Application Of Wide Spectrum Phages Cocktails To Combat Prevalent Capsule-type Klebsiella.Pneumoniae Infection In Mice

Klebsiella pneumoniae(K.pneumoniae) is a common opportunistic pathogen causing considerable morbidity and mortality in humans.Indiscriminate usage of antibiotics has led to rapidly increasing multidrugresistant K.pneumoniae strains,resulting in the inefficacy of antibiotics.Therefore,new alternative antibacterial agents that can supplement conventional antibiotics to control infections caused by drug-resistant bacteria are urgently needed.This study aimed to characterize two novel phages and examine the efficacy and safety of their cocktail against K.pneumoniae strain infection in mice.A total of 180 clinical K.pneumoniae strains were obtained from several hospitals in China,K.pneumoniae strains were identified by polymerase chain reaction(PCR) based on the 16 S rRNA gene.The capsule-types of all K.pneumoniae strains were identified according to the method described by Pan.The strains were cultured in Luria-Bertani(LB)medium at 37 °C.Two extensively drug-resistant strains(strains KP1183 and KP2381)were selected and further used in mice experiments.vB＿KpnP＿IME304(IME304)and vB＿KpnP＿IME335(IME335)were isolated using KP1183 and KP2381 as host strains,respectively.A cocktail consisting of phage IME304 and IME335 was prepared for use in further studies.The obtained high-titer and pure phages were precipitated by adding 10% polyethylene glycol(PEG)8000 and 1 M sodium chloride(Na Cl)to the phage lysate.The morphology of the phages was further visualized and characterized by transmission electron microscopy(TEM)(Japan,JEM-1200EX) of negatively stained preparations.To determine stability of the phages,the test of temperature and pH stability were carried out.The phage genome was sequenced using Illumina Miseq(San Diego,CA,USA) according to the manufacturer’s instructions.The genome was assembled using Newbler 3.0 assembler.Open reading frames(ORFs)were annotated using Rapid Annotation using Subsystem Technology(RAST).The genomic map was generated using CLC Genomics Workbench 3.0.Phage protein genome comparison as performed using Easyfig 2.0.Antimicrobial resistance and bacterial virulence factors were determined from the Res Finder.The phylogenetic tree was constructed using the neighbor-joining method with 1000 bootstrap replicates in MEGA 6.0.Timekill experiments were used to further elucidate a quantitative analysis of phage bactericidal ability.To examine the protective effects of therapy with their cocktail,the previously purified phage IME304 and IME335 were adjusted to a phage titer of 1 × 10~9 CFU/m L,respectively,and the cocktail was generated by mixing the two phages in equal proportions,and then used for further therapeutic evaluation in mice.To further investigated whether our phages and cocktail could prevent and clear K.pneumoniae infections,100 BALB/c mice were also divided into five groups as previously described.Three mice per group were dissected each day,and then the bacterial load and phage count in the three organs were counted until the corresponding bacteria and phages were not detected.To assement immune reactions against their cocktail,the cocktail was introduced intraperitoneally to mice every day for 7 days.The control group was treated with PBS buffer.The liver,spleen,and lung were removed for histopathological observation.Mice were sacrificed and their serum was obtained for further analysis.IgE profile using Mouse Immunoglobulin E(IgE) ELISA Kit(Living,China),and histamine using Mouse Histamine ELISA Kit(Living,China).The capsule-types of a total of 180 K.pneumoniae strains were collected from several Chinese hospitals.The result showed that these strains were clustered into eight capsule-types,and K47 was the dominant capsular type(101/180;58 carbapenem-resistant K.pneumoniae strains out of 101 K47 strains were available),followed by K64(34/180;24 carbapenemresistant K.pneumoniae strains out of 34 K47 strains were available),K15(11/180),K10(7/180),K23(6/180),K24(5/180),K25(10/180),and K28(6/180),respectively.The above capsule-typing results revealed that capsule-types K47 and K64 were the most epidemical strains in the studied Chinese hospitals.On the data of the capsule-typing result,K47 and K64 are the two major epidemic K.pneumoniae capsule-type strains among the 180 strains.These strains were then separately added into the sewage of the Fifth Medical Center of Chinese General Hospital of People’s Liberation Army in Beijing,China.Therefore,the typical strains KP1183(K47)and KP2381(K64)were used as host strains to screen phages.Two novel phages,vB＿KpnP＿IME304(short as IME304,host strain KP1183)and vB＿KpnP＿IME335(short as IME335 host strain KP2381) were isolated and named.Both IME304 and IME335 phages formed transparent circular plaques with a clear boundary on double agar plates after five rounds of purification when incubated with their host strains.Transmission electron microscopy and phylogenetic analysis showed that both phages belonged to the species KP32 virus of the subfamily Autographivirinae.One-step growth experiment was conducted to determine the latent period and burst size of the phages.Both phages had a short latent period and a large burst size and had a wide spectrum of cleavage.Biological property tests revealed their temperature and p H tolerance ranges.In vitro time-kill experiments showed that both phages and their cocktail had strong bactericidal ability,sharply reducing the number of log-phase bacteria to 0 within 100 min.The whole genomes of IME304 and IME335 phages were sequenced using the Illumina Miseq platform(San Diego,CA,USA;Gen Bank accession numbers MK795385 and MN176574,respectively).Their genomes were double-stranded linear DNAs of 40,746 base pairs and 40,361 base pairs,respectively.The genomic arrangements of IME304 and IME335 were highly homologous to KP32,vB＿KpnP＿IME205,and the K30 phage genome,with no significant rearrangements observed.In our in vivo experiments,survival rates were significantly higher in cocktail treated mice than in untreated mice.In particular,the cocktail effectively removed bacteria in the liver,kidney,and spleen of the mice.Furthermore,the safety and therapeutic effects of the cocktail in mice were evaluated.Mice treated with cocktail showed normal serum TBIL,AST,ALT,ALP,Cr,BUN,and LDH levels,with no significant histopathological changes in the liver,spleen,or lungs,and displayed only a slight increase in inflammatory changes.These results showed that the two different capsuletype phages and their cocktail were potential candidates for the treatment of K.pneumoniae infection.Taken together,two different capsule-type phages were isolated for treatment against extensively drug-resistant K.pneumoniae strains.Biological analyses showed that they were stable at a temperature higher than the human body temperature and had good pH stability.Animal experiments showed that the cocktail was effective and safe for the treatment of K.pneumoniae infection in mice.They were shown to be a feasible alternative to antibiotics and could expand the current choices for the treatment of clinical infections.In conclusion,the phages and cocktail showed great potential as alternatives to antibiotics and as medical disinfectants.