Evaluation of Factors Affecting Survival of Escherichia coli in Sea Water

CARLUCCI, A. F. (Rutgers, the State University, New Brunswick, N. J.), P. V. SCARPINO, AND DAVID PRAMER. Evaluation of factors affecting survival of Escherichia coli in sea water. V. Studies with heatand filter-sterilized sea water. Appl. Microbiol. 9:400404. 1961.-The bactericidal action of sea water was measured as the difference in survival of cells of Escherichia coli in untreated and autoclaved portions of water samples. The beneficial effect of sterilization by heat on the survival of E. coli in sea water varied with season and was most marked during summer months, however, the magnitude of the effect differed greatly from sample to sample. The more obvious and commonly suggested explanations for the bactericidal action of sea water were tested experimentally. The pH and salinity of sea water were changed by autoclaving, but the direction of the former was detrimental rather than beneficial and the significance of the latter was not clarified. The survival of cells of E. coli in filtered portions of some water samples was greater than that in untreated portions and equal to that in autoclaved portions, indicating that predators and competitors removed by filtration had contributed significantly to the rapid death of the bacterium in the untreated water. However, in the majority of samples tested, survival of E. coli in autoclaved water was considerably greater than survival in filtered water. The possibility that the beneficial effect of autoclaving over and above that of filtration resulted from inactivation or destruction by heat of bacteriophages and thermolabile toxic substances such as antibiotics was considered. Moreover, the suggestion was tested that the increased survival of E. coli in autoclaved sea water was due to the ability of heat to disrupt and degrade microbial cells and thermolabile compounds I Paper of the Journal Series, New Jersey Agricultural Experiment Station, Rutgers, the State University, Department of Agricultural Microbiology, New Brunswick, N. J. This investigation was supported in part by research grant E1437 from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, U. S. Public Health Service. 2 Present address: Research Department, United Fruit Company, La Lima, Honduras, C. A. 400 and, thereby, to cause an increase in concentration of available nutrients in sea water. It was concluded that the bactericidal action of sea water is not explicable in terms of the destruction or inactivation by heat of bacteriophages or antibiotics. Although added organic matter influenced the survival of E. coli, the test organism was not an effective competitor in sea water and the nutrient levels required to offset the bactericidal action were excessive. Artificial sea water was demonstrated to exert a bactericidal action comparable to that of natural sea water. Low levels of cysteine which favor survival of E. coli in natural sea water had a similar effect in artificial sea water. Nevertheless, it is not at this time possible to conclude that the factors responsible for the bactericidal action of artificial sea water are identical with those responsible in natural sea water. It has been observed repeatedly that bacteria survive to a greater extent in heat-sterilized sea water than in untreated sea water (Nicati and Rietsch, 1885; De Giaxa, 1889; Kiribayashi and Aida, 1934; ZoBell, 1936; Krassilnikov, 1938; Ketchum, Carey, and Briggs, 1949; Vaccaro et al., 1950; Nusbaum and Garver, 1955; Richou, Neant, and Richou, 1955). This thermolabile bactericidal action of sea water varied with season of the year and was greatest during summer months (Vaccaro et al., 1950). Furthermore, the survival time of bacteria in sea water has been extended by filtration, chlorination, and treatment with organic matter as well as by heating (Beard and Meadowcroft, 1935; Krassilnikov, 1938; Vaccaro et al., 1950; Williams, 1950; ZoBell, 1936). However, our present knowledge provides little insight into the nature of the factors responsible for the phenomenon observed. Although numerous explanations for the beneficial effect of complete or partial sterilization on the survival of bacteria in sea water have been suggested (Carlucci and Pramer, 1959), they have not been tested experimentally and the identity of the factors responsible for the more rapid death of bacteria in natural than in sterilized sea water remains to be established. on O cber 2, 2017 by gest ht://aem .sm .rg/ D ow nladed fom SURVIVAL OF E. COLI IN SEA WATER The present report describes the influence of season, pH, salinity, filtration, and organic matter on the bactericidal action of sea water. Moreover, artificial sea water was observed to exert a bactericidal action comparable to that of natural sea water. MATERIALS AND METHODS The bactericidal action of sea water was measured as the difference in survival of cells of Escherichia coli in untreated and autoclaved portions of the same water sample. Detailed descriptions of the methods employed for preparation and use of inocula, treatment and storage of water samples, and of the enumeration and calculation of survival of cells of E. coli in sea water were presented previously (Carlucci and Pramer, 1960a). Sea water samples were heat sterilized by autoclaving at 121 C for 15 min. Sterilization by filtration was performed using glass filter holders and type HA Millipore membranes3 having a pore size of 0.45 ,u. RESULTS AND DISCUSSION Seasonal variation. The bactericidal action of sea water was determined at irregular intervals for 2 years by measuring the survival of cells of E. coli in untreated and autoclaved portions of each water sample. It was first demonstrable in April, increased in magnitude to a maximum in July, then decreased, and was absent from water collected in November. The greatest bactericidal action was exerted by water sampled during July 1957 when the ratio of per cent survival after 48 hr in autoclaved to that in untreated water exceeded 24. These results were consistent with those of Vaccaro et al. (1950) who reported that the bactericidal action of sea water was most pronounced during the summer months and absent in the winter. Water collected during the winter months exerted no bactericidal action and it was not unusual for cells of E. coli to die more rapidly in autoclaved than in untreated portions of these samples. This has been observed (Williams, 1950) but not commented on by previous investigators. pH and salinity. The influence of autoclaving on pH and salinity and the possible contribution of these factors to the bactericidal action of sea water were considered experimentally. Electrometric determinations demonstrated that the reaction of sea water increased from 0.5 to 1.0 pH unit during heat sterilization and that equilibrium was not re-established for 24 to 48 hr. It has been noted previously that the survival of E. coli in sea water varied inversely with hydrogenion concentration (Carlucci and Pramer, 1960b) and on the basis of pH alone sterilization by heat would be expected to have an adverse rather than beneficial effect. Since the survival of E. coli in heat-sterilized water is increased rather than decreased the bactericidal 3 Millipore Filter Corporation, Bedford, Mass. action cannot be attributed to the influence of autoclaving on pH alone. The effects of sterilization by filtration and autoclaving on the salinity of sea water were examined and although there was tendency for filtration to cause a decrease and autoclaving to cause an increase in salinity, the magnitude of the changes never exceeded 1.0%. The nature of the changes was not determined and therefore their significance is not known. Filtration. Water samples collected during July and August of 1957 were divided into three portions. One was sterilized by filtration, the second was sterilized by autoclaving, and the third served as an untreated control. The survival of cells of E. coli in these waters was determined for each of six different collections. The results listed in Table 1 show that in each of the six samples tested E. coli died more rapidly in untreated than in autoclaved water but the magnitude of the effect varied greatly. Likewise, the influence of filtration was not constant. In four of the six experiments survival of the test organism was significantly greater in filtered than in untreated sea water. If it is assumed that the favorable effect of filtration resulted from removal of predatory and competitive organisms, then the numbers and activities of these organisms varied from sample to sample. Waksman and Carey (1935) considered predation as a factor contributing significantly to the death of bacteria in sea water. The decreased survival of E. coli in the filtered portion of water sample no. 5 can be explained by the possible but improbable assumption that a beneficial substance or organism was removed during filtration. In two of the samples tested (no. 1 and 6) the bactericidal action of the water could have been fully accounted for by predatory and competitive organisms that were eliminated by filtration; survival of E. coli was the same in filtered and autoclaved water. In two other samples (no. 3 and 5) filtration had no influence on survival of the test organism and it appeared unlikely that predation and competition contributed significantly to the rapid death of E. coli in the untreated water. In four of the six samples tested (no. 2, 3, 4, and 5), predation and competition were not an adequate explanation for the rapid death of E. coli in untreated water, since survival in autoclaved water was considerably greater than survival in filtered water. TABLE 1. Survival of Escherichia coli in untreated, filtered, and autoclaved portions of six sea water samples collected during July and August, 1957 Survival after 48 hr in sea water sample no.