Study On Risk Factors And Related Mechanism Of Candida Parapsilosis Bloodstream Infection

Background Clinical bloodstream infection caused by non-C.albicans Candida(NCAC)has been growing in recent years.Candida parapsilosis(C.parapsilosis)has become the secondary causative pathogen for candidemia.It deploys various pathogenic factors for infection,which includes adhesion,biofilm and etc.Studies revealed most of C.parapsilosis infection cases are Catheter-related bloodstream infections(CRBSIs).The results of the National Invasive Fungal Drug Resistance Surveillance Network(CHIFNET)also showed that the isolation rate of C.parapsilosis from blood and catheter specimens in patients with invasive candidiasis was first.Such infection can lead to high mortality,morbidity and increased medical costs for patients.Thus,studies on characterization of C.parapsilosis could improve the understanding of its pathogenic mechanism,as well as methods for clinical antiinfection treatment,to lower the mortality.Abundant studies have indicated strong relationship between C.parapsilosis infection and central venous catheters(CVCs),and confirmed that formation of C.parapsilosis biofilm is related to its central venous catheter colonization.Which is also the main invasive mechanism on host mucosal epithelial cells.These studies suggest that biofilm formation may play an important role in BSIs caused by C.parapsilosis.Thus,based on clinical cases and fundamental research,we are trying to explore the risk factors of C.parapsilosis infection,as well as mechanism of its biofilm formation.Part one Analysis of Risk Factors of Candida Parapsilosis Bloodstream Infection in Intensive Care UnitAims By investigating the clinical characteristics of C.parapsilosis candidemia in intensive care unit(ICU)of a tertiary hospital,we analyzed the risk factors for the occurrence of C.parapsilosis bloodstream infections.The results lead to some references for clinical treatment of C.parapsilosis bloodstream infection.Methods The clinical and microbiological data of 267 patients(without duplicate strains)with C.parapsilosis bloodstream infection from January 2013 to March 2019 were retrospectively analyzed.For all the identified patients,we performed a detailed medical record review and retrieved data from the electronic medical records and reports.They were divided into C.albicans group and NCAC group,the latter were subdivided into C.parapsilosis group and other NCAC species.Epidemiological characteristics,clinical features,invasive procedures and outcomes of these patients were analyzed to determine risk factors of candidemia due to C.parapsilosis.Candidemia was diagnosed by clinical information and blood culture,and the identification of Candida species were performed using MALDI-TOF mass spectrometry.Briefly,host factors included age,gender,comorbidities such as circulatory system disease,lung disease,liver disease,kidney disease,neoplastic disease,etc.,the main reason or disease for admission to ICU,APACHE II score(Acute physiology and chronic health evaluation II)within 24 h after ICU admission,ICU length of stay,and outcome.Retrieved medical interventions included total parenteral nutrition(TPN),mechanical ventilation(MV),prior use of medications(broadspectrum antibiotics,hormones,immunosuppressants,etc.Broad-spectrum antibiotics include fourth-generation cephalosporins and carbapenems),abdominal surgeries,intravascular catheterization location(jugular,subclavian and femoral)and duration(> 5 days)considering the risks of infections and the time required by Candida spp.to adhere,colonize and form mature biofilms.Based on these,risk factors for the occurrence of C.parapsilosis bloodstream infections were analyzed and probed.Results A total of 267 cases of Candida bloodstream infection occurred,including 63 cases of C.albicans and 204 cases of non-albicans Candida species.The latter was composed of 104 cases of C.parapsilosis and 100 cases of other non-albicans Candida species(46 cases of C.tropicalis,22 cases of C.glabrata,23 cases of C.guilliermondii,5 cases of C.krusei and 4 cases of C.lusitaniae,C.intermedia,etc),suggesting that C.parapsilosis was the predominant Candida species isolated from cases of candidemia.Univariate analysis indicated that APACHE II scores,CVC,broad-spectrum antibiotics use,TPN more than 3 days and MV were all risk factors of C.parapsilosis candidemias.A binary logistic regression analysis showed that CVC,the use of broad-spectrum antibiotics and APACHE II scores were closely related to C.parapsilosis candidemia,with OR values of 3.913,2.217 and 1.159,respectively.Conclusion C.parapsilosis was the main pathogen among NCAC candidemia in ICU patients,and the isolate rate exceeds that of C.albicans.CVC,the use of broadspectrum antibiotics and APACHE II scores were independent risk factors for the occurrence of C.parapsilosis candidemia.Part two Study on Candida Parapsilosis’ Adhesion and Biofilm Formation CapabilitiesAims Based on in vitro experiments of clinical isolates from central-venous-catheterrelated Candida bloodstream infected patients,we detected and analysis the difference between C.parapsilosis and other Candida strains on adherence,biofilm formation and metabolism activity.Also,several C.parapsilosis strains were also analyzed to probe the critical factors for biofilm formation.Methods Twenty-seven Candida isolates,including seven C.albicans,six C.parapsilosis,five C.guilliermondii,five C.tropicalis,and four C.glabrata,causing CVC related-bloodstream infections(CRBSIs)were investigated.Considering the purity and viability of the microorganisms,all samples were subcultured twice prior to each experiment and assessed using IVD MALDI-TOF mass spectrometry.Their antifungal susceptibilities(Minimal inhibitory concentration,MIC),adhesion properties,and biofilm-forming capabilities were evaluated.According to the ability of yeast cells adhere to fluorescent microspheres of Fluorescence activated cell sorter(FACS),and the adhesion-related parameters(P2)can directly reflect the adhesion ability of Candida isolates,flow cytometry method(FCM)was used to evaluate their adhesion abilities of different clinical Candida isolates.The XTTmenadione direct measurement method(OD450nm)and the crystal violet indirect measurement method(OD630nm)were used to determine the biofilm formation ability,which includes metabolic activities and total amounts of the biofilms.The C.parapsilosis clinical strain CP7,with the strongest biofilm-forming ability and CP5,with medium biofilm-forming ability were selected.The mutant strain CpΔBCR1,with both copies of BCR1 gene deleted and biofilm production impaired,was set as a control in the experiment.Quantitative Real-Time PCR(q RT-PCR)was performed to detect transcriptions of adhesion-related genes(CpALS6,CpALS7)and biofilm formation-related factors(CpEFG1,CpBCR1)in C.parapsilosis strains.Results The MIC results of 27 clinical isolates recovered from CRBSIs showed that all seven C.albicans isolates were sensitive to antifungal agents.For C.glabrata,three isolates were resistant,and one was intermediate to fluconazole(FZ);two isolates were resistant,and one was intermediate to voriconazole(VOR);two isolates were resistant to posaconazole(PZ);and none was resistant to amphotericin B(AMB),caspofungin(CAS),and micafungin(MF).With regard to C.parapsilosis,except that one isolate was resistant to FZ,others were susceptible to antifungal agents.All C.tropicalis isolates were susceptible to AMB,CAS,MF,and PZ,while four were resistant to VOR and the remaining one was resistant to FZ.All C.guillermondii isolates were resistant to both FZ and VOR,whereas no resistance was observed for AMB,CAS,MF,and PZ.To reduce the variability of adherent with microspheres(P2)between distinct Candida isolates and better evaluate the adhesion strength of each isolate,four adhesion profiles were established.Weak(P2: 1-20),medium(P2: 21-30),strong(P2: 31-50),and very strong(P2: >50)according to the flow cytometry results.Based on these adhesion patterns,among the tested C.albicans strains,71.4%(5/7)displayed a strong adhesion profile,while the remaining 28.6%(2/7)exhibited a very strong profile.The majority of C.glabrata strains demonstrated a weak adhesion profile,except for Cgl 1.Almost all C.parapsilosis isolates displayed a very strong profile,and only one strain(Cpa 5)with strong adhesion.The most heterogeneous species were C.guilliermondii and C.tropical clinical strains.Comparing the total biofilm formation of different candida species,CV assay was performed to measure the total biomass of the biofilms at 24 h,the results indicated that C.parapsilosis produced higher volume of biofilms than those of C.glabrata and C.tropicalis.In addition,the XTT results demonstrated that C.guilliermondii and C.parapsilosis had the highest metabolic activities,which are significantly higher than those of C.albicans,C.glabrata and C.tropicalis.The q RT-PCR results showed that expression of two adhesins encoding genes CpALS6 and CpALS7,two biofilm related genes CpEFG1 and CpBCR1,were significantly higher in CP7 clinical strain,compared with that of the BCR1 gene knockout mutant strain.Moreover,the expression of CpALS7,CpEFG1,CpBCR1 in CP5 was higher than that of the mutant strain.Among them,the expression of CpALS6 and CpBCR1 in CP7 is higher than that in CP5.Conclusion Compared with Candida albicans,non-C.albicans Candida species,especially C.parapsilosis,exhibited higher adhesion ability in catheter-related candidemia patients.In addition,C.parapsilosis and C.guilliermondii showed the highest biofilm formation ability.The q RT-PCR results confirmed that,the expressions of CpALS7,CpEFG1 and CpBCR1 may play key roles in the adhesion and biofilm formation processes of C.parapsilosis CRBSIs.Part three Research of Drug Interference on Candida Parapsilosis’ Adhesion and Biofilm Formation CapabilitiesAims To evaluate the inhibitory effects of antifungal agents like Micafungin(MF),Fluconazole(FZ)and N-acetylcysteine(NAC)on adhesion and biofilm formation in C.parapsilosis clinical strains and other Candida species.Meanwhile,to detect the transcription level changes of adhesion and biofilm formation associated genes(CpALS6、CpALS7、CpEFG1 and CpBCR1)when administrated with antifungal agents in different strains.Methods To determine whether antifungal agents can exhibit inhibitory effects on the adhesion,amounts of total biofilm formation and metabolic activities of different clinical Candida isolates from candidemia patients,MF,FZ and NAC were added to the yeast suspensions at different concentrations,respectively.In addition,q RT-PCR was used to detect the transcriptional levels of adhesionrelated genes(CpALS6,CpALS7)and biofilm formation-related factors(CpEFG1,CpBCR1)in BCR1 knockout strain,CP7 and CP5 clinical strains in the presence of MF and NAC.Results MF,FZ and high concentration of NAC reduces the total amount of biofilm formation on C.parapsilosis.For C.albicans,all the tested drugs exhibit inhibitory effects on biofilm formation.For C.glabrata and C.tropicalis,only high concentration MF and NAC exhibit inhibitory effects.For C.guilliermondii,all the tested drugs could inhibit biofilm formation,higher concentration of MF and NAC exhibits stronger inhibitory effects.For adhesion,only high concentration MF exhibits inhibitory effects.Based on results above,we used the MF and NAC treated BCR1-deleted strain,CP5 and CP7 for gene transcription detection.After treated with MF,the transcription level of CpALS6,CpALS7,and CpEFG1 in CP7 were all down regulated,and those of CpALS7 and CpEFG1 in CP5 were also decreased.Following co-incubation with NAC,the expression of CpEFG1 in both CP7 and CP5 clinical strains were decreased,while no significant changes in transcriptional levels of CpBCR1 compared with untreated strain.Conclusion Non-antifungal agent NAC exhibits certain inhibitory effects on clinical isolates’ biofilm formation.MF tended to have stronger inhibitory effects against both adhesion and biofilm formation of different clinical Candida strains from CRBSIs,and the inhibitory effect is dose-dependent.MF can down-regulate the transcription levels of CpALS7 and CpEFG1 in C. parapsilosis clinical strains,which makes it a potential candidate for treatment of C.parapsilosis CRBSIs.