The Establishment Of Caenorhabditis Elegans Model Infection With Klebsiella Pneumonia And Preliminarily Study

1. Background and purposeKlebsiella pneumoniae (KP), a Gram-negative bacterium belonging to the family Enterobacteriaceae is a remarkably successful, versatile and one of the leading human pathogens in causing both nosocomial and community acquired infections. Though,it has been found as a common component of normal microflora of virtually all humans, this opportunistic pathogen can invade host system and cause life-threatening infections in immunocompromised individuals.It can also cause severe pneumonia, bowel diseases,liver abscess, severe bacteraemia and meningitis and inflammation of the urinary tract. In severe cases, pyogenic liver abscess along with meningitis and endophthalmitis are also encountered with a mortality rate of 20-55%. As far as the nosocomial infection of KP is concerned, the prime significance is its initial colonization in the gastrointestinal tract, irrespective of its site of infection. The degree of colonization of KP depends on their ability to form biofilm,which contributes to their persistence and shielding from host defense via oxidative stress and antimicrobial peptides.In recent years, with carbapenem antibiotics for a long time, large, wide use, the carbapenem-resistant Enterobacteriaceae strains increased year by year. CHINET 2014 surveillance of bacterial resistance in china found that the Klebsiella pneumoniae strains were the majority of the carbapenem-resistant Enterobacteriaceae strains.Above 10% of Klebsiella pneumoniae strains were resistant imipenem and meropenem.As CRE strains often characteristiced by extensively drug resistant or pan-drug resistant, the theory of infection may fall into the plight of the drug free.Caenorhabditis elegans is an anatomically simple, genetically tractable nematode.In recent years, the bacteriovorous nematode C. elegans has become a prominent model organism in the study of microbial pathogenesis, innate immunity, and drug discovery that the nematode C. elegans is a facile model host,genetically tractable and short life cycle.As far as concerned,A broad spectrum of Gram-positive,Gram-negative bacteria and Fungi of 40 Species has been reported to kill the C. elegans when substituted for normal laboratory food E.coli OP50.The pathogenesis of the disease is similar to that in mammals.In view of the domestic and foreign about Caenorhabditis elegans infection of the Klebsiella pneumoniae has rarely been reported,extensively drug resistant Klebsiella pneumoniae isolated from clinical patients was used to infect Caenorhabditis elegans in the liquid killing assay and the model may build the basic of analyzing pathogenic mechanis and screening anti-infective compounds.2 Methods2.1 Maintenance of Caenorhabditis elegansNematodes were maintenance according to international standard.2.2 Prepare a synchronized worm populationWash off gravid hermaphrodites with many fertilized embryos in the gonad from up to four 10 cm E.coli OP50 NGM plates with M9 buffer into a 15 mL conical tube.Spin the worms in a clinical centrifuge at 3500r/min for 30 seconds to pellet; aspirate the supernatant until approximately 3.5 mL worm solution remain. Add 1.5 mL of worm bleaching solution. Shake vigorously.8minutes.Quickly wash the embryos 3 times by adding 14 mL of M9 buffer, shaking to resuspend the embryos, and centrifuging to pellet the embryos. After three washes, resuspend the embryos in 4 mL of M9 buffer in a 15 mL conical tube.Rotate 15 mL tubes on a rotator at room temperature overnight. Plating L1-stage worms onto lawns of E.colio nnematode growth medium(NGM)agar media.Worms were grown to sterile,young adults by incubation at 25℃ for 48-52 h.Synchronized L4 stage worms were collected by using M9 solution and were washed several times and used for different assays.2.3 Preparation of Klebsiella pneumoniaStreak Klebsiella pneumonia from a frozen stock onto an LB broth. Grow at 35℃ for 12-18 hours.Separation of a single colony by using the Streak plate method,Grow at 35℃ for 18-20 hours. Inoculate 3-5 mL of LB broth with a single colony of Klebsiella pneumonia. Grow with agitation at 35℃ for 12-16 hours. Determine the density of Klebsiella pneumonia by using Turbidimetric assay.2.4 Nematode-killing assay in liquid mediumBriefly, the age-synchronized nematodes were washed off the plates with M9 buffer, resuspended,and washed in M9 buffer.Approximately 20-30 worms in a volumes of 160 ul were transferred to 96-well plates that contained 40 ul LB broth containing KP(1.5×108 CFU/ml,1.5×107 CFU/ml, I.5×106 CFU/ml),109 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.2.5 Quantification of colonization by CFU assay and Strain identification in worms intestine2.5.1 Quantification of colonization by CFU assayTo quantify the bacterial load by KP, inside the intestine of N2, a bacterial accumulation assay was performed as described earlier.Briefly, a batch of 10 nematodes was exposed to KP at different times and removed the surface bacteria by thoroughly washing with M9. After specified time intervals, the nematodes were washed from the plate thrice with M9 buffer containing 1mM sodium azide to avoid the expulsion of invaded bacteria from the worms’intestine. To the washed nematodes,400 mg of silicon carbide particles (1.0 mm) were added and vortexed at maximum speed for 2-3 minute, 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.2.5.2 Strain identification in worms intestineThe disturbing the worms and releasing the invaded bacteria was performed as described as 2.5.1.The resulted suspension was plated on a LB agar to identify the strain by PHOE-NIX-100 type fully automatic bacteria identification instrument produced by American BD company.2.6 Survival of worms exposed by different drug resistant strains of K. pneumoniaeWorms were exposed by different drug resistant strains of K. pneumoniae 1.5×108 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 K. pneumoniae,109 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.2.7 C.elegans rescuing assaysApproximately 20-30 infected worms in a volumes of 160ul were transferred to 96-well plates that contained 40ul of antibiotics(0、2、8、32 ug/ml meropenem 0、 0.08、0.4、2和10 ug/ml tigecycline),20% of LB medium was used as vehicle control. Each assay was carried out in triplicate.2.8 Quantification of colonization by CFU assay in rescuing assaysBased on the observation of the survival quantity and the state of nematodes,the qantification of colonization by CFU assay in tigecycline treatment was performed as described as 2.5.1 every 48 h.2.9 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 The status of Infection nematodes by XDRKPIn the liquid assay,live worms maintain a sinusoidal posture and can be seen moving their body and pumping their pharyngeal muscles.In contrast,dead worms becomes traight and rigidas they become bloated and do not pump their pharyngeal muscles.3.2 Determination of lethal concentration of nematode infectionThe Caenorhabditis elegans exposed by XDRKP moved more slowly. Different concentrations of XDRKP led to different patterns on the worm death. It showed that the survival rates of XDRKP of 1.5×108 CFU/ml、1.5×107 CFU/ml、1.5×106 CFU/ml and E.coli OP50 control of 109 CFU/ml were 0%、25.31%、83.95%、100% respectively. Kaplane Meier survival curve of Caenorhabditis elegans infected with 1.5×106 CFU/ml of XDRKP and E.coli OP50 were not statistically significant (χ2=0.08,P>0.05).Kaplane Meier survival curve of Caenorhabditis elegans infected with 1.5×108 CFU/ml of XDRKP and E.coli OP50 were statistically significant (χ2=275.98,P<0.001); Kaplane Meier survival curve of C.elegans infected with 1.5×107 CFU/ml of XDRKP and E.coli OP50 were statistically significant (χ2=229.37,P<0.001). It indicated that the survival rates of XDRKP of 1.5×108 CFU/ml and 1.5×107 CFU/ml were higher than E.coli OP50 control.3.3 Quantification of colonization by CFU assay and Strain identification in killing assayTo correlate colonization and proliferation of KP with host killing,the number of bacteria within the Caenorhabditis elegans digestive tract was measured.The variance analysis showed that the difference among different time points was statistically significant (F=1363.389,P<0.001).The SNK test showed that there were very obvious difference between the groups(P<0.05).The resulted suspension was plated on a LB agar to identify the strain was XDRKP.3.4 Survival of worms exposed by different drug resistant strains of K. pneumoniaeThe worms were exposed by three strains of K.Pneumoniae(Strain(A):susceptible; Strain(B):multidrug resistant; Strain(C):extensively drug resistant) and the nematode survival was investigated.It showed that Kaplane Meier survival curve of group A with group B(χ2=56.08,P<0.001),group A with group C(χ2=107.69,P<0.001) and group B with group C(χ2=14.60,P<0.001)were statistically significant.It indicated that the drug resistance of KP is proportional to its pathogenicity.3.5 Antibiotic Treatment Rescues Infected Nematodes3.5.1 Strain MDRKP infected C. elegans by meropenem and tigecycline treatmentWe used the infection protocol described in the previous paragraph to determine whether worms persistently infected with MDRKP could be cured by meropenem(0、 2,8,32ug/ml) treatment and tigecycline(0、0.08、0.4、2、10ug/ml).Kaplane Meier survival curve of Caenorhabditis elegans incured by 2ug/ml meropenem and control group were not statistically significant (χ2=3.028, P=0.08). Kaplane Meier survival curve of Caenorhabditis elegans incured by 8ug/ml meropenem and control group were statistically significant (χ2=21.821, P<0.001).Kaplane Meier survival curve of Caenorhabditis elegans incured by 32ug/ml meropenem and control group were statistically significant(χ2=146.096, P<0.001).It indicated that the survival rates of 8 ug/ml meropenem and 32 ug/ml meropenem were higher than control group.Kaplane Meier survival curve of Caenorhabditis elegans incured by 0.08 ug/ml tigecycline and control group were not statistically significant (χ2=0.116, P=0.73). Kaplane Meier survival curve of Caenorhabditis elegans incured by 0.4 ug/ml tigecycline and control group were statistically significant (χ2=35,462, P<0.001). Kaplane Meier survival curve of Caenorhabditis elegans incured by 2 ug/ml tigecycline and control group were statistically significant (χ2=106.159, P<0.001). Kaplane Meier survival curve of Caenorhabditis elegans incured by 10 ug/ml tigecycline and control group were statistically significant (χ2=264.426, P<0.001).It indicated that the survival rates of 0.4ug/ml,2ug/ml,10ug/ml tigecycline were higher than control group.3.5.2 Strain XDRKP infected C. elegans by meropenem and tigecycline treatmentWe used the infection protocol described in the previous paragraph to determine whether worms persistently infected with XDRKP could be cured by meropenem(0、 2、8、32ug/ml) treatment and tigecycline(0、0.08、0.4、2、10ug/ml).Kaplane Meier survival curve of Caenorhabditis elegans incured by 2ug/ml meropenem and control group were not statistically significant(χ2=0.749, P=0.39). Kaplane Meier survival curve of Caenorhabditis elegans incured by 8ug/ml meropenem and control group were not statistically significant (χ2=1.841, P=0.17).Kaplane Meier survival curve of Caenorhabditis elegans incured by 32ug/ml meropenem and control group were not statistically significant(χ2=0.150, P=0.69).It indicated that 2 ug/ml,8 ug/ml meropenem and 32 ug/ml meropenem did not promote survival rate of infected worm.Kaplane Meier survival curve of Caenorhabditis elegans incured by 0.08 ug/ml tigecycline and control group were not statistically significant (χ2=0.316, P=0.57). Kaplane Meier survival curve of Caenorhabditis elegans incured by 0.4 ug/ml tigecycline and control group were statistically significant (χ2=28.124, P<0.001). Kaplane Meier survival curve of Caenorhabditis elegans incured by 2 ug/ml tigecycline and control group were statistically significant (χ2=94.767, P<0.001). Kaplane Meier survival curve of Caenorhabditis elegans incured by 10 ug/ml tigecycline and control group were statistically significant (χ2=267.963, P<0.001).It indicated that the survival rates of 0.4ug/ml,2ug/ml,10ug/ml tigecycline were higher than control group.3.6 Quantification of colonization by CFU assay in rescuing assaysBased on the observation of the survival quantity and the state of nematodes,the qantification of colonization by CFU assay in tigecycline treatment was performed.The variance analysis showed that the difference among different time points was statistically significant (F=1267.23, P<0.001).The SNK test showed that there were very obvious difference between the groups(P<0.05). Tigecycline could significantly increased the effect of anti-infection.4 Conclusion4.1 The strain of XDRKP isolated from clinical patient was used to infect Caenorhabditis elegans in the liquid killing assay. We had estabilished the infection model of Caenorhabditis elegans-XDRKP sucsessfully.4.2 The worms were exposed by different drug resistance strains of K.Pneumoniae. It indicated that the drug resistance of KP is proportional to its pathogenicity.4.3 MDRKP and XDRKP from clinical patients were used to infect Caenorhabditis elegans and treated by meropenem and tigecycline at different concentrations to investigate the survival rates of infected worms. We found that the drug resistance in Caenorhabditis elegans model were consistent in its in vitro Susceptibility to Antibiotics.