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Water Evaluation for Microbial Pathogens of Station Treated and Groundwater Wells via Chromogenic Culture Media in Riyadh, Saudi Arabia

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* To whom all correspondence should be addressed. Tel.: +966-11-481-3689; Fax: +966-11-481-4750

Water Evaluation for Microbial Pathogens of Station Treated

and Groundwater Wells via Chromogenic Culture Media in

Riyadh, Saudi Arabia

Majed M Albokari

King Abdulaziz City for Science and Technology (KACST), P. O. Box 6086, Riyadh - 114 42, Saudi Arabia.

(Received: 29 September 2013; accepted: 02 December 2013)

Evaluation of water quality for the presence of water borne pathogens has been considered a critical issue with a great concern especially in developing countries, this is a comparative study aims to assess the quality of water treated by the National Water Company and compare it with groundwater wells using chromogenicculture media as a specific, rapid, sensitive and reliable technique for the detection of the presence of microbial pathogens.A total number of 12 samples has been collected randomly and tested, 9 from water stations while the other 3 samples from groundwater wells locatedin city of Riyadh, Saudi Arabia. Some physicochemical parameters have been analyzed including pH with range of 7.1 and 7.3 to 8.1 in treated water and groundwater respectively, TDS for treated water ranged from 243 to 781 ppm which lies within the acceptable range while for the groundwater it showed a remarkable elevation of 636 to 2185 ppm. Results of the chromogenic media revealed the safety of treated water by the National Water Company for the publicuse while for the groundwater, attention is immediately required for such water resources due to the presence of microbial pathogens includingBacillus cereus, Escherichia coli, Staphylococcus aureus and some yeast and moulds.

Key words: Water evaluation, Chromogenic culture media, Microbial pathogens, Physicochemical parameters.

Public health concerns about the availability of sufficient good water quality free of bacterial pathogens safe and clean for drinking

especially in developing countries1.Urban runoffs

is considered one of the major sources of bacteria

affecting groundwaters quality for drinking2.Water

quality is determined mainly through the presence

of members of coliform group such as Escherichia

coli which is extensively used to monitor drinking

water quality and as an indicator for the presence of pathogenic bacteria, whereas World Health Organization declared that drinking water should

be always free of E. coli3-5. Chromogenic culture

media is considered one of the most popular techniques applied for the detection of microbial pathogens nowadays for its sensitivity, accuracy and remarkably low coast, these media are very specific containing different components within its composition which acts like a substrate targeting certain enzymes produced by variety of pathogenic microorganisms to exhibit a distinguished color. In addition, Chromogenic culture media is a rapid technique of detection as it neglect the additional steps of sub-culturing and further biochemical tests thus shortening the time needed for the

pathogenic agent identification6. This study aims

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withdrawn from different water stations for the presence of microbial pathogens and compare it with groundwater wells which is used here as a control.

Materials and Methods

samples collection and sites

Samples were collected from 9 water stations belonging to the National Water Company namely (Al Nassim, Thumamah, Dhahrat laban,

An Nadhim, Exit 7, Irqah, king Abdullah Rod., 2nd

Industrial Area and Al Uraija)and other 3 samples from groundwater wells which are so named after the location from which it was collected(Al karj, Hittin and Umm Al-Hamam), were the coordinates of the locations of both water stations and the groundwater wells sites are presented in Table1. Samples were collected in sterile borosilicate glass bottles with screw cap; pH and TDS were measured using (Ultrameter II™ MYRON l. COMPANY) immediately after taking the samples.

Bacteriological quality testing

Water samples of both station and groundwater wells have been tested for the presence of coliform group, selective pathogenic

bacteria (Bacillus cereus, Listeria spp., vibrio

spp., Staphylococcus spp.and pseudomonas spp.),

yeast and moulds via chromogenic culture media (SAUDI PREPARED MEDIA lAbORATORY CO. lTD), the results of the tests were taken for the average of CFU/100 ml.from three replicate plates used for each chromogenic culture medium in this study, plates were incubated in bINDER bD53 MICRO INCUbATOR(bINDER GmbH inTuttlingen, Germany).

Coliforms and E. coli

A group of chromogenic culture media have been used including:

ChroMagar eCC

Which allows for the rapid and reliable

detection and differentiation of E.coli and other

coliforms, were E.coliappearsas blue colonies

while other coliforms group show as mauve to light purple. According to the manufacturer results appears on the plate after the incubation at 37°Cfor 24 hours.

MacConKeY aGar

A medium used to differentiate between lactose and non- lactose fermenting bacteria, were

E.coli appears as pink to red colonies.According to the manufacturer results appears on the plate after the incubation at 37°Cfor 24 hours.

ChroM agar E. coli

A medium which directly shows the E.coli

colonies in a blue colour thus making the detection and enumeration of this important hygiene indicator as simple as possible. According to the manufacturer results appears on the plate after the incubation at 37°Cfor 24 hours.

leVine eMB agar

A medium based on the detection of

lactose fermenting bacteria, were E.coli appears as

a black colony with metallic sheen. According to the manufacturer results appears on the plate after the incubation at 37°Cfor 24 hours.

Bacillus cereus selective agar

This medium is supplied with Mannitol Egg-Yolk Polymyxin (MYP) which meet the

nutritional needs of B. cereus to grow.According

to the manufacturer B. cereus appears as a bright

pink colonies on the plate after the incubation at

37æ%Cfor 24 hours.

ChroM agar Listeria

A media which differentiate Listeria spp.

according to their phospholipase activity.According

to the manufacturer Listeria spp. appears as blue

colony with white halo on the plate after the incubation at 37°Cfor 48 hours.

ChroM agar Vibrio

This medium helps to easily differentiate Vibrio species such as V. parahaemolyticus, V. vulnificus, V.cholerae and V. alginolyticus, were

according to the manufacturer V. parahaemolyticus

appears as mauve colonies, V. vulnificus and

V.cholerae as blue colonies while V. alginolyticus show as colourless on the plate after the incubation at 37°Cfor 18-24 hours.

ChroM agar Staph

This medium is designed to differentiate

between Staphylococcus spp., were according to the

manufacturer S. aureusappears as purple colonies

while other Staphylococcus spp. ranges from blue

to colourless on the plate after the incubation at 37 °C for 24 hours.

ChroMagarPseudomonas

This medium is used for simultaneous

detection and enumeration of Pseudomonas spp.,

were according to the manufacturer Pseudomonas

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plate after the incubation at 30æ%Cfor 24 hours.

r2a aGar

This medium is made for the detection and

enumeration of bacteria such as E.coli, S.aureusand

B.subtilisfound in treated potable water, prolonged incubation time at lower temperature is needed for the recovery of the stressed and chlorine damaged bacteria from treated waters resulting in higher and more realistic bacterial counts. According to the manufacturer results appears on the plate after

the incubation at 30æ%Cfor 3-5 days.

Yeast and Mould aGar

This medium is recommended for the detection and enumeration of yeast and moulds using a combination of anaerobic/aerobic incubation procedure, as cultures are incubated at

25 æ%C for 3 days under anaerobic conditions and

then for a further 2 days aerobically.

results and disCussion

In this study, water quality of 9 stations and 3 groundwater wells have been evaluated in Riyadh, Saudi Arabia.Sites names along with their coordinates, some physicochemical parameters and bacteriological tests are presented in Table 1., results of water station samples have proved the efficiency of the water treatment processes applied by the National Water Company, as for the physicochemical parameters measured including pH with the range of 7.1 to 8.1 and TDS of 243 to 781 ppm. World Health Organizationguidelines for drinking water quality, pH has to be within the range of 6.5 to 8.5 while for the TDS it is classified into 5 main categories according to the TDS levels: Excellent, less than 300 ppm; good, between 300-600 ppm; fair, between 600-900 ppm; poor, between 900-1200 ppm and unacceptable with value

greater than 1200 ppm7. bacteriological tests also

revealed the absence of coliform group including E.coli as well as other pathogenic bacteria which proves the assumption made by the World Health Organization that the existence of coliform group

especially E. coliin waterindicates the presence of

pathogenic bacteria and vice versa5.

Groundwater samples showed acceptable pH rangedfrom 7.3 to 8.1 and a remarkable elevation in TDS of 636 ppm in Hittingroundwater which is considered of fair quality while Alkarj and Umm Al-Hamam groundwater have exceeded the

limits of the World Health Organization guidelines with 1834and 2185 ppm, respectively, which

isof unacceptable quality7, knowing that these

wells are being used in domestic, livestock and agricultural purposes. bacteriological testsrevealed that groundwater are being contaminated with coliforms as well as some pathogenic agents;

coliform and E.coli have been detected using

variety of chromogenic culture media thus to confirm their existence, CHROMagar ECC medium showed the presence of mauve and blue

coloniesforboth coliforms and E.colirespectively

in the three groundwater samples with33, 25 and 67 CFU in 100 ml. for Al karj, Hittin and Umm Al-Hamam respectively, as this medium is

designated for the detection of b-D-galactosidase

in coliforms and b-D-glucoronidase of E. coli

through their interaction with 5-brmo 6-chlro 3-indol-D-galactosidase and 5-bromo 4-chloro 3-indol -D-gluconoride found within the medium

composition8. MacCONkEY AGAR medium

confirmed the existence of E. coli as pink to red

colonies in the three samples with 18, 13 and 32 CFU/100 ml. for Al karj, Hittin and Umm Al-Hamam respectively, whereas this medium is designated mainly to differentiate between lactose

and non- lactose fermenters9. Further confirmation

CHROMagar E. coli medium resulted in the

presence of 21, 11 and 27CFU/100 ml. for Al karj, Hittin and Umm Al-Hamam respectively. The detection was based on the activity of

â-D-glucoronidase exhibited by E. coli as mentioned

above8. Moreover, the presence of E. coli was

doubled crossed due to its importance as a marker for the presence of other pathogenic agents and this was carried out bylEVINE EMb AGAR

medium.It showedE.colias black colony with a

metallic sheen easily to be recognized with 15, 9 and 23 CFU/100 ml. for Al karj, Hittin and Umm Al-Hamam respectively. Practically,by confirming

the presence of both coliform group and E.coli,

it was clear that these samples are contaminated

with pathogenic agents3, 4, 5. Therefore, a group

of pathogenic bacteria has been tested for their

existence including Bacillus cereus,whichis a

group spore-forming bacteria commonly found in soil,their spores are associated with water and food poisoning as their growth results in the production of enterotoxins causing severe nausea, vomiting

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t

able 1.

Sites, Ph

ysicochemical parameters and

b

acteriological tests for National

W

ater Compan

y and under

ground w

ater wells in Riyadh, Saudi

Arabia

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karj, Hittin and Umm Al-Hamam samples with

11, 2 and 14 CFU/100 ml. respectively, via B.

cereus selective agar medium. Staphylococcus

spp. Specifically S. aureus with its purple colonies

has been detected in the three groundwater water samples with 14, 12 and 18 in Al karj, Hittin and

Umm Al-Hamam respectively, whereas S. aureus

is considered one of the world most prevalent water and food infection as the source of contamination

is mainly by mucous, skin and person hands6.

R2A medium has been used as a final test for the identification of pathogenic bacteria as it is already madefor evaluating the bacteriological quality of treated potable water for the presence of bacterial

species such as E. coli, S. aureus and B. subtilis.

This medium promotes the recovery of stressed and chlorine damaged bacteria through prolonging the incubation time at lower temperatures. Results of

this medium revealed the presence of both E. coli

and S. aureus with 234, 231 and 260 CFU/100

ml. for Al karj, Hittin and Umm Al-Hamam respectively. The enumeration of high counts is believed to be due to the long period of incubation. Other bacterial pathogens tested during this study

have not been detected including Listeria, Vibrio

and Pseudomonas species.

Yeast and Moulds have also been tested for their presence via Yeast and Mould AGAR medium, which uses a combination of anaerobic/ aerobic incubation procedure as a mean of detection. groundwater samples have proved to be contaminated with yeast and moulds with 22, 3 and 30 CFU/100 ml. for Al karj, Hittin and Umm Al-Hamam, respectively. All these results indicate the hazard of using the studied groundwater wells in domestic, livestock and agricultural purposes due to its contamination with different pathogenic agents which may have a harmful impact on its

users5.

ConClusion

National Water Company samples have proved to be with a good quality on both physicochemical and bacteriological levels.The results of this study reflect the great efforts exerted by this company, including its water stations, in providing people with sufficient amounts of safe and clean water for drinking andas well as daily use purposes. On the other hand, results

ofgroundwater wells have showed an unacceptable water quality, which may have a harmful impact on public health indirectly as these waters are being used in domestic, livestock and agricultural uses. This requires an immediate attention for further treatment of such water sources and also for increasing the awareness of people inhabiting those areas of how valuable these resources are and in how to withdraw the maximum benefit out of it.

aCKnowledGMents

The author would like to thank king Abdulaziz City for Science and Technology (kACST) for supporting this work under grant no. 33-374. Also, technical supportprovided by Mr. Ibrahim M. Mashhour and scientific consultancy by Mr. Mohamed S. Hodhod (consultant at Atomic Energy Research Institute) weregreatly appreciated and considerable.

open access

This article is distributed under the terms of the Creative Commons Attribution license which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited.

referenCes

1. levesque b, Pereg D, Watkinson E, Maguire JS, bissonnette l, et al., Assessment of microbiological quality of drinking water from household tanks in bermuda. Can. J. Microbiol 2008; 54: 495-500.

2. Mark Ibekwe A, Shelton Murinda E, Alexandria Graves k., Microbiological Evaluation of Water Quality from Urban Watersheds for Domestic Water Supply Improvement. Int. J. Environ. Res. Public Health 2011; 8: 4460-4476.

3. Annie Rompre´, Pierre Servais, Julia baudart, Marie-Rene´e de-Roubin, Patrick laurent., Detection and enumeration of coliforms in drinking water: current methods and emerging approaches. Journal of Microbiological Methods 2002; 49: 31–54.

4. lye D J., Health risks associated with the consumption of untreated water from household roof catchment system. J. Am. Water Res. Assoc 2002; 38: 1301-1306.

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Geneva. Switzerland, 2006.

6. Tavakoli H, bayat M, kousha A , Panahi P., The Application of Chromogenic Culture Media for Rapid Detection of Food and Water borne Pathogen. American-Eurasian J. Agric. & Environ. Sci. 2008; 4: 693-698.

7. World Health Organization (WHO)., Total dissolved solids in Drinking-water. background document for development of WHO Guidelines for Drinking-water Quality. 2nd ed. Vol. 2. Health criteria and other supporting information. World Health Organization. Geneva. Switzerland, 2003.

8. beauchamp CJ, Simao-beaunoir AM, beaulieu

C, Chalifour FP., Confirmation of E. coli among other thermotolerant coliform bacteria in paper mill effluents, wood chips screening rejects and paper sludges. Water Research 2006; 40 :2452-2462.

9. Gonzalez RD, Tamagnini lM, Olmos PD, de Sousa Gb., Evaluation of a chromogenic medium for total coliforms and Escherichia coli determination in ready-to-eat foods. Food Microbiology 2003; 20: 601-604.

Figure

table 1. Sites, Physicochemical parameters and bacteriological tests for National Water Company and underground water wells in Riyadh, Saudi Arabia

References

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