Co-resistance Study Of A Staphylococcusaureus Strain LZ-01 Isolated From Contaminated Sludge Of The Yellow River To Antibiotics And Heavy Metals

Antibiotics might be the greatest discovery in early 20 th century,which have saved incalculable lives from dangerous infections and alleviated sufferings from injuries.Nowadays there are thousands of different antibiotics used in medical services in cope with various pathogens,as well as agricultural and aquacultural practices for higher yields.Since the occurrence of MRSA(Methicillin-resistant Staphylococcus aureus)in 1960 s,a series of superbugs were found to have pan-drug resistance,like NDM-1,VRE(Vancomycin-resistant Enterococci)and CA-MASA.Abuse of antibiotics was used to be supposed as the major cause but the decreased usage of antibiotics subsequently does not necessarily prevent the spread of antibiotic resistance in clinical.Meanwhile,many studies have found that antibiotic resistance positively correlated to toxic metals or other antimicrobials in environments.Metal-induced antibiotic resistance is supposed to be more prevalent than antibiotic itself,due to the fact that metals were more persistent than antibiotics in nature environment.The metal contamination is hard to eliminate due to the huge areas and scattered distributions,metal-induced antibiotic resistance are enriched either in amount or abundance under metal stress,and incurable diseases caused by resistant pathogens would be prevalent and become a disaster.Therefore,it is essential to fully understand the co-resistant mechanism of metals and antibiotics,and find ways to decelerate the spreading tendency of resistant genes.In this study we isolated a strain from the silt of Yellow River which was polluted by industrial waste water,and this strain showed high heavy metal resistance.It could resisitant to 6 m M metal chromium(Cr),1 m M lead(Pb)and 6 m M manganese(Mn),respectively.This strain also had higher antibiotic resistance levels,with the MIC of 0.51 mg/m L to ampicillin(Amp)and 0.25 mg/m L to chloramphenicol(Chlo).The metals and antibiotics resistance of this strain could be enhanced after the induction by each other.When the strain was induced by 0.50 m M chromium(Cr)or manganese(Mn),the resistant levels to Amp reached 2.5 mg/m L and 1.6 mg/m L respectively.When 0.15 mg/mL Amp or 0.05 mg/mL Chlo was used for induction,the metalresistance of this strain reached 10 m M or 8 m M to chromium(Cr).In addition,the simultaneously occurrence of heavy metals and antibiotics would improve both resistance levels of this strain.This strain could resist to 6 m M Cr,and simultaneously resist to 0.75 mg/m L Amp.After the analysis of 16 S r RNA sequence,this strain was supposed as a novel subspecies of Staphylococcus aureus,and was named Staphylococcus aureus LZ-01 in this study.In order to understand the co-resistance mechanism of S.aureus LZ-01 to heavy metals and antibiotics,we primarily used transcriptome sequencing method to find the response genes under metal chromium stress.Results showed that out of the 2370 annotated genes,the expression of 512 genes was up-regulated.Three possible ways were revealed by the function analysis of up-regulated genes,extracellular reduction,intracellular reduction and efflux all contributed to the chromium resistance of S.aureus LZ-01.Compared with the efflux pathway,the reduction manner was a weak strategy.Then 43 genes were picked out by the criteria that these genes all related to antibiotic resistance,and the expression level of these selected genes were measured by q RT-PCR method under Amp stress.Only several genes were found up-regulated under Amp stress,and benefitted from bioinformatics methods,we attributed the heavy metals and antibiotics co-resistance to the chromosomal operon emr AB,which was vital for co-resistant performance of S.aureus LZ-01.In the experiment,the contribution of emrA/B gene to the metal and antibiotic resistance of S.aureus LZ-01 strain was verified by the method of gene knockout and complementation.The chromium(Cr)and ampicillin resistance(Amp)of mutant strain with emr A gene knockout reduced about 80%,while the emr B gene knockout decreased chromium(Cr)resistance by 30% and ampicillin(Amp)resistance by 60%.Knockout of the emr A/B gene led to the loss of induced resistance and cross-tolerance between metals and antibiotics.The obtained results showed that emr A/B genes in S.aureus LZ-01 strain was very important for its antibiotic and metal co-resistance,there may be three ways assumed for the co-resistance: one is the efflux pump,another is biofilms,and the other is regulation effect.Then we briefly revealed the regulation manners and protein structures of Emr A/B,the results revealed the existence of a two-component regulation system,which could sense the pressures of chromium(Cr)and ampicillin(Amp),and then regulate the gene transcription of emr A/B in its promoter region.The preliminary analysis of the Emr A/B protein structures showed that these two proteins could form a complex structure and functioned as an efflux pump.In this study,the subspecies strain of Staphylococcus aureus with clinical values was concerned,and the metal and antibiotic co-resistance mechanism was thoroughly studied.This is the firstly exposure of emr A/B gene function in gram positive bacteria,and the specific ways to produceco-resistance were put forward.The results of this study supplied valuable reference for the diffusion control of antibiotic resistant genes,and helpful to the design of new antimicrobial drugs.This study also provides scientific support and theoretical basis for elimination of environmental pollution and ecological risk assessment of polluted environments.