The viabilities of five strains of Vibrio vulnificus were evaluated during the storage of the organisms in sterile seawater at 5°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.

Whether Escherichia coli K-12 strain W3110 can enter the "viable but nonculturable" state was studied with sterile and nonsterile water and soil at various temperatures. In nonsterile river water, the plate counts of added E. coli cells dropped to less than 10 CFU/ml in less than 10 days. Acridine orange direct counts, direct viable counts, most-probable-number estimates, and PCR analyses indicated that the added E. coli cells were disappearing from the water in parallel with the number of CFU. Similar results were obtained with nonsterile soil, although the decline of the added E. coli was slower. In sterile water or soil, the added E. coli persisted for much longer, often without any decline in the plate counts even after 50 days. In sterile river water at 37؇C and sterile artificial seawater at 20 and 37؇C, the plate counts declined by 3 to 5 orders of magnitude, while the acridine orange direct counts remained unchanged. However, direct viable counts and various resuscitation studies all indicated that the nonculturable cells were nonviable. Thus, in either sterile or nonsterile water and soil, the decline in plate counts of E. coli K-12 strain W3110 is not due to the cells entering the viable but nonculturable state, but is simply due to their death. MATERIALS AND METHODS Bacterial strains and preparation of inocula. The E. coli K-12 strain used was the standard prototrophic wild-type strain W3110 (1). For the studies requiring a plasmid-bearing strain, strain W3110 was transformed with the plasmid pBR322 (2). This particular plasmid was used to permit the inclusion of ampicillin and tetracycline in the direct viable count (DVC) method and to facilitate the PCR measurements employed. Fresh cultures of the strains W3110 and W3110(pBR322) that had been grown for 14 h in Luria-Bertani (LB) medium [plus ampicillin and tetracycline in the case of W3110(pBR322)] were washed with sterile 0.9% saline, adjusted to the desired cell concentration, and added to the water microcosms in 10 ml of inocula or to the soil microcosms in 3 ml of inocula. Media and chemicals. Levine eosin methylene blue (EMB) agar, tryptone, yeast extract, brain heart infusion medium, and Bacto-agar were obtained from Difco Laboratories (Detroit, Mich.). Cycloheximide, nalidixic acid, acridine orange, 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyltetrazolium chloride (INT), and glycine betaine were obtained from Sigma Chemical Co. (St. Louis, Mo.). LB medium (35) was used to grow the strains; LB agar was LB medium with 15 g of Bacto-agar per liter. Plating of nonsterile soil suspensions on LB agar gave low total counts, mainly because the colonies were often obscured by mats of fungal growth and because of the tendency of many of the soil bacteria to swarm. Use of soil extract (SEC) agar gave higher total counts, and inclusion of cycloheximide prevented both fungal growth and bacterial swarming. Soil extract was prepared by suspending 1 kg of soil and 0.5 g of calcium carbonate in 1 liter of distilled water. The suspension was autoclaved for 60 min...

A new method, called the mixed culture recovery (MCR) method, has been developed to determine whether recovery of culturable bacterial cells from a population of largely nonculturable cells is due to resuscitation of the nonculturable cells from a viable but nonculturable state or simply to growth of residual culturable cells. The MCR method addresses this issue in that it involves the mixing of two easily distinguishable strains (e.g., lactose positive and negative) in such a way that large numbers of nonculturable cells of both strains are present together with a small number of culturable cells of only one strain, performing a nutrient addition resuscitation procedure, and then plating the cells to determine whether both cell types are recoverable. In repeated experiments with strains ofEscherichia coli, Klebsiella pneumoniae,Enterococcus faecalis, Enterobacter aerogenes, and Salmonella choleraesuis, only cells of the culturable strain were recovered after application of various resuscitation techniques. These results suggest that the nonculturable cells were dead and that the apparent resuscitation was merely due to the growth of the remaining culturable cells.

The fate of a derivative of Escherichia coli strain W3110G [pBGH1], a strain used for production of bovine somatotropin, was examined in semi-continuous activated sludge (SCAS) units. A nalidixic acid-resistant derivative of W3110G [pBGH1], strain LBB270 [pBGH1], was used to facilitate tracking. SCAS units (300 ml) containing municipal mixed liquor were operated on a daily cycle of 23 h aeration and 1 h setting followed by decanting of clear supernatant (175 ml) and refilling with fresh primary effluent. SCAS units were inoculated with two concentrations of E. coli LBB270 [pBGH1] and operated for 200 h. Viable levels of E. coli LBB270 [pBGH1] were measured daily in aerated mixed liquor and decanted supernatant. Viable counts in the mixed liquor decreased from 10,000- to 100,000-fold in less than 200 h. Losses of E. coli LBB270 [pBGH1] in decanted supernatants accounted for less than 2-fold of the total losses observed in the SCAS units. The E. coli LBB270 [pBGH1] was not evenly distributed in the mixed liquor, but became preferentially associated with the settleable floc. These results show that E. coli LBB270 [pBGH1] was unable to survive in municipal sludge even when inoculated at concentrations greater than, or comparable to, levels of indigenous microorganisms.

This study examined the transfer of the plasmid pBGH1, an expression vector for bovine somatotropin (BST), from Escherichia coli K-12 strain W3110G [pBGH1] to indigenous microorganisms present in flasks containing Missouri River water. Strain LBB269 is a nalidixic acid-resistant derivative of W3110G which was used as a plasmid-free control strain in these studies. Water samples were inoculated with strains W3110G [pBGH1] and LBB269; after 21 days of incubation the number of viable colony-forming units (CFU) of W3110G [pBGH1] and LBB269 were reduced from an initial level of about 1 x 10(7) CFU per ml to less than 1 CFU per 100 ml. At this time indigenous microbes resistant to both ampicillin and tetracycline (the antibiotic resistance markers on pBGH1) were isolated from 100 ml of water from each of the flasks inoculated with either strain W3110G [pBGH1] or LBB269. Plasmid DNA was isolated from these organisms and examined for sequences containing the gene for BST from pBGH1, using a polymerase chain reaction (PCR) assay. As expected, the day 0 sample from the flask inoculated with E. coli K-12 strain W3110G [pBGH1] gave a positive PCR response and the day 0 sample from the flask inoculated with E. coli K-12 strain LBB269 gave a negative PCR response. All of the day 21 samples containing indigenous microbes isolated from flasks that were inoculated with either W3110G [pBGH1] or LBB269 were negative in the PCR assay, indicating that the target sequence from pBGH1 was not present in any of these indigenous microorganisms.(ABSTRACT TRUNCATED AT 250 WORDS)