Molecular Characterization Of ESBLs-producing Klebsiella Pneumoniae Isolates From A Neonatal Intensive Care Unit

ObjectiveThrough in-depth research on the resistance phenotypes,resistance genes and virulence factors of ESBLs-Kpn strains isolated from children in the neonatal intensive care unit（NICU）,we intend to clarify the resistance mechanism and pathogenic mechanism from the molecular level.It provides molecular basis for the clinical treatment and the course of disease after the special group of newborns infected with ESBLs-Kpn.MethodsFrom January 2017 to December 2018,121 ESBLs-Kpn isolates from bloodstream and respiratory tract of NICU children in a tertiary hospital in Beijing were collected.Instrumental method,pulsed field gel electrophoresis（PFGE）,and multiple sites sequence typing（MLST）were used to detect the drug-resistant phenotype,homology analysis and ST type of the strains.We use real-time PCR（RT-PCR）to detect drug-resistant genes and virulence genesand analyze their virulence characteristics through biofilm formation tests,confocal microscopy three-dimensional biofilm structure imaging and serum killing assay.The molecular characteristics of bloodstream-derived and respiratory tract-derived strains were analyzed through whole genome sequencing（WGS）and bioinformatics methods.ResultsIn vitro drug susceptibility tests of the 121 ESBLs-Kpn strains all showed a similar drug susceptibility spectrum,which was resistant to third-generation cephalosporins,while maintaining sensitivity to carbapenem drugs.121 ESBLs-Kpn strains were divided into 13types by PFGE homology analysis,of which 6 types（A to F）had two or more strains:type A（n=101）,type B（n=4）,type C（n=2）,type D（n=2）,type E（n=3）and type F（n=2）.Corresponding ST classification:type A was ST3330,type B was ST2791,type C was ST290,type D was new ST type,type E was ST37,type F was ST34.Through PFGE and WGS phylogenetic tree analysis,we found that this new type of ST type had low homology with other ST types.8 cases of children had bloodstream infection and respiratory tract infection at the same time.Their molecular typing showed that 7 groups of strains were type A（ST3330）,and 1 group was type B（ST2791）.The screening of drug resistance genes by RT-PCR and WGS showed that the popular clone ST3330 mainly produced CTX-M-3 and TEM-1.The screening of virulence genes showed that the ast A virulence factor was only present in the epidemic clone ST3330.All strains carry various non-specific virulence factors related to siderophore secretionsuch as ent B,ent A,fep C and fep G.All strains carry offensive virulence factors related to fimbriaesuch as yag V/ecp E,yag W/ecp D,yag X/ecp C,yag Y/ecp B,yag Z/ecp A and ykg K/ecp R and omp A multifunctional offensive virulence factors.The biofilm formation ability of the popular clone ST3330 was stronger than other ST strains(median OD_590nm0.709 vs OD_590nm0.327)with P<0.0001（unpaired t-test）which was statistically significant.Confocal laser three-dimensional imaging microscopic visualization of biofilm structure showed that the production of biofilm from the popular clone ST3330 was significantly higher than that of other ST strains.In the new ST-type hole,membrane-like floating matter could be seen,which could not be attached to the plate hole bottom sliver.The fluorescence staining results showed that the biofilm production was extremely high.All six PFGE types isolates showed serum high sensitivity（grade 1）.ConclusionsThe 121 ESBLs-Kpn strains isolated from NICU were mainly ST3330 type,which had clonal transmission and mainly carried bla_CTX-M-3and bla_TEM-1 resistance genes,which was the main drug resistance mechanism.The drug resistance phenotype showed resistance to third-generation cephalosporins.The ST3330 strain carries ast A virulence gene whose product is heat-stable enterotoxin 1.The enterotoxin is not virulent and therefore exhibits low pathogenicity,which is consistent with the good clinical prognosis of most children.All strains carried a variety of virulence factors related to siderophore secretion which met the growth and metabolism requirements of bacteria.At the same time,it carries the virulence factor related to the pili,which helps the bacteria adhere to the host cells and constitutes theinvasiveness of bacteria.The popular clone strain ST3330 had stronger biofilm formation ability than other ST type strains,which may be related to the fact that the strain carried the offensive virulence factor ast A and omp A.The former caused persistent disease,the latter guaranteed the continuous survival of the bacteria by assisting the biofilm formation and its surface activity.The application of confocal microscopy three-dimensional biofilm structure imaging technology provided advantages at the technical level for biofilm related research.All strains were sensitive to serum of healthy adults,suggesting that the virulence of ESBLS-KPN strain was not strong,which was consistent with the good clinical prognosis of the children.A new ST type was discovered.Through phylogenetic tree analysis,it was found that this new type of ST type had low homology with other ST types,suggesting that this type may be infected from outside of the NICU.