Dairrhoeal Diseases

Director

Andamans

Survival ability of Shigellae in natural waters
The study was conducted at Calicut, a diarrhoea-prone rural area in south Andaman. Five natural untreated water sources used for drinking and other domestic purposes were selected for the study. Water samples were collected in a glass bottles from below the surface bimonthly. Water temperature, salinity, pH and oncentrations of calcium (Ca), magnesium (Mg), iron (Fe) and chlorine (Cl2) in mg/L were measured. Total viable counts (TVC) and most-probable number (MPN) of total coliforms and E. coli were estimated. Water samples were sterilised by filtration and were inoculated with 107 colony forming unit (cfu) /mL of a strain of S. dysenteriae type 1 (D117) and a strain of S. flexneri 2a (CC19). The viable counts were enumerated immediately after inoculation and at regular intervals till two consecutive enumerations showed no viable bacteria. Step-wise multiple linear regression was done with the duration of survival of Shigellae in the water samples as the dependant variable and the physical, chemical and bacteriological parameters as the independent variables. The pH of the samples was in the range of 6.3 to 8.2 (mean 7.25 ± 0.45) and the temperature in the range of 220-290 C (mean, 25.50 C ± 1.66). The concentrations of Cl2, Mg, Ca, and Fe (mg/L) were 0-0.2 (mean 0.05 ± 0.05), 0-200 (mean 50.4 ± 14.76), 10-83 (mean 39.43 ± 20.56) and 0-0.5 (mean 0.23 ± 0.29) respectively. The salinity ranged between 0.023 ppt and 0.12 ppt (mean 0.069% ± 0.025). 93.3% of the water samples were positive for E. coli and 100% positive for coliform with MPN of 0 – 180 and 7 – 180 / 100 ml respectively. The TVC ranged between 3.94 x 102 to 1.24 x 105 cfu/ ml. No Shigella spp. was isolated from any of the natural waters.

Fig 1 Growth curve of S. dysenteriae type 1 and S. flexneri in natural water samples	Both the Shigella strains showed an increase in their counts during first 24h followed by gradual decrease (Fig1). The drop in the count was faster in the case of S. dysenteriae than in the case of S. flexneri. S. dysenteriae type 1 survived for 3-6 (mean 4.3 ± 1.5) days and S. flexneri 2a for 14-16 (mean 14.4 ± 2.4) days.

	Dur (SD)	Dur (SF)	TVC	AT	WT	pH	Cl2	Mg	Ca	Fe	Sal	Max (SF)	Max (SD)	24 h (SD)	24 h (SF)
Dur (SD)¹	1.00
Dur (SF)²	0.66	1.00
TVC³	0.13	0.13	1.00
AT⁴	-0.16	-0.24	-0.04	1.00
WT⁵	0.18	0.21	0.13	-0.29	1.00
PH	0.28	0.16	0.45	0.21	0.07	1.00
Cl₂	-0.09	-0.21	-0.23	-0.19	0.27	-0.11	1.00
Mg	0.12	-0.14	0.19	0.08	0.16	0.61	0.02	1.00
Ca	0.22	0.06	0.24	0.39	-0.10	0.32	-0.25	0.31	1.00
Fe	0.07	-0.03	0.34	0.10	0.19	0.23	-0.12	0.52	0.67	1.00
Salinity	-0.27	-0.09	0.19	0.06	0.06	-0.16	0.06	-0.20	0.11	0.12	1.00
Max (SF)⁶	0.34	0.40	-0.04	-0.46	0.05	-0.09	0.12	0.01	-0.16	0.03	-0.30	1.00
Max (SD)⁷	0.48	0.59	0.06	-0.30	0.24	0.10	0.09	0.00	0.05	0.01	-0.17	0.75	1.00
24 h (SD)⁸	0.37	0.47	-0.08	-0.34	0.08	-0.02	0.10	-0.03	-0.15	-0.08	-0.25	0.85	0.90	1.00
24 h (SF)⁸	0.39	0.47	-0.01	-0.40	0.02	0.01	0.07	0.07	-0.07	0.04	-0.35	0.98	0.81	0.88	1.00
Table 1. Correlation matrix of various physico-chemical and bacteriological factors

Table 1 shows the correlation matrix for the 15 variables studied. Duration of survival of S. dysenteriae and S. flexneri had positive relation with a correlation coefficient of 0.66. The durations had positive relations with the concentration of the bacteria in the samples at 24 h and 48 h. In the case of S. flexheri, the duration of survival and maximum concentration of the bacteria also showed a positive relation. There were no other significant correlations.

	Unstandardized		Standardized Beta	t	Significance
	B	Std. Error	Standardized Beta	t	Significance
(Constant)	-5.4648	2.4246		-2.2539	0.0329
Log (Max SD)	1.4886	0.3422	0.6055	4.3503	0.0002
Salinity	-24.3631	12.4999	-0.2724	-1.9491	0.0622
Ca	0.0227	0.0151	0.2107	1.5049	0.1444
Table 2. Multiple linear regression model of duration of survival of S. dysenteriae

The multiple linear regression with the duration of S. dysenteriae as the dependent variable was done in three steps. The final model is summarized in table 2. The coefficient of maximum concentration was stastically significant at 5% level. The coefficient of salinity was negative but the significance was only marginal (0.062) and the coefficient of Ca was not statistically significant.

		Unstandardized Coefficients		Standardized Coefficients	t	Sig.
		B	Std. Error	Beta
	Constant	-23.4228	14.6391		-1.6000	0.1232
	24 h (SF)	0.0000	0.0000	1.8384	2.5524	0.0178
	Max (SF)	0.0000	0.0000	1.3538	1.8673	0.0747
	Mg	-0.1371	0.0507	-0.4807	-2.7058	0.0126
	Water Temp	0.8951	0.3688	0.3534	2.4271	0.0235
	Cl2	-18.9729	12.1819	-0.2291	-1.5575	0.1330
	pH	2.5499	1.7085	0.2771	1.4925	0.1492
	Table 3. Multiple linear regression model of duration of survival of S. flexneri

Table 3 summarizes the final multiple linear regression model with duration of survival of S. flexneri as the dependant variable. The final model contained concentration of S. flexneri at 24 h, maximum concentration achieved, water temperature, Mg, Cl2 and pH. The coefficients for concentration at 24 h, Mg and water temperature were significant at 5% level while the others were not significant. In the case of both S. flexneri and S. dysenteriae, the strongest predictor of the duration of survival was the concentration of the bacteria. Other possible predictors were salinity in the case of S. dysenteriae, Mg in the case of S. flexneri. The range of salinity observed was 0.023 to 0.12 ppt and the duration of survival explained by the change in the salinity is negligible. The maximum concentration of S. dysenteriae (in log base 10 scale) achieved ranged from 4.379 to 7.137 and it had a coefficient of 0.6055. Thus the change in the maximum concentration could explain the difference in the duration of survival up to 1.6 days. The maximum concentration of S. flexneri (in log scale) ranged between 6.255 and 8.477 and with a coefficient of 1.8384, it could explain the difference in the survival up to 4 days. The observed range of Mg (40 – 90 mg/L), can change the duration of survival by 25 days if the other parameters are constant. The range in the duration of survival of S. dysenteriae was 0 – 6 days and none of the physico-chemical parameters studied was a sufficiently strong determinant to cause wide variation in the survival of the bacteria.