| Polymyxin is known as the last resort to treat Gram-negative pathogen infections,which means,the widespread spread of polymyxin-resistant(polymyxinR)bacteria will put humans into the 'pre-antibiotic era'——no antibiotic can treat Gram-negative bacterial infection.The mainly mechanism of polymyxin resistance includes genomic mutations,such as the mutation of the mgrB gene,and the acquisition of the polymyxin resistance gene mcr(the mobilized colistin-resistance gene,mcr).Among PolymyxinR K.pneumoniae of farm,mcr acquired strains are very common while there are few strains of genome mutation.However,the specific research on the distribution of the two strains in the farm environment is unclear.On the other hand,when bacteria acquire drug resistance,they also bring their own fitness costs.However,there has been no breakthrough research progress on the fitness cost associated with polymyxin resistance genes.Therefore,the following two questions are raised:(1)Is the polymyxinR K.pneumoniae in the farm environment dominated by mcr-acquired strains?(2)In the farm environment,does the polymyxinR of K.pneumoniae of genome mutation bring them fitness cost?Therefore,this article takes K.pneumoniae of poultry environment as representative and study on the above two scientific issues.Firstly,isolate K.pneumoniae to obtain the research materials.Collect poultry feces from Anhui Chicken Farm,Huai'an Chicken Farm,Nanjing Qinglong Forest Farm,Xinyang Huangchuan Chicken Farm,Nanjing Xiaowei Street Market,Nanjing Tongwei Street Market and Yunnan Wild Animal Park.Use MacConkey Inositol Atonol Medium in combination with K.pneumoniae-specific gene khe identification and 16S rDNA sequencing.181 K pneumoniae isolates were achieved from fecal samples.Then,the virulence genes and drug resistance genes of these strains were identified by PCR technology,and the drug sensitivity of the strain was determined by minimum inhibitory concentration test of antibiotics.Results showed that virulence genes irp2,and kfu and ?-lactamase gene blaKPC were detected.Secondly,do conjugation to identify whether the polymyxinR gene of K.pneumoniae can transfer.The characteristic of the mcr gene was further investigated through plasmid transfer frequency experiments,antibiotics minimum inhibitory concentration test and multidrug resistant gene identification.The results showed there were 93 strains suitable for transfer experiments,of which polymyxinR of 78 strains were transferable,the detection rate was 83.87%,and 15 were not transferable(genomic mutant strains),detection rate was 16.13%.After identifying mcr gene types,67 strains(85.90%)were identified as positive for mcr-1 gene and 11 strains(14.10%)were positive for mcr-5.This result indicates that the polymyxinR K.pneumoniae isolated from livestock farms is dominated by mcr acquired strains.Thirdly,in order to explain the result of higher rate of the mcr acquired K.pneumoniae in poultry farms.Fitness cost of genomic mutants and mcr acquired strains in these isolates were further compared.The fitness cost of its polymyxinR was tested by the bacterial growth curve measurement,bacterial biofilm experiment and Galler mellnella survival test.The results showed that in LB environment,genomic mutants grew slowly;in fecal liquid,the survival time of genomic mutants is shorter;No significant difference appeared in biofilm formation of two strains;genomic mutant is weaker in virulence test of G.mellonella fatality.In summary,in term of this farm ecological survey,the detection rate of transferable polymyxinR is greater than that of non-transferable in K.pneumoniae isolates.Meanwhile,polymyxinR of non-transferable bring K.pneumoniae fitness cost.The cost is mainly manifested in strain growth rate and strain virulence.The above conclusions explain the phenomenon that polymyxinR of genomic mutants of farms is rare.It has potential meaning in revealing the resistance of polymyxin and controlling the evolution of bacterial resistance. |
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