Bacteriological quality

Bacteriological Quality Hussen Ali, Ketema Bacha and Tsige Ketema

ORIGINAL ARTICLE
Bacteriological Quality and antimicrobial susceptibility of
some isolates of Well Waters Used for Drinking in Jimma
Town, Southwest Ethiopia

Hussen Ali, B.Ed, Ketema Bacha, PhD, Tsige Ketema, MSc
ABSTRACT
Well waters could be polluted with fecal materials due to inadequate protection, depth of
wells, distance and position relative to latrine house, unhygienic practice at the source
and poor environmental sanitation. The objectives of this study were to assess the
current bacteriological safety of well waters being used for drinking and other domestic
uses; and also to evaluate the antibiotic susceptibility patterns of bacterial species
isolated from some of the wells in Jimma town, southwestern Ethiopia. Accordingly,
bacteriological quality and safety, as well as physico-chemical properties of water
samples collected from 30 purposively selected wells were assessed following the
standard microbiological methods. Pure isolates were characterized to species level
using API 20 E kit and evaluated for resistance patterns towards ten antimicrobial
agents being used for human medication. The finding of the study showed that the mean
pH (6.64- 6.8) of all the studied well water samples was within acceptable range (pH=
6.5-8.5), while the mean temperature was not. Likewise, the mean microbial counts
(cfu/ml) of the samples were beyond the value recommended by World Health
Organization. Accordingly, the mean count (cfu/ml) of aerobic mesophilic bacteria
(AMB) was 908 (360 – 1490), while those of Entrobacteriaceae and coliforms were 332
and 234, respectively. A total of 15 representative bacterial species including Aeromonas
hydrophilia (3), Enterobacter cloacae(1), Serratia liqueofaciens(1), Enterobacter
sakazaki(1), Serratia marcesense(1), Klebsella orithinolytica(4), and Escherichia coli(1)
were identified to species level. All isolates were sensitive to Amikacin (AK), Norfloxacin
(Nx), Kanamycin (K), Bacitracin (B) and Tetracyclin (T). On the other hand, all isolates
were resistant to Methicillin (M) and Pencillin G (P). Stains of Klebsella ornithinolytica
showed the highest resistance against most of the antibiotics tested. In general, the well
water samples analyzed in this study were found in unacceptable condition in terms of
bacteriological quality. Moreover, the identification of Escherichia coli in some of the
wells indicates the safety problem of the water for human consumption. Therefore, it
calls for proper disinfection and monitoring of the water sources besides awareness
creation to the well owners.
Key words/phrases: Antimicrobial sensitivity, Bacteriological safety, Jimma, Well water

1Department of Biology, College of Natural Sciences, Jimma University, P. O. Box
378, Jimma, Ethiopia
*[email protected] Tel: +251917800789

Ethiop. J. Educ. & Sc. Vol. 6 No 2

INTRODUCTION
of children in Ethiopia are communicable Well water is one of the potential sources (http://www.unicef.org/ethiopia/wes. html). Bureau, 2008). In developing countries, the health risk associated with drinking water share of well water could be even higher. Thus, it is necessary to make a regular test the safety status of water being used for (Balk et al., 2003). However, improperly constructed or poorly maintained wells and indicators of trouble: bacteria, nitrate, pH, keratoconjunctivitis, respiratory infections, and hepatitis are associated with viruses, availability of pipe lines for tap water in the including groundwater, surface water, and costal water (Borchardt et al., 2003, water in a very intermittent condition. The Borchardt et al., 2004; Xagoraraki et al., poor access to safe water could be due to inconsistency in water supply and/or total Salmonella spp., Shigella spp., pathogenic lack of access to treated water for one or Escherichia coli, Vibrio cholerae, Yersinia many reasons. Therefore, some population enterocolitica, Campylobacter jejuni, and Campylobacter coli can contaminate a drinking water via fecal material, domestic and industrial sewage and agricultural and various purposes such as cleaning utensils, pasture runoff (Geldreich, 1991: Wiggins, washing clothes, bathing and drinking. This 1996). If water used for drinking or bathing contains sufficient numbers of pathogens magnitude of risks associated with frequent such as Pseudomonas aeruginosa and dependence on well water. To this effect, species of Flavobacterium, Acinetobacter, Klebsiella, Serratia, Aeromonas and certain bacteriological quality and safety of well antimicrobial sensitivity patterns of the membranes of the eye, ear, nose and throat et al. (2009) showed the possible isolation MATERIALS AND METHODS
Description of the study area
inadequate sanitation and use of polluted located 353 km southwest of Addis Ababa. water (Epstein et al. 1994). Similarly, about three-quarter of the health problems located at 07o39’ latitude, 36o50’longitude, Bacteriological Quality Hussen Ali, Ketema Bacha and Tsige Ketema

Preliminary survey
Purification and floral assessment
population and the current status of wells After colony counting, 10 to 15 colonies administration of pre-designed structured MacConkey agar, and Violet Red Bile agar (VRBA) plates for further identification. ended questions) and interview. Well water nutrient broth for repeated purification on purposely and a total of thirty well waters nutrient agar (Oxoid). The pure culture of were assessed in this study. The study was aerobic mesophilic bacteria, coliforms and Enterobacteriaceae were streaked on slants of nutrient agar and kept at 4oC for further Sample collection and processing
Characterization of isolates
using sterile flask of 250 ml capacity. From using API 20E kits. The preliminary tests included microscopic observation for cell Physico-chemical analysis
production of catalase enzyme, cytochrome Isolates were further characterized using Microbiological Analysis
From appropriate dilution (1:10), 0.1ml of Marcy I’Etoile, France). The strips were aliquots were spread plated in duplicate on pre-dried surfaces of plates of Violet Red referring to the reading table provided by the manufacturer. The biochemical profiles obtained for the isolates were interpreted the counts of coliforms, aerobic mesophilic Enterobacteriaceae, respectively. Colonies Antimicrobial susceptibility test
32oC for 24 hours. Purplish red colonies surrounded by reddish zone of precipitated susceptibility to different antibiotics on Mueller–Hinton agar (Oxoid) following the
Ethiop. J. Educ. & Sc. Vol. 6 No 2
antibiotic discs; Ampicillin (A, 10µg),
30µg), Methicilin (M, 5 µg), Norfloxacin Inoculation of test plates
Pre-sterilized Mueller–Hinton agar (Oxoid) Preliminary
information
was swabbed with the standardized culture investigated well waters
standard) using sterile cotton swab. The respect to the purpose of using well water, associated risks, and hygienic practices are dispensed onto surface of the inoculated presented in Table 1. Accordingly, all the (Oxoid, Antimicrobial Susceptibility Test System, UK). After 24hrs of incubation at clothes and utensils, and bathing. Some of 30 to 32 oC, diameter of inhibition zones was measured. The strains were classified alternative, though it was not accessible on as sensitive or resistant based on the cut-off regular basis and used only intermittently. Concerning the age and depth of wells, the majority of wells (70%, 21/30) had age of 0 Laboratory Standards). For the purpose of interpretation, those intermediate cases 18/30). Likewise, 70% (21/30) of the wells were considered sensitive (NCCLS, 2000). distance of 11-15 meter from latrine and DATA ANALYSIS
found on equal altitude to the latrine houses
Ethiop. J. Educ. & Sc. Vol. 6 No 2

Table 1. The status of some well waters found in Jimma town, February, 2010
S.
Position of the well relative to latrine · uphill from latrine
Physio-chemical property of well water
The mean pH and temperature of the well waters were 6.71±0.08 and 21.46±1.45,
respectively (Table 2). The variation in temperature and pH values of the well water
samples was not significant (CV<10%).
Table 2. pH and temperature of well waters, Jimma town, February, 2010
Parameters


Microbial count of well waters
The total aerobic mesophilic bacterial count of all the samples analyzed was 907.86 cfu/ml
(360 - 1490). Similarly, the mean count of total coliforms was 234.5 cfu/ml. Moreover,
the mean count of Enterobacteriacea was also high (332 cfu/ml) (Fig. 1).
Bacteriological Quality Hussen Ali, Ketema Bacha and Tsige Ketema
Fig. 1. Microbial load of some well water samples collected from Jimma town,
February, 2010 Where, AMB, aerobic mesophiliic bacteria
A total of 15 representative gram-negative bacterial isolates were further characterized to species level using API 20E kit. The bacteria isolates were Aeromonas hydrophilia, (3), Enterobacter cloacae (1), Serratia liqueofaciens (1), Enterobacter sakazaki (2), Serratia marcesense (1), Klebsella orithinolytica (4), and Escherichia coli (3) (Table 3).
Ethiop. J. Educ. & Sc. Vol. 6 No 2
Table 3. API 20 E profiles of some representative bacterial species isolated from well
water used for drinking, Jimma town, February, 2010
Biochemical Tests for utilization of different substrates
Isolates
Isolates identity
ONPG= ß-galactosidase production, ADH= arginine dihydrolase, LDC= lysine decarboxylase, ODC= ornithine decarboxylase, URE= urease, TDA= tryptophane deaminase, CIT= Citrate, H2S= production of hydrogen sulphide, and IND= indole, GLU= glucose, MAN= mannitol, INO= inositol, VP= Vogus prausker (acetoin production), GEL= Gelatin liquefaction, SOR= sorbitol, RHA= rhaminose, SAC=saccharose, MEL= melibiose, AMY= amylase, and ARA=arabinose, + = positive, - = negative, V = variable, Ju = Jimma University. Bacteriological Quality Hussen Ali, Ketema Bacha and Tsige Ketema
Results of the antibiotic susceptibility pattern of isolates showed that all of the isolates
were sensitive to Amicacin (Ak), Norfloxacin (Nx) and Tetracyclin (T) (Table 4). In
addition, 96.4% (14/15 isolates) were sensitive for Kanamycin (Ka) and Bacitracin (B).
On the other hand, all isolates were resistant to Methicillin (M), Pencillin G (P) and
cephaloxin (Cn) (except Serratia marcesense). Among the isolates, Klebsella
ornithinolytica and Aeromonas hydrophilia showed the highest resistance to most
antibiotics used (seven of the ten antibiotics). It has about 25% of resistance to Ampicillin,
Bacitracin, Erytromicin and Kanamycin. Whereas, Serratia marcesense was sensitive to
almost all antibiotics tested except for Methicillin and Pencillin G. Potentially pathogenic
Escherichia coli identified in this study was sensitive to most of the antibiotics including
Amikacin, Bacitracin, Cephaloxin, Erytromycine, Kanamycin, Norfloxacin, and
Tetracyclin.
Table 4. Antibiotic susceptibility pattern of isolates identified from well waters used for
drinking in Jimma town, February, 2010
Antibiotics and number of isolates resistant or sensitive to the antibiotics
Isolates

Where: A, ampicillin; Ak, amikacin ; B,bacitracin; Cn, Cephaloxin; E, erytromycine;
Ka, kanamycin; M, methicilin; Nx, norfloxacin; P, pencillin G; T, tetracycline;
R, Resistant; S = Sensitive

Ethiop. J. Educ. & Sc. Vol. 6 No 2
A total of seven multiple drug resistance (MDR) patterns were observed. The highest
MDR observed was A/B/Cn/E/Ka/M/P (resistance to ampicillin, bacitracin, cephaloxin,
Erythromycin, kanamycin, methicillin and penicillin G) (Table 5).
Table 5. MDR pattern of bacteria isolated from well water, Jimma town, February, 2010
No of drugs in
MDR patterns
Number of
Percentage (%)
resistant isolates
Where: MDR, Multiple drug resistance ; A, ampicillin; B,bacitracin; Cn, Cephaloxin;
E, erytromycine; Ka, kanamycin; M, methicilin; P, pencillin G

DISCUSSION
area. The distance of wells from latrine is a determining factor for contamination with The majority of private well water owners fecal coliforms. The presence of latrines have been using the water directly without within a radius of 50 m in the vicinity of pumps or wells was identified as being a expose them to infection with water borne risk factor for virus detection in Benin, West Africa (Verheyen et al., 2009). major public health problems, increasing mortality rates among highly susceptible compromised patients) besides its effect on importance of appropriate decision support related non-productive time (Verheyen et systems in these socioeconomic prospering Pollution Prevention Information (2000), The closeness of the well waters to latrine depth less than 20 ft, age greater than 50 (majority of them) on equal altitude to the years and dug types are at risk. In our case, latrine houses along with short depth were although all the wells were younger than the major risk factors observed in the study the afromentioned risk ranges, the fact that Bacteriological Quality Hussen Ali, Ketema Bacha and Tsige Ketema
all the wells were made by digging along
well owners observed during field survey with the practice of utilizing water without waters the possible source of contamination depth from the surface, quality for drinking The microbial counts (cfu/ml) of all the water varies from place to place. Generally, the deeper the well, the better the ground 907) and found beyond the specification set water will be for drinking. The amount of new water flowing into the area also affects guideline (WHO, 1993), the total bacterial counts of a given drinking water should not microbial contaminants, ground water may contain some natural impurities, even with study, the total aerobic mesophilic bacterial cfu/ml) was also above the guideline set for (Environmental Protection Agency, 2002). condition for the proliferation of aerobic Aeromonas hydrophila were encountered at mesophilic bacteria and, thus, contributed to the high microbial counts observed. pH and temperature are among the factors that liqueofaciens (1), Enterobacter sakazaki determine microbial growth (LeChavallier (2), and Serratia marcesense (1). Likewise, et al., 1996). According to the guide line Aeromonas hydrophila are reported to be set by the World Health Organization, the pH of any drinking water should be in the north-east Scotland (Gavriel et al., 1998), other hand, the temperature should also be metropolitan water supply (Burke et al., between 10 to 16oC. With reference to the 1984) where it was found contributing to above specifications, the pH of all well infections of skin and soft tissue (Gold and waters was in acceptable range. However, Salit, 1993). Aeromonas hydrophila are the recorded mean temperature was beyond frequently associated with fish infection being common pathogen of fish although it is one of the most important factors for (LeChavallier et al., 1996), the rise in recommendation to the current 21.46±1.45 Except potentially pathogenic Escherichia coli, all bacteria species identified in this aerobic mesophilic microbes by shortening their lag-phase (Fransolet et al., 1985). Moreover, the absence of microbiological 1993). In terms of the degree of severity, control system of the wells together with the diseases caused by these isolates are poor sanitation, and unhygienic practice of relatively benign when compared to other
Ethiop. J. Educ. & Sc. Vol. 6 No 2
pathogenic bacteria such as Shigella,
and failure to use disinfection are among Salmonella and Vibrio cholera. However, prevalence of contaminants. In addition to microbial contaminants, ground water may waters investigated (this study) are used for contain some natural impurities, even with various infections of skin and the mucous E. coli is a type of fecal coliform bacteria Relatively low resistance to Ampicillin by E. coli was observed in this study. Previous animals and humans. Thus, the isolation of reports by Ahmed et al. (2000) revealed E. coli in well water is a strong indication that Escherichia coli, isolated from urinary of sewage or animal waste contamination. tract of patients were highly resistant to Infection due to E. coli often causes severe Ampicillin. Although all the isolates were sometimes the infection causes non-bloody diarrhea (Environmental Protection Agency resistance to antibiotics (seven out of the elderly, infection with E. coli can also antibiotics important in human medication control infectious disease, especially in cells are destroyed and the kidneys fail developing countries. Resistance to more /library/prevention/ blecoli1.htm). About 2- 7% of all infections lead to this type of isolates possible natural resistance to some hemolytic uremic syndrome is the principal cause of acute kidney failure in children, syndrome are caused by E. coli O157:H7. CONCLUSION
On bases of criteria set by WHO, the well routes of transmission of E. coli is mostly direct animal contact or the consumption of range both in physico – chemical property identification of potentially pathogenic uncooked vegetables (Jay et al., 2005). In bacteria such as E. coli was good indicators general, the possible factors that expose of safety problems. The risk of well water well water sources for contamination with contamination is unavoidable. Therefore, bacterial pathogens could be construction defect, lack of fence, inadequate depth and periodic evaluation of the safety of their distance of wells from the latrine, lack of Furthermore, lack of regular supervision Bacteriological Quality Hussen Ali, Ketema Bacha and Tsige Ketema
suggested
Borchardt, M. A., Haas, N. L. and Hunt, R. Awareness development training should be protecting well water from any source of contamination and on the depth, distance and position of well with respect to latrine. Borchardt, M. A., Bertz, P. D. Spencer, S. In addition, responsible bodies should pay intermittence of potable drinking water in provision of alternative safe water sources. Although not analyzed in this study, some ground water naturally contains dissolved elements such as arsenic, boron, selenium, breakdown of radioactive uranium in soil. present, these natural contaminants could further study on the current status of well waters of the study area with respect to the ACKNOWLEDGEMENTS
material and chemical support given by the Epstein P, Ford T, Puccia C. and Possas, Department of Biology, College of Natural REFERENCES
Fransolet, G., Villers, G., Masschelein, W.
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Gold, W.L, Salit, I.E. (1993). Aeromonas hydrophila infections of skin and review. Clin Infect Dis. 16:69–74. and total coliforms. Appl Environ Hawaii’s Pollution Prevention Information http://www.unicef.org/ethiopia/wes.html. Aeromonas species in foods. J. LeChavallier, M. W., Welch, N.J., Smith, Antimicrobial Disk Susceptibility Tests,222 Approved Standard M2- Bacteriological Quality Hussen Ali, Ketema Bacha and Tsige Ketema
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