Journal of Applied Bacteriology 1992,73,263-268
Coliform bacteria from aquatic sources in Fiji S. Roberts School of Pure and Applied Sciences, University of the South Pacific, Fiji 4000/10/91: accepted 20 March 1992 R o B E R T s . 1992.Coliform bacteria were abundant in water a n d bivalve molluscs in the rivers and present to a lesser extent in t h e coastal areas of Fiji. The rivers fed b y treated sewage were highly polluted. T h e r e was a noticeable increase in concentration of coliforms in bivalve molluscs. It was also found that these bacteria could survive and grow in river water and seawater over a 5-d period, and had a rapid growth rate in nutrient broth under ideal laboratory conditions. T h e y were characterized by the API 20E identification system.
s.
INTRODUCTION
In developing countries the need to examine water supplies for pathogenic bacteria is possibly greater than in developed countries, mainly because the latter usually have comparatively efficient sewage disposal systems. In less developed countries sewage is frequently deposited directly on land or into rivers, lakes, etc., where it relies on the natural flora to render its contents harmless. Unfortunately this does not readily happen and consequently sewage entering a river may contain pathogenic bacteria which will either pose a health problem or alter the ecological balance of the river. In addition, the ability of bivalve molluscs to concentrate and accumulate bacteria is a major concern, especially when the local population rely on these animals as part of their diet. Coastal settlements in particular are at risk because of their diet of oysters, clams and mussels, which are eaten either raw or only lightly cooked. The majority of water-borne infections involve gastrointestinal disorders (Jones & Watkins 1985). Although many infections are of bacterial origin, viral diseases, e.g. viral gastroenteritis and infective hepatitis, are playing a more prominent role, especially when transmitted by shellfish (Brown & Dorn 1977; Kueh & Chan 1985; Pain 1986). The importance of examining water and aquatic animals such as bivalve molluscs for faecal contamination cannot be overemphasized. Table 1 shows the variety of microorganisms that are likely to be encountered in sewage, although some of them may not be found in all countries. Not all coliforms will be associated with the mammalian intestinal tract, as a number of strains are of environmental origin. Some or all may be of faecal origin, and as there is no satisfactory method for differentiating human strains Present address and correspondence to: M r S. Roberts, Plant Science Limited, Firth Court, University of Shefield, Shefield SIO 2TN, UK.
from those of animal origin, an explanation of their presence must be sought. MATERIALS AND METHODS
Samples
Samples were taken at three-monthly intervals over a period of 1 year. T h e water samples were obtained from two rivers on the island of Viti Levu (Fiji), and from Table 1 Pathogenic agents that may be present in sewage*
Bacteria Leptospira icterohaemorrhagiae Campylobacter spp. Pseudomonas aeruginosa Brucella spp. Enteropathogenic E . coli Shigella spp. Salmonella typhi Salm. paratyphi Klebsiella spp. Yersinia enterolytica Yersinia pestis Vibrio cholerae Aeromonas hydrophila Bacillus anthracis Clostridium spp. Listeria monocytogenes Erysipelothrix rhusiopathiae Mycobacterium tuberculosis Mycobacterium paratuberculosis Staphylococcus aureus
Fungi Candida albicans
* See Jones & Watkins 1985.
Viruses Adenovirus Enterovirus New Enterovirus Poliovirus Echovirus Coxsackievirus Norwalk Virus Rotavirus Reovirus Parvovirus Intestinal parasites Entamoeba histolytica Acanthamoeba spp. Naegleria spp. Giardia lamblia Cryptosporidia spp. Ascaris lumbricoides Trichuris trichuria Ancylostoma duodenale Necator americanus Schistosoma mansoni Diphyllobothrium latum Taenia spp. ~~
264 S . R O B E R T S
Fig. 1 Laucala Bay, Fiji, showing sites H and I
25
0
Km
-P
REWA RIVER
The water samples were diluted depending upon the The shellfish origin of the sample, from lo-' to were scrubbed clean under running water, and all debris and barnacles were removed. T h e two halves of the shell were separated by a sterile scalpel and the juices were discarded. Ten animals were homogenized in a Waring blender and 10 g of homogenate were suspended in 100 ml of normal sterile saline. Dilutions were made from 10- to
'
Bacteriological examination LAUCALA B A Y
Enumeration Fig. 2 Rewa River flowing into Laucala Bay, Fiji
L A U C A L A BAY
Fig. 3 Vatuwaqa River, Fiji
Laucala Bay (Figs 1-3). T h e bivalve molluscs examined were the freshwater clam, Batissa violacea from the Rewa river, and the mangrove oyster, Crassostrea mordax from Laucala Bay.
T h e five-tube Most Probable Number (MPN) method was used, with 10, 1.0 and 0.1 ml volumes and MacConkey Broth (Oxoid) (Anon. 1983). Tubes showing acid and gas after incubation at 37°C for 24 h were subcultured into Brilliant Green Lactose Bile Salts Broth (BGBS, Oxoid) and incubated at 44.5"C for 24 h. Tubes showing gas were further subcultured into Tryptone Water (Oxoid) and incubated at 44.5"C. These samples were tested for indole production by Kovac's method (Anon. 1983). Gas production and indole formation at 44.5"C were considered confirmation of Escherichia coli. The presumptive M P N counts of coliforms and E. coli in 100 ml of water or per gram of shellfish were estimated from the probability tables (Anon. 1983).
Identification
Samples were taken from the positive tubes of BGBS and plated on MacConkey Agar. Colonies were selected and
COLIFORMS IN FIJI 265
purified by subculture. The organisms were then examined microscopically and biochemically by the API 20E system.
Table 2 Most probable number (MPN) of coliform bacteria in the Vatuwaqa River
MPN/100 ml water Growth curves
The bacteria were grown under various conditions and monitored by periodically plating varying dilutions of samples (0.1 ml) on appropriate media. The growth of the bacteria was monitored in nutrient broth (Oxoid) with and without 3.0% NaCl (w/v), at 37°C and 24°C. The culture medium (200 ml) was inoculated with 3.0 ml of an overnight broth culture. This was diluted lo-’ and lo-* in sterile saline and 0.1 ml of each dilution was plated on nutrient agar (NA) and incubated at 30°C overnight. The remaining culture was incubated at either 24°C or 37°C in a shaking water bath. After 30 min, 1.0 ml of the culture was removed, diluted to lo-’ and and 0.1 ml plated on NA and incubated at 30°C overnight. This process was repeated every 60 min for 5 h, increasing the dilutions at each stage to take into account the multiplication of the bacteria. The number of colony forming units (cfu) was estimated and the number of bacteria per ml of original culture calculated. Survival in river water and seawater
The bacteria were grown overnight in nutrient broth at 30°C. T h e cells were centrifuged at 10 000 g and the supernatant fluid was discarded. The pellet was washed twice in sterile saline, centrifuged and resuspended in either 100 ml of sterile river water or seawater (sterilized by autoclaving at 115°C for 20 min). Ten ml of this suspension were added to 200 ml of sterile river or seawater and incubated at 24°C in a shaking water bath for 5 d. At intervals, samples were removed and plated on nutrient agar either with or without 3.0% NaCl (w/v) and incubated at 30°C overnight. The number of viable bacteria was estimated. The experiment was repeated 14 d later with the same strains under identical conditions and in duplicate.
Site
Date
Coliforms
E . coli
D
Jun SeP Dec Mar
2.8 x 1.7 x 1.7 x 1.6 x
lo7 107 lo7 10’
2.8 1.7 1.7 9.0
x x x x
lo7 107 lo7 107
E
Jun SeP Dec Mar
1.7 x 2.3 5.0 x 2.6 x
lo6 107 106 lo7
1.7 x 1.3 x 2.0 x 1.3 x
lo6 107 106 lo7
F
Jun SeP Dec Mar
9.0 1.7 5.0 8.0
x x x x
104 lo4 102 103
4.0 x 103 9.5 x 103 0 3.5 x 103
G
JUn SeP Dec Mar
1.3 1.4 1.1 1.7
x 104 x 103 x 104
103 103 10’ lo4
downstream (site F) and at the mouth of the river the numbers were reduced even further (as low as 1.1 x lo3). T h e Rewa River gave a different set of results (Table 3). The highest figure obtained was 3.5 x 103/100 ml, but as Table 3 Most probable number (MPN) of coliform bacteria in the Rewa River and Laucala Bay
MPN/100 mi water
MPN/g shellfish
Site
Date
Coliforms
E . coli
Coliforms
A
Jun Sep Dec Mar
2.5 x lo3 25 9.0 x 10’ 9.0 x lo2
1 2 2 25
1.3 x 1.3 x 3.5 x 3.5 x
lo7 lo4 103 10’
Jun Sep Dec Mar
3.5 x lo3 25 5.5 x 10’ 1.6 x lo3
0 25 50 1
2.5 1.1 3.5 1.7
104 103 103 10’
Dec Mar
5.5 x 10’ 9.0 x 10’
9 7
8.0 x 10’ 2.5 x 10’
Jun Sep Dec Mar
50 35 0 1.3 x 10’
0 0 0 0
9.0 x 1.6 x 7.0 2.5 x
Jun Sep Dec Mar
11 25 0 14
0 0 0 0
9.0 x 103 1.4 x lo4 1.1 x lo4 5.0 x 103
B
RESULTS
High counts of coliforms were obtained in the Vatuwaqa River (Table 2). This was to be expected, as the effluent from the Raiwaqa Treatment Plant flows into this river. At sites D and E the coliform numbers were equal to the E. coli count in most cases, although occasionally there was a slight discrepancy between faecal coliforms and E. coli numbers. The MPN of coliforms in the effluent (site D) was 1.7 x lo7 to 1.6 x 108/100 ml. At the point where the effluent flows into the river (site E) it was 1.7 x lo6 to 2.6 x 107/100 ml. Fewer coliforms were detected further
3.5 x 3.0 x 4.0 x 1.7 x
x lo4
C
H
I
x x x x
104 105 103 103
E. coli 1.3 x lo6 3.5 x 103
o
80
o 1.7 x 102 90 80
50 50 2.0
102
2.0
102
o
o o o
2.0 x 102
o
266 S. R O B E R T S
few as 25/100 ml were found. It was noticeable that there were more coliforms in freshwater clams than in the surrounding water, and although there were fewer E. coli in the water samples, the concentration was again increased in the clams. Very few coliforms were found in Laucala Bay and in some samples none were found (Table 3). However, there was a greater concentration in oysters at both of the sites. Escherzchia coli was not found in any of the water samples and a maximum of 200/g of tissue in the oysters. Thirty-three strains of E. cola were characterized by the API 20E method. Of these 91% were biotype 1 and the remainder were probably biotype 2. Escherichia coli 1 should give a positive reaction for lysine decarboxylase (1,DC). Seven strains gave negative results with API 20E but of these four were positive when checked by tube methods, Isolates which were negative for either L D C or ornithine decarboxylase (ODC) may be biotype 2. One strain resembled Yersinia intermedia but it was motile at both 25°C and 37°C and urease-negative. Some strains were non-motile. All the strains examined produced gas from lactose in BGBS broth at 44.5"C. The generation time of the two E . coli strains examined varied according to temperature and salt concentration. Strain 204 (E. coli 2) had a mean generation time (MGT) of 8 min at 37"C, but 14.5 min at 24°C. In salt broth the M G T was 19 min. Strain 320 ( E . coli 1) gave a more realistic result of 19 min at 37°C but at 24°C the M G T was only 20 min and in salt broth was 21 min. Strain 204 grew more rapidly than strain 320 in river water as it did in the nutrient broth. Figure 4 shows that both of these strains can survive and grow in river water and seawater over a 5 d period. T h e numbers appeared to level off slightly after 3-4 d in river water, but not in seawater where there was a steady growth rate. T h e results correlate quite well with the growth of these bacteria in simulated conditions in nutrient broth at 24°C. DISCUSSION
There was a variation in the numbers of coliforms, not only a t the various sites, but also a t different times of the year. There did not appear to be a seasonal variation, however, which is to be expected, as there are only two seasons in Fiji, with little difference in water temperature. Variation in bacterial numbers may reflect changes in effluent concentration and distance from known sewage outfalls. There was a greater concentration of coliforms in the Vatuwaqa River as this river receives effluent directly from the Raiwaqa Treatment Plant. The Rewa river is much larger in area and depth and so consequently any discharge will be influenced initially by a dilution factor. Also, it receives only the overflow from irrigation ditches
to5 I 0
1
I
I
I
I
I
2
3
4
5
Time ( d )
Fig. 4 Growth of Escherichia coli in river and seawater at 24°C for 5 d ; E. coli 1 in river A, and seawater 0; E. coli 2 in river A,
and seawater adjacent to site A. In Laucala Bay very few coliforms were detected and no E. coli. This area receives sewage directly from the Kinoya Treatment Plant, but the combined effect of dilution, salt concentration, sunlight and predation will drastically reduce the number of coliforms. This may also explain why the oysters had fewer coliforms than the freshwater clams. In the Vatuwaqa and Rewa Rivers dispersal of the bacteria will be more or less in one direction, i.e. downstream, whereas in Laucala Bay dispersal will occur in several directions, but predominantly down the coastline away from the rivers and from the oyster populations being sampled. The final effluents at both Kinoya and Raiwaqa Treatment Plants are known to contain large numbers of faecal coliforms. In a study done at the Kinoya plant the average faecal coliform count was 2.0 x lo5 faecal coliforms/1OO ml effluent (Anon. 1982) and at Raiwaqa, the average faecal coliform count was 9.5 x 105/100 ml effluent (Anon. 1984). I n this study the average count over a 12 month period was 5.5 x lo7 coliforms/100 ml. In the U K the guidelines for shellfish grown for consumption is 2-5 E. coli/g of flesh (Yoovidhya & Fleet 1981). After depuration 100 g of tissue should contain fewer than 230 E . cola (Perkins et al. 1980; Pain 1986). At the mouth of the Vatuwaqa River the
C O L I F O R M S I N FIJI 267
average count was 1.1 x 104/100 ml. This is five times the standard set for shellfish growing areas and 100 times the bathing water standard set by the World Health Organisation (Anon. 1977). Local inhabitants fish, bathe and wash clothes in this river. The effectiveness of E . coli as an indicator organism has been demonstrated repeatedly (Andrews & Presnell 1972; Wolf 1972; Anderson & Baird-Parker 1975; Owens 1978). Evison & James (1973) suggested that E. coli was a good indicator in temperate climates but is less satisfactory in tropical waters because the bacteria could survive for several months. The work of Owens (1978) who estimated the coliforms around Penang Island, Malaysia, however, does not support this view. Escherichia coli would not die as rapidly in river water, and if there is a continual input of sewage into the river the mortality rate will be balanced by the new load. There would not be the combined effect of sunlight and salt, the osmotic pressure is less, and there would be a higher concentration of nutrients through leaching from the soil. The E . cola isolated in this study had a rapid generation time, i.e. E. coli biotype 1 was 19 min, but E . coli biotype 2 was only 8 min at 37°C. It has previously been shown that E . coli has a generation time of 16 rnin (Mason 1935), Vibrio natriegens 9.8 min (Eagon 1961) and V . parahaemolyticus 10 min (Barrow 1973). The E. coli could also survive for 5 d in both river and seawater, but again biotype 2 grew more rapidly than biotype 1 (Fig. 4). Flint (1987) showed that the increase in the numbers of E . coli in river water was never more than double the inoculum. However, he found that 90% of the E. coli survived for 260 d at 25°C without the addition of an extra carbon source but at 37°C the bacteria only survived 60 d. In this study a multiple tube test was used for the detection of gas production at 44.5"C. Although atypical E . coli are usually suppressed in BGBS broth, if they do grow, they will not produce indole at this temperature (Qadri et al. 1974). Thomas & Jones (1971) showed that 3.7% of colonies examined from 44°C tubes were irregular type 11, i.e. acid and gas but not indole at 44°C. In the Vatuwaqa River, 6*60/0of the strains did not produce indole. As with all enumeration methods, the multiple tube method has a large sampling error. T h e final figure could be three times higher or one-third lower than the M P N (Anon. 1983). In the majority of cases, however, it is better to be on the side of safety and accept the higher figure. The discrepancy could result from a number of factors, such as incubation temperature, inaccurate dilution and contamination of cultures. Even if the figure is accurate for a particular sample, it will not necessarily be representative of the whole body of water. For instance, a figure of 10000 coliforms/100 ml of water could be 10 or even 100 times higher or lower a few metres downstream. Each small area
of a river will have its own ecosystem, and mixing of the micro-organisms in the river will depend on currents, tides, rainfall, sedimentation, etc. Traditional methods of enumeration are based on the production of visible signs of growth. However, this may be inadequate for environmental samples and may not detect all viable bacteria present. Bacteria which fit into this category have been labelled 'viable but non-culturable' (Xu et al. 1982). This phenomenon is thought to be a survival strategy adopted by Gram-negative species in response to unfavourable conditions such as low nutrient concentration. This could account for the low numbers in Laucala Bay which may be subjected to nutrient starvation, osmotic stress, etc. T h e lethal effect of light could be aggravated by the high salinity of the water in Laucala Bay. T h e water in the rivers would be more turbid, contain less salt and contain u.v.-absorbing substances such as humic acid (Davis & Evison 1991). T h e survival of any micro-organism in an environment in which it is not indigenous will obviously be dependent upon its ability to withstand certain physical, chemical and biological conditions which are different from those encountered in its natural habitat (Lim & Flint 1989). Survival techniques may possibly be transferred to other organisms in the environment. For microbes to pass on their genes to the indigenous population, they must persist in the environment for a long period of time and at a high density. It has been shown that the ability to accept or to release extracellular DNA is favoured when the cells face survival problems (Chao & Feng 1990). The flesh of bivalve molluscs would be ideal in this situation. Anomalies were occasionally encountered with biochemical reactions, but this seems to be a common problem in the isolation of bacteria from the environment. T h e API 20E results were very consistent except for the lysine decarboxylase (LDC) and amygdalin tests, which were not always reliable. This can lead to confusion, but comparisons with conventional methods often produce the correct result. Several authors have evaluated the API 20E kit. Holmes et al. (1978) found that the overall disagreement between API 20E and conventional tests was 7%. It is not a comparison of individual tests that is important but the accuracy of the final identification (Robertson & MacLowry 1975). Holmes et al. (1978) showed that 88% of enteric bacteria could be identified correctly by API but 10% remained unidentified and 2% were incorrectly identified. If faecal pollution is considered to be a problem, the river should be monitored at various sites periodically throughout the year, particularly in populated areas. This is especially important if the river is being used as a source of drinking or bathing water. Many coliforms are free living in freshwater, e.g. Klebsiella spp. and Enterobacter spp., but E . coli is found only in animals, and its occurrence is a result of faecal contamination.
268 S. R O B E R T S
REFERENCES A N D E R S O NJ ,. M . & B A I R D - P A R K EA R.,C . (1975) A rapid and direct plate method for enumerating Escherichia coli biotype 1 in food. Journal of Applied BacterioloRy 39, 111-1 17. A N D R E W S , W . H . & P R E S N E L LM, . W . (1972) Rapid recovery of Escherichia coli from estuarine water. Applied Microbiology 23, 521-523. A N O N . (1977) Health Criteria and Epidemiology Studies Related to Coastal Water Pollution. Copenhagen : World Health Organisation. A N O N . (1982) Report on Receiving Water Study. Kino.ya Sewage Treatment Plant. Fiji : Public Works Department. A N O N . (1983) The Bacteriological Examination of Drinking Water Supplies 1982. Reports on Public Health and Medical Subjects No. 71. London: HMSO. A N O N . (1984) Water Quality Audit. Long Term Monitoring of Kinoya S T P Discharge. Fiji : Public Works Department. B A R R O WG, . I . (1973) Marine microorganisms in food poisoning. In The Microbiological Safety of Food. Proceedings of the Eighth International Symposium on Food Microbiology ed. Hobbs, B.C. & Christian, J.H.B. pp. 181-196. London: Academic Press. B R O W N ,L . P . & D O R N , C . R . (1977) Fish, shellfish and human health. Journal of Food Protection 40, 712-717. C H A O ,W . L . & F E N G R , . L . (1990) Survival of genetically engineered Escherichia coli in natural soil and river water. Journal of Applied Bacteriology 68, 31%325. D A V I SC , . M . & E V I S O NL , . M . (1991) Sunlight and the survival of enteric bacteria in natural waters. Journal of Applied Bacterrology 70, 265-274. E A G O NR , . G . (1961) Pseudomonas natriegens, a marine bacterium with a generation time of less than 10 minutes. Journal of Bacteriology 83, 736-737. E V I S O NL, . M . & J A M E S , A . (1973) A comparison of the distribution of intestinal bacteria in British and East African water sources. Journal of Applied Bacteriology 36, 109-1 18. F L I N T ,K .P . (1987) The long-term survival of Escherichia coli in river water. Journal of Applied Bacteriology 63, 261-270. , . R . & L A P A G ES,. P . (1978) HOLMES,B . , W I L L C O XW Identification of Enterobacteriaceae by the API 20E system. Journal of Clinical Pathology 31, 22-30 J O N E S F, . & W A T K I N SJ,. (1985) The water cycle as a source of pathogens. In Microbial Aspects of Water Management.
Journal of Applied Bacteriology, Symposium Supplement 59, 27s-36s. K U E H , C . S . W . & C H A N , K . Y . (1985) Bacteria in bivalve shellfish with special reference to the oyster. Journal of Applied Bacteriology 59, 41-47. , . P . (1989) The effects of nutrients on L I M , C . H . & F L I N TK the survival of Escherichia coli in lake water. Journal of Applied Bacteriology 66, 559-569. M A S O NM , . M . (1935) A comparison of maximal growth rates of various bacteria under optimal conditions. Journal of Bacteriology 29, 103-110. O W E N SJ,. D . (1978) Coliform and Escherichia coli bacteria in seawater around Penang Island Malaysia. Water Research 12, 365-370. P A I N ,S . (1986) Are British shellfish safe to eat? New Scientist No. 1523,2%33. PERKINSF , . O . , H A V E N ,D . , M O R A L E S - A L A M O R ,. & R H O D E SM , . W . (1980) Uptake and elimination of bacteria in shellfish. Journal of Food Protection 43, 124-126. QADRI, P . B . , B U C K L EK , . A . & E D W A R D SR.A. , (1974) Rapid methods for the determination of faecal contamination of oysters. Journal of Applied Bacteriology, 37, 7-14. ROBERTSON E ., A . & M A C L O W R YJ ., D . (1975) Construction of an interpretive pattern directory for the API 10s kit and analysis of its diagnostic accuracy. Journal of Clinical Microbiology 1, 515-520. T H O M AK S.,L . & J O N E S , A . M . (1971) Comparison of methods of estimating the numbers of Escherichia coli in edible mussels and the relationship between the presence of salmonellas and E. coli. Journal of Applied Bacteriology 34, 717-735. WOLF, H . W . (1972) The coliform count as a measure of water quality. In Water Pollution Microbiology ed. Mitchell, R. pp. 333-345. New York: Wiley. X u , H . S . , R O B E R T SN., , S I N G L E T OFN.,L . , A T T W E L L , R . W . , G R I M E SD , . J . & C O L W E L LR, . R . (1982) Survival and viability of nonculturable Escherichia coli and Vibrio cholerae in the estuarine and marine environment. Microbial Ecology 8, 313-323. YOOVIDHYA T ,. & F L E E TG, . H . (1981) An evaluation of the A-1 most probable number and the Anderson and Baird-Parker plate count methods for enumerating Escherichia coli in the Sydney rock oyster Crassostrea commercialis. Journal of Applied Bacteriology 50, 519-528.
Coliform bacteria from aquatic sources in Fiji S. Roberts School of Pure and Applied Sciences, University of the South Pacific, Fiji 4000/10/91: accepted 20 March 1992 R o B E R T s . 1992.Coliform bacteria were abundant in water a n d bivalve molluscs in the rivers and present to a lesser extent in t h e coastal areas of Fiji. The rivers fed b y treated sewage were highly polluted. T h e r e was a noticeable increase in concentration of coliforms in bivalve molluscs. It was also found that these bacteria could survive and grow in river water and seawater over a 5-d period, and had a rapid growth rate in nutrient broth under ideal laboratory conditions. T h e y were characterized by the API 20E identification system.
s.
INTRODUCTION
In developing countries the need to examine water supplies for pathogenic bacteria is possibly greater than in developed countries, mainly because the latter usually have comparatively efficient sewage disposal systems. In less developed countries sewage is frequently deposited directly on land or into rivers, lakes, etc., where it relies on the natural flora to render its contents harmless. Unfortunately this does not readily happen and consequently sewage entering a river may contain pathogenic bacteria which will either pose a health problem or alter the ecological balance of the river. In addition, the ability of bivalve molluscs to concentrate and accumulate bacteria is a major concern, especially when the local population rely on these animals as part of their diet. Coastal settlements in particular are at risk because of their diet of oysters, clams and mussels, which are eaten either raw or only lightly cooked. The majority of water-borne infections involve gastrointestinal disorders (Jones & Watkins 1985). Although many infections are of bacterial origin, viral diseases, e.g. viral gastroenteritis and infective hepatitis, are playing a more prominent role, especially when transmitted by shellfish (Brown & Dorn 1977; Kueh & Chan 1985; Pain 1986). The importance of examining water and aquatic animals such as bivalve molluscs for faecal contamination cannot be overemphasized. Table 1 shows the variety of microorganisms that are likely to be encountered in sewage, although some of them may not be found in all countries. Not all coliforms will be associated with the mammalian intestinal tract, as a number of strains are of environmental origin. Some or all may be of faecal origin, and as there is no satisfactory method for differentiating human strains Present address and correspondence to: M r S. Roberts, Plant Science Limited, Firth Court, University of Shefield, Shefield SIO 2TN, UK.
from those of animal origin, an explanation of their presence must be sought. MATERIALS AND METHODS
Samples
Samples were taken at three-monthly intervals over a period of 1 year. T h e water samples were obtained from two rivers on the island of Viti Levu (Fiji), and from Table 1 Pathogenic agents that may be present in sewage*
Bacteria Leptospira icterohaemorrhagiae Campylobacter spp. Pseudomonas aeruginosa Brucella spp. Enteropathogenic E . coli Shigella spp. Salmonella typhi Salm. paratyphi Klebsiella spp. Yersinia enterolytica Yersinia pestis Vibrio cholerae Aeromonas hydrophila Bacillus anthracis Clostridium spp. Listeria monocytogenes Erysipelothrix rhusiopathiae Mycobacterium tuberculosis Mycobacterium paratuberculosis Staphylococcus aureus
Fungi Candida albicans
* See Jones & Watkins 1985.
Viruses Adenovirus Enterovirus New Enterovirus Poliovirus Echovirus Coxsackievirus Norwalk Virus Rotavirus Reovirus Parvovirus Intestinal parasites Entamoeba histolytica Acanthamoeba spp. Naegleria spp. Giardia lamblia Cryptosporidia spp. Ascaris lumbricoides Trichuris trichuria Ancylostoma duodenale Necator americanus Schistosoma mansoni Diphyllobothrium latum Taenia spp. ~~
264 S . R O B E R T S
Fig. 1 Laucala Bay, Fiji, showing sites H and I
25
0
Km
-P
REWA RIVER
The water samples were diluted depending upon the The shellfish origin of the sample, from lo-' to were scrubbed clean under running water, and all debris and barnacles were removed. T h e two halves of the shell were separated by a sterile scalpel and the juices were discarded. Ten animals were homogenized in a Waring blender and 10 g of homogenate were suspended in 100 ml of normal sterile saline. Dilutions were made from 10- to
'
Bacteriological examination LAUCALA B A Y
Enumeration Fig. 2 Rewa River flowing into Laucala Bay, Fiji
L A U C A L A BAY
Fig. 3 Vatuwaqa River, Fiji
Laucala Bay (Figs 1-3). T h e bivalve molluscs examined were the freshwater clam, Batissa violacea from the Rewa river, and the mangrove oyster, Crassostrea mordax from Laucala Bay.
T h e five-tube Most Probable Number (MPN) method was used, with 10, 1.0 and 0.1 ml volumes and MacConkey Broth (Oxoid) (Anon. 1983). Tubes showing acid and gas after incubation at 37°C for 24 h were subcultured into Brilliant Green Lactose Bile Salts Broth (BGBS, Oxoid) and incubated at 44.5"C for 24 h. Tubes showing gas were further subcultured into Tryptone Water (Oxoid) and incubated at 44.5"C. These samples were tested for indole production by Kovac's method (Anon. 1983). Gas production and indole formation at 44.5"C were considered confirmation of Escherichia coli. The presumptive M P N counts of coliforms and E. coli in 100 ml of water or per gram of shellfish were estimated from the probability tables (Anon. 1983).
Identification
Samples were taken from the positive tubes of BGBS and plated on MacConkey Agar. Colonies were selected and
COLIFORMS IN FIJI 265
purified by subculture. The organisms were then examined microscopically and biochemically by the API 20E system.
Table 2 Most probable number (MPN) of coliform bacteria in the Vatuwaqa River
MPN/100 ml water Growth curves
The bacteria were grown under various conditions and monitored by periodically plating varying dilutions of samples (0.1 ml) on appropriate media. The growth of the bacteria was monitored in nutrient broth (Oxoid) with and without 3.0% NaCl (w/v), at 37°C and 24°C. The culture medium (200 ml) was inoculated with 3.0 ml of an overnight broth culture. This was diluted lo-’ and lo-* in sterile saline and 0.1 ml of each dilution was plated on nutrient agar (NA) and incubated at 30°C overnight. The remaining culture was incubated at either 24°C or 37°C in a shaking water bath. After 30 min, 1.0 ml of the culture was removed, diluted to lo-’ and and 0.1 ml plated on NA and incubated at 30°C overnight. This process was repeated every 60 min for 5 h, increasing the dilutions at each stage to take into account the multiplication of the bacteria. The number of colony forming units (cfu) was estimated and the number of bacteria per ml of original culture calculated. Survival in river water and seawater
The bacteria were grown overnight in nutrient broth at 30°C. T h e cells were centrifuged at 10 000 g and the supernatant fluid was discarded. The pellet was washed twice in sterile saline, centrifuged and resuspended in either 100 ml of sterile river water or seawater (sterilized by autoclaving at 115°C for 20 min). Ten ml of this suspension were added to 200 ml of sterile river or seawater and incubated at 24°C in a shaking water bath for 5 d. At intervals, samples were removed and plated on nutrient agar either with or without 3.0% NaCl (w/v) and incubated at 30°C overnight. The number of viable bacteria was estimated. The experiment was repeated 14 d later with the same strains under identical conditions and in duplicate.
Site
Date
Coliforms
E . coli
D
Jun SeP Dec Mar
2.8 x 1.7 x 1.7 x 1.6 x
lo7 107 lo7 10’
2.8 1.7 1.7 9.0
x x x x
lo7 107 lo7 107
E
Jun SeP Dec Mar
1.7 x 2.3 5.0 x 2.6 x
lo6 107 106 lo7
1.7 x 1.3 x 2.0 x 1.3 x
lo6 107 106 lo7
F
Jun SeP Dec Mar
9.0 1.7 5.0 8.0
x x x x
104 lo4 102 103
4.0 x 103 9.5 x 103 0 3.5 x 103
G
JUn SeP Dec Mar
1.3 1.4 1.1 1.7
x 104 x 103 x 104
103 103 10’ lo4
downstream (site F) and at the mouth of the river the numbers were reduced even further (as low as 1.1 x lo3). T h e Rewa River gave a different set of results (Table 3). The highest figure obtained was 3.5 x 103/100 ml, but as Table 3 Most probable number (MPN) of coliform bacteria in the Rewa River and Laucala Bay
MPN/100 mi water
MPN/g shellfish
Site
Date
Coliforms
E . coli
Coliforms
A
Jun Sep Dec Mar
2.5 x lo3 25 9.0 x 10’ 9.0 x lo2
1 2 2 25
1.3 x 1.3 x 3.5 x 3.5 x
lo7 lo4 103 10’
Jun Sep Dec Mar
3.5 x lo3 25 5.5 x 10’ 1.6 x lo3
0 25 50 1
2.5 1.1 3.5 1.7
104 103 103 10’
Dec Mar
5.5 x 10’ 9.0 x 10’
9 7
8.0 x 10’ 2.5 x 10’
Jun Sep Dec Mar
50 35 0 1.3 x 10’
0 0 0 0
9.0 x 1.6 x 7.0 2.5 x
Jun Sep Dec Mar
11 25 0 14
0 0 0 0
9.0 x 103 1.4 x lo4 1.1 x lo4 5.0 x 103
B
RESULTS
High counts of coliforms were obtained in the Vatuwaqa River (Table 2). This was to be expected, as the effluent from the Raiwaqa Treatment Plant flows into this river. At sites D and E the coliform numbers were equal to the E. coli count in most cases, although occasionally there was a slight discrepancy between faecal coliforms and E. coli numbers. The MPN of coliforms in the effluent (site D) was 1.7 x lo7 to 1.6 x 108/100 ml. At the point where the effluent flows into the river (site E) it was 1.7 x lo6 to 2.6 x 107/100 ml. Fewer coliforms were detected further
3.5 x 3.0 x 4.0 x 1.7 x
x lo4
C
H
I
x x x x
104 105 103 103
E. coli 1.3 x lo6 3.5 x 103
o
80
o 1.7 x 102 90 80
50 50 2.0
102
2.0
102
o
o o o
2.0 x 102
o
266 S. R O B E R T S
few as 25/100 ml were found. It was noticeable that there were more coliforms in freshwater clams than in the surrounding water, and although there were fewer E. coli in the water samples, the concentration was again increased in the clams. Very few coliforms were found in Laucala Bay and in some samples none were found (Table 3). However, there was a greater concentration in oysters at both of the sites. Escherzchia coli was not found in any of the water samples and a maximum of 200/g of tissue in the oysters. Thirty-three strains of E. cola were characterized by the API 20E method. Of these 91% were biotype 1 and the remainder were probably biotype 2. Escherichia coli 1 should give a positive reaction for lysine decarboxylase (1,DC). Seven strains gave negative results with API 20E but of these four were positive when checked by tube methods, Isolates which were negative for either L D C or ornithine decarboxylase (ODC) may be biotype 2. One strain resembled Yersinia intermedia but it was motile at both 25°C and 37°C and urease-negative. Some strains were non-motile. All the strains examined produced gas from lactose in BGBS broth at 44.5"C. The generation time of the two E . coli strains examined varied according to temperature and salt concentration. Strain 204 (E. coli 2) had a mean generation time (MGT) of 8 min at 37"C, but 14.5 min at 24°C. In salt broth the M G T was 19 min. Strain 320 ( E . coli 1) gave a more realistic result of 19 min at 37°C but at 24°C the M G T was only 20 min and in salt broth was 21 min. Strain 204 grew more rapidly than strain 320 in river water as it did in the nutrient broth. Figure 4 shows that both of these strains can survive and grow in river water and seawater over a 5 d period. T h e numbers appeared to level off slightly after 3-4 d in river water, but not in seawater where there was a steady growth rate. T h e results correlate quite well with the growth of these bacteria in simulated conditions in nutrient broth at 24°C. DISCUSSION
There was a variation in the numbers of coliforms, not only a t the various sites, but also a t different times of the year. There did not appear to be a seasonal variation, however, which is to be expected, as there are only two seasons in Fiji, with little difference in water temperature. Variation in bacterial numbers may reflect changes in effluent concentration and distance from known sewage outfalls. There was a greater concentration of coliforms in the Vatuwaqa River as this river receives effluent directly from the Raiwaqa Treatment Plant. The Rewa river is much larger in area and depth and so consequently any discharge will be influenced initially by a dilution factor. Also, it receives only the overflow from irrigation ditches
to5 I 0
1
I
I
I
I
I
2
3
4
5
Time ( d )
Fig. 4 Growth of Escherichia coli in river and seawater at 24°C for 5 d ; E. coli 1 in river A, and seawater 0; E. coli 2 in river A,
and seawater adjacent to site A. In Laucala Bay very few coliforms were detected and no E. coli. This area receives sewage directly from the Kinoya Treatment Plant, but the combined effect of dilution, salt concentration, sunlight and predation will drastically reduce the number of coliforms. This may also explain why the oysters had fewer coliforms than the freshwater clams. In the Vatuwaqa and Rewa Rivers dispersal of the bacteria will be more or less in one direction, i.e. downstream, whereas in Laucala Bay dispersal will occur in several directions, but predominantly down the coastline away from the rivers and from the oyster populations being sampled. The final effluents at both Kinoya and Raiwaqa Treatment Plants are known to contain large numbers of faecal coliforms. In a study done at the Kinoya plant the average faecal coliform count was 2.0 x lo5 faecal coliforms/1OO ml effluent (Anon. 1982) and at Raiwaqa, the average faecal coliform count was 9.5 x 105/100 ml effluent (Anon. 1984). I n this study the average count over a 12 month period was 5.5 x lo7 coliforms/100 ml. In the U K the guidelines for shellfish grown for consumption is 2-5 E. coli/g of flesh (Yoovidhya & Fleet 1981). After depuration 100 g of tissue should contain fewer than 230 E . cola (Perkins et al. 1980; Pain 1986). At the mouth of the Vatuwaqa River the
C O L I F O R M S I N FIJI 267
average count was 1.1 x 104/100 ml. This is five times the standard set for shellfish growing areas and 100 times the bathing water standard set by the World Health Organisation (Anon. 1977). Local inhabitants fish, bathe and wash clothes in this river. The effectiveness of E . coli as an indicator organism has been demonstrated repeatedly (Andrews & Presnell 1972; Wolf 1972; Anderson & Baird-Parker 1975; Owens 1978). Evison & James (1973) suggested that E. coli was a good indicator in temperate climates but is less satisfactory in tropical waters because the bacteria could survive for several months. The work of Owens (1978) who estimated the coliforms around Penang Island, Malaysia, however, does not support this view. Escherichia coli would not die as rapidly in river water, and if there is a continual input of sewage into the river the mortality rate will be balanced by the new load. There would not be the combined effect of sunlight and salt, the osmotic pressure is less, and there would be a higher concentration of nutrients through leaching from the soil. The E . cola isolated in this study had a rapid generation time, i.e. E. coli biotype 1 was 19 min, but E . coli biotype 2 was only 8 min at 37°C. It has previously been shown that E . coli has a generation time of 16 rnin (Mason 1935), Vibrio natriegens 9.8 min (Eagon 1961) and V . parahaemolyticus 10 min (Barrow 1973). The E. coli could also survive for 5 d in both river and seawater, but again biotype 2 grew more rapidly than biotype 1 (Fig. 4). Flint (1987) showed that the increase in the numbers of E . coli in river water was never more than double the inoculum. However, he found that 90% of the E. coli survived for 260 d at 25°C without the addition of an extra carbon source but at 37°C the bacteria only survived 60 d. In this study a multiple tube test was used for the detection of gas production at 44.5"C. Although atypical E . coli are usually suppressed in BGBS broth, if they do grow, they will not produce indole at this temperature (Qadri et al. 1974). Thomas & Jones (1971) showed that 3.7% of colonies examined from 44°C tubes were irregular type 11, i.e. acid and gas but not indole at 44°C. In the Vatuwaqa River, 6*60/0of the strains did not produce indole. As with all enumeration methods, the multiple tube method has a large sampling error. T h e final figure could be three times higher or one-third lower than the M P N (Anon. 1983). In the majority of cases, however, it is better to be on the side of safety and accept the higher figure. The discrepancy could result from a number of factors, such as incubation temperature, inaccurate dilution and contamination of cultures. Even if the figure is accurate for a particular sample, it will not necessarily be representative of the whole body of water. For instance, a figure of 10000 coliforms/100 ml of water could be 10 or even 100 times higher or lower a few metres downstream. Each small area
of a river will have its own ecosystem, and mixing of the micro-organisms in the river will depend on currents, tides, rainfall, sedimentation, etc. Traditional methods of enumeration are based on the production of visible signs of growth. However, this may be inadequate for environmental samples and may not detect all viable bacteria present. Bacteria which fit into this category have been labelled 'viable but non-culturable' (Xu et al. 1982). This phenomenon is thought to be a survival strategy adopted by Gram-negative species in response to unfavourable conditions such as low nutrient concentration. This could account for the low numbers in Laucala Bay which may be subjected to nutrient starvation, osmotic stress, etc. T h e lethal effect of light could be aggravated by the high salinity of the water in Laucala Bay. T h e water in the rivers would be more turbid, contain less salt and contain u.v.-absorbing substances such as humic acid (Davis & Evison 1991). T h e survival of any micro-organism in an environment in which it is not indigenous will obviously be dependent upon its ability to withstand certain physical, chemical and biological conditions which are different from those encountered in its natural habitat (Lim & Flint 1989). Survival techniques may possibly be transferred to other organisms in the environment. For microbes to pass on their genes to the indigenous population, they must persist in the environment for a long period of time and at a high density. It has been shown that the ability to accept or to release extracellular DNA is favoured when the cells face survival problems (Chao & Feng 1990). The flesh of bivalve molluscs would be ideal in this situation. Anomalies were occasionally encountered with biochemical reactions, but this seems to be a common problem in the isolation of bacteria from the environment. T h e API 20E results were very consistent except for the lysine decarboxylase (LDC) and amygdalin tests, which were not always reliable. This can lead to confusion, but comparisons with conventional methods often produce the correct result. Several authors have evaluated the API 20E kit. Holmes et al. (1978) found that the overall disagreement between API 20E and conventional tests was 7%. It is not a comparison of individual tests that is important but the accuracy of the final identification (Robertson & MacLowry 1975). Holmes et al. (1978) showed that 88% of enteric bacteria could be identified correctly by API but 10% remained unidentified and 2% were incorrectly identified. If faecal pollution is considered to be a problem, the river should be monitored at various sites periodically throughout the year, particularly in populated areas. This is especially important if the river is being used as a source of drinking or bathing water. Many coliforms are free living in freshwater, e.g. Klebsiella spp. and Enterobacter spp., but E . coli is found only in animals, and its occurrence is a result of faecal contamination.
268 S. R O B E R T S
REFERENCES A N D E R S O NJ ,. M . & B A I R D - P A R K EA R.,C . (1975) A rapid and direct plate method for enumerating Escherichia coli biotype 1 in food. Journal of Applied BacterioloRy 39, 111-1 17. A N D R E W S , W . H . & P R E S N E L LM, . W . (1972) Rapid recovery of Escherichia coli from estuarine water. Applied Microbiology 23, 521-523. A N O N . (1977) Health Criteria and Epidemiology Studies Related to Coastal Water Pollution. Copenhagen : World Health Organisation. A N O N . (1982) Report on Receiving Water Study. Kino.ya Sewage Treatment Plant. Fiji : Public Works Department. A N O N . (1983) The Bacteriological Examination of Drinking Water Supplies 1982. Reports on Public Health and Medical Subjects No. 71. London: HMSO. A N O N . (1984) Water Quality Audit. Long Term Monitoring of Kinoya S T P Discharge. Fiji : Public Works Department. B A R R O WG, . I . (1973) Marine microorganisms in food poisoning. In The Microbiological Safety of Food. Proceedings of the Eighth International Symposium on Food Microbiology ed. Hobbs, B.C. & Christian, J.H.B. pp. 181-196. London: Academic Press. B R O W N ,L . P . & D O R N , C . R . (1977) Fish, shellfish and human health. Journal of Food Protection 40, 712-717. C H A O ,W . L . & F E N G R , . L . (1990) Survival of genetically engineered Escherichia coli in natural soil and river water. Journal of Applied Bacteriology 68, 31%325. D A V I SC , . M . & E V I S O NL , . M . (1991) Sunlight and the survival of enteric bacteria in natural waters. Journal of Applied Bacterrology 70, 265-274. E A G O NR , . G . (1961) Pseudomonas natriegens, a marine bacterium with a generation time of less than 10 minutes. Journal of Bacteriology 83, 736-737. E V I S O NL, . M . & J A M E S , A . (1973) A comparison of the distribution of intestinal bacteria in British and East African water sources. Journal of Applied Bacteriology 36, 109-1 18. F L I N T ,K .P . (1987) The long-term survival of Escherichia coli in river water. Journal of Applied Bacteriology 63, 261-270. , . R . & L A P A G ES,. P . (1978) HOLMES,B . , W I L L C O XW Identification of Enterobacteriaceae by the API 20E system. Journal of Clinical Pathology 31, 22-30 J O N E S F, . & W A T K I N SJ,. (1985) The water cycle as a source of pathogens. In Microbial Aspects of Water Management.
Journal of Applied Bacteriology, Symposium Supplement 59, 27s-36s. K U E H , C . S . W . & C H A N , K . Y . (1985) Bacteria in bivalve shellfish with special reference to the oyster. Journal of Applied Bacteriology 59, 41-47. , . P . (1989) The effects of nutrients on L I M , C . H . & F L I N TK the survival of Escherichia coli in lake water. Journal of Applied Bacteriology 66, 559-569. M A S O NM , . M . (1935) A comparison of maximal growth rates of various bacteria under optimal conditions. Journal of Bacteriology 29, 103-110. O W E N SJ,. D . (1978) Coliform and Escherichia coli bacteria in seawater around Penang Island Malaysia. Water Research 12, 365-370. P A I N ,S . (1986) Are British shellfish safe to eat? New Scientist No. 1523,2%33. PERKINSF , . O . , H A V E N ,D . , M O R A L E S - A L A M O R ,. & R H O D E SM , . W . (1980) Uptake and elimination of bacteria in shellfish. Journal of Food Protection 43, 124-126. QADRI, P . B . , B U C K L EK , . A . & E D W A R D SR.A. , (1974) Rapid methods for the determination of faecal contamination of oysters. Journal of Applied Bacteriology, 37, 7-14. ROBERTSON E ., A . & M A C L O W R YJ ., D . (1975) Construction of an interpretive pattern directory for the API 10s kit and analysis of its diagnostic accuracy. Journal of Clinical Microbiology 1, 515-520. T H O M AK S.,L . & J O N E S , A . M . (1971) Comparison of methods of estimating the numbers of Escherichia coli in edible mussels and the relationship between the presence of salmonellas and E. coli. Journal of Applied Bacteriology 34, 717-735. WOLF, H . W . (1972) The coliform count as a measure of water quality. In Water Pollution Microbiology ed. Mitchell, R. pp. 333-345. New York: Wiley. X u , H . S . , R O B E R T SN., , S I N G L E T OFN.,L . , A T T W E L L , R . W . , G R I M E SD , . J . & C O L W E L LR, . R . (1982) Survival and viability of nonculturable Escherichia coli and Vibrio cholerae in the estuarine and marine environment. Microbial Ecology 8, 313-323. YOOVIDHYA T ,. & F L E E TG, . H . (1981) An evaluation of the A-1 most probable number and the Anderson and Baird-Parker plate count methods for enumerating Escherichia coli in the Sydney rock oyster Crassostrea commercialis. Journal of Applied Bacteriology 50, 519-528.
