Determination of some Antibiotic Resistances genes by Polymerase Chain Reaction of Lactic Acid Bacteria Isolates from Local Dairy Products

Abstract

Background: This study sheds light on the current resistance situation in local dairy product associated microorganisms Like Lactic Acid Bacteria (LAB) and will provide a basis for further experiments concerning gene transferability.
Objective: Isolation of Lactic Acid Bacteria (LAB) from local dairy products that have resistance phenotypes to antibiotics, and detection by PCR presence of certain known antibiotic resistance genes.
Materials and methods: This study was conducted during the period from 1st of September 2016 to the end of January 2017. Two hundred samples from local dairy products were collected as 50 samples from each raw milk, sour-milk, yogurt, cream and soft cheese. All isolates were identified according to biochemical characters, antimicrobial sensitivity test and MIC of the isolates was done by the Kirby–Bauer disc diffusion and micro dilution methods respectively. PCR amplification genes associated with resistance to β-lactam antibiotics (bla Z) gene, erythromycin (ermB), genes, and tetracycline ribosomal protection proteins (tet M), were done by PCR.
Results: The results showed that the most common bacteria presents in all dairy source samples were Lactobacillus as followed:75(31.9%), 40(17%), 30(12.7%) and 90(38.2%) in yogurt, white cheese, cream and sour milk respectively. Lactococcus isolates was appeared as 20(36.3%) and 15(27.27%) in yogurt and creams respectively and 10(18.1%) in each of white cheese and sour milk. Streptococcus was found as 45(40.9%) in white cheese and 30(27.2%) in yogurt, while in cream and sour milk was found as 27(24.5%) and 8(7.2%) respectively, whereas the total number of Lactococcus and Pediococcus were 55(12.6%) and 36(8.25%) respectively. The physiological characters and phenotypic identification was found that out of the 436 LAB isolates; Lactobacillus isolates were the dominant genus appeared followed by Streptococcus species. Isolates of LAB demonstrated different profiles of antibiotic resistance, all Lb. delbrukii, Lb. plantarum and Lb. fermentum were resistance to penicillin G, while showed a variable susceptibility rates to other antibiotics. The detection of tet M and erm B and bla Z resistance genes in LAB isolates showed that some isolates harbor tet M and/or erm B and bla Z genes. Twenty isolates of Lactobacillus delbrukii showed the presence of tet M and erm B and 17 isolates harbor bla Z genes corresponding to their resistance phenotypes. As well as 10 isolates of Lb. plantarum showed these gens in some of them as follow; five of the ten possessed the resistance tet M gen and 4 isolates have erm B while 8 isolates showed the penicillin G resistance gen. From the ten isolates of Lb. fermentum; only 4 isolates harbor tet M gene and 5 isolates possessed erm B gens while 9 isolates had the resistance gens bla Z. The isolates Pedicoccus sp. and Lc. lactis showed only presence of tet M gens in 8 and 7 isolates respectively. On the other hand Strep. thermophilus (20 isolates); the band of the detected gens appeared in 18 isolates for tet M and 15 for the erm B, but a β-lactamase gene detected in only 4 of these resistant isolates. Leu. mesntroids (10 isolates) showed 6 and 4 isolates possessed bla Z and tet M gen respectively but only one isolates showed to possessing the gene erm B.
Conclusion: This study had established that wide varieties of LAB the most common bacteria presents in all local dairy source samples were lactobacillus species which show high resistance properties to amoxicillin and ampicillin. Lactococcus lactis isolates showed resistance to tetracycline and all Leuconostoc cremoris isolates showed sensitivity to all tested antibiotics except, some isolates resisted to tetracycline and chloramphenicol. While Leuconostoc mesntroids had variable resistance to tetracycline, chloramphenicol, erythromycin, vancomycin, amoxicillin, ampicillin and penicillin G. All Streptococcus thermophilus isolates were resisting to tetracycline. The genes tet M, erm B and bla Z were detected in Lactic acid bacterial isolates.