OCCURRENCE OF YERSINIA ENTEROCOLITICA IN DRINKING …

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Sahota et al. World Journal of Pharmaceutical Research

OCCURRENCE OF YERSINIA ENTEROCOLITICA IN DRINKING

WATER IN THE ABSENCE OF INDICATOR ORGANISM

Sahota P*, Sharma N, Kirandip and Pandove G

Department of Microbiology, Punjab Agricultural University, Ludhiana, Punjab,

India.

ABSTRACT

The drinking water quality with respect to bacteriological and

physicochemical examination was done for 238 drinking water

samples from different water utilities (municipal corporation supply

taps, submersible pumps, filter samples, water tanks, storage tanks,

water coolers, etc.) of urban areas of Ludhiana, Punjab, India. Standard

methods were used for the analysis of pH, TDS, while E. coli, Yersinia

spp. determination was carried out using BWTK and Multiple Tube

Technique. Yersinia spp. was detected in 164 (68.91%) water samples.

Statistical correlation was examined between the bacteriological and

physicochemical parameters and found to be independent from each other. Difference

between mean in each case, high standard deviation indicate that the distribution is widely off

normal and exhibit an asymmetric distribution. The occurrence of E. coli was also not found

to be associated with the presence or absence of Yersinia enterocolitica. So, E. coli was not

found to be a suitable indicator for Yersinia spp. The findings of the current study reports the

occurrence of Y. enterocolitica in drinking water and suggesting that there is need for further

surveillance studies to understand the global epidemiology of emerging pathogens.

Keywords: BWTK, correlation, indicator orgainsm, urban areas, drinking water, Yersinia

enterocolitica.

INTRODUCTION

Safe water supplies and environmental sanitation are vital for protecting the environment,

improving health and alleviating property. Deterioration in water quality below the

established standards is mainly due to human activities and untreated or inadequately treated

wastes discharge in the environment. The World Health Organisation (WHO) estimated that

Article Received on 01 April 2014, Revised on 22 April 2014, Accepted on 15 May 2014

*Correspondence for Author

Dr.(Mrs.) Param Pal Sahota,

Sr.Microbiologist, Deptt. of

Microbiology, PAU,Ludhiana-

141004, Punjab.

World Journal of Pharmaceutical ReseaRch SJIF Impact Factor 5.045

Volume 3, Issue 4, 529-542. Research Articl ISSN 2277 – 7105


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1.1 billion people globally drink unsafe water and 88% of diarrheal diseases in the world is

attributed to unsafe water, sanitation and hygiene 20. The contamination of water supplies

often with raw sewage is a likely route of spread of each disease. Drinking water released

into the distribution system becomes altered during its passage through pipes, open

reservoirs, standpipes and storage tanks. Transient negative pressure and pipeline leak events

provide a potential portal for the entry of ground water into treated drinking water; and

permit faecal indicators and microbial pathogens present in the water and soil exterior to

enter the distribution system 9.

Ideally the finding of such an indicator bacteria should denote the possible presence of all

relevant pathogens. The indicator organisms presently used for the monitoring of drinking

water in many countries are total coliforms, faecal coliforms and/or E. coli, although the

reliance on indicator organisms as the main source of information about the safety of drinking

water is under review in many jurisdictions as the spectrum of waterborne diseases is

expanding. The survival of disease causative agent under different environmental conditions

represents one of the factors which determine the spread of diseases between the consumers

of contaminated water or food. Owing to the long survival and/ or growth of some strains of

Yersinia spp. in water, E. coli (or thermotolerant coliforms) is not a suitable indicator for the

presence or absence of this organism in drinking water 21.

Yersinia enterocolitica, the causative agent of yersiniosis, has been associated with water-

borne diseases and was isolated from human as well as non-human sources including water7,

19. The disease is characterized by symptoms of gastroenteritis and/or vomiting with

abdominal pain. Yersinia infections has been etiologically incriminated in a broad range of

clinical entities, which include acute mesenteric lymphadenitis, terminal ileitis,

gastroenteritis, septicaemia, non-suppurative arthritis, erythema nodosum, and localized

abscesses in the liver and spleen 17, 23.

In developing countries, 2.2 million people die every year from diseases associated with a

lack of safe drinking water, inadequate sanitation and poor hygiene 22. Rapid urbanisation,

changing lifestyles and socio-economic factors have led to an increase in the levels of

pollution in the waters. The urban cities have inadequate drainage networks which get

clogged frequently, resulting in water logging and health hazards. Ludhiana and its suburbs

(Punjab, India) suffers a severe drinking water supply crisis. Apart from quantitative

shortages, the quality of drinking water is becoming a serious public health issue for the past


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few years. Budha Nala, an important drainage line of Ludhiana district, passes through the

Ludhiana city, parallel to Sutlej and carries large volume of untreated sewerage water and

industrial effluents of the city. Most urban areas are serviced by a municipal water

distribution system that originates from local reservoirs or canals. The Municipal Corporation

is responsible for providing pure and safe drinking water to city dwellers on a regular and

continuous basis, and for regularly disposing of sewerage. However, the city dwellers often

find that the water is contaminated because of leaks in the pipelines, imperfect water taps and

misuse. The only reliable way to link water quality and health problems in urban areas where

a large population is exposed would be to perform the surveillance which contributes to the

protection of public health by promoting improvement of the quality, quantity, accessibility,

coverage, affordability and continuity of drinking water supplies.

The objective of this study was to determine the frequency of contamination as tested for

with indicators of bacterial and physico-chemical water quality parameters, occurrence of

yersiniae in drinking water, evaluating their association with the coliform fecal indicator

bacteria, characterization and study of its virulence factors.

MATERIAL AND METHODS

Study area

Ludhiana is the largest city in Punjab both in terms of area and population. The Municipal

Corporation has divided the whole city into ‘declared areas’ and ‘undeclared areas’. The

municipal corporation supplies domestic water to the declared areas while undeclared areas

have installed their own private water supply systems.

Sampling and physicochemical analyses

A total of 238 drinking water samples were collected from 185 municipal corporation supply

taps, 18 submersible pumps, 11 filter samples and 24 from water tanks, storage tanks, water

coolers, etc. from different water utilities from January 2010 to July 2011. The water samples

used for analysis were collected in sterile 1 L glass bottles, treated with sodium thiosulfate to

inactivate any residual halogen compound present in the sample (Na2S2O3 concentration of

18 mg/L neutralizes upto 5 mg of free (residual) chlorine per litre). The samples containing

high concentration of zinc and copper were treated with EDTA at concentration of 372 mg/L

to reduce metal toxicity 18. The samples were transported in refrigerated containers at 4˚C,

and analyzed within 24 h. The pH and TDS were determined using a portable calibrated pH

meter (Hanna Instruments) and HiMedia’s TDS meter, respectively.


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Enumeration and identification techniques

Total coliform content were determined by using the bacteriological water testing kit

(BWTK) developed in the Department of Microbiology, PAU, Ludhiana, India 15 and by the

most probable number (MPN) method per litres as specified by the standard methods IS-

10500-1991 BIS New Delhi, India.

The concentration of Yersinia spp. was determined from BWTK, used as primary enrichment

broth for water sample. An inoculum from the broth was further streaked onto Yersinia

selective agar (YSA) supplemented with cefsulodin, triclosan and novobiocin, incubated

overnight at 25˚ and 37˚C. Distinct convex colonies with deep red centres and white to

translucent periphery, characteristic bull’s eye morphology that were 2 to 3 mm in diameter

were considered as presumptive Yersiniae colonies. Yersinia enterocolitica MTCC 3099,

biotype 1A was used as a referral positive control. Positive isolates were further identified on

the basis of gram staining, motility (at 25˚ and 37˚C) and morphology under microscope. The

culture was stored at 4˚C and maintained on nutrient agar slants and in nutrient broth. For

each experiment, cultures were taken from the frozen stocks and subcultured only once to

curb the changes in virulence of the enteropathogen due to repeated passage of the culture.

Characterization of Yersinia isolates

Suspected colonies were subjected to the fermentation of 21 sugars using carbohydrate (25

mg) discs of HiMedia, Mumbai. The sugars were adonitol, arabinose, cellobiose, dextrose,

dulcitol, fructose, galactose, inositol, inulin, lactose, maltose, mannitol, mannose, melibiose,

raffinose, rhamnose, salicin, sorbitol, sucrose, trehalose and xylose. The positive strains were

submitted to complementary biochemical tests for the determination of the species. The

strains were identified as Yersinia enterocolitica on the basis of urease, indole, hydrogen

sulphide, phenylalanine deaminase, gelatinase, catalase, lysine decarboxylase, ornithine

decarboxylase, arginine dihydrolase production, orthonitrophenyl-β-galactosidase (ONPG),

methyl red (MR), voges-proskauer’s (VP), triple sugar iron (TSI), citrate utilization, nitrate

reduction, oxidase tests.

Pathogenicity tests

The isolated Yersinia spp. were tested for haemolytic acitivity on agar base supplemented

with 5% sheep erythrocytes. Pathogenicity was tested further by performing phenotypic tests

like congo red dye uptake 13, binding of crystal violet 3 and autoagglutination test 8 to

differentiate between the pathogenic and the non pathogenic strains.


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Statistical analysis

Mean, standard deviation, variance and coefficient of variation of water samples were

calculated statistically of all the bacteriological and physicochemical parameters (MPN

index, pH, TDS, E. coli and Yersinia spp.). MPN index was compared with pH, TDS using

Pearson value of correlation at 1% level of significance. Measurements of bacteria (E. coli

and Yersinia spp.) were dichotomized as 0 for nondetection and 1 when measured as in

detection limits. For comparison between the dichotomous variables, the chi-squared

coefficient as per Yates correction factor was calculated at P <0.01 to evaluate the

relatedness.

RESULTS AND DISCUSSION

A randomized prospective drinking water survey was conducted over a period of 18 months

from the endemic area of gastroenteritis, urban and semi-urban areas of Ludhiana, Punjab,

India. A total of 238 drinking water samples were analyzed, 185 from Municipal Corporation

taps, 18 from submersible pumps, 11 filter samples and 24 from water coolers, water tanks,

storage tanks, etc., of which 66% (157) samples were positive for the occurrence of total

coliforms.

Out of 157 bacteriologically contaminated samples, 28.15% showed highest MPN index from

the adjoining areas of Budha Nala; Dharampura, New Madho Puri, Dhandari Kalan, New

Shakti Nagar, Basti Jodhewal. The large volume of domestic and industrial wastewater has

converted the Budha Nala to a virtually sewage drain and this resulted in the deterioration of

drinking water quality in the surrounding areas. The possible reason of high level of

microbial contamination in adjoining areas could be due to poor water storage conditions,

distribution lines, untreated water, sewage, poor hygiene, crowded living conditions with

inadequate sewage facilities. Other reasons for contamination may be the crossing over of

sewage pipes with fresh water supply lines, wrong alignment of water pipelines, illegal water

and sewerage connections, non chlorination of water reservoir due to lackadaisical attitude of

Municipal Corporation authorities. This suggests the reason for high prevalence of

waterborne diseases such as typhoid fever, diarrhea and dysentery.

A summary of the bacteriological and physicochemical data for the water samples is shown

in Table 1. The average pH ranged from 6.83 - 7.75 and the average TDS was between

234.64 - 447. The data showed the maximum variation for MPN index ranged from 9 to 2400

followed by level of E. coli, Yersinia spp., TDS and minimum variation was observed in pH.


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Difference between mean in each case, high standard deviation indicate that the distribution

is widely off normal and exhibit an asymmetric distribution. Measurements of bacteria (E.

coli and Yersinia spp.) were dichotomized as 0 for nondetection and 1 when measured as in

detection limits. Thus to conduct the surveillance of drinking water of a city, random

sampling is desirable as one particular area or locality does not represent the whole

population.

MPN value was statistically compared with pH and TDS using Pearson value of correlation at

1% level of significance (Table 2). pH value showed only upto 28% correlation with the

MPN index at P=0.01 which is not considered to be statistically significant, and TDS also did

not show any significant value. Thus, no correlation was found and it was concluded that the

bacteriological parameter (MPN index) is independent of physicochemical parameters (pH

and TDS). So, to interpret the quality of water, these parameters must be considered and

studied separately.

Identification of E. coli

For the isolation of E. coli, drinking water sample was inoculated in BWTK and MacConkey

tube and presumptive positive results were confirmed based on the ability of the organisms to

ferment by showing colour change in the kit and MPN tubes. Positive water samples were

then streaked on MacConkey lactose agar plates (37˚C, 48 h) and were further confirmed

with MUG medium after incubation for 24 h and observed yellow or yellowish brown

colonies under a UV lamp.

Isolation, identification and biochemical characterization of Yersinia enterocolitica from

drinking water.

For primary enrichment, test water sample (10 ml) was inoculated in BWTK and MacConkey

tubes and incubated at 37°C for 12 h. Water samples positive for Yersinia spp. formed

superficial film on the liquid media in the kit and tube, were streaked on Yersinia Selective

Supplement Medium (Cefsulodin 7.50mg/500ml Medium, Triclosan 2.0mg/500ml Medium,

Novobiocin 1.25mg/500ml) and incubated at 28°C and 37°C for 24 h. The cultural

characterization of presumptive isolates of Yersinia spp. showed convex colonies with 2-3

mm diameter, characteristic bull’s eye morphology with deep red centres and white to

translucent periphery. Typical Y. enterocolitica colonies on selective media that responded

positively to the mannitol and urease tests were counted and scored as Y. enterocolitica. They

were all gram negative rods, motile at 25˚C and non motile at 37˚C when observed under


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microscope. The morphology of the isolates were also observed on HiChrome UTI agar and

nutrient agar which showed circular, smooth, convex colonies with glistening surface and

were easily emulsifiable. In nutrient broth, some strains of Yersinia enterocolitica produced

uniform turbidity with some green colouration in the medium. On MacConkey’s lactose agar,

all the isolated and test strains produced colourless, raised convex colonies having entire

margin. The colonies were small when incubated at 25˚C and were large when incubated at

37˚C (Table 3).

Yersinia spp. were present in 68.91% (164) drinking water samples tested bacteriologically

contaminated, of which Y. enterocolitica was the most frequently isolated. The isolation rate

of Yersiniae was nearly same in rainy (Jul-Sep) months and winter (Dec-Feb) months (Fig

1). The possible reasons may be due to its ability to survive for extended periods and even

multiply when water temperatures are low. The presence of Yersinia tends to be poorly

correlated with the levels of coliforms and HPC bacteria 5, 6. This might to a large extent be

due to the different temperature preference of these bacteria; whereas fecal coliform counts

are lower in winter, the occurrence of Y. enterocolitica is more frequent in colder months 10.

The preference for colder months is also reflected by the fact that the majority of yersiniosis

cases are reported from cooler regions in Europe and North America.

For identification to the species level, presumptive colonies were taken out of the pure culture

obtained and were then subjected to the fermentation of 21 sugars, of which, isolates were

able to ferment the following sugars: cellobiose, dextrose, fructose, mannitol, mannose,

melibiose, raffinose, and sucrose while negative for adonitol, arabinose, dulcitol, inulin,

inositol, lactose, maltose, salicin and xylose (Table 4). All the isolates were then

biochemically characterized and were positive for urease, methyl red, nitrate reduction,

orthonitrophenyl-β-galactosidase (ONPG), ornithine decarboxylase and catalase while 83%

of the strains showed indole-positive test. Citrate, triple sugar iron (TSI) utilization,

hydrolysis of esculin, voges-proskauer’s (VP), phenylalanine deaminase, oxidase, lysine

decarboxylase, arginine dihydrolase, gelatinase and H2S were not produced (Table 5). Strains

of Y. enterocolitica can be differentiated from Y. pseudotuberculosis with a positive result for

urease, fermentation of sucrose and negative reactions for rhamnose and melibiose

fermentation 1.


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Correlation between the occurrence of E. coli and Yersinia spp.

The relationship between the occurrence of Yersinia spp. and E. coli was studied on the basis

of their presence or absence with respect to each other by chi-square test using Yates

correction factor at one degree of freedom. All the drinking water samples divided into 4

sections- samples in which both the organisms were absent and vice-versa, and the samples in

which one organism is absent and other is present as shown in Table 6. For statistical

comparison, chi-square test between presence or absence of the organisms (Yersinia spp. and

E. coli) was used, which is considered a valid hypothesis that there is no association between

the occurrence of 2 populations (or organisms). Calculated value at one degree of freedom

was 0.039 which is less than the one-tailed P value (0.4219) at P<0.01. This result showed

that the association between the occurrence of two organisms (Yersinia spp. and E. coli) is

considered not to be statistically significant, i.e., E. coli is not a suitable indicator to show the

presence of Yersinia spp.

Different authors have also found that coliform count did not correlate with the occurrence of

Yersinia spp. Schiemann 16 in his study on surface and well water also found that many

samples that showed positive results for Y. enterocolitica were negative for coliforms.

Determination of pathogenicity of Y. enterocolitica

Our study revealed that all the strains of Y. enterocolitica showed haemolytic activity, with a

halo diameter. Production of hemolysin was performed in nutrient agar plates with 5% of

sheep blood incubated at 35+0.5˚C for 24 and 48 h. The haemolytic activity is strongly

associated with enterotoxin production 12.

All the isolates of Yersinia enterocolitica were positive for congo red dye uptake and crystal

violet binding (Table 7). The ability to take up dye is associated with the presence of

virulence plasmid. The virulent plasmid associated determinants have been used to

distinguish between virulent and avirulent strains, including colony morphology, crystal

violet binding, congo red uptake, autoagglutination, hydrophobicity, mannose resistant

haemagglutination and serum resistance 2,4,14. The congo red pigmentation assay provides a

simple and efficient means of screening for virulence.

The autoagglutination of Y. enterocolitica is dependent on the virulence plasmid. All the

isolates were tested by inoculating and incubating two tubes of tissue culture medium, one

was incubated at 37˚C and the second at 28˚C. All the strains showed autoagglutination at


537


37˚C while the tube remained turbid at 28˚C. Mazigh et al 11 also reported the positive

autoagglutination test for small colonies and negative for large colonies of Y. enterocolitica.

Table 1 Average, maximum and minimum of bacteriological and physicochemical

properties of drinking water samples.

Parameters Mean

(Std. error) Std.

Deviation Min value Max value

Coefficient of

variation MPN index (MPN units) 505.19 (+ 41.07) 633.54 9 2400 125.41

pH (pH units) 7.29 (+ 0.03) 0.46 5.9 8.5 6.31

TDS (ppm) 340.82 (+ 6.89) 106.18 7 880 31.15 Yersinia spp. (cfu/ ml) 0.67 (+ 0.030) 0.463 0 1 69.10

E.coli (cfu/ ml) 0.43 (+ 0.032) 0.494 0 1 114.88

Table 2: Pearson value of correlation (r), along with levels of statistical significance

between various parameters

Parameters MPN P value

pH 0.280159 <0.0001*

TDS -0.09686 0.1362

* Test of significance at 1% level (P=0.01)

Table 3 Morphological and cultural characteristics of Y. enterocolitica isolates from

drinking water samples.

Morphological and cultural tests

Observations

Gram’s reaction Gram negative

Motility At 25˚C At 37˚C

Motile Non-motile

Shape Rods

CIN (Yersinia Selective Medium)

Bull’s eye morphology with deep red centers and translucent periphery


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HiChrome UTI agar circular, smooth, convex colonies, with glistening surface

Trypticase soya agar circular, smooth, convex colonies and were easily emulsifiable

Nutrient agar circular, smooth, convex colonies, with glistening surface, 1-2 mm in diameter, entire or slightly crenated edges and were easily emulsifiable

Mac Conkey’s lactose agar At 25˚C At 37˚C

Small colourless, raised convex colonies having entire margin Large colourless, raised convex colonies having entire margin

Nutrient broth Yersinia enterocolitica produced uniform turbidity with some green colouration by some strains

Bacteriological Water Testing Kit (BWTK)

Forms superficial layer on the kit

Table 4 Comparison of fermentation of various sugars by isolates of Y. Enterocolitica.

Sugars utilization No. of isolates Y. enterocolitica (%)

1. Adonitol - (100%) 2. Arabinose + (17%), - (83%) 3. Cellobiose + (67%), - (33%) 4. Dextrose + (50%), - (50%) 5. Dulcitol - (100%) 6. Fructose +(50%), - (50%) 7. Galactose +(17%),- (83%) 8. Inulin - (100%) 9. Inositol - (100%) 10. Lactose - (100%) 11. Maltose +(17%),- (83%) 12. Mannitol + (100%) 13. Mannose +(67%),- (33%) 14. Melibiose +(50%),- (50%) 15. Raffinose +(50%),- (50%) 16. Rhamnose +(17%),- (83%) 17. Salicin - (100%) 18. Sorbitol +(17%),- (83%) 19. Sucrose +(67%),- (33%) 20. Trehalose +(17%),- (83%) 21. Xylose - (100%)


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Table 5 Biochemical characteristics of Y. Enterocolitica.

Biochemical Characteristics No. of isolates Y. enterocolitica (%)

1. Urease test + (100%) 2. Indole test + (83%) 3. Citrate utilization - (100%) 4. Triple Sugar Iron (TSI) test - (100%) 5. Esculin hydrolysis at (28°C) At 37°C

- (100%) - (100%)

6. Methyl Red (MR) test + (100%) 7. Voges-Proskauer’s (VP) test - (100%) 8. Phenylalanine deaminase agar

test - (100%)

9. Nitrate reduction test + (100%) 10. Oxidase test - (100%) 11. Orthonitrophenyl-β-

galactosidase (ONPG) + (100%)

12. Lysine decarboxylase - (100%) 13. Ornithine decarboxylase + (100%) 14. Arginine dihydrolase - (100%) 15. Gelatinase production - (100%) 16. H2S production - (100%) 17. Catalase test + (100%)

Table 6 Relationship of occurrence of Yersinia spp. with E. coli (chi-square test using

Yates correction).

E. coli

Yersinia spp.

Absent Present

Absent 41 33

Present 87 77 χ2 value (Calculated value) = 0.039 (at 1 degree of freedom)

Table value (P value) = 0.4219 (P<0.01)

So, there is no association between the occurrence of Yersinia spp. and E. coli.


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Table 7 Pathogenicity tests

Pathogenicity tests Observations

Congo red dye uptake Red coloured colonies

Crystal violet binding Violet coloured colonies

Autoagglutination test An irregularly edged layer of agglutinated

bacteria which formed flocculate covering at the

bottom of the tube with clear supernatant fluid

Figure 1: Percentage of occurrence of Yersinia enterocolitica in various months

CONCLUSION

Few developed countries have included emerging pathogens in their standards for drinking

water and hence require occurrence of emerging pathogens in water and foods to be reported

to authorities for the safety of health but these pathogens are not included in water and food

standards of any developing country. The water samples of study area were highly polluted

bacteriologically. These results also demonstrated the presence of widespread, biochemically

and pathogenically characterized, waterborne Y.enterocolitica in urban areas of Punjab. The

detection of Y.enterocolitica in drinking water sources studied represent a public health

concern, and must be taken into account for assessing the quality of drinking water. We

would like to recommend the following important points: proper sanitary survey, design and

implementation of water and/ or sanitation projects; regular disinfection, maintenances and

supervisions of water sources and regular bacteriological assessment of all water sources for

drinking should be planned and conducted. Systems using surface water sources are required


541


to disinfect to ensure that all bacterial contamination (such as E. coli and Yersinia spp.) is

inactivated.

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