| Antibiotic resistance(AR)is considered as a new type of environmental pollution,and has become a major threat to human health.Two characteristics of AR pollution make it difficult to eliminate:First,under certain selection pressure of antibiotics,microorganisms(including pathogens)can carry resistance to spread and accumulate in water,air and soil,and lead to difficulties for treating patients infected with the pathogens;Second,antibiotics resistance genes(ARGs)are relatively stable in nature and can persist in environment for a long time,as well as cannot be removed completely by conventional sewage treatment systems.These ARGs enter microorganisms by information exchange and spread to humans and animals.To solve the problem of AR pollution,the monitoring of AR in the environment and the study of AR mechanism are two very important basic works.Hospitals are pool area of pathogens,and also the largest unit of antibiotic use.Multidrugresistant bacteria in hospitals were discharged into the wastewater through the sewer,these ARBs may spread into the surrounding environment,causing serious AR problems.In this context,hospital wastewater and hospital wards were selected as AR monitoring units to evaluate the AR level of hospital environment,and daptomycin(DAP),the "last-resort"antibiotic,was selected to explore its AR mechanism,hoping to provide more basic data for solving the AR problem.The main research contents and results are as follows:(1)The common potential pathogens in hospital wastewater were preliminarily investigated and ARBs were isolated and identified to provide basic data and materials for the evaluation of AR level in the next stage.The samples were collected from hospital wastewater before and after treatment during one year.The "last-resort antibiotics":polymyxin E(PME),tigecycline(TGC),vancomycin(VAN),daptomycin(DAP),meropenem(MEM),ceftazidimeavibatam(CZA),were used to investigate the common potential pathogens and drug resistance in four seasons.It is shown that:①High levels of Enterobacteriaceae and Enterococcus(104～105 CFU/mL)and intermediate level(102～103 CFU/mL)of Pseudomonas and Staphylococcus were found in untreated wastewater.②The abundance of common potential pathogens showed seasonal variation.The abundance of most potential pathogens was always high in spring,low in summer,and medium in autumn and winter.③The AR ratio of common potential pathogens to the last-resort antibiotics is different.The average AR level of Enterobacteriaceae to PME is the highest,while the average AR level to TGC is still very low.Most Enterococcus is still susceptible to VAN,but its AR rates to TGC and DAP are high.The AR level of Pseudomonas to the last-resort antibiotic is still relatively low.The AR rates to DAP.TGC and VAN.the last resort antibiotics angainst Staphylococcus,are all high,suggesting that resistance to several last resort antibiotics has reached worrying levels in hospital settings;④REP-PCR could effectively remove duplicate strains,and about 40%of redundant strains were removed from 1384 single strains,and 809 unique strains were obtained,thus providing more reliable survey data;⑤Based on the results of isolation and identification,we understand the general community characteristics of common potential pathogens in hospital wastewater:Enterobacteriaceae strains were the main Gram-negative bacteria;The main sources of Gram-positive bacteria were Enterococcus strains.These results suggest that there is a close relationship between pathogens in hospital wastewater and nosocomial infection.(2)Based on the isolated and identified strains,the AR level of the current major antibiotics were comprehensively analyzed and evaluated.AR characteristics of the current mainstream last-line antibiotics,including polymycin E(PME),tigecycline(TGC),linezolid(LZD),daptomycin(DAP)and vancomycin(VAN),were highlighted and their risk of transmission was assessed.It is shown that:①AR phenotype analysis showed that most of the potential pathogens in hospital wastewater were multi-drug resistant(MDR).The proportion of MDR strains in Enterobacteriaceae and Enterococcus was up to 74.93%and 98.18%,respectively.②The multiple antibiotics resistance indice(MAR Indice)was more than twice as high as previous reported results,suggesting that the AR pollution of hospital wastewater was becoming more and more serious.③Several potential pathogens contribute most to MAR Indice,including Escherichia,Klebsiella and Proteus in Enterobacteriaceae,and E.faecium and E.faecalis in Enterococcus,and they should be the key bacteria for monitoring AR contamination.④The analysis of antibiotics resistance determinants showed that class I integron and class II integron had high detection ratio,suggesting that they might be the main mobile genetic elements(MGEs)of ARGs transmission in hospital wastewater.⑤The AR of last line antibiotics deserves high attention.Most of the pathogens showed multiple drug resistance,and the proportion of AR phenotype and genotype was as high as 33%～69%.Most of Enterobacteriaceae strains(77.81%)were non-susceptible to PME,and almost all(97.95%)Enterococcus were non-susceptible to DAP.Some strains with VAN and LZD resistance showed a potential risk of transmission.(3)The changes of microbial community in hospital wastewater before and after treatment were compared by 16S high-throughput sequencing.The effects of wastewater treatment procedures on AR were evaluated from the changes of the abundance,diversity and function of microbial community.It is shown that:①At phylum level and genus level,the abundance of microbial community in hospital wastewater changed significantly,especially in summer and spring,and the structure became more simple;②α diversity of microbial community decreased at phylum level and genus level,but the variation was not significant.The variation was more obvious in summer and spring than in autumn and winter.The result of β diversity showed that the internal structure of the community changed significantly.③The analysis of microbial function showed that treatment procedure of wastewater had little effect on function of microbial community at both phylum level and genus level,suggesting that microbial community was relatively stable.④In addition,the results based on plate culture showed that no bacteria could be isolated from the wastewater after treatment,suggesting that the wastewater treatment system could effectively remove common culturable potential pathogens,but did not destroy the functional stability of microbial community in wastewater.Taken together,treatment systems of hospital wastewater may not be able to prevent the spread of AR,and effective removal techniques for ARGs are necessary.(4)MDR E.Faecium with DAP and VAN resistance was analyzed through genomic sequencing and comparative genomics to understand the genetic basis of its multiple resistance.The mechanism of DAP resistance was investigated by molecular cloning,point mutation,twodimensional thin layer chromatography,cardiolipin fluorescence probe,transmission electron microscopy and survival assays.It is shown that:①Most of the ARGs of this strain were located on the chromosome,but the plasmid pEF332-2 carried both vanA and vanM gene clusters,which was associated with high VAN resistance and high transmission risk of VAN resistance.②The I269T mutation of cls gene may reduce the negative charge on cytomembrane surface by reducing the content of cardiolipin in outer membrane of cytomembrane,thus reducing the adherence to DAP-Ca2+ and producing DAP resistance;It is also possible to transfer DAP from septum to other location through the redistribution of cardiolipin,resulting in DAP resistance;The two modes may also synergistically confer DAP resistance.③P201S mutation of gdpD gene did not change DAP resistance,but changed amino lipid composition of cytomembrane.④The analysis of epidemiological characteristics showed that this MDR E.faecium belongs to the CC17(clade Al)clone complex associated with the hospital outbreak.VanA gene cluster in the plasmid pEF332-2 is located in both the prophage and genomic island,and vanM gene cluster may have spread nationwide,suggesting a high risk of transmission.Surveillance of AR to MDR E.Faecium in hospitals should be strengthened.In conclusion,the overall level of AR in the hospital environment is relatively high.The high proportion of MDR bacteria and the high level of MAR Indice indicate that the AR pollution in hospital wastewater has been very serious.The AR to last-line antibiotics deserves great attention,with a high proportion of strains resistant to two or more last-line antibiotics;A few last lines are at risk of being broken,such as DAP and PME;VanM-the new VAN reistance gene showed signs of rapid diffusion;The AR level to LZD was moderate;TGC is still an effective antibiotic in the treatment of infections by Gram-negative and Gram-positive bacteria,with a low rate of resistance.Wastewater treatment systems are effective at killing common potential pathogens,but they will not eliminate and stop the spread of AR,so the risk of AR spreading remains.A clinical strain of MDR E.faecium with VAN and DAP resistance also reflects the high plasticity and the potential of AR transmission.Studies on AR mechanism revealed that a new mutation of cls gene may confer DAP resistance by multiple patterns,and the characteristics of its chromosome and plasmid suggest high transmission potential.Based on the results of this study,it is necessary to strengthen the monitoring of multiple resistance pollution in hospital environment,and develop effective sewage treatment technology to remove ARGs. |
PDF Full Text Request