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

Tezera Liku Bekele, Page Keith, Rhakimova Adelina, Salisbury Vyvyan, Davenport Victoria

University of Southampton, Southampton, UK.
University of the West of England, Bristol, UK.

DOI: 10.4236/aim.2014.42013                

Abstract

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.

Keywords

Epithelial; Protection; Neisseria meningitides; Lactobacillus; Innate; Lux

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	S. J. Gray, C. L. Trotter, M. E. Ramsay, M. Guiver, A. J. Fox, R. Borrow, R. H. Mallard and E. B. Kaczmarski, “Epidemiology of Meningococcal Disease in England and Wales 1993/94 to 2003/04: Contribution and Experiences of the Meningococcal Reference Unit,” Journal of Medical Microbiology, Vol. 55, No. 7, 2006, pp. 887-896. http://dx.doi.org/10.1099/jmm.0.46288-0
[2]	D. S. Stephens, B. Greenwood and P. Brandtzaeg, “Epidemic Meningitis, Meningococcaemia, and Neisseria Meningitidis,” The Lancet, Vol. 369, No. 9580, 2007, pp. 2196-2210. http://dx.doi.org/10.1016/S0140-6736(07)61016-2
[3]	P. Brandtzaeg, “Pathogenesis and Pathophysiology of Invasive Meningococcal Disease,” In: M. Frosch and M. C. J. Maiden, Eds., Handbook of Meningococcal Disease: Infection Biology, Vaccination, Clinical Management, Weinheim: Wiley-VCH; John Wiley Distributor, Chichester, 2006, pp. 427-480.
[4]	M. Sadarangani and A. J. Pollard, “Serogroup B Meningococcal Vaccines—An Unfinished Story,” Lancet Infect Disease, Vol. 10, No. 2, pp. 112-124. http://dx.doi.org/10.1016/S1473-3099(09)70324-X
[5]	K. A. Cartwright, J. M. Stuart, D. M. Jones and N. D. Noah, “The Stonehouse Survey: Nasopharyngeal Carriage of Meningococci and Neisseria lactamica,” Epidemiol Infect, Vol. 99, No. 3, 1987, pp. 591-601. http://dx.doi.org/10.1017/S0950268800066449
[6]	M. Howitz, L. Lambertsen, J. B. Simonsen, et al., “Morbidity, Mortality and Spatial Distribution of Meningococcal Disease, 1974-2007,” Epidemiological Infect, Vol. 137, No. 11, 2009, pp. 1631-1640. http://dx.doi.org/10.1017/S0950268809002428
[7]	R. Gold, I. Goldschneider, M. L. Lepow, T. F. Draper and M. Randolph, “Carriage of Neisseria meningitidis and Neisseria lactamica in Infants and Children,” The Journal of Infectious Diseases, Vol. 137, No. 2, 1978, pp. 112-121. http://dx.doi.org/10.1093/infdis/137.2.112
[8]	P. G. Coen, K. Cartwright and J. Stuart, “Mathematical Modelling of Infection and Disease Due to Neisseria meningitidis and Neisseria lactamica,” International Journal of Epidemiology, Vol. 29, No. 1, 2000, pp. 180-188. http://dx.doi.org/10.1093/ije/29.1.180
[9]	A. T. Vaughan, L. S. Brackenbury, P. Massari, V. Davenport, A. Gorringe, R. S. Heyderman and N. A. Williams, “Neisseria lactamica Selectively Induces Mitogenic Proliferation of the Naive B Cell Pool via Cell Surface Ig,” Journal of Immunology, Vol. 185, No. 6, 2010, pp. 3652-3660. http://dx.doi.org/10.4049/jimmunol.0902468
[10]	L. B. Tezera, J. Hampton, S. K. Jackson and V. Davenport, “Neisseria lactamica Attenuates TLR-1/2-Induced Cytokine Responses in Nasopharyngeal Epithelial Cells Using PPAR-Gamma,” Cell Microbiology, Vol. 13, No. 4, 2011, pp. 554-568. http://dx.doi.org/10.1111/j.1462-5822.2010.01554.x
[11]	Strahinic, M. Busarcevic, D. Pavlica, J. Milasin, N. Golic and L. Topisirovic, “Molecular and Biochemical Characterizations of Human Oral Lactobacilli as Putative Probiotic Candidates,” Oral Microbiological Immunology, Vol. 22, No. 2, 2007, pp. 111-117. http://dx.doi.org/10.1111/j.1399-302X.2007.00331.x
[12]	S. Skovbjerg, K. Roos, S. E. Holm, E. Grahn Håkansson, F. Nowrouzian, M. Ivarsson, I. Adlerberth and A. E. Wold, “Spray Bacteriotherapy Decreases Middle Ear Fluid in Children with Secretory Otitis Media,” Archives of Disease in Childhood, Vol. 94, No. 2, 2009, pp. 92-98. http://dx.doi.org/10.1136/adc.2008.137414
[13]	S. Guglielmetti, V. Taverniti, M. Minuzzo S. Arioli, I. Zanoni, M. Stuknyte, F. Granucci, M. Karp and D. Mora, “Oral Bacteria as Potential Probiotics for the Pharyngeal Mucosa,” Applied and Environmental Microbiology, Vol. 76, No. 12, 2010, pp. 3948-3958. http://dx.doi.org/10.1128/AEM.00109-10
[14]	S. Guglielmetti, V. Taverniti, M. Minuzz, S. Arioli, I. Zanoni, M. Stuknyte, F. Granucci, M. Karp and D. Mora, “A Dairy Bacterium Displays in Vitro Probiotic Properties for the Pharyngeal Mucosa by Antagonizing Group A Streptococci and Modulating the Immune Response,” Infection and Immunity, Vol. 78, No. 11, 2010, pp. 4734-4743. http://dx.doi.org/10.1128/IAI.00559-10
[15]	L. Petricevic, K. J. Domig, F. J. Nierscher, I. Krondorfer, C. Janitschek, W. Kneifel and H. Kiss, “Characterisation of the Oral, Vaginal and Rectal Lactobacillus Flora in Healthy Pregnant and Postmenopausal Women,” The European Journal of Obstetrics & Gynecology and Reproductive Biology, Vol. 160, No. 1, 2012, pp. 93-99. http://dx.doi.org/10.1016/j.ejogrb.2011.10.002
[16]	S. Ahrne, S. Nobaek, B. Jeppsson I. Adlerberth, A. E. Wold and G. Molin, “The Normal Lactobacillus Flora of Healthy Human Rectal and Oral Mucosa,” Journal of Applied Microbiology, Vol. 85, No. 1, 1998, pp. 88-94. http://dx.doi.org/10.1046/j.1365-2672.1998.00480.x
[17]	C. Badet and N. B. Thebaud, “Ecology of Lactobacilli in the Oral Cavity: A Review of Literature,” The Open Microbiology Journal, Vol. 2, 2008, pp. 38-48. http://dx.doi.org/10.2174/1874285800802010038
[18]	A. C. Senok and V. O. Rotimi, “The Management of Clostridium difficile Infection: Antibiotics, Probiotics and Other Strategies,” Journal of Chemotherapy, Vol. 20, No. 1, 2008, pp. 5-13.
[19]	M. E. Falagas, G. I. Betsi, T. Tokas and S. Athanasiou, “Probiotics for Prevention of Recurrent Urinary Tract Infections in Women: A Review of the Evidence from Microbiological and Clinical Studies,” Drugs, Vol. 66, No. 9, 2006, pp. 1253-1261. http://dx.doi.org/10.2165/00003495-200666090-00007
[20]	C. L. Abad and N. Safdar, “The Role of lactobacillus Probiotics in the Treatment or Prevention of Urogenital Infections-A Systematic Review,” Journal of Chemotherapy, Vol. 21, No. 3, 2009, pp. 243-252.
[21]	L. Pascual, F. Ruiz, W. Giordano and I. L. Barberis, “Vaginal Colonization and Activity of the Probiotic Bacterium Lactobacillus fermentum L23 in a Murine Model of Vaginal Tract Infection,” Journal of Medical Microbiology, Vol. 59, No. Pt 3, 2010, pp. 360-364.
[22]	R. R. Spurbeck and C. G. Arvidson, “Lactobacillus jensenii Surface-Associated Proteins Inhibit Neisseria gonorrhoeae Adherence to Epithelial Cells,” Infection and Immunity, Vol. 78, No. 7, 2010, pp. 3103-3111. http://dx.doi.org/10.1128/IAI.01200-09
[23]	S. H. Huang, L. He, Y. Zhou, C. H. Wu and A. Jong, “Lactobacillus rhamnosus GG Suppresses Meningitic E. coli K1 Penetration across Human Intestinal Epithelial Cells in Vitro and Protects Neonatal Rats against Experimental Hematogenous Meningitis,” International Journal of Microbiology, Vol. 2009, 2009, Article ID: 647862. http://dx.doi.org/10.1155/2009/647862
[24]	L. Maudsdotter, H. Jonsson, S. Roos and A. B. Jonsson “Lactobacilli Reduce Cell Cytotoxicity Caused by Streptococcus pyogenes by Producing Lactic Acid That Degrades the Toxic Component Lipoteichoic Acid,” Antimicrob Agents Chemother, Vol. 55, No. 4, 2011, pp. 1622-1628. http://dx.doi.org/10.1128/AAC.00770-10
[25]	M. Rogosa, R. F. Wiseman, J. A. Mitchell, M. N. Disraely and A. J. Beaman, “Species Differentiation of Oral Lactobacilli from Man including Description of Lactobacillus salivarius nov spec and Lactobacillus cellobiosus nov spec,” Journal of Bacteriology, Vol. 65, No. 6, 1953, pp. 681-699.
[26]	M. E. Colloca, M. C. Ahumada, M. E. Lopez and M. E. Nader-Macias, “Surface Properties of Lactobacilli Isolated from Healthy Subjects,” Oral Disease, Vol. 6, No. 4, 2000, pp. 227-233. http://dx.doi.org/10.1111/j.1601-0825.2000.tb00118.x
[27]	P. Koll, R. Mandar, H. Marcotte, H. E. Leibur, M. Mikelsaar and L. Hammarstrom, “Characterization of Oral Lactobacilli as Potential Probiotics for Oral Health,” Oral Microbiology and Immunology, Vol. 23, No. 2, 2008, pp. 139-147. http://dx.doi.org/10.1111/j.1399-302X.2007.00402.x
[28]	P. Manzoni, G. Lista, E. Gallo, P. Marangione, C. Priolo, P. Fontana, R. Guardione and D. Farina, “Routine Lactobacillus rhamnosus GG Administration in VLBW Infants: A Retrospective, 6-Year Cohort Study,” Early Human Development, Vol. 87, No. S1, 2011, pp. S35-S38. http://dx.doi.org/10.1016/j.earlhumdev.2011.01.036
[29]	M. A. Bartelt and J. L. Duncan, “Adherence of Group A Streptococci to Human Epithelial Cells,” Infection and Immunity, Vol. 20, No. 1, 1978, pp. 200-208.
[30]	H. Sjolinder and A. B. Jonsson, “Imaging of Disease Dynamics during Meningococcal Sepsis,” PLoS One, Vol. 2, No. 2, 2007, Article ID: e241. http://dx.doi.org/10.1371/journal.pone.0000241
[31]	H. Shaw and S. Falkow, “Model for Invasion of Human Tissue Culture Cells by Neisseria gonorrhoeae,” Infection and Immunity, Vol. 56, No. 6, 1988, pp. 1625-1632.
[32]	E. B. Williams Jr. and A. D. Spencer, “Serum Lactic Dehydrogenase Activity in Intestinal Necrosis and Other Disease States—A Clinical Study,” Journal of the National Medical Association, Vol. 53, 1961, pp. 556-561.
[33]	A.-L. Nieminen, G. J. Gores, J. M. Bond, R. Imberti and J. J. Lemasters, “A Novel Cytotoxicity Screening Assay Using a Multiwell Fluorescence Scanner,” Toxicology and Applied Pharmacology, Vol. 115, No. 2, 1992, pp. 147-155. http://dx.doi.org/10.1016/0041-008X(92)90317-L
[34]	S. J. Beard, V. Salisbury, R. J. Lewis, J. A. Sharpe and A. P. MacGowan, “Expression of Lux Genes in a Clinical Isolate of Streptococcus pneumoniae: Using Bioluminescence to Monitor Gemifloxacin Activity,” Antimicrob Agents Chemotherapy, Vol. 46, No. 2, 2002, pp. 538-542. http://dx.doi.org/10.1128/AAC.46.2.538-542.2002
[35]	L. Dunn, M. Virji and E. R. Moxon, “Investigations into the Molecular Basis of Meningococcal Toxicity for Human Endothelial and Epithelial Cells: The Synergistic Effect of LPS and pili,” Microbial Pathogenesis, Vol. 18, No. 2, 1995, pp. 81-96. http://dx.doi.org/10.1016/S0882-4010(95)90085-3
[36]	M. I. Fowler, K. Y. Yin, H. E. Humphries, J. E. Heckels and M. Christodoulides, “Comparison of the Inflammatory Responses of Human Meningeal Cells Following Challenge with Neisseria lactamica and with Neisseria meningitidis,” Infection and Immunity, Vol. 74, No. 11, 2006, pp. 6467-6478.
[37]	L. A. Dieleman, M. S. Goerres, A. Arends, D. Sprengers, C. Torrice, F. Hoentjen, W. B. Grenther and R. B. Sartor, “Lactobacillus GG Prevents Recurrence of Colitis in HLA-B27 Transgenic Rats after Antibiotic Treatment,” Gut, Vol. 52, No. 3, 2003, pp. 370-376. http://dx.doi.org/10.1136/gut.52.3.370
[38]	M. E. Baldassarre, N. Laforgia, M. Fanelli, A. Laneve, R. Grosso and C. Lifschitz, “Lactobacillus GG Improves Recovery in Infants with Blood in the Stools and Presumptive Allergic Colitis Compared with Extensively Hydrolyzed Formula Alone,” The Journal of Pediatrics, Vol. 156, No. 3, 2010, pp. 397-401. http://dx.doi.org/10.1016/j.jpeds.2009.09.012
[39]	R. Paolillo, C. Romano Carratelli, S. Sorrentino, N. Mazzola and A. Rizzo, “Immunomodulatory Effects of Lactobacillus plantarum on Human Colon Cancer Cells,” International Immunopharmacology, Vol. 9, No. 11, 2009, pp. 1265-1271. http://dx.doi.org/10.1016/j.intimp.2009.07.008
[40]	E. O. Petrof, E. C. Claud, J. Sun, T. Abramova, Y. Guo, T. S. Waypa, S. M. He, Y. Nakagawa and E. B. Chang, “Bacteria-Free Solution Derived from Lactobacillus plantarum Inhibits Multiple NF-kappaB Pathways and Inhibits Proteasome Function,” Inflammatory Bowel Diseases, Vol. 15, No. 10, 2009, pp. 1537-1547. http://dx.doi.org/10.1002/ibd.20930
[41]	J. H. Pollack, A. S. Ntamere and F. C. Neuhaus, “DAlanyl-Lipoteichoic Acid in Lactobacillus casei: Secretion of Vesicles in Response to Benzylpenicillin,” Microbiology, Vol. 138, No. 5, 1992, pp. 849-859. http://dx.doi.org/10.1099/00221287-138-5-849
[42]	H. G. Kim, S. Y. Lee, N. R. Kim, Y. Lee, M. Y. Ko, B. J. Jung, C. M. Kim, J. M. Lee, J. H. Park, S. H. Han and D. K. Chung, “Lactobacillus plantarum Lipoteichoic Acid Down-Regulated Shigella flexneri Peptidoglycan-Induced Inflammation,” Molecular Immunology, Vol. 48, No. 4, 2011, pp. 382-391. http://dx.doi.org/10.1016/j.molimm.2010.07.011
[43]	H. G. Kim, N. R. Kim, M. G. Gim, J. M. Lee, S. Y. Lee, M. Y. Ko, J. Y. Kim, S. H. Han and D. K. Chung, “Lipoteichoic Acid Isolated from Lactobacillus plantarum Inhibits Lipopolysaccharide-Induced TNF-alpha Production in THP-1 Cells and Endotoxin Shock in Mice,” Journal of Immunology, Vol. 180, No. 4, 2008, pp. 2553-2561.
[44]	P. Matzinger, “The Evolution of the Danger Theory. Interview by Lauren Constable, Commissioning Editor,” Expert Review of Clinical Immunology, Vol. 8, No. 4, 2012, pp. 311-317.
[45]	D. S. Stephens and M. M. Farley, “Pathogenic Events during Infection of the Human Nasopharynx with Neisseria meningitidis and Haemophilus influenzae,” Clinical Infectious Diseases, Vol. 13, No. 1, 1991, pp. 22-33. http://dx.doi.org/10.1093/clinids/13.1.22
[46]	Y. Kim, K. S. Han, J. Y. Imm, S. Oh, S. You, S. Park and S. H. Kim, “Inhibitory Effects of Lactobacillus acidophilus lysates on the Cytotoxic Activity of Shiga-Like Toxin 2 Produced from Escherichia coli O157:H7,” Letters in Applied Microbiology, Vol. 43, No. 5, 2006, pp. 502-507. http://dx.doi.org/10.1111/j.1472-765X.2006.02005.x
[47]	P. Johns, S. L. Pereira, A. E. Leonard, P. Mukerji, R. A. Shalwitz, L. Dowlati, R. R. Phillips, M. S. Bergana, J. D. Holton and T. Das, “Cytoprotective Agent in Lactobacillus bulgaricus Extracts,” Current Microbiology, Vol. 54, No. 2, 2007, pp. 131-135. http://dx.doi.org/10.1007/s00284-006-0256-6
[48]	K. M. Burkholder and A. K. Bhunia, “Salmonella enterica Serovar Typhimurium Adhesion and Cytotoxicity during Epithelial Cell Stress Is Reduced by Lactobacillus rhamnosus GG,” Gut Pathogens, Vol. 1, No. 1, 2009, p. 14. http://dx.doi.org/10.1186/1757-4749-1-14
[49]	P. Banerjee, G. J. Merkel and A. K. Bhunia, “Lactobacillus delbrueckii ssp. Bulgaricus B-30892 can Inhibit Cytotoxic Effects and Adhesion of Pathogenic Clostridium difficile to Caco-2 Cells,” Gut Pathogens, Vol. 1, No. 1, 2009, p. 8. http://dx.doi.org/10.1186/1757-4749-1-8
[50]	L. J. Fooks and G. R. Gibson, “In Vitro Investigations of the Effect of Probiotics and Prebiotics on Selected Human Intestinal Pathogens,” FEMS Microbiology Ecology, Vol. 39, No. 1, 2002, pp. 67-75. http://dx.doi.org/10.1111/j.1574-6941.2002.tb00907.x
[51]	D. C. St Amant, I. E. Valentin-Bon and A. E. Jerse, “Inhibition of Neisseria gonorrhoeae by Lactobacillus Species that Are Commonly Isolated from the Female Genital Tract,” Infection and Immunity, Vol. 70, No. 12, 2002, pp. 7169-7171. http://dx.doi.org/10.1128/IAI.70.12.7169-7171.2002
[52]	M. S. Princivalli, C. Paoletti, G. Magi, C. Palmieri, L. Ferrante and B. Facinelli, “Lactobacillus rhamnosus GG Inhibits Invasion of Cultured Human Respiratory Cells by prtF1-Positive Macrolide-Resistant Group A Streptococci,” Letters in Applied Microbiology, Vol. 48, No. 3, 2009, pp. 368-372. http://dx.doi.org/10.1111/j.1472-765X.2008.02540.x
[53]	Z. H. Liu, T. Y. Shen, P. Zhang ,Y. L. Ma and H. L. Qin, “Lactobacillus plantarum Surface Layer Adhesive Protein Protects Intestinal Epithelial Cells against Tight Junction Injury Induced by Enteropathogenic Escherichia coli,” Molecular Biology Reports, Vol. 38, No. 5, 2011, pp. 3471-3480. http://dx.doi.org/10.1007/s11033-010-0457-8
[54]	M. I. Fowler, R. O. Weller, J. E. Heckels and M. Christodoulides, “Different Meningitis-Causing Bacteria Induce Distinct Inflammatory Responses on Interaction with Cells of the Human Meninges,” Cellular Microbiology, Vol. 6, No. 6, 2004, pp. 555-567. http://dx.doi.org/10.1111/j.1462-5822.2004.00382.x
[55]	C. Rydberg, A. Mansson, R. Uddman, K. Riesbeck and L. O. Cardell, “Toll-Like Receptor Agonists Induce Inflammation and Cell Death in a Model of Head and Neck Squamous Cell Carcinomas,” Immunology, Vol. 128, No. 1, 2009, pp. e600-e611. http://dx.doi.org/10.1111/j.1365-2567.2008.03041.x
[56]	H. Tettelin, N. J. Saunders, J. Heidelberg, et al., “Complete Genome Sequence of Neisseria meningitidis Serogroup B Strain MC58,” Science, Vol. 287, No. 5459, 2000, pp. 1809-1815. http://dx.doi.org/10.1126/science.287.5459.1809
[57]	Q. Hao, Z. Lu, B. R. Dong, C. Q. Huang and T. Wu, “Probiotics for Preventing Acute Upper Respiratory Tract Infections,” Cochrane Database of Systematic Reviews, No. 9, 2011, Article ID: CD006895.