| Polymyxin is considered to be the"last line of defense"for the treatment of multi-drug-resistant gram-negative bacteria infections,however,the emergence of polymyxin resistance gene mcr-1,which is plasmid-carried and horizontally transferable seriously threatens the clinical application of the drug.Most studies have shown that,the intestinal tract of food-animals is an important place for the emergence and development of the mcr-1 gene.However,little is known about the distribution and spread characteristics of this gene in food-animal intestinal flora.In this study,we used intestinal content of a single pig as the research object,carried out large-scale bacteria strain isolation and identification,established the polymyxin-resistant bacteria pool.In order to investigate the distribution characteristic and spread pattern of the mcr-1 in the intestinal flora,PCR was used to detect mcr-1 gene,and Matrix-assisted Laser Analysis Ionization Time-of-Flight Mass spectrometry(MALDI-TOF MS)technology was used for identification and genetic relationship analysis of the mcr-1-positive strains.On this basis,whole-genome sequencing(WGS)and bioinformatics analysis are used to interpret molecular mechanism of mcr-1 transmission.This study has important scientific value for understanding the emergence and development of mcr-1 gene in swine intestinal flora and provide useful information for its prevention and control.In this study,fresh fecal sample of a single pig.was collected and screened for polymyxin-resistant bacteria with antibiotic supplementary plates,With the MALDI-TOF MS identification,a total of 410 polymyxin-resistant strains were obtained,including 70 Escherichia coli(17.2%),19 Klebsiella pneumoniae(4.6%),26 Proteus mirabilis(11.0%),82Pasteurella aeruginosa(20%)and 193(47.2%)unidentified strains.PCR results showed that 282 of these strains were mcr-1positive,including the 70 E.coli,19 K.pneumoniae and the 193 unidentified result strains.The protein fingerprints cluster analysis of the mcr-1 positive strains showed that the 70 E.coli strains were divided into 13 subtypes,and 19 K.pneumoniae strains were divided into 3 subtypes,of which 17 strains belonging to one subtype(89.5%),The protein fingerprints of the193 unidentified bacteria showed high similarity,indicating that all these strains not only belongs to same bacteria species but also same clone.The co-existence of the diversity and clonality of mcr-1-positive strains suggests that the mcr-1 gene has both clonal transmission and horizontal transfer in the pig intestinal flora.The representative strain GY-402 of the-above-mentioned unidentified mcr-1 carrying bacteria was selected to conduct in-further research on the genetic environment and spreading mechanism of the mcr-1 gene.Using Illumina Hiseq 2000 and Min ION sequencing platforms,the complete genome of GY-402 was obtained.Species identification using JSpecies WS online server platform showed that strain GY-402 is a new species of Actinobacillus genus.Bioinformatics analysis showed that mcr-1 gene is located on a novel integrative conjugative element element ICEAsp1,which is 72 978 bp in length.mcr-1and 9 other drug resistance genes were identified in the multi-drug resistance(MDR)region of the ICEAsp1.Inverse PCR showed that ICEAsp1 elements could form circular cyclization intermediates.Conjugation experiments showed that ICEAsp1 could transfer into E.coli and Pasteurella multocida recipient strains,with transfer frequency 5.8×10-8 and 4.3×10-9,respectively.The susceptibility test of the transconjugant and corresponding original strains revealed that the resistance genes on the ICEAsp1 can result in MIC increases of the corresponding antibiotics.Further analysis of the MDR structure of the multidrug resistance region of ICEAsp1 showed that one ISVsa5 and four ISApl1 elements are scattered among the 9drug resistance genes,and the four ISApl1 elements in this region can be combined in pairs to form different composite transposon.In addition,a new chloramphenicol resistance gene was identified in the MDR region,named cat T.Gene cloning and functional verification showed that cat T is an active chloramphenicol resistance gene,belonging to the new Cat-A subfamily A-17.In summary,this study explored the distribution characteristics and spread mechanisms of mcr-1 gene in pig intestinal flora.The coexistence of typing diversity and clonality of mcr-1-carrying strains suggests that distribution of this gene could be mediated by both horizontal transfer and clonal transmission in the pig intestinal flora.The discovery of a new ICE element carrying the mcr-1 gene in a new Actinobacillus bacteria indicates that the mcr-1 gene has a wider species distribution and more diverse transmission vectors,further studies are warranted to investigate the distribution of other important resistance genes in different food-animals intestinal flora. |
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