The aim of this study was to determine the species distribution of Staphylococcus, Gram negative bacteria (GNB) and the occurrence of Methicillin Resistant Staphylococci (MRS) and Extended-Spectrum β-lactamase- (ESBL-) producing GNB. Bacterial culture of 300 clinical mastitis milk samples from 30 different farms across different regions of Tunisia during four seasons was realized. The obtained results showed the presence of high frequency of the tested samples with a positive growth for bacteria (64%). In addition a high recovery rate of Staphylococci and/or GNB in these clinical mastitis milk samples (87%) was detected. In addition, a high percentage of GNB (68.2%) compared to Staphylococcus species (32%) was noted. Moreover, a significant variation of the number of these bacteria according to the farm location, the seasons, and cows age was detected. The highest percentage was observed in the North of Tunisia during the winter and the spring seasons in adult cows with a dominance of GNB growth. Coagulase negative Staphylococci (CNS) (n=11) and GNB (n=16) species were identified. Escherichia coli (E. coli) was the most frequently found bacterium followed by Klebsiella pneumoniae. The dominant Staphylococcus isolates was S. xylosus followed by S. aureus the major pathogen isolated. Methicillin resistance was confirmed by the presence of the mecA gene in 3 S. aureus and 14 CNS isolates; all of these isolates were lacking the mecC gene. Various species of GNB, resistant to cefotaxime, were detected (n=15). ESBLs were detected on selective medium in 10 E. coli and 4 K. pneumoniae. All ESBL producers strains carry the blaCTX-M. The presence of different resistant mastitis pathogens in dairy farms may complicate therapeutic options and contaminated animals could become zoonotic agent reservoir for human.
Spore-forming bacteria, principally Bacillus species, are important contaminants of milk. Because of their high heat resistance, Bacillus species spores are capable of surviving the heat treatment process of milk and lead to spoilage of the final product. To determine the factors influencing the contamination of milk, spore-forming bacteria occurrence throughout the UHT milk production line during winter, spring, and summer was studied. The obtained results confirm that the total viable rate decreases rapidly throughout the production line of UHT milk showing the efficiency of thermal treatments used. However, the persistent high rate of spore-forming bacteria indicates their high heat resistance, especially in spring and summer. In addition, a significant variation of the quality of raw milk according to the location of the collecting centers was revealed. The molecular identification showed a high degree of diversity of heat-resistant Bacillus species, which are isolated from different milk samples. The distribution of Bacillus species in raw milk, stored milk, bactofuged milk, pasteurized milk, and UHT milk were 28, 10, 16, 13, and 33%, respectively. Six Bacillus spp. including Bacillus licheniformis (52.38%), Bacillus pumilus (9.52%), Bacillus sp. (4.76%), Bacillus sporothermodurans (4.76%), Terribacillus aidingensis (4.76%), and Paenibacillus sp. (4.76%) were identified in different milk samples.
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