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Objective To analyze the characteristics of toxin, the PCR-ribotyping(RT) and the multilocus sequence typing(MLST) of Clostridium difficile strains isolated from China-Japan Friendship Hospital in order to provide a basis for monitoring the outbreak of nosocomial Clostridium difficile infection.Methods A total of 321 samples were collected from the patients with suspected Clostridium difficile infection(CDI) in China-Japan Friendship Hospital(CJFH) during 2012 to 2013.All Clostridium difficile strains were isolated and identified by the standard phenotypic culture method.Cytotoxicity test was performed to detect toxin B.Toxin genes (tcdA and tcdB) and binary toxin genes (cdtA and cdtB) harbored by those strains were analyzed.RT and MLST were used for homologous analysis.Clinical data of the patients were collected to analyze the isolation rate of Clostridium difficile in different populations.Results Forty-eight strains of Clostridium difficile were isolated from 46 patients with diarrhea and three of them were isolated from the same patient.The incidence of CDI among all patients, outpatients and inpatients were 14.3%(46/321), 12.8%(5/39) and 14.5%(41/282), respectively.Toxin B was detected in all of the strains as indicated by the cytotoxicity test.Strains of sequence type 1(ST1) showed the strongest cytotoxicity of all the isolated Clostridium difficile strains.Ten out of the 48 strains (20.8%) were tcdA(-)/tcdB(+) strains, which belonged to either ST37 or ST81.The results of RT and MLST were consistent in assigning the strains into nine types, in which the predominant type was ST1/RT027 accounting for 27.1% (13/48).All of the ST1/RT027 strains presented a toxin gene profile of tcdA(+)/tcdB(+) and cdtA(+)/cdtB(+).Most of the ST1/RT027 strains were isolated from the Traditional Chinese Medicine Department of Respiratory, where smallnosocomial outbreaks of ST1/RT027 strain infection might happen.Conclusion CDI diagnosed in CJFH mainly belongs to nosocomial infection.Most of the isolated strains harbor tcdA(+)/tcdB(+) genes.Surveillance for the outbreaks of CDI caused by ST1/RT027 strains over producing toxins A and B should be strengthened in hospitals.
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Objective To investigate the correlation between serum procalcitonin (PCT) levels and infection sites,as well as between PCT and bacterial species in gram negative (G-) bacteria induced sepsis,so as to provide rationale for therapeutic strategy of using antibiotic in sepsis.Methods The data of patients with sepsis admitted in Emergency Department and ICU from January 2014 to June 2015 were retrospectively analyzed.The blood culture of G-bacteria and PCT detection were carried out simultaneously within 24 hours after admission.The clinical data was analyzed to find out the correlation between PCT levels and infection sites,as well as between PCT levels and pathogenic bacterial species.Results A total of 187 specimens (came from 162 patients) were enrolled in the study with a median age of 70 years old and a median sequential organ failure assessment (SOFA) score of 4.PCT levels were found to be associated with bacterial species.PCT level caused by Escherichia coli bacteremia infection was higher than that caused by Acinetobacter baumannii bacteremia and Burkholderia cepacia bacteremia infection (4.62 ng/mL vs.2.44 ng/mL;4.62 ng/mL vs.0.81 ng/mL;P ＜ 0.05).Receiver operating characteristic (ROC) analysis showed an area under the curve (AUC) for PCT was 0.61 to discriminate Escherichia coli infection from Acinetobacter baumannii infection and an AUC was 0.66 to discriminate Escherichia coli infection from Burkholderia cepacia infection.When the cutoff point of PCT was 30.32 ng/mL,it could predict Escherichia coli infection rather than Acinetobacter baumannii infection with 94.10％ specificity,90.00％ positive predictive value and positive likelihood ratio for 4.24.When the cutoff point of PCT was 8.01 ng/mL,it could predict Escherichia coli infection rather than Burkholderia cepacia infection with 85.70％ specificity,93.94％ positive predictive value,and positive likelihood ratio for 3.01.When PCT cutoff value reached 47.31 ng/mL,the specificity and positive predictive value were both 100.00％.PCT level caused by urinary tract infection was higher than that caused by pulmonary infection (11.58 ng/mL vs.2.07 ng/mL,P ＜ 0.05),and the AUC was 0.69.When the cutoff point of PCT was 32.11 ng/mL,it could predict Escherichia coli infection rather than Acinetobacter baumannii infection with 90.60％ specificity,86.18％ negative predictive value and positive likelihood ratio for 3.68.Conclusions PCT elevation in G-bacteria induced sepsis might be associated with infection sites and bacterial species.
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Objective To design a multiplex PCR for simultaneous identification and toxigenic type characterization of Clostridium difficile isolates. MethodsThree pairs of primers were designed for the amplification of a species-specific internal fragment of the tpi( triose phosphate isomerase) gene, an internal fragment of the tcdB ( toxin B) gene, and an internal fragment of the tcdA ( toxin A) gene. Twenty-one standard strains including Clostridium difficile ATCC 9689 and 47 isolates of Clostridium difficile were applied for the assessment of detection limit, specificity and detections of the multiplex PCR, respectively. Toxin A and Toxin B of 47 isolates were analyzed by ELISA. ResultsThe detection limit for DNA concentration of the multiplex PCR was 0.5 pg/μl. The specificity was determined to be 100％. Among the results of 47 isolates detected by multiplex PC R, 37 strains were tpi ( + )/tcdA (+)/tcdB ( + ), 10 strains were tpi ( + )/tcdA (-)/tcdB ( - ). Tpi ( + )/tcdA ( - )/tcdB ( + ) was not found. The toxin detection of 47 isolates by ELISA showed that 20 isolates were positive and 27 isolates were negative. Twenty isolates of toxin (+) by ELISA were all tpi( +)/tcdA( +)/tcdB(+) by multiplex PCR. ConclusionThe multiplex PCR method combined diagnosis and toxigenic type characterization contributes to the diagnosis for Clostridium difficile infection.
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To investigate the gene typing, molecular characteristics of virulence and resistance associated gene of Clostridium difficile from clinical isolates in China, the genes tcdA,tcdB of toxin A and B, cdtA,cdt B of binary-toxin, and erm B of clindamycin resistance were detected by conventional PCR. Genotyping of toxic C. difficile were conducted by means of analysis of 16s-23s internal spacer region polymorphism with PCR assay. Then the antibiotic resistance of toxic C. difficile to ampiciline, clindamycin, metronidazole and vancomycin was conducted with E-test. It was found that 8 toxic C. difficile strains were demonstrated out of 12 clinical isolates, in which 5 strains were tcdA+ and tcdB+, and 3 strains tcdA- and tcdB+, accounting for 62.5% and 37.5% respectively. Binary-toxin genes detection were negative in all the strains. Clindamycin resistance associated gene ermB was positive in 4 out of 8 toxic C. difficile strains, accounting for 50%. 8 toxic isolates were typed into 4 gene types, the dominant type was ZR I,accounting for 62.5%. Resistance rate of 8 toxic C. difficile strains against ampiciline(AC), clindamycin(CM), metronidazole(MZ) and vancomycin(VA) was 37.5%,87.5%,12.5%, and 0 respectively. No isolates belonged to ribotype 027 or 078. Isolation rate of toxic C. difficile is high to 66.7%. There is obvious gene polymorphism in clinical isolates of Chinese toxic C.difficite, and ZR I is preponderant genotype in 4 genotypes. C. difficile shows some resistance to ampiciline, clindamycin, metronidazole, but susceptive to vancomycin.