| Elizabethkingia genus is an opportunistic pathogen with intrinsic multidrug-resistant phenotype.It has been occasionally associated with clinical infections and high mortality,especially in neonate and immunocompromised patients.In recent years,Elizabethkingia spp.has been reported to cause meningitis-like infectious disease in frogs,which is highly contagious and fatal.It has been one of the most serious epidemic diseases in farmed frogs in China.However,the taxonomy of Elizabethkingia genus was incomplete until recent years,and the species in this genus were commonly misidentified,which resulted in the lack of knowledge about this organism,including the epidemiological studies and the drug resistance mechanism.In this study,we collected a large number of diseased frogs,after muti-molecular identification to clarify the Elizabethkingia species related to this frog disease,whole genome sequence,comparative genomics and genotyping were conducted to study the phylogenetic and epidemiological relatedness between Elizabethkingia strains.Besides,we studied the molecular diversity of chromosomal metallo-?-lactamase genes in Elizabethkingia genus.The main results and conclusions are as follows:Elizabethkingia miricola was first reported to be the etiology of meningitis-like infectious disease in frogs.To figure out the etiology of this disease,bacteria isolation,microscopic parasites examination,PCR test for fungi and viruses were conducted in the diseased frogs collected from different farms.The diagnostic tests indicated that Batrachochytrium dendrobatidis?Bd?,ranaviruses and parasute are not the etiological agents of this disease.Bacterial infections?190/213?were confirmed in the etiological examination.The dominant strains were identified as E.miricola based on 16S r DNA and gyr B.Histopathologic examination showed severe meningitis with denatured,incrassate meninges.Inflammatory infiltrates,moderate multifocal gliosis and damaged neuronal cells were observed in the cerebellum.Experimental pathogenicity testing of E.miricola FL160902 was conducted by various infection routes,including intramuscular injection,immersion infection and cohabitation with infected frogs.E.miricola FL160902 is highly pathogenic and contagious for frogs,especially by immersion infection,indicating that contaminated water and the infected individuals can be the source of infection.The classification system of Elizabethingia genus and the zoonotic potential of E.miricola were discussed based on comparative genomics.Whole genome sequence was conducted to study the phylogenetic relatedness of different species in Elizabethingia genus.According to the phylogenetic analysis,we modified the classification system of this genus.The UBII subgroups branched separately supports the view that they are different Elizabethkingia species.We propose that UBII are not E.meningoseptica,the UBII:1 group is E.anophelis,and UBII:2 group is E.miricola.Comparative genomics demonstrated that the E.miricola CSID 3000517120 isolated from human and the strains with amphibian origins reveals a mixed subset and shared similar virulence-associated genes profiles,revealing that E.miricola is a potential zoonotic pathogen,which rise concerns for public health.PFGE and Rep-PCR were used to study the molecular epidemiology of 62 different E.miricola isolates.These two methods both can distinguish the isolates with different source roughly.However,more strains were distinguishable by PFGE than Rep-PCR.A total of 28 PFGE types were shown in all the strains,with 8 main different PFGE cluster.The cluster results were highly related with the origins,including frog species?Rana nigromaculata,Rnan catesbeiana and Quasipaa spinosa?and locations?Hunan,Hubei and Fujian province?.Some strains from the different farms were in the same type,even for the strains isolated from different frog species,indicating that this pathogen can be transmitted between different farms and different species.This result provided some theoretical basis to the disease prevention.We found the molecular diversity of chromosomal metallo-?-lactamase genes in Elizabethkingia genus.Twenty-seven resistance genes were predicted in the WGS of FL160902,including 2 chromosome-borne metallo-?-lactamase genes.These two genes were named as bla B-16 and bla GOB-19 based on amino acid sequence and their carbapenems resistance function.Besides,a total of 23 and 32 novel Bla B and Bla GOBvariants were found in the Elizabethkingia genomes available in NCBI.Cloning of representative bla B and bla GOBinto E.coli DH5?resulted in increased and diverse MICs to most?-lactams,indicating that naturally occurring variants have distinct resistance phenotypes for intrinsic genes.12 and 15 clusters were assigned in 39 Bla B and 51Bla GOBvariants based on the amino acid identities and phylogenetic studies.The variants from the same cluster all belonged to one Elizabethkingia species or one Elizabethkingia cluster.Phylogenetic study of the variants from different species suggested potential inter-species dissemination of MBLs genes among Elizabethkingia species. |
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