| Background and objectiveBloodstream infections caused by carbapenem-resistant Acinetobacter baumannii（CRAB）are serious and challenging to treat clinically.Epidemiologic surveillance studies have been conducted to CRAB,which is still dominated by the CC92 complex in Asian,but there are differences in CRAB detection rates,sequence types（STs）,virulence abilities and antibiotic resistance genes（ARGs）in various regions and hospitals.Due to these characteristics,during the CRAB pandemic,individualized management is crucial to control the infection.The purpose of this study is to analyze the molecular epidemiological characteristics of CRAB isolates isolated from blood cultures in Guangzhou and Foshan hospitals,evaluate vitro efficacy of antibiotic combination therapy and investigate the clinical features,providing further evidence for early warning surveillance and treatment of CRAB bloodstream infections in the region.MethodsFrom January 2019 to June 2021,CRAB isolates isolated from blood cultures were collected in three tertiary care hospitals in the Guangzhou and Foshan region.Multi-site sequence typing（MLST）was performed by PCR to analyze the genetic relationship between strains.The strains obtained by typing were tested for abdominal infection of Galleria mellonella,then were divided into high virulence group and non-high virulence group according to the experimental results.Core genome multi-locus sequence typing（cg MLST）,virulence genes and antibiotic resistance genes of the strains in the high virulence group were subsequently analyzed using whole-genome sequencing（WGS）.The MICs of common antimicrobials were determined using the broth microdilution method.Combination therapy in vitro was performed based on the single drug susceptibility results to evaluate the activity of different antimicrobial combinations（colistin-based combinations and tigecycline-based combinations）against different genotypes of CRAB isolates isolated from blood cultures.The clinical and microbiological characteristics of CRAB were analyzed by collecting clinical data and antibiotic susceptibility testing.ResultA total of 10 ST types were identified in 31 isolates of CRAB,which were assigned to CC92 clones by e BURST,and no new ST types were identified.The predominant population was ST208,accounting for 22.5%,followed by ST1145（12.9%）,ST457（12.9%）,ST1417（12.9%）,and ST1486（12.9%）.31 isolates of CRAB were divided into high virulence group（n=24）and non-high virulence group（n=7）according to Galleria mellonella model.The resistance rate of 31 isolates of CRAB to cephalosporins（including enzyme inhibitors）,quinolones and aminoglycosides was 77%-100%,to tigecycline and minocycline were 25.8%and64.5%respectively,and all isolates were susceptibility to colistin.The resistance rates of high virulence group strains to both minocycline and tigecycline were lower than those of non-high virulence group strains,which were 54.2%vs.100%and 20.8%vs.42.9%,respectively.Combination therapy showed that colistin-based combinations had good antibacterial activity against 31 isolates of CRAB,of which colistin combined with minocycline and colistin combined with meropenem had the strongest synergistic and additive activity in vitro（the synergistic and additive ratio both>80%）,the synergistic and additive activity in vitro of colistin combined with cefoperazone/sulbactam was weaker（the synergistic and additive ratio<60%）.Among tigecycline-based combinations,the best synergistic and additive activity was observed in tigecycline combined with meropenem（ratio>75%）,while combined with colistin or cefoperazone/sulbactam showed mainly additive and indifferent activity.Genomic analysis showed that the detection rates of eps A and ptk were higher in the high virulence group than in the non-high virulence group,which were 91.6%vs.42.8%and 83.3%vs.0%,respectively.The proportion of isolates carrying both eps A and ptk with G.mellonella survival rates≤10%and≤30%were 61.1%and 100%,respectively,while the proportion of isolates carrying only one of the above virulence genes with G.mellonella survival rates≤10%and≤30%were 22.2%and 66.7%,respectively,indicating that the virulence of isolates carrying only eps A and ptk virulence gene was weaker than isolates carrying both eps A and ptk,while the virulence of isolates carrying neither eps A and ptk was significantly weaker（50%-90%survival rate of G.mellonella）.In addition,all highly virulent isolates carried virulence genes related to biofilm（ade FGH,bap,bfm RS,omp A,pga ABCD）,iron（ent E,bas-bau-bar）,phospholipase（plc D）,Chaperone-usher（csu AB/ABCDE）,Quorum Sensing System（aba I）and type VI secretion system（T6SS）,and all carried the same type of O-antigen surface polysaccharides（OCL1）.aba R genes were absent in ST457.In addition,highly virulent isolates mostly carried carried 9-17 resistance genes,carbapenemase(bla OXA-23,bla OXA-66),cephalosporinase(bla ADC-73),β-lactam genes(bla TEM-12),aminoglycoside（ant（3’’）-IIa,aph（3’’）-Ib,aph（6）-Id,aph（3’）-Ia,arm A）,macrolide（mph（E）,msr（E））,tetracycline（tet（B））and sulfonamide（sul1,sul2）were detected>80%.In addition to the above genes,some isolates also detected aminoglycoside（aac（6’）-Ib’,aad A1）and chloramphenicol（cat B8）,while one isolate of ST457 was not detected mph（E）,msr（E）and arm A.The bla OXA-23gene was harboured by Tn2008 in ST369 and one isolate of ST208 with 17 resistance genes,and by Tn2006 in other isolates.WGS analysis revealed that 24 highly virulent isolates in this study had similar virulence gene profile and resistance gene profile with 24 highly virulent isolates in our previous study（2012-2015）.According to cg MLST,48 isolates could be divided into 9 clusters,and it was observed that ST457,ST208 and ST1633 highly virulent isolates had longer epidemic clone and spread in Guangdong Province.Clinical data show that CRAB bloodstream infections are more frequent in elderly males,with a higher incidence of septic shock（79.2%vs.14.3%,P=0.004）and higher mortality（66.7%vs.14.3%,P=0.028）in high virulence group than in non-high virulence group.Patients with poorer clinical outcomes were more likely to have invasive procedures（P=0.01）such as central venous placement,tracheal intubation or dissection,urinary catheterization,and gastric tube（P<0.05）,and also more likely to have septic shock（P<0.001）.No significant correlation was found between ptk and eps A virulence gene carriage and patient outcome,which may be related to the fact that patient clinical outcome is regulated by multiple factors.ConclusionsCRAB isolates isolated from blood cultures in Guangzhou and Foshan hospitals is still predominantly prevalent in the CC92 complex,with a detection rate of 77.4%（24/31）of highly virulent isolates,which carry abundant resistance genes to commonly used antimicrobials and have a high resistance rate to commonly used clinical antimicrobials,while were susceptibility to colistin.Colistin combined with meropenem had good antibacterial activity against 31 isolates of CRAB.The virulence genes eps A and ptk may be potential molecular markers for early warning of highly virulent CRAB.Meanwhile,the highly virulent epidemic clones ST457,ST208and ST1633 have spread for a long time within Guangdong Province and deserve nosocomial surveillance. |
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