United States Environmental Protection Agency Risk Reduction Engineering Laboratory Cincinnati OH 45268 Research and Development EPA/600/S2-89/019 Mar. 1990 &EPA Project Summary Protozoan Sources of Spontaneous Coliform Occurrence in Chlorinated Drinking Water Emmett B. Shotts, Jr. and Richard E. Wooley The spontaneous occurrence of conforms in chlorinated drinking waters has resulted in concern over their potential source and mecha- nism(s) of Introduction into water de- livery systems. Previous observations related to protozoa! resistance to chlorine coupled with the ingestion of bacteria by this group of organisms prompted an in-depth study of bacterial-protozoan interaction in the presence of chlorlnatlon. Bacterial organisms used were either from previous "spontaneous coliform outbreaks" or potential human pathogens. The protozoans utilized were laboratory adapted and wild strains of protozoans found naturally In water reservoirs. These organisms were established In axenic cocultures and the effect of various concentrations of chlorination and exposure were determined. It was found that, alone, the bacteria were killed by from 0.25-1 mg/L of chlorine; however, when ingested by protozoans, these same bacteria survived in significant con- centrations from levels of chlorina- tion 4 to 10 times the above dose. These findings provide a possible and plausible explanation of how bacteria may survive chlorination and appear in water delivery systems. It further suggests a need for increased awareness in developing improved methods of processing drinking water that include the removal of both viable and dead protozoans from entering the distribution system. This Project Summary was devel- oped by EPA's Risk Reduction Engi- neering Laboratory, Cincinnati, OH, to announce key findings of the research project that Is fully documented in a separate report of the same title (see Project Report ordering Information at back). Introduction The spontaneous occurrence of coli- forms in chlorine-treated drinking water has generated a great deal of concern, particularly in areas where reservoir water is sand filtered and chlorinated. Prior work on this problem addressed the production of chlorine resistant clones of bacteria and also the inability to readily isolate chlorine-injured bacteria. It was the purpose of this investigation to related the sparing and enhancement qualities noted in Legionella-Tetrahymena interaction into this emerging problem in water delivery systems. This was ac- complished by evaluating the bacterial (coliforms isolated from outbreaks)-proto- zoan (both laboratory and wild strains) relationship to determine if ciliated protozoans that naturally occur in water impoundments could provide protection against chlorination for engulfed bacteria, thus allowing the bacteria to be released spontaneously by the protozoan at a later time in the water distribution system. To achieve this, techniques were used that involved enumeration of bacteria follow- ing protozoal engulfment with subsequent ------- chlorination. Chlorination was done on aliquots both before sonication and after sonication, respectively, thus providing appropriate controls. From this research, it was determined that protozoa did provide protection against chlorination to engulfed coliform bacteria, in some cases, for at least 24 hr from initial exposure of 2-4 mg/L of chlorine although protection to exposures of 20 mg/L of chlorine was noted in some interactions. Materials and Methods This investigation examined the effects of free and combined chlorine upon selected protozoan-bacterial cocul- tures that might be found in drinking water resources. Comparative inactiva- tion studies were done on bacteria alone and in cocultures with protozoans under chlorine demand-free conditions that simulated those of routine water treat- ment. Selected coliforms previously recovered from "spontaneous occur- rences" in water distribution systems were obtained from the Risk Reduction Engineering Laboratory, USEPA, Cin- cinnati, OH. These include Klebsiella pneumonia, K. oxytoca, Enterobacter cloacae, E. agglormerans, Citrobacter freundii and Escherichia coli. Other organisms subsequently used included Campylobacter jejuni, Legionella gor- manii, Salmonella typhimurium, Yersinia enterocolitica, and Shigella sonneii. These bacteria were studied in coculture with Tetrahymena pyriformis (primary organism used because of its ease in handling) (ATCC 30327), Acan- thamobea castellanii (ATCC 30234), and Bodo edax (ATCC 30903). Axenic cultures of the latter resulted in limited success; subsequent engulfment studies were not fruitful and it was removed from the study. Near the end of the study, a wild strain of Cylidium was studied in coculture with £ coli to establish that the relationship extended beyond laboratory strains of protozoans. All studies were carried out using chlorine demand free buffer (CDFB) and were considered as chlorine demand free (CDF) when the chlorine con- centration was less than 0.01 mg/L free available chlorine as measured by DPD colorimetric method read in a B & L Spectronic 2000 spectrophotometer.* All chlorine estimations were standardized to chlorine quality control samples from "Mention of trade names and commercial products does not constitute endorsement or recom- mendation for use USEPA, Environmental Monitoring Sys- tems Laboratory, Cincinnati. Subsequent study of the experimental components (i.e., protozoa and bacteria alone) were done prior to coculture studies where concentrations of 1 x 104 of the respective bacteria and protozoan were allowed to interact. Following exposure to various con- centrations of chlorine at time intervals, bacterial viability and numbers were determined by plate counts using mT7 agar where possible to permit maximum recovery of possible chlorine-damaged cells. Prior to plating, the chlorine containing suspension of coculture was neutralized with sodium thiosulfate, centrifuged, and resuspended in sterile culture medium for sonication to release the bacteria from the protozoa. Samples were obtained and observed for engulfment, vacuole formation, and multiplication by electron microscopy (EM). Results As noted in Table 1, a great variability existed among bacteria isolated as to their survival within respective protozoan hosts. It was readily established that all bacteria unprotected by protozoan engulfment were dead at 1 mg/L of chlorine or less. The protozoa, on the other hand, were viable after 2 to 5 times this chlorine dose. These data indie that although Tetrahymena was killec 2 mg/L of chlorine, the bacteria con ued to replicate within the protozc Similarly, the same thing happened the Acanthamoeba, and, although re tance to chlorine was not demonstra in the wild type Cylidium, viable bact< were present after 24 hr. It should noted that in no case were more than 10 organisms noted ingested in protozoan, yet large percentages bacterial survival were obtained p viding evidence of bacterial replicat within both the viable and dead prc zoans. Discussion and Conclusions These studies have demonstrated alternate explanation to chlorine res tance as a mechanism of bactei introduction into water delivery syster This study further demonstrates tl although protozoans may be killed duri chlorination, viable bacteria may still introduced into water delivery systems These occurrences have been denru strated not only with isolates frc previous spontaneous coliform occi rences but also with several prims human pathogens of gastrointestir disease and also with Leg/one gormanii. In the latter case, the inti duction of viable organisms into t delivery system with subsequent temp* Table 1. Relative Survival of Selected Bacteria Engulfed by Protozoans and Exposec Varying Amounts of Chlorination for 24 Hours.* Protozoans* Bacteria& K. pneumonia K. oxytoca Ent. cloacae Ent. agglormerans Cit. freundii Esc. coli Camp, jejuni Leg. gormanii Sal. typhimurium Yer. enterocolitica Shig. sonneii Tetrahymena pyriformis 2/20* 1120 0.51100 2/70 1/30 4/60 0.25/10 4/50 2/65 4/100 41100 Acanthamoeba castellanii 10/80 4/20 2/80 2/100 0/0 1/100 -@ — Cylidium spp. - : — - — 'Percentage survival of engulfed bacteria at highest chlorine concentrations following 24 exposure. Tetrahymena resisted 1 mg/L chlorine for 24 hr; Acanthamoeba resisted 10 mg/L chlorine 24 hr. &AII bacteria (alone) were killed at 1 mg/L chlorine except for C. jejunt and Ent. cloacae (0 mg/L chlorine). * First number denotes highest concentration of chlorine in mg/L showing surviving bacterii 24 hr; second number represents the percentage of the original bacterial inoculum (1 x I surviving. @Not done ------- ature selection in hot water tanks could provide an explanation related to the spread of this disease in bath waters. In other studies, L pneumophila has been shown to act similarly. From the standpoint of gastrointestinal disease, it is suggested that, without further processing, drinking water that is sand filtered and chlorinated for human use presents a potential source of infection. This risk could be reduced by further processing of the water prior to distribution. In summary, a mechanism present in nature has been demonstrated whereby bacterial-protozoan interaction negates usual methods of water treatment for many supply systems and, despite chlorination, presents the possibility of the introduction of measurable amounts of potentially pathogenic bacteria into drinking water delivery systems. The full report was submitted in fulfillment of CR812833-01 by the Uni- versity of Georgia Research Foundation under the sponsorship of the U.S. Envi- ronmental Protection Agency. ------- Emmett B. Shotts, Jr. and Richard E. Wooley are with the University of Georgia, Athens, GA 30602. Eugene W. Rice and Harry 0. Nash are the EPA Project Officers (see below). The complete report, entitled " Protozoan Sources of Spontaneous Coliform Oc- currence in Chlorinated Drinking Water,"(0rder No. PB 89-190 292IAS; Cost: $13.95, sub/ect to change) will be available only from: National Technical Information Service 5285 Port Royal Road Springfield, VA 22161 Telephone: 703-487-4650 The EPA Project Officers can be contacted at: Risk Reduction Engineering Laboratory U.S. Environmental Protection Agency Cincinnati, OH 45268 United States Environmental Protection Agency Center for Environmental Research Information Cincinnati OH 45268 MALTY Official Business Penalty for Private Use $300 EPA/600/S2-89/019 u.s.OFFICIAL MAIL: U.S.POSIASf]" s 0 .2 5 : S00S°E»VIB PMTECTICH. CHICAGO ------- |