Establishment Of An Infection Model Using Caenorhabditis Elegans-multidrug Resistant Acinetobacter Baumannii

1 purposeUsing Caenorhabditis elegans-mutidrug resistance Acinetobacter baumannii to establish an infection model.And the model is basis for study of pathogenicity mechanism and screening anti-infective compounds.2 Methods2.1 Prepare a synchronized worm populationWash off gravid hermaphrodites into a 15 mL conical tube.Then washing,centrifugal,abandon the supernatant,and reserve 3.5 mL worm solution.Add 1.5 mL of worm pyrolysis solution.Then,using M9 buffer to wash the embryos three times,the embryos were placed in the 20? overnight to incubation.Plating L1-stage worms onto the nematode growth medium media at 20? for 48h,then the synchronized L4 stage worms were collected.2.2 Preparation of infected platesThe bacteria to be tested were diluted to 1.2 × 10~9CFU/mL,100 ?L of the bacteria suspension was taken to the 10 cm plate,and the bacteria solution was uniformly coated on the NGM plate with a sterile coated bar,but not on the edge of the plate.Dried at room temperature for 20min,placed at 37? for 8h,and then cooled to room temperature.2.3 Nematode-killing assay in solid mediumTen L4 nematodes were transferred on each infected plate.In order to prevent the growth of N2 nematodes,FUDR was added to the infected plate,and E.coli OP50 was the experimental control group.Each group contained 10 infected plates The 20 ? culture every 24 hours to observe the survival of a nematode,when the body stiffness and no response to touch is determined to die.2.4 Nematode-killing assay by different concentrations of MRDABApproximately 20-30 worms were transferred to 96-well plates,each hole was added with 40?L LB solution that containing a certain concentration of bacteria,160?L M9 buffer and 0.2 mmoml/L FUDR.The infection group was joinned with 40 ul LB broth containing Ab(1.2×107 CFU/ml,1.2×108 CFU/ml,1.5×10~9 CFU/ml),1.2×10~9CFU/ml E.coli OP50 was used as a negative control,and 20%of LB medium was used as vehicle control.The plates were incubated at 20? and scored for live and dead worms at least every 24 h and each assay was carried out in triplicate.2.5 Survival of worms exposed by different drug resistant strains of A.baumanniiWorms were exposed by different drug resistant strains of A.baumannii.1.2×10~9 CFU/ml,Approximately 20-30 worms in a volumes of 160 ul were transferred to 96-well plates that contained 40 ul LB broth containing different drug resistant strains of A.baumannii,1.2×10~9 CFU/ml E.coli OP50was used as a negative control.Experiments were performed at least thrice at 20? and 20%of LB medium was used as vehicle control.The plates were incubated at 20? and scored for live and dead worms at least every 24 h and each assay was carried out in triplicate.2.6 Bacteria Counting and Identification of Nematode IntestinalNematodes were infected with 1.2 × 10~9cfu/mL MDRAB.Ten nematodes which had been infected for 24h were picked and the worms were washed three times with sodium azide solution to inhibit the bacteria from the bacteria.The nematodes were incubated with 200 ?l of M9 buffer in a 1.5mlEP tube,and then add sterilized quartz sand after a 2?3min shock,which disturbed the worms and release the invaded bacteria in the suspension of M9.The resulted suspension was serially diluted and plated on a Hicrome Klebsiella isolation agar(Himedia)to determine the CFU.2.7 C.elegans rescuing assaysFollow the procedure of 2.4,infected the L4 nematodes for 24 h,and washed the infected nematodes with M9 buffer four times and then collected them well.About 20?30 infected nematodes were transferred to 96-well plates(each well were added by 40?L LB solution,160ul M9 buffer,0.2mmoml/LFUDR and a certain amount of antibiotics),and were cultured at 20?.The rats in the experimental group were treated with different concentrations of antibiotics:meropenem(0,16,32,64ug/ml)and tigecycline(0,8,16 and 32ug/ml);Negative control group:without any antibiotics;The first row of negative control,without any antibiotics,followed by the addition of different concentrations of antibiotics,nematode infection treatment,observed once every 24 times the survival of nematodes.T2.8 Statistical analysis.The analysis was carried out using SPSS20.0.To compare the entire survival curves in nematode killing assays,we used a Kaplan-Meier.In order to perform pairwise comparison between two different strains,we used a log-rank test.Difference of measurement data was compared with completely random design analysis of variance.Comparison of the two was used by SNK test analysis.If P<0.05,it indicated that the difference was significant,P>0.05,the difference was not significant.3 RESULTS3.1 Nematode-killing assay in solid mediumThe phenomenon of lethal nematode on the MDR-Ab plate was More obviousd than that of the OP50 plate,and died at 4 to 6 days after infection.The worms of the E.coli OP50 group generally died only after 3 weeks.However,Ab-nematode solid lethal experiments have the following shortcomings,it is difficult to collect stable and reliable data.3.2 Nematode-killing assay in liquid medium3.2.1 Nematode-killing assay by different concentrations of MRDABThe results showed that there was no significant difference in the survival curve of 1.2×107cfu/mL MDRAB group and E.coliOP50 control group(?~2 = 0.148,P =0.70),suggesting that the concentration of bacteria was not significantly lethal to nematode;1.2×108,1.2×10~9cfu/mL MDRAB survival rate were lower than E.coliOP50 control group,and the survival curve and the control group were statistically significant(?~2 were 38.47,90.18,P<0.001),indicating bacteria MDRAB in the range of 1.2 x 108?1.2 × 10~9 cfu/mL had the effect of infection on nematodes.The survival curves of the two nematodes were statistically different(x2 = 21.91,P<0.001)when the 1.2 × 10 8 and 1.2 × 10~9cfu/mL MDRAB groups were Compared with each other,and suggested that the lethal effect of 1.2 × 10~9cfu/mL MDRAB on the nematodes was more Significantly than the former.3.2.2 Survival of worms exposed by different drug resistant strains of A.baumanniiThe results showed that the average survival time of A,B,C and D were 9.4,9.7,9.5 and 15.4 days.The log-rank test was used to compare the levels.There was no significant difference in the mortality of nematodes between different degrees of resistance to Acinetobacter baumannii(P>0.05);However,there were statistically significant differences in the survival curves of group A,B and C(P<0.05);Suggesting that sensitive,multidrug-resistant,pan-resistant Acinetobacter baumannii can shorten nematode life.However,different drug resistance of Acinetobacter baumannii in nematode infection model no significant difference in virulence.3.3 Bacteria Counting and Identification of Nematode IntestinalThe nematode lysate was tested for bacterial identification and susceptibility testing.It was confirmed that MDRAB was confirmed.After the nematodes were infected with MDRAB 4h,8h,12h,24h respectively,and then the infected nematodes were cracked,and the supernatant was subjected to bacterial plate count.The amount of bacteria in the nematodes were(0.22 ± 0.02)× 105,(0.54 ± 0.03)x 105,(1.69±0.02)× 105,(3.22 ± 0.60)×105 cf/ml respectively,Using One-Way ANOVA analysis,we found that there were significant differences in the number of intestine bacteria in nematodes infected at different time points(F=27.32,P<0.001),which Indicated that MDRAB could grow and reproduce in the intestinal tract of the nematodes,and cause nematode infection to death.3.4 C.elegans rescuing assaysWhen treated with different concentrations of meropenem for nematode infection with MDRAB,16 ug/mL meropenem was found to have little effect(?~2 =2.093,P = 0.148)compared with the untreated group.32 ug/mL and 64 ug/mL of meropenem could increase the survival rate of nematodes.The survival rate of nematodes was 51.7%and 62.8%after 10 days respectively,and the survival rate was 2?3 times(P<0.001)compared with untreated group.(?~2 = 7.129,P<0.05).The results showed that there were statistically significant differences in the survival curves between the two groups(?~2 = 7.129,P<0.05).It was suggested that meropenem was effective in the treatment of nematodes in the range of 32?64ug/mL,and the therapeutic effect was positively correlated with the drug concentration.Treatment of MDRAB-infected nematodes with different concentrations of tigecycline showed that the median survival time of infected nematodes increased with the increase of the concentration of tigecycline.The log-rank test showed that there was no significant difference in survival curve between 8ug/ml tigecycline treatment group and control group(?~2=0.105?1.4,P=0.746);The survival curve of 16 ug/ml and 32ug/ml tigecycline treatment group compared with the blank control group were both statistically significant(?~2=81.290?124.302,P?<0.001;).Which suggested that tigecycline was effective in the treatment of nematodes in the range of 16?32ug/mL,and the therapeutic effect was positively correlated with the drug concentration.4 Conclusion4.1 Nematode liquid infection method compared to solid infection method,the former to ensure better experimental operability,repeatability and stability.4.2 MDRAB can infect nematodes.However,there was no significant difference in the toxicity of different resistant strains of Acinetobacter baumannii to nematodes.4.3 The survival rate of nematodes was positively correlated with the concentration of antimicrobial agents,which was consistent with the results of in vitro susceptibility test.