Isolation,Characterization,Mechanisms Of Pathogenicity And Macrolid Resistance Of S. Gallolyticus Subsp. Pasteurianus

Streptococcus gallolyticus subsp.pasteurianus,also named as S.bovis II/2,is a main infectious pathogen in hospitals.It mainly causes neonatal meningitis,bacteremia or even death.It could also infect animals and lead to high mortality.From 2010 to 2013,several nature outbreaks have taken place in six different duck farms in Hubei or Guangxi province,and caused~20% of death rate.In this study,bacteria were isolated from the brains or spleens of dead ducklings from the outbreaks,and the isolated bacteria were identified as S.gallolyticus subsp.pasteurianus using biochemical approaches,16 S r RNA gene sequencing,and animal regress experiment.Furtherly,the mechanisms of pathogenicity and macrolide resistance were explored.1.Isolation and identification of Streptococcus gallolyticus subsp.pasteurianusGram-staining revealed Gram-positive in pairs or short chains,antigen detected revealed positive staining of group D Lancefield antigen.The isolates were identified as S.gallolyticus subsp.pasteurianus(S.bovis ?/2)by phenotypical characterization and 16 S r RNA gene sequencing.Theses isolates were designated as AL101002,GX130304,GX130307,GX130630,GX130723 and GX130809.Subcutaneously inoculation of all six isolates to ducklings lead to similar symptoms,mortality rate and pathological changes to those by natural outbreaks.Further examination using AL101002 strain: The Gram-staining of spleens,livers and kidneys revealed the presence of Gram-positive cocci within macrophages around a white pulp in spleen,sinusoids or sinusoidal endothelial cells and glomerulus.The finding was confirmed by IHC.TEM revealed the presence of replicating coccoid bacteria within the phagosomes of degenerating macrophages in spleen.In conclusion,these six strains were Streptococcus gallolyticus subsp.pasteurianus.2.The pathogenicity of Streptococcus gallolyticus subsp.pasteurianus on ducklings7-day-old ducklings were inoculated with AL101002 to examine the pathogenecity of Streptococcus gallolyticus subsp.pasteurianus.At 5d post infection,typical clinical symptoms began to display on the 7-day-old ducklings,100% infection rate and 52.38%(22/42)of mortality rate wer observed.At 1d to 5d post infection,bacteria were isolated from each organs and blood.After 5d,bacteria could not be isolated from the blood,but from kidney and bursa at 7d.Gross examination showed the swelling spleen,thickened pericardium like trichocardia,liver covered with caseating substance and serious meminges congestion.Histopathological examination revealed no basic spleen structure but myocarditis substantiality 5d post infection.The lymphocytes of bursa decreased significantly 7d post infection.Meningitis was observed 3-9 d post infection.Other organs revealed heterophilic granulocytes and infiltrated macrophages.TUNEL staining showed significantly increased necrotic cells in the infected group than control group.Ultrastructural pathology showed replicating coccoid bacteria within the spleen macrophages 1d,3d,5d post infection.Furthermore,CD68 staining showed macrophages nearnecrotic area.Fluorescent quantitative PCR showed increased TNF-? and IL-1? in infected brain,increased necroptosis facter RIPK1 and MLKL,and IL-1??IL-6 in infected spleen.These data indicated acute inflammation in brain or spleen.In conclusion,Streptococcus gallolyticus subsp.pasteurianus mainly targets the immune organs and leads to macrophages necroptosis in spleen.In addition,this bacterium can also invade meninges and cause meningitis.3.Mechanisms of macrolide resistanceMIC testing were performed by agar-dilution method.Six isolates showed multidrug resistance and especially high level macrolide resistance(erythromycin MIC = 1024 mg/L,clarithromycin MIC = 512 mg/L).The mechanistic study of macrolide resistance revealed:A.Plasmid and mef A or mef E genes were absent in the six isolates.Efflux inhibitor PA?N or CCCP could not reduce macrolide MIC significantly.These results suggested that macrolide resistance in all six isolates were not induced by efflux pump.B.No mutation was detected in the ribosomal proteins L4 and L22,while mutations in individual 23 S r RNA induced macrolide MIC? 32 mg/L.These results demonstrated that the high macrolide resistance was not induced by mutations in L4,L22 or 23 S r RNA.C.Ribosomal methylase erm B and erm T,and tetracycline resistance genes tet M and tet L were found in the genomic DNA of all six isolates.Four isolates harbored the lincomycin resistance genes Lnua,and one isolate contained the gentamycin resistance gene.The flanking sequences of erm B or erm T were amplified by PCR based on AL101002 genomic DNA sequencing.Two resistance gene clusters of 5.731 kb and of 11.244 kb(accession number KU511281 and KU511280)were identified,respectively.D.Both erm B and erm T were expressed in the six isolates.Erm B and Erm T expression were tested by Western blotting with or without erythromycin.In the presence of erythromycin,both erm genes were expressed in all six isolates,while in the abscence of erythromycin,neither erm gene was expressed.Using His-tag containing Erm B and Erm T fusion clones in E.coli strain,Western blot analysis revealed express Erm B and Erm T proteins in the presence of erythromycin,while less expression was observed in the abscence of erythromycin due to leaky expression off the lac Z promoter.E.Contribution of erm B and erm T to macrolide resistance in E.coli A leader peptide was identified upstream of erm B and erm T.Cotransformation of both erm B and erm T yielded much higher macrolide MICs than those MICs obtained by individual erm genes,no matter if the leader peptide was included upstream of individual erm gene.The macrolide MIC values were comparable to those obtained in all six isolates when both erm genes were cotransformed and the leader peptide was recruited.Without the leader peptide,macrolide MIC values were twice as much as those with the leader peptide.Co-transformation of these two genes,regardless of the presence of the leader peptide,all tested MIC values were reduced by half comparing those without the leader peptide.These results demonstrated that the the presence of both erm B and erm T was responsible for high macrolide resistance in all six isolates,and that the expression of both erm genes may be attenuated by the leader peptideIn summary,the conclusions from this study were as follows:A.Six Streptococcus gallolyticus subsp.pasteurianus isolates were isolated for the first time from ducklings spleen and brain.These bacteria can caused meningitis and splenic macrophage necroptosis.B.erm B and erm T were present in the genomes of all six isolates.Their expression were macrolide inducible and conferred high macrolide resistance in these isolates.The data of this study provided theoretical basis for drug resistance monitoring,clinical diagnosis and treatment as well as the development of antibacterial synerists regarding Streptococcus gallolyticus subsp.pasteurianus.