Extracellular Components in Culture Media of Mycobacterium Avium Subspecies and Staphylococci with Implications for Clinical Microbiology and Blood Culture

Peilin Zhang^1, , Lawrence M. Minardi¹, J. Todd Kuenstner¹, Stephen M. Zekan¹ and Rusty Kruzelock²

¹PZM Diagnostics, LLC, Charleston, WV, USA

²West Virginia Reginal Technology Park, South Charleston, WV, USA

Cite this paper:
Peilin Zhang, Lawrence M. Minardi, J. Todd Kuenstner, Stephen M. Zekan and Rusty Kruzelock. Extracellular Components in Culture Media of Mycobacterium Avium Subspecies and Staphylococci with Implications for Clinical Microbiology and Blood Culture. American Journal of Infectious Diseases and Microbiology. 2016; 4(6):112-117. doi: 10.12691/ajidm-4-6-1

Abstract

Rapid culture of Mycobacterium avium subspecies paratuberculosis (MAP) from patients remain a challenge. During the process of developing a rapid culture method for MAP, we found extracellular bacterial DNA, RNA and proteins for MAP, MAH (Mycobacterium avium subspecies hominissuis) and other bacteria such as Staphylococcus aureus, and Staphylococcus pseudintermedius in the supernatants of our liquid culture media. When cultured on the solid media plate, there are a limited number of colonies developed for MAP and MAH disproportionate to the expected numbers of mycobacterial growth. The presence of extracellular components within the liquid culture media raises the possibility of an unknown biological process or a cell death process during the culture that has not been previously described. Based on the presence of extracellular bacterial DNA, RNA and proteins in the liquid media of various bacterial/mycobacterial cultures, and the fact that automated clinical blood culture system consistently gives rise to a low yield, we tested 62 blood culture specimens that have been reported negative by a routine automated blood culture method after 5 days of incubation. We used high sucrose culture media and molecular diagnostic techniques to test these negative culture bottles, and we found a large percentage of bacterial growth by high sucrose culture media (32%) and by molecular PCR amplification using 16S rDNA primers and DNA sequencing (69%). The bacteria identified by PCR/sequencing methods from these culture-negative bottles are diverse and may play roles in pathogenesis of these clinically ill patients. The sensitivity of detection by an alternative culture media and the molecular PCR/sequencing method are significantly higher than by routine automated blood culture. We suspect the low yield of automated blood culture in hospital setting may result from cell death of bacteria under the culture conditions. Our study demonstrates a potential alternative bacterial growth/death pattern in liquid media and provides an area of needs for more sensitive and faster diagnostic tools to guide clinical practice and improve the outcome of sepsis management.

Keywords:
Mycobacterium Paratuberculosis Hominissus Programed cell death Automated blood culture Blood culture sensitivity PCR 16S rDNA sequencing

This work is licensed under a Creative Commons Attribution 4.0 International License. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

References:

[1]	Chiodini, R.J., Van Kruiningen, H.J., Merkal, R.S., Thayer, W.R., Jr. and Coutu, J.A. (1984). J Clin Microbiol 20, 966-71.
[2]	Behr, M., Collins, DM. (2010). Paratuberculosis: Organism, Disease, Control, CABI.
[3]	Mameli, G., Cossu, D., Cocco, E., Frau, J., Marrosu, M.G., Niegowska, M. and Sechi, L.A. (2015). J Neuroimmunol 280, 66-8.
[4]	Niegowska, M., Rapini, N., Piccinini, S., Mameli, G., Caggiu, E., Manca Bitti, M.L. and Sechi, L.A. (2016). Sci Rep 6, 22266.
[5]	Niegowska, M., Paccagnini, D., Mannu, C., Targhetta, C., Songini, M. and Sechi, L.A. (2016). J Diabetes Res 2016, 5842701.
[6]	Sambrook, J., Fritsch, E.F., Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.
[7]	Skulachev, V.P. (1999). Biochemistry (Mosc) 64, 1418-26.
[8]	Basra, S., Anany, H., Brovko, L., Kropinski, A.M. and Griffiths, M.W. (2014). Arch Virol 159, 2659-74.
[9]	Foley-Thomas, E.M., Whipple, D.L., Bermudez, L.E. and Barletta, R.G. (1995). Microbiology 141 (Pt 5), 1173-81.
[10]	Fan, X., Abd Alla, A.A. and Xie, J. (2016). J Biomol Struct Dyn 34, 233-8.
[11]	Fan, X., Xie, L., Li, W. and Xie, J. (2014). BMC Genomics 15, 243.
[12]	Uchiya, K., Takahashi, H., Yagi, T., Moriyama, M., Inagaki, T., Ichikawa, K., Nakagawa, T., Nikai, T. and Ogawa, K. (2013). PLoS One 8, e71831.
[13]	Tran, Q.T. and Han, X.Y. (2014) J Clin Microbiol 52, 1201-6.
[14]	Zhang, P., Minardi, L.M., Kuenstner, J.T. and Kruzelock, R. (2015). Gastroenterol Rep (Oxf) 1, 1-4.
[15]	Horan, K.L., Freeman, R., Weigel, K., Semret, M., Pfaller, S., Covert, T.C., van Soolingen, D., Leao, S.C., Behr, M.A. and Cangelosi, G.A. (2006). J Clin Microbiol 44, 783-9.
[16]	Rice, K.C. and Bayles, K.W. (2008) Microbiol Mol Biol Rev 72, 85-109, table of contents.
[17]	Tagawa, M., Udaka, S. (1978). Agricultural and Biological Chemistry 42, 1157-1166.
[18]	Tagawa, M., Udaka, S. (1980). Agric. Bjol. Chem. 44, 1867-1875.
[19]	Mattman, L.H. (2001) Cell Wall Deficient Forms, Stealth Pathogens, CRC Press.
[20]	Gutierrez, A., Zapater, P., Juanola, O., Sempere, L., Garcia, M., Laveda, R., Martinez, A., Scharl, M., Gonzalez-Navajas, J.M., Such, J., Wiest, R., Rogler, G. and Frances, R. (2016). Am J Gastroenterol.
[21]	Gutierrez, A., Frances, R., Amoros, A., Zapater, P., Garmendia, M., Ndongo, M., Cano, R., Jover, R., Such, J. and Perez-Mateo, M. (2009). Inflamm Bowel Dis 15, 508-14.
[22]	Pier, G.B., Grout, M., Zaidi, T., Meluleni, G., Mueschenborn, S.S., Banting, G., Ratcliff, R., Evans, M.J. and Colledge, W.H. (1998). Nature 393, 79-82.
[23]	Lyczak, J.B. and Pier, G.B. (2002). Infect Immun 70, 6416-23.
[24]	Sakamoto, K., Mori, Y., Takagi, H., Iwata, H., Yamada, T., Futamura, N., Sago, T., Ezaki, T., Kawamura, Y. and Hirose, H. (2004). Nutrition 20, 372-6.
[25]	Kuna, A.T. (2013). Biochem Med (Zagreb) 23, 28-42.