| Pullorum disease (PD) is a severe systemic infection caused by Salmonella enterica subspecies enterica serovar Pullorum (S. Pullorum). PD is rare in the poultry industry of developed countries and mainly distributing in the developing countries, such as Africa and Asia. S. Pullorum is one of the pathogenic bacteria seriously affecting the poultry industry in China. Antimicrobial therapy is being used as an important measure to control PD, however, the widespread use and abuse have contributed to the increasing occurrence of resistant bacteria in poultry, which can be transmitted to humans via the food chain. Bacterial resistance can be divided into innate resistance and acquired drug resistance. The acquired resistance genes are often located on mobile elements and the plasmids with these genes are important for dissemination of antimicrobial resistance. Until now, the research about characteristics and mechanism of resistance in S.pullorum isolates are still limited.To characterize the prevalence and mechanism of resistance in S. Pullorum isolates, the resistant characteristics and multiple drug resistance status of the isolates between 1962 and 2015 were investigated, and also analyzed the isolates between home and abroad. The resistance genotype and its correlation with phenotype were analyzed through blasting the information of whole genome sequencing with NCBI. We also identified a specific resistance plasmid, named pSPUR, and explored its resistance and dissemination mechanism.1. The characteristics of antimicrobial resistance in S. Pullorum isolates in ChinaThis study determined the minimum inhibitory concentration (MIC) of 423 S. Pullorum isolates between 1962 and 2015 to 18 kinds of antimicrobial agents in China. The resistance rate of NAL was the highest, up to 69.3% and the next was TET (43.8%). The prevalence of resistance to the antimicrobials of AMP, AMC, SXT, CFZ, CIP and AZT was higher, which ranged approximately from 5.2% to 22.0%. Low levels of resistance to EFT, MEM, AMK, CHL and OLA were detected with less than 1%. Luckily, no resistant strain to KAN, GEN, ENR and NIT was found.In terms of time, the resistant trend of isolates to AZT, TET and NAL was increasing gradually. The strains resistant to AZT, TET and NAL mainly distributed after 1997 and the resistant rate of NAL was 100% at 2009 and stayed at this level with slight fluctuation. In 2006, the isolate resistant to CIP appeared and the resistant level stayed low and stable. And the resistant rate of (3-lactam antibiotics was reduced firstly and then increased,In this study, the multi-drug resistance of S. Pullorum isolates in China showed an increasing trend. The multi-drug resistant strains were only appearing in the 1990s with a rate of 13.3%. Then the multi-drug resistance rates gradually increased and the spectrum were also more complex. The rates increased from 12.2% in 1990s to 37.4% in 2000s and then to 47.3% in 2010s together with appearance of the strains resistant to 4 to 5 kinds of antimicrobials. The multi-drug resistant strains were mainly resistant to the β-lactam antibiotics, tetracycline, sulfonamides and quinolones.We also compared the resistance of the isolates between our country and the abroad. Only 1 and 2 resistant strains were detected among those from Europe and South America, while the rates were significantly higher in our country at the same time period, ranging from 7.1% to 28.6% and 3.6% to 28.6%, respectively.2. Predicting the resistance genes of 50 S.Pullorum isolates based on whole genome sequencesBLASTn was used to identify the presence of relevant resistance gene loci from the reference ARDB database in the raw contigs for 50 S. pullorum isolates from China. Matches with>80% identity at the nucleotide level and representing a match of>80% of the reference gene length were retained; this included partial matches with>80% sequence homology over 80% of the reference gene length, but distributed over several contigs. All the 50 isolates had the MDR efflux pumps and transporter (Acrb-TolC, MdtK, MdtG, EmrD, MdfA, SdeY) and the resistance gene of kasugamycin ksga. Antimicrobial genes, aph33ib, tetA, sull/2 and blaTEM-1 were identified associated with the resistance of STR, TET, SXT and penicillins and among the 17 isolates had at least one of these genes, the genotype of 16 strains was in concordance with the phenotype and the coincidence rate was 100%. Phenotypic and genotypic correlation was 100% for STR and NAL, and 92% for TET. All isolates resistant to SXT had the gene sull and the accordance rate was 100%. S83F was the main mutation in the gyrA gene and all the isolates had this mutation showed resistant to NAL. Also the MIC value of CIP was higher. The mutation in other point of gyrA and parE didn’t show resistance to the quinolones. The quinolones and the mutation in the 83 point of gyrA was the most important.3. Identification of a specific resistance plasmid pSPUR in S. Pullorum isolatesThis study also identified a new specific resistant plasmid pSPUR with the size of 30 to 50 kb contained in the strains with MIC value of≥512μg/ml. The structure of these plasmids was similar and this plasmid was not reported previously. This plasmid contains main resistant gene of blaTEM-1 and four kinds of transponsases (IS1, IS240, Tn3 and InsA). A transconjugant was obtained successfully with the recipient of S06004 which may be contributed to the species-specific conjugation of the plasmid. The transconjugant showed 1024-and 32-fold increases in the MICs of AMP and AMC compared with parent strain S06004. The transformants showed 512,128-256 and 16-256 fold increases in the MICs of AMP, AMC and CFZ compared with J53 and the MIC value was also increased compared to the donors. This plasmid has the transferable ability and may disseminate the resistance of penicillin in S. Pullorum isolates. |
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