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Enterococci in the New Zealand environment: Implications for water quality monitoring
e:>
Pergamon
War. Sci. T.ch. Vol. 35, No. 11-12, pp. 325-331.1997. <0 1997 IAWQ. Published by Elsevier Science Ltd Pnnted in Greal Britain.
PH: S0273-1223(97)00280-1
0273-1223197517'00 + 0-00
ENTEROCOCCI IN THE NEW ZEALAND ENVIRONMENT: IMPLICATIONS FOR WATER QUALITY MONITORING S. A. Anderson*, S. J. Turner* and G. D. Lewis*'** • Molecular Genetics & Microbiology Research Group. School ofBiological Sciences, University ofAuckland, PrivaM Bag 92019, Auckland. New Zealand •• School ofEnvironmental & Marine Sciences, University ofAuckland, Private Bag 92019, Auckland, New Zealand
ABSTRACf Faecal enlerococci ecology outside the hosl is of great relevance when using these organisms as indicators of water qualily. As a complement to New Zealand epidemiological studies of bathing water quality and health risk. a study of the environmental occurrence of mese organisms has been undertaken. Specific concerns over the use of enterococci derive from the unique Situation in New Zealand which has few chlorinated sewage effluents, a high ratio of grazing animals to humans, and significant inputs of animal processing effluents into the environment. Human and animal faecal wastes are the main sources. with I06.10 7cful100m1 found in human sewage. Analysis of domestic and feral animal faeces found enterococci in the range of 10 J. l06cfulg with considerable variation between species. The laller observations support the notion that a considerable proportion of the load in urban/rural catchments and waterways (typically 102..10 3 enterococci cfullOOmI) is derived from non·human sources. Previous studies of enterococci quiescence in marine/fresh waters indicate that they enter a non·growth phase, exposure to sunlight markedly reducing culturability on selective and non.selective media. Enterococci were also found to surviVe/multiply within specific non· faecal environments. Enterococci on degrading drift seaweed at recreational beaches exceeded seawater levels by 2-4 orders of magnitude, suggesting mat expansion had occurred in this permissive environment with resultant potential to contaminate adjacent sand and water. These studies suggest that multiple sources, environmental persistence, and environmental expansion of enterococci within selected niches add co?siderable complexity 10 the interpretation of water quality data. @ 1997 IAWQ. Published by Elsevier SCience Ltd
KEYWORDS Enterococci; water quality; environment; sources; survival; expansion. INTRODUCTION In New Zealand, the Provisional Guidelines for recreational waters (DoH, 1992), based on USEPA criteria, recommend the use of enterococci for quality monitoring. Enterococci, or the umbrella group the faecal streptococci (FS), have been used as indicators as they are present in the faeces of warm·blooded animals (Geldreich and Kenner 1969), are unable to multiply in sewage effluents (Slanetz and Bartley, 1965) and exhibit the ability to survive for longer periods in water than colifonns (Evison and Tosti, 1981). However some notes of caution over the use of FS as an indicator have been expressed. The abundance of FS o~ vegetation and insects was noted by Clausen et al. (1977) suggesting that Group D streptococci originating 32S
326
S. A. ANDERSON er al.
from plams would compromise the use of these organisms as faecal pollution indicators. Earlier research (Geldreich el al., 1964) suggested that the non-faecal species formed a negligible component of the bacterial load in waters with human faecal contamination. In New Zealand concern has been expressed regarding the use of enterococci as indicators for marine/fresh waters as there is (I) extensive reliance on oxidation ponds as primary means of wastewater treatment and (2) a high ratio of grazing animals to humans with significant animal waste inputs to most waters. However, current guidelines (DoH, 1992) are based on US studies of sites predominantly impacted by human effluents treated by processes such as activated sludge and chlorination (Cabelli, 1980; Bandaranayake el al., 1995). The variable range of enterococci sources in New Zealand, compared to overseas situations, raises concern over the validity of the enterococci/pathogen relationship in waters. We present a review of recent studies to clarify aspects of the presence, survival, and expansion of enterococci in the environment as well as commenting on the possible implications for water quality monitoring programmes. OCCURRENCE OF ENTEROCOCCI IN WATER AND WASTEWATER Studies conducted over the past 18 months have recorded enterococci from a variety of water sources (pristine water catchment areas to final effluent and raw sewage) (Figure I). The expected trend in enterococci levels is apparent from heavily contaminated wastewater (> 107cfulIOOml), to 'unimpacted' marine waters with
102
ABC
0
E
F
G
H
J
Sample
K
L
---
1--
f-
M
N
nn 0
Median enterococci ~m~
for recreational
_era (35 cfull00ml)
P
Seawater
----------------------11> Pristine
Wastewater
Figure I. Enterococci levels in water samples from various sources collected from within the Auckland region between October 1994 and May 1996. Results graphed in order of decreasing enterococci levels. [Sample tocations: (Ai Piggery - raw un screened effluent; (B) Raw Sewage - North Shore wastewater treatment plant; (e) Dairy shedraw effluent; (D) Dairy shed - primary pond discharge; (E) Dairy shed - secondary pond discharge; (F) Oxidation pond effluent - North Shore wastewater treatment plant; (G) Final effluent - Wellsford Oxidation Ponds; (H) Cascade Stream - Waitakere Ranges; (I) Wainamu Stream - Waitakere Ranges; 0) Wait; Stream - Waitakere Ranges; (K) Piggery - secondary pond wastewater; (L) Rural stream· Rodney district; (M) Rural streamWellsford; (N) Wenderholm Beach; (0) Wenderholm Beach; (P) Mission Bay. The direction of the arrow represents the spectrum of water quality observed from wastewater to 'pristine' water samples].
Enterococci in the New Zealand environment
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SOURCES
Enterococci in animal faece$ - apart from direct human sources, the largest potential input of faecal enterococci into New Zealand marine/fresh waters comes from animals. These inputs are frequent, widespread, largely non-point sources and have a variable impact on receiving waters. In order to quantify and identify enterococci from a range of animal sources, 46 faecal samples were collected from the Auckland region and analysed by spread-plating homogenised samples onto mE agar. Levels recorded (Table I) indicate a high probability that urban and rural catchments and hence waterways in the region contain elevated levels of enterococci from non-human sources e.g. it is apparent that birds exhibit a consistently high enterococci load «I06cfulg recorded for some species). In many instances the faecal load, particularly ducks and gulls, is deposited directly into, or adjacent to, water bodies resulting in extremely high 'non-human' faecal enterococci levels in the affected waters (K Taylor (1994) Canterbury Regional Council - personal communication). By comparison, enterococci levels recorded from cattle and sheep were lower in terms of mean recovery but are likely, because of the large volume of faecal material generated. to have a significant impact on the quality of pastoral runoff. The potential for faecal indicator bacteria to leach into groundwater has been demonstrated by Martin and Noonan (1987) where faecal indicator bacteria were detected 1O-15m beneath soils used previously for effluent irrigation and it is likely that rural streams and groundwater sources are significantly impacted by these animal-sourced enterococci. The levels of enterococci observed in feral animals (rats, possums and mice) indicate that these species may be important contributors to the background levels of enterococci recorded in undeveloped catchment areas and freshwater streams. Considerable intraspecies variability in levels. as indicated by variation of <3 orders of magnitude between individual faecal samples, results in a poor ability to predict the faecal enterococci load of any animal. Factors that can account for this observed variability include sample age, sample location, feed source, and animal health. Table I. Enumeration of enterococci in animal faeces by culture on mElEIA media Animal type
Enterococci cfulg faeces (wet weight) Range Geometric mean
Domestic spp 1.I3x10· Dog 1.65x10' .2.5xI03 Cattle 9.65xl
NO l.85xl
1.21x10·
No samples Median NO 1.84xl02 NO 5.63xl~
1.34xl0·
1.25x I06 3.38x10' NO 4.23x103
2.26x106 3.96x106 NO l.77xIO·
NO 8.17x103 3.36xI0·
NO 6.95x103 1.80xlO3
I 13
1 3 7 2 2 1
2 I
10 3
ENTEROCOCCI SURVIVAL The survival capability of any faecal bacteria in water will have great bearing on their utility as indicators. To be of use for water quality monitoring an indicator of faecal pollution must persist in the environment for as long as the pathogens they are intended to monitor. Studies have indicated the ability of enterococci to survive for extended periods in the environment. Specific gene probe detection methodology that utilises a non-selective culturing step to recognise the presence of enterococci within experimental fresh/sea water
328
S. A. ANDERSON et al.
microcosms (Anderson et al., 1995) has shown that detection of sunlight-exposed Enterobacter faecalis by probing was significantly greater than recovery on selective media and that enterococci remained culturable for significantly longer periods (4-24h) than E. coli. Although counts declined over time with sunlight exposure, the gene probe detection method was significantly more effective at detecting stressed enterococci, indicating an enterococci quiescent response. Studies on the persistence of faecal indicator bacteria in the environment have found that solar radiation, salinity (Davies and Evison, 1991; Barcina et al., 1990) and temperature (Evison and Tosti, 1981) contribute to declining recovery in marine/fresh waters. Alkan et al. (1995) demonstrated that their survival at the bottom of a column microcosm was better than E. coli in marine waters. These and other studies have demonstrated not only the persistence of enterococci in the environment. but also the ability to persist in a quiescent state. Consequently, the use of enterococci as a water qUality indicator has yet to be adequately resolved but it is likely that their recorded usefulness as an indicator of viral risk to human health largely relates to both extended survival and retention of culturability. EXPANSION OF ENTEROCOCCI IN THE ENVIRONMENT Over the course of this investigation enterococci have been recorded from a number of diverse environments. These include drift seaweed on bathing beaches and leaf litter in forested areas. Enterococci in leaf litter from two areas of native bush were analysed by spread-plating homogenised samples onto selective mE agar. At both sites they were isolated from most of the samples collected with mean levels of 8.4cfulg (range 0-4.7xlQl cfulg) and 83cfulg (range 0-3.3xI02cfulg) recorded. showing that variable but potentially high levels of enterococci can occur on leaf litter). Seaweed, sand and seawater from two bathing beaches located in the Auckland region were studied during summer and winter (Anderson et al., 1996) with enterococci recovered in each medium on most occasions (Table 2). Table 2. Summary of seaweed monitoring data from two recreational beaches Site
Enterococci Faecal coliforms Wenderholm Mission Bay Wenderholm Mission Bay Summer Winter Summer Summer Winter Summer (Feb 1995) (July 1995) (Feb 1995) (Feb 1995) (July 1995) (Feb 1995) I 22:1:14 3:1:6 26:1:1 164:1'15 2:1:9 3,467:1:13 2 ND. I. 125 ND.2:ND 130.1:6 ND:1:367 14'1:1 104:1:16 3 9:1:1 1:8.ND 967:12:1 ND:ND'IO 90:16:1 31:1:1 NO • not detected; (seaweed from within decaying mass. generally moist and decaying; 2sand from areas adjacent to degrading seaweed, the top 2·5cm of sand sampled; 3seawater at knee depth in direct line with each beach site; presented as ratios e.g. 22: 1: 14 = seaweed. sand, seawater ratio. (Anderson et oJ. 1996) Significant variability was found between beach sites and between the seaweed, sand, and seawater sources. Seaweed-sourced enterococci and faecal coliforms were found On occasion to exceed seawater levels by 2-4 orders of magnitude. Generally, where enterococci and faecal coliforms have been recorded from the degrading seaweed. levels were found to be more elevated in the summer than in winter e.g. at Mission Bay, site 3 seaweed sample contained >900x more enterococci than the seawater sample (ratio 967:12:1). The same is true for faecal coliforms for which levels in seaweed from Mission Bay site I sample exceeded sand levels by over 3,OOOx. Interestingly. the enterococci recovered were types often associated with faecal sources with the majority being Enterobacter faecalis and E faecium. Although earlier studies have reported the occurrence of enterococci on plants (Mundt, 1961; Geldreich and Kenner, 1969), the occurrence of enterococci on seaweed has not been reported. However, Koop et al (1982) noted the primary role of coccoid bacteria in biodegradation and carbon flow based on kelp (Ecklonia maxima) debris in a sandy beach microcosm. They noted initial colonisation by cocci bacteria (not identified) along the junctions of epidennaJ cell walls leads to lysis and release of cell contents, the lysed cells then being colonised by bacterial rods. The possible origin of enterococci on the drift seaweed has not yet being elucidated although Clausen et oJ. (1977) highlighted several possibilities. The presence of enterococci on plant material may be due to direct contamination by animals and insects (Geldreich et al., 1964). Alternatively they may exist on plants in an epiphytic relationship or as temporary residents capable of limited growth/reproduction (Mundt,
Enterococci in the New Zealand environment
329
1961). The elevated levels in seaweed are an indication that possible expansion has occurred in this growthpennissive environment. Wenderholm and Mission Bay beaches are considered to be of good water quality (ARC Environment, unpublished data). The recovery of enterococci in reasonably high numbers from seaweed at some sites suggests that ambient water quality is not causally related to enterococci occurrence expansion of enterococci in these sites may be occurring. Thus there is potential for these enterococci to be introduced into adjacent waters by resuspension and wash-off effects. An earlier report by Clausen et al. (1977) also recognised that 'the incidence of faecal streptococci on plants is of concern for two reasons. streptococci present on plants may be introduced into soil and water, and that detection in water of populations of Group D Streptococci would invalidate the use of these organisms as indicators of faecal pollution', Seaweed m
Seawa~r
Sand
1
1111111
A
A
mm
2
m
m m
323
12
16
A
A
C
CB
B
Figure 2. Agarose gel electrophoresis of San digested genomic DNA from enterococci. [Enterococci were isolated from seaweed, sand, and seawater from Wenderholm Beach, Site I of the winter survey. Lane numbers indicate matching restriction patterns. A selection of the isolates were identified using the Rapid 1032 Strep System (bio Meiurex, France). (A) Ent.faecalis, (B) Ent.faecium, (e) Ent. casseliflavus, (D) Em. hirae. Molecular size markers (lane m) (I·kb ladder Life Technologies) (Anderson et al. 1996)1.
To test the notion that enterococci were surviving/expanding in these environments a seleclion of enterococci (from seaweed, sand and seawater) were characterised by restriction enzyme analysis techniques (Figure 2) (Anderson et al., 1996). Genetically identical (clonal) populations (as indicated by identical restriction profiles) dominate in the seaweed isolates as only three different profiles were observed (Figure 2, profiles 1-3), Sand isolates were more variable with eight different restriction profiles, two being repeated (Figure 2, profiles 4 and 5). Interestingly. restriction profile I. the most frequent seaweed profile. was recorded in the sand, The restriction profiles for the water were also variable and no similarities to the profiles observed from the seaweed and sand were observed. The presence of clonal enterococci populations in decaying seaweed strongly suggests that active replication or selection is occurring within this medium. The mixture of enterococci genotypes found in the sand and water suggests that the bacteria are derived or accumulated not only from the seaweed but also other from sources (e.g. direct faecal contamination and runoff), CONCLUSION The ahove studies were conducted to elucidate the occurrence of enterococcI In the New Zealand environment and the potential of non-human-sourced enterococci to significantly confound interpretation of the sanitary significance of monitoring results. The high levels of enterococci found in animal faeces (>I06efulg) and the ability of enterococci to persist in stressful environments and replicate in more permissive environments means it is likely that catchments and waterways contain elevated levels of
330
S. A. ANDERSON et al.
enterococci from non-human sources. Three major sources of enterococci in the environment can be identified: human faeces, animal faeces and those which occur through environmental expansion of populations from these or other sources. These last populations are determined to be those individuals or clones not directly sourced from humans or animal faeces, which are present on organic material and replicate in these environments. The demonstrated ability of enterococci to survive (as quiescent bacteria) and to expand or replicate in permissive environments (decaying vegetation) potentially allows delayed dispersal of enterococci into waterways and catchment areas. This in turn has implications for monitoring programmes and interpretation of water quality monitoring data. Seaweed and leaf-litter associated enterococci will produce positive results in routine water quality analysis and will contribute to final assessments of bathing water quality. It is possible that these enterococci could artificially degrade water quality estimates in situations where faecal wastes are not a major influence. Results of a national bathing water epidemiological study (Bandaranayake et at.. 1995) indicated that of the three bacteriological water quality indicators tested (faecal coliforms, E. coli and enterococci) enterococci was the most strongly associated with illness risk. However in the light of this discussion we must carefully consider the implications of the epidemiological study results. Enterococci levels recorded from the bathing waters sampled were very low (majority of values <35cfu/IOOml). On analysis of this data, enterococci occurrence was found to be significantly associated with respiratory symptoms rather than gastrointestinal illness which has traditionally been associated with human faecal contamination. Two possible scenarios arise when integrating the the epidemiological study results with environmental data on the occurrence of enterococci: 1.
2.
It is unclear whether enterococcI In the epidemiological studies were from human, animal or environmental sources. The pathogen:indicator relationship would be expected to vary for these different sources, destabilising the relationship between enterococci and illness observed in the epidemiological study. The relationship between enterococci and illness may be stable because enterococci levels and illness risk agent are both of non-faecal origin but their occurrence in water is similarly affected by external factors such as rainfall, e.g. respiratory symptoms may be due to an allergen transported to the environment, along with enterococci, by a rainfall event.
It has been demonstrated that enterococci are abundant and persistent in the New Zealand environment and that there is a clear need for local/regional authorities to consider the occurrence of these bacteria within water quality monitoring programmes, particularly where a source of faecal contamination is not apparent. ACKNOWLEDGMENTS This work was funded in part by the Auckland Regional Council. REFERENCES Alkan, U., Elliot, D. J. and Evison, L. M. (1995). Survival of enteric bacteria in relation 10 simulated solar radiation and other environmenial factors in marine warers. Wat. Res., 29, 2071-2081. Anderson, S. A., Lewis, G. D. and Pearson, M. N. (1995). Use of gene probes for the detection of quiescent enteric bacteria in marine and fresh waters. Wat. Sci. Tech., 31(5-6),291-298. Anderson, S. A., Lewis, G. D. and Turner, S. J. (1996). Drift seaweed - a permissive environment for the survival and growth of enterococci. (In preparation). APHA (1992). Standard Methods for the Examination of Water and Wastewater. 18th edition Washington DC. Bradfield, M. J. P. (1996). Faecal conlaminalion of Kerikeri and Piha catchments: enterococci as an indicator of faecal conlaminalion. MSc Thesis, University of Auckland. Bandaranayake. D. R., Salmond, C. E., Turner, S. J., McBride, O. B., Lewis, O. D. and Till, D. O. (1995). Health Effects of Bathing at Selected New Zealand Marine Beaches. Ministry for the Environment. 98p. Barcina, I., Gonzalez, J. M.,lriberri, J. and Egea, L. (1990). Survival strategy of E. coli and Enterococcwfaecalis in iIIuminaled fresh and marine systems. J. AppL Bact., 68, 189-198. Cabelli, V. J. (1980). Health Effects Criteria for Marine Recreational Waters. US Environmental Pro1ection Agency. EPA-6O
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Clausen, E. M., Green. B. L. and Litsky, W. (1977). Faecal streptococci: Indicators of pollution. In: Bacterial indicatorslHealth hazards associated with water. Hoadley, A.W., Dutka. BJ. eds. ASTM STP. 635.247-264. Davies. C. M. and EVison, L. M. (1991). Sunlight and the survival of enteric bacteria in natural waters. J. Appl. Bacteriol. 70.265274. Department of Health (1992). Provisional microbiological water quality guidelines for shellfish gathering waters in New Zealand. Prepared by McBride, G. B., Cooper, A. B. and Till, D. G.• Public Health Services. New Zealand Department of Health. Evison. L. M. and Tosti, E. (1981). An appraisal of bacterial indIcators of pollution in seawater. Prog. Water Tech., 13(1). 591599. Geldreich. E. E., Kenner, B. A. and Kabler. P. W. (1964). Occurrence of coliforms. faecal coliforms and streptococci on vegetation and insects. Appl. Environ. Microbiol., 12,63-69. Geldreich, E. E. and Kenner. B. A. (1969). Concepts of faecal streptococci in stream pollution. J. Wat. Pollut. Control Fed.. 41, R336-R352. Koop. K., Newell, R. C. and Lucas. M. I. (1982). Biodegradation and carbon flow based on Kelp (Ecklonia maxima) debris in a sandy beach microcosm. Marine Ecology - Progress Series 7, 315-326. Martin, G. N. and Noonan, M. J. (1977). Effects of domestic wastewater disposal by land irrigation on groundwater quality of the Central Canterbury Plains. Water and soil Technical Publication 7. Published for the National Water and Soil Conservation Organisation by Ministry of Works and Development, Wellington. 25p. Mundt. J. O. (1961). Occurrence of enterococci: bud. bloom and soil studies. Appl. Microbiol., 9, 541-544. SJanetz. L. W. and Bartley, C. H. (1965). Survival of faecal streptococci in water. Health Lab. Sci.• 2,142-148.
Pergamon
War. Sci. T.ch. Vol. 35, No. 11-12, pp. 325-331.1997. <0 1997 IAWQ. Published by Elsevier Science Ltd Pnnted in Greal Britain.
PH: S0273-1223(97)00280-1
0273-1223197517'00 + 0-00
ENTEROCOCCI IN THE NEW ZEALAND ENVIRONMENT: IMPLICATIONS FOR WATER QUALITY MONITORING S. A. Anderson*, S. J. Turner* and G. D. Lewis*'** • Molecular Genetics & Microbiology Research Group. School ofBiological Sciences, University ofAuckland, PrivaM Bag 92019, Auckland. New Zealand •• School ofEnvironmental & Marine Sciences, University ofAuckland, Private Bag 92019, Auckland, New Zealand
ABSTRACf Faecal enlerococci ecology outside the hosl is of great relevance when using these organisms as indicators of water qualily. As a complement to New Zealand epidemiological studies of bathing water quality and health risk. a study of the environmental occurrence of mese organisms has been undertaken. Specific concerns over the use of enterococci derive from the unique Situation in New Zealand which has few chlorinated sewage effluents, a high ratio of grazing animals to humans, and significant inputs of animal processing effluents into the environment. Human and animal faecal wastes are the main sources. with I06.10 7cful100m1 found in human sewage. Analysis of domestic and feral animal faeces found enterococci in the range of 10 J. l06cfulg with considerable variation between species. The laller observations support the notion that a considerable proportion of the load in urban/rural catchments and waterways (typically 102..10 3 enterococci cfullOOmI) is derived from non·human sources. Previous studies of enterococci quiescence in marine/fresh waters indicate that they enter a non·growth phase, exposure to sunlight markedly reducing culturability on selective and non.selective media. Enterococci were also found to surviVe/multiply within specific non· faecal environments. Enterococci on degrading drift seaweed at recreational beaches exceeded seawater levels by 2-4 orders of magnitude, suggesting mat expansion had occurred in this permissive environment with resultant potential to contaminate adjacent sand and water. These studies suggest that multiple sources, environmental persistence, and environmental expansion of enterococci within selected niches add co?siderable complexity 10 the interpretation of water quality data. @ 1997 IAWQ. Published by Elsevier SCience Ltd
KEYWORDS Enterococci; water quality; environment; sources; survival; expansion. INTRODUCTION In New Zealand, the Provisional Guidelines for recreational waters (DoH, 1992), based on USEPA criteria, recommend the use of enterococci for quality monitoring. Enterococci, or the umbrella group the faecal streptococci (FS), have been used as indicators as they are present in the faeces of warm·blooded animals (Geldreich and Kenner 1969), are unable to multiply in sewage effluents (Slanetz and Bartley, 1965) and exhibit the ability to survive for longer periods in water than colifonns (Evison and Tosti, 1981). However some notes of caution over the use of FS as an indicator have been expressed. The abundance of FS o~ vegetation and insects was noted by Clausen et al. (1977) suggesting that Group D streptococci originating 32S
326
S. A. ANDERSON er al.
from plams would compromise the use of these organisms as faecal pollution indicators. Earlier research (Geldreich el al., 1964) suggested that the non-faecal species formed a negligible component of the bacterial load in waters with human faecal contamination. In New Zealand concern has been expressed regarding the use of enterococci as indicators for marine/fresh waters as there is (I) extensive reliance on oxidation ponds as primary means of wastewater treatment and (2) a high ratio of grazing animals to humans with significant animal waste inputs to most waters. However, current guidelines (DoH, 1992) are based on US studies of sites predominantly impacted by human effluents treated by processes such as activated sludge and chlorination (Cabelli, 1980; Bandaranayake el al., 1995). The variable range of enterococci sources in New Zealand, compared to overseas situations, raises concern over the validity of the enterococci/pathogen relationship in waters. We present a review of recent studies to clarify aspects of the presence, survival, and expansion of enterococci in the environment as well as commenting on the possible implications for water quality monitoring programmes. OCCURRENCE OF ENTEROCOCCI IN WATER AND WASTEWATER Studies conducted over the past 18 months have recorded enterococci from a variety of water sources (pristine water catchment areas to final effluent and raw sewage) (Figure I). The expected trend in enterococci levels is apparent from heavily contaminated wastewater (> 107cfulIOOml), to 'unimpacted' marine waters with
102
ABC
0
E
F
G
H
J
Sample
K
L
---
1--
f-
M
N
nn 0
Median enterococci ~m~
for recreational
_era (35 cfull00ml)
P
Seawater
----------------------11> Pristine
Wastewater
Figure I. Enterococci levels in water samples from various sources collected from within the Auckland region between October 1994 and May 1996. Results graphed in order of decreasing enterococci levels. [Sample tocations: (Ai Piggery - raw un screened effluent; (B) Raw Sewage - North Shore wastewater treatment plant; (e) Dairy shedraw effluent; (D) Dairy shed - primary pond discharge; (E) Dairy shed - secondary pond discharge; (F) Oxidation pond effluent - North Shore wastewater treatment plant; (G) Final effluent - Wellsford Oxidation Ponds; (H) Cascade Stream - Waitakere Ranges; (I) Wainamu Stream - Waitakere Ranges; 0) Wait; Stream - Waitakere Ranges; (K) Piggery - secondary pond wastewater; (L) Rural stream· Rodney district; (M) Rural streamWellsford; (N) Wenderholm Beach; (0) Wenderholm Beach; (P) Mission Bay. The direction of the arrow represents the spectrum of water quality observed from wastewater to 'pristine' water samples].
Enterococci in the New Zealand environment
327
SOURCES
Enterococci in animal faece$ - apart from direct human sources, the largest potential input of faecal enterococci into New Zealand marine/fresh waters comes from animals. These inputs are frequent, widespread, largely non-point sources and have a variable impact on receiving waters. In order to quantify and identify enterococci from a range of animal sources, 46 faecal samples were collected from the Auckland region and analysed by spread-plating homogenised samples onto mE agar. Levels recorded (Table I) indicate a high probability that urban and rural catchments and hence waterways in the region contain elevated levels of enterococci from non-human sources e.g. it is apparent that birds exhibit a consistently high enterococci load «I06cfulg recorded for some species). In many instances the faecal load, particularly ducks and gulls, is deposited directly into, or adjacent to, water bodies resulting in extremely high 'non-human' faecal enterococci levels in the affected waters (K Taylor (1994) Canterbury Regional Council - personal communication). By comparison, enterococci levels recorded from cattle and sheep were lower in terms of mean recovery but are likely, because of the large volume of faecal material generated. to have a significant impact on the quality of pastoral runoff. The potential for faecal indicator bacteria to leach into groundwater has been demonstrated by Martin and Noonan (1987) where faecal indicator bacteria were detected 1O-15m beneath soils used previously for effluent irrigation and it is likely that rural streams and groundwater sources are significantly impacted by these animal-sourced enterococci. The levels of enterococci observed in feral animals (rats, possums and mice) indicate that these species may be important contributors to the background levels of enterococci recorded in undeveloped catchment areas and freshwater streams. Considerable intraspecies variability in levels. as indicated by variation of <3 orders of magnitude between individual faecal samples, results in a poor ability to predict the faecal enterococci load of any animal. Factors that can account for this observed variability include sample age, sample location, feed source, and animal health. Table I. Enumeration of enterococci in animal faeces by culture on mElEIA media Animal type
Enterococci cfulg faeces (wet weight) Range Geometric mean
Domestic spp 1.I3x10· Dog 1.65x10' .2.5xI03 Cattle 9.65xl
NO l.85xl
1.21x10·
No samples Median NO 1.84xl02 NO 5.63xl~
1.34xl0·
1.25x I06 3.38x10' NO 4.23x103
2.26x106 3.96x106 NO l.77xIO·
NO 8.17x103 3.36xI0·
NO 6.95x103 1.80xlO3
I 13
1 3 7 2 2 1
2 I
10 3
ENTEROCOCCI SURVIVAL The survival capability of any faecal bacteria in water will have great bearing on their utility as indicators. To be of use for water quality monitoring an indicator of faecal pollution must persist in the environment for as long as the pathogens they are intended to monitor. Studies have indicated the ability of enterococci to survive for extended periods in the environment. Specific gene probe detection methodology that utilises a non-selective culturing step to recognise the presence of enterococci within experimental fresh/sea water
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microcosms (Anderson et al., 1995) has shown that detection of sunlight-exposed Enterobacter faecalis by probing was significantly greater than recovery on selective media and that enterococci remained culturable for significantly longer periods (4-24h) than E. coli. Although counts declined over time with sunlight exposure, the gene probe detection method was significantly more effective at detecting stressed enterococci, indicating an enterococci quiescent response. Studies on the persistence of faecal indicator bacteria in the environment have found that solar radiation, salinity (Davies and Evison, 1991; Barcina et al., 1990) and temperature (Evison and Tosti, 1981) contribute to declining recovery in marine/fresh waters. Alkan et al. (1995) demonstrated that their survival at the bottom of a column microcosm was better than E. coli in marine waters. These and other studies have demonstrated not only the persistence of enterococci in the environment. but also the ability to persist in a quiescent state. Consequently, the use of enterococci as a water qUality indicator has yet to be adequately resolved but it is likely that their recorded usefulness as an indicator of viral risk to human health largely relates to both extended survival and retention of culturability. EXPANSION OF ENTEROCOCCI IN THE ENVIRONMENT Over the course of this investigation enterococci have been recorded from a number of diverse environments. These include drift seaweed on bathing beaches and leaf litter in forested areas. Enterococci in leaf litter from two areas of native bush were analysed by spread-plating homogenised samples onto selective mE agar. At both sites they were isolated from most of the samples collected with mean levels of 8.4cfulg (range 0-4.7xlQl cfulg) and 83cfulg (range 0-3.3xI02cfulg) recorded. showing that variable but potentially high levels of enterococci can occur on leaf litter). Seaweed, sand and seawater from two bathing beaches located in the Auckland region were studied during summer and winter (Anderson et al., 1996) with enterococci recovered in each medium on most occasions (Table 2). Table 2. Summary of seaweed monitoring data from two recreational beaches Site
Enterococci Faecal coliforms Wenderholm Mission Bay Wenderholm Mission Bay Summer Winter Summer Summer Winter Summer (Feb 1995) (July 1995) (Feb 1995) (Feb 1995) (July 1995) (Feb 1995) I 22:1:14 3:1:6 26:1:1 164:1'15 2:1:9 3,467:1:13 2 ND. I. 125 ND.2:ND 130.1:6 ND:1:367 14'1:1 104:1:16 3 9:1:1 1:8.ND 967:12:1 ND:ND'IO 90:16:1 31:1:1 NO • not detected; (seaweed from within decaying mass. generally moist and decaying; 2sand from areas adjacent to degrading seaweed, the top 2·5cm of sand sampled; 3seawater at knee depth in direct line with each beach site; presented as ratios e.g. 22: 1: 14 = seaweed. sand, seawater ratio. (Anderson et oJ. 1996) Significant variability was found between beach sites and between the seaweed, sand, and seawater sources. Seaweed-sourced enterococci and faecal coliforms were found On occasion to exceed seawater levels by 2-4 orders of magnitude. Generally, where enterococci and faecal coliforms have been recorded from the degrading seaweed. levels were found to be more elevated in the summer than in winter e.g. at Mission Bay, site 3 seaweed sample contained >900x more enterococci than the seawater sample (ratio 967:12:1). The same is true for faecal coliforms for which levels in seaweed from Mission Bay site I sample exceeded sand levels by over 3,OOOx. Interestingly. the enterococci recovered were types often associated with faecal sources with the majority being Enterobacter faecalis and E faecium. Although earlier studies have reported the occurrence of enterococci on plants (Mundt, 1961; Geldreich and Kenner, 1969), the occurrence of enterococci on seaweed has not been reported. However, Koop et al (1982) noted the primary role of coccoid bacteria in biodegradation and carbon flow based on kelp (Ecklonia maxima) debris in a sandy beach microcosm. They noted initial colonisation by cocci bacteria (not identified) along the junctions of epidennaJ cell walls leads to lysis and release of cell contents, the lysed cells then being colonised by bacterial rods. The possible origin of enterococci on the drift seaweed has not yet being elucidated although Clausen et oJ. (1977) highlighted several possibilities. The presence of enterococci on plant material may be due to direct contamination by animals and insects (Geldreich et al., 1964). Alternatively they may exist on plants in an epiphytic relationship or as temporary residents capable of limited growth/reproduction (Mundt,
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1961). The elevated levels in seaweed are an indication that possible expansion has occurred in this growthpennissive environment. Wenderholm and Mission Bay beaches are considered to be of good water quality (ARC Environment, unpublished data). The recovery of enterococci in reasonably high numbers from seaweed at some sites suggests that ambient water quality is not causally related to enterococci occurrence expansion of enterococci in these sites may be occurring. Thus there is potential for these enterococci to be introduced into adjacent waters by resuspension and wash-off effects. An earlier report by Clausen et al. (1977) also recognised that 'the incidence of faecal streptococci on plants is of concern for two reasons. streptococci present on plants may be introduced into soil and water, and that detection in water of populations of Group D Streptococci would invalidate the use of these organisms as indicators of faecal pollution', Seaweed m
Seawa~r
Sand
1
1111111
A
A
mm
2
m
m m
323
12
16
A
A
C
CB
B
Figure 2. Agarose gel electrophoresis of San digested genomic DNA from enterococci. [Enterococci were isolated from seaweed, sand, and seawater from Wenderholm Beach, Site I of the winter survey. Lane numbers indicate matching restriction patterns. A selection of the isolates were identified using the Rapid 1032 Strep System (bio Meiurex, France). (A) Ent.faecalis, (B) Ent.faecium, (e) Ent. casseliflavus, (D) Em. hirae. Molecular size markers (lane m) (I·kb ladder Life Technologies) (Anderson et al. 1996)1.
To test the notion that enterococci were surviving/expanding in these environments a seleclion of enterococci (from seaweed, sand and seawater) were characterised by restriction enzyme analysis techniques (Figure 2) (Anderson et al., 1996). Genetically identical (clonal) populations (as indicated by identical restriction profiles) dominate in the seaweed isolates as only three different profiles were observed (Figure 2, profiles 1-3), Sand isolates were more variable with eight different restriction profiles, two being repeated (Figure 2, profiles 4 and 5). Interestingly. restriction profile I. the most frequent seaweed profile. was recorded in the sand, The restriction profiles for the water were also variable and no similarities to the profiles observed from the seaweed and sand were observed. The presence of clonal enterococci populations in decaying seaweed strongly suggests that active replication or selection is occurring within this medium. The mixture of enterococci genotypes found in the sand and water suggests that the bacteria are derived or accumulated not only from the seaweed but also other from sources (e.g. direct faecal contamination and runoff), CONCLUSION The ahove studies were conducted to elucidate the occurrence of enterococcI In the New Zealand environment and the potential of non-human-sourced enterococci to significantly confound interpretation of the sanitary significance of monitoring results. The high levels of enterococci found in animal faeces (>I06efulg) and the ability of enterococci to persist in stressful environments and replicate in more permissive environments means it is likely that catchments and waterways contain elevated levels of
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enterococci from non-human sources. Three major sources of enterococci in the environment can be identified: human faeces, animal faeces and those which occur through environmental expansion of populations from these or other sources. These last populations are determined to be those individuals or clones not directly sourced from humans or animal faeces, which are present on organic material and replicate in these environments. The demonstrated ability of enterococci to survive (as quiescent bacteria) and to expand or replicate in permissive environments (decaying vegetation) potentially allows delayed dispersal of enterococci into waterways and catchment areas. This in turn has implications for monitoring programmes and interpretation of water quality monitoring data. Seaweed and leaf-litter associated enterococci will produce positive results in routine water quality analysis and will contribute to final assessments of bathing water quality. It is possible that these enterococci could artificially degrade water quality estimates in situations where faecal wastes are not a major influence. Results of a national bathing water epidemiological study (Bandaranayake et at.. 1995) indicated that of the three bacteriological water quality indicators tested (faecal coliforms, E. coli and enterococci) enterococci was the most strongly associated with illness risk. However in the light of this discussion we must carefully consider the implications of the epidemiological study results. Enterococci levels recorded from the bathing waters sampled were very low (majority of values <35cfu/IOOml). On analysis of this data, enterococci occurrence was found to be significantly associated with respiratory symptoms rather than gastrointestinal illness which has traditionally been associated with human faecal contamination. Two possible scenarios arise when integrating the the epidemiological study results with environmental data on the occurrence of enterococci: 1.
2.
It is unclear whether enterococcI In the epidemiological studies were from human, animal or environmental sources. The pathogen:indicator relationship would be expected to vary for these different sources, destabilising the relationship between enterococci and illness observed in the epidemiological study. The relationship between enterococci and illness may be stable because enterococci levels and illness risk agent are both of non-faecal origin but their occurrence in water is similarly affected by external factors such as rainfall, e.g. respiratory symptoms may be due to an allergen transported to the environment, along with enterococci, by a rainfall event.
It has been demonstrated that enterococci are abundant and persistent in the New Zealand environment and that there is a clear need for local/regional authorities to consider the occurrence of these bacteria within water quality monitoring programmes, particularly where a source of faecal contamination is not apparent. ACKNOWLEDGMENTS This work was funded in part by the Auckland Regional Council. REFERENCES Alkan, U., Elliot, D. J. and Evison, L. M. (1995). Survival of enteric bacteria in relation 10 simulated solar radiation and other environmenial factors in marine warers. Wat. Res., 29, 2071-2081. Anderson, S. A., Lewis, G. D. and Pearson, M. N. (1995). Use of gene probes for the detection of quiescent enteric bacteria in marine and fresh waters. Wat. Sci. Tech., 31(5-6),291-298. Anderson, S. A., Lewis, G. D. and Turner, S. J. (1996). Drift seaweed - a permissive environment for the survival and growth of enterococci. (In preparation). APHA (1992). Standard Methods for the Examination of Water and Wastewater. 18th edition Washington DC. Bradfield, M. J. P. (1996). Faecal conlaminalion of Kerikeri and Piha catchments: enterococci as an indicator of faecal conlaminalion. MSc Thesis, University of Auckland. Bandaranayake. D. R., Salmond, C. E., Turner, S. J., McBride, O. B., Lewis, O. D. and Till, D. O. (1995). Health Effects of Bathing at Selected New Zealand Marine Beaches. Ministry for the Environment. 98p. Barcina, I., Gonzalez, J. M.,lriberri, J. and Egea, L. (1990). Survival strategy of E. coli and Enterococcwfaecalis in iIIuminaled fresh and marine systems. J. AppL Bact., 68, 189-198. Cabelli, V. J. (1980). Health Effects Criteria for Marine Recreational Waters. US Environmental Pro1ection Agency. EPA-6O
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Clausen, E. M., Green. B. L. and Litsky, W. (1977). Faecal streptococci: Indicators of pollution. In: Bacterial indicatorslHealth hazards associated with water. Hoadley, A.W., Dutka. BJ. eds. ASTM STP. 635.247-264. Davies. C. M. and EVison, L. M. (1991). Sunlight and the survival of enteric bacteria in natural waters. J. Appl. Bacteriol. 70.265274. Department of Health (1992). Provisional microbiological water quality guidelines for shellfish gathering waters in New Zealand. Prepared by McBride, G. B., Cooper, A. B. and Till, D. G.• Public Health Services. New Zealand Department of Health. Evison. L. M. and Tosti, E. (1981). An appraisal of bacterial indIcators of pollution in seawater. Prog. Water Tech., 13(1). 591599. Geldreich. E. E., Kenner, B. A. and Kabler. P. W. (1964). Occurrence of coliforms. faecal coliforms and streptococci on vegetation and insects. Appl. Environ. Microbiol., 12,63-69. Geldreich, E. E. and Kenner. B. A. (1969). Concepts of faecal streptococci in stream pollution. J. Wat. Pollut. Control Fed.. 41, R336-R352. Koop. K., Newell, R. C. and Lucas. M. I. (1982). Biodegradation and carbon flow based on Kelp (Ecklonia maxima) debris in a sandy beach microcosm. Marine Ecology - Progress Series 7, 315-326. Martin, G. N. and Noonan, M. J. (1977). Effects of domestic wastewater disposal by land irrigation on groundwater quality of the Central Canterbury Plains. Water and soil Technical Publication 7. Published for the National Water and Soil Conservation Organisation by Ministry of Works and Development, Wellington. 25p. Mundt. J. O. (1961). Occurrence of enterococci: bud. bloom and soil studies. Appl. Microbiol., 9, 541-544. SJanetz. L. W. and Bartley, C. H. (1965). Survival of faecal streptococci in water. Health Lab. Sci.• 2,142-148.