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

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.

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