Microbial quality, diversity and antibiotic susceptibility

African Journal of Microbiology Research Vol. 5(3), pp. 198-210, 4 February, 2011 Available online http://www.academicjournals.org/ajmr DOI: 10.5897/AJMR10.338 ISSN 1996-0808 ©2011 Academic Journals Full Length Research Paper

Microbial quality, diversity and antibiotic susceptibility profiles of bacterial isolates from borehole water used

by schools in Greater Giyani Municipality, Mopani District, South Africa

A. Samie1*, T. E. Makonto2, J. Odiyo2, P. O. Ouaboi-Egbenni1, P. Mojapelo3 and

P. O. Bessong1

1Department of Microbiology, School of Mathematical and Biological Sciences University of Venda, Private Bag X5050, Thohoyandou 0950, Limpopo, South Africa.

2Department of Hydrology, School of Environmental Sciences, University of Venda, South Africa. 3Department of Chemistry, School of Mathematical and Biological Sciences, University of Venda, South Africa.

Accepted 12 August, 2010

In the present study, the microbial quality of several boreholes, used by rural schools in Greater Giyani Municipality, was assessed over a six months period to determine their safety for human consumption and to highlight the potential occurrence of water-borne diseases. The microbiological quality of the water sources was performed using the membrane filtration technique, while standard culture methods were used for bacteria isolation. The MicroScan was used for identification and antibiotic susceptibility profiles of isolated potentially pathogenic bacteria. The results obtained indicated that the water quality of the boreholes was poor over the study period (June to October, 2009). Indicator organisms were higher than the acceptable maximum limits prescribed by the South African Department of Water Affairs and Forestry (DWAF) and the World Health Organisation. Numerous organisms that are potential enteric pathogens such as Vibrio spp, Shigella species and Klebsiella spp, were isolated throughout the study period. There was high resistance to many antibiotics particularly ampicillin (92%), cefazolin (95.9%), cefuroxime (91.7%) and cefoxitin (92.6%). The most active antibiotics were tobramycin with resistance level of 8.7% and levofloxacin (23.0%). The study indicates that water from the studied boreholes was not suitable for human consumption and may pose a serious threat to the health of consumers and therefore calls for urgent intervention. High level of antibiotic resistance is of concern in the management of infections caused by these organisms. Further studies are needed to identify the sources of contamination in order to curb the negative effect of contaminated water particularly in children. Key words: Antibiotic resistance, bacterial indicators, children, water quality, borehole, schools, South Africa.

INTRODUCTION South Africa is a water scarce country, and groundwater from either private or public boreholes is the main water source in many rural areas for cooking, drinking, and general cleaning (Shirinda, 2008). In some primary schools, boreholes are used to provide drinking water to school children and communities around the school when *Corresponding author. E-mail: [email protected].

there are no other water sources available. Previous studies in some rural areas of Limpopo Province in South Africa have reported poor quality of ground water consumed by the population (Potgieter et al., 2007). However, there are very little published data available on microbiological properties of water resources used in public schools in rural areas of Limpopo Province, where children might be at high risk of diarrheal diseases (Maake, 2007). Elsewhere, outbreaks of diarrhoea have been reported in rural areas of Limpopo Province due to

contaminated borehole water (Bessong et al., 2009). It is therefore important to assess the characteristics of groundwater resources particularly in rural communities where groundwater is used on a daily basis.

In the Greater Giyani municipality situated in the eastern part of the Limpopo Province, drinking water is provided by the Giyani Water Works that was initially designed for section A location at Giyani in the 1960s (Shirinda, 2008). The population increase in Giyani area resulted in the plant failure to treat and pump sufficient surface water for the bulk supply of several villages including Bode, Dzingidzingi, Maswanganyi and Hlaniki. Because of this, groundwater became the predominant source of water for domestic use and other purposes in these rural communities. The microbiological quality of water used in these villages has not been studied and there are no data on the diversity and antibiotic susceptibility profiles of bacterial isolates from these villages.

Children are generally more vulnerable to intestinal pathogens and it has been reported that about 1.1 million children die every year due to diarrheal diseases (Steiner et al., 2006). It is therefore important to determine the quality, microbial diversity and antibiotic susceptibility profiles of microbial isolates from water sources consumed by the school children, because they are vulnerable to different kinds of diseases since their immune systems are still developing.

In Malawi 3000 children were infected with diarrhoea in 2005 and 1000 of them died (Pritchard et al., 2007). The latter study reported that 43% of the population obtain water from wells, streams and other unreliable water sources leaving them prone to water related diseases including cholera. The aim of this study was to assess the total quality of borehole water used in 6 schools in Greater Giyani Municipality of South Africa and to determine the diversity and antibiotic susceptibility profiles of potential bacterial pathogen isolated from these sources. MATERIALS AND METHODS Characteristics of the study area The study area is located at the outskirts of Giyani Town, in Greater Giyani Municipality of Mopani District of Limpopo Province. The study area is located between latitudes (S 23°16’7” and S 23°20’8’’) and longitudes of (E 030°30’02.6’’ and E 030°38’19.6’’) (Figure 1). The area is characterized by warm, dry, frost free and subtropical climate with summer rainfall. The temperature ranges from a minimum of 14 - 17°C and a maximum average of 28 - 30°C (Mopani District Municipality (MDM), accessed 30 June, 2008). The river that flows within the area of study is Klein Letaba. Nsami Dam which is on this river within the study area, is used to supply water for irrigation and domestic purposes (Maake, 2007). There is a relatively large groundwater resource in this catchment, estimated to be about 30% of the current utilization in the sub-area. The contribution of groundwater to available water in the Klein Letaba sub-area is estimated to be 9 million m³/a (Steyn, 2004).

Samie et al. 199 Identification of schools with boreholes and spatial mapping of the distribution Different schools in Greater Giyani Municipality were visited with a view to formulating research questions. The questions asked centered on the availability of boreholes in schools, their uses, water reservoirs such as tanks, if there is prior treatment of borehole water before use and any other related concern within the school community. For the purpose of the study, only 20% of the schools that have and use borehole water in Greater Giyani Municipality were selected for analysis using stratified random sampling technique. Global positioning systems (GPS) was used to obtain the geographical coordinates of identified boreholes. The coordinates obtained were plotted in arc view to generate the spatial distribution of contaminated and non contaminated boreholes. Microbiological analysis of the borehole water samples This was done by investigating the presence of indicator organisms such as total coliforms, enterococci, faecal coliform and heterotrophs, and detecting pathogenic bacteria such as Salmonella, Shigella, Vibrio species, Campylobacteria and Escherichia coli. Collection of water samples All the sampling points were selected within the chosen schools. Samples were collected for four months (June, August, September and October). The borehole water sources that were selected were those that were used for drinking and for other domestic purposes such as cleaning. The following steps were followed when sampling water for microbiological analysis (As previously described by Rice (1998)): 500 ml Nalgene glass sampling bottles were used. Sampling bottles were presterilized in an autoclave for field use. A burner was used to sterilize the faucet of the borehole source and water was left to run for 3 - 4 min before collection. Collected samples were kept at 4°C in the cooler box packed with ice and transported to the laboratory for analysis within six hours. Assessment of total microbial quality Preparation of culture media The media used include: McConkey Agar (for enterobacteriacea isolation), m-Enterococcus Agar (for the isolation of enterococci), mEndo agar (For the determination of total coliform), MFC Agar (for the determination of fecal coliform) and Plate Count Agar (For the determination of heterotrophic count). The media were prepared a day before going to the sampling site. The media were prepared in accordance with the manufacture‘s instruction (Heyns Lab Supplies) depending on the volume needed. After preparation, media were allowed to cool, and then dispersed into Petri dishes. Membrane filtration and culture Microbial quality assessment was done using the standard membrane filtration technique as described by Ziel et al. (1998). Briefly, samples (100 ml) were filtered through 47 mm microsep membrane filter paper of 0.45 µm pore size. Using sterile forceps, the membrane filters were removed from the filtration cup and transferred to the Petri dishes of defined sizes containing the appropriate media for the culture of bacteria of interest.

For total coliform, the plates were incubated at 37ºC for 24 h; for

200 Afr. J. Microbiol. Res.

Figure 1. Orientation map of study area within Greater Giyani Municipality.

faecal coliform and heterotrophs the plates were incubated at 37°C for 48 h; and for enterococci bacteria the plates were incubated at 44.5°C for 24 h. After 24 h of incubation, number of bacterial colonies was determined using BOECO colony counter and expressed as colony forming units (CFU) per 100 ml.

According to Klein and Bickmell (1995) the maximum

number of colonies that can accurately be counted on a plate is usually 300. Therefore, the counts for plates with over 300 colonies were regarded as numerous. Ten fold serial dilutions were made in order to obtain countable plates. 1 ml of sample was added to 9 ml of sterilized distilled water. The bacterial suspension was mixed by rotating between the hands, and 1 ml of the suspension

was transferred to 9 ml of sterile distilled water labelled 10-

2. The same procedure for mixing was employed for 10-a and 10-4. 1 ml of the sample dilution was poured into agar plate (with media of interest). A sterile spreader was used to evenly distribute the inoculum. After agar has hardened for about 5 min, the plates were inverted and incubated at 37°C for 24 h. After incubation, colonies were counted

Samie et al. 201

Figure 2. Spatial distribution of sampled boreholes in Greater Giyani Municipality.

using colony counter. Isolation and identification of selected pathogenic bacteria The membrane filtration method as described under total microbial quality was employed, however the filters were placed aseptically on TCBS, Campylobacter Blood Free Agar, Salmonella-Shigella Agar and m-Endo Agar to isolate Vibrio, Campylobacter, Salmonella, Shigella and E. coli respectively. Incubation of Campylobacter was in microphilic conditions at 44.5°C, while the other organisms were grown under aerobic condition at 37°C for 24 to 48 h (Rompre et al., 2001; Alam et al., 2003).

Presumptive identification of colonies was determined on the basis of cultural characteristics. Green colonies on TCBS indicated Vibrio; yellow and cream white colonies on Campylobacter Blood Free Agar indicated Campylobacter; brown, green, yellow, pink and cream white colonies on Salmonella-Shigella Agar indicated Salmonella and Shigella species and pink and cream white colonies on m-Endo Agar indicated E. coli. Subculturing was performed in order to obtain pure colonies by streaking into fresh plates. Pure colonies were incubated at 37°C for 24 h. Identification of bacteria and antibiogram determination The isolates were subcultured on fresh MacConkey agar plates. The identity of the isolates as well as their sensitivity to different antibiotics was determined using Autoscan analyzer (Dade Behring/Siemens). The procedure was followed according to manufacturer’s instruction using the MicroScan Negative Combo Panels 50 (Bulik et al., 2010).

RESULTS Borehole water quality and spatial distribution Twenty of the 100 schools in the Greater Giyani Municipality were visited in preliminary survey to identify schools with boreholes that could constitute the area of study. 30% (6) of the 20 schools were chosen for the study. The distribution of borehole water quality showed that poor water quality is likely to occur in schools which use borehole water only (no surface water from the bulk supply). The polygon diagram in Figure 2 indicates this cluster. The cluster could have been influenced by the position of the boreholes which are close to the pit latrines. Total microbial quality of boreholes used by schools in Greater Giyani Municipality Generally, the microbial water quality in all sites got poorer as there was an increase in all microbial indicator counts throughout the study period. Table 1 summarizes the results of the microbial quality of the sampled points. Faecal coliform counts from June to September varied between 8 and 9 x 103 cfu/100 ml. However, according to DWAF (1996) the maximum limit for no risk of faecal coliform is 0 cfu/100 ml. Total coliform counts ranged


Table 1. The total microbial quality of borehole water sources used by schools in Greater Giyani Municipality.

Name of sampling point Dates of sample collection

Total

coliforma

Heterotrophs a Feacal

coliform a

Enterecocci a

Limit for no riskb 0 - 5 cfu/100 ml 0 - 100 cfu/100 ml 0 cfu/100 ml 0 cfu/100 ml

Khomisani Primary School borehole water from Storage Tank 1

05/06/2008 40 78 ND ND

01/08/2008 11 49 8 0

03/09/2008 540 543 240 0

08/10/2008 ND ND ND ND

Khomisani Primary School borehole water from Storage Tank 2

05/06/2008 180 280 ND ND

01/08/2008 220 230 204 0

03/09/2008 ND ND ND ND

08/10/2008 ND ND ND ND

Holapondo High School borehole water from Storage Tank

05/06/2008

01/08/2008

25

129

156

213

ND

50

ND

0

03/09/2008 169 312 109 0

08/10/2008 4.9 x 104 2.84 x 105 9 x 103 0

Maswanganyi Primary School borehole 1 water from Storage Tank 1

05/06/2008 49 654 ND ND

01/08/2008 376 877 107 0

03/09/2008 1.17 x 103 2.76 x 103 1.07 x 103 9

08/10/2008 5.7 x 104 6.5 x 104 4.9 x 103 51

Maswanganyi Primary School borehole 2 water from Storage Tank 2

05/06/2008 156 208 ND ND

01/08/2008 239 336 102 2

03/09/2008 73 248 48 0

08/10/2008 6.92 x 104 2.52 x 105 3.7 x 103 0

Nyanisi High School borehole water from Storage Tank

05/06/2008 264 378 ND ND

01/08/2008 91 339 64 0

03/09/2008 50 144 81 0

08/10/2008 4.17 x 104 9.6 x 104 1 x 103 0

Hlaniki High School borehole water from Storage Tank

05/06/2008 61 200 ND ND

01/08/2008 288 296 36 0

03/09/2008 424 2.10 x 103 31 0

08/10/2008 5.6 x 103 3.83 x 104 1 x 102 0

Macema High School borehole water from Storage Tank 1

05/06/2008 94 156 ND ND

01/08/2008 246 284 114 0

03/09/2008 157 276 134 1

08/10/2008 1.9 x 103 4.0 x 105 5 x 102 0


05/06/2008 156 208 ND ND

01/08/2008 218 222 118 5

03/09/2008 236 280 234 1

08/10/2008 4.8 x 103 6 x 103 1.9 x 103 5 a: The unit is: -cfu/100ml; b: According to DWAF, (1996); ND indicates that analysis was not done.

between 11 and 6.92 x 104 cfu/100 ml. The counts exceeded the 5 cfu/100 ml which is the maximum recommended limit (DWAF, 1996). The heterotrophs counts exceeded the recommended maximum limit of 100 cfu/100 ml (DWAF, 1996) except for borehole water in Storage Tank 1 in Khomisani Primary School in winter. The heterotrophs counts reached a maximum of 4.0 x 10- cfu/100 ml. The enterococci counts were mostly within the recommended minimum limit of 0 cfu/100 ml except at Maswanganyi Primary School and Macema High School where they exceeded the maximum limit in some months. Isolated microorganisms Table 2 shows the bacteria detected from each of the sampling point and at each testing time. Overall, Enterobacter cloacae was the most frequently occurring organism (23.7%) (Table 3). A total of 40 bacteria types composed of a total of 190 bacteria were identified with the microscan system (Table 3). Further analysis revealed the dominance of specific enteric bacteria in each site of the study. E. cloacae, Vibrio hollisae and Klebsiella pneumonia with frequencies of 3 (23.1%), 2 (15.4%) and 2 (15.4%) respectively, were the most common organisms in Khomisani Primary School borehole water from Storage Tank 1. Enterobacter aerogenes with a frequency of 3 (13.1%) was the most frequent organism at Maswanganyi High School borehole 1 water from Storage Tank 1. Cedecea lapagae with frequency of 6 (26.1%) was the most frequently occurring organism at Maswanganyi Primary School borehole 2 water from Storage Tank 2. E. cloacae with frequencies of 8 (34.8%), 11 (47.8%), 8 (44.4%) and 11 (40.7%) respectively was the most frequently occurring organism in Hlaniki Primary School borehole water from Storage Tank, Nyanisi Primary School borehole water from Storage Tank, Holapondo High School borehole water from Storage Tank and Macema High School borehole water from Storage Tank 1. Vibrio fluvialis with a frequency of 6 (19.4%) was the most frequently occurring organism in Macema high school borehole water from Storage Tank 2.

Disease causing organisms such as Serratia marcescens, Pseudomonas aeruginosa, Serratia fonticola, V. fluvialis, Shigella species and Yersinia pseudotuberculosis were identified (Table 2). Important pathogens, some of which are those that are listed above, were detected in Macema High School borehole water from Storage Tank 1, Macema High School borehole water from Storage Tank 2, Maswanganyi Primary School borehole 1 water from Storage Tank 1 and Maswanganyi Primary School borehole 2 water from Storage Tank 2 throughout the sampling period (that is, from 1st to 4th sampling). During 2nd, 3rd and 4th sampling important pathogens were detected at Holapondo High

Samie et al. 203 School borehole water from Storage Tank and Nyanisi High School borehole water from Storage Tank. During 2nd and 3rd sampling, important pathogens were detected at Khomisani Primary School borehole water from Storage Tank 1. During 2nd sampling important pathogens were detected in Khomisani Primary School borehole water from Storage Tank 2.

There is seasonal variation in the number of organisms. During early winter (June), few organisms were detected. The effect of elevated temperature explains the high number of organisms during August, September and October. August is towards the end of winter and had relatively elevated temperature averaging 25°C. The temperature increases gradually from September (spring) to October (summer).

From all the boreholes, members of the Enterobacteriacae family (E. cloacae, Enterobacter amnigenus, E. aerogenes, Enterobacter agglomerans and Enterobacter cancenogenous) were identified in high numbers as compared to other organisms. The identification of this kind of bacteria in water justifies the contamination of water by faecal material from warm blooded animals (humans). This further justifies the fact that one of the potential sources of borehole water pollution is sewerage. Antibiotic sensitivity of identified bacteria isolates Most bacteria identified in this study were sensitive to Levofloxacin and resistant to cefazolin. Other antibiotics to which some of the organisms were also sensitive include amicacin, ciprofloxacin, meropenem and tobramycin. Other antibiotics to which resistance was observed include amox/K clav, ampicillin, aztreonam, cefepime, cefotaxime, cefoxitim, ceftazidime, ceftriaxone, cefuxoxime, ertapenem, Pip/Tazo, piperacillin, tetracycline, ticar/K clav and trimeth/sulfa. Table 4 indicates the detailed results of the antibiotic sensitivity of the identified bacteria isolates. DISCUSSION Water meant for human consumption should be safe and acceptable and must be free from all pathogenic organisms. In South Africa, microbial quality of water is guided by the water quality guidelines or standards of the Department of Water Affairs and Forestry (DWAF, 1996). According to guidelines for drinking water quality, the results of the present study indicated that all the borehole water sources tested were of poor microbiological quality. The present study indicated very high level of contamination of borehole water used by school children in rural areas in the Giyani region as well as a high diversity of bacterial organisms and high levels of antibiotic resistance by the isolated organisms.


Table 2. Organisms detected from the borehole water sources used by schools in Greater Giyani Municipality, and their frequency of occurrence during each sampling period.

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Samie et al. 205

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Serratia fonticola (1)

Cedecea lapagei (1)

Cedecea neteri (2)

Vibrio fluvialis (4)

Enterobacter cloacae (1)

Cedecea davisae (1)

Klebsiella pneumoniae (1)

Chromobacterium violaceum(1)

Enterobacter gergoviae (2)

Enterobacter cancerogenous(1)

Pseudomonas aeruginosa(1)


Klebsiella pneumoniae (1)

Klebsiella ozaenae (1)

Klebsiella rhinosclerromatis(1)


Pseudomonas aeruginosa (2)

Enterobacter aerogenes (2)



Rolstonia paucula (1)

Yersinia pseudotuberculosis (1)

Yokenella regensburgei (1)

Numbers in brackets ( ) represent the frequency of species identified; ND indicates that the analyses were not done. There was no water at the point of sampling due to technical problems.

In the present study, general water quality of boreholes in different schools was very poor with very high counts of bacterial indicators. Similar results have been described by other authors (Obi et al., 2004; Potgieter et al., 2007) on studies done in different rural communities of South Africa. However, the sources of this high contamination in the boreholes have not been investigated. Several sources of contamination could be suggested and could include the possibility of contamination from pit latrines. In fact, the construction of boreholes in the rural areas does not always respect the location regulations to make sure that these boreholes are not situated close to pit latrines; for example, which might be sources of contamination. Previous studies in Zimbabwe indicated that pit latrines were microbiologically impacting on groundwater quality up to 25 m lateral distance (Dzwairo et al., 2006). Boreholes used as sources of water in most schools in Greater Giyani Municipality are generally located next to pit latrines. For example in Khomisani Primary School the distances between the borehole and pit latrines were found to be 40, 46, 70 and 80 m which were below the allowable distance of 100 m

in sandy soil (DWAF, 2004). Other schools in the region hosting boreholes were also more or less close to pit latrines. These include Holapondo High School (120 m), Maswanganyi Primary School (70 and 170 m), Nyanisi High School (190 m), Hlaniki Primary School (59 m) and Macema High School (160 m). Considering the schools far away from the pit latrines (>100 m), the impact of the sanitation is not likely to be highly pronounced except if there is high hydraulic variation in terms of groundwater level that promote high transport of pollutants towards the borehole. However, this hypothesis remains to be demonstrated.

According to the South African Water Quality Guidelines set by DWAF in 1996, the results of the present study mostly exceeded the limits of microbiological quality for all the boreholes. Therefore, the water is not suitable for human consumption. Drinking water from the boreholes can pose serious health effects to consumers. Some sites did not show any contamination from enterococci bacteria. For site 1 (Khomisani Primary School borehole water from Storage Tank 1) and site 2 (Khomisani Primary School borehole water from Storage Tank 2) the poor microbiological quality may be due to very many

closely spaced septic systems in a limited area (Lyle and Raymond, 1998). For all the boreholes, contamination may be due to lack of sewer pipe for discharge of sanitary waste into the treatment plant (Giyani sewage treatment plant). Lack of sewer lines results in underground disposal of sewage into the aquifer. This results in loss of microbiological quality of groundwater. Pit latrines located next to groundwater sources may be cause of contamination into the underground aquifer. Lack of sanitary education has resulted in poor microbiological quality of groundwater. This is because the schools just locate the pit latrines without measuring the distance between the borehole and the pit latrines.

In a study in Kenya, water sources mainly wells close to pit latrines (within 15 m) were found to be contaminated with all the wells (100%) found to be containing total as well as fecal coliforms (thermo tolerant) while tap water was not contaminated, indicating the possibility of pit latrines being a major source of contamination in this case (Kimani-Murage and Ngindu, 2007). However, the authors also suggested that contamination through surface runoff during rains was also plausible, as indiscriminate excreta disposal

Samie et al. 207

Table 3. Each of the bacteria types isolated all the sampling points, their frequency and percentage in relation to the total number of organisms. Organism Frequency % of organisms Achromobacter species (Group VD-1,2) 2 1.1 Achromobacter xylosoxidans 1 0.5 Achromobacteria xyloxidans subsp xyloso 1 0.5 Alcaligenes 1 0.5 Burkholderia (pseudomonas). Cepacia 4 2.1 Cedecea davisae 8 4.2 Cedacea lapagei 13 6.8 Cedecea neteri 6 3.2 Cedecea species 1 0.5 Chromobacterium violaceum 12 6.3 Citrobacter freudii complex 2 1.1 Enterobacter aerogenes 11 5.8 Enterobacter agglomerans 1 0.5 Enterobacter agglomerans group 2 1.1 Enterobacter amnigenus 1 0.5 Enterobacter cancerogenous 5 2.6 Enterobacter cloacae 45 23.7 Enterobacter gergoviae 3 1.6 Hafria alvei 3 1.5 Klebsiella pneumoniae 8 4.2 Klebsiella ozaenae 1 0.5 Klebsiella rhioscleromatis 3 1.6 Klyvera crycrescens 1 0.5 Proteus mirabilis 2 1.1 Proteus peneri 1 0.5 Proteus vulgaris 1 0.5 Pseudomonas aeruginosa 7 3.7 Ralstonia paucula 4 2.1 Salmonella/Arizona 2 1.1 Serratia fonticola 6 3.2 Serratia marcescens 6 3.2 Serratia odorifera 1 0.5 Serratia rubidaea 1 0.5 Shigella species 1 0.5 Stenotrophomonas maltophillia 1 0.5 Vibrio damsela 1 0.5 Vibrio fluvialis 15 7.9 Vibrio hollisae 2 1.1 Yersinia pseudotuberculosis 2 1.1 Yokenella regensburgei 1 0.5 Total 190 100

particularly by children was also common. Nyati (2004) showed that the quality of borehole water supplies in Zimbabwe showed a seasonal fluctuation, with higher coliform counts in the wet season from November to March, while municipal and mining compound water supplies were of satisfactory microbial and chemical quality. Although, all the water sources tested in the

present study were contaminated, the sources of contamination though speculated remains to be confirmed.

Different types of organisms have been identified in water sources in a number of studies. These include pathogenic bacteria such as Salmonella, Vibrio cholerae, Yersinia pseudotuberculosis, Cedecea neteri, detected in


Table 4. Antibiotic sensitivity of the identified bacteria isolates in Greater Giyani Municipality.

Antibiotic Resistant (%) Indeterminate (%) Sensitive (%) Amikacin 73 (33.6) 25 (11.5) 111 (51.2) Amox/K Clav 197 (90.8) 3 (1.4) 9 (4.1) Ampicillin 199 (91.7) 5 (2.3) 6 (2.8) Aztreonam 168 (77.4) 12 (5.5) 30 (13.8) Cefazolin 208 (95.9) 2 (0.9) Cefepime 147 (67.7) 16 (7.4) 47 (21.7) Cefotaxime 157 (72.4) 35 (16.1) 18 (8.3) Cefoxitin 201 (92.6) 3 (1.4) 6 (2.8) Ceftazidime 133 (61.3) 7 (3.2) 70 (32.3) Ceftriaxone 148 (68.2) 41 (18.9) 21 (9.7) Cefuroxime 199 (91.7) 5 (2.3) 6 (2.8) Ciprofloxacin 82 (37.8) 12 (5.5) 116 (53.5) Ertapenem 157 (72.4) 21 (9.7) 32 (14.7) Gentamicin 84 (38.7) 90 (41.5) 7 (3.2) Imipenem 83 (38.2) 29 (13.4) 98 (45.2) Levofloxacin 50 (23.0) 32 (14.7) 128 (59.0) Meropenem 73 (33.6) 12 (5.5) 125 (57.6) Pip/Tazo 143 (65) 18 (8.3) 48 (22.1) Piperacillin 161 (74.2) 17 (7.8) 31 (14.3) Tetracycline 162 (75.7) 16 (7.4) 32 (14.7) Ticar/K Clav 151 (69) 25 (11.5) 34 (15.7) Tobramycin 84 (8.7) 26 (12.0) 100 (46.1) Trimeth/sulfa 191 (88.0) 19 (8.8)

Resistant means that the organisms can withstand the effect of the antibiotic; Sensitive means that the organisms cannot withstand the effect of the antibiotic (that is, the organisms can be treated with the antibiotic); Indeterminate means that the organisms may either be resistant or sensitive to the antibiotics.

borehole water sources in the present study. Similar bacterial profile was described in Nigeria where Escherichia coli, Klebsiella spp., Proteus spp., Enterobacter spp., Pseudomonas spp., and Staphylococcus aureus were isolated from samples from boreholes (Ibe and Okplenye, 2005). However, S. aureus was not tested for in the present study. In a study in Finland, shallow groundwater down to a depth of 16.2 m on average contained more biomass and cultivable microorganisms than did deep groundwater, except in a zone at a depth of approximately 300 m where the average biomass and number of cultivable microorga-nisms approached those of shallow groundwater (Pedersen et al., 2008). The presence of such bacteria might result in diseases and poor health of the children consuming that water. Y. pseudotuberculosis is known to cause fever and acute abdominal pains disease outbreak in school children.

The present study has indicated high levels of antibiotic resistance among all the bacterial isolates. The high resistance of the different microorganisms to carbapemems including imipenem (38%) and meropenem (33.6%) is of great concern. Studies in Uganda have described lower antibiotic resistance with

less than 50% resistance to ampicillin (Soge et al., 2009) while the present study found 92% resistance to ampicillin. Antibiotic resistance to gentamicin was 39 and 34% to amikacin. These resistance rates are quite high compared to those previously described among organisms isolated from river water where resistance to amikacin was less than 10% and resistance to gentamicin was less than 25% (Obi et al., 2004). In a recent study in Poland, high resistance to erythromycin was observed among enterococci isolated from surface water reaching resistance level of 50% (Luczkiewicz et al., 2010). In another study in Alice, South Africa, V. fluvialis showed 100, 90, 70 and 80% resistances to trimethoprim, penicillin, cotrimoxazole and streptomycin, respectively, while 92, 82 90 and 100% of cephalothin resistances were exhibited by Vibrio vulnificus, Vibrio parahaemolyticus, V. fluvialis and Vibrio metschnikovii respectively (Okoh and Igbinosa, 2010). Increase of antibiotic resistance has also been observed among organisms isolated from water samples in Brazil were high indices of resistance to Imipenem, Cephalothin and Ampicillin were observed (Vieira et al., 2010). High numbers of indicator and pathogenic bacteria were also detected as the temperature increased. Considering the

long term impact of diarrhea in children (Guerrant et al., 2008), it is imperative that actions have to be taken in order to correct the quality of water in the boreholes consumed by children. Interventions such as the implementation of point of use water treatment could be advocated as has been recommended elsewhere.

According to the results obtained, it can be concluded that the borehole water used by school children at Khomisani primary school, Nyanisi high school, Holapondo high school, Maswanganyi Primary School, Hlaniki Primary School and Macema High School is of poor microbial quality. It is recommended that a possible follow up would be to identify the sources of contamination and it is also recommended as a short term solution that could be to disinfect the water in the storage reservoir tank before distribution through the school taps. There is also need to carry out a comprehensive social study to determine the number of people suffering from diseases or illnesses related to the microbial water quality problems identified in the area of study. This will provide information on the actual health problems on ground as well as contribute to the use of untreated groundwater in schools. This will lead to recommendation of realistic remediation methods for each specific health problem. Information obtained would be valuable in the design and implementation of intervention strategies if required. This type of study is even more important, because many of the schools rely on borehole water as the only source of water. This will enable the provision of data available to indicate that groundwater does not meet the national guidelines of water for human consumption in the study area unless treated before use. ACKNOWLEDGEMENTS The authors are grateful to the school administrators as well as the local leaders for their cooperation. REFERENCES Alam MJ, Miyoshi S, Shanoda S (2003). Studies on pathogenic Vibrio

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Microbial quality, diversity and antibiotic susceptibility

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