Virulence Analysis Of Klebsiella Pneumoniae With Different Drug Resistance Phenotypes In Caenorhabditis Elegans Infection Model

1 PurposeTo establish the C.elegans-Klebsiella pneumoniae infection model by using a new method,solid culture method;use this model to compare the virulence of Klebsiella pneumoniae with different drug resistance phenotypes;analyze the mechanism of virulence and further explore the relationship between drug resistance and virulence.2 Method2.1 Nematode synchronizationThe gravid hermaphrodites,which contain many fertilized eggs visible in the uterus,were collected into a centrifuge tube.And then the gravid hermaphrodites were washed and centrifuged and the supernatant were aspirated until approximately 3.5 mL liquid were remained.1.5 mL of bleach solution were added into the centrifuge tube and then shake the centrifuge tube vigorously.The eggs were washed quickly by M9 buffer three times and were hatched at 20? overnight.The L1 worms were pipetted onto NGM-OP50 plate.The worms were incubated 48 h at 20? until they reach the L4 larval stage.2.2 Preparation of infected platesK.pneumoniae strains were suspended at a concentration of 105 cfu/mL.10?L of bacterial suspension were pipetted onto 3.5 cm SK agar plates and were spread on the culture slightly.SK agar plates were dried at room temperature and were inoculated at 37? for 8 h.The plates should be cooled to room temperature before used.2.3 Nematode killing assayA certain concentration of 5-fluoro-2-deoxyuridine(FUDR)was pipetted onto the bare agar.10 age-matched L4 hermaphrodite worms were picked onto the prepared infected plates.The nematode fed with E.coli OP50 was used as experimental control group.All the plates were incubated at 25 ?.Live worms were scored each day.A worm was considered dead when it no longer shake head,chew and other movements and showed a straight line.2.4 Survival of nematodes exposed by different drug resistant strains of Klebsiella pneumoniaeAccording to the susceptibility test results in vitro,the strains were divided into three groups:sensitive group,multi-drug resistance group,carbapenem resistance group.Nematodes were exposed by different drug resistant strains of Klebsiella pneumoniae.10 age-matched L4 hermaphrodite worms were transferred onto the prepared infected plates.At least three replicates repeated each time were performed for each selected strain.The nematodes fed with E.coli OP50 was used as a control group.Plates were then incubated at 25 ? and scored each day for live worms.2.5 Counting and Identification of Bacteria in NematodesAt a fixed time point,the nematodes infected with Klebsiella pneumoniae were collected and washed three times with sodium azide.Five nematodes were picked into an EP tube,which contained steriled PBS solution and quartz sand.The worms were disturbed and released bacteria.Serial dilutions of the homogenized solutions were then plated to count the live bacteria and identification.2.6 Detection of Klebsiella pneumoniae virulence factorsString test and target genes(including capsular serotypes and virulence genes)were detected in all of the stains.2.7 Statistical AnalysisAll of the experiments were repeated 3 times.Statistical analysis are carried out by using IBM SPSS Statitics 19.0.Survival analysis was performed by using Kaplan-Meier method and Log-rank test was used to analyze the significant difference between groups.Difference of measurement data was compared with completely random design analysis of variance.LSD test analysis was used to comparison each other.When P<0.05,the difference was obvious.3 Result3.1 Establishment of Caenorhabditis elegans-Klebsiella pneumoniae infection modelThe results showed that compared to the control group,the death of nematodes was more obvious in the experimental group.LT50(d)of nematodes in the experimental group and the control group was 8.000 ± 0.769,11.000 ± 0.458.There was a significant difference in the survival curves(x2=27.519,P<0.05).After 24,48,and 72 h of infected with K.pneumoniae,the amount of bacterial in the nematode were 1.949×104cfu/mL,0.911 × 105cfu/mL,1.523×106cfu/mL,respectively.There was significant difference in the number of bacterial at different time(F=37.192,P<0.001).The LSD test showed that there was significant difference in number of bacterial at each time point(P<0.05),which showed that K.pneumoniae could colonized in nematodes,and eventually led to nematodes' death.The identification and drug susceptibility result of bacterial showed that the bacteria in nematodes were consistent with the initial.3.2 Pathogenic ability comparison of Klebsiella pneumoniae with different resistant phenotypesThe LT50(d)in the control group(E.coli OP50)was 11.000±0.458.While the LT50(d)of sensitive group,multidrug-resistance group(MDRKP)and carbapenem-resistance group(CRKP)were 10.000±0.231,9.000±0.080,and 9.000±0.130,respectively.There were significant differences in the lethal time when compared to MDRKP and CRKP(P<0.05).The lethal time(d)of three groups were 5.50,3.86,and 3.56,respectively.After 72 hours,the number(106cfu/mL)of bacterial in nematodes of three groups were 1.478±0.861,1.6222±0.524,and 1.4667±0.739,respectively.There was no significant difference between the three groups(F=0.137,P=0.872).3.3 Detection of Klebsiella pneumoniae virulence factorsIn the 10 isolates of Klebsiella pneumoniae,only KP3 was positive for the ST test and the rmpA gene.The rest of the strains were negative for ST test and virulence gene tests.4 Conclusion4.1 A novel experimental method,solid culture method,has been used to establish the C.elegans-Klebsiella pneumoniae infection model.4.2 The resistant strains are slightly more pathogenic than susceptible strains,they can colonize in nematodes for a long time and lead to the death of C.elegans in a shorter time.4.3 No virulence factors have been found in drug-resistant strains in our study,which suggests that there may be other mechanisms leading to virulence enhanced.