Study On Molecular Characteristics Of Virulence And Resistance Of Klebsiella Pneumoniae From Mastitis Cows In Hubei

Klebsiella pneumoniae（KP）is an important zoonotic pathogen that can cause pneumonia,sepsis and other diseases in humans and various animals.KP have been widely studied in human medicine,but there are relatively few reports in animals.KP mainly causes mastitis in dairy herds and causes huge economic losses to the dairy industry.With the continuous increase of KP isolation rate,the enhancement of toxicity and the continuous emergence of resistance,it is urgent to study the pathogenicity,molecular epidemic characteristics and traceability of this bacteria.In this study,the epidemiological investigation,the molecular characteristics of virulence and drug resistance,and the traceability of KP were carried out on five typical farms in Hubei.In this way,the molecular mechanism of virulence and antibiotic resistance of KP,as well as its potential sources of infection and transmission routes,will be revealed.This provides a theoretical basis for the precise prevention and treatment of cow mastitis,and provides a scientific basis for the rational use of antibiotics in clinical practice and to reduce the prevalence and antibiotic resistance of KP in dairy farming.1.Epidemiological investigation of Klebsiella pneumoniaeA total of 887 samples were collected from five typical dairy farms in Hubei for the separation and identification of KP to investigate the prevalence of KP,during 2019 and2020.Including nipple milk,nipple skin swabs,anal swabs from healthy cows,subclinical mastitis and clinical mastitis cows and environmental samples（feed,bedding,etc.）.The results showed that a total of 239 KP（26.9%）were identified.The isolation rates of KP in milk from subclinical mastitis cows and clinical mastitis cows were 24.5%and 25.4%,respectively.There was no significant difference in the isolation rate of KP in milk,nipple skin swabs and anal swabs between healthy and mastitis cows,which may be due to the fact that most of the mastitis cows are in the treatment or recovery period.The differences in the separation rate between different farms may be related to the stocking density and breeding environment.2.Genetic evolution analysis of Klebsiella pneumoniaeSerotype identification and genotyping of 239 KP were performed by wzi sequencing and Multi Locus Sequence Typing（MLST）.The genetic evolution of KP was analyzed by constructing an evolutionary tree and a minimum spanning tree to trace its potential infection source and transmission route.The results showed that a total of 101 wzi allele types（32 capsular K types）and 100 STs were identified,including 5 new wzi allele type and 25 new STs.However,the wzi allele types of 40 isolates and the STs of 30 isolates are still unknown.Evolutionary analysis indicated that strains of the same serotype also differ in genome,and strains of the same genotype also have different serotypes.The 100 STs mainly constituted 2 clonal complexes CC20 and CC200,in which KP from milk,skin swabs,feed,litter and feces from healthy and mastitis cows were distributed.This indicated that the KP from milk had homology with the KP from environmental.In the comparative analysis with the isolates from different countries and different hosts in the Pub MLST database,it was found that 58 and 67 STs were isolated from different countries and different hosts,respectively.Of which 60 STs have been isolated in humans.The strains of these genotypes have the risk of spreading across regions and species.3.Virulence characteristics of Klebsiella pneumoniaeThe virulence of KP was characterized by string test,PCR detection of 23 virulence factors and mouse pathogenicity test,and its possible pathogenic mechanism was analyzed.The results of the string test were all negative.Sixteen of 23 virulence genes were positive detected by PCR.The detection rate of fimbriae synthesis-related gene（fim H,mrk D）,lipopolysaccharide-related gene（uge,wab G）,iron uptake system（ent B,iut A,iro N）and urease-related gene ure A were over 85%.99.2%（237/239）of KP carried at least five virulence genes.Besides,3 isolates carrying iuc A,which related to high virulence.Two isolates carrying iuc A with different serotypes,and 2 multidrug-resistant isolates from milk without iuc A were selected for pathogenicity test in mice.The results showed that the clinical symptoms of infected mice in the 10⁸CFU/m L and 10⁹CFU/m L concentration groups were mainly depression,rough coat,closed eyes,etc.After autopsy,the main lesions that can be observed are enlargement and nodules of the liver and spleen,pulmonary congestion and so on.The results of hematoxylin-eosin staining on pathological tissues included necrosis of hepatocytes,splenic macrophage hyperplasia,and different numbers of red blood cells were observed in the alveolar cavity,showing different degrees of hemorrhage and the 10⁹CFU/m L group also showed necrosis.The LD₅₀of the four strains were all>10⁸CFU/m L,and they were all non-toxic.No hv KP was found in this study.4.Molecular mechanisms of resistance in Klebsiella pneumoniaeThe susceptibility of 239 KP to 17 antibiotics was determined by the micro-broth dilution method,and the ESBLs-producing KPs were screened.PCR was used to determine the prevalence of 70 resistance genes,3 integrons,5 efflux pumps and 2regulatory genes,and the correlation between resistance genes and resistance phenotypes was analyzed.The results showed that KP was highly sensitive to meropenem（MEM）and polymyxin（CT）,but completely resistant to ampicillin（AMP）,highly resistant to sulfisoxazole（SOX）（94.6%）.The multidrug resistance rate was 43.9%.The resistance rate of KP to ceftriaxone（CRO）,streptomycin（STR）and gentamicin（GEN）in mastitis milk is significantly higher than that in healthy milk,which may due to the use of antibiotics in five farms.The 93 resistance spectrum types were found in 239 KP,of which AMP-SOX（23.9%）accounted for the most.A total of 17 ESBLs-producing KP（7.1%）were screened out of 239 isolates,of which 10 strains（58.8%）were from the milk of mastitis cows.ESBLs-producing KP are completely resistant to AMP,cephalothin（CEP）,ceftiofur（EFT）,CRO,and SOX.The resistance rate to STR,KAN and GEN was also more than 50%.All 17 ESBLs-producing KP were multidrug-resistant.PCR results showed that 32 resistance genes,1 integron gene,4 efflux pump genes and 2 regulatory genes were positive.The detection rates of bla _TEM,bla _SHV,str A,str B,aad A1,aac（6’）-Ib-cr all exceeded 50%.The coincidence rates of different classes of antibiotics and resistance genes were all over 50%.Different degrees of positive correlation were observed in ceftiofur,ceftriaxone,amoxicillin/clavulanate,streptomycin,kanamycin,tetracycline,doxycycline,florfenicol and their corresponding resistance genes.However,sulfonamides and ciprofloxacin were not found to be significantly associated with resistance genes.However,SOX,sulfamethoxazole/trimethoprim（SXT）and ciprofloxacin were not significantly associated with a single resistance gene.The resistance of KP to these 3 drugs may also be mediated by other mechanisms.5.Whole Genome Sequencing of Klebsiella pneumoniaeWhole genome sequencing was performed on 17 ESBLs-producing strains and 33non-ESBLs-producing strains to analyze their resistance and association with Virulence gene epidemic characteristics.The results showed that KP carried a variety of virulence and drug resistance genes.The virulence factors were mostly adhesin,iron uptake and secretion system,and the resistance genes were mostlyβ-lactam and aminoglycoside resistance genes.At least one ESBLs and fosfomycin resistance gene was detected in ESBLs-producing strains.Among the 15 ESBLs-KP strains carrying the bla _CTX-Mgene,bla _CTX-M-15was the most prevalent.The coincidence rates of PCR results of virulence genes and whole-genome sequencing results were all>90%.Except for bla _SHVand aac（6’）-Ib-cr,the coincidence rates of other genes were all≥70%.82%of the 50 KP strains carried plasmid replicons,a total of 17 plasmid replicon types were obtained,of which Inc FIB（K）accounted for 36%.This study assessed the contamination of KP in 5 dairy farms in Hubei,revealed its molecular characteristics of resistance and virulence as well as its potential sources of infection and transmission routes.It provides a theoretical basis for the precise prevention and treatment of cow mastitis,and a scientific basis for clinical rational drug use and reduction of bacterial resistance.