Reemergence of covert bacteria Bacillus pumilus and Brevibacillus sp. in microbe-freed grape and watermelon stocks attributable to occasional autoclaving-defying residual spores from previous cycles

Abstract

Covert bacteria-harboring long-term micropropagated culture of grape that was sanitized of associated microbes as assessed through the indexing of tissue and medium for several passages (Thomas and Prakash (2004) In Vitro – Plant 40:603–607) showed reemergence of bacteria in a section of cultures (3/40) during the subsequent screening. Similarly, long-term propagated triploid watermelon cultures showed bacterial reentry in some sub-stocks (2/14) during their extended monitoring post-antibiotic treatment (Thomas et al. (2006) Plant Cell Tiss Org Cult, 85:317–329). Each of the three grape sub-stocks showed single bacterium, identified as Bacillus pumilus (×2) or Brevibacillus sp. (×1) based on 16S rDNA sequence analysis with 100% similarity to ‘ARBG2’ and ‘ARBG1’ isolates respectively, retrieved originally from grape stocks as long-term alcohol-surviving spore-forming covert contaminants. The two isolates from watermelon included Brevibacillus sp. (ARBG1) and B. pumilus (AuRB-WM1), the latter distinct from the ‘ARBG2’ isolate. The chance of bacterial re-entry through endophytic survival or during culture handling appeared remote on account of the tissue indexing employed and the care taken. Monitoring the autoclaves for proper functioning using chemical, physical and biological indicators, sealing the vessels to protect from microscopic vectors and scrutinizing the autoclaved medium for microbial growth all pointed the contaminant source to occasional vessel-adhering residual spores from the previous covertly contaminated cycle(s) that were not recognized as contaminated. Such spores withstood the standard autoclaving either due to their high heat resistance or some conditions typical to tissue culture vessels, and the proportion of vessels showing carry-over inoculum increased with the number of passages of culturing with the organism. The problem due to these hardy spore-forming bacteria was circumvented through double autoclaving of culture vessels with the first one a day prior to the medium preparation