The Survival of Shigella in Sewage12

The Survival of Shigella in Sewage12 I. An Effect of Sewage and Fecal Suspensions on Shigella flexneri WEN LAN Lou WANG, STUART G. DUNLOP AND ROBERT G. DE BOER Department of Microbiology, University of Colorado School of Medicine, Denver, Colorado Received for publication August 19, 1955 Leiguarda, Peso, and Kempny (198) reported that no Shigella were isolated during their river water studies in Argentina, although 67 per cent of the samples showed Salmonella. Gispen and Gan (195) also reported the frequent isolation of Salmonella from river water in Indonesia, but Shigella were not recovered. During a five-year study of irrigation water in Colorado, the present authors likewise failed to isolate Shigella despite the frequent recovery of Salmonella (Dunlop, Twedt and Wang, 1952). The isolation of Shigella organisms from stool specimens also has not been satisfactory. Kligler, Oleinik, and Czazkes (193) stated that the primary reason for this failure of isolation of Shigella from cases of dysentery appeared to be the age of the stool. Stools cultured shortly after they were voided gave more positives than when culture was delayed. These authors suggested that the presence of Shigella bacteriophage was probably responsible for the rapid death of dysentery bacteria in stools. As Shigella organisms are associated frequently with man's exereta, the danger of consuming raw fruits and vegetables irrigated with sewage-polluted water is recognized. A study, therefore, has been undertaken to investigate the survival of Shigella in sewage and sewage-contaminated irrigation water, and to explore the methods used for isolation of these organisms. MATERIALS AND METHODS Preliminary studies indicated that a large number of Shigella flexneri (Shigelli paradysenteriae) were required to be added to raw sewage before recovery of these organisms could be made. An investigation was undertaken to study the cause of the difficulties in recovering these organisms from raw sewage. In order to eliminate the competition of growth of the other organisms naturally present in sewage, autoclaved sewage was used for the survival studies with S. fiexneri. A known number of S. flexneri II (culture obtained from the Communicable Disease Center, Chamblee, Georgia) was added to 1 Presented in part at the national meeting of the Society of American Bacteriologists in New York City, May 11, 1955. 2 This study was supported by a grant (E-52) from the National Institutes of Health, Public Health Service, Department of Health, Education and Welfare. 3 sterile sewage and their survival was determined by streaking duplicate plates of SS agar (Difco) with.1 ml of the inoculated sewage, serially diluted, at certain periods up to 96 hours. LB agar (Bertani, 1951), which contained no inhibitive constituents, was used for comparison. The sewage specimens were collected from the Denver sewage disposal plant and from two other plants, Thornton and Westminster, which represented only domestic sewage. Later, studies were extended to fecal specimens collected from various individuals. For the dilution of all suspensions,.1 per cent of bovine albumin in distilled water was used, as the bacteria were found to be stable in bovine albumin during the dilution period (Fenner, Martin and Pierce, 198). When the period of contact exceeded 2 hours, air contaminants occasionally occurred in the samples during manipulation. One unit of penicillin per ml of sample was added to avoid this complication. Tests made indicated that S. flexneri II was resistant to 5 to 25 units of penicillin per ml of broth. RESULTS Effect of sterile sewage. The recovery of S. flexneri II from sterile sewage (Denver sewage plant) at to 72 or 96 hours is shown in table 1. As indicated by LB agar, S. flexneri II multiplied in these samples in 6 to 2 hours. On the other hand, from the determination with SS agar, these organisms appeared to decline sharply after exposure to sewage for 6 hours. It may be noted in table 1 that each sample of sewage acted somewhat differently; the general pattern, however, was similar. A total of 18 samples of sewage from the Denver plant showed similar results. Figure 1 shows the results of a typical experiment graphically. As media without inhibitive constituents, such as LB agar, permit the growth of many organisms present in sewage, Shigella usually are overgrown and cannot be isolated. SS agar, on the other hand, is rather routinely used for the isolation of Shigella as well as Salmonella. Therefore, it seemed important to investigate the cause of the failure to grow S. flexneri II on SS agar. Further experiments were carried out using sewage from different sewage disposal plants containing only domestic sewage. Table 2 shows the results of 2 typical experi-

SURVIVAL OF SHIGELLA IN SEWAGE. I 1956] 35 TABLE 1. Comparison of the counts per ml of Shigella flexneri II on LB and SS agar after exposure to sterile sewage for periods up to 96 hours Test #1 Test #2 Test %3 LB SSt LB SS LB SS 5,3, 7,2 5,3 7,6 3,8 6 15, <51 23, 1 6,2 3,6 2 2,2, 22, 96, 3 21, <5 8 1,,,8, 1, 95 16, < 5 72 17,, 6,8, 1,, 2, 27, < 5 96 - _ - 3,2, 1, -I ox IL 5 * LB = a noninhibitive medium (Bertani, 1951). I No growth on duplicate plates with.1 ml inoculation. - O 3- NO GROWTH ON DUPLICATE PLATES WITH. I ML. INOCULATION o~~ I ~~~ 6 2 5 72 FIG. 1. Comparison of the growth curves of Shigella flexneri II in sterile sewage from Denver as determined by LB and SS agar. ments. S. flexneri II lost the ability to grow on SS agar after 6 hours exposure to sterile sewage and regained this ability after 2 hours. On LB agar, the organisms grew well and a normal growth was exhibited. Twentyfive more samples of domestic sewage showed a similar effect. Effect of fecal suspensions. The last group of experiments showed that this effect, demonstrated on SS agar, was also associated with domestic sewage. Studies were made to determine whether fecal suspensions would show this same phenomenon. Figure 2 shows LB TABLE 2. Comparison of the counts per ml of Shigella flexneri II on LB and SS agar after exposure to sterile domestic sewage for periods up to 72 hours w Sewage from Thornton Sewage from Westminster LB* SSt LB SS,9 1,5 3,6 1,7 1 3,6 3,5 98 6,3 <51 5,2 <5 2 1, <5 11, <5 8 3,7, 2, 2,6, 12, 72 1,, 9,3, 12,, 7,5, * LB = a noninhibitive medium (Bertani, 1951). $ No growth on duplicate plates with.1 ml inoculation. 5- co _ /tno GROWTH ON DUPLICATE PLATES O V WITH.1 ML. INOCULATION 2-2 72 FIG. 2. Comparison of the growth curves of Shigella flexneri II in sterile fecal suspension as determined by LB and SS agar. that the factor was also present in sterile fecal suspensions. The organisms multiplied in this medium in 2 hours, as determined by subculturing to LB agar. Using SS agar, the number of organisms appeared to drop off after 6 hours exposure. The organisms again regained the ability to grow on SS agar after 2 to 8 hours exposure. Effect of heat. To test whether heat in autoclaving was responsible for the production of this effect, bacteriafree fecal filtrates were tested along with autoclaved fecal suspensions. These filtrates were shown to be free of specific bacteriophage for S. flexneri II. The results (table 3) show that after the organisms were exposed to

36 W. L. L. WANG, S. G. DUNLOP AND R. G. DE BOER [VOL. TABLE 3. Comparison of the counts per ml of Shigella flexneri II on LB and SS agar after exposure to autoclaved fecal suspensions and filtered fecal suspensions Test # 1 Test %2 Autoclaved Filtered Autoclaved Filtered LB* Sst LB 6,1 2,7 5,8 6 8,2 2 8,8 2 35,, 6,2, -t * LB = a noninhibitive medium (Bertani, 1951). t Contaminated with gram positive rods. TABLE. Failure of Shigella flexneri II to develop resistance to the sewage or fecal effect during 2 to 96 hours exposure; comparison of counts per ml on LB and SS agar of original and derived cultures exposed to sewage or fecal suspension Sewage Fecal Suspension Original culture Derived culture %1* Original culture Derived culture #2* LBt SSt LB SS LB SS LB SS 5,2 2,,8 2,5,5 1,7 5, 1,6 6 7, 2 9, 1 6, <5 5, <5 2 72, 17, 1,2, 6, 3,9, 1,1, 1,8, 5, 8 23,, 8,7, 22,, 17,, 12,, 7,6, 8,8, 1,, * Organisms isolated as SS agar from sterile sewer or fecal samples which had been inoculated 2 to 96 hours previously with Shigella flexneri II. t LB = a noninhibitive medium (Bertani, 1951). SS = Difco SS agar. No growth on duplicate plates with.1 ml inoculation. either filtered or autoclaved specimens for 6 hours, the growth on SS agar dropped to very low levels; but the organisms were either stable or multiplied as determined by LB agar. However, at 2-hour periods, the organisms multiplied in autoclaved suspensions and dropped off in filtered fecal suspensions, as indicated by both LB and SS agar. The drop in number of organisms in filtered specimens was possibly due to the removal of nutritional requirements for the organisms. Nevertheless, the results indicated that the organisms were affected at 6 hours in both autoclaved and filtered specimens, and that heat was not responsible for the formation of the observed effect. Variant phenomenon. In order to find out whether the organisms growing on SS agar after being in fecal suspensions or sewage for 2 to 96 hours were variants of the stock strain of S. flexneri II, these organisms on SS agar were isolated from several specimens and reinoculated into the same or different samples of sterile sewage or fecal specimens and recovery studies were made as usual. The original stock strain of S. flexneri II was used as a control. Table indicates that the derived organisms were affected by the sterile sewage or fecal suspension just as much as the stock strain. Similar results were obtained with 7 additional samples. Other Shigella flexneri. The effect of sterile sewage on the various types of S. Jlexneri was also tested twice. S. flexneri III failed to grow on both LB and SS agar after 6 hours exposure to sewage. The effect on S. flexneri I and IV was the same as noted previously with type II. S. flexneri V and VI did not grow on SS agar at all. Effect of ions. Experiments presented thus far showed that sterile sewage and fecal suspensions contained factors which could cause S. flexneri II, inoculated into these media, to lose their ability to grow on SS agar. As this factor could very well be responsible for the difficulties encountered in the isolation of Shigella from sewage or fecal specimens, attempts have been made to study the nature of this phenomenon and, if possible, to eliminate it. It had been noted previously in some studies with Shigella bacteriophage that CaCl2 (2.5 X 13M) exerted a slight inhibitory effect on S. flexneri II during the early growth phase in LB broth when the organisms were subsequently transferred to SS agar. This suggested that certain salts or ions in the exposure medium, such as sewage, feces or broth, might be responsible. Although CaC12 had an inhibitory effect, NaCl, on the other hand, was found to be essential for subsequent

1956] SURVIVAL OF SHIGELLA IN SEWAGE. I 37 -J a. ) 'I " w~/s 2-~ ~ ~ ~ ~ ~ -S - 6 d :i 8 FIG. 3. The effect of adding sodium chloride (.15 M) and sodium chloride and phosphates (K2HPO-.17 M and KH2PO -.8 M) on the survival of Shigella flexneri II in sterile sewage. growth on SS agar. Studies, therefore, were made to reverse this effect by adding different ions into sterile sewage or fecal specimens. Figure 3 shows the effects of adding NaCl, K2HPO and KH2PO (or Na2HPO plus NaH2PO) and NaCl to sterile sewage along with S. flexneri II. As compared with the controls with no salts added the addition of NaCl alone to sterile sewage resulted in a partial reversal of the inhibitory effect at 6 hours and recoveries of organisms declined sharply after 6 hours, as indicated by both LB and SS agar. The addition of NaCl plus either sodium or potassium phosphates achieved striking reversal. Further experiments indicated that potassium or sodium phosphates alone added to sterile sewage also reversed this effect. In the presence of NaCl, lowering the ph of sewage from 9. or 9.5 to 7.5 with HCI, H2S or potassium phthalate resulted in complete recovery of the organisms on SS agar. As for fecal suspensions, figure shows that the effect on S. flexneri II was reversed by the addition of NaCl alone. The combinations of NaCl and sodium or potas- J. (L& 2LID C, -j FIG.. The effect of adding sodium chloride (.15 M) on the survival of Shigella flexneri II in sterile fecal suspension. -J CL \ LAdding NaCI P en2-\ rx CONTROL SS ~~~~~~~and I_II 3 ' 7 8 FIG. 5. The effect of adding sodium chloride (.15 M) and phosphates (K2HPO-.17 M and KH2PO-.8 M) after the 6-hour exposure of Shigella flexneri II in sterile sewage. sium phosphates were also tried and showed similar reversal. It appeared that only NaCl was necessary as the ph of sterile fecal suspensions was normally about 7.. The minimum concentration of NaCl needed for this reversal was about.35 M. Potassium chloride reacted the same as NaCl. Therefore, the effect of K2HPO orna2hpo (.17 M) and KH2PO or NaH2PO

38 W. L. L. WANG, S. G. DUNLOP AND R. G. DE BOER [VOL. (.8 M) in combination (.25 M) is probably due to the concentration of potassium or sodium ions (.2 M). As under natural conditions in sewage, the organisms have been exposed for periods up to 6 hours or more before samples are taken, it was interesting to try the effect of adding NaCl and P after such exposure but prior to transfer to SS agar. Figure 5 shows that the reversal is almost immediate. However, a recovery period of at least one hour is more desirable. DISCUSSIOX The results showed that S. flexneri II added to sterile sewage or to a fecal suspensioni would not grow on SS agar when transferred after 6 to 2 hours exposure. On the other hand, transfers to LB agar indicated that the organisms were either viable or even multiplying in these specimens. Some unknown factor or factors in sewage or fecal suspensions affected the organisms so that they grew only on media without inhibitive constituents. After a long exposure period, 2 to 8 hours, the organisms, however, regained the ability to grow on SS agar. This phenomenon was also true but to less extent when desoxycholate citrate agar (Difco) and MacConkey's agar (Difco) were tested. It was noted that the effect was the greatest during the lag or early logarithmic period of growth. This suggests that the organisms are more sensitive to the inhibitive agents in these media during these phases. In a few samples of sewage, the number of organisms declined after 6 to 2 hours exposure as determined by both LB and SS agar. Was this effect similar to the phenomenon already described? Heinmets, Taylor and Lehman (195) reported that suspensions of Escherichia coli, strain B/8, which have been sterilized by the action of heat, chlorine, Zephiran chloride, and ethyl alcohol, contained viable cells when incubated with various metabolites of the tricarboxylic acid cycle. It is obvious that the present concepts of sterility are arbitrary. From the observations made in this report, it may also be stated that sewage or fecal specimens, negative for Shigella when cultured on SS agar, may, in fact, still contain viable cells if a less inhibitive medium could be used. However, the overgrowth of other organisms in sewage or fecal specimens on noninhibitive media usually renders the isolation of Shigella impossible, and some more selective medium is required. It is interesting to note that the addition of NaCl reversed this effect in fecal suspensions, and that the reaction was almost immediate. For sewage, adjusting the ph from 9. to 9.5 to about 7.5 was necessary for the reversal effect of NaCl. It was also noted that S. flexneri II in a broth without NaCl added would not grow well on SS agar. It seems that NaCl or KCl protected the organisms in the exposure medium, and they could then grow on SS agar normally. Adding NaCl inlto SS agar did not show any protective effect, however. The mechanism of this action is not yet determined. It may be that these cations, sodium or potassium, are absorbed to the surface of the negatively charged bacteria in the sewage or fecal suspensions, and thus the organisms become more resistant to the surface action of the anionic bile salts present in the inhibitive media. The reversal action of NaCl may also be reflected in the use of formalinized buffered glycerol saline for the preservation of dysenteric stool specimens (Kligler, Oleinik and Czazkes, 193). It may be that the role of buffered saline is to give protective action to the organisms so that they will grow better on SS agar. SUMMARY Shigella flexneri II, inoculated into sterile sewage or fecal suspensions, appeared to decline sharply in numbers, as indicated by transfer to SS agar after 6 to 2 hours exposure in the sewage. No decline was noted when transfers were made to a medium without inhibitive constituents. Autoclaving the sewage or fecal suspension was shown not to be responsible for the production of this effect and no variant phenomenon was involved. This observed effect was reversed by adding NaCl (or KCl) into fecal suspensions. With sewage, NaCl (or KCl) also reversed the effect, but only when the ph was first adjusted to about 7.5. Sodium or potassium phosphate was equally as effective as NaCl or KCl in reversing the effect. It is suggested that the sodium or potassium ions are the essential elements in the reversal. REFERENCES BERTANI, G. 1951 Studies on lysogenesis. I. The mode of phage liberation by lysogenic Escherichia coli. J. Bacteriol., 62, 293-3. DUNLOP, S. G., TWEDT, R. M., AND WANG, W. L. L. 1952 Quantitative estimation of Salmonella in irrigation water. Sewage Ind. Wastes, 2, 115-12. FENNER, F., MARTIN, S. P., AND PIERCE, C. H. 199 The enumeration of viable tubercle bacilli in cultures and infected tissue. N. Y. Acad. Sci. Annals, 52, 751-76. GISPEN, R., AND GAN, K. H. 195 The antibacterial effect of river water on Shigella shigae in connection with the presence of corresponding antagonists and bacteriophages. Antonie Van Leeuwenhoek. J. Microbiol. Serol., 16, 373-385. HEINMETS, F., TAYLOR, W. W., AND LEHMAN, J. J. 195 The use of metabolites in the restoration of the viability of heat and chemically inactivated Escherichia coli. J. Bacteriol., 67, 5-12. KLIGLER, I. J., OLEINIK, E., AND CZAZKES, I. 193 Improved technic for isolation of dysentery bacteria from stools by formaldehyde inactivation of bacteriophage. Am. J. Public Health, 33, 682-68. LEIGUARDA, R. H., PEso,. A., AND KEMPNY, J. C. 198 Investigacion de bacterias de los generos Salmonella y Shigella en agua del Rio de la Plata. Assoc. (Inter- American) Ingenieria Sanitaria, 2, 153-163.