Description of the study area
The study was conducted in two areas in central Oromia, namely Bishoftu and Sebeta towns. Bishoftu town is located in East Shewa zone of the Oromia regional state, and the area is located at a latitude and 40°E longitude of 8°45′N 38°59′E at an altitude of 1920 m above sea level in central high lands of Ethiopia (39). Sebeta (Oromo: Sabbataa) town is located in the Oromia special zone surrounding Finfinne (Addis Ababa) of the Oromia region, Ethiopia; approximately 25 km southwest of Addis Ababa with a latitude and longitude of 8°54′40″N 38°37′17″E and an altitude of 2,356 m above sea level (40). Farmers in the vicinity of Bishoftu and Sebeta practice a mixed crop and livestock farming system (41).
Study design and sampling strategy
A cross-sectional study was conducted from February to May 2015 to isolate S. aureus from milk collected from randomly selected lactating dairy cows found in smallholder dairy farms of Bishoftu and Sebeta towns. A convenience sampling strategy was used to select 23 smallholder dairy farms (14 from Bishoftu and 9 from Sebeta). A total of 209 randomly selected lactating dairy cows, 114 from Bishoftu and 95 from Sebeta, were used for sampling.
A pretested questionnaire (see additional file 4) (translated to local languages; Afaan Oromo and Amharic) was also used to assess the type of antibiotic commonly prescribed in the study dairy farms (n= 23) by interviewing veterinarians or farm animal health professionals (if getting contacted) or a farm manager or farm owner or farm supervisor, by auditing the medicines or empty bottles found in the farms, and by collecting data from the patient case book available in each of the selected dairy farms at the study area. We also assessed professional's perception about brands of antibiotics and their prescribing practices of penstrep, the antibiotics commonly used in the dairy farms and at veterinary clinics in the study areas. The veterinarian’s perception of antibiotic quality and clinical efficacy in comparison with brand antibiotics, country of manufacturer, and knowledge of antibiotic and its use, were assessed. Both private and government employee clinicians were selected for the interview. A total of 30 volunteer veterinarians who are practicing drug prescribing in the government and or private veterinary clinics or rendering ambulatory private clinical services in the study area were included in the study.
Following the recommendations by Newton et al (2009) guidelines for field surveys of quality of medicines (42), three different brands of penstrep were collected from randomly selected veterinary pharmacy located in the Bishoftu town. All three brands (see Table 3) were legally registered and approved for use in veterinary medicine in Ethiopia. The brands were selected by surveying for their availability in the veterinary clinics and or pharmacies/drug stores in the study area, and by assessing the most widely prescribed brands by veterinary clinicians and the most commonly found in the smallholder dairy farms. All the selected products were evaluated and passed the recommended assessment for pharmaceutical product characteristics: drug name, company name, country of production, batch number, legal permission, preparation, color inspection, manufacturing and expiry dates, and availability of label inserts (43).
Table 3. Characteristics by strength, country of origin and average price of brands of penstrep used for efficacy evaluation against S. aureus.
Laboratory examinations
The isolation and identification of S. aureus from the dairy milk samples were done following the Standard Operating Procedures recommended for microbiological technique (44). Then, the three brands of penstrep, mentioned in table 1, were used for efficacy evaluation. Both the microbiological analysis of the sample and efficacy evaluation were performed at Addis Ababa University College of Veterinary Medicine and Agriculture (AAU-CVMA), Bishoftu.
Preparation and impregnations of the antibiotic disks
The standard commercial disk for both penicillin (10 units) and streptomycin (10µg) were obtained commercially (Oxoid, UK). Known volume of different brands of penstrep was diluted at the time of disc preparation using sterile distilled water to obtain the working solution equivalent to the concentrations of the commercial standard disks. Tubes filled with sterile distilled water were used to make dilution till the concentration of the antibiotic solutions reaching10µg (20 µl) (45).
The sterile disks were placed in petri dishes and a fixed volume of 20µl or 0.02ml solution of each prepared brands of penstrep was loaded on each disc one by one using a pipette.
The impregnated discs were arranged in separate plates and dried by placing them in an incubator at a temperature of 37°C for 2 hours (45).
In-vitro antimicrobial susceptibility testing
The efficacy of the prepared antibiotics was tested against the test organism using recommended test protocols for the Kirby-Bauer disc diffusion method. In this study, a day-old young subculture of the test organisms S. aureus (n = 43) isolated from cow milk were used. The antibiotic susceptibility patterns of the isolates to different brands of penstrep were evaluated using the agar-disk diffusion method on MHA (5,30,46).
The inoculums were prepared from the cultures and were matched for turbidity with 0.5 McFarland suspension, and spread onto an MHA plates. The prepared antibiotic discs were aseptically placed on the inoculated agar plate along with the commercially available discs for comparison of the efficacy of the prepared discs. The plates were then incubated at 37 °C overnight and then measured for a diameter of the zones of inhibition (5), which is related to minimum inhibitory concentration (MIC) for that particular bacterium/antimicrobial combination; the zone directly correlates inversely with the MIC of the test bacterium. However, this depends on the concentration of the antibiotic in the disk and its infusibility (47).
Interpretation of disc diffusion testing results was performed following the Clinical and Laboratory Standards Institute (CLSI). For the three brands of penstrep, streptomycin 10µg and penicillin-G 10-unit disks were used as a standard disc. Finally, the diameters of the zone of inhibition around the disks were measured to the nearest millimeter using a digital caliper, and the isolates were classified as susceptible, intermediate and resistant according to the interpretative standards of CLSI (2015) and Magiorakos et al (2012) (48,49).
Statistical analysis
Data were entered into a Microsoft Excel dataset created specifically for the study using Microsoft Office 365 and statistical analyses were conducted using RStudio software Version 3.4. Categorical variables were expressed by proportions and their significance was assessed, when appropriate, using Chi-square ( χ2) test or Fisher’s exact test. This is used to test potential associations between categorical variables and P-value of ≤ 0.05 was considered as statistically significant.
Questions were used to assess veterinarians’ knowledge about antibiotics and their use (n= 6) and perception about veterinary antibiotics quality and brand prescribing (n = 9) (The results of the antibiotic knowledge and perception about veterinary antibiotics quality and brand prescribing questions were expressed by the values of either 0 (not correct) or 1 (correct) or on a five-point Likert scale “Strongly agree/agree” or “Strongly disagree /disagree.” The percentage of correct answers for each knowledge questions and the statements Strongly agreed/Agreed by the respondents were calculated.
Continuous variables were expressed by means and standard deviations (SD) and assessed for statistical significance using Kruskal-Wallis chi-square test. Descriptive statistics were computed to summarize a data set for efficacy evaluation of different brands of penstrep. The results of the zone of inhibition recorded by the tested antibiotics and interpreted as an intermediate resistance (IR) was expressed by the value of resistance (R).