Evaluation Of Drug Resistance And Virulence Of Klebsiella Pneumoniae From Different Host Origins

Klebsiella pneumonia（KP）is a zoonotic pathogen that triggers the occurrence of infection within immunocompromised hosts.KP isolates have emerged being highly virulent and/or multidrug-resistant worldwide.As KP strains are being increasingly reported for being drug-resistant and hypervirulent,KP is imposing a threat to the public health and healthcare system across the whole world.Current studies conducted to investigate the KP transmission between humans and animals are still limited.Current studies conducted to investigate zoonotic and reverse zoonotic transmission of KP between humans and animals are still limited.The aim of this study is to preliminarily investigate the underlying molecular mechanism of KP in drug resistance and hyper-virulence by studying drug resistance,pathogenicity,molecular characteristics and genetic correlations of KP from animals and human origins,with sample size:pig（n=11）,cattle（n=11）,ducks（n=7）and humans（n=10）.The epidemic transmission possibility of KP between animals and humans are also discussed,which provides reference values for epidemiological analysis and conceptual basis for public surveillance,prevention and control of KP.1.Genetic evolution characteristics of Klebsiella pneumoniaeCapsular serotype and Sequence type of animal and human origin are obtained through the implementation of next-generation whole-genome sequencing.Comparison and identification of homologous genes contribute to the construction of the phylogenetic tree,analysis of genetic connection among stains and evaluation of the possibility of KP transmission between humans and animals.The result shows animal and human KP are scattered in the phylogenetic tree with limited clustering among hosts.A total of 19 wzi allele types（7 capsular K types）and 20 STs were identified form 39 KP strains,including new ST6431 and ST6432 found in pig origin.ST2410-wzi274 has been detected in human,and animal strains,and there is a risk of cross-regional and cross-species transmission.A highly virulent-associated K2 capsular type was detected in human KP-HB21H30772.2.Drug resistance characteristics of Klebsiella pneumoniaeThe susceptibility of 39 KP strains to 20 types of antibiotics is assessed through the microboth dilution method.ESBLs test and m CIM test were performed to detect the production of ESBLs and carbapenemase,and analyze the drug resistance profile of KP within various hosts.Crystal violet staining is used to examine the biofilm forming capability of KP.Drug-resistance and molecular epidemiological characteristics of KP were identified through second-generation whole genome sequencing.The results showed that multidrug resistance was serious,and 92.31%of the strains were multidrug-resistant bacteria.KP is highly resistant to sulfamethoxazole（SOX）and ciprofloxacin（CIP）,but highly sensitive to amikacin（AK）and polymyxin（CT）.Drug resistance characteristic of KP shown within different hosts are evidently different.The drug resistance rate of human KP to third-generation cephalosporin antibiotics（ceftriaxone,ceftiofur）,carbapenem antibiotics（meropenem）and new-generation antibiotics（tigecycline,amikacin）was found significantly higher than that of animal origin（p<0.001）.ESBLs-producing strains and carbapenemase-producing strains were not detected in animal origin.Seven ESBLs-producing strains were screened out of 10 human KP strains with bla _SHV.From ESBLs-producing strains,four strains,which carried KPC resistance genes were found resistant to carbapenem antibiotics.Among those four strains,m CIM test result of bla _KPC-71 carrying KP-HB21H30535 showed negative while of the other three bla _KPC-2 strains,m CIM test result showed positive.Evaluation of biofilm formation ability revealed that 89.74%of KP showed the capability of biofilm formation,and the biofilm formation ability of KP in animal origin was higher than that of human origin.Next-generation whole-genome sequencing analysis showed that KP strains carried a variety of antibiotic-resistance genes and multiple efflux pump genes.among which,bla _SHV,sul,Arn T and ept B were shown with higher detection rates.Drug resistance phenotype and the coincidence rate of the drug-resistance-associated gene are different among different host origins.The coincidence rate of cattle KP was higher than that of other KP origins.According to the correlation analysis result of drug resistance and drug resistance genes,the aminoglycoside resistance phenotype is strongly correlated with associated drug-resistance genes,also rmt B showed a positive correlation with amikacin resistance.Additionally,there were varying levels of correlations between the resistance of KP to other antibiotics and drug resistance-associated genes,such as ceftiofur,ceftriaxone,tetracycline,doxycycline,etc.3.Virulence characteristics of Klebsiella pneumoniaeThe virulence characteristics of KP were analyzed highly viscous phenotypic test,pathogenicity test in mice and genotype analysis of KP strains.It was shown that the detection rate of high viscosity phenotype was low with positive rate being 14.29%.A significant difference in the detection rate of KP viscosity phenotype was found in different host origin with highest detection rate 36.36%shown in pig origin followed by detection rate in human and duck origin being 20%and 14.29%respectively.In cattle origin,KP viscosity phenotype detection rate showed negative.High-viscosity-associated genes rmp A and mag A were not detected in all KP strains.KP carries a variety of virulence genes,but the carrier rate of highly virulence genes（peg-344,iro B,iuc A,rmp A,rmp A2）was low.It is shown that in duck origin,gene markers of peg-344,iro B and iuc A were detected in both KP00520 and KP00523,and in human origin,KP-HB21H30772 was shown carrying iro B and iuc A genes.Within 9 KP strains from pig origin,iuc A gene was found.No highly virulent genes were detected in cattle KP and rmp A and rmp A2 genes were absent in all strains.Based on the ST type,serotype and overall high-virulence-gene distribution,10 KP strains were selected for pathogenicity testing in mice.It is observed that the mice infected with KP developed symptoms of depression,rough coat,loss of appetite and diarrhea and in severe cases,death occurred within 48 hours.Autopsy and histopathological examination found that KP mainly caused damage to the lung,liver,spleen,and kidney.KP-HB21H30772,a serotype of K2 showed high virulence,LD₅₀=4.88×10³ CFU.K-QNE20WY293 and K-DXH20e226 were suspected to be highly virulent capsular serotypes K54 and K64,but the virulence was low,and the LD₅₀ was 1.65×10⁷ CFU and1.87×10⁷ CFU respectively.It is found that 2 KP strains of human origin,KP-HB21H30786KP-HB21H30845 which belong to ST11,showed low virulence(LD₅₀=1.31×10⁷～5.6×10⁷CFU).KP00520 carries three highly virulence genes,but has low virulence(LD₅₀=2.31×10⁷CFU).K-210902y113M-5（pig）,K-20210828y196M-4（pig）,KP00522（duck）,K-QNE20e240（cattle）and KP-HB21H30740（human）which belong to ST240showed the difference in virulence to mice.KP in pig origins demonstrated slightly higher virulence(LD₅₀=1.87×10⁶～7.83×10⁶ CFU)compared with that in KP from human,duck and cattle origins(LD₅₀=9.12×10⁶～2.29×10⁷ CFU).It is deduced that different host origins may affect the pathogen invasion and pathogenicity to some extent.K-20210828y196M-4is a highly viscous strain while there was no significant difference in virulence found between K-20210828y196M-4 strain(LD₅₀=7.83×10⁶ CFU)and K-210902y113M-5 strain(LD₅₀=1.87×10⁶ CFU).