Effect And Mechanism Of Escherichia Coli Ampicillin Resistance On Phage Lysis

Antibiotic-resistant bacteria are a common threat to human and animal health.E.coli is an important food-borne opportunistic pathogen.The treatment failure caused by antibiotic resistance of E.coli is increasing.The World Health Organization listed 12 drug-resistant pathogens of greatest concern for human health in 2017,the most important of which are Acinetobacter baumannii,Pseudomonas aeruginosa,and Enterobacteriaceae.Because the development of bacterial antibiotic resistance is much faster than the development of new antibiotics,it is urgent to develop alternative therapies.Bacteriophages are regarded as potential biotherapeutic agents in the fight against resistant bacteria due to their host specificity,no side effect on patients and potential for constantly evolution to fight resistance developed by bacteria.Although a large number of phage monotherapy or combination therapy has been carried out,it is still unclear whether the lytic effect of phage on bacteria will be affected by rapidly increasing antibiotic resistance.In this study,the phage vB_EcoM-ep3 and E.coli CVCC1418,which were preserved in the laboratory,were the main research objects,and the molecular mechanism of ampicillin resistance affecting phage lysis of E.coli was sought.First,we found that E.coli strain O78-6,which has more complex antibiotic resistance,was significantly more sensitive to phage vB_EcoM-ep3 than the strain CVCC1418,which also belongs to the O78 serogroup.The spontaneous mutant ampicillin-resistant strain CVCC1418 AmpR and ciprofloxacin-resistant mutant CVCC1418 CipR were obtained through antibiotic acclimation.The lytic effect of phage vB_EcoM-ep3 was detected.The results showed that compared with the parent strain CVCC1418,the phage sensitivity of ampicillin-resistant mutant CVCC1418 AmpR increased,while the phage sensitivity of the ciprofloxacin-resistant mutant CVCC1418 CipR did not change significantly.The minimum inhibitory concentration of ampicillin on a large number of clinical isolates of E.coli stored in the laboratory was detected,and 34 of them were selected according to the MIC and divided into the ampicillin low-resistant group(MIC?25 mg / L,a total of 15 Strains)and ampicillin high resistance group(MIC?800 mg / L,19 strains in total),81 strains of phage were isolated from these strains as host bacteria,and the lytic range of these phages to 34 strains of E.coli was detected.The relationship between the lysis efficiency of these phages and the minimum inhibitory concentration of ampicillin to their host bacteria was analyzed,the results show that the increase in ampicillin resistance is closely related to the increase in the sensitivity of E.coli to phages,and this phenomenon is more obvious in the O78 serogroup strains,suggesting that these isolates may have the common LPS involved in phage binding.However,not all O78-derived phages are capable of infecting all O78 serogroup strains,suggesting that other phage receptors may be involved and that these phage receptors have different expression and / or accessibility in these isolates.In order to explore the mechanism by which ampicillin resistance promotes phage lysis of E.coli,iTRAQ technology was used to analyze the proteomic differences between the parental strain CVCC1418 and the ampicillin-resistant mutant strain CVCC1418 AmpR.iTRAQ proteomics experiments identified 54 differentially expressed proteins in CVCC1418 AmpR compared with CVCC1418,of which 31 proteins were up-regulated and 23 proteins were down-regulated.A large number of differentially expressed proteins related to metabolic pathways in CVCC1418 AmpR have changed;multiple differential proteins related to chemotaxis,signal transduction,and flagella synthesis of bacteria so that bacteria can respond to external stimuli in time;the expressions of multiple outer membrane porin are significantly different,which accelerate antibiotic efflux and reduce antibiotic intake;?-lactamase AmpC is significantly upregulated to promote the hydrolysis of ampicillin and fundamentally makes the strain resist to the bactericidal effect of ampicillin.It can be seen from the proteomics results that the bacteria activate multiple mechanisms to fight against the killing of antibiotics.At the same time,transposon random insertion system was used to construct transposon random insertion mutation library of strain CVCC1418 AmpR,67 mutants were screened whose phage vB_EcoM-ep3 sensitivity were reduced and the transposon insertion sites of these strains were identified as ampC or ampR.Because the expression of AmpC was significantly up-regulated in the strain CVCC1418 AmpR in the results of iTRAQ proteomics,AmpC was selected as the main target of subsequent research.RT-PCR confirmed that the transposon insertion sites of mutants 14 and 53 were indeed ampC,and the sensitivity of both mutants to ampicillin was significantly increased compared with CVCC1418 AmpR.To further determine the effect of AmpC on phage lysis of E.coli,we constructed an AmpC overexpression strain CVCC1418 pAmpC.Compared with the parent strain CVCC1418,the phage lysis efficiency and adsorption efficiency of CVCC1418 pAmpC were significantly increased.Then we tried to construct an ampC knockout strain.Many attempts in CVCC1418 AmpR were unsuccessful,but the ampC was successfully knocked out in K12 MG1655 to obtain the strain K12?AmpC,at the same time a complementation strain K12?AmpCpAmpC was constructed.The phage lysis effect was compared in these strains.The knockout of ampC gene significantly inhibited the phage lysis efficiency and adsorption efficiency.After ampC complementation,the phage lysis efficiency increased to some extent.Since the major membrane proteins that directly affect phage adsorption in E.coli are usually outer membrane proteins,the yield of outer membrane proteins that are affected by changes in AmpC expression were tested.The outer membrane protein OmpA transcription level increased with increased AmpC expression.As OmpA has been reported as a phage receptor,we speculated that AmpC may affect phage lysis by regulating OmpA expression,which maybe the receptor of phages.In order to determine whether OmpA is the receptor of phage vB_EcoM-ep3 on the host bacteria surface,a pull-down test was performed,and mass spectrometry was used to identify receptors on the surface of E.coli that may interact with phage vB_EcoM-ep3 receptor binding protein Dpo41.Three putative phage receptors were identified: lipoprotein LPP,outer membrane protein OmpA,and enolase Eno.In order to further determine the phage receptor,the identified proteins are prokaryotic expressed and purified,co-immunoprecipitation were carried out to detected whether these proteins bound to Dpo41.The results showed that OmpA bound to Dpo41,which means OmpA was the receptor for Dpo41 on the surface of E.coli.Therefore,the upregulation of AmpC in the strain CVCC1418 promotes the expression of the phage receptor OmpA.The increased phage receptor on the surface of the bacteria provides more opportunities for the phage to bind to their host,resulting in an increase in phage lysis efficiency.Our research proves that even against,whose antibiotic resistance is rapidly increasing,phages can still effectively play a bactericidal role,explaining to some extent the mechanism by which phage-antibiotic combination can significantly inhibit the development of antibiotic resistance.It provides theoretical and experimental basis for the synergistic effect of phage-antibiotics combined application in infectious diseases.