ISOLATION AND CHARACTERIZATION OF LYTIC BACTERIOPHAGES INFECTING SOME BATHOGENIC BACTERIA

Abstract

This study was aimed to isolate and characterize bacteriophages infecting some pathogenic bacteria. The E. coli O157:H7 wild type strain 93111 and B. cereus ATCC 33013 - specific phages were successfully isolated. Four B. cereus phages (BCP1, BCP2, BCP3 and BCP4) were isolated from clay soil samples, whereas four E. coli phages (ECP1, ECP2, ECP3 and ECP 4) were isolated from sewage and Nile water. The morphological characterization of isolated phages suggests that the phages of infecting B. cereus could be considered as members of Siphoviridae or Myoviridae families. The morphology of E.coli phages confirms their classification to Tectiviridae, Corticoviridae, Siphoviridae or

Podoviridae families. The genome size of the isolated B. cereus and E. coli phagesranged between 28.7 - 33.6 kbp and 34.5- 41.5, respectively. The four isolated B. cereus phages exhibited very limited host spectrum, infecting only 2 of 9 Bacillus spp. The ECP2 has the ability to infect eight strains of Escherichia coli including five strains of shiga toxin producing E. coli and three nontoxigenic E. coli strains. The SDS-PAGE analysis of phage proteins revealed that the BCP3 and ECP1 had five protein bands with molecular weights ranging from 37.0 to 110.0 and from 34.0 to 112.0 kDa, respectively. The effect of some stress conditions on the BCP1 and ECP2 survivability was evaluated. These conditions comprised UV radiation, high and low temperatures, acidic and alkaline pH, and some essential oils. After 75 min exposure to the UV light with a wavelength of 254.0 nm, the BCP1 and ECP2 retained 58.7 and 10.89% of the lytic activity, respectively. With the HTST pasteurization, the BCP1 and ECP2 retained 81.9% and 90.4% lytic competence, respectively. On the other hand, the two phages lost their infection activity entirely after boiling for 10, 20 and 30 minutes. The survivability of BCP1 and ECP2 was not affected significantly under refrigeration conditions at 4.0ºC for nine months. During the storage period, the BCP1 and ECP2 counts were fluctuated between 8.70 ± 0.37 to 9.79 ± 0.55 log10 and 9.023 ± 0.27 to 10.02 ± 0.41, respectively. The BCP1 appeared to be extremely unstable at very vigorous acidic and alkaline environments as its infection ability was lost completely at pH 1, 3 and 13. On the other hand, the ECP2 lost its activity entirely at pH 1 and 79.96% at pH 13. The greatest inhibition of the BCP1 lytic activity was recorded with the garlic oil (0.004 mg/mL) followed by ginger oil (0.11 mg/mL) as the phage activity has reduced by 11.12 and 9.64%, respectively. The clove, garlic and ginger oils diluted by 50% had weak significant antiviral effect against ECP2, whereas only clove oil with a concentration of 20 % had a significant effect.

Keywords:Bacillus cereus,Escherichia coliO157:H7, Bacteriophage, Characterization,Host range, Phage stability