Microbiological and molecular study of lactococcus lactis subsp. lactis isolated from cow milk

Abstract EN

In this study a total of one hundred forty five-milk sample were collected during the period (July to November 2013) from different fields in Erbil and Duhok province.Screening revealed that 45 out of 145 samples of crude cow milk (31%) were Lactococcus lactis subsp.

lactis.

Characterization of these selected isolates were carried out using morphological, biochemical, API 20 system and further confirmation was achieved by PCR assay.

Antibiotic sensitivity test performed for L.lactis subsp.

lactis against thirteen antibiotics.The results showed that the isolates appeared to be varied in their resistance, the number and resistance percent for antibiotics used were as follow: Cephalothin 95.55 %, Ampicillin93.33 %, Amoxicillin / clavulanic acid (AMC) 93.33 %,Sulfamethoxazole/Trimethoprim 91.11 %, Cefotaxime sodium 84.44 %,Clindamycin 77.77 %,Ceftriaxone 71.11 %, Nitrofurantoin 62.22 %,Gentamycin 53.33 %, Vancomycin 40 %,Ciprofloxacin 35.55,Chloramphenicol 24.44%, and Imipenem 2.22 %.

All isolates extracts exhibited marked activity against five pathogenic bacteria includes Bacillus subtilis, Pseudomonas aeroginosa, Echerichia coli, Staphylococcus aureus and Clostridium perfringes using agar well diffusion method.

Isolate 15 and 41 exert maximum activity (20mm) and (16mm) against B.

subtilis and S.

aureus, whereas isolate 20 showed highest activity (13mm) against P.

aeroginosa.

Generally, all aforementioned isolates were active against gram positive and Gram–negative bacteria.

In addition, the effect of pH, and temperature t were studied on isolates 10, 26, and 42 extract (bacteriocin)activities against some pathogenic bacteria.

The results showed that isolate 42 displayed highest activity at pH 2.5 and temperature at – 4°C against pathogenic bacteria under study.

One highly resistance isolate was chosen (L42) for transformation test to determine the location of the antibiotic resistance genes by genetic transformation using the laboratory strain E.

coli DH5 – α with plasmid DNA purified from this isolates.

The transformation process conducted successfully and the results showed that the resistance genes for Nitrofurnation, Chloramphenicol, Cefotaxime sodium, and Vancomycin were located on plasmid DNA while for others were located on chromosomal DNA