The Molecular Typing And Antibiotic Resistance Among Proteus From Acute Diarrheal Patients

Objective:Proteus species play an important role in the foodborne disease,and the strains used in this study were isolated from the stool samples of acute diarrhea patients in the diarrhea outpatient clinic of a hospital in Second Hospital of Tianjin Medical University, then PFGE typing and antimicrobial susceptibility testing were carried out.PCR and DNA sequencing were also performed to characterize the class 1 integron、class 2 integron and the left and right junction of Genomic Island, in order to identify the homologous of isolates、antibiotic resistance situation and the possible dissemination mechanism of drug resistance, providing data reference for the spread of multiple antibiotic resistant Proteus from food and environment to the human body via food chain, and prevention and control of foodborne disease and reasonable use of antibiotics. Method:1、The strains of Proteus were from the the stool samples of acute diarrhea patients in the diarrhea outpatient clinic of a hospital from May to October 2013 in Second Hospital of Tianjin Medical University,and then plate-culture and PCR were carried out to identify the isolates.2、PFGE: extraction the whole genome DNA of Proteus strains, the strains were genotyped by PFGE of Sfi I-digested chromosomal DNA.3、Susceptibility Testing:Kirby-Bauer disk diffusion method was used to detect the antibiotic resistance of Proteus on 18 kinds of antimicrobial drugs(Ampicillin、chlorampheniel、streptomyen、trimethoprim-sulfalllethoxazolete、traeyelin、ceftriaxon、imipenem、ciprofloxacin and so on) which commonly used in clinical,and to analyse the drug resistance.4、Integrases and the left and right junction of Genomic Island in the Proteus were amplificated by polymerase chain reaction(PCR),and analysed the sequence of class 2 integron integrase gene and their relationships to antibiotic resistance. Results:1、62 strains of Proteus(48 P.mirabilis,8 P.vulgaris,2 P.penneri and 4 P.myxofaciens)were isolated by PCR and biochemical identification,and the tuf gene PCR assay could identify the Proteus.spp isolated from the stool samples of acute diarrhea patients rapidly.2、The 62 Proteus isolates were classified into 51 PFGE types, and the PFGE genotypes of the strains the similarity was from 40% to 100%.There were 5、3、3、2、2、2strains respectively belonged to the same genotypes,and these strains were isolated in the near time,indicating that the same clone strains caused the aggregative infection in this region.3、The drug resistance in Proteus isolated from diarrhea patients was a serious issue. Multiple antibiotic resistant strains(26%) and ACSSu T resistant strains(16%)were found, and a few strains were insensitivity to some important antibiotics, such as cephalosporin、imipenem and fluoroquinolones.4、Of the 50 Proteus isolates tested, 54%(27/50) and 32%(16/50) isolates carried class 1 and 2 integrons, respectively, and Class 1 and class 2 integrons were all detected in13(26%) of 50 Proteus isolates. In the 16 class 2 integrons, of the 6 class 2 integrons the internal stop codons in the int I2 genes were all mutated from TAA to the glutamine codon CAA. 36%(18/50) strains were also positive for the left and right junction sequence of genomic island.5、The proportion of class 1 integron(92.3%), class 2 integron(61.5%) and also carrying the two types of integrons(53.8%) in multiple antibiotic resistant strains were significantly higher than that of non-multidrug resistant strains(P values were 0.001、0.014、0.023, respectively)6、The proportion of class 2 integron(87.5%) and also carrying the class 1and 2 integrons and linked sequence(50%) in ACSSu T resistant strains were significantly higher than that of non- ACSSu T resistant strains(P values were 0.001、0.016, respectively). Conclusion:1、The traditional culture combined with PCR can detect the Proteus rapidly and accurately, and could be helpful to clinical diagnosis and treatment timely. And the aggregative infection caused by the same clone Proteus strains in this region may not be rare.2、The multiple drug resistance and ACSSu T resistant strains isolated from the stool samples of diarrhea patients in this region was a serious issue,and a few a few strains were insensitivity to some important antibiotics, such as cephalosporin、imipenem and fluoroquinolones. The proportion of class 1 and class 2 integrons was higer, and associated with multiple drug resistance and ACSSu T resistance.3、The proportion of linked sequence of genomic island in Proteus isolated from diarrhea patients in this region was higer, and the class 2 integrase were mutated in some strains, implying that functional int I2 genes were present. And the effect of complete resistant island and functional class 2 integrase in the strains need to further study.4、In this region,the foodborne Proteus was under obvious antimicrobial pressure. The threat to the clinical need to pay attention, and should continue to carry out the relevant research.