Tn916-Tn1545-like elements in Clostridiumdifficile clinical isolates
Abstract number: 1134_03_271
Spigaglia P., Carucci V., Barbanti F., Mastrantonio P.
In C. difficile, tetracycline resistance is predominantly due to a tet(M) gene. This gene has been shown to be carried by Tn5397 in the clinical strain C. difficile 630, whereas by a Tn916-like element in the environmental strain C. difficile 42373. These two elements are related, but very different in their integration/excision module and can not be co-present in the same cell. The aim of the study was to examine Clostridiumdifficile clinical isolates for the presence of the Tn916 and Tn1545 like elements.
Detection of tet(M), erythromycin resistant gene erm(B) and the integrase gene int (markers for the Tn916) was performed by PCR, as well as the analysis of the genetic arrangement of the elements. The amplified fragments were used as probes for hybridisation assays on genomic DNA of C. difficile isolates, after digestion with HindIII. The nucleotide sequence of tet(M) genes was also analysed. Antibiotic susceptibility of the strains was assessed by the E-test method.
Eighteen C. difficile isolates were positive for tet(M) and int and the signals obtained using these genes as probes overlapped. Ten isolates showed one hybridising band of either 6.3, 9.0 or 14 kb. Seven isolates showed two hybridising bands of 6.3 and 9.0 kb and one strain two banda of 6.0 and 9.0 kb, indicating the presence of two copies of a Tn916-Tn1545 like elements. Heterogeneity in these elements was observed. The E-test results indicated that isolates with two copies of tetracycline resistance elements were resistant (88%) or inducibly resistant to tetracycline (12%), whereas isolates with one copy were resistant (36%), inducibly resistant (18%) or susceptible (46%) to this antibiotic. Three isolates were also erm(B)-positive and resistant to erythromycin. The bands obtained using tet(M) and erm(B) probes overlapped at 9.0 kb, indicating the presence of a Tn1545-like element. The association of these genes was also confirmed by PCR. Seven different alleles were identified sequencing the tet(M) gene of prototype strains among those examined.
Heterogenic elements of the Tn1545Tn916 family are harboured by C. difficile clinical isolates showing different phenotypes for tetracycline. These elements carry different tet(M) alleles and can be found in one or two copies in C. difficile chromosome. Tn1545-like elements, have been detected for the first time in this pathogenic bacterium.
|Session name:||XXIst ISTH Congress|
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