also increased the sensitivity of mice to systemic infection and altered gene expression profiles in the liver. CONCLUSIONS: We adopted a systems approach to identify the effects of HF diet upon L. monocytogenes infection through analysis of host responses and microbiota changes (both pre- and post-infection). Overall, the results indicate that short-term consumption of a westernized diet has the capacity to significantly alter host susceptibility to L. monocytogenes infection concomitant with changes to the host physiological landscape. The findings suggest that diet should be a consideration when developing models that reflect human infectious disease.

Analysis of the isoprenoid biosynthesis pathways in Listeria monocytogenes reveals a role for the alternative 2-C-methyl-D-erythritol 4-phosphate pathway in murine infection.

Most bacteria synthesize isoprenoids through one of two essential pathways which provide the basic building block, isopentyl diphosphate (IPP): either the classical mevalonate pathway or the alternative non-mevalonate 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. However, postgenomic analyses of the Listeria monocytogenes genome revealed that this pathogen possesses the genetic capacity to produce the complete set of enzymes involved in both pathways. The nonpathogenic species Listeria innocua naturally lacks the last two genes (gcpE and lytB) of the MEP pathway, and bioinformatic analyses strongly suggest that the genes have been lost through evolution. In the present study we show that heterologous expression of gcpE and lytB in L. innocua can functionally restore the MEP pathway in this organism and confer on it the ability to induce Vgamma9 Vdelta2 T cells. We have previously confirmed that both pathways are functional in L. monocytogenes and can provide sufficient IPP for normal growth in laboratory media (M. Begley, C. G. Gahan, A. K. Kollas, M. Hintz, C. Hill, H. Jomaa, and M. Eberl, FEBS Lett. 561:99-104, 2004). Here we describe a targeted mutagenesis strategy to create a double pathway mutant in L. monocytogenes which cannot grow in the absence of exogenously provided mevalonate, confirming the requirement for at least one intact pathway for growth. In addition, murine studies revealed that mutants lacking the MEP pathway were impaired in virulence relative to the parent strain during intraperitoneal infection, while mutants lacking the classical mevalonate pathway were not impaired in virulence potential. In vivo bioluminescence imaging also confirmed in vivo expression of the gcpE gene (MEP pathway) during murine infection.

Pseudomonas aeruginosa RsmA plays an important role during murine infection by influencing colonization, virulence, persistence, and pulmonary inflammation.

The ability of Pseudomonas aeruginosa to cause a broad range of infections in humans is due, at least in part, to its adaptability and its capacity to regulate the expression of key virulence genes in response to specific environmental conditions. Multiple two-component response regulators have been shown to facilitate rapid responses to these environmental conditions, including the coordinated expression of specific virulence determinants. RsmA is a posttranscriptional regulatory protein which controls the expression of a number of virulence-related genes with relevance for acute and chronic infections. Many membrane-bound sensors, including RetS, LadS, and GacS, are responsible for the reciprocal regulation of genes associated with acute infection and chronic persistence. In P. aeruginosa this is due to sensors influencing the expression of the regulatory RNA RsmZ, with subsequent effects on the level of free RsmA. While interactions between an rsmA mutant and human airway epithelial cells have been examined in vitro, the role of RsmA during infection in vivo has not been determined yet. Here the function of RsmA in both acute and chronic models of infection was examined. The results demonstrate that RsmA is involved in initial colonization and dissemination in a mouse model of acute pneumonia. Furthermore, while loss of RsmA results in reduced colonization during the initial stages of acute infection, the data show that mutation of rsmA ultimately favors chronic persistence and results in increased inflammation in the lungs of infected mice.

Development of multiple strain competitive index assays for Listeria monocytogenes using pIMC; a new site-specific integrative vector.

BACKGROUND: The foodborne, gram-positive pathogen, Listeria monocytogenes, is capable of causing lethal infections in compromised individuals. In the post genomic era of L. monocytogenes research, techniques are required to identify and validate genes involved in the pathogenicity and environmental biology of the organism. The aim here was to develop a widely applicable method to tag L. monocytogenes strains, with a particular emphasis on the development of multiple strain competitive index assays. RESULTS: We have constructed a new site-specific integrative vector, pIMC, based on pPL2, for the selection of L. monocytogenes from complex samples. The pIMC vector was further modified through the incorporation of IPTG inducible markers (antibiotic and phenotypic) to produce a suite of four vectors which allowed the discrimination of multiple strains from a single sample. We were able to perform murine infection studies with up to four EGDe isolates within a single mouse and showed that the tags did not impact upon growth rate or virulence. The system also allowed the identification of subtle differences in virulence between strains of L. monocytogenes commonly used in laboratory studies. CONCLUSION: This study has developed a competitive index assay that can be broadly applied to all L. monocytogenes strains. Improved statistical robustness of the data was observed, resulting in fewer mice being required for virulence assays. The competitive index assays provide a powerful method to analyse the virulence or fitness of L. monocytogenes in complex biological samples.

Genome-Wide Search for Genes Required for Bifidobacterial Growth under Iron-Limitation.

Bacteria evolved over millennia in the presence of the vital micronutrient iron. Iron is involved in numerous processes within the cell and is essential for nearly all living organisms. The importance of iron to the survival of bacteria is obvious from the large variety of mechanisms by which iron may be acquired from the environment. Random mutagenesis and global gene expression profiling led to the identification of a number of genes, which are essential for Bifidobacterium breve UCC2003 survival under iron-restrictive conditions. These genes encode, among others, Fe-S cluster-associated proteins, a possible ferric iron reductase, a number of cell wall-associated proteins, and various DNA replication and repair proteins. In addition, our study identified several presumed iron uptake systems which were shown to be essential for B. breve UCC2003 growth under conditions of either ferric and/or ferrous iron chelation. Of these, two gene clusters encoding putative iron-uptake systems, bfeUO and sifABCDE, were further characterised, indicating that sifABCDE is involved in ferrous iron transport, while the bfeUO-encoded transport system imports both ferrous and ferric iron. Transcription studies showed that bfeUO and sifABCDE constitute two separate transcriptional units that are induced upon dipyridyl-mediated iron limitation. In the anaerobic gastrointestinal environment ferrous iron is presumed to be of most relevance, though a mutation in the sifABCDE cluster does not affect B. breve UCC2003's ability to colonise the gut of a murine model.