Establishment And Application Of Dual PCR Method For Porcine Klebsiella Pneumoniae And Escherichia Coli

Klebsiella pneumoniae is an important zoonotic pathogen.The drug resistance and pathogenicity of human clinical isolates have been studied more than veterinary clinical isolates.Although there are a few reports on porcine Klebsiella pneumoniae,most of them took the Klebsiella pneumoniae complex as the research object,resulting in our limited understanding of the harm of Klebsiella pneumoniae sensu stricto(Kp1).Combined with Klebsiella pneumonia and Escherichia coli in growth characteristics,dyeing,biochemical phenotype has a certain similarity,rely on veterinary clinical preliminary identification of the suspected E.coli infection during the diagnosis,possible to Klebsiella pneumonia infection cases mistaken for E.coli infection.Therefore,there is an urgent need to develop a method for the detection of Kp1 and E.coli in clinical samples.1.In this study,we compared the homology of 1618 Kp1 and 3245 E.coli whole genome sequences in NCBI database,screened the conserved genes within species and specific genes between species,and established a dual PCR method for simultaneous detection of Kp1 and E.coli.The sensitivity of this method to Kp1 and E.coli was up to 10 CFU.2.Dual PCR was used to detect the isolated samples of 69 suspected cases of E.coli infection,among which 12 samples were Kp1 positive,and 7 strains of Kp1 were isolated.3.Dual PCR was used to detect 201 commercial pig tissue samples,8 Kp1 positive samples were detected,and 5 strains of Kp1 were isolated.4.MLST typing of 12 Kp1 isolates showed that 7 known sequences were ST1,ST11,ST14,ST25,ST35,ST45,ST828 and 3 new sequences.There were 2 strains of ST25 and ST45,and 1 strain of the rest.The above 7 known sequences were all common in clinical practice,among which ST1,ST11,ST14,ST25 and ST828 were isolated from pigs for the first time.These results suggest that porcine Kp1 is highly likely to infect humans and is a zoonotic pathogen.