Recovery of hydrogen peroxide-sensitive culturable cells of Vibrio vulnificus gives the appearance of resuscitation from a viable but nonculturable state

Abstract

The viabilities of five strains of Vibrio vulnificus were evaluated during the storage of the organisms in sterile seawater at 5 degrees C. The number of CFU was measured by plate count methods on rich media. The total cell numbers were determined by direct microscopic count methods. The titer of CFU declined logarithmically to undetectable levels over a period of 2 to 3 weeks, while the total cell numbers were unchanged. Midway through each study, higher culturable cell counts began to be observed on plates containing catalase or sodium pyruvate; during the latter stages of the study, the plate counts on such media were up to 1,000-fold higher than those on unsupplemented plates. Because autoclaving is known to generate hydrogen peroxide in rich media, and because catalase and sodium pyruvate are known to eliminate hydrogen peroxide, it appears that the conditions of the experiments led to the selection of a hydrogen peroxide-sensitive culturable cell subpopulation. At the time of the final stage of the decline in viability of each culture, hydrogen peroxide-sensitive cells were the only culturable cells present. Warming samples of the cultures to room temperature led to the growth of these residual culturable cells, utilizing nutrients provided by the nonculturable cells. The cells that grew recovered hydrogen peroxide resistance. When mixtures of culturable and nonculturable cells were diluted to the point where only nonculturable cells were present, or when the hydrogen peroxide-sensitive culturable cells had declined to undetectable levels, warming had no effect; no culturable cells were recovered. Warming has been reported to "resuscitate" nonculturable cells. Recognition of the existence of hydrogen peroxide-sensitive culturable cell populations, as well as their ability to grow to high levels in the warmed seawater microcosms, leads instead to the conclusion that while warming permits culturable cells to grow, it has no effect on nonculturable cells.

FIG. 1

Decline of V. vulnificus strains in sterile seawater at 5°C. Colony counts on HI plates (●) and on HI-80 plates (■) are shown. (A) Decline of strain MLT403; (B) decline of strain C7184-opaque; (C) decline of strain VV1009. The declines of strains MLT367 and 2400-112 were very similar to the declines of strains C7184-opaque and VV1009, respectively (data not shown). Each point is the mean of values from duplicate microcosms. The standard errors averaged 20% of the presented values when the CFU per milliliter were greater than 100.

FIG. 2

Growth curves (■) of V. vulnificus strains C7184-opaque (A) and MLT367 (B). Also shown are the plate counts for the two strains on HI plates (▴) and HI-80 plates (●) after cold ASW microcosm samples had been shifted to room temperature. The strain C7184-opaque sample was taken on day 25, and the strain MLT367 sample was taken on day 26. Strains MLT403, VV1009, and 2400-112 exhibited similar responses (data not shown). After the HI plate counts increased to the level of the HI-80 plate counts, the counts were essentially identical, and only the HI-80 plate counts are shown past that point.