ISOLATION AND IDENTIFICATION OF PATHOGENIC STAPHYLOCOCCI AND E. COLI FROM RAW BOVINE MILK COLLECTED FROM MILK COOPERATIVE CENTERS IN HAWASSA, SOUTHERN ETHIOPIA

Background: Pathogenic microorganisms commonly isolated from milk and milk products pose a serious threat to human health. Escherichia coli and Staphylococci are the major pathogens isolated from milk. The objective of this study was to isolate and identify pathogenic Staphylococci and E. coli from raw bovine milk collected from milk cooperative centers found in Hawassa City, Southern Ethiopia. Result: The overall prevalence of pathogenic species of Staphylococci and E. coli was found to be 35.16 % and 8.59 % (n=384), respectively. From the total 384 raw milk samples examined, 1.56 % was found to be positive for both pathogenic species of Staphylococci and E. coli . The prevalence of pathogenic species of Staphylococci was found to be 33.33 %, 36.05 % and 39.21 % milk collected from Arsi Negele, Hawassa and Shashemene, respectively. Whereas, the prevalence of E. coli was found to be 9.68 %, 8.84 % and 3.92 %) milk collected from Arsi Negele, Hawassa and Shashemene, respectively. The study results showed a relative higher prevalence of pathogenic species of Staphylococci in raw milk collected from Shashemene than raw milk collected from Arsi Negele and Hawassa. However, the difference was statistically insignificant (p > 0.05). Conclusion: Higher isolation rate of E. coli and pathogenic species of Staphylococci in raw milk samples collected from different milk cooperative centers in Hawassa could be associated to mastitis, poor udder preparation, poor milker’s hygiene, poor milk handling practices, poor environmental sanitation and sanitation of milking equipment. Overall, this study showed that pathogenic species of Staphylococcus and E. coli are prevalent in raw milk produced and consumed in the area. Therefore, awareness should be created to producers and raw milk collecting cooperatives on strict preventive measures of raw milk contamination.


Background
Milk provided for human beings should not contain any pathogenic microorganisms (Bertu et al., 2010). However, microbes are isolated and identified from milk and milk products causing a critical health problem to human beings. Milk constitute a diverse biochemical compound as well as it has high water activity and nutritional value which serves as a favorable medium for growth and multiplication of microorganisms (Parekh and Subhash, 2008). Microbes which are commonly isolated from milk includes Escherichia coli, Staphylococcus aureus, Salmonella Typhimurium, Listeria monocytogens, Mycobacterium, Campylobacter, Leptospira, Clostridium, Pseudomonas aeruginosa and Proteus species (Abeer et al., 2012;LeJeune and Rajala, 2009). Milk could be contamination by microorganisms during the preharvest period by the infected lactating animals and during the post-harvest period during milking by milkers, milk handlers, unsanitary utensils and milking equipment's (Parekh and Subhash, 2008;LeJeune and Rajala, 2009).
Staphylococcus species found everywhere in the environment of dairy cattle. The major reservoir of Staphylococci is the infected mammary gland of lactating cows. At the time of milking these organisms transmitted from infected cow to healthy cow during milking through contaminated milker's hands and teat cup liners (Radostits et al., 2007;Mayada and Fatma, 2013). Escherichia coli is a natural flora of the intestines of animals and humans. However, it is commonly recovered from food staffs which could affects the public health due to the possible presence of enteropathogenic and/or toxigenic strains. These enteropathogenic and toxigenic strains of Escherichia coli lead to critical gastrointestinal disorder. The presence of E. coli in milk and milk products is a reliable indicator of milk contamination by manure, soil and contaminated water (Quinn et al, 2002;Radostits et al., 2007).
In Ethiopia, milk produced from smallholder farms is marketed and distributed without any form of pasteurization and quality control measures. According to different reports in Ethiopia, of the total milk production, 71 -97 % of milk is consumed through an informal market without checking the quality measures (Tsehay, 2002). So far, there is no any study conducted on isolation and identification of pathogenic Staphylococci and Escherichia coli in dairy cooperative milk collection centers in Hawassa City. Therefore, the present study was planned to isolate and identify pathogenic Staphylococci and E. coli from raw cow milk collected from dairy cooperative milk collection centers of Hawassa City.

Study Area
The study was conducted in and around Hawassa town. Hawassa is the capital city of Sidama Zone and SNNPRs, which is located in the northern part of SNNPRs and 275 km south of Addis Ababa with a total human population of 157,879. Geographically it lies between 7 0 3'1.35"N latitude

Sample source
The target populations were all raw cows' milk from individual dairy cooperative milk collection centers which are ready for human consumption in Hawassa town. The milk collection cooperative centers those collecting milk from Hawassa and surroundings, from Arsi Negele and Shashemene towns were included in this study.

Study Design
A cross -sectional bacteriological study was conducted from November 2016 to April 2017 to isolate and identify pathogenic Staphylococci and E. coli from raw cows' milk collected from milk cooperative centers in Hawassa town.

Sampling Method
Simple random sampling technique was conducted to select milk cooperative collection centers and to select milk containers to take appropriate raw milk samples.

Sample Size Determination
The desired sample size required for this study was determined depending on the expected prevalence of pathogenic staphylococci and E. coli in the study area and the desired absolute precision. The sample size was computed using the formula given in Thrusfield (2005)  Since there was no previous study regarding the prevalence of pathogenic staphylococci and E. coli in the area, 50 % expected prevalence was used to determine the sample size. Using desired 95 % confidence interval, 5 % precision and 50 % expected prevalence, the necessary numbers of raw milk samples needed to isolate and identify pathogenic staphylococci and E. coli were 384.

Milk sample collection and transportation
Attempts were made to prevent contamination and cross contamination in the course of sample collection. Approximately 5 ml of raw milk samples were collected into sterile screw capped bottles.
The milk samples then were held in an icebox with ice packs and transported to Microbiology Laboratory of School of Veterinary medicine, Hawassa University. All samples were clearly labeled with date of sampling, type of sample and with the name of milk collection cooperatives. In laboratory, raw milk samples were cultured immediately or stored at +4 o C for a maximum of 24 hour until they were inoculated onto a standard bacteriological media (NMC, 1999).

Bacteriological isolation and identification
Bacteriological examination was done according to the Quinn et al. (2002). A loop full of milk samples were streaked on to sterile blood agar plates and MacConkey agar plates using the quadrant streaking method. Then the plates were incubated aerobically at 37°C for 24 to 48 hours and they were examined for growth, morphology, pigmentation and haemolytic characteristics of the colonies. For further identification the colonies suggested colonies of pathogenic staphylococci were subcultured onto sterile Trypticase Soya Agar (TSA) plates. All the suspected cultures of Staphylococci species were subjected to Gram's staining and examined using light microscope under oil immersion objective to appreciate their morphology, cell arrangement and the gram reactions of the isolate. So that the suggested colonies of Staphylococci species that showed gram positive cocci occurring in bunched, grape like irregular clusters were taken as presumptive colonies of Staphylococci species. All colonies those were considered as presumptive colonies of Staphylococci species were subjected to catalase test, oxidase, motility and OF tests. All isolates which were gram positives cocci arranged irregularly as bunch of grapes, did not show growth on MacConkey agar plates, catalase positives, oxidase negatives; non motile and facultative anaerobes were considered as isolates of Staphylococci species (Quinn et al., 2002).
Colonies those were considered as isolates of genus Staphylococci were further differentiated into pathogenic and opportunistic pathogenic species by tube coagulase test. The test was performed by adding a heavy colony of pure culture from Trypticase Soya Agar to a 0.5 ml of rabbit plasma. After mixing the suspension of bacteria with rabbit plasma by gentle rotation, the tubes were incubated at 37 0 C aerobically for 4 -24 hours and examined for any degree of clotting of plasma within the tube.
Those isolates which were positive in tube coagulase test were considered as pathogenic species of Staphylococci (S. aureus, S. intermedius and most strains of S. hyicus). However, those isolates which were negative in tube coagulase test were considered as non pathogenic species of Staphylococci (mainly S. saprophyticus and S. epidemics and can be others) (Quinn et al., 2002).
The pathogenic species of Staphylococci were differentiated by inoculating their colonies on Mannitol Salt Agar (MSA) plates. So that the colonies of pathogenic staphylococci were inoculated onto MSA plates and incubated at 37 0 C aerobically for 24 -48 hours. The plates were examined after 24 hours of incubation for the presence or absence of growth and if there was a colour change from red to yellowish in the medium due to bacterial growth. The presence of growth and colour change from red to yellow were regarded as confirmative identification of the salt tolerant staphylococci which ferment mannitol. Those colonies of pathogenic staphylococci which grew on MSA and developed a yellowish discoloration of the medium were considered as Staphylococci aureus and 11 -89 % stains of S. intermedius. While those colonies of pathogenic staphylococci which grew on MSA whereas colonies that develop without fermenting sugar mannitol were considered as S. hyicus (Quinn et al., 2002).
Isolation of E. coli was done by inoculation of appropriate milk samples on to sterile blood agar plates and MacConkey agar plates. All the inoculated plates were incubated at 37 0 C aerobically for 24 -48 hours. Then colonies which were grayish, round, discrete haemolytic on blood agar plates and which grew on MacConkey agar plates by fermenting lactose (having pinkish colonies) were considered as presumptive colonies of E. coli. For primary identification of Escherichia coli; presence and absence of bacterial growth on MacConkey agar, colony characteristics (size, colour) and presence or absence of lactose fermentation on MacConkey agar plates, catalase, oxidase, motility and OF tests were noted and recorded. Those colonies which were round, discrete haemolytic and developed bright pink colour colonies on MacConkey agar plates; and those were positive in catalase test, negative in oxidase test, motile and facultative anaerobes were subcultured on Brilliant Green Agar (BGA) plates for their further characterization. Those isolates which grew on BGA plates and developed yellowish colonies were considered as the colonies of E. coli and were subjected for further identification by secondary biochemical tests (Quinn et al., 2002) Secondary biochemical tests such as indole test, methyl red test, Voges-Proskeur test and citrate utilization test(I/MR/VP/C) were performed as a presumptive identification of Escherchia coli. Those isolates were positive in indole test, positive in methyl red test, negative in Voges -Proskeur and citrate utilization tests were identified as the colonies of E. coli.

Data Management and Analysis
The data generated from this study were entered and managed in Microsoft Excel. All the data analysis was done using Statistical Package for Social Sciences (SPSS) software version 20. Descriptive stastics such as percentages and frequency distribution were used to describe the nature and the characteristics of data. The overall prevalence of pathogenic staphylococci and E. coli isolates was analyzed using percentages. The association of the risk factor with prevalence of pathogenic staphylococci and E. coli isolates was computed by Pearson's chi-square (χ 2 ) test. In all this analysis, comparison having P -value less than 0.05(P < 0.05) at 5 % level of significance were considered as statistically significant.

Overall Isolation rate of Pathogenic Staphylococci and E. coli
A total of 384 raw milk samples were collected and bacteriologically investigated to determine the isolation rate of pathogenic staphylococci and E. coli. Out of the total 384 raw milk samples examined, 186 (48.44 %), 147(38.28 %) and 51(13.28 %) origin were found from Arsi Negele, Hawassa and Shashemene, respectively as shown in Table 1 below. Out of the total 384 raw milk samples examined 162(42.18 %) raw milk samples were found to be positive. Out of 162 (42.18 %) positive raw milk samples 78(20. 31 %), 62(16.14 %), 22(5.72 %) were found to be from raw dairy milk samples collected from selected raw milk cooperative collection centers in Hawassa which primary origin were found from Arsi Negele, Hawassa and Shashemene towns respectively as shown in Table 1 below.

Association of the Overall Isolation rate of Staphylococci and E. coli Isolates in Relation to Raw Milk Samples Origin
The isolation rate of pathogenic staphylococci and E. coli was found to be different in raw milk samples collected from three towns. It was observed that out of the total 186 raw milk samples collected from Arsi Negele by milk cooperatives found in Hawassa, 33.33 % (62/186) and 9.68 % (18/186) were found to be positives for pathogenic staphylococci and E. coli, respectively. Whereas from the total of 147 raw milk samples collected from selected raw milk collection cooperatives found in Hawassa town which origin was Hawassa itself, 36.05 % (53/147) and 8.84 % (13/147) were found to be positive for pathogenic staphylococci and E. coli respectively. Finally, from the total of 51 raw milk samples collected from selected milk collection cooperatives found in Hawassa town which origin was Shashemene, 39.21 % (20/51) and 3.92 % (2/51) were found to be positive for pathogenic staphylococci and E. coli, respectively. The result showed a relative higher isolation rate of pathogenic staphylococci isolates in raw milk samples collected from selected raw milk collection cooperatives found in Hawassa town which origin was found from Shashemene (39.21%) than from raw milk samples collected from selected raw milk collection cooperatives in Hawassa which origin was found in Arsi Negele town (33.33%) and Hawassa town (36.05%). But the difference was statistically insignificant (P = 0.62; χ 2 = 4.46) as shown in Table 2 below.

DISCUSSION
A total of 384 raw cows' milk samples was collected from selected milk cooperative centers in Hawassa town. The result of the current study showed that the overall isolation rate of pathogenic Staphylococci was found to be 35.16 % which was comparable to the findings of Mekuria et al. (2014), Abera et al. (2013) and Addis et al. (2011) who reported 35.20 % in Hawassa, 42.14 % in Adama and 39.50 % in Debre Zeit, respectively. However, the prevalence of pathogenic staphylococci in this study was relatively lower than the report of Kundu et al. (2013), Mekibib et al. (2010), Demme andAbegaz (2015); and Takle and Birhe (2015) (2012); study conducted in Shashemene (28.10 %) and Zerihun et al. (2013); study conducted in and around Addis Ababa (28.8%). This difference in prevalence of pathogenic staphylococci in raw cows' milk might be arise from the differences in farm management and husbandry practices, breed, environmental conditions, milk handling and/or differences in study methodology and sensitivity of tests employed by the investigators. The high prevalence of staphylococci might be partly explained by presence of this agent on the skin and mucus membranes of various parts of the animal body (Quinn et al., 2002) and their contagious nature, especially S. aureus (Radostits et al., 2007).
The overall prevalence of Escherichia coli was found to be 8.59 %. The present finding was closely related with the works of Etifu (2012) (2015), Mekibib et al. (2010) and Dieser et al. (2014) who reported 3.80 % in Sidama Zone, 4.57 % in Holeta town and 2.10% in Argentinean, respectively. However, the current finding was lower than the work of Soomro et al (2002) in Tandajom, Asmahan and Warda (2011) in Khartoum, Worku et al (2012) in Borona pastoral community and Demme and Abegaz (2015) in Addis Ababa who reported the prevalence of E. coli in raw cows' milk 51.66%, 37.00 %,12.91 % and 18.60 % respectively. Such difference in prevalence might be arise from differences in farm management and husbandry practices, breed, environmental conditions, milk handling and transportation and/or differences in study methodology and sensitivity of tests employed by the investigators. The higher prevalence of Escherichia coli in raw milk is presumably due to the fact that E. coli is the commonest environmental contaminant, which is closely associated with hygiene (Radostits et al., 2007). Moreover, the existence of high numbers of E. coli in milk also indicates the relatively poor quality of milk, related with substandard hygiene of the farm management, poor milk collection and poor transportation system to the market (FAO, 1990).

CONCLUSION
The current study showed that the higher prevalence of pathogenic species of Staphylococci and E.
coli in raw milk produced and distributed in the study area. This indicated that raw milk contamination by pathogenic species of Staphylococci and E. coli found to be the major problem to reduce the quality of raw milk in the area. The higher isolation rate of E. coli and pathogenic species of Staphylococci in raw milk taken from randomly selected cooperative centers found in Hawassa could be associated with poor udder preparation before milking, milkers' hygienic condition, poor milk handling and transportation practices, poor environmental sanitation and poor sanitation of milking equipment.
Overall, this study showed that pathogenic species of Staphylococcus and E. coli are prevalent in raw milk produced and consumed in the area; and the majority of raw dairy milk produced and consumed in the area was poor in quality.