Genetic Variation Of Hydrogen Sulfide Negative Salmonella Enterica And The Antibiotic Resistance Mechanism Of Hydrogen Sulfide In Salmonella Enterica

Salmonella enterica remained one of the most foodborne pathogen causing bacterial gastroenteritis worldwide.Through contaminated food and water,human could get infection,diarrhea and death even worse.Due to the characteristic of hydrogen sulfide?H₂S?production,the detection of H₂S was the preliminary way to identify and differentiate Salmonella and other intestinal bacteria.However,numerable H₂S-negative Salmonella isolates were identified and reported worldwide in recent years.The phs operon was critical for the reduction of thiosulfate to hydrogen sulfide and contains three genes?phsA,phsB,and phsC?encoding thiosulfate reductases in Salmonella.The previous studies have reported that the mutations in phsA gene may lead to the inability of Salmonella to produce H₂S.The multi-drug resistance Salmonella has emerged around the world.And not only that,the emergence of H₂Snegative Salmonella became frequent,especially in China.According to the previous studies,H₂S was not only a byproduct of bacterial sulfur metabolism,but also was considered to play an important role in bacterial resistance,such as Escherichia coli and Salmonella.It not only suggested that gas molecules are recognized as a new drug resistance,but also revealed the complexity of bacterial antibiotic resistance mechanisms.However,the molecular mechanism of H₂S participating in drug resistance has been still unknown.In our laboratory,90 H₂S-negative Salmonella isolates were collected based on a national Salmonella monitoring system between 2006 and 2013.The aim of this study was to reveal the evolution regularity between H₂S-negative and H₂S-positive Salmonella isolates,and provide a reference and scientific basis for the prevention and control the spread of Salmonella in China.At the same time,the expound about the biological function of H₂S involved in antibiotic-resistance in Salmonella would not only make a possibility for new targets and ideas of the treatment,but also provide the scientific basis for the treatment and prevention and control of multi-drug resistant Salmonella.The H₂S-negative and H₂S-positive Salmonella isolates were subjected to antimicrobial susceptibility testing.The aim was to analyze the characteristics of antibiotic-resistance of 90 H₂S-negative Salmonella isolates,and the rate of antibioticresistance between H₂S-negative and H₂S-positive isolates with the same serotype.Besides,quinolone-resistant and multi-drug resistant H₂S-negative Salmonella would be pay more attention in this study.Finally,we hope to provide a reasonable basis for the rational selection of antibiotics in the treatment of Salmonella infection.All identified H₂S-negative Salmonella isolates were tested against 21 different antibiotics for antimicrobial susceptibility testing.The results showed that the resistance rate of H₂S-negative Salmonella to ampicillin,ticarcillin,gentamicin,cotrimoxazole,tetracycline and chloramphenicol were higher than that of H₂S-positive Salmonella.The H₂S-negative or H₂S-positive Salmonella isolates showed a general susceptive to cephalosporins,imipramine,amikacin and aztreonam.There were differences in antimicrobial susceptibility of H₂S-negative and H₂S-positive Salmonella isolates which with the same serotype,including S.Choleraesuis,S.Enteritidis and S.Typhimurium.There were 13 Salmonella isolates resistant or intermediate sensitivity to quinolone.Further analysis showed the key mutations in the gyrA or parC gene may responsible for the resistance to quinolone.In addition,the H₂S-negative S.Choleraesuis displayed highest antibiotics resistance rate among the 14 serotypes and 76% H₂S-negative S.Choleraesuis isolates were MDR.Pulsed-field gel electrophoresis?PFGE?and multilocus sequence typing?MLST?were the common methods for studying the genetic evolution of bacteria.By MLST analysis,the 90 H₂S-negative Salmonella isolates were divided into 22 STs,with two new STs,ST1960 and ST1961.H₂S-negative S.Enteritidis isolates formed two clonal complexes?CC?,including CC92 and CC1960,while other serotypes of H₂S-negative Salmonella formed one CC,respectively.In addition,the H₂S-negative S.Senftenberg,S.Choleraesuis,and S.Paratyphi B isolates possessed the different STs of the H₂Spositive isolates with the corresponding serotype.Compared to the H₂S-negative Salmonella isolates,the H₂S-positive S.Enteritidis and S.Typhimurium isolates own more STs.Other serotypes of H₂S-negative and H₂S-positive isolates possessed same STs,respectively.By PFGE analysis,47 H₂S-negative isolates and 40 H₂S-positive isolates were divided into three main distinct groups with approximately 50% similarity.Some H₂S-negative and H₂S-positive isolates were not in the same cluster,suggesting they may evolve from diverse sources,including S.Enteritidis and S.Paratyphi B.In contrast,some H₂S-negative and H₂S-positive isolates were in the same cluster,including S.Typhimurium,S.Derby,S.Agona,and S.Thompson,indicating they may had a closely genetic relationship.Interestingly,the H₂S-negative S.Enteritidis isolates from Guangzhou and the H₂S-negative S.Gallinarum isolates from Shanghai had the similar PFGE profiles and same ST,and the H₂S-negative S.Paratyphi A isolate from Yuxi had the same PFGE profiles and ST with H₂S-negative S.Paratyphi A isolates from Shanghai,indicating that the H₂S-negative Salmonella may be likely to spread across different regions.Moreover,the H₂S-negative S.Derby and S.Typhimurium isolates from livestock had a closely genetic relationship with the H₂S-positive isolates from human,respectively.Counterpart,the H₂S-negative S.Meleagridis isolated from water or human showed the same ST and PFGE bands.These phenomena indicated that the H₂S-negative Salmonella may infect human or animals by contaminating food.The phs genetic operon was amplified by PCR and sequenced for analyzing the reason of H₂S phenotype deletion in H₂S-negative Salmonella.By the analysis of phs operon,we found 77 isolates?85%?have variety of mutations in phs operon,of which 74 isolates were detected mutations in phsA gene.Among them,three mainly kinds of mutations type had been detected in phsA gene: the frameshift mutation at position 760 or 1087,the missense mutation at position 1624;four mainly kinds of mutation type had been detected in phsB gene: including the missense mutation at position 164,314,319,373;two mainly kinds of mutation type had been detected in phsC gene: including the nonsense mutation at position 577 and the missense mutation at position 754.These mutation leading to the gene product incomplete or amino acid sequence changes may cause the Salmonella unable to produce H₂S.In general,the sequences of the H₂Snegative Salmonella isolates phs operon had instability and diversity.Interestingly,the sequences tend to be stable between the same serotypes.The phsA gene deletion mutant strain of S.Typhimurium ATCC14028 was constructed by using no-SCAR?Scarless Cas9 Assisted Recombineering?system.The wild-type strain was recovered as black colonies on SS agar,while the ?phsA mutant strain was recovered as colorless colonies.The result was confirmed by using the lead acetate.These results suggested that the ?phsA mutant strain was unable to consume thiosulfate to produce H₂S,so it was unable to react with the indicator to produce black precipitate.The growth curve of wild-type and mutant strain with the stimulation of common antibiotics?norfloxacin,gentamicin,kanamycin,and tetracycline?or H2O2 were detecting.The results showed that the growth level of wild-type strain was significantly higher than that of the ?phsA mutant,suggesting that the wild-type strain could defense against the antibiotic through producing H₂S.Under the stimulation of the antibiotic or hydrogen peroxide,the ROS with oxidative damage in the wild-type strain was significantly lower than that of the ?phsA mutant,indicating that H₂S defense against antibiotics by reacting with ROS.In conclusion,the studies of H₂S-negative Salmonella isolates were carried out in this study.By antimicrobial susceptibility testing,we found the different in antibioticresistance H₂S-negative and H₂S-positive Salmonella.By the analysis of the genetic polymorphism and variation between H₂S-negative and H₂S-positive Salmonella,we found that two newly ST of H₂S-negaitive Salmonella,and some H₂S-negative and H₂S-positive isolates may evolve from diverse sources.By the exploration for the function of H₂S defense against antibiotics in Salmonella,we found that H₂S increases Salmonella resistance to oxidative stress and antibiotics by removing the ROS.These founding provided an important basis for the appropriate use of antibiotics,the trace to the infectious source and the effective prevention of Salmonella transmission.In addition,these results provided new ideas for the study of H₂S as potential antibioticresistance targets in bacterial resistance.