Studies on Pathogenic Halophiles Ii. Salt Requirements for Growth and Survival Yukio Yamazi, Tsuneo Kozima, Tsuyoshi Shiba and Tiuichi Ishizeki

Japan. J. Microb., Vol. 3, No. 1, 1959. UDC: 576.8:546.33.13

STUDIES ON PATHOGENIC HALOPHILES

II. SALT REQUIREMENTS FOR GROWTH AND SURVIVAL

YUKIO YAMAZI, TSUNEO KOZIMA, TSUYOSHI SHIBA ANDTIUICHI ISHIZEKI Department of Hygiene and Bacteriology,National Hygienic Laboratory, Tokyo, Japan SADAYOSHI HATTA Department of Hygiene, Nippon Medical College,Tokyo, Japan

(ReceivedDecember 24, 1958)

Halophiles are a group of bacteria which grow well only in media containing more than 2 per cent salt. Few studies have been carried out on the effect on en- vironmental conditions established by salt on the pathogenic halophiles. Sodium chloride can be replaced by certain other salts for some halophiles but not for others.(1-4) According to Shoop(5)facultative halophilic bacteria grow well in media containing other salts than sodium chloride, but obligate halophilic bacteria speci- fically require sodium chloride. The purpose of this paper is to present results of an investigation on the salt requirements of two pathogenic halophile strains, N4 and EB102, with particular re- ference to the replacement of sodium chloride with a variety of cations and anions. N4 and EB102 strains have been isolated respectively by Takikawa(6) and Fujino et al.(7) from cases of food-poisoning, and have been identified as Pseudomonas.

(6,8) MATERIALS ANDMETHODS Cultures The cultures used in this experiment were same as used in report 1(8). Experimental procedure Salt requirements were examined using solutions of sodium chloride, sodium nitrate, sodium bromide, sodium iodide, sodium sulfate, potassium chloride, magnesium chloride and magnesium sulfate. Concentrated stock solutions of the salts were prepared and working solutions for each salt were made from the stock solutions. Working solution with concentrations of 0 to 20 M in 2 M steps was prepared for every salt examined. One volume of the working solution for a particular salt was added to 9 volumes of 1.1 per cent peptone and adjusted to pH 7.6 to 7.8. Each tube was added with 5 ml of each test medium, and sterilized by autoclaving. After cooling, the tubes were allowed to stand at least 4 hours before inoculation. This was done to ascertain that no precipitation of any material occurred. The inoculum used in this experiment was one drop of a twice-washed 24 hour culture grown in peptone solution containing 3 per cent sodium chloride. Inoculum 34 YAMAZI, KOZIMA, SHIBA, ISHIZEKI AND HATTA Vol. 3, No. 1

was added to 5 ml test medium, as described above. The inoculated tubes were incubated for 24 hours at 37•Ž. Immediately following the incubation interval, the cell density of the media was determined by a Kotaki AKA electrophotometer using a 470 mƒÊ filter. Previously we confirmed that N4 and EB102 grew well respectively at pH 5.6 to 7.8 and 6.4 to 8.2. Takikawa observed that the optimal pH for the growth of N4 strain was 6.6 to 7.8(6). Therefore, the test media in this experiment were adjusted to pH 7.6 to 7.8.

RESULTS

Response of N4 and EB102 to sodium chloride The growth of N4 and EB102 strains in peptone solutions containig various amounts of sodium chloride is shown in figure 1. N4 grew well in 0.2 to 1.2 M sodium chloride, EB102 in 0.2 to 1.0 M; they did not grow in peptone solutions with no sodium chloride and with more than 1.6 to 1.8 M. When these incubations were made in anaerobic conditions, where air was replaced by hydrogen or carbon dioxide, neither halophile grew well and in carbon dioxide they grew only in a strictly limited concentratson of salt.

Figure 1. Effect of NaCl Concentration on the Growth of Strain N4 and EB102

Response to cation and anion substitution Figure 2 and 3 show the growth of the halophiles in media containing other salts than sodium chloride. When chloride was substituted with other anions, N4 did not grow in sodium iodide, but grew well in 0.4M sodium nitrate and in 0.2 to 0.4M sodium bromide or sodium sulfate. When sodium was substituted by some other cations, 0.2 to 0.6 M potassium chloride or magnesium chloride gave a good growth of N4. Therefore it may be concluded that the opt- imum concentration of these substituted salts to the growth of N4 were, if any, lower and more strictly limited than that of sodium chloride. The effect of magnesium sulfate on the growth of N4 was similar to that of sodium chloride. In figure 2 and 3, •gNa2SO4+MgC12•h means that each molecular concentration of sodium sulfate and magnesium chloride in the medium is 1/2 of each molecular concentration number plotted in the figures. Magnesium chloride and sodium sulfate, or sodium chloride and magnesium sulfate are considered to exist in media containing sodium sulfate and magnesium chloride. It may be concluded that each of magne- January, 1959 STUDIES ON PATHOGENIC HALOPHILES 35

Figure 2. Effect of Concentration of saltes on the Figure 3. Effect of Concentration of salts on the Growth of Strain N4 Growth of Strain EB102

Figuse 4. Effect of Salt Concentration with 0.05M Figure 5. Effect of Salt Concentration with 0.05M NaCl on the Growth of Strain N4 NaCl on the growth of Strain EB102 36 YAMAZI, KOZIMA, SHIBA, ISHIZEKI AND HATTA Vol. 3, No. 1 sium chloride, sodium sulfate and sodium chloride are not antagonized respectively by sodium sulfate, magnesium chloride and magnesium sulfate but magnesium sulfate is antagonized by sodium chloride. This is realized by observing the maximum concentration of each salt for growth of N4 in figure 4. When sodium chloride was substituted with sodium iodide, sodium nitrate and sodium sulfate, these substituted salts did not support the growth of EB102 at all. But EB102 grew weakly in a medium containing 0.4 M sodium bromide or sodium chloride. When sodium was substituted with some other cations, EB102 grew well only in 0.2 M magnesium chloride and weakly in 0.2 to 0.8M potassium chloride, and the maximum concentration of these two salts to the growth of EB102 was lower than that of sodium chloride. In media containing magnesium sulfate EB102 behaved with a growth similar to that observed in sodium chloride. Consequently it may be concluded that magnesium sulfate is as adequate as sodium chloride for the growth of halophiles employed in this experiment. The experimental results with media containing magnesium chloride plus sodium sulfate suggest that sodium chloride, magnesium chloride and magnesium sulfate behave similarly with N4. But it is concluded that with EB102 strain the adverse effect of sodium sulfate is antagonized by magnesium chloride, because EB102 does not grow in a medium containing sodium sulfate alone.

SUMMARY AND DISCUSSION

The best growth of N4 and EB102 strains was observed in the presence of 0.4 to 1.2M (2.3 to 7.0 per cent) sodium chloride. This agrees with Takikawa's result(6). Consequently these halophiles can be classified with the facultative halophiles according to Flannery(9). In this experiment, anaerobic culture was performed in almost 100 per cent carbon dioxide or hydrogen, and it was observed that the growth of N4 and EB102 was poor and the peak of the growth curve occurred with lower salt concentrations. The influence of gases upon halophilic bacteria was of special interest to Stuart et at.(10), who made a study in which varying amount of several gases were substituted for air, concluding that red-pigmented halophiles have a preference for reduced oxygen tension. Flannery(9) repeated the work quantitatively with a nonpigmented halophile, and concluded that oxygen tension has little influence on the growth response of the organism. Sodium chloride, magnesium sulfate, potassium chloride , magnesium chloride, sodium bromide, sodium sulfate and sodium nitrate were employed in our investigation of salt requirements of pathogenic halophiles. From the experimental results it is evident that the sodium chloride requirement of the halophile strains is not specific . With salts in which anion substitution was made, chloride, bromide , sulfate and nitrate were found to contribute to the survival of N4 in the presence of sodium . Iodide not to contribute to survival. Chloride and bromide contributed to survival of EB102 in the presence of sodium, but sulfate, nitrate and iodide did not . When cation substitutions were made, sodium, magnesium and potassium contributed to the survival of both strains in the presence of chloride. The degree of the beneficial January, 1959 STUDIES ON PATHOGENIC HALOPHILES 37

effect of ions is described above. Magnesium was more favourable to these organisms than sodium in the presence of sulfate. It was observed that the maximum concentration allowing growth of halophiles was around 1.6M even for the most effective salts. Although our results of salt requirements resemble those of Takikawa(6), some differences are observed which can be ascribed to differences of the experimental methods. Eisenberg(11) and Holm et al.(22) observed that the toxic effect to anions in the presence of sodium for non-halophiles, was in order of toxicity, fluoride, iodide, nitrate, phosphate, bromide, sulfate and chloride ions. A similar order of toxicity was observed in our experiment, except for sulfate and bromide. According to Winslow et al.(13), an antagonistic effect between salts may be explained by antagonism between cations. In our experiment in which halophiles were incubated in media containing one of various salts plus 0.05M sodium chloride, the toxicity of iodium, sulfate, nitrate, and bromide was observed to be antagonized by chloride. This may be due to the supporting effect of a chloride ion concentration as low as 0.05M as an important growth promoting factor for these halphiles. The results of our experiment do not seem to agree with an opinion of Flannery et al.(4,9) that sodium ions are more important than chloride ion for the growth of V. costicolus, but it must be noted that no cations generally considered to inhibit enzymes were used in our experiment. Lefebre et al.(14) suggested that a certain osmotic pressure may be required by halophilic bacteria, and that if this be so other salts would act similarly and satisfy the requirement. Lamanna et al.(15) insisted on the importance of choosing a mea- sure to separate the effects of osmotic pressure and chemical activity of an environ- ment required by halophiles. Though nonelectrolytes (sugars) were used extensively by investigators of osmotic pressure phenomena, it was found that the sugars would not substitute for the salt required by V. costicolus.(4,9) Richter(9) proposed that sodium chloride has at least two functions to perform for halophilic bacteria; one function is of a nutrition and the other is of a regulator of osmotic pressure. Outline of our conclusions on salt requirements of pathogenic halophiles is following: Chloride, bromide, sulfate and nitrate contributed to the well-being of N4 strain in the presence of sodium and iodium did not contribute to it. Chloride and bromide contributed to that of EB102 strain in the presence of sodium, and sulfate, nitrate and iodium did not. Sodium, magnesium, and potassium contributed to the well-being of both strains in the presence of chloride. The degree of that contribu- tional effect of the ions was observed in order of above description. While magnesium was more favourable to these organisms than sodium in the presence of sulfate. Sodium ion and chloride ion contributed to the well-being of these organisms, but the sodium ion was more important of the two. These investigations on the effect of cations and anions were made by a substitution method. In addition, the experiment was performed with respective media added with also 0.05 M sodium chloride. 38 YAMAZI , KOZIMA, SHIBA, ISHIZEKI AND HATTA Vol. 3, No. 1

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