| [Objective]1.To evaluate the antimicrobial susceptibility of commonly-used clinical antibiotics and eravacycline in the clinical isolates of extensive ?-lactamase(ESBL)-producing Klebsiella pneumoniae and further study the distribution characteristics of the resistance genes,virulence genes and the MLST typing.2.To investigate the relationship between eravacycline susceptibility and resistance genes,virulence genes as well as MLST typing in the clinical isolates of ESBL-producing Klebsiella pneumoniae.[Methods]1.Isolates Collection and identification of bacterial strains: 122 clinical isolates of Klebsiella pneumoniae were collected during January 2016 to September 2017 in Shenzhen Nanshan people's hospital.The identification of these strains was determined by BD Phoenix TM-100 system and the E.coli ATCC25922 was used as the control strain.2.ESBL Determinations and Antimicrobial Susceptibility testing: Determination of ESBL-producing Klebsiella pneumoniaeisolates and antimicrobial susceptibility testing of various antibiotics,including tetracycline,levofloxacin,cefepime,cotrimoxazole,piperacillin/tazobactam,gentamycin,were performed by BD Phoenix TM-100 system according to the CLSI standard.The MICs(minimum inhibitory concentration)of eravacycline,Imipenem and Meropenem were determined by the agar dilution method.3.Detection of resistance genes and virulence factors: Virulence determinants,including capsule K1/K2/ K5/ K20/ K54/ K57,Rmpa,Rmpa2,Wcag,Aero,and resistance genes,containing SHV,TEM,CTX,OXA,were amplified by polymerase chain reaction(PCR)assay using previously described primers and procedures.The PCR products were being purified and sequenced if necessary.The typing of SHV,TEM,CTX was confirmed according to recomendations on the https://blast.ncbi.nlm.nih.gov/Blast.cgi website.4.MLST typing: MLST procedure was carried out to determine the sequencing type(ST)of the clinical isolates as previously described.The strains were defined through amplifying and sequencing of seven housekeeping genes(rpob?gapa?mdh?pgi?phoe?tonb)by PCR,and the STs were assigned to each isolate after matching in the designated website(http://www.mlst.net/).5.Distribution characteristics of antimicrobial resistant genes,virulence genes and MLST typing: The prevalence of the ESBL specific resistant genes,including SHV,TEM,CTX,OXA and the virulence genes,including capsule K1/K2/ K5/ K20/ K54/ K57,Rmpa,Rmpa2,Wcag,Aero,in 122 ESBL-producing Klebsiella pneumoniae isolates were analyzed.The characteristics of MLST distribution in these striains were also assessed.6.The correlation of eravacycline susceptibility with the antimicrobial resistant genes,virulence genes,MLST typing: In order to explore the correlation of resistance genes,virulence genes,MLST typing and eravacycline susceptibility,five levels of eravacycline MIC values were divided with ? 0.5 ?g/ml,1 ?g/ml,2 ?g/ml,4 ?g/ml and 8 ?g/ml,respectively.Subsequently,the detection rates of the antimicrobial resistant genes and virulence genes in the isolates with each levels of eravacycline MIC values were calculated correspondingly and their differences among groups with different levels of eravacycline MIC values were compared.[Results]1.Bacterial origins and antimicrobial susceptibility test results: 122 ESBL-producing Klebsiella pneumoniae strains were collected from different clinical specimens,including fleas(72,59.0%),urine(16,13.1%),bronchial secretions(11,9.0%)and blood(10,8.2%)respectively.The resistant rates of tetracycline,levofloxacin,cefepime,compound sulfamethoxazole and gentamycin in these isolates were 88.5%,40.1%,86%,99.2%,83.6%and 58.1%respectively and that of piperacillin/tazobactam,imipenem and eravacycline were29.5%,0.8%,12.2% respectively.All strains kept susceptible to meropenem.2.Resistant genes,virulence factors profiles and MLST typing: The frequecncy of SHV,TEM,CTX,OXA in these ESBL-producing klebsiella pneumonia were 99.2%(121/122),54.9%(67/122),91.8%(112/122),11.5%(14/122)respectively and the prevance of capsule K1,K2,K20,K54 and K57 were 20.5%(25/122),5.7%(7/122),1.6%(2/122),3.2%(4/122) and 2.5%(3/122)respectively.Meanwhile,the detected rates of Rmpa,Rmpa2,Wcag and Aero were 18%(22/122),14.8%(18/122),18%(22/122)and 21.3%(26/122)respectively.The ST analysis by MLST typing indicated that total 44 STs in these isolates were obtained,including ST11(5.7%,7/122),ST23(5.7%,7/122),ST133(9.8%,12/112),ST35(5.7%,7/122),ST15(4.1%,5/122).3.Relationship between eravacycline susceptibility and resistant genes,virulecnegenes:The total rate of eravancycline non-susceptibility(MIC?4?g/ml)in these isolates was 31.9%.The positive rates of ESBL-sepcific resistant genes,including SHV,TEM,CTX,OXA,in eravancycline non-susceptible(MIC?4?g/ml)and susceptible(MIC?2 ?g/ml)groups were62.8%/37.2%,67.2%/32.8%,67.9%/32.1%,50%/50% respectively.In eravacycline non-susceptible group,the prevalences of capsule K1,K2,K20,K54 and K57 were 30.1%(12/39),10.3%(4/39),2.6%(1/39),2.6%(1/39)and 2.6%(1/39)respectively,whereas that of Rmpa,Rmpa2,Wcag and Aero were 28.2%(11/39),20.5%(8/39),20.5%(8/39),and 28.2%(11/39)respectively.4.Relationship between eravacycline susceptibility and MLST types: The major STs in these strains were ST11(5.7%,7/122),ST23(5.7%,7/122),ST133(9.8%,12/112),ST35(5.7%,7/122),ST15(4.1%,5/122),and the prevalence of ST11,ST15,ST23,ST35 and ST133 were 71.4%(5/7),20%(1/5),42.8%(3/7),41.7%(5/12)respectively in eravancycline non-susceptible group.All clinical klebsiella pneumonia isolates with ST35 type in this study maintained sensitive to eravacycline.The virulence genes of K1,Rmpa,Rmp2,Wcag and Aero were all positive in ST23 type..[Conclusions]1.Clinical ESBL-producing Klebsiella pneumoniae isolates in our hospital exhibit a comparatively high frequency of eravacycline nonsusceptibility.The ESBL-specific resistant genes most commonly detected in this study are SHV and CTX and the most commonly capsule serotypeare includes K1 and K2;the top two STs types by MLST types are ST11 and ST133.2.ESBL-producing Klebsiella pneumoniae isolates carrying the antimicrobial resistant genes facilitate to show the eravacycline MIC creep compared with that without resistant genes.Our data indicate that the virulence genes of K1,Rmpa,Rmp2,Wcag and Aero in eravacycline-nonsusceptible klebsiella strains are still commonly detected,suggesting the transmission risks of the kelbsiella with the co-existence risks of multidrug resistance and virulence factors in this area.3.The major ST types of ESBL-producing Klebsiella pneumoniae isolates in our hospital are ST11,ST23,ST133,ST15 and ST35.The Klebsiella pneumoniae strains with eravacycling MIC over-than 4?g/ml showed the clonality of ST11,ST23 and ST133,indicating the clonal aggregation of eravacycline resistance in this species And strain ST23 may be a highly virulent and resistant strain. |
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