A competitive microflora increases the resistance of Salmonella typhimurium to inimical processes: evidence for a suicide response

Abstract

The presence of a viable competitive microflora at cell densities of 10(8) CFU ml-1 protects an underlying population of 10(5) CFU of Salmonella typhimurium ml-1 against freeze injury. The mechanism of enhanced resistance was initially postulated to be via an RpoS-mediated adaptive response. By using an spvRA:: luxCDABE reporter we have shown that although the onset of RpoS-mediated gene expression was brought forward by the addition of a competitive microflora, the time taken for induction was measured in hours. Since the protective effect of a competitive microflora is essentially instantaneous, the stationary-phase adaptive response is excluded as the physiological mechanism. The only instantaneous effect of the competitive microflora was a reduction in the percent saturation of oxygen from 100% to less than 10%. For both mild heat treatment (55 degrees C) and freeze injury this change in oxygen tension affords Salmonella a substantive (2 orders of magnitude) enhancement in survival. By reducing the levels of dissolved oxygen through active respiration, a competitive microflora reduces oxidative damage to exponential-phase cells irrespective of the inimical treatment. These results have led us to propose a suicide hypothesis for the destruction of rapidly growing cells by inimical processes. In essence, the suicide hypothesis proposes that a mild inimical process leads to the growth arrest of exponential-phase cells and to the decoupling of anabolic and catabolic metabolism. The result of this is a free radical burst which is lethal to unadapted cells.

FIG. 1

The induction of RpoS as measured by an spvRA::luxCDABE reporter in S. typhimurium LT2. Induction time for RpoS was derived from the intersection of lines drawn through the stationary and exponential portions of the growth (▪, log₁₀ optical density [OD]) and bioluminescence (⧫, log₁₀ relative light unit [RLU]) curves.

FIG. 2

Survival of S. typhimurium LT2(pSB100) after freeze-thaw without competitors. The dashed line represents the control adjusted to subtract the growth which occurred during freezing of the experimental sample. ◊, growth in 0.1% peptone water; ○, control culture incubated in LB; •, experimental culture subjected to freezing; ▾|, samples frozen; ▾⋮, samples thawed. At time zero, all samples were diluted 1:10 into LB. The difference in survival between the control and freeze-thawed cultures was determined from the lines of best fit. A 239- ± 65-fold drop in viability represents the survival of 0.42% (± 0.13%) of the original cell number. Each point represents the mean of three independent replicates; the error bars represent 1 standard deviation from the mean.

FIG. 3

Survival of S. typhimurium LT2(pSB100) after freeze-thaw without competitors (○, •), in the presence of 10⁸ CFU of viable competitors ml⁻¹ (▵, ▴) and in the presence of heat-killed competitors (□, ▪). Open symbols represent control cultures, while filled symbols represent the experimental cultures. ----, adjusted controls. The lines of best fit are through points which represent the mean of three independent replicates and take account of error bars with one standard deviation. For clarity the error bars are not shown. The data for ▵ after 150 min are not presented as the cultures entered stationary phase. Since there is no statistical difference between the Salmonella populations without competitors (circles) or in the presence of heat-killed competitors (squares), the dashed and solid lines show the means of both data sets. The inclusion of a viable competitive microflora increases the survival of S. typhimurium from 0.34% (± 0.13%) (•, ▪) to 4.9% (± 0.53%) (▴). (B) The percent dissolved oxygen in cultures of 10⁵ CFU of S. typhimurium LT2 ml⁻¹, alone () or in the presence of 10⁷ CFU of viable competitors ml⁻¹ (.....) or 10⁸ CFU of viable competitors ml⁻¹ (----).

FIG. 4

Survival of E. coli BJ4(pSB100) (○, •) and of RpoS⁻ E. coli BJ4 L1(pSB100) (□, ▪) after freeze-thaw in the presence of 10⁸ CFU of viable competitors ml⁻¹. Open symbols represent the control cultures, while filled symbols represent the experimental cultures. Since there is no statistical difference between the E. coli populations, the lines show the means of both. The mean difference between the control and experimental cultures is 27-fold, equivalent to 4.06% survival.