title: Clostridium innocuum is a significant vancomycin-resistant pathogen for extraintestinal clostridial infection.
abstract: OBJECTIVE
  Extra-intestinal clostridial infection (EICI) is rare but can be fatal. Traditional phenotypic methods can only assign many of the Clostridium species to the genus level.
  
  
  METHODS
  A total of 376 non-repetitive Clostridium isolates from sterile sites were collected and subjected to matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) Biotyper analysis and 16S rRNA sequencing. Antimicrobial susceptibility was determined, and clinical characteristics of the patients were assessed. Clostridium innocuum isolates were characterized by genome sequencing and genotyping. We used molecular and cellular methods to explore the virulence and resistance mechanisms of C.innocuum.
  
  
  RESULTS
  Clostridium innocuum was the second most common species to cause EICI, only next to Clostridium perfringens. All Clostridium isolates showed susceptibility to clindamycin, metronidazole, penicillin, piperacillin and ampicillin-sulbatam, while C. innocuum isolates were invariably resistant to vancomycin. Among 24 patients with EICI caused by C. innocuum, two (8.3%) had diarrhoea, three (12.5%) had soft-tissue infection, six (25%) had appendicitis and four (16.7%) each had shock and gastrointestinal perforation. The 30-day mortality was 16.7%. The C. innocuum isolated from different sites could not be separated from one another by genotyping. No known toxin genes were identified in the genome of C. innocuum but the species expressed cytotoxicity to epithelial cells. d-Alanine-d-alanine ligase, alanine racemase and d-alanyl-d-alanine carboxypeptidase are three main genes responsible for vancomycin resistance in C. innocuum.
  
  
  CONCLUSIONS
  Vancomycin-resistant C. innocuum is a previously unrecognized, yet prominent, cause for EICI. Genome analysis showed that the species could carry a lipopolysaccharide-like structure that is associated with cytotoxicity to cells in vitro.
authors.affiliation: Department of Laboratory Medicine, Chang Gung Memorial Hospital, Taoyuan, Taiwan; Department of Medical Biotechnology and Laboratory Science, Chang Gung University, Taoyuan, Taiwan.
authors.forename: J-H
authors.initials: JH
authors.lastname: Chia
authors.affiliation: Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China; Institute of Translational Medicine, Zhejiang University School of Medicine, Hangzhou, China.
authors.forename: Y
authors.initials: Y
authors.lastname: Feng
authors.affiliation: Department of Laboratory Medicine, Chang Gung Memorial Hospital, Taoyuan, Taiwan; Department of Medical Biotechnology and Laboratory Science, Chang Gung University, Taoyuan, Taiwan; Molecular Infectious Disease Research Centre, Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Taoyuan, Taiwan.
authors.forename: L-H
authors.initials: LH
authors.lastname: Su
authors.affiliation: Department of Laboratory Medicine, Chang Gung Memorial Hospital, Taoyuan, Taiwan; Department of Medical Biotechnology and Laboratory Science, Chang Gung University, Taoyuan, Taiwan.
authors.forename: T-L
authors.initials: TL
authors.lastname: Wu
authors.affiliation: Molecular Infectious Disease Research Centre, Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Taoyuan, Taiwan.
authors.forename: C-L
authors.initials: CL
authors.lastname: Chen
authors.affiliation: Molecular Infectious Disease Research Centre, Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Taoyuan, Taiwan.
authors.forename: Y-H
authors.initials: YH
authors.lastname: Liang
authors.affiliation: Molecular Infectious Disease Research Centre, Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Taoyuan, Taiwan; Division of Paediatric Infectious Diseases, Department of Paediatrics, Chang Gung Children's Hospital, Chang Gung University College of Medicine, Taoyuan, Taiwan. Electronic address: chchiu@adm.cgmh.org.tw.
authors.forename: C-H
authors.initials: CH
authors.lastname: Chiu
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D002648:Child; D003013:Clostridium / Q000737:chemistry / Q000145:classification / Q000187:drug effects* / Q000302:isolation & purification*; D003015:Clostridium Infections / Q000382:microbiology* / Q000473:pathology*; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004275:DNA, Ribosomal / Q000737:chemistry / Q000235:genetics; D005260:Female; D005838:Genotype; D060005:Genotyping Techniques; D006801:Humans; D008297:Male; D008826:Microbial Sensitivity Tests; D008875:Middle Aged; D010641:Phenotype; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D017422:Sequence Analysis, DNA; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D020713:Vancomycin Resistance; D000073336:Whole Genome Sequencing; D055815:Young Adult
chemical_list: D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D012336:RNA, Ribosomal, 16S
keywords: 16S rRNA sequencing; Clostridium innocuum; Extra-intestinal clostridial infection; MALDI-TOF; Vancomycin resistance