Antimicrobial Susceptibility Profiles of Bacteria Isolated from Traditionally Fermented Bovine Milk from Selected Farms of Kajiado-Central Sub-County, Kenya

G, gentamycin, and streptomycin. Klebsiella spp. were highly resistant to vancomycin (100%) and were sensitive to gentamycin (100%) and streptomycin (100%). Conclusion: The study showed a high prevalence of AMR in bacteria isolated from traditional milk consumed by the pastoralists and thus there is a high risk of zoonotic spread of the pathogenic bacteria. There is a need to educate the local households on strategies to minimize the occurrence of AMR in animals and also improve hygiene practices in the preparation of traditionally fermented milk.


Introduction
Globally, in both human and veterinary medicine, the efficacy of antibiotics is diminishing, posing a global challenge due to the widespread emergence of resistance, resulting in infections that are challenging to treat.The World Health Organization estimates that currently close to 5 million deaths annually are associated with antimicrobial resistance (AMR) and the United Nations (UN) predicts that by 2050, up to 10 million deaths could be caused by microbes having AMR 1,2 .The cost of AMR to national and international economies and their health systems is significant as it affects the productivity of the patients and their caretakers through prolonged hospital stays and the need for more expensive and intensive care 3 .Several studies project that AMR would cost more than $1 trillion annually by 2050 worldwide 1,2,4 .Thus, AMR is a key priority area identified by both national and international agencies and is mushrooming as a silent pandemic 5 .While poorly quantified, the burden of AMR is comparably higher in low-income countries including Kenya and the wider Sub-Saharan Africa 6 .
Although antibiotic use in livestock has largely contributed to improving their health and productivity, it has also played a significant role in the evolution of resistant bacterial strains 5 .Regarding zoonotic-resistant strains, they can end up getting transmitted from animals to human beings through products such as milk 7 .The burden of AMR has not been well studied amongst pastoralists, who are people who keep herds of animals under nomadic settings and consume products such as milk emanating from these animals.Bovine milk has over a long time been considered nature's single most complete food and is one of the most valuable and regularly consumed foods in many households 7 .Amongst the milk products consumed, traditional fermented milk is popular amongst pastoralists in Africa.The improper handling of raw milk and milk products including consumption of the same without proper formal or informal education on milk hygiene serves as a threat to the rising challenge 8 .Due to the poor storage facilities in the traditional setup, proliferation of the milk pathogens may ensue.In a recent study 9 , amongst the Maasai pastoralists in Kajiado Kenya, the traditional fermented milk was found to be contaminated with bacterial species including Klebsiella spp.(15.79%),Coagulase Negative Staphylococcus (14.91%),Pseudomonas spp.(14.04%),Staphylococcus aureus (11.4%),Salmonella (S.) typhi (7.89%), and Escherichia coli (4.39%).Studies conducted in Brazil, Egypt, Nigeria, and Zambia in traditionally fermented dairy products revealed that Staphylococcus spp., Listeria monocytogenes, Streptococcus spp., Pseudomonas spp., Escherichia coli (E.coli), Klebsiella spp., and Salmonella spp., among other bacteria, are prevalent contaminants in milk [10][11][12][13] .
This contamination of milk coupled with the use and misuse of antibiotics in the treatment of various diseases in the livestock have contributed to the build-up of antibioticresistant strains of some of the known zoonotic pathogens 14 .To date, only a handful of studies on the burden of AMR in bacterial isolates from traditional milk consumed by communities in Africa 15,16 .This study therefore aimed at determining the antimicrobial susceptibility profiles of bacteria isolated from traditionally fermented Maasai milk against commercial antibiotics and the risk factors that could be associated with the problem of AMR.

Ethical approval
The ethical approval was obtained from the JKUAT Institutional Ethical Review Committee (JKU/2/4/896B).The livestock farmers consented to participate in the study.

Study area
We did the study between May and October 2021 The study was conducted between October and December 2020 in Kajiado Central Sub-County, Kenya, which is characterized as semi-arid with an average annual temperature of 18.9°C.The region experiences an annual rainfall of 500 mm, following a bimodal pattern with short rains occurring from October to December and long rains from March to May.The population of the sub-county stands at 161,862 individuals and has an area of 4,239.50km².The predominant population is Maasai pastoralists keeping various livestock such as cattle, sheep goats, and donkeys.The latest census shows that the number of cattle in Kajiado Central Sub County was 95,534 17 .

Study design and sample size determination
A cross-sectional study design was implemented, collecting fermented indigenous cow milk from households randomly selected for participation.The sample size, determined using Cochran's formula, was set at 114 for the study of small population samples 19 .The farmers were randomly selected from the list provided by extension officers from the Directorate of Veterinary Services, Kajiado County, Kenya.

Fermented milk sample collection
At the household level, the fermented milk samples were stored in traditional milk guards locally known as Enkukuri.Each guard contained milk from one cow.The milk samples were collected aseptically and aliquoted in sterile 20ml vials, tightly closed, labeled, and transported into the JKUAT Microbiology laboratory in a cool box.In the laboratory, the milk was stored at -4 o C until it was analyzed.

Determination of pathogenic bacteria
The samples were cultured in selective media for the determination of bacteria present in the milk as described previously 9 .After incubation, individual pure colonies were identified and characterized using standard bacteriological methods.The isolates were frozen at -20 o C until further analyzed.

Antibiotic sensitivity testing
The present study employed the Kirby-Bauer Disk diffusion susceptibility test method 20 .Antimicrobial susceptibility testing was specifically conducted for the isolated Staphylococcus spp., E. coli, Klebsiella spp., and S. typhi.which were randomly chosen and were assumed to have zoonotic potential.Muller Hilton Agar plates were inoculated with pure culture isolates of the isolated suspensions with their concentration determined using 0.5% McFarland's standard.These bacteria were selected because they were assumed to have pathogenic potential.Inoculation was done by the spread plate method.The antibiotic disks were placed on the inoculated agar surface and the culture plates were then incubated while inverted at 37 o C for 24 hours 20 .The antibiotic disks (HI Media Ltd, India) used were tetracycline (30ug), gentamycin(10ug), chloramphenicol (50µg), ampicillin (10µg), vancomycin (20 ug), streptomycin (10ug), penicillin G (10IU) and kanamycin (30ug).The areas where the growth of bacteria was inhibited during susceptibility testing were measured with a ruler and interpreted based on the 2019 guidelines from the Clinical and Laboratory Standards Institute (CLSI) 21 .The outcomes were documented as either resistant or susceptible to the particular antibiotics.

analysis
The data was entered into a Microsoft Excel (Microsoft, USA) spreadsheet and subsequently transferred to Statistical Package for the Social Sciences (IBM, USA) for comprehensive statistical analysis.The analyzed data were presented in the form of percentages and frequencies, and the results were organized into tables.The proportion of bacterial isolates resistant to antibiotics was calculated as the number of isolates resistant to at least one antibiotic divided by the total number of bacterial isolates.

Results
A total of 6 pathogenic bacteria species were isolated from the milk and these included Streptococcus spp.(43.86%),Klebsiella spp.(15.79%),Coagulase Negative Staphylococcus (14.91%),Pseudomonas spp.(14.04%),Staphylococcus aureus (11.4%), S. typhi (7.89%) and E. coli (4.39%).These results are described in a previous publication 20 Among these bacteria, an evaluation of the antimicrobial susceptibility profiles was conducted specifically for the isolated S. aureus, Coagulase Negative Staphylococcus, E. coli, Klebsiella spp., and S. typhi.These bacteria, known to be prevalent in milk, were presumed to have zoonotic potential.

Discussion
This study was aimed at assessing the antimicrobial susceptibility profiles of bacteria present in fermented milk produced and consumed by Maasai pastoralists in Kajiado Central Sub-County, Kenya.A previous publication has already discussed the prevalent bacteria in the fermented milk 20 in the study area.These bacteria included S. aureus, Coagulase negative Staphylococcus, E. coli, S. typhi, Streptococcus spp., Lactobacillus spp., Pseudomonas spp.and Klebsiella spp.In the current study, Staphylococcus spp., Coagulase-negative Staphylococcus, E. coli, S. typhi, and Klebsiella spp.were selected randomly for antimicrobial susceptibility tests.
In this study, S. aureus revealed the highest resistance to Oxytetracycline and Streptomycin.Locally, studies carried out on fresh milk also showed that S. aureus had high resistance to Streptomycin at 46.9% 9 .High antibiotic resistance levels in S. aureus isolated from milk were also documented against oxytetracycline in India (98%) 21 and Italy (84%) 22 .In the present study, S. aureus displayed resistance to Kanamycin at rates close to that reported in India (60%) 21 but higher resistance was reported in Italy (88.0%) 22 .According to the local veterinary officers (personal communications) in Kajiado County, oxytetracycline and streptomycin are the commonly used antibiotics for the treatment of cattle diseases in the study area.Most often, these drugs are administered without professional prescription, and drug withdrawal periods are often not observed.This may lead to the accumulation of these drugs in the milk and can lead to the emergence of drug resistance.On the positive side, the S. aureus isolates in the current study were all sensitive to gentamycin and vancomycin and moderately sensitive to chloramphenicol and penicillin.A similar study carried out in the study area on fresh milk showed that the isolated S. aureus was sensitive to gentamicin (81.3%) and vancomycin (84.4%) 9 .In Ethiopia, isolates of S. aureus from milk were highly sensitive to gentamycin and chloramphenicol 23 .
Coagulase-negative staphylococci are currently posing a threat to global health due to increased cases of antibiotic resistance and being frequently associated with nosocomial infections 24,25 .The Coagulase Negative Staphylococcus species isolated from the current study were shown to be highly sensitive to ampicillin, chloramphenicol, penicillin G, gentamycin, and streptomycin.Sensitivity to tetracycline and oxytetracycline was moderate.These findings are in agreement with a study carried out in South Africa on unpasteurized cow milk locally consumed where all the isolates were generally susceptible to vancomycin, streptomycin, and ampicillin 26 .Another study carried out on goat milk sold in Juja Sub-County, Kenya recorded similar findings where CoNS had high sensitivity against gentamycin, chloramphenicol, and streptomycin 27 .However, these findings contradict those of similar studies carried out in Kajiado County, Kenya where CoNS resistance to vancomycin, gentamycin, and streptomycin varied from 3.7% to 32.7% 9 .
In the current study, the E. coli isolates exhibited multi-drug resistance patterns against vancomycin, tetracycline, oxytetracycline, and ampicillin.These results are comparable to a study carried out in dairy farms on bovine mastitis in Bangladesh 28 , where resistance to both ampicillin and oxytetracycline was high.In Ethiopia, E. coli isolates from milk were found to be resistant to Ampicillin 70% 29 and Tetracycline 40% 29 .Furthermore, a study carried out on E. coli isolated from raw milk in Northern Kenya indicated that 55% of them were resistant to Tetracycline 30 .However, in the current study, all E. coli isolates were susceptible to kanamycin, penicillin G, gentamycin, and streptomycin.The presence of E. coli in the finished products may be a result of poor techniques during milk handling and processing and is generally regarded as an indicator of fecal contamination 31,32 .
In the present study, the isolated Klebsiella spp.exhibited high sensitivity to vancomycin, streptomycin, gentamycin, chloramphenicol, kanamycin, and penicillin G.These findings are comparable to a study carried out in Egypt which involved buffalo and cow mastitis milk 33 .However, the latter study recorded a high prevalence of Klebsiella spp.resistant to Ampicillin (100%), chloramphenicol (83%), streptomycin (82.6%), and penicillin G (78.3%).The findings of the current study is also close to a study carried out in Pakistan where a Klebsiella spp.had high resistance to vancomycin (95%) 34 .Klebsiella spp.such as K. pneumoniae and K. oxytoca are important causes of bovine mastitis, and there is zoonotic potential for transmission to humans where it can cause serious morbidities 35 .
The findings from the present study indicated that S.
typhi isolates exhibited high sensitivity to chloramphenicol, penicillin G, gentamicin, and streptomycin.However, the bacteria demonstrated the highest resistance levels to ampicillin, vancomycin, oxytetracycline, tetracycline, and kanamycin.These findings are comparable to studies carried out in Ethiopia where Salmonella spp.resistance to ampicillin and tetracycline was 66.7-94.4% and 52.8%, respectively 36,37 .In Iran, Salmonella spp.isolates from milk were resistant to chloramphenicol (21.42%), ampicillin, and tetracycline (42.6%) 38 .Sporadic outbreaks of antibiotic-resistant S. typhi are common, especially in resource-poor settings 39 .Since humans are the only source of S. typhi infection the milk could have been contaminated through poor milk hygiene and thus there is a need for sanitation improvements in this community.

Conclusion
The present study showed that several pathogenic bacteria present in fermented milk manifested antimicrobial resistance to various antibiotics.The S. aureus, Coagulase-Negative Staphylococci, E. coli, Klebsiella spp., and S. typhi showed the highest sensitivity to gentamycin, chloramphenicol and penicillin G.There was marked resistance of the isolates to oxytetracycline and tetracycline and this could be due to wide usage of the drugs in Kajiado County to treat animal diseases, such as mastitis.There is a need to educate the farmers concerning the proper use of antibiotics and the dangers that may arise as a result of the use and misuse of these antibiotics.These isolates can be transmitted to man since amongst most pastoralist communities, processing of milk products does not involve pasteurization before fermentation and is also stored in traditional condition for an indefinite time.Public health education could also go a long way in uplifting the general sanitary conditions in the farms, especially in regards to waste disposal and provision of clean water for domestic use and milking of animals.Further studies should also be undertaken to determine the zoonotic potential of the isolates and the implications of possible transfer to the local human population.

Table 1 .
Antibiotic sensitivity profiles of Staphylococcus aureus, coagulase-negative Staphylococcus, and Escherichia coli isolated from traditional milk of cattle from Kajiado Central Sub-County, Kenya

Table 2 .
Antibiotic sensitivity profiles of Klebsiella spp.and Salmonella typhi isolated from traditional milk of cattle from Kajiado Central Sub-County, Kenya