Oral Lactobacillus plantarum NCIMB 8825 inhibits adhesion, invasion and metabolism of Neisseria meningitidis serogroup B and affords anti-inflammatory and cytotoxic protection to nasopharyngeal epithelial cells

In this study, we investigate the potential for oral Lactobacilli (LB) to afford innate protection against nasopharyngeal coloniser Neisseria meningitidis serogroup B (NmB), which causes the bulk of UK meningococcal disease. Oral isolates of L. plantarum, L. salivarious, L. casei, L. rhamnosus, L. gasseri and gut probiotic L. rhamnosus GG were assessed for their ability to suppress nasopharyngeal epithelial inflammatory responses to pathogenic NmB. The specificity of attenuation was examined using TLR 2 ligand, Pam3Cys, and early response cytokine IL1β; and the mechanism of attenuation was explored using heat-killed organisms and conditioned medium. Pro-inflammatory IL-6 and TNFα cytokine secretion was quantified by ELISA and associated cell death was quantified by PI staining and LDH release. NmB adhesion, invasion and metabolism were determined using standard gentamicin protection with viable counts, and bioluminescence, respectively. L. plantarum and L. salivarious suppressed IL-6 and TNFα secretions from NmB-infected epithelial cells. LB did not need to be alive and could suppress using secretions, which were independent of TLR2 or IL1β receptor signalling. L. plantarum, in particular, reduced NmB-induced necrotic cell death of epithelial monolayers. Like L. salivarious, it significantly inhibited NmB adhesion but uniquely L. plantarum abolished NmB invasion. Using bioluminescence as a reporter of pathogen metabolism, L. plantarum and its secretions were found to inhibit NmB metabolism during cell invasion assays. We conclude that oral L. plantarum and its secretions could be used to help reduce the burden of meningococcal disease by removing the intracellular nasopharyngeal reservoir of NmB

Construction and evaluation of multisite recombinatorial (Gateway) cloning vectors for Gram-positive bacteria

<p>Abstract</p> <p>Background</p> <p>The Gateway recombinatorial cloning system allows easy and rapid joining of DNA fragments. Here we report the construction and evaluation of three different Gram-positive vectors that can be used with the Multisite Gateway cloning system to rapidly produce new gene arrangements in plasmid constructs for use in a variety of Gram-positive bacteria.</p> <p>Results</p> <p>Comparison of patterns of reporter gene expression with conventionally constructed clones show that the presence of residual recombination (att) sites does not have an effect on patterns of gene expression, although overall levels of gene expression may vary. Rapid construction of these new vectors allowed vector/gene combinations to be optimized following evaluation of plasmid constructs in different bacterial cells and demonstrates the benefits of plasmid construction using Gateway cloning.</p> <p>Conclusion</p> <p>The residual <it>att </it>sites present after Gateway cloning did not affect patterns of promoter induction in Gram-positive bacteria and there was no evidence of differences in mRNA stability of transcripts. However overall levels of gene expression may be reduced, possibly due to some post-transcriptional event. The new vectors described here allow faster, more efficient cloning in range of Gram-positive bacteria.</p

Use of bioluminescent Escherichia coli O157:H7 to study intra-protozoan survival of bacteria within Tetrahymena pyriformis

A method was developed that enabled real-time monitoring of the uptake and survival of bioluminescent Escherichia coli O157 within the freshwater ciliate Tetrahymena pyriformis. Constitutively bioluminescent E. coli O157 pLITE27 was cocultured with T. pyriformis in nutrient-deficient (Chalkley's) and in nutrient-rich (proteose peptone, yeast extract) media. Non-internalised bacteria were inactivated by addition of colistin, indicated by a decline in bioluminescence. Protozoa were subsequently lysed with Triton X-100 which lead to a further drop in bioluminescence, consistent with release of live internal bacteria from T. pyriformis into the colistin-containing environment. Bioluminescence measurements for non-lysed cultures indicated that internalised E. coli O157 pLITE27 cells were only slowly digested by T. pyriformis, in both media, over the time period studied. The results suggest that bioluminescent bacteria are useful tools in the study of bacterial intra-protozoan survival. © 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved

Discrepancy between viable counts and light output as viability measurements, following ciprofloxacin challenge of self-bioluminescent Pseudomonas aeruginosa biofilms

Objectives: To utilize bioluminescence to follow the effect of ciprofloxacin challenge on Pseudomonas aeruginosa biofilms. Methods: The Sorbarod continuous perfusion culture system was used for the cultivation of biofilms of a self-bioluminescent strain of P. aeruginosa PAO1. Biofilms were challenged with ciprofloxacin (5 mg/L) in the perfusing medium for 3 h and allowed to recover to pre-challenge population levels before initiation of a second 3 h challenge. In addition to determining eluate and biofilm cell survival by conventional viable plate counts, light output was monitored via a luminometer and a low-light-level ICCD camera, to give an indication of metabolism. The effect of drug challenge on biofilm structure was investigated using an environmental scanning electron microscope, which allowed discernment of changes to the three-dimensional biofilm architecture. Results: On challenge with ciprofloxacin, eluate light output measurements declined to a lesser extent than viable counts for the same samples and also indicated that post-challenge recovery of the biofilm metabolism did not occur as rapidly as suggested by viable count data. Photon detection by ICCD camera allowed real-time, non-invasive imaging of metabolic activity within intact biofilms. Conclusions: The application of a bioluminescent reporter strain to biofilm research provides valuable real-time positional data on the efficacy of anti-biofilm treatment strategies. © The Author 2005. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved

Evaluation of Biophotonic Imaging To Estimate Bacterial Burden in Mice Infected with Highly Virulent Compared to Less Virulent Streptococcus pneumoniae Serotypes▿ †

Bioluminescence imaging is an innovative, noninvasive tool to analyze infectious disease progression under real-life conditions in small laboratory animals. However, the relevance of bioluminescence imaging to monitor invasive compared to noninvasive bacterial infections of the lung has not been examined so far. In the current study, we systematically evaluated the importance of bioluminescence imaging to monitor pneumococcal disease progression by correlating biophotonic signals with lung bacterial loads in two mouse strains (BALB/c, C57BL/6) infected with either self-glowing, bioluminescent serotype 19 Streptococcus pneumoniae causing focal pneumonia or serotype 2 S. pneumoniae causing invasive pneumococcal disease. The best correlations between bioluminescence signals and lung CFU counts were observed in BALB/c mice compared to C57BL/6 mice just on day 3 after infection with invasive serotype 2 S. pneumoniae, while excellent correlations between photon counts and bacterial loads were observed in isolated lungs of BALB/c and C57BL/6 mice, irrespective of the employed pneumococcal serotype. Moreover, good correlations between biophotonic signals and CFU counts were also observed in mice upon infection with serotype 19 S. pneumoniae causing focal pneumonia in mice, again with best correlation values obtained for BALB/c mice at day 3 postinfection. Collectively, we show that the relevance of biophotonic imaging to monitor S. pneumoniae-induced lung infections in mice is largely influenced by the disease model under investigation. The provided data may be important for studies of infectious diseases

Construction and evaluation of multisite recombinatorial (Gateway) cloning vectors for Gram-positive bacteria-2

<p><b>Copyright information:</b></p><p>Taken from "Construction and evaluation of multisite recombinatorial (Gateway) cloning vectors for Gram-positive bacteria"</p><p>http://www.biomedcentral.com/1471-2199/8/80</p><p>BMC Molecular Biology 2007;8():80-80.</p><p>Published online 19 Sep 2007</p><p>PMCID:PMC2039747.</p><p></p> ●; no sites) were grown to mid-log phase in Tris Minimal Succinate medium with 5 μg mlCm and 0.5% xylose. Bacteria were washed and resuspended in an equal volume of medium containing 5 μg mlCm and 1% glucose. Aliquots were placed in a 96 well microtitre plate and incubated at 37°C in a Tecan Genios Pro. Panel A: fluorescence (solid symbols, (Relative Fluorescence Units; RFU) and absorbance (open symbols) were measured at 10 min intervals. Plasmids used were pSB3004 (□, ■; 4 sites), pSB3002 (□, ▲; 2 sites) and pSB3000 (○, ●; no sites). Panel B: luminescence (solid symbols, Relative Light Units; RLU) and absorbance (open symbols) were measured at 10 minute intervals. Plasmids used were pSB3014 (□, ■; 4 sites), pSB3012 (□, ▲; 2 sites) and pSB3010 (○, ●; no sites). Data is presented as % maximal signal to allow direct comparison of repression kinetics despite the fact that light levels from each construct were different

Construction and evaluation of multisite recombinatorial (Gateway) cloning vectors for Gram-positive bacteria-3

<p><b>Copyright information:</b></p><p>Taken from "Construction and evaluation of multisite recombinatorial (Gateway) cloning vectors for Gram-positive bacteria"</p><p>http://www.biomedcentral.com/1471-2199/8/80</p><p>BMC Molecular Biology 2007;8():80-80.</p><p>Published online 19 Sep 2007</p><p>PMCID:PMC2039747.</p><p></p>operon under the control of the Ppromoter (see Table 2). Cells were grown in MWB at 37°C and luminescence (closed symbols, Relative Light Units; RLU)) and growth measurements (open symbols) were taken at intervals. Reporter gene data are presented as RLU/ODto account for increasing cell number during the experiment

Construction and evaluation of multisite recombinatorial (Gateway) cloning vectors for Gram-positive bacteria-1

<p><b>Copyright information:</b></p><p>Taken from "Construction and evaluation of multisite recombinatorial (Gateway) cloning vectors for Gram-positive bacteria"</p><p>http://www.biomedcentral.com/1471-2199/8/80</p><p>BMC Molecular Biology 2007;8():80-80.</p><p>Published online 19 Sep 2007</p><p>PMCID:PMC2039747.</p><p></p> ●; no sites) were grown to mid-log phase in Tris Minimal Succinate medium with 5 μg mlCm and then diluted 1/20 into fresh medium supplemented with 0.5% xylose. Triplicate samples were placed in a 96 well microtitre plate and incubated at 37°C in a Tecan Genios Pro. Panel A: fluorescence (solid symbols, Relative Fluorescence Units; RFU) and absorbance (open symbols) were measured at 10 min intervals. Plasmids used were pSB3004 (□, ■; 4 sites), pSB3002 (□, ▲; 2 sites) and pSB3000 (○, ●; no sites). Panel B. luminescence (solid symbols, Relative Light Units; RLU)) and absorbance (open symbols) were measured at 10 min intervals. Plasmids used were pSB3014 (□, ■; 4 sites), pSB3012 (△, ▲; 2 sites) and pSB3010 (○, ●; no sites). Data is presented as % maximal signal to allow direct comparison of expression kinetics despite the fact that light levels from each construct were different

Construction and evaluation of multisite recombinatorial (Gateway) cloning vectors for Gram-positive bacteria-0

<p><b>Copyright information:</b></p><p>Taken from "Construction and evaluation of multisite recombinatorial (Gateway) cloning vectors for Gram-positive bacteria"</p><p>http://www.biomedcentral.com/1471-2199/8/80</p><p>BMC Molecular Biology 2007;8():80-80.</p><p>Published online 19 Sep 2007</p><p>PMCID:PMC2039747.</p><p></p>; 4 sites) were grown in LB medium containing 0.5% (w/v) xylose at 37°C. Growth (OD 600 nm; open symbols) and luminescence (Relative Light Units; RLU closed symbols) were monitored over time. Reporter gene data are presented as RLU/ODto account for increasing cell number during the experiment