ABSTRACT
UNLABELLED: Molecular fingerprinting methods are currently used to study microbial communities by culture independent approaches. They are proposed as identification tool owing to the availability of rapid automated methods. The 16S rRNA gene (16S rDNA) is an efficient marker for bacterial identification and microbial communities analysis. However, the 16S rDNA polymorphism among strains of the same species is an underestimated pitfall of the fingerprinting approaches. AIM OF THE STUDY: We studied the 16S rDNA variability among strains of three bacterial species of medical interest. MATERIAL AND METHODS: Total DNA was extracted from clinical isolates of Pseudomonas aeruginosa (N=20), Clostridium difficile (N=20) and Enterobacter cloacae (N=14). The Polymerase Chain Reaction (PCR) products obtained with consensus primers flanking the 16S rDNA variable regions V3 and V6-V7-V8 were separated by Temporal Temperature Gradient gel Electrophoresis (TTGE). DNA extracted from TTGE bands were sequenced and analysed. RESULTS: All the isolates of P. aeruginosa and of C. difficile displayed one single TTGE band with constant migration distances suggesting that there was no 16S rDNA polymorphism among strains in these two species. Oppositely, the isolates of E. cloacae gave complex TTGE patterns formed by multiple bands with variable migration distances. These patterns corresponded to 16S rRNA genes variable in a single genome as well as among strains of the species. CONCLUSION: Intra-species and/or intragenomic variability of 16S rDNA should be taken into account for pertinent interpretation of molecular fingerprint. For this purpose, a comprehensive description of the polymorphism of this marker is necessary.
Subject(s)
Clostridioides difficile/genetics , DNA Fingerprinting/methods , DNA, Bacterial/genetics , DNA, Ribosomal/genetics , Enterobacter cloacae/genetics , Genome, Bacterial , Pseudomonas aeruginosa/genetics , RNA, Ribosomal, 16S/genetics , Clostridioides difficile/isolation & purification , DNA, Bacterial/isolation & purification , DNA, Ribosomal/isolation & purification , Enterobacter cloacae/isolation & purification , Humans , Nucleic Acid Denaturation , Pseudomonas aeruginosa/isolation & purification , RNA, Bacterial/genetics , RNA, Bacterial/isolation & purification , RNA, Ribosomal, 16S/isolation & purification , Reproducibility of Results , Species SpecificityABSTRACT
Two species of medical interest belong to the genus Ochrobactrum, Ochrobactrum anthropi and Ochrobactrum intermedium. They are members of the microbiota of soil and an increasing number of works report the isolation of O. anthropi from clinical specimen, especially from immunocompromised patients and nosocomial infection. Involving of each species in human infection is poorly estimated due to unclear differential phenotypic characters. We performed 16S rDNA sequencing for identification of 20 clinical isolates of Ochrobactrum sp. to the species level. Then, we studied the phenotype of each isolate especially, morphology, culture onto different media and at different temperatures, biochemical characters and antibiotics resistance pattern. Colony morphology after growth onto Trypticase-Soy and McConkey agar, culture at 45 degrees C onto Trypticase-Soja agar, presence of urease, and netilmycin, tobramycin and colistin resistance allowed identification of species. Ribotyping using HindIII and EcoRI gave a supplementary criterion for species determination but did not allow typing at the infra-species level. In contrast, Pulsed-Field Gel Electrophoresis showed high degree of polymorphism between strains and proved the clonality of certain isolates. Thus, this method could be a useful tool for molecular epidemiology of Ochrobactrum infections.
