The Development Of Antibiotic-resistance Bacteria Under Photocatalytic Stimulation

With the excessive use of antibiotics,the occurrence,persistence,and transfer of antibiotic-resistance bacteria(ARB)have posed great threat on the global human public health.Particularly,antibiotic resistance is a serious problem in developing countries,where many patients infected with antibiotic-resistance pathogens are face to the situation where no medicine can cure the diseases.It was reported that,if the problem of antibiotic-resistance is not effectively controlled,by 2050,more than 10 million people will die each year from infections mediated by antibiotic-resistance pathogens.Generally speaking,the acquisition of antibiotic-resistance by bacteria is mainly through two pathways.One is the overuse antibiotics in the clinical and the continuous induction and screening of antibiotic-resistance bacterial mutants.Another is the acquisition of specific antibiotic-resistance genes(ARGs)by horizontal or vertical transfer.At present,a large number of studies that focus on the development and evolution of ARB are limited to the clinical and microbial community levels.However,in the natural environment,bacteria exposed to residual antibiotics can also evolve and lead to enrichment or maintenance of ARB.Except to the induction of antibiotics in the environment,bacteria also suffered from a series of other adverse growth conditions or potentially lethal factors such as light,heat,nutrition stress,metal ions.Bacteria could arouse their stress system and change the expression patterns of genes with different functions as well as the structure or physiological state of cells after encountering these adverse stimuli,which so as to make bacteria perform certain tolerance to external stimuli.Correspondingly,the emergence of bacterial stress response and physiological changes might be closely related to their antibiotic-resistance.Therefore,the purpose of this study is to reveal how ARB responded to photocatalytic(PC)interface and elucidate its subsequent development,persistence,transfer and genetic stability.(1)Before studying how bacterial stress response to the PC stimulation,the damage of bacteria under PC stimulation was firstly analyzed.The biofilm(BF),the main existing form of bacteria in the various nature environments,was obtained by artificial culture and exposed to the PC stimulation.The changes in bacterial activity were observed using flow cytometer and fluorescence microscope.The changes in bacterial motility was measured to analyze the damage degree of bacterial capsule structure during the damage process.In addition,the extracellular polymer substance(EPS)content of the bacteria was analyzed.Further,the changes of quorum sensing and barrier effects of BF were also analyzed during PC stimulation process.The results shown that,30%of bacteria were inactivated after 4.5 h of PC stimulation.It could be seen from the growth trajectory of the bacteria on the swimming agar that the motility of the bacteria also began to increase significantly,indicating that the structure of the bacterial biofilm was seriously damaged.Correspondingly,the number of planktonic bacteria were increased.Through the analysis of quorum sensing,it can be seen that the PC stimulation could obviously eliminate the quorum sensing phenomenon.In addition,the barrier effects of BF were significant reduced.These results indicated that the PC oxidation could eliminate the vitality and characteristic of BF.(2)After revealing the damage that occurs when the bacteria were PC stimulated,from the perspective of gene,protein,physiological and biochemical,a systematical method has been established to further study how the ARB responded to the stimulation of PC interface.The bacterial stress responses were determined through the analysis of the gene expression change(including oxidative stress,envelope stress,SOS stress,nutrition impairment).Besides,the level of bacterial intracellular reactive oxygen species(ROSs)and antioxidant enzymes including catalase(CAT)and superoxide dismutase(SOD)were assay.In addition,the changes in bacterial morphology were also observed.Furthermore,a series of changes of bacteria exposed to PC stimulation and antibiotic stimulation were comparatively studied.The results shown that the intracellular ROSs level and the activities of CAT and SOD increased significantly after PC stimulation,indicating that the bacteria suffered oxidative damage.The morphology analysis showed that the bacteria also broke after a long time of stimulation.However,within the induction period of 30 min,the bacteria could still survive and arouse a variety of stress responses.Moreover,these stress responses were very similar to those caused by antibiotics,indicating that these stresses related genes associated with antibiotic resistance could respond not only to antibiotics but also to photocatalytic stimuli.These results also suggests that there might have some potential link between photocatalytic stimulation and bacterial antibiotic-resistance.(3)Furthermore,the long-term phototatalytic induction and cross-induction mediated by phototatalytic and antibiotic were established respectively to investigate if the antibiotic-resistance could be induced by the photocatalytic stimulation.Besides,the mechanisms related to antibiotic-tolerance was assayed such as the mutation rate,expression of efflux pump,formation of biofilm.The results showed that the photocatalytic stimulation cannot contribute to the occurrence of ARB directly.However,the time needed to inactivate 99%of bacteria increased from 30 to 150 min for polymyxin(PB),55 to 135 min for tetracycline(TET),14 to 40 min for ciprofloxacin(CIP),22 to 35 min for streptomycin(SM),and 29 to 45 min for azithromycin(AZI).Besides,the mutation rate was increased for 3-5 fold change The activity of efflux pump was improved so that reducing the accumulation of antibiotics in bacterial intracellular,which help bacteria escape from the attack of antibiotics.Afterwards,the bacteria with higher antibiotic-tolerance could evolve to antibiotic-resistance faster with subsequent antibiotic selection.The fold change of antibiotic-resistance level was up to 32 for PB,32 for TET,16 for CIP,16 for SM and 4 for AZI,which are higher than those of normal bacteria without pre-induction.(4)After that,we further investigated how the oxidation pressure posed by photocatalytic interface affect the persistence of bacterial antibiotic resistance.Two kinds of antibiotic-resistance bacterial strains related to polymyxin(PB)and cefotaxime(CTX)both in type of chromosomal mutation and ARGs plasmid mediated were employed to investigate their fade or maintenance mechanism under Sub-PC stimulation through competitive growth with antibiotic-susceptible bacteria(ASB).The fitness cost of ARB and ASB were compared through competitive fitness assay and followed by respiration rate measurement,ROSs generation and anti-oxidation enzyme activities.Furthermore,during competitive growth,the antibiotic susceptibilities of community were also determined using minimal inhibitory concentration(MIC)method,and the abundance of ARGs-encoded plasmid in bacterial community was also quantified through quantify polymerase chain reaction(Q-PCR)assay.Results revealed that the fitness cost of both two types of ARB decreased under PC stimulation as compared with Luria-Broth(LB)broth condition.Plate counting revealed that antibiotic resistance mutant exhibited 6-7 days longer maintenance under the sub-PC conditions than that under the LB condition.Besides,for plasmid-encoded ARB,the average abundance of ARGs in bacteria community was maintained at 0.38 copies for ctx-1 and 0.58 copies for mcr-1 under the sub-PC condition,which were higher than that under the LB condition(0.29 copies for ctx-1 and 0.45 copies for mcr-1).This study suggested that photocatalytic interface could cause longer-term maintenance of antibiotic resistance,which could provide an insightful understanding that how anthropogenic sterilization technologies or stressor may alleviate or spread bacterial antibiotic resistance(5)Furthermore,a complex antibiotic-resistance bacterial community(ARBC)combined with five different constructed ARBs was applied to study the effects of PC on the mutually transfer of ARGs plasmids in the ARBC and changes in diversity of antibiotic resistance,change of different ARGs plasmid abundance in ARBC,antibiotic resistance level of ARBC as well as genetic stability of ARGs.The results showed that,mutually transfer of ARGs occurred with all possible multi-ARB such as double-ARB,triplet-ARB,quadruple-ARB and quintet-ARB emerged.With PC inactivation,more than 90%of ARB was transformed to ASB with ARGs discarding.Among residual ARB,double-ARB was dominant and accounted for 99%.Nevertheless,the abundance of ARGs changed different,where decrease for gem and tet,increase for kan,no obviously change for ctx and pb.Besides,the alternation of resistance level was consistent with the alternation of ARGs abundance.Correspondingly,there existed point mutation for ctx,kan,pb after PC stimulation.These results suggested that the transfer and genetic stability of ARGs could select under PC stimulation.In all,in this study,the effects of PC stimulation on the development,persistence,transfer and genetic stability of ARB were comprehensively elucidated.Among them,PC stimulation could trigger a series of stress responses of bacteria and mediate some functional expression or physiological changes to improve the antibiotic-tolerance.In addition,PC conditions can reduce the fitness cost of ARB and lead to longer maintenance.In the bacterial community system,PC stimulation could control the mutual transfer of different ARGs in the community and affect the stability of certain ARGs to a certain extent.This thesis provides a more in-depth and comprehensive understanding of the development and changes of ARB in the environmental process and provides guidance for the solution of antibiotic-resistance problem.