Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 14-Day Trial for You or Your Team.

Learn More →

Bacterial contaminations of raw cow’s milk consumed at Jigjiga City of Somali Regional State, Eastern Ethiopia

Bacterial contaminations of raw cow’s milk consumed at Jigjiga City of Somali Regional State,... Background: Milk is a compensatory part of daily diet especially for the expectant mothers as well as growing children. It is virtually a sterile fluid when secreted into alveoli of udder. However, beyond this stage of production, microbial contamination might generally occur from different sources. Methods: A cross-sectional study was carried out from March 2013-January 2014 in Jigjiga city to assess bacterial contamination of raw milk meant for human consumption and to determine antimicrobial susceptibility patterns of the isolates. A total of 120 raw milk samples were aseptically collected from different sampling points that were hypothesized to be a source of potential contaminations. Data were analyzed using SPSS version 17 computer software. P-value of <0.05 was taken as statistical significance. Results: Overall, the organisms identified and their prevalence rates were Escherichia coli 70(58 %), Staphylococcus aureus 29(24.2 %), Shigella Sp. 21 (17.5 %), Proteus sp. 9 (7.5 %) and Salmonella sp. 4 (3.3 %). The isolation rates of these identified bacteria from each sampling points are statistically significant in E. coli and Proteus sp. (P < 0.05). High antibiotic resistance for E. coli isolates were observed to Doxycycline (42.3 %) and Ampicillin (30 %). Shigella sp. was resistant to Ampicillin (38.1 %). Salmonella sp. isolates were highly resistant to Amoxicillin (50 %). Out of a total of 29 S.aureus isolates, high resistance rate was observed to penicillin G 27(93.1 %) followed by tetracycline 20(69 %), and very low level of resistance to vancomycin 2(6.9 %) and rifampicin 1(3.4 %). Multidrug resistance was also observed in 55.2 % of the total isolates. Conclusions: Considering the high rate of raw milk contamination with the above isolated bacteria, sanitary practice during collecting, transporting and vending is recommended since the consumption of unpasteurized milk may inflict an important public health risk. Keywords: Bacterial contamination, Critical sampling points, Raw milk, Antibiotic, Jigjiga Background milk handlers, and poor sanitation practices associated Milk is used throughout the world as a human food at with milking and storage equipments (Garedew et al. least one form or more. It is virtually a sterile fluid when 2012). Milk is largely made up of water, within which a secreted into alveoli of udder. However, beyond this wide range of nutrients including vitamins, proteins, fats stage of production, microbial contamination might gen- and carbohydrates are suspended. These rich nutritional erally occur from different sources (Mennane et al. contents, the production and processing procedures in 2007). Conditions for contamination of raw milk at dif- commercial milk production render it susceptible to con- ferent critical points are due to less hygienic practices in tamination by a host of pathogenic microbes that could pre-milking udder preparation, sub-optimal hygiene of cause diseases in humans. Therefore, milk is known to be an efficient vehicle for transmission of disease causing agents to humans (Garedew et al. 2012). The demand of * Correspondence: melese1985@gmail.com consumers for safe and high quality milk has placed a sig- Faculty of Health Science, Department of Nursing, Woldia University, P.O.Box nificant responsibility on dairy producers, retailers and 400, Woldia, Ethiopia Full list of author information is available at the end of the article © 2016 The Author(s). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Reta et al. International Journal of Food Contamination (2016) 3:4 Page 2 of 9 manufacturers to produce and market safe milk and milk production from cows, camels and goats. The study pop- products (Adesiyun et al. 1995; Mennane et al. 2007). Milk ulations were raw cow’s milk from individual farmers’ and milk products have important role in feeding the rural cows, milk collectors, and milk venders in Jigjiga city. and urban population of Ethiopia owing to its high nutri- tional value. It is produced daily, sold for cash or readily Collection of raw milk samples at critical sampling points processed. It is a cash crop in the milkshed areas that en- and transportation ables families to buy other foodstuffs and significantly Milk samples were collected from points considered to contributing to the household food security (Abebe et al. be associated with contamination (critical sampling 2012). Lack of refrigeration facilities at farm and house- points). The sampling points were the teat during milk- hold level in developing countries of tropical regions with ing, milking buckets at farm level, transport containers, high ambient temperature implies that raw milk will easily and selling point up on arrival at the market. Overall, be spoiled during storage and transportation (Godefay and 120 raw milk samples were analyzed: of these, 30 raw Molla 2000). Milk and milk products may carry toxic me- milk samples were from teat, 30 from milking buckets, tabolites of different pathogenic organisms growing in it. 30 from storage containers, and 30 from selling point up Ingestion of such products contaminated with these me- on arrival at the markets. During sampling of raw milk tabolites cause food poisoning for consumers. On the directly from teats, the udder and teats were cleaned other hand the ingestion of viable pathogenic bacteria and dried before sampling; each teat end was scrubbed along with the food product leads to food borne infection gently with cotton swabs moistened with 70 % ethyl al- (Aneja et al. 2002). The disease causing bacteria in the cohol. The first 3-4 streams of milk were discarded, and milk are Salmonella sp. Mycobacterium bovis, Corynebac- approximately 10 ml of milk was collected into sterile terium sp., Clostridium perfringens, Yersinia enterocolitica, sampling bottles. The other raw milk samples were col- Coxiella burnetii, Brucella, Staphylococcus sp., Campylo- lected in the morning following standard safety proce- bacter jejuni, Mycobacterium avium, Listeria sp., Escheri- dures. Prior to sampling from milking buckets and chia coli, and coliforms (Fadaei 2014; Olatunji et al. 2009). transport containers, the milk was thoroughly mixed by The total coliforms, E. coli and other enteric bacteria are shaking and 25 ml of milk was transferred into a sterile reliable indicators of fecal pollution generally in insanitary screw capped bottle. Transportation of samples to the conditions of water, food, milk and other dairy products. Ethiopian Somali Regional Laboratory was immediately Recovery of E. coli from food is an indicative of possible conducted for further processing using ice packs follow- presence of enteropathogenic and/or toxigenic micro- ing the standard safety procedures (Robinson 2002). organism which could constitute a public health hazard (Soomro et al. 1996). These microorganisms are usually Bacterial identification and isolation from milk samples associated with food borne diseases and outbreaks, as re- Detection of E.coli: All the samples positive for E. coli corded by official health organizations (Bouazza et al. contamination were confirmed using Gram’s staining, 2012). The presence of these pathogenic bacteria in milk cultural and biochemical examinations. The samples appeared as main public health concerns, especially for were inoculated on MacConkey Agar (Difco laboratories, those people who still drink unpasteurized raw milk USA) and incubated aerobically at 37°c for 24 h. The (Claeys et al. 2013). Despite this, the aim of this study was plates were observed for the growth of E. coli. A single, to determine the presence of contaminating microorgan- isolated colony was picked and sub-cultured again on isms and their antibiotic resistance patterns in the raw MacConkey agar for purification of the isolate. Simul- milk produced by individual farmers, collectors and milk taneously another single colony with similar characters vendors in Jigjiga city, eastern Ethiopia. was picked for the preparation of smear and stained with Gram’s stain for the examination of staining and mor- Methods phological characters of the isolate using bright field Study area, design and study period microscope. The cultural characteristics of the isolates A cross-sectional study was conducted in Jigjiga city were confirmed by inoculating the pure colonies on from March 2013-January 2014. Jigjiga is the capital city Blood Agar (Oxoide, Germany), Nutrient Agar (Oxoid of Ethiopian Somali Regional State located at 628 km CM0003, Basingstoke, England), Nutrient Broth and east of Addis Ababa at 9° 20’ north latitude and 42° 47’ Violet Red Bile Agar (Oxoid CM107). Biochemical tests east longitude. The altitude of the district ranges from were performed to confirm the E. coli using catalase test, 900–1600meters above sea level and receives an annual Simmon’s Citrate Agar, sugar fermentation on Triple rainfall of 300–500 mm with the mean minimum and Sugar Iron Agar(Oxoid CM0277, Basingstoke, England), maximum annual temperatures of 20°c and 28°c res- Gelatin liquefaction, Indole Production, Nitrate reduc- pectively (CSA 2003). The community in this region is tion, Urease production, Voges proskaur, Methyl red and pastoral and agro-pastoralist and there is large milk Presumptive test. Detection of Salmonella Sp: The Reta et al. International Journal of Food Contamination (2016) 3:4 Page 3 of 9 isolation and identification involves three steps; 1 ml of Sp: One (1 ml) of milk sample was enriched in 10 ml of milk was pre-enriched with 9 ml of buffered peptone Buffer peptone water aseptically and incubated at 37°c for water (Oxoid CM509, Basingstoke, England) and incu- 24 h. Inoculum from the enrichment broth was streaked bated for 24 h at 37°c. A portion (0.1 ml) of the pre- on Hektoen Enteric Agar (HEA) and MacConkey Agar enriched cultured was transferred to 10 ml of selenite (Difco laboratories, USA) and incubated at 37°c for 24 h. cysteine broth (Merck) and incubated at 37°c for 24 h The cultures were identified on the basis of their morpho- respectively. Finally, from the selective enrichment logical, and biochemical characteristics. media the sample was inoculated on to xylose lysine deoxycholate (XLD) agar (Oxoid CM0469, Basingstoke, Antimicrobial susceptibility testing England) and incubated at 37°c for 24 h. Characteristic The antimicrobial susceptibility patterns of the above Salmonella colonies, having a slightly transparent zone detected bacteria were carried out following the Kirby- of reddish color and a black center were sub-cultured on Bauer disc diffusion method on Mueller Hinton agar nutrient agar and confirmed biochemically using triple (Oxoid CM0337 Basingstoke, England) as described by sugar iron agar (TSI)(Oxoid CM0277, Basingstoke, the Clinical and Laboratory Standards Institute (CLSI England), Christensen’s urea agar (Oxoid CM53, 2008). The criteria used to select the antimicrobial Basingstoke, England), lysine iron agar (LIA) (Oxoid agents tested were based on the availability and fre- CM381, Basingstoke, England), Voges Proskauer (VP), me- quency of prescription for the management of bacterial thyl red (MR)(Micromaster Thane, India), and Indole tests infections in animals as well as for human in Ethiopia (Becton Dickinson, USA) (Hendriksen 2003). Detection of and on the basis of their different structures and mecha- S.aureus: Gram staining was performed (Cruikshank et al. nisms of action. Antimicrobial susceptibility test was 1975) and Gram-positive cocci that occurred in clusters performed for all S. aureus isolates according to the cri- under the microscope were subjected to preliminary bio- teria of the Clinical and Laboratory Standards Institute chemical tests (the catalase and oxidase tests). The iden- (CLSI 2008). For susceptibility test for S. aureus, one tities of the isolates were confirmed based on positive anti-microbial from each subclass of antimicrobials results for the DNase test, beta haemolytic patterns on which were commonly used for treatment of bovine blood agar enriched with 5 % (v/v) sheep blood and the mastitis or considered as important antimicrobial agents coagulase slide test for S. aureus using the (PROLD Diag- for human were selected for antibiogram based on the nostics, Canada). The slide agglutination test was per- criteria of Clinical and Laboratory Standards Institute formed according to the manufacturer’s instructions. (CLSI 2008). Thus, antimicrobials used for treatment of Briefly, cells from a pure colony were placed on the clean bovine mastitis included in this study were erythro-mycin area of the slide using a sterile toothpick and a drop of the (E/15 μg), cephalothin (KF/30 μg), penicillin-G(10unit), PROLD reagent was added. These were mixed using the sulphoxazole-trimethoprim (SXT/25 μg), amoxicillin- toothpick and the isolates were identified based on the clavulinic acid (AMC/30 μg), chloroamphenicol (C/ formation of agglutination. An isolates that formed agglu- 30 mg), (Oxoid), tetracycline (TE/30 μg) and gentamicin tination were recorded as S. aureus and maintained at 4°c (CN/10 μg) (Biomerioux). Antimicrobials not used for in 30 % glycerol for further characterization by antibiotic treatment of bovine mastitis but important for human susceptibility testing. Detection of Shigella Sp: Speci- were oxacillin (OX/1 μg), vancomycin (VA/30 μg), clinda- mens were plated directly on primary media: Salmonella- mycin (DA/10 μg) and rifampicin (RD/5 μg) (Oxoid). Shigella agar (Merck) and Selenite F broth (Mast Diagnos- Finally, the diameters of the zone of inhibition around the tics DM 210, Mast Diagnostics, UK). For those negative disks were measured to the nearest millimeter using samples on primary sold media, sub-culturing from en- rulers, and the isolates were classified as susceptible, inter- richment broth to primary media was performed to im- mediate and resistant (CLSI 2008). E. coli ATCC 25922 prove recovery of the isolates. All of the inoculated media was used as a quality control organism for the antimicro- were incubated at 37°c for 18-24h. The non-black colonies bial susceptibility test (Hendriksen, 2002). Moreover, iso- observed on the center were suspected positive test for lates showing resistance to three or more antimicrobial Shigella sp. and Klingler Iron Agar (KIA) was used for bio- subclass were considered as multidrug resistant. chemical differentiation of Shigella from other coliform bacteria. Colonies of suspected Shigella was inoculated on Statistical analysis Salmonella-shigella Agar plate (Merck), deoxycholate cit- The collected data for bacterial contamination analysis rate agar (DCA) (Oxoid CM 35; Oxoid Ltd, UK) and incu- were entered and analyzed using SPSS version 17 com- bated at 37°c for 24 h. Growth of suspected Shigella sp. puter software. Accordingly, descriptive statistics such as change in color butt of media its color(red) to yellow and percentages and frequency distribution was used to de- red slope remained as it is because Shigella sp. is lactose scribe/present bacterial isolates and antimicrobial sus- fermenter in anaerobic condition. Detection of Proteus ceptibility which was expressed as percent of resistant, Reta et al. International Journal of Food Contamination (2016) 3:4 Page 4 of 9 intermediate and susceptible. P-value <0.05 was taken as The observations made in the present study clearly cut-off for statistical significance. proved that S. aureus showed resistance to all antimicro- bials tested except for Rifampicin and Vancomycin. These indicate that the problem is highly distributed and Results disseminated. Moreover, the overall resistance of S. Isolated bacterial species aureus isolates, to Vancomycin, Rifampicin, Clindamycin Overall, five bacterial targets were identified in the milk and Gentamycin showed less than 25 % of resistance. sampled in the study area. The bacteria so identified and The highest resistance rate was observed in Penicillin their isolation rate were E.coli 70(58 %), Salmonella sp. (93.1 %), followed by Tetracycline (69 %). On the other 4(3.3 %), Shigella sp. 21(17.5 %), Staphylococcus aureus hand, about 55.2 % (16/29) of S.aureus isolates were 29 (24.2 %) and Proteus Sp. 9(7.5 %). These are indicative found to be multidrug resistant (Table 3). of significant contamination of milk and important hu- man pathogens. The most prevalent organism overall MAR phenotypes of S. aureus was E. coli, while the least prevalent was Salmonella sp. Multiple antibiotic resistance (MAR) phenotypes were In this study, the contamination degree of milk by the determined for S. aureus (Table 4). The predominant isolated bacteria is utterly worsened at each critical sam- MAR phenotypes for S. aureus isolated from this study pling point. High contamination level was observed at area were PG-TE -Ox and PG-TE-AC-E-SXZ-Ox in market point sampled milk. The difference in isolation 24.1 % and 17.2 % of the isolates, respectively. Furthermore, rate across market chain (critical sampling points) is sta- MAR phenotypes PG-TE- AC-Ox, PG-TE-AC-E-SXZ-Ox- tistically significant in E. coli. (P= 0.00) and Proteus sp. CN, PG-TE-AC-E-SXZ-Ox- CN-CH and PG-TE-AC-E- (P = 0.016) (Table 1). SXZ-Ox- CN- VA were obtained in 3.4 % of the isolates. Results of the present study revealed that 49 (40.8 %) Also PG-TE-AC 6.9 %, PG-TE-Ox- KF 13.8 % and PG-TE- of milk sampled had at least two different bacterial or- AC-E-Ox 6.9 % were the MAR phenotypes for S. aureus ganisms, 6 (12 %) from the udder, 10(20.4 %) milking isolated from this study area (Table 4). bucket, 12 (24.5 %) from storage container and 21 It is thus evident that MAR S. aureus was isolated (42.9 %) from the market point (Fig. 1). from all critical sampling points. However, among the isolates from this study area 55.2 % of the isolates de- Antimicrobial susceptibility of the bacterial isolates velop MAR. Among all MAR phenotypes of S. aureus, The antimicrobial susceptibility tests of the bacterial 40.3 % of them were resistance to six different antibi- isolates were grossly very variable. About 76.1 % E. coli otics and 7.2 % were resistance to seven antibiotics. Fifty isolates were resistant and it had the highest resistance four percent (54 %) of them were resistance to 3 or 4 rates to Doxycycline, Ampicillin and Gentamycin antibiotics. (42.3 %, 30 % and 30 %) respectively. A quarter of E. coli isolates (25.4 %) were multidrug resistant (≥3drugs). Discussion Similarly higher antimicrobial resistance (74.6 %) was re- The outcome of our study revealed that 84.1 % of milk corded against Salmonella sp. isolates as well. The high- samples were contaminated with at least one bacterium est resistance rate to Salmonella sp. was observed in that comprised of E. coli, Salmonella sp., Shigella sp., S. Amoxicillin (50 %). The highest resistance rate to aureus, and Proteus Sp., with isolation rates of 70(58 %), shigella was observed in Ampicillin (38.1 %). All shigella 4(3.3 %), 21(17.5 %), 29(24.2 %), and 9(7.5 %), respectively. isolates were highly susceptible to Co-trimoxazole(81 %). The levels of contamination with each isolated bacteria About 14 % of Shigella isolates were multidrug resistant- were higher across critical sampling points (from teats, fairly better than E.coli isolates. All Proteus sp. isolates Milking bucket, transportation container, and at market were 66.7 % sensitive to Ciprofloxacin and showed points). Similar findings reported by Daka et al.(2012) (55.6 %) resistance to Ampicillin (Table 2). revealed that the level of contamination with S.aureus Table 1 Occurrence of isolated bacteria across critical sampling points (n = 120) of milk collected from Jigjiga city Bacteria Isolate No. of positive sample (%) P- value isolated No(%) Udder (n = 30) Collection Bucket(n = 30) Storage Material(n = 30) Market point (n = 30) E. coli 70(58 %) 9(30 %) 16(53.3 %) 22(73.3 %) 23(76.7 %) 0.00 Salmonella sp. 4(3.3 %) 1(3.3 %) 0(0.0 %) 1(3.3 %) 2(6.7 %) 0.140 Shigella sp. 21(17.5 %) 4(13 .3 %) 3(10 %) 4 (13.3 %) 10(33.3 %) 0.069 S. aureus. 29(24.2 %) 6(20 %) 7(23.3 %) 6(20 %) 10(33.3 %) 0.727 Proteus sp. 9(7.5 %) 0(0.0 %) 2 (6.7 %) 3(10 %) 4(13.3 %) 0.016 Reta et al. International Journal of Food Contamination (2016) 3:4 Page 5 of 9 Fig. 1 Isolation of mixed bacteria from milk sampled at different sampling points (n = 120), Jigjiga city were higher in milk obtained from teat(17.9 %), Milking (24.4 %), S. aureus (38.2 %), and Salmonella sp. (2 %), from bucket at farm level(25.7 %), storage containers at milk apparently normal milk samples from different critical sam- collection center(26.9 %) and from transportation con- plingpoints. It is knownthatevenwhendrawnunderasep- tainer(21.8 %). Conditions for contamination of raw milk tic condition, milk always contains microorganisms which at different critical points are due to less hygienic prac- are derived from the milk ducts in the udder, in addition tices in pre-milking udder preparation, sub-optimal hy- contaminants coming from milking utensils and human giene of milk handlers, and poor sanitation practices handlers (Solomon et al. 2013). Higher isolation frequen- associated with milking and storage equipments, higher cies, especially for E. coli across market chain was observed environmental contamination during transportation or in the current study as compared to similar studies per- contamination during waiting along the roadside (Garedew formed to assess bacteriological quality of raw milk in et al. 2012). Based on observations made during the collec- Ethiopia (Tassew and Seifu 2011; Tiruneh 1996). This tion of samples, we therefore report that improper hygiene might be due to poor and unhygienic bedding condition in and poor farm management practices contributed to the the majority of farms and absence of teat dipping and disin- presence of these isolated bacteria in the milk. In this study fection practices in the current study. These practices have area milk was obtained from animals by washing their been known as critical components of mastitis prevention hands and/or the utensils and containers used. In certain and control program in dairy herds (Galton et al. 1986). cases, untreated groundwater was used to wash the con- Other findings by different researchers confirm that E. coli tainers that were used for milking. This may have contrib- grow well in milk and hence endanger its keeping milk uted to the high level of enteric bacteria and S.aureus quality (Frazeir and Westhoff 1988). E. coli and coliforms isolated. Improving the hygienic conditions of the milking are often used as indicator microorganisms, and the environment and/or utensils may reduce the prevalence of presence of E. coli in milk samples implies a risk that entropatogenic as well as S.aureus in milk and prevent its other enteropathogenic bacteria may be present in the transmission to humans. Olatunji et al (2009) in Nigeria sample (Najib 2003; Olatunji et al. 2009; Arafa and had reported that higher isolation frequencies of E. coli Soliman 2013). Table 2 Antibiotic sensitivity pattern of bacterial isolates in milk samples collected from Jigjiga city Antimicrobial E. coli(n = 70) Salmonella sp.(n = 4) Shigella sp.(n = 21) Proteus sp.(n = 9) R (%) I (%) S (%) R(%) I (%) S (%) R (%) I (%) S (%) R (%) I (%) S (%) Amoxicillin 15(21.4) 31(44.3) 24(34.3) 2(50) 1((25) 1(25) 2(9.5) 11(52.4) 8(38.1) 1(11.1) 6(66.7) 2(22.2) Ampicillin 21(30) 28(40) 21(30) 1(25) 1(25) 2(50) 8(38.1) 8(38.1) 5(23.8) 5(55.6) 3(33.3) 1(11.1) Ciprofloxacin 7(10) 17(24.3) 46(65.7) 1(25) 1(25) 2(50) 2(9.5) 6(28.6) 13(61.9) 0(0.0) 3(33.3) 6(66.7) Co-trimoxazole 12(17.1) 22(31.4) 36(51.4) 1(25) 2(50) 1(25) 0(0.0) 4(19.0) 17(81) 1(11.1) 3(33.3) 5(55.6) Chloramphenicol 15(21.4) 26(37.1) 29(41.4) 1(25) 2(50) 1(25) 3(14.3) 7(33.3) 11(52.4) 1(11.1) 5(55.6) 3(33.3) Gentamycin 21(30) 35(50) 14(20) 1(25) 2(50) 1(25) 4(19.0) 10(47.6) 7(33.3) 2(22.2) 4(44.4) 3(33.3) Doxycycline 30(42.9) 27(38.6) 13(18.5) 1(25) 2(50) 1(25) 6(28.6) 10(47.6) 5(23.8) 2(22.2) 3(33.3) 4(44.4) R Resistance, I Intermediate, S Sensitive, n number Reta et al. International Journal of Food Contamination (2016) 3:4 Page 6 of 9 Table 3 Antimicrobial susceptibility pattern of S. aureus isolates (n = 29) from milk samples collected from Jigjiga city Antimicrobial Susceptible number (%) Intermediate number (%) Resistant number (%) Pencillin (P) 1(3.4) 1(3.4) 27(93.1) Chloroamphenicol (CH) 11(37.9) 10(34.5) 8(27.6) Cephalothin (KF) 15(51.7) 5(17.2) 9(31.0) Gentamycin (CN) 10(34.5) 12 (41.4) 7(24.1) Erythromycin (E) 5(17.2) 17(58.6) 7(24.1) Clindamycin (DA) 9(31.0) 16(55.2) 4(13.8) Tetracycline (TE) 5(17.2) 4 (13.8) 20(69) Amoxicillin + clavulanic 19(65.5) 0(0.0) 10(34.5) Rifampicin (RD) 27(93.1) 1 (3.4) 1(3.4) Oxacillin (OX) 18(62.1) 2(6.9) 9(31.0) Vancomycin (VA) 24(82.8) 3(10.3) 2(6.9) Sulphamethoxazole-timethoprim (SXZ) 19(65.5) 3 (10.3) 7(24.1) Our results indicated that 24.2 % of the samples were presence of S. aureus in the milk. In this study low positive for S.aureus. This is a favorable finding because, salmonella sp. isolation rate with 3.3 % was found. for human health some strains of S. aureus are capable Junaidu et al (2011), Forough et al. (2012) and Sanaa et of producing heat stable enterotoxins (Asperger 1994). al.(2005) had reported comparable findings with 2.17 %, A comparable finding to our result was reported by 4 % and 1.43 % prevalence respectively. Addis et al. Abebe et al. (2013) that S.aureus prevalence was 15.5 % (2011) reported a prevalence of 10.7 % from raw milk in raw milk samples. In contrast to this, different lite- which is higher than the present report. In the other ratures revealed a very significant isolation rate of S. study by Addis et al. (2011) from 195 dairy cows tested aureus from raw milk samples (Olatunji et al. 2009; 28.6 % were positive from milk samples. Akoachere et Pourhassan and Taravat-Najafabadi 2011; Mohanty et al. al.(2009) in Cameroon reported a high prevalence (27 %) 2013; Sanaa et al. 2005). Although the prevalence of S. of Salmonella among cattle. This may be due to the differ- aureus has been reported to vary with the size and geo- ence in the living condition, like housing conditions, feed- graphic region of the area sampled, a high proportion of ing habits, types of feed given for the cattle, of the two these bacteria in milk relates to poor hygiene practices. cattle populations. The detection of Salmonella in 3.3 % of Based on observations made during the collection of the samples tested indicates that the degree of prevalence samples, we therefore report that improper hygiene and of the pathogen in raw milk in jigjiga is relatively higher poor farm management practices contributed to the than originally believed. Although contamination of dairy products currently accounts for a small percentage of foodborne illness, it is clear that raw milk consumption Table 4 The predominant MAR phenotypes for S. aureus and the consumption of products made with raw milk isolated from milk samples (n = 29) collected from Jigjiga city present some risk. Although proper pasteurization MDR patterns Phenotype Number Percentage observed minimizes these risks to the public, there is a small but Three PG-TE-AC 2 6.9 growing group of people that consume unpasteurized milk or milk products, either for practical or cultural reasons, PG-TE -Ox 7 24.1 or because of perceived health benefits (Karns et al. 2005). Four PG-TE-Ox- KF 4 13.8 Although the levels of Salmonella in the milk samples PG-TE- AC-Ox 1 3.4 tested here seemed to be very low and the infectious Five PG-TE-AC-E-Ox 2 6.9 dose for this organism is low, the potential for this Six PG-TE-AC-E-SXZ-Ox 5 17.2 organism to grow in improperly stored raw milk and Seven PG-TE-AC-E-SXZ-Ox- CN 1 3.4 in products made from raw milk presents a public health risk, particularly to susceptible members of the Eight PG-TE-AC-E-SXZ-Ox- CN- CH 1 3.4 population. PG-TE-AC-E-SXZ-Ox- CN- VA 1 3.4 The isolation rates of proteus Sp. in this study (7.5 %) The percentage representations of the phenotypes were obtained by dividing is comparable with the report by Junaidu et al (2011) the number of a particular phenotype by the total number of multiple antibiotic resistant isolates identified in a given area. VA Vancomycin, PG with 8.69 % prevalence. Most of the organisms identified Penicillin G, SXZ Sulphamethoxazole-timethoprim, E Erythromycin, Ox Oxacillin, in this study were enteric bacteria indicating probable AC Amoxicillin-Clavulanic Acid, TETetracycline, KF Cephalothin; CN Gentamycin, CH Chloroamphenicol faecal contamination of the milk as a result of poor Reta et al. International Journal of Food Contamination (2016) 3:4 Page 7 of 9 hygiene. The practice of pooling milk from different S. aureus isolates to vancomycin, rifampicin, clindamycin sources by traders, and the absence of pasteurization and gentamycin showed less than 25 % of resistance and generally observed among them could increase the risk this is similar with the report of Ma et al. (2006) from the posed by such organisms. dairy farm in Taiwan. The reason why these antimicrobials In the present study, Doxycycline had the highest re- were less resistant might be they are not frequently used sistance rates in E. coli. In contrast to this, fairly higher in the study area in veterinary services, and perhaps in hu- resistant rate was recorded in Ampicillin (100 %) and man medicine. Similar suggestion was given by Jaims et Amoxicillin (42.11 %) (Thaker et al. 2012). On the other al.(2002) that the development of antimicrobial resistance hand the highest resistance rate for Salmonella sp. in is nearly always as a result of repeated therapeutic and/or this report was observed to Amoxicillin (50 %). Different indiscriminate use of them. However, the present study researchers reported antimicrobial resistant Salmonella has demonstrated the existence of alarming level of resist- isolates of milk in their previous studies from Ethiopia ance of S. aureus to commonly used antimicrobials (pen- (Molla et al. 2003; Mekonnen et al. 2005) and from cillin G and tetracycline) in the study area. This is due to other countries (White et al. 2001). Forough et al. (2012) the fact that tetracycline and penicillin are frequently and reported that salmonella sp. isolates were resistance to improperly used antimicrobials in animal and human Ampicillin (42.58 %), Tetracycline (42.58 %) and Nalidixic treatment. The results were in accordance with reports acid (78.57 %). Addis et al. (2011) reported a high resist- from earlier studies in other countries (Jakee et al. 2008; ance rate salmonella isolates to ampicillin (100 %). The re- Edward et al. 2002; Gentilini et al. 2002) suggesting a pos- markable degree of resistance to many drugs represents sible development of resistance from prolonged and indis- public health hazard due to the fact that food borne out- criminate usage of some antimicrobials. This is in contrast breaks would be difficult to treat and this pool of MDR with the report of Ma et al. (2006) on his report with re- Salmonella in food supply represents a reservoir for the spect to pencillin and tetracycline in Taiwan. This is not transferable resistant genes (Diaze De Aguayo et al. 1992). surprising because penicillin G and tetracycline are the The reasons for the recovery of antimicrobial resistance most commonly used antimicrobials for the treatment of Salmonella isolates were most likely due to the indiscrim- infection or mastitis in veterinary practice in Ethiopia. inate use of antimicrobials (WHO 1988), self-medication Moreover, penicillin resistance is plasmatic and, it spread and administration of sub therapeutic dose of antimi- out very quickly to several other strains. Pereira et al. crobials to livestock for prophylactic purpose (Acha and (2009) showed that 70 to 73 % of S. aureus strains isolated Szyfers 2001). Antimicrobial use in animal production from various foods were resistant to β-lactam such as pen- systems has long been suspected to be a cause of the cillin and ampicillin. Staphylococci are frequently isolated emergence and dissemination of antimicrobial resistant from bovine mastitis which is one of the most common Salmonella (Forough et al. 2012). causes for the use of antimicrobial in lactating dairy cows. In this study the highest resistance rate for shigella Similarly, the present investigation indicated that the re- sp. was observed to Ampicillin (38.1 %) followed by sistance pattern of penicillin was found to be 93.1 % Doxycycline (28.6 %). In contrast to our finding Sanaa et (Table 3) which is similar to the finding made by Tariku et al. (2005) reported that shigella isolated from raw milk al.(2011) (87.2 %) in Ethiopia, Landin (2006) (80 %) in were sensitive to Gentamycin (64.3 %) followed by Sweden, Gooraninejad et al. (2007) (57 %) in Iran and Chloramphenicol (92.1 %), and the highest antimicrobial Myllys et al.(1998) (50 %) in Finland. This is in contrast to resistant pattern was observed in Ampicillin and Amoxi- findings observed by Adesiyun (1994) who reported 23 % cillin (92.9 %, 92.9 %) followed by penicillin (42.9 %). In of resistance to pencillin G in West India. agreement with our result Ayalu et al. (2011) reported Moreover, the present study showed the resistance of that shigella isolates were 100 % resistant to Ampicillin and S. aureus to tetracycline (69 %), amoxicillin-clavulinic Amoxicillin but sensitive to Chloramphenicol, Gentamicin, acid (34.5 %), oxacillin (31 %), cephalothin (31 %), chlor- and Norfloxacin(41.2 %, 88.2 %, and 94.1 %) respectively. amphenicol (27.6 %), sulphamethoxazole-trimethoprim Shiferaw et al. (2012) reported that 74 % shigella isolates (24.1 %), erythromycin (24.1 %), gentamycin (24.1 %), were resistant to Ampicillin, and 58 % to Streptomycin. On clindamycin (13.8 %) observed in milk samples taken the other hand, All the Shigella isolates were resistant to from dairy cows in jigjiga city. This is in accordance with Ampicillin, 94 % to Tetracycline, and 82 % to Ciprofloxacin the findings of Tariku et al.(2011) who reported resist- in a report by Debdutta et al. (2012). ance of S. aureus to amoxicillin-clavulinic acid (46 %), The observations made in the present study clearly chloroamphenicol (16 %), vancomycin (3 %), but it dis- proved that S. aureus showed resistance to all antimicro- agree with the observation made by Tariku et al. (2011) bials tested except for rifampicin and Vancomycin in the case of tetracycline(0 %), Co-trimoxazole (0 %) (Table 3). These indicate that the problem is highly distrib- and clindamycin (4 %) in dairy farms in Jimma town. uted and disseminated. Moreover, the overall resistance of The probable explanation could be that S. aureus strains Reta et al. International Journal of Food Contamination (2016) 3:4 Page 8 of 9 have the capacity to change their resistance behavior to human commensal, and multidrug resistant S. aureus may the exposed antimicrobials. present without clinical illness. However, when they cause With a particular emphasis to tetracycline, the present infection they are extremely serious. Furthermore, dairy observation agrees with preliminary finding conducted cows become infected with multidrug resistant S. aureus, by Bayhun (2008) (55.3 %). However, apparent difference therefore diagnosis of S. aureus does not have implication was observed in the report of Tariku et al.(2011) (0 %). for treatment only but also it indicates zoonotic trans- This is due to the fact that tetracycline is the most com- mission since it becomes reservoir for human infection. In monly used antimicrobial in the treatment of infections practice of indiscriminate use of drugs should be con- in the livestock sector in Ethiopia. Moreover, tetracycline trolled. Further studies that could incorporate isolation of is widely used as growth factors in veterinary medicine milk contaminating bacteria to the species level should be for livestock rearing as well in the treatment of bacterial done to evaluate the imminent danger posed by microbes infection occurring in human medicine (Ardic et al. from milks. 2005). Furthermore, the resistance profile of S. aureus to Acknowledgements amoxicillin-clavulinic acid and oxacillin in milk samples Jigjiga University is greatly acknowledged for funding and providing all was found to be high. This is due to the fact that resist- rounded technical assistance for the smooth accomplishment of this research. The authors are also highly indebted to Ethiopian Somali Regional Health ance of S. aureus to pencillin G, amoxicillin and oxacillin Bureau, Regional Public Health and Research Laboratory for laboratory facilities. may be attributed to the production of β-lactamase, an enzyme that inactivates pencillin and closely related Authors’ contributions antimicrobial. It is believed that about 50 % of mastitis MA carried out the conception of the research concept and designed the methodology, data analysis and interpretation and preparation of the causing S. aureus produces β-lactamase (Green and manuscript for publication. TW carried out the laboratory work, sample Bradely 2004). Likewise, S. aureus showed resistance to collection and revision of the manuscript. AN critically revised the proposal, vancomycin and clindamycin. This might indicate trans- designed the methodology, and reviewed the manuscript. All authors read and approved the final manuscript. fer of resistant strain among environment, livestock and human since this antimicrobials are not used in veterin- Competing interests ary practice. The authors declare that there is no financial or non-financial competing The MAR phenotypes (Table 4) obtained in the study interest from anybody or institute. We also want to assure that we did not receive any technical assistant in developing the research concept or correlated with the percentage of antibiotic resistance. preparation of the manuscript. Although the development of resistance to a particular antibiotic depends on the level of exposure to the antimi- Author details Faculty of Health Science, Department of Nursing, Woldia University, P.O.Box crobials, (Rychlik et al. 2006) there are many other factors 400, Woldia, Ethiopia. College of Natural Sciences, Department of Biology, that are involved. We are therefore suggesting that mo- 3 Wolkite University, P.O.Box 07, Wolkite, Ethiopia. College of Veterinary lecular methods be used to characterize these isolates for Medicine, Department of Veterinary Microbiology and Public Health, Jigjiga University, P.O.Box 1020, Jigjiga, Ethiopia. the presence of antibiotic-resistance determinants, which may provide data to support our conclusions. S. aureus is Received: 3 February 2016 Accepted: 13 May 2016 normally resident in humans; therefore, the S. aureus present in the cow’s milk may have resulted from trans- References mission from humans, which raises questions regarding Abebe B, Zelalem Y, Ajebu N. Hygienic and microbial quality of raw whole cow’s the hygiene practices followed. milk produced in Ezha district of the Gurage zone, Southern Ethiopia. Wudpecker J Agric Res. 2012;1(11):459–65. Abebe M, Daniel A, Yimtubezinash W, Genene T. Identification and antimicrobial Conclusions susceptibility of S. aureus isolated from milk samples of dairy cows and nasal This study revealed that raw cow’s milk in the study area swabs of farm workers in selected dairy farms around Addis Ababa, Ethiopia. could be an important source of infection with a wide Afr J Microbiol Res. 2013;7(27):3501–10. Acha PN, Szyfers B. Zoonoses and Communicable Diseases Common to Man and range of organisms, particularly enteropathogens. An im- rd Animals: Bacteriosis and Mycosis. 3 ed.Vol I. Washington DC: Pan American portant source of microbial contamination of the milk is Health Organization; 2001. p. 233–46. faecal pollution probably from cow dung. There is the Addis Z, Kebed N, Worku Z, Gezahegn H, Yirsa A, Kassa T. Prevalence and antimicrobial resistance of Salmonella isolated from lactating cows and in need for instituting effective control measures to protect contact humans in dairy farms of Addis Ababa. BMC Infect Dis. 2011;11:222–8. public health. This includes mandatory milk pasteurization Adesiyun A. Characteristics of S. aureus strains isolated from bovine mastitic milk: by traders and improved hygienic handling of the Bacteriophage and antimicrobial agent susceptibility and enterotoxigenecity. J Vet Med. 1994;42:129–39. commodity during milking, ensuring milking is not done Adesiyun AA, Webb L, Rahman S. Microbiological quality of raw cow’s milk at on cow dung. The occurrence of multidrug resistance S. collection centers in Trinidad. J Food Prot. 1995;58:139–46. aureus should be under consideration during selection of Akoachere TKJ, Tanih FN, Ndip ML, Ndip RN. Phenotypic Characterization of Salmonella Typhimurium Isolates from Food-animals and Abattoir Drains in antimicrobials for treatment of mastitis especially if the Buea, Cameroon. J Health Popul Nutr. 2009;27(5):612–8. possibility exists in the transfer of resistance in or between Aneja RP, Muthur BN, Chandan RC, Banerejee AK. Technology of Indian milk microbial species. Moreover, S. aureus is a common products. New Delhi: Dairy Indian Yearbook; 2002. p. 183–96. Reta et al. International Journal of Food Contamination (2016) 3:4 Page 9 of 9 Arafa M, Soliman M. Bacteriological Quality and Safety of Raw Cow’s Milk and Junaidu AU, Salihu MD, Tambuwal FM, Magaji AA, Jaafaru S. Prevalence of Fresh Cream. Slov Vet Res. 2013;50(1):21–30. Mastitis in Lactating Cows in some selected Commercial Dairy Farms in Ardic N, Ozyurt M, Sareyyupoglu B. Investigation of erythromycin and tetracycline Sokoto Metropolis. Adv Appl Sci Res. 2011;2(2):290–4. resistance genes in Methicillin-resistant Staphylococci. Int J Antimicrob Karns JS, Van Kassel JS, McKluskey BJ, Perdue M. Prevalence of Salmonella enteric Agents. 2005;26:213–8. in bulk tank milk from US dairies as determined by Polymerase Chain Asperger H. Stapylococcus aureus. In: The Significance of Pathogenic Microorganisms Reaction. J Dairy Sci. 2005;88:3475–9. in Raw Milk, International Dairy Federation. Brussels: IDF; 1994. p. 24–42. Landin H. Treatment of mastitis in Swedish dairy production (in Swedish with English summary). Svensk Veterina¨rtidning. 2006;58:19–25. Ayalu AR, Berhanu S, Jemal Y, Gizachew A, Sisay F, Jean MV. Antibiotic Ma Y, Chang SK, Chou CC. Characterization of bacterial susceptibility isolates in susceptibility patterns of Salmonella and Shigella isolates in Harar, Eastern sixteen dairy farms in Taiwan. J Dairy Sci. 2006;1:55–6. Ethiopia. J Infect Dis Immun. 2011;3(8):134–9. Mekonnen H, Workineh S, Bayleyegne M, Moges A, Tadele K. Antimicrobial Bayhun S. Beta-lactamase activities and antibiotic resistance comparison of susceptibility profile of mastitis isolates from cows in three major Ethiopian Staphylococcus aureus isolated from clinic and food material (M.Sc Thesis). dairies. Med Vet. 2005;176(7):391–4. Gazi University, Institute of Science Technology;2008. p.126 Mennane Z, Ouhssine M, Khedid K, Elyachioui M. Hygienic Quality of Raw Cow’s Bouazza F, Hassikou R, Ohmani F, Hmmamouchi J, Ennadir J, Qasmaoui A, et al. Milk Feeding from Domestic. Int J Agri Biol. 2007;9(1):1560–8530. Hygienic quality of raw milk at Sardi breed of sheep in Morocco. Afr J Mohanty NN, Das P, Pany SS, Sarangi LN, Ranabijuli S, Panda HK. Isolation and Microbiol Res. 2012;6(11):2768–72. antibiogram of Staphylococcus, Streptococcus and E. coli isolates from clinical Claeys WL, Cardoen S, Daube G, Block JD, Dewettinck K, Katelijne Dierick K, et al. and subclinical cases of bovine mastitis. Vet World. 2013;6(10):739–43. Herman Raw or heated cow milk consumption. Rev Risks benefits Food Molla B, Alemayehu D, Salah W. Source and distribution of Salmonella serovars Control. 2013;31:251–62. isolated from food animals, slaughter house personnel and retail meat Clinical and Laboratory Standards Institute (CLSI). Performance Standards for products in Ethiopia: 1997-2002. Ethiopia J Health Dev. 2003;17:63–70. Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated From Myllys V, Asplund K, Brofeld E, Hirevela-Koski V, Honkanen-Buzalski T. Bovine Animals. Clin Lab Stand Inst. 2008;28:M31–A3. Mastitis in Filand in 1988 and 1995. Changes in Prevalence and Antimicrobial Cruikshank R, Duguid JP, Marmoin BP, Swain RH. Medical microbiology. 12th ed. resistance. Acta Vet Scand. 1998;39:119–26. New York: Longman Group Limited; 1975. p. 34. Najib G. Risk assessment of dairy products, Consumer committee seminar,Msca CSA (Central Statistical Authority). Central Statistical Authority, Federal Democratic Tour Municipality MM Service, Columbia USA. 2003. Republic of Ethiopia, Central Statistical Investigatory, Statistical Report. Olatunji EA, Ahmed I, Ijah UJ. Evaluation of microbial qualities of skimmed milk Addis Ababa: Central Statistical Authority of Ethiopia; 2003 th (nono) in Nasarawa State, Nigeria. Proceeding of the 14 Annual Conf. of Daka D, Gebresilassie S, Yihdego D. Antibiotic-resistance Staphylococcus aureus th th Ani.Sc. Asso. of Nig. (ASAN) LAUTECH Ogbomoso, Sept. 14 -17 , 2009. 2009. isolated from cow’s milk in the Hawassa area, South Ethiopia. Ann Clin Pereira V, Lopes C, Castro A, Silva J, Gibbs P, Teixeira P. Characterization for Microbiol Antimicrob. 2012;11:26. Enterotoxins production, virulence factors and antibiotic susceptibility of S. Debdutta B, Sugunan AP, Haimanti B, Thamizhmani R, Sayi DS, Thanasekaran K, et al. aureus isolates from various food in Portugal. Food Microbiol. 2009;26:278–82. Antimicrobial resistance in Shigella -rapid increase & widening of spectrum in Pourhassan M, Taravat-Najafabadi ART. The spatial distribution of bacterial Andaman Islands, India. Indian J Med Res. 2012;135(3):365–70. pathogens in raw milk consumption on Malayer City, Iran. Shiraz E Med J. Diaze De Aguayo ME, Duarte AB, Montes De Oca Canastillo F. Incidence of 2011;12:2–10. multiple antibiotic resistant organisms isolated from retail milk products in Robinson RK. Dairy Microbiology Handbook: The Microbiology of Milk and Milk Hermosillo, Mexico. J Food Prot. 1992;55(5):370–3. Products. 3rd ed. USA: John Wiley & Sons, Inc; 2002. p. 51–305. Edward M, Anna K, Michal K, Henryka L, Krystyna K. Antimic-robial susceptibility Rychlik I, Gregorova D, Hradecka H. Distribution and function of plasmids in of staphylococci isolated from mastitic cows. Bull Vet Inst. 2002;46:289–94. Salmonella enterica. Vet Microbiol. 2006;112(1):1–10. Fadaei A. Bacteriological Quality of Raw Cow Milk in Shahrekord, Iran. Veterinary Sanaa OY, Nazik EA, Ibtisam EM, Zubeir EL. Incidence of Some Potential World. 2014;7(4):240–3. Pathogens in Raw Milk in Khartoum North (Sudan) and Their Susceptibility to Forough T, Elahe T, Manochehr M, Ebrahim R, Rafie S. Occurrence and Antibiotic Antimicrobial Agents. J Anim Vet Adv. 2005;4(3):356–9. Resistance of Salmonella spp Isolated from Raw Cow’s Milk from Shiferaw B, Solghan S, Palmer A, Joyce K, Barzilay EJ, Krueger A, Cieslak P. Shahahrekord, Iran. Int J Microbiol Res. 2012;3(3):242–5. th Antimicrobial susceptibility patterns of Shigella isolates in Foodborne Frazeir WC, Westhoff DT. Food Microbiology (4 ed). Singapore: Mcgraw-Hill Book Diseases Active Surveillance Network (FoodNet) sites, 2000-2010. Clin Infect Company; 1988. p. 419–28. Dis. 2012;54 Suppl 5:S458–63. Galton DM, Petersson LG, Merril WG. Effects of Pre- Milking Udder Preparation on Solomon M, Mulisa M, Yibeltal M, Desalegn G, Simenew. Bacteriological quality of Bacterial Counts of in Milk and on Teat. J Dairy Sci. 1986;69:260–6. bovine raw milk at selected dairy farms in Debre Zeit town, Ethiopia. Compr Garedew L, Berhanu A, Mengesha D, Tsegay G. Identification of gram-negative J Food Sci Technol Res. 2013;1(1):1–8. bacteria from critical control points of raw and pasteurized cow milk consumed Soomro AH, Arain MA, Khaskheli M, Bhuto B. Isolation of E. coli from raw milk at Gondar town and its suburbs, Ethiopia. BMC Public Health. 2012;12:950. and milk products in relation to public health sold under market conditions Gentilini E, Danamiel A, Betancor M, Rebuelto MR, Fermepin RM, Detorrest RA. at Tandonjam, Pakistan. J Nutr. 1996;1(3):151–2. Antimicrobial susceptibility of coagulase-negative Staphylococci isolated Tariku S, Jemal H, Molalegne B. Prevalence and susceptibility assay of from bovine Mastitis in Argentina. J Dairy Sci. 2002;85:1913–7. Staphylococcus aureus isolated from bovine mastitis in dairy farms in Jimma Godefay B, Molla B. Bacteriological quality of raw milk from four dairy farms and town South West Ethiopia. J Anim Vet Adv. 2011;10:745–9. milk collection center in and around Addis Ababa, Berl. Münch Tierarztl Tassew A, Seifu E. Microbial Quality of Raw Cow’s Milk Collected from Farmers Wschr. 2000;113:1–3. and Dairy Cooperatives in Bahir Dar Zuria and Mecha District, Ethiopia. Agric Gooraninejad S, Ghorbanpoor M, Salati AP. Antibiotic Susceptibility of Staphylococci Biol J N Am. 2011;2(1):29–33. isolated from bovine sub-clinical mastitis. Pak J Biol Sci. 2007;10:2781–3. Thaker HC, Brahmbhatt MN, Nayak JB. Study on occurrence and antibiogram Green M, Bradely A. Clinical Forum- S. aureus mastitis in cattle UK. VET. 2004;9:4. pattern of E. coli from raw milk samples in Anand, Gujarat, India. Vet World. Hendriksen RS. A global Salmonella surveillance and laboratory support project 2012;5(9):556–9. of the World Health Organization: Laboratory Protocols (Susceptibility testing Tiruneh Z. A Study on Bovine Subclinical Mastitis at Stella Dairy Farm, DVM of Salmonella using disk diffusion). 2002. p. 3. Thesis. Addis Ababa: Faculty of Veterinary Medicine, Addis Ababa University; Hendriksen RS. A global Salmonella surveillance and laboratory support project 1996. p. 30–45. of the World Health Organization: Laboratory Protocols (Isolation of White DG, Zhao S, Sudler R, Ayers S, Friedman S, Chen S, et al. The isolation of Salmonella). 2003. p. 4. antibiotic-resistant Salmonella from retail ground meats. New Engl J Med. Jaims E, Montros LE, Renata DC. Epidemiology of drug resistance; the case of 2001;345(16):1147–54. Staphylococcus aureus and Coagulase negative Staphylococci infections. Salud World Health Organization. Salmonellosis Control: The Role of Animal and Publica Mex. 2002;44:108–12. Product Hygiene, Technical Report Series, 774. Geneva: WHO; 1988. Jakee J, Ata S, Nagwa M, Bakry SA, Zouelfakar EE, Gad El-Said WA. Characteristics of S. aureus strains isolated from human and animal sources. Am-Euras J Agric Environ Sci. 2008;4:221–9. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png International Journal of Food Contamination Springer Journals

Bacterial contaminations of raw cow’s milk consumed at Jigjiga City of Somali Regional State, Eastern Ethiopia

Loading next page...
 
/lp/springer-journals/bacterial-contaminations-of-raw-cow-s-milk-consumed-at-jigjiga-city-of-BAZNGSnOK7

References (74)

Publisher
Springer Journals
Copyright
Copyright © 2016 by The Author(s).
Subject
Chemistry; Food Science
eISSN
2196-2804
DOI
10.1186/s40550-016-0027-5
Publisher site
See Article on Publisher Site

Abstract

Background: Milk is a compensatory part of daily diet especially for the expectant mothers as well as growing children. It is virtually a sterile fluid when secreted into alveoli of udder. However, beyond this stage of production, microbial contamination might generally occur from different sources. Methods: A cross-sectional study was carried out from March 2013-January 2014 in Jigjiga city to assess bacterial contamination of raw milk meant for human consumption and to determine antimicrobial susceptibility patterns of the isolates. A total of 120 raw milk samples were aseptically collected from different sampling points that were hypothesized to be a source of potential contaminations. Data were analyzed using SPSS version 17 computer software. P-value of <0.05 was taken as statistical significance. Results: Overall, the organisms identified and their prevalence rates were Escherichia coli 70(58 %), Staphylococcus aureus 29(24.2 %), Shigella Sp. 21 (17.5 %), Proteus sp. 9 (7.5 %) and Salmonella sp. 4 (3.3 %). The isolation rates of these identified bacteria from each sampling points are statistically significant in E. coli and Proteus sp. (P < 0.05). High antibiotic resistance for E. coli isolates were observed to Doxycycline (42.3 %) and Ampicillin (30 %). Shigella sp. was resistant to Ampicillin (38.1 %). Salmonella sp. isolates were highly resistant to Amoxicillin (50 %). Out of a total of 29 S.aureus isolates, high resistance rate was observed to penicillin G 27(93.1 %) followed by tetracycline 20(69 %), and very low level of resistance to vancomycin 2(6.9 %) and rifampicin 1(3.4 %). Multidrug resistance was also observed in 55.2 % of the total isolates. Conclusions: Considering the high rate of raw milk contamination with the above isolated bacteria, sanitary practice during collecting, transporting and vending is recommended since the consumption of unpasteurized milk may inflict an important public health risk. Keywords: Bacterial contamination, Critical sampling points, Raw milk, Antibiotic, Jigjiga Background milk handlers, and poor sanitation practices associated Milk is used throughout the world as a human food at with milking and storage equipments (Garedew et al. least one form or more. It is virtually a sterile fluid when 2012). Milk is largely made up of water, within which a secreted into alveoli of udder. However, beyond this wide range of nutrients including vitamins, proteins, fats stage of production, microbial contamination might gen- and carbohydrates are suspended. These rich nutritional erally occur from different sources (Mennane et al. contents, the production and processing procedures in 2007). Conditions for contamination of raw milk at dif- commercial milk production render it susceptible to con- ferent critical points are due to less hygienic practices in tamination by a host of pathogenic microbes that could pre-milking udder preparation, sub-optimal hygiene of cause diseases in humans. Therefore, milk is known to be an efficient vehicle for transmission of disease causing agents to humans (Garedew et al. 2012). The demand of * Correspondence: melese1985@gmail.com consumers for safe and high quality milk has placed a sig- Faculty of Health Science, Department of Nursing, Woldia University, P.O.Box nificant responsibility on dairy producers, retailers and 400, Woldia, Ethiopia Full list of author information is available at the end of the article © 2016 The Author(s). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Reta et al. International Journal of Food Contamination (2016) 3:4 Page 2 of 9 manufacturers to produce and market safe milk and milk production from cows, camels and goats. The study pop- products (Adesiyun et al. 1995; Mennane et al. 2007). Milk ulations were raw cow’s milk from individual farmers’ and milk products have important role in feeding the rural cows, milk collectors, and milk venders in Jigjiga city. and urban population of Ethiopia owing to its high nutri- tional value. It is produced daily, sold for cash or readily Collection of raw milk samples at critical sampling points processed. It is a cash crop in the milkshed areas that en- and transportation ables families to buy other foodstuffs and significantly Milk samples were collected from points considered to contributing to the household food security (Abebe et al. be associated with contamination (critical sampling 2012). Lack of refrigeration facilities at farm and house- points). The sampling points were the teat during milk- hold level in developing countries of tropical regions with ing, milking buckets at farm level, transport containers, high ambient temperature implies that raw milk will easily and selling point up on arrival at the market. Overall, be spoiled during storage and transportation (Godefay and 120 raw milk samples were analyzed: of these, 30 raw Molla 2000). Milk and milk products may carry toxic me- milk samples were from teat, 30 from milking buckets, tabolites of different pathogenic organisms growing in it. 30 from storage containers, and 30 from selling point up Ingestion of such products contaminated with these me- on arrival at the markets. During sampling of raw milk tabolites cause food poisoning for consumers. On the directly from teats, the udder and teats were cleaned other hand the ingestion of viable pathogenic bacteria and dried before sampling; each teat end was scrubbed along with the food product leads to food borne infection gently with cotton swabs moistened with 70 % ethyl al- (Aneja et al. 2002). The disease causing bacteria in the cohol. The first 3-4 streams of milk were discarded, and milk are Salmonella sp. Mycobacterium bovis, Corynebac- approximately 10 ml of milk was collected into sterile terium sp., Clostridium perfringens, Yersinia enterocolitica, sampling bottles. The other raw milk samples were col- Coxiella burnetii, Brucella, Staphylococcus sp., Campylo- lected in the morning following standard safety proce- bacter jejuni, Mycobacterium avium, Listeria sp., Escheri- dures. Prior to sampling from milking buckets and chia coli, and coliforms (Fadaei 2014; Olatunji et al. 2009). transport containers, the milk was thoroughly mixed by The total coliforms, E. coli and other enteric bacteria are shaking and 25 ml of milk was transferred into a sterile reliable indicators of fecal pollution generally in insanitary screw capped bottle. Transportation of samples to the conditions of water, food, milk and other dairy products. Ethiopian Somali Regional Laboratory was immediately Recovery of E. coli from food is an indicative of possible conducted for further processing using ice packs follow- presence of enteropathogenic and/or toxigenic micro- ing the standard safety procedures (Robinson 2002). organism which could constitute a public health hazard (Soomro et al. 1996). These microorganisms are usually Bacterial identification and isolation from milk samples associated with food borne diseases and outbreaks, as re- Detection of E.coli: All the samples positive for E. coli corded by official health organizations (Bouazza et al. contamination were confirmed using Gram’s staining, 2012). The presence of these pathogenic bacteria in milk cultural and biochemical examinations. The samples appeared as main public health concerns, especially for were inoculated on MacConkey Agar (Difco laboratories, those people who still drink unpasteurized raw milk USA) and incubated aerobically at 37°c for 24 h. The (Claeys et al. 2013). Despite this, the aim of this study was plates were observed for the growth of E. coli. A single, to determine the presence of contaminating microorgan- isolated colony was picked and sub-cultured again on isms and their antibiotic resistance patterns in the raw MacConkey agar for purification of the isolate. Simul- milk produced by individual farmers, collectors and milk taneously another single colony with similar characters vendors in Jigjiga city, eastern Ethiopia. was picked for the preparation of smear and stained with Gram’s stain for the examination of staining and mor- Methods phological characters of the isolate using bright field Study area, design and study period microscope. The cultural characteristics of the isolates A cross-sectional study was conducted in Jigjiga city were confirmed by inoculating the pure colonies on from March 2013-January 2014. Jigjiga is the capital city Blood Agar (Oxoide, Germany), Nutrient Agar (Oxoid of Ethiopian Somali Regional State located at 628 km CM0003, Basingstoke, England), Nutrient Broth and east of Addis Ababa at 9° 20’ north latitude and 42° 47’ Violet Red Bile Agar (Oxoid CM107). Biochemical tests east longitude. The altitude of the district ranges from were performed to confirm the E. coli using catalase test, 900–1600meters above sea level and receives an annual Simmon’s Citrate Agar, sugar fermentation on Triple rainfall of 300–500 mm with the mean minimum and Sugar Iron Agar(Oxoid CM0277, Basingstoke, England), maximum annual temperatures of 20°c and 28°c res- Gelatin liquefaction, Indole Production, Nitrate reduc- pectively (CSA 2003). The community in this region is tion, Urease production, Voges proskaur, Methyl red and pastoral and agro-pastoralist and there is large milk Presumptive test. Detection of Salmonella Sp: The Reta et al. International Journal of Food Contamination (2016) 3:4 Page 3 of 9 isolation and identification involves three steps; 1 ml of Sp: One (1 ml) of milk sample was enriched in 10 ml of milk was pre-enriched with 9 ml of buffered peptone Buffer peptone water aseptically and incubated at 37°c for water (Oxoid CM509, Basingstoke, England) and incu- 24 h. Inoculum from the enrichment broth was streaked bated for 24 h at 37°c. A portion (0.1 ml) of the pre- on Hektoen Enteric Agar (HEA) and MacConkey Agar enriched cultured was transferred to 10 ml of selenite (Difco laboratories, USA) and incubated at 37°c for 24 h. cysteine broth (Merck) and incubated at 37°c for 24 h The cultures were identified on the basis of their morpho- respectively. Finally, from the selective enrichment logical, and biochemical characteristics. media the sample was inoculated on to xylose lysine deoxycholate (XLD) agar (Oxoid CM0469, Basingstoke, Antimicrobial susceptibility testing England) and incubated at 37°c for 24 h. Characteristic The antimicrobial susceptibility patterns of the above Salmonella colonies, having a slightly transparent zone detected bacteria were carried out following the Kirby- of reddish color and a black center were sub-cultured on Bauer disc diffusion method on Mueller Hinton agar nutrient agar and confirmed biochemically using triple (Oxoid CM0337 Basingstoke, England) as described by sugar iron agar (TSI)(Oxoid CM0277, Basingstoke, the Clinical and Laboratory Standards Institute (CLSI England), Christensen’s urea agar (Oxoid CM53, 2008). The criteria used to select the antimicrobial Basingstoke, England), lysine iron agar (LIA) (Oxoid agents tested were based on the availability and fre- CM381, Basingstoke, England), Voges Proskauer (VP), me- quency of prescription for the management of bacterial thyl red (MR)(Micromaster Thane, India), and Indole tests infections in animals as well as for human in Ethiopia (Becton Dickinson, USA) (Hendriksen 2003). Detection of and on the basis of their different structures and mecha- S.aureus: Gram staining was performed (Cruikshank et al. nisms of action. Antimicrobial susceptibility test was 1975) and Gram-positive cocci that occurred in clusters performed for all S. aureus isolates according to the cri- under the microscope were subjected to preliminary bio- teria of the Clinical and Laboratory Standards Institute chemical tests (the catalase and oxidase tests). The iden- (CLSI 2008). For susceptibility test for S. aureus, one tities of the isolates were confirmed based on positive anti-microbial from each subclass of antimicrobials results for the DNase test, beta haemolytic patterns on which were commonly used for treatment of bovine blood agar enriched with 5 % (v/v) sheep blood and the mastitis or considered as important antimicrobial agents coagulase slide test for S. aureus using the (PROLD Diag- for human were selected for antibiogram based on the nostics, Canada). The slide agglutination test was per- criteria of Clinical and Laboratory Standards Institute formed according to the manufacturer’s instructions. (CLSI 2008). Thus, antimicrobials used for treatment of Briefly, cells from a pure colony were placed on the clean bovine mastitis included in this study were erythro-mycin area of the slide using a sterile toothpick and a drop of the (E/15 μg), cephalothin (KF/30 μg), penicillin-G(10unit), PROLD reagent was added. These were mixed using the sulphoxazole-trimethoprim (SXT/25 μg), amoxicillin- toothpick and the isolates were identified based on the clavulinic acid (AMC/30 μg), chloroamphenicol (C/ formation of agglutination. An isolates that formed agglu- 30 mg), (Oxoid), tetracycline (TE/30 μg) and gentamicin tination were recorded as S. aureus and maintained at 4°c (CN/10 μg) (Biomerioux). Antimicrobials not used for in 30 % glycerol for further characterization by antibiotic treatment of bovine mastitis but important for human susceptibility testing. Detection of Shigella Sp: Speci- were oxacillin (OX/1 μg), vancomycin (VA/30 μg), clinda- mens were plated directly on primary media: Salmonella- mycin (DA/10 μg) and rifampicin (RD/5 μg) (Oxoid). Shigella agar (Merck) and Selenite F broth (Mast Diagnos- Finally, the diameters of the zone of inhibition around the tics DM 210, Mast Diagnostics, UK). For those negative disks were measured to the nearest millimeter using samples on primary sold media, sub-culturing from en- rulers, and the isolates were classified as susceptible, inter- richment broth to primary media was performed to im- mediate and resistant (CLSI 2008). E. coli ATCC 25922 prove recovery of the isolates. All of the inoculated media was used as a quality control organism for the antimicro- were incubated at 37°c for 18-24h. The non-black colonies bial susceptibility test (Hendriksen, 2002). Moreover, iso- observed on the center were suspected positive test for lates showing resistance to three or more antimicrobial Shigella sp. and Klingler Iron Agar (KIA) was used for bio- subclass were considered as multidrug resistant. chemical differentiation of Shigella from other coliform bacteria. Colonies of suspected Shigella was inoculated on Statistical analysis Salmonella-shigella Agar plate (Merck), deoxycholate cit- The collected data for bacterial contamination analysis rate agar (DCA) (Oxoid CM 35; Oxoid Ltd, UK) and incu- were entered and analyzed using SPSS version 17 com- bated at 37°c for 24 h. Growth of suspected Shigella sp. puter software. Accordingly, descriptive statistics such as change in color butt of media its color(red) to yellow and percentages and frequency distribution was used to de- red slope remained as it is because Shigella sp. is lactose scribe/present bacterial isolates and antimicrobial sus- fermenter in anaerobic condition. Detection of Proteus ceptibility which was expressed as percent of resistant, Reta et al. International Journal of Food Contamination (2016) 3:4 Page 4 of 9 intermediate and susceptible. P-value <0.05 was taken as The observations made in the present study clearly cut-off for statistical significance. proved that S. aureus showed resistance to all antimicro- bials tested except for Rifampicin and Vancomycin. These indicate that the problem is highly distributed and Results disseminated. Moreover, the overall resistance of S. Isolated bacterial species aureus isolates, to Vancomycin, Rifampicin, Clindamycin Overall, five bacterial targets were identified in the milk and Gentamycin showed less than 25 % of resistance. sampled in the study area. The bacteria so identified and The highest resistance rate was observed in Penicillin their isolation rate were E.coli 70(58 %), Salmonella sp. (93.1 %), followed by Tetracycline (69 %). On the other 4(3.3 %), Shigella sp. 21(17.5 %), Staphylococcus aureus hand, about 55.2 % (16/29) of S.aureus isolates were 29 (24.2 %) and Proteus Sp. 9(7.5 %). These are indicative found to be multidrug resistant (Table 3). of significant contamination of milk and important hu- man pathogens. The most prevalent organism overall MAR phenotypes of S. aureus was E. coli, while the least prevalent was Salmonella sp. Multiple antibiotic resistance (MAR) phenotypes were In this study, the contamination degree of milk by the determined for S. aureus (Table 4). The predominant isolated bacteria is utterly worsened at each critical sam- MAR phenotypes for S. aureus isolated from this study pling point. High contamination level was observed at area were PG-TE -Ox and PG-TE-AC-E-SXZ-Ox in market point sampled milk. The difference in isolation 24.1 % and 17.2 % of the isolates, respectively. Furthermore, rate across market chain (critical sampling points) is sta- MAR phenotypes PG-TE- AC-Ox, PG-TE-AC-E-SXZ-Ox- tistically significant in E. coli. (P= 0.00) and Proteus sp. CN, PG-TE-AC-E-SXZ-Ox- CN-CH and PG-TE-AC-E- (P = 0.016) (Table 1). SXZ-Ox- CN- VA were obtained in 3.4 % of the isolates. Results of the present study revealed that 49 (40.8 %) Also PG-TE-AC 6.9 %, PG-TE-Ox- KF 13.8 % and PG-TE- of milk sampled had at least two different bacterial or- AC-E-Ox 6.9 % were the MAR phenotypes for S. aureus ganisms, 6 (12 %) from the udder, 10(20.4 %) milking isolated from this study area (Table 4). bucket, 12 (24.5 %) from storage container and 21 It is thus evident that MAR S. aureus was isolated (42.9 %) from the market point (Fig. 1). from all critical sampling points. However, among the isolates from this study area 55.2 % of the isolates de- Antimicrobial susceptibility of the bacterial isolates velop MAR. Among all MAR phenotypes of S. aureus, The antimicrobial susceptibility tests of the bacterial 40.3 % of them were resistance to six different antibi- isolates were grossly very variable. About 76.1 % E. coli otics and 7.2 % were resistance to seven antibiotics. Fifty isolates were resistant and it had the highest resistance four percent (54 %) of them were resistance to 3 or 4 rates to Doxycycline, Ampicillin and Gentamycin antibiotics. (42.3 %, 30 % and 30 %) respectively. A quarter of E. coli isolates (25.4 %) were multidrug resistant (≥3drugs). Discussion Similarly higher antimicrobial resistance (74.6 %) was re- The outcome of our study revealed that 84.1 % of milk corded against Salmonella sp. isolates as well. The high- samples were contaminated with at least one bacterium est resistance rate to Salmonella sp. was observed in that comprised of E. coli, Salmonella sp., Shigella sp., S. Amoxicillin (50 %). The highest resistance rate to aureus, and Proteus Sp., with isolation rates of 70(58 %), shigella was observed in Ampicillin (38.1 %). All shigella 4(3.3 %), 21(17.5 %), 29(24.2 %), and 9(7.5 %), respectively. isolates were highly susceptible to Co-trimoxazole(81 %). The levels of contamination with each isolated bacteria About 14 % of Shigella isolates were multidrug resistant- were higher across critical sampling points (from teats, fairly better than E.coli isolates. All Proteus sp. isolates Milking bucket, transportation container, and at market were 66.7 % sensitive to Ciprofloxacin and showed points). Similar findings reported by Daka et al.(2012) (55.6 %) resistance to Ampicillin (Table 2). revealed that the level of contamination with S.aureus Table 1 Occurrence of isolated bacteria across critical sampling points (n = 120) of milk collected from Jigjiga city Bacteria Isolate No. of positive sample (%) P- value isolated No(%) Udder (n = 30) Collection Bucket(n = 30) Storage Material(n = 30) Market point (n = 30) E. coli 70(58 %) 9(30 %) 16(53.3 %) 22(73.3 %) 23(76.7 %) 0.00 Salmonella sp. 4(3.3 %) 1(3.3 %) 0(0.0 %) 1(3.3 %) 2(6.7 %) 0.140 Shigella sp. 21(17.5 %) 4(13 .3 %) 3(10 %) 4 (13.3 %) 10(33.3 %) 0.069 S. aureus. 29(24.2 %) 6(20 %) 7(23.3 %) 6(20 %) 10(33.3 %) 0.727 Proteus sp. 9(7.5 %) 0(0.0 %) 2 (6.7 %) 3(10 %) 4(13.3 %) 0.016 Reta et al. International Journal of Food Contamination (2016) 3:4 Page 5 of 9 Fig. 1 Isolation of mixed bacteria from milk sampled at different sampling points (n = 120), Jigjiga city were higher in milk obtained from teat(17.9 %), Milking (24.4 %), S. aureus (38.2 %), and Salmonella sp. (2 %), from bucket at farm level(25.7 %), storage containers at milk apparently normal milk samples from different critical sam- collection center(26.9 %) and from transportation con- plingpoints. It is knownthatevenwhendrawnunderasep- tainer(21.8 %). Conditions for contamination of raw milk tic condition, milk always contains microorganisms which at different critical points are due to less hygienic prac- are derived from the milk ducts in the udder, in addition tices in pre-milking udder preparation, sub-optimal hy- contaminants coming from milking utensils and human giene of milk handlers, and poor sanitation practices handlers (Solomon et al. 2013). Higher isolation frequen- associated with milking and storage equipments, higher cies, especially for E. coli across market chain was observed environmental contamination during transportation or in the current study as compared to similar studies per- contamination during waiting along the roadside (Garedew formed to assess bacteriological quality of raw milk in et al. 2012). Based on observations made during the collec- Ethiopia (Tassew and Seifu 2011; Tiruneh 1996). This tion of samples, we therefore report that improper hygiene might be due to poor and unhygienic bedding condition in and poor farm management practices contributed to the the majority of farms and absence of teat dipping and disin- presence of these isolated bacteria in the milk. In this study fection practices in the current study. These practices have area milk was obtained from animals by washing their been known as critical components of mastitis prevention hands and/or the utensils and containers used. In certain and control program in dairy herds (Galton et al. 1986). cases, untreated groundwater was used to wash the con- Other findings by different researchers confirm that E. coli tainers that were used for milking. This may have contrib- grow well in milk and hence endanger its keeping milk uted to the high level of enteric bacteria and S.aureus quality (Frazeir and Westhoff 1988). E. coli and coliforms isolated. Improving the hygienic conditions of the milking are often used as indicator microorganisms, and the environment and/or utensils may reduce the prevalence of presence of E. coli in milk samples implies a risk that entropatogenic as well as S.aureus in milk and prevent its other enteropathogenic bacteria may be present in the transmission to humans. Olatunji et al (2009) in Nigeria sample (Najib 2003; Olatunji et al. 2009; Arafa and had reported that higher isolation frequencies of E. coli Soliman 2013). Table 2 Antibiotic sensitivity pattern of bacterial isolates in milk samples collected from Jigjiga city Antimicrobial E. coli(n = 70) Salmonella sp.(n = 4) Shigella sp.(n = 21) Proteus sp.(n = 9) R (%) I (%) S (%) R(%) I (%) S (%) R (%) I (%) S (%) R (%) I (%) S (%) Amoxicillin 15(21.4) 31(44.3) 24(34.3) 2(50) 1((25) 1(25) 2(9.5) 11(52.4) 8(38.1) 1(11.1) 6(66.7) 2(22.2) Ampicillin 21(30) 28(40) 21(30) 1(25) 1(25) 2(50) 8(38.1) 8(38.1) 5(23.8) 5(55.6) 3(33.3) 1(11.1) Ciprofloxacin 7(10) 17(24.3) 46(65.7) 1(25) 1(25) 2(50) 2(9.5) 6(28.6) 13(61.9) 0(0.0) 3(33.3) 6(66.7) Co-trimoxazole 12(17.1) 22(31.4) 36(51.4) 1(25) 2(50) 1(25) 0(0.0) 4(19.0) 17(81) 1(11.1) 3(33.3) 5(55.6) Chloramphenicol 15(21.4) 26(37.1) 29(41.4) 1(25) 2(50) 1(25) 3(14.3) 7(33.3) 11(52.4) 1(11.1) 5(55.6) 3(33.3) Gentamycin 21(30) 35(50) 14(20) 1(25) 2(50) 1(25) 4(19.0) 10(47.6) 7(33.3) 2(22.2) 4(44.4) 3(33.3) Doxycycline 30(42.9) 27(38.6) 13(18.5) 1(25) 2(50) 1(25) 6(28.6) 10(47.6) 5(23.8) 2(22.2) 3(33.3) 4(44.4) R Resistance, I Intermediate, S Sensitive, n number Reta et al. International Journal of Food Contamination (2016) 3:4 Page 6 of 9 Table 3 Antimicrobial susceptibility pattern of S. aureus isolates (n = 29) from milk samples collected from Jigjiga city Antimicrobial Susceptible number (%) Intermediate number (%) Resistant number (%) Pencillin (P) 1(3.4) 1(3.4) 27(93.1) Chloroamphenicol (CH) 11(37.9) 10(34.5) 8(27.6) Cephalothin (KF) 15(51.7) 5(17.2) 9(31.0) Gentamycin (CN) 10(34.5) 12 (41.4) 7(24.1) Erythromycin (E) 5(17.2) 17(58.6) 7(24.1) Clindamycin (DA) 9(31.0) 16(55.2) 4(13.8) Tetracycline (TE) 5(17.2) 4 (13.8) 20(69) Amoxicillin + clavulanic 19(65.5) 0(0.0) 10(34.5) Rifampicin (RD) 27(93.1) 1 (3.4) 1(3.4) Oxacillin (OX) 18(62.1) 2(6.9) 9(31.0) Vancomycin (VA) 24(82.8) 3(10.3) 2(6.9) Sulphamethoxazole-timethoprim (SXZ) 19(65.5) 3 (10.3) 7(24.1) Our results indicated that 24.2 % of the samples were presence of S. aureus in the milk. In this study low positive for S.aureus. This is a favorable finding because, salmonella sp. isolation rate with 3.3 % was found. for human health some strains of S. aureus are capable Junaidu et al (2011), Forough et al. (2012) and Sanaa et of producing heat stable enterotoxins (Asperger 1994). al.(2005) had reported comparable findings with 2.17 %, A comparable finding to our result was reported by 4 % and 1.43 % prevalence respectively. Addis et al. Abebe et al. (2013) that S.aureus prevalence was 15.5 % (2011) reported a prevalence of 10.7 % from raw milk in raw milk samples. In contrast to this, different lite- which is higher than the present report. In the other ratures revealed a very significant isolation rate of S. study by Addis et al. (2011) from 195 dairy cows tested aureus from raw milk samples (Olatunji et al. 2009; 28.6 % were positive from milk samples. Akoachere et Pourhassan and Taravat-Najafabadi 2011; Mohanty et al. al.(2009) in Cameroon reported a high prevalence (27 %) 2013; Sanaa et al. 2005). Although the prevalence of S. of Salmonella among cattle. This may be due to the differ- aureus has been reported to vary with the size and geo- ence in the living condition, like housing conditions, feed- graphic region of the area sampled, a high proportion of ing habits, types of feed given for the cattle, of the two these bacteria in milk relates to poor hygiene practices. cattle populations. The detection of Salmonella in 3.3 % of Based on observations made during the collection of the samples tested indicates that the degree of prevalence samples, we therefore report that improper hygiene and of the pathogen in raw milk in jigjiga is relatively higher poor farm management practices contributed to the than originally believed. Although contamination of dairy products currently accounts for a small percentage of foodborne illness, it is clear that raw milk consumption Table 4 The predominant MAR phenotypes for S. aureus and the consumption of products made with raw milk isolated from milk samples (n = 29) collected from Jigjiga city present some risk. Although proper pasteurization MDR patterns Phenotype Number Percentage observed minimizes these risks to the public, there is a small but Three PG-TE-AC 2 6.9 growing group of people that consume unpasteurized milk or milk products, either for practical or cultural reasons, PG-TE -Ox 7 24.1 or because of perceived health benefits (Karns et al. 2005). Four PG-TE-Ox- KF 4 13.8 Although the levels of Salmonella in the milk samples PG-TE- AC-Ox 1 3.4 tested here seemed to be very low and the infectious Five PG-TE-AC-E-Ox 2 6.9 dose for this organism is low, the potential for this Six PG-TE-AC-E-SXZ-Ox 5 17.2 organism to grow in improperly stored raw milk and Seven PG-TE-AC-E-SXZ-Ox- CN 1 3.4 in products made from raw milk presents a public health risk, particularly to susceptible members of the Eight PG-TE-AC-E-SXZ-Ox- CN- CH 1 3.4 population. PG-TE-AC-E-SXZ-Ox- CN- VA 1 3.4 The isolation rates of proteus Sp. in this study (7.5 %) The percentage representations of the phenotypes were obtained by dividing is comparable with the report by Junaidu et al (2011) the number of a particular phenotype by the total number of multiple antibiotic resistant isolates identified in a given area. VA Vancomycin, PG with 8.69 % prevalence. Most of the organisms identified Penicillin G, SXZ Sulphamethoxazole-timethoprim, E Erythromycin, Ox Oxacillin, in this study were enteric bacteria indicating probable AC Amoxicillin-Clavulanic Acid, TETetracycline, KF Cephalothin; CN Gentamycin, CH Chloroamphenicol faecal contamination of the milk as a result of poor Reta et al. International Journal of Food Contamination (2016) 3:4 Page 7 of 9 hygiene. The practice of pooling milk from different S. aureus isolates to vancomycin, rifampicin, clindamycin sources by traders, and the absence of pasteurization and gentamycin showed less than 25 % of resistance and generally observed among them could increase the risk this is similar with the report of Ma et al. (2006) from the posed by such organisms. dairy farm in Taiwan. The reason why these antimicrobials In the present study, Doxycycline had the highest re- were less resistant might be they are not frequently used sistance rates in E. coli. In contrast to this, fairly higher in the study area in veterinary services, and perhaps in hu- resistant rate was recorded in Ampicillin (100 %) and man medicine. Similar suggestion was given by Jaims et Amoxicillin (42.11 %) (Thaker et al. 2012). On the other al.(2002) that the development of antimicrobial resistance hand the highest resistance rate for Salmonella sp. in is nearly always as a result of repeated therapeutic and/or this report was observed to Amoxicillin (50 %). Different indiscriminate use of them. However, the present study researchers reported antimicrobial resistant Salmonella has demonstrated the existence of alarming level of resist- isolates of milk in their previous studies from Ethiopia ance of S. aureus to commonly used antimicrobials (pen- (Molla et al. 2003; Mekonnen et al. 2005) and from cillin G and tetracycline) in the study area. This is due to other countries (White et al. 2001). Forough et al. (2012) the fact that tetracycline and penicillin are frequently and reported that salmonella sp. isolates were resistance to improperly used antimicrobials in animal and human Ampicillin (42.58 %), Tetracycline (42.58 %) and Nalidixic treatment. The results were in accordance with reports acid (78.57 %). Addis et al. (2011) reported a high resist- from earlier studies in other countries (Jakee et al. 2008; ance rate salmonella isolates to ampicillin (100 %). The re- Edward et al. 2002; Gentilini et al. 2002) suggesting a pos- markable degree of resistance to many drugs represents sible development of resistance from prolonged and indis- public health hazard due to the fact that food borne out- criminate usage of some antimicrobials. This is in contrast breaks would be difficult to treat and this pool of MDR with the report of Ma et al. (2006) on his report with re- Salmonella in food supply represents a reservoir for the spect to pencillin and tetracycline in Taiwan. This is not transferable resistant genes (Diaze De Aguayo et al. 1992). surprising because penicillin G and tetracycline are the The reasons for the recovery of antimicrobial resistance most commonly used antimicrobials for the treatment of Salmonella isolates were most likely due to the indiscrim- infection or mastitis in veterinary practice in Ethiopia. inate use of antimicrobials (WHO 1988), self-medication Moreover, penicillin resistance is plasmatic and, it spread and administration of sub therapeutic dose of antimi- out very quickly to several other strains. Pereira et al. crobials to livestock for prophylactic purpose (Acha and (2009) showed that 70 to 73 % of S. aureus strains isolated Szyfers 2001). Antimicrobial use in animal production from various foods were resistant to β-lactam such as pen- systems has long been suspected to be a cause of the cillin and ampicillin. Staphylococci are frequently isolated emergence and dissemination of antimicrobial resistant from bovine mastitis which is one of the most common Salmonella (Forough et al. 2012). causes for the use of antimicrobial in lactating dairy cows. In this study the highest resistance rate for shigella Similarly, the present investigation indicated that the re- sp. was observed to Ampicillin (38.1 %) followed by sistance pattern of penicillin was found to be 93.1 % Doxycycline (28.6 %). In contrast to our finding Sanaa et (Table 3) which is similar to the finding made by Tariku et al. (2005) reported that shigella isolated from raw milk al.(2011) (87.2 %) in Ethiopia, Landin (2006) (80 %) in were sensitive to Gentamycin (64.3 %) followed by Sweden, Gooraninejad et al. (2007) (57 %) in Iran and Chloramphenicol (92.1 %), and the highest antimicrobial Myllys et al.(1998) (50 %) in Finland. This is in contrast to resistant pattern was observed in Ampicillin and Amoxi- findings observed by Adesiyun (1994) who reported 23 % cillin (92.9 %, 92.9 %) followed by penicillin (42.9 %). In of resistance to pencillin G in West India. agreement with our result Ayalu et al. (2011) reported Moreover, the present study showed the resistance of that shigella isolates were 100 % resistant to Ampicillin and S. aureus to tetracycline (69 %), amoxicillin-clavulinic Amoxicillin but sensitive to Chloramphenicol, Gentamicin, acid (34.5 %), oxacillin (31 %), cephalothin (31 %), chlor- and Norfloxacin(41.2 %, 88.2 %, and 94.1 %) respectively. amphenicol (27.6 %), sulphamethoxazole-trimethoprim Shiferaw et al. (2012) reported that 74 % shigella isolates (24.1 %), erythromycin (24.1 %), gentamycin (24.1 %), were resistant to Ampicillin, and 58 % to Streptomycin. On clindamycin (13.8 %) observed in milk samples taken the other hand, All the Shigella isolates were resistant to from dairy cows in jigjiga city. This is in accordance with Ampicillin, 94 % to Tetracycline, and 82 % to Ciprofloxacin the findings of Tariku et al.(2011) who reported resist- in a report by Debdutta et al. (2012). ance of S. aureus to amoxicillin-clavulinic acid (46 %), The observations made in the present study clearly chloroamphenicol (16 %), vancomycin (3 %), but it dis- proved that S. aureus showed resistance to all antimicro- agree with the observation made by Tariku et al. (2011) bials tested except for rifampicin and Vancomycin in the case of tetracycline(0 %), Co-trimoxazole (0 %) (Table 3). These indicate that the problem is highly distrib- and clindamycin (4 %) in dairy farms in Jimma town. uted and disseminated. Moreover, the overall resistance of The probable explanation could be that S. aureus strains Reta et al. International Journal of Food Contamination (2016) 3:4 Page 8 of 9 have the capacity to change their resistance behavior to human commensal, and multidrug resistant S. aureus may the exposed antimicrobials. present without clinical illness. However, when they cause With a particular emphasis to tetracycline, the present infection they are extremely serious. Furthermore, dairy observation agrees with preliminary finding conducted cows become infected with multidrug resistant S. aureus, by Bayhun (2008) (55.3 %). However, apparent difference therefore diagnosis of S. aureus does not have implication was observed in the report of Tariku et al.(2011) (0 %). for treatment only but also it indicates zoonotic trans- This is due to the fact that tetracycline is the most com- mission since it becomes reservoir for human infection. In monly used antimicrobial in the treatment of infections practice of indiscriminate use of drugs should be con- in the livestock sector in Ethiopia. Moreover, tetracycline trolled. Further studies that could incorporate isolation of is widely used as growth factors in veterinary medicine milk contaminating bacteria to the species level should be for livestock rearing as well in the treatment of bacterial done to evaluate the imminent danger posed by microbes infection occurring in human medicine (Ardic et al. from milks. 2005). Furthermore, the resistance profile of S. aureus to Acknowledgements amoxicillin-clavulinic acid and oxacillin in milk samples Jigjiga University is greatly acknowledged for funding and providing all was found to be high. This is due to the fact that resist- rounded technical assistance for the smooth accomplishment of this research. The authors are also highly indebted to Ethiopian Somali Regional Health ance of S. aureus to pencillin G, amoxicillin and oxacillin Bureau, Regional Public Health and Research Laboratory for laboratory facilities. may be attributed to the production of β-lactamase, an enzyme that inactivates pencillin and closely related Authors’ contributions antimicrobial. It is believed that about 50 % of mastitis MA carried out the conception of the research concept and designed the methodology, data analysis and interpretation and preparation of the causing S. aureus produces β-lactamase (Green and manuscript for publication. TW carried out the laboratory work, sample Bradely 2004). Likewise, S. aureus showed resistance to collection and revision of the manuscript. AN critically revised the proposal, vancomycin and clindamycin. This might indicate trans- designed the methodology, and reviewed the manuscript. All authors read and approved the final manuscript. fer of resistant strain among environment, livestock and human since this antimicrobials are not used in veterin- Competing interests ary practice. The authors declare that there is no financial or non-financial competing The MAR phenotypes (Table 4) obtained in the study interest from anybody or institute. We also want to assure that we did not receive any technical assistant in developing the research concept or correlated with the percentage of antibiotic resistance. preparation of the manuscript. Although the development of resistance to a particular antibiotic depends on the level of exposure to the antimi- Author details Faculty of Health Science, Department of Nursing, Woldia University, P.O.Box crobials, (Rychlik et al. 2006) there are many other factors 400, Woldia, Ethiopia. College of Natural Sciences, Department of Biology, that are involved. We are therefore suggesting that mo- 3 Wolkite University, P.O.Box 07, Wolkite, Ethiopia. College of Veterinary lecular methods be used to characterize these isolates for Medicine, Department of Veterinary Microbiology and Public Health, Jigjiga University, P.O.Box 1020, Jigjiga, Ethiopia. the presence of antibiotic-resistance determinants, which may provide data to support our conclusions. S. aureus is Received: 3 February 2016 Accepted: 13 May 2016 normally resident in humans; therefore, the S. aureus present in the cow’s milk may have resulted from trans- References mission from humans, which raises questions regarding Abebe B, Zelalem Y, Ajebu N. Hygienic and microbial quality of raw whole cow’s the hygiene practices followed. milk produced in Ezha district of the Gurage zone, Southern Ethiopia. Wudpecker J Agric Res. 2012;1(11):459–65. Abebe M, Daniel A, Yimtubezinash W, Genene T. Identification and antimicrobial Conclusions susceptibility of S. aureus isolated from milk samples of dairy cows and nasal This study revealed that raw cow’s milk in the study area swabs of farm workers in selected dairy farms around Addis Ababa, Ethiopia. could be an important source of infection with a wide Afr J Microbiol Res. 2013;7(27):3501–10. Acha PN, Szyfers B. Zoonoses and Communicable Diseases Common to Man and range of organisms, particularly enteropathogens. An im- rd Animals: Bacteriosis and Mycosis. 3 ed.Vol I. Washington DC: Pan American portant source of microbial contamination of the milk is Health Organization; 2001. p. 233–46. faecal pollution probably from cow dung. There is the Addis Z, Kebed N, Worku Z, Gezahegn H, Yirsa A, Kassa T. Prevalence and antimicrobial resistance of Salmonella isolated from lactating cows and in need for instituting effective control measures to protect contact humans in dairy farms of Addis Ababa. BMC Infect Dis. 2011;11:222–8. public health. This includes mandatory milk pasteurization Adesiyun A. Characteristics of S. aureus strains isolated from bovine mastitic milk: by traders and improved hygienic handling of the Bacteriophage and antimicrobial agent susceptibility and enterotoxigenecity. J Vet Med. 1994;42:129–39. commodity during milking, ensuring milking is not done Adesiyun AA, Webb L, Rahman S. Microbiological quality of raw cow’s milk at on cow dung. The occurrence of multidrug resistance S. collection centers in Trinidad. J Food Prot. 1995;58:139–46. aureus should be under consideration during selection of Akoachere TKJ, Tanih FN, Ndip ML, Ndip RN. Phenotypic Characterization of Salmonella Typhimurium Isolates from Food-animals and Abattoir Drains in antimicrobials for treatment of mastitis especially if the Buea, Cameroon. J Health Popul Nutr. 2009;27(5):612–8. possibility exists in the transfer of resistance in or between Aneja RP, Muthur BN, Chandan RC, Banerejee AK. Technology of Indian milk microbial species. Moreover, S. aureus is a common products. New Delhi: Dairy Indian Yearbook; 2002. p. 183–96. Reta et al. International Journal of Food Contamination (2016) 3:4 Page 9 of 9 Arafa M, Soliman M. Bacteriological Quality and Safety of Raw Cow’s Milk and Junaidu AU, Salihu MD, Tambuwal FM, Magaji AA, Jaafaru S. Prevalence of Fresh Cream. Slov Vet Res. 2013;50(1):21–30. Mastitis in Lactating Cows in some selected Commercial Dairy Farms in Ardic N, Ozyurt M, Sareyyupoglu B. Investigation of erythromycin and tetracycline Sokoto Metropolis. Adv Appl Sci Res. 2011;2(2):290–4. resistance genes in Methicillin-resistant Staphylococci. Int J Antimicrob Karns JS, Van Kassel JS, McKluskey BJ, Perdue M. Prevalence of Salmonella enteric Agents. 2005;26:213–8. in bulk tank milk from US dairies as determined by Polymerase Chain Asperger H. Stapylococcus aureus. In: The Significance of Pathogenic Microorganisms Reaction. J Dairy Sci. 2005;88:3475–9. in Raw Milk, International Dairy Federation. Brussels: IDF; 1994. p. 24–42. Landin H. Treatment of mastitis in Swedish dairy production (in Swedish with English summary). Svensk Veterina¨rtidning. 2006;58:19–25. Ayalu AR, Berhanu S, Jemal Y, Gizachew A, Sisay F, Jean MV. Antibiotic Ma Y, Chang SK, Chou CC. Characterization of bacterial susceptibility isolates in susceptibility patterns of Salmonella and Shigella isolates in Harar, Eastern sixteen dairy farms in Taiwan. J Dairy Sci. 2006;1:55–6. Ethiopia. J Infect Dis Immun. 2011;3(8):134–9. Mekonnen H, Workineh S, Bayleyegne M, Moges A, Tadele K. Antimicrobial Bayhun S. Beta-lactamase activities and antibiotic resistance comparison of susceptibility profile of mastitis isolates from cows in three major Ethiopian Staphylococcus aureus isolated from clinic and food material (M.Sc Thesis). dairies. Med Vet. 2005;176(7):391–4. Gazi University, Institute of Science Technology;2008. p.126 Mennane Z, Ouhssine M, Khedid K, Elyachioui M. Hygienic Quality of Raw Cow’s Bouazza F, Hassikou R, Ohmani F, Hmmamouchi J, Ennadir J, Qasmaoui A, et al. Milk Feeding from Domestic. Int J Agri Biol. 2007;9(1):1560–8530. Hygienic quality of raw milk at Sardi breed of sheep in Morocco. Afr J Mohanty NN, Das P, Pany SS, Sarangi LN, Ranabijuli S, Panda HK. Isolation and Microbiol Res. 2012;6(11):2768–72. antibiogram of Staphylococcus, Streptococcus and E. coli isolates from clinical Claeys WL, Cardoen S, Daube G, Block JD, Dewettinck K, Katelijne Dierick K, et al. and subclinical cases of bovine mastitis. Vet World. 2013;6(10):739–43. Herman Raw or heated cow milk consumption. Rev Risks benefits Food Molla B, Alemayehu D, Salah W. Source and distribution of Salmonella serovars Control. 2013;31:251–62. isolated from food animals, slaughter house personnel and retail meat Clinical and Laboratory Standards Institute (CLSI). Performance Standards for products in Ethiopia: 1997-2002. Ethiopia J Health Dev. 2003;17:63–70. Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated From Myllys V, Asplund K, Brofeld E, Hirevela-Koski V, Honkanen-Buzalski T. Bovine Animals. Clin Lab Stand Inst. 2008;28:M31–A3. Mastitis in Filand in 1988 and 1995. Changes in Prevalence and Antimicrobial Cruikshank R, Duguid JP, Marmoin BP, Swain RH. Medical microbiology. 12th ed. resistance. Acta Vet Scand. 1998;39:119–26. New York: Longman Group Limited; 1975. p. 34. Najib G. Risk assessment of dairy products, Consumer committee seminar,Msca CSA (Central Statistical Authority). Central Statistical Authority, Federal Democratic Tour Municipality MM Service, Columbia USA. 2003. Republic of Ethiopia, Central Statistical Investigatory, Statistical Report. Olatunji EA, Ahmed I, Ijah UJ. Evaluation of microbial qualities of skimmed milk Addis Ababa: Central Statistical Authority of Ethiopia; 2003 th (nono) in Nasarawa State, Nigeria. Proceeding of the 14 Annual Conf. of Daka D, Gebresilassie S, Yihdego D. Antibiotic-resistance Staphylococcus aureus th th Ani.Sc. Asso. of Nig. (ASAN) LAUTECH Ogbomoso, Sept. 14 -17 , 2009. 2009. isolated from cow’s milk in the Hawassa area, South Ethiopia. Ann Clin Pereira V, Lopes C, Castro A, Silva J, Gibbs P, Teixeira P. Characterization for Microbiol Antimicrob. 2012;11:26. Enterotoxins production, virulence factors and antibiotic susceptibility of S. Debdutta B, Sugunan AP, Haimanti B, Thamizhmani R, Sayi DS, Thanasekaran K, et al. aureus isolates from various food in Portugal. Food Microbiol. 2009;26:278–82. Antimicrobial resistance in Shigella -rapid increase & widening of spectrum in Pourhassan M, Taravat-Najafabadi ART. The spatial distribution of bacterial Andaman Islands, India. Indian J Med Res. 2012;135(3):365–70. pathogens in raw milk consumption on Malayer City, Iran. Shiraz E Med J. Diaze De Aguayo ME, Duarte AB, Montes De Oca Canastillo F. Incidence of 2011;12:2–10. multiple antibiotic resistant organisms isolated from retail milk products in Robinson RK. Dairy Microbiology Handbook: The Microbiology of Milk and Milk Hermosillo, Mexico. J Food Prot. 1992;55(5):370–3. Products. 3rd ed. USA: John Wiley & Sons, Inc; 2002. p. 51–305. Edward M, Anna K, Michal K, Henryka L, Krystyna K. Antimic-robial susceptibility Rychlik I, Gregorova D, Hradecka H. Distribution and function of plasmids in of staphylococci isolated from mastitic cows. Bull Vet Inst. 2002;46:289–94. Salmonella enterica. Vet Microbiol. 2006;112(1):1–10. Fadaei A. Bacteriological Quality of Raw Cow Milk in Shahrekord, Iran. Veterinary Sanaa OY, Nazik EA, Ibtisam EM, Zubeir EL. Incidence of Some Potential World. 2014;7(4):240–3. Pathogens in Raw Milk in Khartoum North (Sudan) and Their Susceptibility to Forough T, Elahe T, Manochehr M, Ebrahim R, Rafie S. Occurrence and Antibiotic Antimicrobial Agents. J Anim Vet Adv. 2005;4(3):356–9. Resistance of Salmonella spp Isolated from Raw Cow’s Milk from Shiferaw B, Solghan S, Palmer A, Joyce K, Barzilay EJ, Krueger A, Cieslak P. Shahahrekord, Iran. Int J Microbiol Res. 2012;3(3):242–5. th Antimicrobial susceptibility patterns of Shigella isolates in Foodborne Frazeir WC, Westhoff DT. Food Microbiology (4 ed). Singapore: Mcgraw-Hill Book Diseases Active Surveillance Network (FoodNet) sites, 2000-2010. Clin Infect Company; 1988. p. 419–28. Dis. 2012;54 Suppl 5:S458–63. Galton DM, Petersson LG, Merril WG. Effects of Pre- Milking Udder Preparation on Solomon M, Mulisa M, Yibeltal M, Desalegn G, Simenew. Bacteriological quality of Bacterial Counts of in Milk and on Teat. J Dairy Sci. 1986;69:260–6. bovine raw milk at selected dairy farms in Debre Zeit town, Ethiopia. Compr Garedew L, Berhanu A, Mengesha D, Tsegay G. Identification of gram-negative J Food Sci Technol Res. 2013;1(1):1–8. bacteria from critical control points of raw and pasteurized cow milk consumed Soomro AH, Arain MA, Khaskheli M, Bhuto B. Isolation of E. coli from raw milk at Gondar town and its suburbs, Ethiopia. BMC Public Health. 2012;12:950. and milk products in relation to public health sold under market conditions Gentilini E, Danamiel A, Betancor M, Rebuelto MR, Fermepin RM, Detorrest RA. at Tandonjam, Pakistan. J Nutr. 1996;1(3):151–2. Antimicrobial susceptibility of coagulase-negative Staphylococci isolated Tariku S, Jemal H, Molalegne B. Prevalence and susceptibility assay of from bovine Mastitis in Argentina. J Dairy Sci. 2002;85:1913–7. Staphylococcus aureus isolated from bovine mastitis in dairy farms in Jimma Godefay B, Molla B. Bacteriological quality of raw milk from four dairy farms and town South West Ethiopia. J Anim Vet Adv. 2011;10:745–9. milk collection center in and around Addis Ababa, Berl. Münch Tierarztl Tassew A, Seifu E. Microbial Quality of Raw Cow’s Milk Collected from Farmers Wschr. 2000;113:1–3. and Dairy Cooperatives in Bahir Dar Zuria and Mecha District, Ethiopia. Agric Gooraninejad S, Ghorbanpoor M, Salati AP. Antibiotic Susceptibility of Staphylococci Biol J N Am. 2011;2(1):29–33. isolated from bovine sub-clinical mastitis. Pak J Biol Sci. 2007;10:2781–3. Thaker HC, Brahmbhatt MN, Nayak JB. Study on occurrence and antibiogram Green M, Bradely A. Clinical Forum- S. aureus mastitis in cattle UK. VET. 2004;9:4. pattern of E. coli from raw milk samples in Anand, Gujarat, India. Vet World. Hendriksen RS. A global Salmonella surveillance and laboratory support project 2012;5(9):556–9. of the World Health Organization: Laboratory Protocols (Susceptibility testing Tiruneh Z. A Study on Bovine Subclinical Mastitis at Stella Dairy Farm, DVM of Salmonella using disk diffusion). 2002. p. 3. Thesis. Addis Ababa: Faculty of Veterinary Medicine, Addis Ababa University; Hendriksen RS. A global Salmonella surveillance and laboratory support project 1996. p. 30–45. of the World Health Organization: Laboratory Protocols (Isolation of White DG, Zhao S, Sudler R, Ayers S, Friedman S, Chen S, et al. The isolation of Salmonella). 2003. p. 4. antibiotic-resistant Salmonella from retail ground meats. New Engl J Med. Jaims E, Montros LE, Renata DC. Epidemiology of drug resistance; the case of 2001;345(16):1147–54. Staphylococcus aureus and Coagulase negative Staphylococci infections. Salud World Health Organization. Salmonellosis Control: The Role of Animal and Publica Mex. 2002;44:108–12. Product Hygiene, Technical Report Series, 774. Geneva: WHO; 1988. Jakee J, Ata S, Nagwa M, Bakry SA, Zouelfakar EE, Gad El-Said WA. Characteristics of S. aureus strains isolated from human and animal sources. Am-Euras J Agric Environ Sci. 2008;4:221–9.

Journal

International Journal of Food ContaminationSpringer Journals

Published: May 26, 2016

There are no references for this article.