| Due to the overuse and misuse of antibiotics,antibiotic resistance is an urgent threat to global health.The emergence of multidrug-resistant and pan-resistant strains is increased and has been a challenge to humans,animals and environment.Among the infections caused by Gram-negative bacteria,the increased prevalence of carbapenem-resistant Klebsiella pneumoniae(CRKP),which belongs to the family Enterobacteriaceae and is an opportunistic pathogen commonly associated with community-and healthcare-associated infections,has greatly compromised the efficacy of carbapenem antibiotics.Among the sequence types of K.pneumoniae(Kp),ST11 was demonstrated as a predominant clone in China.Colistin is a drug of last resort for the treatment of many multidrug-resistant Gramnegative bacteria,including Kp.Unfortunately,bacteria readily acquire colistin resistance through envelope modifications that change the charge and possibly other physicochemical properties of the outer membrane.These modifications include the addition of aminoarabinose and phosphoethanolamine to lipid A and the polysaccharide core of lipopolysaccharides(LPS)by spontaneous mutations or through the horizontal transfer of mobile elements.The fact that the enzymes responsible for these modifications are tightly regulated in most Enterobacteriaceae suggests that their expression comes with a fitness cost.However,to date,the fitness cost associated with these modifications remains poorly understood.Phage therapy is the use of bacteriophages-viruses that infect and replicate within a bacterium-to treat pathogenic bacteria,which had a short history in the premolecular era of Western medicine.Now,the global antibiotic resistance crisis and a new appreciation for the importance of the human microbiota have led to a resurgence of interest in phage therapy,not only in the classic sense of treating bacterial infections but also for its potential role in modulating microbiota.In this study,a long-tailed bacteriophage NJS1 was isolated from domestic sewage with a host Kp isolate.It belongs to the Webervirus of Tunavirinae with dsDNA genome of 49,292bp(MH445453)and specifically infects 46.88%(15/32)clinical K.pneumoniae isolates.One-step growth curve analysis showed a short latency period of approximately 10 min with a burst size of about 75 PFU/cell.Further research found that colistin-resistant Kp(colistinR Kp)conferred by a horizontally transferred mcr-1-containing plasmid was more likely to be adsorbed and infected by ΦNJS1.We then compared the levels of phage adsorption and lysis on the Kp strains containing Δmcr-1 and Δmcr-1+mcr-1 constructs and found that it is specifically the mcr-1 gene in the pHNSHP45 plasmid that contributes to phage sensitivity of Kp.The alteration of phage sensitivity also results fromΔmgrB and ΔmgrBWT.To examine whether the enhanced phage sensitivity associated with colistin resistance described above is common among Kp-phage interactions,we introduced the mcr-1 plasmid into six distinct Kp clinical isolates and compared their levels of phage sensitivity to those of their parental strains.We found that they all except one strain displayed colistin resistancedependent hypersensitivity to the ΦNJS1.In addition,we also found that other four lytic Kp phages were also more virulent against colistinR strains than against colistinS strains.ColistinR Escherichia coli MG1655(mcr-1)also showed increased sensitivity to Myoviridae Plvir than wildtype strain.Taken together,these data suggest that the acquisition of colistin resistance in Kp does indeed come with a fitness cost:increased susceptibility to lytic phage infection.Adsorption of a tailed phage to its host begins with a reversible event of cell surface recognition,which then coordinates with irreversible binding that ultimately leads to phage DNA release.All the strains that can be lysed by ΦDNJS1 produce a nonmucoid phenotype and ΦDNJS1 can infect A1806(Δwza)but not its mucoid wildtype,indicating capsular polysaccharides may protect from ΦNJS1 infection.Periodate and acetic acid-acetate buffer treatment and transposon library screen showed that lipopolysaccharide played an important role in ΦDNJS1 adsorption and infection.Long chain of O-antigen may provide an effective barrier against phage infectivity by determining ΔwyE sensitivity to ΦNJS1.Outer membrane proteins BtuB,FepA and TIGR02099 family membrane protein affect ΦNJS1 infection and there maybe more than one receptor for ΦNJS1 adsorption.Further phage adsorption and desorption tests show that colistinR Kp are more likely to be adsorbed byΦNJS1 due to increased reversible adsorption levels.We assayed the surface charges of Kp and phage particles by taking zeta potential measurements.The isoelectric point of ΦDNJS1 was about 2.5 and Kp cells with acquired colistin resistance from either ΔmgrB or the mcr-1 plasmid have less negative zeta potentials in comparison with the wild-type cells or with mcr1 deleted plasmid(colistins).These data imply that a decrease in the net negative surface charge of colistinR cells promotes the electrostatic interaction between bacteriophage and bacteria,allowing for rapid phage attachment to host cells followed by irreversible binding and infection.Given the therapeutic potential of phage therapy on colistinR bacteria,we investigated phage lysis ability using biofilm lysis model,a wax moth(Galleria mellonella)larvae model and a mouse colonization model.Biofilm formation has been shown previously to promote resistance to antibiotics and to be important for bacterial pathogenesis in many contexts.We found that enhanced phage sensitivity associated with colistin resistance also occurs in Kp biofilms.We then used G.mellonella larva model,which is a simple system for evaluating Kp pathogenesis and gene expression,to examine the in vivo therapeutic efficacy of phage against colistinR Kp.We found that Kp with mcr-1 plasmid showed increased pathogenicity than wildtype maybe caused by modification of the virulence factor LPS.However,ΦNJS1 protected Kp(mcr-1)infected larvae more effectively than those infected with Kp lacking mcr-1 in both an immediate and a delayed treatment model.Similarly,ΦNJS1 can kill colistinR Kp more effectively than colistins Kp in mouse colonization model and the use of phage decreased colistin resistance of colistinR Kp in vivo.A better understanding of these fitness costs may lead to new treatment approaches that minimize the emergence and spread of colistin-resistant pathogens in human and environmental reservoirs. |
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