Abstract:

Streptococcus pneumoniae is Gram-positive alpha haemolytic bacteria that commonly found in the nosphrynax of elderly people and young children; it causes approximately 2 million deaths mostly children under age of 5 year and people over 60 years of age. Most important diseases caused by S. pneumoniae are including pneumonaie, meningitis and bacteraemia. The pathogens can be transmitted through contact. Streptococcuspneumoniae obtains its energy mainly carbohydrates through fermentation process. However, in some situations where there are limited sugars or in the presence of galactose the homolactic fermentation is shifted to mixed fermentation in which in addition to lactate, ethanol, formate and acetate are formed. In this study, the role of lactate (lactic acid) and formate (formic acid) in bacterial competition and cytotoxicy was investigated. We hypothesised that lactic acid and formic acid are able to contribute to the virulence of streptococcus pneumoniae. Bacteria were grown on either BHI or BAB. The killing assay was done by exposing various acids on S.pneumoniae as control then lactic acid producing bacteria and non-acid producing bacteria was tested with these acids. Growth assay experiment was done followed by cytotoxicity test using A549 epithelial cells incubated for 24h. The effect of lactic acid for killing assay was significant. Similar effect was seen when lactic acid was exposed to A549 cells. However, a hydrochloric acid was unable to inhibit the growth of bacteria. This study concludes that lactic acid produced by Streptococcus pneumoniae is a potential virulence factor and may contribute to Streptococcus invasive disease.

Keywords: Streptococcal, pneumonia, Gram-positive immunocompromised, microorganisms BAB, Lactate, fermentation

References:

[1]. AlonsoDeVelasco, E., Verheul, A.F., Verhoef, J. and Snippe, H. (1995) Streptococcus pneumoniae: virulence factors, pathogenesis, and vaccines. Microbiol Rev 59, 591-603.

[2]. Alakomi, H.L., Skytta, E., Saarela, M., Mattila-Sandholm, T., Latva-Kala, K. and Helander, I.M. (2000) Lactic acid permeabilizes gram-negative bacteria by disrupting the outer membrane. Appl Environ Microbiol 66, 2001-2005.

[3]. Berry, A.M., Lock, R.A., Thomas, S.M., Rajan, D.P., Hansman, D. and Paton, J.C. Cloning and nucleotide sequence of the Streptococcus pneumoniae hyaluronidase gene and purification of the enzyme from recombinant Escherichia coli. Infect Immun 62, 1101-1108.

[4]. Barbara, A., Bannister, S.H. and Gillespie, J.J.(2006) Infection: Microbiology and management 3 rd edition. Blackwell publishing Ltd, Oxford UK.

[5]. Charlier, C., Cretenet, M., Even, S. and Le Loir, Y. (2009) Interactions between Staphylococcus aureus and lactic acid bacteria: an old story with new perspectives. Int J Food Microbiol 131, 30-39.

[6]. Christopher David (2003) The role of hydrogen peroxide production in the biology of Streptococcus pneumoniae, University of Pennsylvania. USA

[7]. Ciapetti, G., Cenni, E., Pratelli, L. and Pizzoferrato, A. (1993) In vitro evaluation of cell/biomaterial interaction by MTT assay. Biomaterials 14, 359-364.

[8]. Cotter, P.D. and Hill, C. (2003) Surviving the acid test: responses of gram-positive bacteria to low pH. Microbiol Mol Biol Rev 67, 429-453, table of contents.

[9]. Fayol-Messaoudi, D., Berger, C.N., Coconnier-Polter, M.H., Lievin-Le Moal, V. and Servin, A.L. (2005) pH-, Lactic acid-, and non-lactic acid-dependent activities of probiotic Lactobacilli against Salmonella enterica Serovar Typhimurium. Appl Environ Microbiol 71, 6008-6013.

[10]. Greenwood, D. & Barer, M., (2007). Medical microbiology: a guide to microbial infections: pathogenesis, immunity, laboratory diagnosis, and control. 17th ed. Edinburgh; London: Churchill Livingstone/Elsevier.

[11]. Hibbing, M.E., Fuqua, C., Parsek, M.R. and Peterson, S.B. Bacterial competition: surviving and thriving in the microbial jungle. Nat Rev Microbiol 8, 15-25.

[12]. Hayreh,M.M., Hayreh, S. S., Baumbach, G. I., Cancila, P., Martin-Amat, G., and Tephly, T. R. (1980). Ocular toxicity of methanol: An experimental study. In Neurotoxicity of the Visual System (W. Merigan and B. Weiss, Eds.), pp. 35–53. Raven Press, New York.

[13]. Hoskins, J., Alborn, W.E., Jr., Arnold, J., Blaszczak, L.C., Burgett, S., DeHoff, B.S., Estrem, S.T., Fritz, L., Fu, D.J., Fuller, W., Geringer, C., Gilmour, R., Glass, J.S., Khoja, H., Kraft, A.R., Lagace, R.E., LeBlanc, D.J., Lee, L.N., Lefkowitz, E.J., Lu, J., Matsushima, P., McAhren, S.M., McHenney, M., McLeaster, K., Mundy, C.W., Nicas, T.I., Norris, F.H., O'Gara, M., Peery, R.B., Robertson, G.T., Rockey, P., Sun, P.M., Winkler, M.E., Yang, Y., Young-Bellido, M., Zhao, G., Zook, C.A., Baltz, R.H., Jaskunas, S.R., Rosteck, P.R., Jr., Skatrud, P.L. and Glass, J.I. Genome of the bacterium Streptococcus pneumoniae strain R6. J Bacteriol 183, 5709-5717.

[14]. Jeong, J.K., Kwon, O., Lee, Y.M., Oh, D.B., Lee, J.M., Kim, S., Kim, E.H., Le, T.N., Rhee, D.K. and Kang, H.A. (2009) Characterization of the Streptococcus pneumoniae BgaC protein as a novel surface beta-galactosidase with specific hydrolysis activity for the Galbeta1- 3GlcNAc moiety of oligosaccharides. J Bacteriol 191, 3011-3023.

[15]. Kadioglu, A., Weiser, J.N., Paton, J.C. and Andrew, P.W. (2008) The role of Streptococcus pneumoniae virulence factors in host respiratory colonization and disease. Nat Rev Microbiol 6, 288–301

[16]. Kaijalainen, Tarja, (2006) Identification of Streptococcus pneumoniae. The National Public Health Institute

[17]. Kim, Y.H., Anirban, J.M., Song, H.Y., Seo, H.S. and Lee, B.T. In vitro and in vivo evaluations of 3D porous TCP-coated and non-coated alumina scaffolds. J Biomater Appl 25, 539-558.

[18]. Lanie, J.A., Ng, W.L., Kazmierczak, K.M., Andrzejewski, T.M., Davidsen, T.M., Wayne, K.J., Tettelin, H., Glass, J.I. and Winkler, M.E. Genome sequence of Avery's virulent serotype 2 strain D39 of Streptococcus pneumoniae and comparison with that of unencapsulated laboratory strain R6. J Bacteriol 189, 38-51.

[19]. Leppanen, V.M., Merckel, M.C., Ollis, D.L., Wong, K.K., Kozarich, J.W. and Goldman, A. (1999) Pyruvate formate lyase is structurally homologous to type I ribonucleotide reductase. Structure 7, 733-744.

[20]. Li, S., Kelly, S.J., Lamani, E., Ferraroni, M. and Jedrzejas, M.J. (2000) Structural basis of hyaluronan degradation by Streptococcus pneumoniae hyaluronate lyase. EMBO J 19, 1228- 1240.

[21]. Lock, R.A., Paton, J.C. and Hansman, D. (1988) Purification and immunological characterization of neuraminidase produced by Streptococcus pneumoniae. Microb Pathog 4, 33-43.

[22]. Lysenko, E.S., Lijek, R.S., Brown, S.P. and Weiser, J.N. Within-host competition drives selection for the capsule virulence determinant of Streptococcus pneumoniae. Curr Biol 20, 1222-1226.

[23]. Margolis, E., Yates, A. and Levin, B.R. The ecology of nasal colonization of Streptococcus pneumoniae, Haemophilus influenzae and Staphylococcus aureus: the role of competition and interactions with host's immune response. BMC Microbiol 10, 59.

[24]. Maudsdotter, L., Jonsson, H., Roos, S. and Jonsson, A.B. Lactobacilli reduce cell cytotoxicity caused by Streptococcus pyogenes by producing lactic acid that degrades the toxic component lipoteichoic acid. Antimicrob Agents Chemother 55, 1622-1628.

[25]. Mosmann, T. (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65, 55-63.

[26]. Nagaoka, S., Murata, S., Kimura, K., Mori, T. and Hojo, K. (2010) Antimicrobial activity of sodium citrate against Streptococcus pneumoniae and several oral bacteria. Applied microbiology 51.546-551

[27]. Paton, J.C., Berry, A.M., Lock, R.A., Hansman, D. and Manning, P.A. (1986) Cloning and expression in Escherichia coli of the Streptococcus pneumoniae gene encoding pneumolysin. Infect Immun 54, 50-55.

[28]. Pericone, C.D., Overweg, K., Hermans, P.W. and Weiser, J.N. (2000) Inhibitory and bactericidal effects of hydrogen peroxide production by Streptococcus pneumoniae on other inhabitants of the upper respiratory tract. Infect Immun 68, 3990-3997.

[29]. Pericone, C.D., Park, S., Imlay, J.A. and Weiser, J.N. (2003) Factors contributing to hydrogen peroxide resistance in Streptococcus pneumoniae include pyruvate oxidase (SpxB) and avoidance of the toxic effects of the fenton reaction. J Bacteriol 185, 6815-6825.

[30]. Raftari, M., Jalilian, F.A., Abdulamir, A.S., Son, R., Sekawi, Z. and Fatimah, A.B. (2009) Effect of Organic Acids on Escherichia coli O157:H7 and Staphylococcus aureus Contaminated Meat. Open Microbiol J 3, 121-127.

[31]. Ross, J.J., Saltzman, C.L., Carling, P. and Shapiro, D.S. (2003) Pneumococcal septic arthritis: review of 190 cases. Clin Infect Dis 36, 319-327.

[32]. Sakurazawa, T. and Ohkusa, T. (2005) Cytotoxicity of organic acids produced by anaerobic intestinal bacteria on cultured epithelial cells. J Gastroenterol 40, 600-609.

[33]. Skrivanova, E., Marounek, M., Benda, V., and Brezina, P. (2006) Susceptibility of Escherichia coli, Salmonella sp. and Clostridium perfringens to organic acids and monolaurin. Veterinarni Medicina, 51,81-88

[34]. Shakhnovich, E.A., King, S.J. and Weiser, J.N. (2002) Neuraminidase expressed by Streptococcus pneumoniae desialylates the lipopolysaccharide of Neisseria meningitidis and Haemophilus influenzae: a paradigm for interbacterial competition among pathogens of the human respiratory tract. Infect Immun 70, 7161-7164.

[35]. Tatusov, R.L., Mushegian, A.R., Bork, P., Brown, N.P., Hayes, W.S., Borodovsky, M., Rudd, K.E. and Koonin, E.V. (1996) Metabolism and evolution of Haemophilus influenzae deduced from a whole-genome comparison with Escherichia coli. Curr Biol 6, 279-291.

[36]. Terra, V.S., Homer, K.A., Rao, S.G., Andrew, P.W. and Yesilkaya, H. Characterization of novel beta-galactosidase activity that contributes to glycoprotein degradation and virulence in Streptococcus pneumoniae. Infect Immun 78, 348-357.

[37]. Tettelin, H., Nelson, K.E., Paulsen, I.T., Eisen, J.A., Read, T.D., Peterson, S., Heidelberg, J., DeBoy, R.T., Haft, D.H., Dodson, R.J., Durkin, A.S., Gwinn, M., Kolonay, J.F., Nelson, W.C., Peterson, J.D., Umayam, L.A., White, O., Salzberg, S.L., Lewis, M.R., Radune, D., Holtzapple, E., Khouri, H., Wolf, A.M., Utterback, T.R., Hansen, C.L., McDonald, L.A., Feldblyum, T.V., Angiuoli, S., Dickinson, T., Hickey, E.K., Holt, I.E., Loftus, B.J., Yang, F., Smith, H.O., Venter, J.C., Dougherty, B.A., Morrison, D.A., Hollingshead, S.K. and Fraser, C.M. (2001) Complete genome sequence of a virulent isolate of Streptococcus pneumoniae. Science 293, 498-506.

[38]. Treichel, J.L., Henry, M.M., Skumatz, C.M., Eells, J.T. and Burke, J.M. (2004) Antioxidants and ocular cell type differences in cytoprotection from formic acid toxicity in vitro. Toxicol Sci 82, 183-192.

[39]. Tong, H.H., Blue, L.E., James, M.A. and DeMaria, T.F. (2000) Evaluation of the virulence of a Streptococcus pneumoniae neuraminidase-deficient mutant in nasopharyngeal colonization and development of otitis media in the chinchilla model. Infect Immun 68, 921-924.

[40]. Yang, J., Naik, S.G., Ortillo, D.O., Garcia-Serres, R., Li, M., Broderick, W.E., Huynh, B.H. and Broderick, J.B. (2009) The iron-sulfur cluster of pyruvate formate-lyase activating enzyme in whole cells: cluster interconversion and a valence-localized [4Fe-4S]2+ state. Biochemistry 48, 9234-9241.

[41]. Yesilkaya, H., Spissu, F., Carvalho, S.M., Terra, V.S., Homer, K.A., Benisty, R., Porat, N., Neves, A.R. and Andrew, P.W. (2009) Pyruvate formate lyase is required for pneumococcal fermentative metabolism and virulence. Infect Immun 77, 5418-5427.