Characterization of endogenous plasmids from Lactobacillus salivarius UCC118.

The genome of Lactobacillus salivarius UCC118 comprises a 1.83-Mb chromosome, a 242-kb megaplasmid (pMP118), and two smaller plasmids of 20 kb (pSF118-20) and 44 kb (pSF118-44). Annotation and bioinformatic analyses suggest that both of the smaller plasmids replicate by a theta replication mechanism. Furthermore, it appears that they are transmissible, although neither possesses a complete set of conjugation genes. Plasmid pSF118-20 encodes a toxin-antitoxin system composed of pemI and pemK homologs, and this plasmid could be cured when PemI was produced in trans. The minimal replicon of pSF118-20 was determined by deletion analysis. Shuttle vector derivatives of pSF118-20 were generated that included the replication region (pLS203) and the replication region plus mobilization genes (pLS208). The plasmid pLS203 was stably maintained without selection in Lactobacillus plantarum, Lactobacillus fermentum, and the pSF118-20-cured derivative strain of L. salivarius UCC118 (strain LS201). Cloning in pLS203 of genes encoding luciferase and green fluorescent protein, and expression from a constitutive L. salivarius promoter, demonstrated the utility of this vector for the expression of heterologous genes in Lactobacillus. This study thus expands the knowledge base and vector repertoire of probiotic lactobacilli.

Multireplicon genome architecture of Lactobacillus salivarius.

Lactobacillus salivarius subsp. salivarius strain UCC118 is a bacteriocin-producing strain with probiotic characteristics. The 2.13-Mb genome was shown by sequencing to comprise a 1.83 Mb chromosome, a 242-kb megaplasmid (pMP118), and two smaller plasmids. Megaplasmids previously have not been characterized in lactic acid bacteria or intestinal lactobacilli. Annotation of the genome sequence indicated an intermediate level of auxotrophy compared with other sequenced lactobacilli. No single-copy essential genes were located on the megaplasmid. However, contingency amino acid metabolism genes and carbohydrate utilization genes, including two genes for completion of the pentose phosphate pathway, were megaplasmid encoded. The megaplasmid also harbored genes for the Abp118 bacteriocin, a bile salt hydrolase, a presumptive conjugation locus, and other genes potentially relevant for probiotic properties. Two subspecies of L. salivarius are recognized, salivarius and salicinius, and we detected megaplasmids in both subspecies by pulsed-field gel electrophoresis of sizes ranging from 100 kb to 380 kb. The discovery of megaplasmids of widely varying size in L. salivarius suggests a possible mechanism for genome expansion or contraction to adapt to different environments.

Correlation of probiotic Lactobacillus salivarius growth phase with its cell wall-associated proteome.

Lactobacillus salivarius subsp. salivarius UCC118 is a probiotic bacterium that was originally isolated from human intestinal tissues and was subsequently shown in a pilot study to alleviate symptoms associated with mild-moderate Crohn's disease. Strain UCC118 can adhere to animal and human intestinal tissue, and to both healthy and inflamed ulcerative colitis mucosa, irrespective of location in the gut. In this study, an enzymatic technique has been combined with proteomic analysis to correlate bacterial growth phase with the presence of factors present in the cell wall of the bacterium. Using PAGE electrophoresis, it was determined that progression from lag to log to stationary growth phases in vitro correlated with increasing prominence of an 84kD protein associated with in vitro adherence ability. Isolated proteins from the 84kD band region were further separated by two-dimensional electrophoresis, resolving this band into 20 individual protein spots at differing isoelectric points. The protein moieties were excised, trypsin digested and subjected to tandem mass spectrometry. The observed proteins are analogous to those reported to be associated with the Listeria monocytogenes cell-wall proteome, and include DnaK, Ef-Ts and pyruvate kinase. These data suggest that at least some of the beneficial attributes of probiotic lactobacilli, and in particular this strain, may be due to nonpathogenic mimicry of pathogens and potentially be mediated through a form of attenuated virulence.

Lactobacillus and bifidobacterium in irritable bowel syndrome: symptom responses and relationship to cytokine profiles.

BACKGROUND & AIMS: The aim of this study was to compare the response of symptoms and cytokine ratios in irritable bowel syndrome (IBS) with ingestion of probiotic preparations containing a lactobacillus or bifidobacterium strain. METHODS: Seventy-seven subjects with IBS were randomized to receive either Lactobacillus salivarius UCC4331 or Bifidobacterium infantis 35624, each in a dose of 1 x 10 10 live bacterial cells in a malted milk drink, or the malted milk drink alone as placebo for 8 weeks. The cardinal symptoms of IBS were recorded on a daily basis and assessed each week. Quality of life assessment, stool microbiologic studies, and blood sampling for estimation of peripheral blood mononuclear cell release of the cytokines interleukin (IL)-10 and IL-12 were performed at the beginning and at the end of the treatment phase. RESULTS: For all symptoms, with the exception of bowel movement frequency and consistency, those randomized to B infantis 35624 experienced a greater reduction in symptom scores; composite and individual scores for abdominal pain/discomfort, bloating/distention, and bowel movement difficulty were significantly lower than for placebo for those randomized to B infantis 35624 for most weeks of the treatment phase. At baseline, patients with IBS demonstrated an abnormal IL-10/IL-12 ratio, indicative of a proinflammatory, Th-1 state. This ratio was normalized by B infantis 35624 feeding alone. CONCLUSIONS: B infantis 35624 alleviates symptoms in IBS; this symptomatic response was associated with normalization of the ratio of an anti-inflammatory to a proinflammatory cytokine, suggesting an immune-modulating role for this organism, in this disorder.

Bacterial DNA within granulomas of patients with Crohn's disease--detection by laser capture microdissection and PCR.

OBJECTIVES: We previously reported the use of laser capture microdissection (LCM) and PCR to detect the presence of Mycobacterium paratuberculosis DNA in granulomas of patients with Crohn's disease. While this does not imply a cause-effect relationship, it may influence the disease process because bacterial DNA has immunomodulatory effects. The aim of this study was to determine whether DNA from nonmycobacterial commensals, such as Escherichia coli, is also increased in the granulomas of Crohn's disease. METHODS: Archival tissue from 15 surgical cases of Crohn's disease and 10 non-Crohn's granulomatous bowel disease controls were examined. Granulomas were isolated using LCM, and the extracted DNA was examined for presence of E. coli DNA by nested PCR amplification of a 135 base-pair segment of the uidA gene. RESULTS: E. coli DNA was detected in microdissected granulomas in 12/15 Crohn's disease patients and in 1/10 non-Crohn's control granulomas (p < 0.001). Also, E. coli DNA was detected in 8/15 Crohn's full-thickness sections and in 4/10 control full-thickness sections. CONCLUSIONS: E. coli DNA may be detected more frequently in Crohn's granulomas than in other non-Crohn's bowel granulomas. This may indicate a tendency for lumenal bacteria to colonize inflamed tissue, or may be due to increased uptake of bacterial DNA by gut antigen presenting cells. In light of previous detection of M. paratuberculosis DNA in Crohn's granulomas, the nonspecific nature of the type of bacterial DNA present in granulomas is evidence against any one bacterium having a significant causative role in Crohn's disease.

Prevalence of bone marrow micrometastases in esophagogastric cancer patients with and without neoadjuvant chemoradiotherapy.

BACKGROUND: Bone marrow micrometastases are present in a high proportion of patients undergoing curative resection for esophagogastric cancer. The incorporation of preoperative systemic therapies into these patients' treatment is widely practiced. This study investigates the effect of neoadjuvant chemoradiotherapy (CRT) on the incidence of micrometastases and the viability of detected tumor cells. MATERIALS AND METHODS: Rib bone marrow was obtained from patients (n = 106) in three centers, who were selected for potentially curative resection. Patients received neoadjuvant CRT plus surgery (n = 55), or surgery alone (n = 51). To detect micrometastases, mononuclear cells were isolated from fresh marrow and immediately stained immunohistochemically with an anti-cytokeratin-18 antibody using the APAAP technique. Tumor cell viability was assessed by immunohistochemical staining of marrow cell cultures for cytokeratin-positive cells. RESULTS: Micrometastases were detected in fresh marrow in 42% (23/55) of patients who received neoadjuvant CRT plus surgery, and in 67% (34/51) of patients treated with surgery alone. Viable tumor cells were demonstrated in 10 of 18 marrow cultures from CRT plus surgery cases. In this patient subset, combination of results of staining fresh and cultured marrow significantly increased micromet detection to 78%. CONCLUSIONS: A significant proportion of patients with esophagogastric cancer have disseminated viable tumor cells at time of surgery, irrespective of pre-operative treatment. The use of marrow culture in parallel with fresh marrow staining may increase the detection of micrometastases. The persistence of tumor cells resistant to systemic therapy may explain why these regimens fail in a majority of patients.

Neurokinin-1 receptor (NK-1R) expression is induced in human colonic epithelial cells by proinflammatory cytokines and mediates proliferation in response to substance P.

We have previously shown that the receptor for substance P (SP), neurokinin-1 receptor (NK-1R), is a marker of human mucosal but not peripheral mononuclear cells. In the present study, we investigate NK-1R expression in the human colonic mucosa in vivo, particularly in the epithelial cells. We investigate the influence of proinflammatory Th1 cytokines and SP on expression and function of NK-1R in colonic epithelial cells in vitro. Using in situ hybridization to detect NK-1R mRNA, and immunohistochemistry to detect NK-1R protein, colonic epithelial cells were found to express NK-1R in vivo. In contrast, colon epithelial cell lines (Caco-2, HT29, SW620, T84) were negative for NK-1R mRNA and protein. However, stimulation with a proinflammatory cytokine cocktail containing IFN-gamma, TNF-alpha, and IL-1beta, caused induction of NK-1R expression. Expression of NK-1R in human colonic epithelial cells in vivo may therefore reflect cytokine conditioning by the mucosal microenvironment. SP did not alter ion transport in monolayers of cytokine-treated T84 cells. While SP stimulated epithelial ion transport in colonic mucosae ex vivo, this was not a direct effect of SP on the epithelial cells, and appeared to be neurally mediated. However, SP (10(-10)-10(-8) M) elicited a dose-dependent proliferative effect on cytokine-stimulated, but not unstimulated, SW620 cells. Proliferation of the epithelial cells in response to SP was mediated specifically via cytokine-induced NK-1R, since an NK-1R-specific antagonist (Spantide 1) completely blocked SP-mediated proliferation in the cytokine-treated cells. Our results therefore demonstrate that proinflammatory cytokines induce expression of NK-1R in human colonic epithelial cell lines, and that SP induces proliferation of the epithelial cells via cytokine-induced NK-1R.

Genotoxicity of fecal water in a free-living Irish population.

The alkaline single-cell gel electrophoresis (comet) assay was used to investigate the genotoxicity of fecal water (FW) isolated from 47 Irish subjects using Caco-2 colonocytes as target cells. Two methods of comet assay analysis were compared to determine the extent of DNA damage and to categorize the samples as having no, low-to-moderate, or high genotoxicity. FW was isolated from stool samples by centrifugation and tested for its ability to induce DNA damage in Caco-2 cells. DNA damage was assessed using the comet assay by measuring the extent of DNA migration from the nucleus (microns, tail length) or by classifying the nuclei into five different categories depending on their morphology. Data collected from the two methods were used to categorize the FW samples on the basis of their genotoxic activity. Both methods showed good agreement. There was an approximately 50:50 split, with half the samples having some level of genotoxic activity and half having no genotoxicity. About one-third of the samples were considered to be highly genotoxic. There was a trend for low pH of the FW to be associated with increased DNA damage, but this was not significant. The results presented in this report show a relatively high incidence of genotoxic FW in samples derived from a free-living Irish population. Our data demonstrate the suitability of classifying nuclei on the basis of their morphology as a means of determining DNA damage. This procedure is very rapid and, therefore, advantageous in analyzing a large number of slides in the absence of an image analysis system.

Characterization of the genetic locus responsible for the production of ABP-118, a novel bacteriocin produced by the probiotic bacterium Lactobacillus salivarius subsp. salivarius UCC118.

ABP-118, a small heat-stable bacteriocin produced by Lactobacillus salivarius subsp. salivarius UCC118, a strain isolated from the ileal-caecal region of the human gastrointestinal tract, was purified to homogeneity. Using reverse genetics, a DNA fragment specifying part of ABP-118 was identified on a 10769 bp chromosomal region. Analysis of this region revealed that ABP-118 was a Class IIb two-peptide bacteriocin composed of Abp118alpha, which exhibited the antimicrobial activity, and Abp118beta, which enhanced the antimicrobial activity. The gene conferring strain UCC118 immunity to the action of ABP-118, abpIM, was identified downstream of the abp118beta gene. Located further downstream of abp118beta, several ORFs were identified whose deduced proteins resembled those of proteins involved in bacteriocin regulation and secretion. Heterologous expression of ABP-118 was achieved in Lactobacillus plantarum, Lactococcus lactis and Bacillus cereus. In addition, the abp118 locus encoded an inducing peptide, AbpIP, which was shown to play a role in the regulation of ABP-118 production. This novel bacteriocin is, to the authors' knowledge, the first to be isolated from a known human probiotic bacterium and to be characterized at the genetic level.