Økologisk byfornyelse og spildevandsrensning, 45 – Overlevelse af enterokokker og protozoen Cryptosporidium parvum i urin fra mennesker

Survival of Enterococci and Protozoan Cryptosporidium Parvum in Human Urine

Summary and conclusions

The background for the studies described in this report was the outcome of a previous project financed by the Danish EPA "Evaluation of the possibilities and constraints for re-circulating nutrients from cities to agriculture – microbiological studies of stored urine from urine separating toilets" (Dalsgaard and Tarnow, 2001). The former study indicated that some indicator bacteria could multiply in stored urine from separation toilets, and, thus, questioned the usefulness of using such indicators for assessing the hygienic quality of stored urine. Furthermore, a low number of protozoan oocysts (eggs) of Cryptosporidium parvum were found in a few urine storage tanks. These oocysts survived long-term storage and could infect mice in experimental infections. To follow up these finding, the Danish EPA wished to initiate controlled laboratory experiments for further assessments of these problems, with the aim of obtaining information that could possibly be used to define hygienic guidelines and suggest required storage times for human urine to be used in agri- and horticulture.

The studies were divided into two parts. The first part investigated the question of possible (re)growth of enterococci and total viable counts at 37°C in stored, separated human urine, and the second part investigated the survival of the protozoa C. parvum’s in stored, separated human urine.

Bacteriological part

Urine samples for the experiments were collected from four different localities and urine storage tanks representing densely populated urban housing areas (localities 1 and 2), and from garden allotments (localities 3 and 4). The urine was stored at 4, 10 and 20°C for up to six months. The pH, the ammonia concentration and microbiological parameters were measured at one month intervals. The microbiological quality of the urine was assessed based on numbers of the indicator bacteria enterococci, total viable counts at 37°C and suspected numbers of thermotolerant coliforms.

The pH values were stable around 8.5 to 9 and highest in urine from localities 3 and 4, where the use of water for flushing the toilets had been smaller. The ammonia concentration was also stable, with the highest concentrations found in urine from localities 3 and 4 (approx. 8 ppm for urine collected from open storage tanks and 16-17 ppm in urine from closed storage tanks). The higher pH and ammonia concentration in the relatively more concentrated urine from these localities correlated well with lower initial bacterial counts and reduced bacterial survival.

After 1-4 months storage, a general reduction was found in the number of enterococci, to below the detection limit (1 cfu per ml.). However, after 5-6 months a slight increase in numbers of enterococci was seen after storage at 4, 10 and 20° C. This indicates that enterococci can survive and/or multiply during long-term storage of human urine at different temperatures. Enterococci may therefore be poor indicators of faecal pollution of urine, because their occurrence will then indicate a longer survival of pathogens than is really the case. It should therefore be considered to use E. coli as an indicator of faecal pollution, because the survival of E. coli seems more sensitive to the urine environment compared with enterococci.

As bacterial genuses and species other than Enterococcus may grow on Slanetz & Bartley agar, it was important to determine the genus and species of the different bacterial colony types that grew on the agar plates. Furthermore, it was important to determine if only few bacterial types (clones) or many different bacterial types could survive and multiply in stored urine. Typing by the Pulsed Field Gel Electroforesis technique (PFGE; "DNA finger printing") was used to show if few or many different bacterial types were isolated on Slanetz & Bartley agar.

All 34 bacterial isolates selected from the Slanetz & Bartley agar plates could be identified as belonging to the genus Enterococcus by traditional biochemical testing and PCR. The majority of tested isolates from locality 1 were E. faecium with an identical PFGE type A. This indicates that these isolates are closely related, i.e. the isolates may originate from the same person and/or the isolates belong to the same clone that has multiplied in the urine. Thus, it looks as if an identical E. faecium strain showed better survival and/or ability to multiply in the urine flasks stored at the three temperatures. The origin of E. faecium is normally seen as being strictly faecal, and it may only to a very limited degree be present and survive outside the gastro-intestinal tract.

PFGE typing of E. faecium isolates from locality 2 revealed three different PFGE types, which indicates that different E. faecium strains were present in the urine where they survived long-term storage and/or had the ability to multiply. All 12 bacterial isolates tested from locality 2 were identified as E. gallinarum, which is normally associated with poultry. E. gallinarum was isolated from both urine that originated from open and closed urine storage tanks at the three different temperatures under study, and all isolates had an identical PFGE type F. This suggests that an identical E. gallinarum strain showed increased survival and/or the ability to multiply in the urine flasks stored at the three different temperatures. It could not be determined if the E. gallinarum strains were introduced into the urine storage tanks through human faeces or from the soil environment, e.g. through leaks in the storage tanks.

Only a limited reduction was seen in total viable counts at 37° C during the six months storage. Slight increases in viable counts were seen in some urine flasks suggesting bacterial multiplication. The use of total viable counts at 37° C to assess the hygienic quality of stored urine seems limited because of an apparent ability for naturally occurring bacteria to multiply.

Parasitological part

Urine was collected and analyzed from two different localities representing densely populated urban areas (localities 1 and 2). Urine was stored at 4, 10 and 20°C with measurements of pH and ammonia being made monthly together with analyses of the microbiological quality, which was determined by enumerating the indicator bacteria: enterococci, total viable counts at 37°C, and suspected thermotolerant coliforms. Semi-permeable capsules containing C. parvum oocysts were inoculated into the urine, and samples were obtained for analysis after 14 days, one month, and after two months storage. The viability of the oocysts was then determined in colour staining assays, and the infectivity of the oocysts was assessed in experimental infection of mice.

Oocysts of C. parvum survived for a relatively short time in human urine, <14 days at 20° C and 1-2 months at 10 and 4° C. It was not possible to infect mice with oocysts from urine that had been stored for 14 days at 20° C, one month at 10° C and two months at 4° C. Thus, a good correlation was found between the results from the viability testing and the infection experiments with mice. The assessment of lack of viability (<2% viable oocysts) with the colour staining assay, which makes it possible to differentiate between live and dead oocysts, seems then to be a good indicator of the oocysts’ ability to infect mice. Oocysts that can infect mice would normally also be able to infect humans.

In contrast to the previous Danish study (Dalsgaard and Tarnow, 2001), but in agreement with a Swedish study, it was shown that urine that had been stored for a minimum of two months did not contain viable and infective C. parvum oocysts. Two months storage would also eliminate eggs (ocysts) of Giardia, because this protozoa is seen as being more sensitive to environmental stress than Cryptosporidium. Accordingly, this project did not include any survival studies of Giardia.