Microb Ecol (1988) 15:203-215

MICROBIAL ECOLOGY 9 Springer-VerlagNew York Inc. 1988

Ecology of Vibrio cholerae in the Freshwater Environs of Calcutta, India G. Balakrish Nair,* B. L. Sarkar, S. P. De, M. K. Chakrabarti, R. K. Bhadra, and S. C. Pal National Institute of Cholera and Enteric Diseases, Beliaghata, Calcutta, 700 010, India Abstract. Seasonal incidence of Vibrio cholerae was monitored for a year in a man-made freshwater lake, an open sewage canal, and a pond composed o f rainwater accumulations, located in Calcutta. V. cholerae was found in all sites. It exhibited a distinct bimodal seasonal cycle in the lake with a primary peak in August-September and a secondary peak in May-June. Correlation with environmental parameters revealed that temperature and, to a certain extent, pH were the important factors governing the densities o f V. cholerae. In the lake, sediment samples harbored high densities o f V. cholerae immediately after months when peak counts were observed in plankton, suggesting a cycle of cells between sediment and water. At the other sampling areas, no defined seasonality was observed. Instead, high counts o f V. cholerae were observed at these severely polluted sites throughout the study period, including the winter months. All the 15 water samples passed via the ligated loop of rabbits yielded pure cultures o f V. cholerae, indicating that the rabbit intestine selects out V. cholerae from a mixed flora. Uniformly high isolation rates of V. cholerae were observed from brackish water and freshwater species of export quality prawns. V. cholerae was found to be abundant and was represented by 32 individual Louisiana State University (LSU) serovars, including two new serovars. The 01 serovar could not be isolated from any of the samples examined in this study. It was concluded that V. cholerae non-01 is c o m m o n in the freshwater environs o f Calcutta. Introduction

The recent claim that Vibrio cholerae 01, the causative agent o f epidemic cholera, has a natural aquatic reservoir is supported by the sporadic isolations of this organism from the environment in different geographic areas [7, 17, 25] and has generated a great deal of interest. On the basis o f the presence of V. cholerae in water, sediment, and shellfish in the Chesapeake Bay, and since its presence did not correlate with fecal coliforms, Kaper et al. [14] concluded that the organism was an autochthonous resident o f estuaries. Laboratory studies on * Present address: Dr. G. Balakrish Nair, Guest Research Fellow, National Children's Medical Research Center, 3-35-31, Taishido, Setagaya-ku,Tokyo 154, Japan.

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V. cholerae s e e m to i n d i c a t e t h a t t h i s s p e c i e s is w e l l a d a p t e d t o s u r v i v e a n d p r o l i f e r a t e u n d e r c o n d i t i o n s s i m u l a t i n g t h e n a t u r a l e n v i r o n m e n t [2, 9, 10, 18, 33, 34]. R e c e n t s t u d i e s h a v e a l s o d e m o n s t r a t e d t h e e x i s t e n c e o f a v i a b l e b u t n o n c u l t u r a b l e f o r m o f V. cholerae [5] t h a t a p p a r e n t l y c a n s u r v i v e u n d e r a d v e r s e e n v i r o n m e n t a l c o n d i t i o n s a n d m a y p l a y a n i m p o r t a n t r o l e in t h e e p i d e m i o l o g y o f c h o l e r a [ 11]. S u c h r e p o r t s h a v e , i n fact, b e e n t h e " g e n e s i s " f o r t h e p r o p o s a l of a new model of cholera epidemiology which purports that aquatic reservoirs m i g h t b e t h e m e c h a n i s m b y w h i c h c h o l e r a e n d e m i c i t y is m a i n t a i n e d i n a g i v e n a r e a [ 19]. For several centuries, cholera has ravaged the gangetic plains of eastern India, the traditional home of Asiatic cholera. Though several studies on diverse aspects of cholera have been conducted in Calcutta, a notable exception has been the lack of a defined ecological study of this organism. Most previous s t u d i e s o n t h e i n c i d e n c e o f V.. cholerae in a q u a t i c b o d i e s i n C a l c u t t a h a v e b e e n limited either to epidemiological investigations or to qualitative studies which d i d n o t d e a l w i t h d e t a i l s o f t h e effects o f a s s o c i a t e d e n v i r o n m e n t a l p a r a m e t e r s . A l s o , t h e r e h a s b e e n n o a t t e m p t to s t u d y t h e v a r i a t i o n i n d e n s i t i e s o f t h e o r g a n i s m i n t h e e n v i r o n m e n t i n r e l a t i o n t o s e a s o n s . T h e p r e s e n t s t u d y was, t h e r e f o r e , u n d e r t a k e n t o e x p l o r e t h e e c o l o g y o f V. cholerae i n t h e f r e s h w a t e r environs of Calcutta. Materials and Methods

Sampling Sites Four sampling sites (Fig. 1) located in the eastern parts of Calcutta (longitude, 88~ latitude, 22~ and representative of three different aquatic bodies were sampled once a month from July 1984 to June 1985. Site 1 was located in a man-made freshwater lake occupying 39.5 acres. The lake has a subterranean source. Sites 2 and 3 were in an open sewage canal meandering through the city of Calcutta, which receives both domestic and industrial effluents. Site 2 was in an upstream area of the canal, whereas site 3 was located further north about 2 km downstream of site 2. Site 4 was located in a pond of approximately 20 square meters chiefly comprising rainwater accumulations. The pond is extensively used for bathing and domestic purposes by the human population residing at its periphery. On the basis of hydrographical events, which are chiefly influenced by the monsoons, the study period could be demarcated into four seasons, i.e., monsoon (July to September), postmonsoon (October to November), winter (December to February), and summer (March to June).

Collection of Environmental Samples Water, sediment, and plankton were sampled at site 1, whereas at sites 2, 3, and 4, only water samples were monitored. Planktonic organisms were harvested by towing a horizontal plankton net (bolting silk No. 20; mesh size, 77/~m) for ca. 30 min at a depth of ca. 0.5 m below the surface. They were then transferred into sterile, wide-mouth glass bottles. Presterilized glass bottles were used to collect water samples just below the water surface. A Petersen Grab, which was thoroughly rinsed and air-dried before use, was employed to collect bottom sediments.

Physical and Chemical Parameters Methods used for the measurement of physical and chemical parameters, i.e., temperature, salinity, dissolved oxygen, turbidity, and pH, have been previously described [22].

Vibrio cholerae in Calcutta Environs

205

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Fig. 1.

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Map of Calcutta Corporation area indicating the sampling sites.

Bacteriological Methods Total viable aerobic, heterotrophic counts (TVC) were determined on nutrient agar (Difco) without added sodium chloride by the spread plate technique. The plates were incubated at room temperature for 48 hours before colonies were counted. The method used for enumeration of V. cholerae was the three-tube most-probable-number procedure described by Kaper et al. [14]. Appropriate decimal dilutions o f homogenized plankton, water, and sediment were serially prepared to cover a dilution range between I and 10 -~ using a sterilized water sample from the collection area as diluent. Alkaline peptone water (APW) containing peptone (Difco; 10 g/liter) and NaCI (10 g/liter) at pH 8.6 was used as the enrichment medium. Larger volumes (100 ml) of the water samples were filtered through membrane filters (0.45 um pore size; Millipore Corp., Bedford, Massachusetts) and then introduced into 50 ml single-strength broth while 100 ml o f sediment samples were directly introduced into 200 ml of single-strength broth. The 10 ml sample volumes o f water and sediment were introduced into 10 ml o f double-strength broth. After incubation for 18 hours at 37~ the enrichment broth cultures were plated on thiosulfate citrate bile-salts sucrose agar (TCBS; Eiken Chemical Co. Ltd., Japan) and incubated for 24 hours at 37~ Typical colonies (fiat sucrosepositive and negative colonies with elevated centers) were picked and presumptively identified

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using the multitest screening medium (J. B. Kaper, Ph.D. thesis, University of Maryland, College Park, USA, 1979). The presumptive identifications were confirmed by additional biochemical tests using the API 20E system (La Balme-Les-Grottes, Vercieu, France). Most-probable-number values of samples yielding confirmed isolates of V. cholerae were determined from published tables [1]. During the course of this study, we resorted to an unconventional procedure to isolate V. cholerae. In this method, 100 ml water samples obtained from different points located in site 1 were filtered through 0.45 #m membrane filters (Millipore Corp., Bedford, Massachusetts). The filters were immediately washed in 5 ml of phosphate-buffered saline (PBS; 0.425% NaCl, 1.5275% NazHPO4, 0.244% KHEPO4, pH 7.2), and 1 ml of this was directly introduced into the ligated ileal loop of adult male rabbits with a negative control containing only PBS. This was done with the rationale that the intestinal milieu of rabbits would perhaps be the most efficient environment to select out strains of culturable and/or nonculturable V. cholerae 01, if any. After 12 hours, the rabbits were sacrificed, and an index of fluid accumulation was derived from the ratio of loop fluid volume-toloop length. A portion of the fluid accumulated in each loop and a swab from the loops were plated on TCBS. Colonies were picked and characterized as described above.

Prawn Samples One hundred and thirty-one specimens belonging to five different species of brackish and freshwater export quality prawns cultured in paddy fields (locally known as "bhery culture") were sampled from June to November 1985. Freshly caught prawn samples, provided by the Marine Products Export Development Authority (MPEDA), Calcutta, were harvested using standard equipment and immediately transferred to individual polythene bags. Samples were brought to the laboratory in portable ice chests (< 5~ At the laboratory, sand and detritus adhering to the specimens were washed away with sterile saline. A 1 g portion taken from the caudal region of each sample was aseptically pulverized and introduced into 10 ml of APW and incubated for 18 hours at 37~ Subsequently, two loopfuls of the broth were plated on TCBS and incubated for 24 hours at 37~ Colonies were picked and identified as described above.

Serotyping Serological analysis was carried out at two stages using polyvalent V. cholerae 01 antiserum prepared at our institute. At all times, before initiating the serological analysis, the reactivity of the antiserum was checked with standard clinical strains of V. cholerae 01. The first stage involved the spot inoculation of randomly picked, single, typical V. cholerae-like colonies from the selective medium onto nutrient agar plates. The growth obtained after overnight incubation at 37~ was used to perform the serotyping. In the second stage, isolates that yielded a positive response in the multitest presumptive identification medium (alkaline slant/acid butt) were screened for agglutination in the polyvalent 01 antiserum. These strains were further rechecked for agglutination in 01 antiserum after biochemical characterization. This strategy permitted us to screen more than 10,000 individual presumptive and confirmed V. cholerae cultures during the entire period of study. A representative number of V. cholerae non-01 cultures were also serotyped by Dr. R. Siebeling, Louisiana State University, Baton Rouge, Louisiana, USA.

Results The range of variations in environmental parameters and in TVC monitored a t t h e f o u r s a m p l i n g s i t e s a r e g i v e n i n T a b l e 1. S u r f a c e w a t e r a n d s e d i m e n t t e m p e r a t u r e s a t a l l s i t e s v a r i e d b e t w e e n 21 a n d 36~ w i t h h i g h e r t e m p e r a t u r e s recorded during the summer months. Salinity at all the four sites was found

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Table 1. Range of variations in environmental parameters and total viable aerobic, heterotrophic counts (TVC) at the four sampling sites during the period of investigation Source of Site sample Temp (~ 1 2 3 4

Plankton Water Sediment Water Water Water

. 23-33 21-32 24-36 24-36 21-35

.

Salinity (%~)

Turbidity (K)

. 0.02-0.1 -0.03-0.4 0.1-0.5 0.03-0.2

.

. 1.3-66.6 -3.0-45.9 2.0-56.6 5.3-12.1

pH

Dissolved oxygen (ml/liter)

TVC (CFU/ml) x 104

. 7.3-9.0 6.2-7.7 7.0-8.6 6.9-8.3 7.4-8.8

2.1-16.1 -0-8.9 0-13.4 5.8-16.9

10-6,600 4-540 9.6-1,800 57-3,100 6.9-8,800 7.1-1,600

- Not applicable

to be u n i f o r m l y low ( < 0.5%0) with no a p p a r e n t seasonal fluctuations. Eutrophic conditions were indicated by alkaline p H values ranging between 8.2 a n d 9.0 in water samples at site 1 during the s u m m e r m o n t h s . C o r r e s p o n d i n g s e d i m e n t p H values at this site were, however, m u c h lower during the s a m e period. T h e r e was a similar seasonal fluctuation in p H o f water samples, though less defined, at sites 2, 3, a n d 4. D i s s o l v e d oxygen values varied erratically without a defined seasonal trend. Essentially anoxic conditions were o b s e r v e d at the heavily polluted sites 2 and 3 f r o m S e p t e m b e r 1984 to M a r c h 1985 a n d in June 1985. Variations in turbidity did not fall into a readily discernible seasonal pattern. Highest T V C were o b s e r v e d during the s u m m e r and p o s t m o n s o o n m o n t h s at all sites, whereas m i n i m u m counts were e n c o u n t e r e d during the winter m o n t h s . Seasonal incidence and counts o f V. cholerae in the various e n v i r o n m e n t a l s a m p l e s m o n i t o r e d at the four sites during the entire period o f investigation are presented in T a b l e 2. V. cholerae was f o u n d at all the sites a n d exhibited a distinct b i m o d a l seasonal cycle at site 1 with a p r i m a r y p e a k in A u g u s t S e p t e m b e r and a secondary p e a k in M a y - J u n e . D u r i n g the winter m o n t h s , the counts o f F. cholerae declined a n d r e m a i n e d below detectable levels in p l a n k t o n a n d in the water samples at site 1 in D e c e m b e r . S e d i m e n t samples at site 1 h a r b o r e d highest densities o f V. cholerae i m m e d i a t e l y after m o n t h s when p e a k counts were o b s e r v e d in plankton, suggesting a cycle o f cells between these two niches. A distinct seasonal cycle, as evident in site 1, was not o b s e r v e d at sites 2, 3, a n d 4. Instead, relatively high counts o f 1I. cholerae were o b s e r v e d throughout the study period, including the winter m o n t h s . Overall densities o f F. cholerae at these three sites were, however, highest during the s u m m e r m o n t h s as c o m p a r e d to the other seasons. All the 15 m e m b r a n e - f i l t e r e d w a t e r s a m p l e s passed via the ligated loop o f adult rabbits yielded V. cholerae. T h e average fluid a c c u m u l a t i o n (mostly h e m orrhagic) ranged between 0.87 and 1.14. T h e striking o b s e r v a t i o n was that the rabbit intestinal milieu did, indeed, select o u r F. cholerae f r o m a m i x e d flora o f the water samples as reflected b y the pure cultures obtained on plating the fluid a c c u m u l a t e d a n d swabs taken f r o m the loops on TCBS. N o n e o f the 21 isolates o f V. cholerae r e c o v e r e d by this m e t h o d agglutinated with polyvalent 01 antiserum. Instead, the following 1I. cholerae non-01 L S U serovars were recovered: CC (5 strains) a n d M M , J and V (one strain each).

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Table 2. Seasonal incidence and counts of V. cholerae measured between July 1984 and June 1985 in environmental samples at the four sampling sites Most-probable-number counts" Site 1 2 3 4

Source of sample July '84 Plankton Water Sediment Water Water Water

4 15 210 2,100 430 21

Monsoon

Post-monsoon

Winter

Aug.

Sept.

Oct.

Nov.

Dec.

Jan. '85

Feb.

2,400 93 240 460 750 1,200

930 1,500 3,900 2,400 4,600 240

110 150 75 2,100 210 93

0.93 ND b ND ND ND ND

Ecology ofVibrio cholerae in the freshwater environs of Calcutta, India.

Seasonal incidence ofVibrio cholerae was monitored for a year in a man-made freshwater lake, an open sewage canal, and a pond composed of rainwater ac...
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