| Human salmonellosis is a major health problem worldwide. Salmonella infection occurs mainly through contaminated food and water sources, which results in gastroenteritis and other severe infections, including enteric fever and bacteremia. Self-healing gastroenteritis is the most common disease caused by Salmonella. It leads to fever and diarrhea. The duration of the fever is within two days and the diarrhea would not last seven days. The typical symptoms of typhoid fever were fever, headache, diarrhea and abdominal pain. Typhoid fever also will cause fatal injury of respiratyory system, liver, spleen, and nervous system.Salmonella is a kind of gram negative bacillus with flagella. The genus Salmonella mainly involves three types of antigen structure:somatic antigen (O antigen); flagellin protein (H antigen); and surface antigen. Currently,64 O and 114 H antigens have been identified. It can be divided into more than 2,500 serotypes by the Kauffmann-White serotyping scheme. Different Salmonella serotypes would cause different diseases. Enteric fever was caused by S. Typhi and S. Paratyphi. Bacteremia was always caused by S. Choleraesuis. In addition, during foodborne disease outbreaks by Salmonella, it often caused by one or more sortypes of Salmonella. We can find the soure of the outbreak according to the serotype of Salmonlla to control the outbreak and prevent the salmonellosis spreading. Thus, to serotype Salmonella rapidly and accurately is of great benefice to diagnosis and treat salmonellosis, investigate for Salmonella outbreaks and surveil for Salmonella serotypes.Matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) is a new technology of soft ionization mass spectrometry developed in recent years, which mainly ionize the ribosomal protein of microorganism. Then, the specific protein fingerprint profiles were produced and taken to find the matched profile in the established mass profiles database. Thus, we could acquire the identification results rapidly. MALDI-TOF MS has several advantages of easy processing, rapid detection and high-throughput, which has been widely used in clinical microbiology.Liquid suspension array technology was a multifunctional liquid phase analysis platform, which was known as xMAP (flexible multi-analyte profiling), liquid array or suspension array because many reactions occurred in microsphere suspending in liquid phase. As a new and high-throughput biochip technology, xMAP combined flow cytometry, laser technology and fluid technology together. The coding microspheres were suspended in the liquid as detecting vector. Consequentely, the xMAP has several advantages of high speed, wide detection range, strong sensitivity and specificity. For the genes ecoding O and H antigens of Salmonella, corresponding primers and probes were designed. After multiple PCR, the microspheres coupled with various probes were hybridized with PCR amplification products. Then, the fluorescenc signal was detected to determine correspongding O and H antigens of Salmonella. Recently, the Luminex Corporation has developed a bead-based suspension array assay, the xMAP Salmonella serotyping assay (xMAP SSA), that can determine the complete serotype. The application of Luminex xMAP SSA is still at the exploratory stage and no relevant reports were presented in China. The study included two parts:Part I. Rapid identifying and serotyping of Salmonella by MALDI-TOF MS and liquid suspension arrayThe traditional serotyping method plays an important role in identifying serovars of Salmonella for a long time. However, for this type of serotyping, production and application of hundreds of antisera, which, in turn, requires rigid quality control. To overcome these problems, we use MALDI-TOF MS and the liquid suspention array technology—Luminex xMAP Salmonella Serotyping Assay (xMAP SSA) to create a method to identify and serotype Salmonella rapidly. Additionally, we evaluate application value of the method in Southern China. Two hundred and five Salmonella isolates from diarrhea patients in Southern China were identified by MALDI-TOF MS and the micriobial identification system—Vitek 2 at genus level. Then they were serotyped by xMAP SSA in parallel with the traditional serotyping. For discordant results between the xMAP SSA and the traditional serotyping method, PCR and sequencing for gene fljB and fliC were considered to be arbitrated methods to validate the serotyping results. All Salmonella isolates were correctly identified by MALDI-TOF MS in accordance of Vitek 2. It took average five hours to identify one Salmonella isolate by Vitek 2, while MALDI-TOF MS only need five minutes. MALDI-TOF MS could derectly identify Salmonella isolates culturing in selective medium, but Vitek 2 could not. Forty serotypes were identified among 205 isolates; the most prevalent serotypes identified were Salmonella Enteritidis (n=42,20.49%), Salmonella Stanley (n=32,15.61%), Salmonella I 4,5,12:i:-(n=29,14.15%), and Salmonella Typhimurium (n=24,11.71%) and Salmonella Derby (n=9,4.39%). One hundred and ninety-five (95.1%,195/205) isolates were serotyped completely by xMAP SSA, while ten stereotypes were partially detected as their O or H antigens weren’t included in the assay. The xMAP SSA correctly identified 192 (98.5%, 192/195) isolates as the H antigens of three strains were falsely identified. The flagellar antigens of five isolates could not be detected by the traditional serotyping assay. Five nonmotile and three monophasic strains, which possessed flagellar antigen genes that weren’t expressed, were completely serotyped by xMAP SSA; however, these isolates were left undetected by the traditional method. xMAP SSA does not need phase inversion and automatically analysis data. Comparing with the method of combing Vitek 2 with traditional serotyping, the method of combining MALDI-TOF MS with xMAP SSA could decrease 79 hours and 2/3 consuming cost averagely. The accuracy rate of the method of combining MALDI-TOF MS and xMAP SSA was up to 98.5%. Combing MALDI-TOF MS with xMAP SSA, with high-throughput characteristics, provides an accurate rapid and cost-effective serotyping system that dramatically strengthens the capability of clinical and public health laboratories for Salmonella serotyping. It is suitable to identify the serovars of Salmonella in Southern China.Part Ⅱ. Antimicrobial resistance patterning and molecular typing for Salmonella isolated from diarrhea cases in Guangzhou from January 2013 to May 2014Salmonella is a main pathogen that causes foodborne diseases, which was pathogenic to human and animals.In Guangzhou (China), Salmonella was one of the first four pathogens which resulted in the food poisoning over the years. Matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) is a new technology of soft ionization mass spectrometry developed in recent years. MALDI-TOF MS is gradually applied to rapidly identify bacteria and fungi. It aslo was applied for Salmonella subtyping. To investigate the serotype distribution, antimicrobial resistance and molecular epidemiological characteristics of Salmonella isolated from diarrhea cases in Guangzhou from 2013 to 2014, and to investigate the clinical application values of MALDI-TOF MS for Salmonella subtyping, all Salmonella isolates were serotyped by traditional serological method. Antibiotic susceptibility was determined by disk diffusion method (KB). Subtyping for Salmonella Stanley, Salmonella Enteritidis, Salmonella Typhimurium and Salmonella Ⅰ 4,5,12:i:-was performed by pulsed field gel electrophoresis (PFGE) and MALDI-TOF MS.38 serotypes were identified among 179 Salmonella strains and the top four serotypes were Salmonella Enteritidis (21.2%), Salmonella Stanley (17.3%), Salmonella Salmonella Ⅰ 4,5,12:i:-(16.2%) and Typhimurium (10.1%). The resistance rate of nalidixic acid, macrodantin, tetracycline and ampicillin was highest with respectively 44.7%,42.5%,39.7% and 35.8% for all Salmonella strains. The susceptibility rate of ciprofloxacin was only 20.7%. The resistance was also observed with ceftazidime (7.3%), cefotaxime (9.5%), ceftriaxone (9.5%) and cefepime (7.3%). Among 179 strains,72 (40.2%) strains were multidrug resistant (MDR) to three or more antimicrobials. There were 54 (46.6%) MDR strains among the most four common serotypes of Salmonella Stanley, Salmonella Enteritidis, Salmonella Typhimurium and Salmonella Ⅰ 4,5,12:i:-. Two Salmonella Typhi isolates were not resistant to ciprofloxacin and cephalosporins. Different Salmonella serotypes produced different antimicrobial resistant profiles. When considered a type of more than 90% similarity as a same type,27 Salmonella Stanley,21 Salmonella Enteritidis,21 Salmonella Typhimurium and 13 Salmonella Ⅰ 4,5,12:i:-were respectively typed to 13、10、11、0 PFGE profiles and 26、20、 21、13 MS profiles. When considered a type of 100% similarity as a same type,27 Salmonella Stanley,21 Salmonella Enteritidis,21 Salmonella Typhimurium and 13 Salmonella I 4,5,12:i:- were respectively typed to 25,15,17 and 13 PFGE profiles. All those identical PFGE types almost presented different resistant profiles. Salmonella Stanley, Salmonella Enteritidis, Salmonella Typhimurium and Salmonella I 4,5,12:i:-were main serotypes from diarrhea cases in Guangzhou from 2013 to 2014. All Salmonella strains were typed to multiple different PFGE profiles, suggesting the diarrhea cases were distributed sporadically. A worrying percentage of MDR strains has been a prevalent problem in the study. The situation about increasing resistance to quinolones and cephalosporins was observed, especially along with decreased susceptibility to ciprofloxacin. Comparing with PFGE, more types were obtained by MALDI-TOF MS for subtyping of 82 Salmonelal isolates, suggesting MALDI-TOF MS has higher discernibility. For MALDI-TOF MS, it is a new method with great potientical ablitity to verify pathogens and trace to the source during Salmonella outbreaks. |
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