Molecular Epidemiology And Drug Resistance Analysis Of Clostridium Difficile Carried By The Healthy Population

Objective: Clostridium difficile is a strictly anaerobic, Gram-positive, spore-forming rod, mainly spreading of the fecal-oral and is the common pathogens for Clostridium difficile associated diarrhea(CDAD). Asymptomatic colonization is rare(prevalence, 1%-7%) in healthy adults, yet Clostridium difficile colonization can be frequent in infants(prevalence, 2%-75%) and usually in an asymptomatic fashion. Clostridium difficile can be changed into a resilient spore form when exposed to heat, drought, alcohol, or an aerobic environment. When excreted with feces, spores can persist in the environment for months, and germinate again when the environment turns favorable. Spores of Clostridium difficile can play a role in the transmission of the disease. Generally, Clostridium difficile infection is associated with the use of antibiotic and hospitalization. However, it is investigated that the prevalence and incidence of Clostridium difficile infection has been changing especially community-acquired Clostridium difficile infection increasing. Related researches have been carried on healthy population of Clostridium difficile in foreign countries, yet little has been reported in China. To understand the situation on healthy population colonization prevalence and to characterize these strains, in terms of their virulence factors and their phylogenetic diversity, a screening of Clostridium difficile in the stools of healthy population in Hebei Province has been conducted. It can provide the basis for the transmission of Clostridium difficile.Methods:1 The sources of the human samples examined for intestinal carriage of Clostridium difficile were:(i) stool specimens of 36 infants;(ii)stool specimens of 1661 children in three kindergartens;(iii)stool specimens of 1654 community-dwelling healthy adults;(iv)stool specimens of 348 healthcare workers during September 2013-September 2014. None of the subjects had had diarrhea.2 Stool specimens were screened for Clostridium difficile by Enrichment Spore Culture Method. Identification of colonies was performed on the basis of morphology, odor, and biochemical identification.3 DNA was extracted by a genomic DNA extraction kit according to the manufacturer’s instructions, in addition, tcd A, tcd B, cdt A, cdt B, 16 Sr DNA detected by a 5-plex polymerase chain reaction.4 Specific oligonucleotide primers complementary to the 3’end of the 16 S r RNA gene(5’-GTGCGGCTGGATCACCTCCT-3’) and the 5’ end of the 23 S r RNA gene(5’-CCCTGCACCCTTAATAACTTGACC-3’) were used to amplify the variable-length intergenic spacer region. The bands were ananlysed with Quantity One software. A total of 226 toxigenic isolates of Clostridium diffile from healthy population in Hebei Province were obtained to investigate epidemic relationships via multilocus sequence typing. We compared the genotypes of Clostridium diffile isolates from healthy population to those of isolates causing disease in adults.5 The minimum inhibitory concentrations(MICs) of 8 antimicrobial agents, including vancomycin, metronidazole, fidaxomicin, rifaximin, levoflo- xacin, ciprofloxacin, meropenem, and chloramphenicol were determined using the agar dilution method recommended by the Clinical and Laboratory Standards Institute(CLSI).Results:1 Of the thirty-six enrolled infants, 9(25%) carried Clostridium difficile, with 7(19.4%) carrying a toxigenic isolate. Factors that may influence Clostridium difficile colonization were analyzed for these 36 infants. Food diversification was the only factor that was significantly associated with the Clostridium difficile carrier state.A total of 1661 children(sex male/female 1.34) in three kindergartens were tested for faecal Clostridium difficile presence. Clostridium difficile was isolated in the stools of 222 children(13.4%). One hundred and forty-six of the Clostridium difficile carriers were colonized with a toxigenic strain. The carriage rates in children in three kindergartens were 13.6%(127/931), 13.1%(79/600), 12.3%(16/130) repectively. When analyzed according to age group, the carriage rates were 25%, 0, 13.9%, 18.0%, 11.9%, 12.4% and 6.3% in infants and children aged 0, 1, 2, 3, 4, 5 and 6 years old, respectively. The prevalence of intestinal carriage among 1654 community-dwelling healthy adults was 5.5%(91/1654), 60 of which were toxin positive. A prevalence of asymptomatic Clostridium difficile carriage was of 22 in 348 healthcare workers, 13 of which were toxin positive.2 Twenty-four PCR ribotypies were identified, 12 types of adult patients with CDI in addition to one type(1 strains), the rest of types were founded in healthy population. A total of 27 sequence types(STs) were generated, of which ST-286, 289 and 290 were novel. Our study highlighted that STs-54, 3, 35, 2 were the dominant STs. In addition, no correlation was found between the genotype and host population origin. ST-2, 3, 35, 37, 53, 54, 55 were detected from both healthy population and adult patients with CDI.3 All isolates were sensitive to the vancomycin, metronidazole, fidaxomicin and rifaximinin. However, high-level resistance to levofloxacin and ciprofloxacin was found, the resistance rates were 39.8% and 98.3%, respectively. 1.45% of the Clostridium difficile isolates were resistant to meropenem, 2% to chloramphenicol.Conclusions:1 The carrying rates of infants and children in this study are very low compared with the foreign reports. Food diversification is significantly associated with the Clostridium difficile carrier state of infants. However, the carrying rates of community-dwelling healthy adults and healthcare workers are similar to foreign reports.2 Currently our study highlight that ST54 and ST3 are the dominant STs in healthy individuals of Hebei Province.ST54 and ST3 are the dominant STs in healthy infants and ST54 is the dominant ST in healthy adults.3 Some strains carried by healthy population have homology with the strains caused by CDI. These results indicate the asymptomatic colonization population may constitute a potential reservoir of Clostridium difficile and spores.The healthy population may be the main source of community-acquired CDI.4 All isolates are sensitive to the vancomycin, metronidazole, fidaxomicin and rifaximinin, with a narrow range of MICs. The strains have a low level resistance to meropenem and chloramphenicol while have a high level resistance to levofloxacin and ciprofloxacin.