The Drug Resistance Surveillance And Genotyping Research Of Nontyphoidal Salmonella Isolated From Patients With Acute Diarrhea

Objective:To investigate the serotypes and antibiotic resistance situation of non-typhoid Salmonella in our region. Application Pulsed-field gel electrophoresis (PFGE) and Multilocus sequence typing (MLST) to identify gene sequences homologous of isolates, understand clearly the popular genotype in our region, determine the genetic relationships between different strains, providing data reference for future tracking traceability of non-typhoid Salmonella pathogen infection in the region, also offer help to prevention and control and treat of non-typhoid Salmonella infection.Method:1. Serotyping and Susceptibility Testing:The strains isolated from stool specimens of patients with acute diarrhea in intestinal outpatient in Second Hospital of Tianjin Medical University, strains were collected from May to October period of2012and2013year. Serum diagnosis of Salmonella serotypes identified in accordance with the procedure provided by the Thai S&A, serotype were determined based on the obtained serotype antigens. K-B method were used to detect the antibiotic resistance of non-typhoid Salmonella on17kinds of antimicrobial drugs.2. PFGE:Extracted the whole genome DNA of non-typhoid Salmonella strains, the strains were genotyped by PFGE of XbaI-digested chromosomal DNA, after analysis with restriction endonuclease Xbal, then Salmonella enteritidis were genotyped by PFGE of BlnI-digested chromosomal DNA.3. MLST:amplified core fragment (510bp-894bp) of seven housekeeping genes (thrA, pure, sucA, hisD, aroC, hemD, dnaN) of non-typhoid Salmonella strains respectively, the sequencing results were submitted to the International MLST database website (http://www.mlst.net), then we obtained allele maps and the corresponding Sequence Type (ST) of strains.Results:1. A total of45NTS strains were isolated, serotyping involved five group,12serum types, all within A-F group. Advantage serogroups was D group and B group, with0:4and0:9dominated. Domestic common serotypes includes:enteritidis, typhimurium, agona, Senftenberg, Derby, Braenderup; infrequent serotype includes: Mbandaka, Montevideo; rare serotype has:Kouka, Grampian, Fillmore, Oritamerin. Superiority serotype was S. enteritidis and S. typhimurium.2. Antibiotic susceptibility:for the45test strains nalidixic acid sensitive rate was lowest, followed by streptomycin, gentamicin, ampicillin, tetracycline, piperacillin, chloramphenicol. For levofloxacin, amikacin, amoxicillin/clavulanicacid and cephalosporins the sensitive rate was greater than90%, for ciprofloxacin, ceftriaxone, cefotaxime, Trimethoprim/sulfamethoxazole, Aztreonam and Imipenem sensitive rate was100%. Nalidixic acid resistant strains than sensitive strains have reduced sensitivity to ciprofloxacin and levofloxacin. All sensitive strains was24.44%,44.44%single drug resistant strains and multiple drug resistance was17.78%. Typhimurium serotype with multiple drug resistance was relatively common, they resistanted to ampicillin, chloramphenicol, tetracycline and streptomycin, was ACST resistance phenotype.3. PFGE results:PFGE produced23kinds of graph types, stripe number was between12to20. Divided into four big clustering (A, B, C, D), the clustering then included different subclusterings. There were11kinds of PT types including two or more isolates. S. enteritidis and S. typhimurium existed advantages PFGE type PT2and PT18respectively. PT2, PT6, PT20and PT22were isolated both in2012and2013year.6strains PT2and4strains PT18were isolated from one same date respectively, and6strains of type PT2S. enteritis were confirmed as an infection outbreak finally. The same genotype of S. enteritidis divided by XbaI were divided into three genotypes by BlnI. We used PFGE typing to predicte Serotype, consistent rate of forecasted serotype and actual serotype was70.9%.4MLST results:45test strains included13STs,14strains Enteritis serotype were ST11,Typhimurium serotype including ST19and ST34,1strains Kouka serotype and4strains Senftenberg serotype were ST14,4strains Agona serotype were ST13,3strains Braenderup serotype were ST22, and3strains Derby serotype include1strain ST40and2strains ST71,2strains Fillmore serotype was ST46,1strain Grampian serotype was ST358,1strain Mbandaka serotype was ST413,2strains Montevideo serotype were ST26,1strain Oritamerin serotype was ST1516. ST11was most common, followed by ST34. The relationships between the STs were far, ST40and ST71Belonged to the same serotype but genetically unrelated, and genetic-related ST14includes both Kouka serotype and Senftenberg serotype.Conelusion:1. Serotypes of local NTS showed diversity, Enteritis and Typhimurium serotype were the major serotype, we detected some domestic uncommon serotypes (Mbandaka, Montevideo) and rare serotypes (Kouka, Grampian, Fillmore, Oritamerin).2. This data showed NTS rarely resistant to three generations of cephalosporins, but presented higher prevalence of nalidixic acid resistance and reduced susceptibility to FQs, posed a threat to clinical experience administration of FQs. Most of S. typhimurium MDR presented ACST resistant phenotype.3. PFGE display PT2and PT18were predominated types, included11same clone groups. PT2, PT6, PT20and PT22cloning strains presented cross-annual existence. Confirmed a S.enteritis infection outbreak and suggested a S.typhimurium infection outbreak incidents. For S.enteritis, XbaI-BlnI double enzyme digestion PFGE could improve the resolving power. PFGE typing had predictive value on serotyping.4. MLST classification show ST11and ST34were main ST types in our study. Serotyping confused unrelated strains in genetics, STs may better reflect the relationship between the genetic evolution of NTS.5. We should further enlarge and strengthen the resistance surveillance and Sub-type research of NTS in Tianjin.