| Klebsiella is an important conditionally pathogenic bacterium of the family Enterobacteriaceae,which plays an important role in the spread of drug resistance and pathogenicity,and has a potential zoonotic risk.In recent years,there have been increasing clinical reports on multidrug-resistant,hypervirulent,and even multidrug-resistant hypervirulent strains of Klebsiella.Infection caused by Klebsiella has become an important public health issue worldwide,posing a serious threat to the health of humans and animals.Due to its distinct relationship with humans,studying the differences in the same pathogenic bacteria carried by wild animals and companion animals is more conducive to exploring the impact of human activities on animal microbiota,and providing a basis for reasonable control of antibiotic use and effective prevention and control of the spread of zoonotic pathogens.However,at present,there were few reports about the resistance and pathogenicity of Klebsiella from wild animals and companion animals at home and abroad.In order to better ascertain the drug resistance and pathogenicity of Klebsiella carried by wild animals and companion animals in China,this study collected animal samples which including migratory bird,captive giant panda,working dog and companion animal from six provinces in China.Klebsiella strains were isolated and purified by selective culture medium.Identification and drug resistance phenotype of Klebsiella isolates were completed by the species-specific gene amplification of Klebsiella and BD PhoenixTM-100 automated identification and susceptibility testing system.Extended spectrumβ-lactamases resistance genes,virulence genes and capsular serotypes were further detected by PCR.Conducted string tests on Klebsiella isolates,and determine the hypervirulent Klebsiella strain based on the PCR results of the hypermucoviscosity phenotype,virulence genes,and capsule serotype.Conducted whole genome sequencing and bioinformatics analysis on isolates with multiple drug resistance phenotypes or carrying virulence genes,further studied the resistance genes,virulence genes,capsule serotypes,plasmid types,and ST types of Klebsiella isolates,and analyze the correlation between drug resistance and virulence of the strains.In this study,203 Klebsiella strains（10.8%）were isolated from 2648 samples（feces or anal swabs or throat swabs）,the isolation rates of Klebsiella from different animal sources were consistent with the relationships between animal and human.The isolates include 159 K.pneumoniae（78.3%）,41 K.oxytoca（20.1%）,two K.aerogenes（0.9%）and one K.ozaenae（0.4%）.The identification results of drug susceptibility showed that:96 strains isolated from migratory birds were only resistant to tetracycline（3.1%）and ampicillin（47.9%）.Strains isolated from captive giant pandas,working dogs and companion animals were resistance to 11classes or more antibiotics,among which pet cat-derived isolates had the most severe degree of multidrug resistance,up to 66.7%.64.7%（22/34）were ESBLs Klebsiella pneumoniae in multidrug-resistant strains,and four extended-spectrum beta-lactamases resistance genes(bla OXA,bla TEM,bla CTX-M and bla SHV),one Amp C beta-lactamase gene(bla DHA-1)and a broad-spectrum beta-lactamase gene(bla LAP-2)were detected in ESBLs strains by PCR detection and whole-genome sequencing.90.9%（20/22）of ESBLs Klebsiella pneumoniae carried the bla CTX-M gene,and bla CTX-M-3 was the dominant type（59.0%,13/20）.The sequencing analysis results of the whole genome framework map of MDR strains showed that the carrier rates of drug-resistant genes such asβ-lactams,tetracyclines,sulfonamides,chloramphenicol,aminoglycosides,fosfomycin and so on were high,and the drug resistance phenotypes of these strains were basically consistent with the drug resistance genotypes.70.5%of MDR strains carrying quinolone resistant genes showed sensitivity to quinolone antibiotics.The plasmid types of 42 sequencing strains were mainly Inc FII（K）（42.8%）and Inc FIB（K）（35.7%）.The results of string test showed that captive giant panda-derived K.pneumoniae strain 41and pet dog-derived K.pneumoniae strain HP259K have the hypermucoviscosity phenotype.Combined with PCR results of virulence genes,capsular serotypes and genome framework analysis,9.3%（19/203）of the isolates carried key virulence genes,including the mucoid factor encoding gene rmp A（0.9%）and the iron uptake and transport genes ybt S（3.9%）,iro N（0.9%）,iro B（0.9%）,iuc A（1.4%）,iut A（4.9%）,fyu A（2.9%）,irp1（2.4%）,irp2（1.4%）.The detection rates of hypervirulence related capsular serotypes K2,K5,K54 and K57 in 203 isolates were0.9%,0.4%,0.4%and 0.9%,respectively.Among the 42 whole genome framework map sequencing strains,K31（16.6%）was the dominant capsular serotype,and K31 strains were resistant to 4-7 classes antibiotics.The results of other capsular serotypes were scattered.Combining the hypermucoviscosity phenotype,the PCR results of virulence genes and capsular serotypes,two K.pneumoniae isolates（41 and HP259K）were defined as hypervirulent strains.The results of multilocus sequence typing analysis showed that there were 27 ST subtypes among the 42 whole-genome sequencing strains,of which ST2891 was relatively concentrated（14.2%,6/42）,all of which were multidrug-resistant strains isolated from pet cat feces.Other ST subtypes were more dispersed.ST subtypes closely related to human infection were found in isolates from both wild and companion animals,including high-risk ST307,ST147,and ST661.The correlation analysis between drug resistance and virulence showed that the correlation coefficient between the drug resistance classes and the key virulence gene distribution of the isolate was-0.571*（P<0.05）,with significant differences,the more key virulence genes the strain carries,the lower the probability of drug resistance occurring.In summary,this study conducted a background investigation on the resistance and pathogenicity of Klebsiella isolated from different anima sources,confirming that wild animals and companion animals in close contact with humans were potential spreaders and reservoirs of multidrug-resistant pathogenic Klebsiella.Correlation analysis showed that there was an negative correlation between the distribution of resistance and virulence genes in animal-derived Klebsiella.This study provided a data basis for the study of drug resistance and pathogenicity of Klebsiella from different animal sources in China,suggested the risk of Klebsiella transmission from wild animals to humans and the risk of community transmission caused by Klebsiella carried by companion animals.It also provided guidance for clinical antibiotics use in animals and helped to prevent and control the widespread spread of multidrug-resistant Klebsiella between humans and animals. |
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