| Klebsiella is a common and important conditionally pathogenic gram-negative bacterium,which can cause severe pneumonia,septicemia and mastitis in cattle breeding industry,and has a high incidence and mortality of related diseases.Klebsiella pneumoniae is a major pathogen of nosocomial and community acquired infections,which can cause a variety of diseases such as human pneumonia,meningitis and suppurative liver abscess,posing a serious threat to global animal husbandry and human public health and causing great economic losses.Due to the irregular use of antibiotics,the problem of drug resistance of Klebsiella pneumoniae is becoming more and more serious,and some strains combine high virulence phenotype with drug resistance phenotype,which poses a serious challenge to the development of animal husbandry industry.Therefore,it is imperative to develop new therapies that complement or even replace antibiotics.Lytic phage is a promising green biological control agent targeting Klebsiella pneumoniae.With highly specific characteristics,it can mediate the rapid lytic death of host bacteria,but does not affect human or animal cells,showing high biological safety.Compared with phage,the endolysin encoded by phage have the advantages of wider bactericidal range and less resistance to bacteria,so they are considered as antibacterial agents with great potential.In view of this,this study carried out isolation,identification,biological characteristics and comparative genomics research on phages of effective lytic Klebsiella strains,evaluated the in vitro and in vivo bacteriostatic effects of phages on Klebsiella strains,and systematically analyzed the evolution characteristics of endolysogens,so as to provide theoretical basis for further rational utilization of phage resources and lay the foundation for the development of phage therapy.In this study,34 strains of Klebsiella were isolated from human feces,sputum samples,animal feces from zoos,bovine nose swabs from a cattle farm in Baoding,Malay pangolins intercepted by customs and wild birds during 2020-2023,including 22 strains of Klebsiella pneumoniae.Multi-locus sequence typing showed ST23,ST36,ST101,ST111,ST133,ST290,ST629,ST1035 and ST1565.9 strains of Klebsiella variicola,including ST347,ST3972,ST4115 and ST4394.3 strains of Klebsiella quasipneumoniae,including ST2355,ST2558 and ST4115.Using Klebsiella strain isolated in this study as host bacteria,a total of 48 strains of phage were isolated from sewage,human feces,and animal feces.Based on host characteristics,the re-screening was conducted,and the whole genome sequencing and comparative genomics studies were conducted on four of the phages to clarify their species taxonomic status and phylogenetic relationship.The results showed that the four strains belonged to Autographiviridae,and pKP-M186-2.1 and pKV-BS375-3.1 belonged to Molineuxvirinae.The phage pKP-M212-2.1 and pKP-M 186-2.2 were classified in the genus Drulisvirus;The genome length of phage was 42 894 bp(pKP-M186-2.1),45 617 bp(pKV-BS375-3.1),44 728 bp(pKP-M 186-2.2)and 42 673 bp(pKP-M212-2.1),respectively.None carried antibiotic resistance genes or virulence genes.The morphology of the phage was observed by transmission electron microscopy.The host range,temperature,acid-base stability and one-step growth curve of the phage were characterized.The analysis of biological characteristics showed that the phage pKP-M212-2.1 had a diameter of about 2.5 mm and an outer translucent halo of about 5 mm.Head diameter about 61 nm,tail length about 19 nm;Klebsiella pneumoniae M212-2(ST23)with K1 capsule serotype cleaved and Klebsiella pneumoniae BS433-2 with the same ST type cleaved;It can maintain stable activity at-20～37℃ and pH 6～10.The incubation period was less than 10 min and the lysis period was 70 min.Phage pKP-M 186-2.1 and pKP-M 186-2.2 from different sources could lysate the same strain(M186-2).The head diameter of pKP-M186-2.1 was about 60 nm,and the tail length was about 17 nm.The incubation period was about 20 min,and the lysis period lasted to 60 min.pKP-M 186-2.2 was similar to pKP-M 186-2.1 in morphology,with a head diameter of about 52 nm and a tail length of 12 nm.The incubation period was less than 10 min,and the lysis period lasted to 60 min.The two bacteriophages had a narrow host range and only lysed the host bacteria.Both bacteriophages maintained stable activity at-20～37℃ and pH 6～10.The head diameter of phage pKV-BS375-3.1 was about 68 nm,and the tail length was 17 nm.pKV-BS375-3.1 can lyse 5 strains of Klebsiella variicola(all ST4115)and 2 strains of Klebsiella quasipneumoniae(all ST2355),as well as 1 strain of Klebsiella pneumoniae(ST101)from Malay pangolin.The titer remained stable at-20～37℃,completely lost its activity after incubation at 60℃ for 1 h,and maintained stable activity at pH 6-10.The incubation period was less than 10 min,and the lysis period lasted to 50 min.The bacteriostatic effect of phage in vitro was measured.The results showed that the single phage could significantly inhibit the proliferation of bacteria within 3-6 h.Bacteriophage pKP-M 186-2.1 and pKP-M 186-2.2 resistant mutant strains showed increased sensitivity to multiple antibiotics.The bacteriostatic effect of cocktail prepared by 7 strains of phage was better than that of pKP-M 186-2.1 and pKP-M 186-2.2.Phage cocktail combined with cefuroxime and levofloxacin could completely inhibit bacterial growth and proliferation within 24 h,and there was no significant difference in bacteriostatic effect when the dosage of the two antibiotics was reduced from 8 μg/mL to 2 μg/mL and 0.5μg/mL to 0.25 μg/mL,respectively.The larvae of G.mellonella larvae infected with multiple drug resistant Klebsiella variicola BS375-3 were protected by phage,and the survival rate of the larvae infected without phage treatment was only 10%(1/10)within 96 h.The survival rates of larvae treated with pKV-BS375-3.1(MOI were 100,10 and 1)reached 80%,40%and 40%within 96 h.The analysis of Endolysin based on the amino acid sequence of Endolysin gene showed that Endolysin showed diversity in the phage of Klebsiella,and was conserved within the genus.The type of endolysin had no obvious correlation with the region.In conclusion,the four strains of phage of the family Autographiviridae in this study were stable and did not carry drug resistance genes or virulence factors.pKV-BS375-3.1 is a wide host spectrum phage that can lysate a variety of ST types of Klebsiella,and does not harm the animal body.pKV-BS375-3.1 has a good protective effect on the larvae of G.mellonella infected with Klebsiella variicola,and has the potential to be used as a biological control agent for phages.A single phage can completely inhibit bacterial proliferation within a certain period of time,and the phage cocktail can prolong the inhibition time.Phage cocktail combined with antibiotics can completely inhibit bacterial proliferation within 24 h,and the application of phage cocktail can achieve the same bacteriostatic effect while reducing the concentration of antibiotics.The endolysin protein of Klebsiella phage showed diversity among genus and conserved within genus,and there was no obvious correlation between the protein type and region. |
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