Isolation and characterization of lytic bacteriophages, a potential alternative for bovine mastitis control
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Date
2018
Authors
Noguda, Thembakazi
Journal Title
Journal ISSN
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Publisher
University of the Free State
Abstract
Mastitis is a common disease affecting dairy herds, with high occurance incidents and tremendous economic losses associated with it. Varieties of infectious agents are associated with mastitis, but bacteria are responsible for most of the cases. Apart from large numbers and heterogeneity between bacterial species associated with mastitis, it is an infectious disease and the process of milk production makes it easy for the disease to spread and difficult to control. Good milking hygiene, antibiotic therapy during lactation and dry off and chemical teat dips are some of the measures used in its control and treatment of mastitis. However, more cases of antibiotic therapy in treatment and control end up in failure and antimicrobial resistance is the reason attributed to this.
The main goal of this study was to investigate the diversity of bacterial species causing mastitis in South African dairy farms, determine their antimicrobial susceptibility profile and isolate lytic bacteriophages for these species as potential alternative for the control of mastitis caused by bacterial pathogens. Milk samples from mastitis and normal cows were analysed using traditional isolation methods on non-selective and selective differential agar plates, identification by standard biochemical tests. The following bacterial species were predominantly found: Staphylococcus aureus and members of Coagulase-negative Staphylococcus group (Staphylococcus chromogenes, Staphylococcus haemolyticus, Staphylococcus xylosis, Staphylococcus epidermis, Staphylococcus hominis, Staphylococcus hycus, Staphylococcus capitis, Staphlococcus sciuri), Streptococcus dysgalactiae, Streptococcus uberis, Escherichia coli, Klebsiella spp., Pseudomonas spp., Enterococcus spp., Enterobacteriacea spp., Proteus spp., Citrobacter spp., Bacillus spp., Bacillus pumilus, Acinetobacter spp., Lactococcus lactis and Pasteurella spp.
For their antimicrobial susceptibility evaluation, disk-diffusion and broth microdilution methods were used. The highest sensitivity was found on cephalosporins (cefuroxime 94% and cephalexin 65%) and aminoglycosides (streptomycin 82% and kanamycin 82%), tetracycline 65%, bacitracin 59%, novobiocin 59% and amplicillin 53% and resistance to polymyxin B (53%), penicillin (53%), ampicillin (41%), bacitracin (41%), novobiocin (41%). Intermediate resistance was found for neomycin (47%). Also, different teat dip disinfectants with different active ingredients were evaluated. Products tested were, Deosan Teat Form (Chlorhexidine based), Mastocide (Chlorhexidine digluconate based), Deosan Iodel Gel and locally available chemical pre- and post-milking teat dip (Citric acid monohydrate 0.42% ppm m/v and Dodecyl benzene sulphonic acid based) and milking equipment sanitiser Perosan (peracetic acid based). All teat dips products were found to be effective, with 100% lethal effects at the recommended application rate in inhibiting growth of all mastitis associated causative strains tested. While the local chemical distribitor chemical Perosan acid sanitizers was ineffective at the concentration of 0.4% and double these manufacture recommended concentration for both Gram-negative and positive isolates at exposure time of 5 minutes. Deosan Perosan was also ineffective at manufacture recommended working concentration 0.5% and at double this concentration for Gram-negative. The MIC for these strains was predicted to be >0.75% and for Gram-positive strains, their MICs were between 0.75%-0.19%.
For isolation of lytic bacteriophages, 12 lytic phages were successfully isolated from cow manure for the following species S. aureus, Coagulase-negative Staphylococcus, Streptococcus spp., Corynebacterium sp., Acenetobacterium sp. and E. coli. All these phages formed clear round plaques with size between 1-2 mm in diameter and titer between 108- 10¹² PFU/ml. Their TEM morphology characteristics showed that they belong to Myoviridae family. Spot test and efficiency of plaque formation were used to determine phage host ranges and most showed a broad host range, they were able to lyse other strains from the same species and strains from other species showing their potential for phage therapy application.
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Keywords
Dissertation (M.Sc. (Food Science))--University of the Free State, 2018, Mastitis, Treatment, Antimicrobial susceptibility profile, Lytic bacteriophages, Control