The Study Of Tracing And Monitoring On Contamination Of Salmonella In Seafood

Salmonella, the important foodborne bacteria, accounts for most foodborne bacterialenteritis worldwidely. Annual infection cases caused by Salmonella are16million in thewhole world, while600,000of them died. Hence it seems quite critical to develop fastdetection and typing method on Salmonella for food hygiene safety and economy concerns.The current study investigated to1) develop a fast real-time PCR method for Salmonelladetection in seafoods;2) utilize multilocus sequence typing (MLST) method for Salmonellatyping;3) gauge the antimicrobial resistance of Salmonella isolated from seafoods. Theaforementioned results are full of social and economic significance in terms of providingtechnical supports for the prevention and control of Salmonella in seafoods.The main contents and results are as followings:1. Establishment and application of real-time PCR method for Salmonella detection inseafoodsPrimers and TaqMan probe pointing at the outer membrane gene Ompc were designedfor Salmonella detection in seafoods. The rapid real-time PCR detection method onSalmonella was finally developed after optimizing the reaction system and conditions.Subsequently specificity and sensitivity assay were performed to evaluate the feasibility of theabove real-time PCR method. Thirty-two reference strains including Salmonella enteritidis,Salmonella typhimurium, Salmonella cholerae, Vibrio Parahaemolyticus, Vibrio cholerae,E.coli O157:H7, Staphylococcus aureus, Listeria.monocytogenes etc., were used forspecificity assay. The results revealed that all Salmonella strains were positive, whereas thenon-Salmonella strains were found to be negative. These indicated this real-time PCR methodwas highly specific. Sensitivity assay was performed via detecting target gene by real-timePCR after10times dilution of the Ompc-based recombinant plasmid. The detection limit wasrevealed as1×101copy/μL, indicating the established real-time PCR had high sensitivity.Real-time PCR probing Ompc was applied for Salmonella detection in120samples fromZhoushan aquatic product processing factory and zhoushan local market. The nationalstandard method was also carried out in parallel for comparison. Two detection methods were consistent, with12Salmonella strains isolated from120samples. The detection rate was10%(12/120). Besides, the established real-time PCR was also introduced in200fresh porksamples (room temperature preservation50, chilled preservation150). Salmonella detectionrate in fresh pork samples was8.0%(4/50), while that of chilled pork was11.3%(17/150).Totally21samples were found to be positive with the detection rate10.5%(21/200). Whenusing the national standard method for verification, the two results were in agreement witheach other.2. Multilocus sequence typing of Salmonella isolated in seafoodsSeven house-keeping genes (aroC、 dnaN、 hemD、hisD、pure、sucA and thrA) wereused for MLST typing in46Salmonella isolates from seafoods as referenced from the MLSTwebsite (http://mlst.ucc.ie/). DNA star.Lasergene.v7.1software was used for data analysis inorder to ascertain the genetic relationship among46Salmonella isolates and elucidate thepredominant sequence types in Salmonella strains isolated from seafoods in Zhoushan city.MLST results indicated that46strains of Salmonella could be divided into20sequencetypes. The dominant sequence type was ST11, with12strains grouping into this cluster,accounting for26.09%(12/46). The following primary sequence type was ST34, having5strains accouting for10.9%(5/46). Four strains grouped in ST155, accounting for8.7%(4/46). Sequence types ST14and ST19, having3strains, accounted for6.5%(3/46). Eachsequence type of ST46, ST64, ST185and ST1500had2strains, accouting for4.4%(2/46)respectively. Besides, all the following sequence types ST29, ST106, ST197, ST203, ST413,ST1291, ST1542, ST1561, ST1808, ST1809and ST1810had one strain with detection rateof2.2%(1/46). Noteworthily, three new sequence types named ST1808, ST1809andST1810were found in the current study.3. Antimicrobial resistance of Salmonella isolates from seafoodsVitek-2compact system was used to characterize the drug resistance of46Salmonellaisolates to18common antibiotics. The results revealed that most of the isolates wereresistant to the test antimicrobials and some of them had multi-drug resistance. Theresistance against cefazolin, cefotetan, amikacin, gentamicin and tobramycin exhibited the highest (100.0%), followed by nitrofurantoin(73.9%), ampicillin (39.1%), ampicillin/sulbactam (39.1%), trimethoprim/sulfamethoxazole (17.4%) and ciprofloxacin(4.3%). Theresistance to levofloxacin, ceftazidime and ceftriaxone is2.2%. Besides, all the isolates weresensitive to ertapenem and imipenem. Strikingly fluoroquinolones and third-generationcephalosporin resistant isolates were delineated in the current study. In the multi-drugresistant pattern,16strains exhibited resistance to6antimicrobials, accounting for thelargest population with rate of34.4%(16/46). Twelve strains were found to be resistant tofive antibiotics, accouting for26.1%(12/46). All the Salmonella isolates exhibited multidrug-resitance (resistant to at least2antimicrobials) pattern. This study implicated that moreefficient measures should be taken to monitor the Salmonella in seafoods.