The standard for determining the number of infectious particles was the pUC18 construct with the 4867 bp DNA fragment of bacteriophage φ53. It was determined that the penicillinase plasmid occurs on average in three copies per cell (exact value deduced by qPCR is
2.98). This value correlates with the expected copy number for such a large plasmid per cell (Novick, 1990). Based upon absolute quantification of the blaZ gene by qPCR, the number of copies of this gene in 1 mL of transducing phage lysate was determined as 1216. An analogous approach enabled determining the number 2.108 × 106 infectious phage particles in the lysate. Comparing the aforementioned values, we determined the approximate ratio of transducing particles to number of infectious phages to be 1 : 1700. selleck chemicals The number of transducing virions carrying blaZ (2.71 × 104) deduced from the qPCR data and from the titer of infectious phages in the lysate (4.7 × 107 PFU mL−1), as well as the number of acquired transductants Forskolin nmr (720 CFU mL−1), enabled determining the effectiveness of transduction as 2.7%. Transduction effectiveness was determined on the multiplicity of infection level of 0.16, when the probability of introducing more plasmids into a single recipient cell and superinfection of the created transductants followed by their elimination is very low. In further experiments, we explored the possibility for disseminating
antibiotic resistance genes by the φJB prophage induced from donor lysogenic cells prepared by lysogenization of strain 07/759. Using UV radiation, a transducing lysate with titer 8.6 × 105 PFU mL−1 was prepared from the lysogenic strain 07/759 (φJB+) and was used successfully to transfer the 31 kb penicillinase plasmid into the strain 07/235 with frequency 2.3 × 10−6 CFU/PFU. The genotype of transductants was determined in the same
way as of the transductants obtained by propagated phage lysates. Another donor strain used for these experiments was the lysogenic transductant 07/235 (φ80α+) containing the φ80α prophage and 27 kb penicillinase plasmid of the 08/986 strain described above. The objective was to clarify the hypothesis whether by receiving a plasmid and integration of φ80α phage 07/235 became a new Resminostat potential donor capable of transferring the plasmid into other strains after induction of the φ80α prophage. The induced lysate with titer of 1.6 × 106 PFU mL−1 was used for transducing plasmid into the RN4220 strain, which successfully received it with a frequency of 3.1 × 10−6 CFU/PFU. This shows that if the transductant is lysogenized, the plasmid can be very effectively mobilized. Transduction experiments with induced lysates proved that prophages that abundantly occur in a number of clinical strains can play an important role in transferring plasmids. Transduction of a resistance plasmid from one strain into others may be a quite efficient way of spreading antibiotic resistance.