PCR ribotype 078 has recently emerged as a hypervirulent C. difficile
]. Previously published MLVA studies have shown that all PCR ribotype 078 strains are closely related [3
], irrespective of human or porcine origin [16
], fostering the notion that PCR ribotype 078 infection could be a zoonosis. Recently, the full genome sequence of a C. difficile
PCR ribotype 078 strain was published [5
]. This M120 strain was shown to contain a unique insert of approximately 100 kilobases. In this paper we show that this insert is a transposable element, Tn6164
. It is not representative for all PCR ribotype 078 strains. On the contrary, we found that the majority of the PCR ribotype 078 strains do not contain the element. Moreover, some strains contain only half of the element. So, three different kinds of PCR ribotype 078 can now be distinguished: Those with a full length element, those with half the element, and those with no element at all. Tn6164
was exclusively found in tetracycline resistant PCR ribotype 078 strains, isolated from humans. We tested a collection of other PCR ribotypes, of which none contained the element. Since we only tested 1 strain per PCR ribotype, we cannot rule out the possibility that Tn6164
is present in other PCR ribotypes. We covered the whole genomic spectrum of C. difficile
since we tested multiple samples of each genetic clade previously identified [10
]. In addition, Tn6164
has not been found in any other C. difficile
genome that has been published so far than M120.
contained a tet
(44) gene, we could not demonstrate increased tetracycline resistance of strains containing the element. Previously, it has been shown that this gene, present on a homologues resistance island, is active in C. fetus
]. In C. difficile
, the copresence of the tet
(44) gene on Tn6164
and the tet
(M) gene on the Tn6190
in one bacterium does not result in an increased resistant phenotype. Also the spectinomycin and streptomycin resistance genes did not result in a phenotype, despite the presence of two potential aminoglycoside resistance genes (ant(9)Ia)
) on Tn6164
(see Figure and Table ). We do not know if the resistance genes are expressed in M120. However, since we show the presence of the circular intermediate transposon DNA, some activity of transposon related genes is expected.
Since we have only found Tn6164
in strains also containing Tn6190
, it is possible that Tn6164
transfer is dependent on Tn6190
. Further research is needed to investigate the possibility of Tn6190
-dependent transfer of Tn6164
. In addition, remarkably, Tn6164
(the whole or half the element) was significantly (p = 0.01) more found in strains isolated from humans than in strains isolated from pigs. Although the same strains circulate in humans and pigs [16
], and also Tn6190
circulates in pig strains [16
], we did not find any porcine strain that contained the element. We have no explanation for this difference.
None of the transconjugants tested showed the presence of Tn6164
, but all contained Tn6190
. These results indicate that Tn6164
has a (much) lower transfer frequency than Tn6190.
Nevertheless, a complete set of proteins, required for transfer, is present on Tn6164
. Loss of Tn6190
or introduction of another selection marker in Tn6164
] could prove to be a strategy to further study the capability of conjugative transfer of this element.
has integrated intergenically between homologs of the 630 ORFs CD0406 and CD0437, a tRNA methyltransferase and a hypothetical protein respectively. In strain 630, this target site is occupied by the conjugative transposon CTn2
]. There is no significant homology between Tn6164
. The empty target site is present in many sequenced strains of C. difficile.
However, no other mobile genetic elements have been reported to integrate at this site.
It was impossible to phenotypically distinguish strains containing Tn6164 from strains without the element. Although we have no transcriptional data available of the genes that are located on Tn6164 it is clear that it could provide an advantage under certain circumstances. In this respect it is interesting to note that the patients suffering from an element-containing strain are suggested to undergo a more severe illness than patients with a strain not containing Tn6164. However, because of the low number of strains containing the insert no multivariate analysis could be carried out. Therefore, we cannot rule out that these data are biased. Further research is needed to confirm this observation.
Isolates containing the full element originated from all over Europe, including Ireland, England, Norway, Germany, Bulgaria, Greece and the Netherlands, whereas isolates containing half the element were only found in the United Kingdom, Spain and the Netherlands. MLVA showed genetic relatedness between most of the strains, although no epidemiologic link between the strains from different countries could be found. It has recently been shown that PCR ribotype 078 strains show a lot less heterogeneity in MLVA than for instance PCR ribotype 027 or PCR ribotype 017 [36
]. This could indicate a higher level of relatedness, or it could mean that the mechanism behind the MLVA variability is different in PCR ribotype 078 strains than in other PCR ribotypes [16
Altogether, we show the presence of a 100 kb transposon in some C. difficile
PCR ribotype 078 strains. Although we could not show any evolutionary benefits of the transposon, it could very well serve as a reservoir of antibiotic resistance [26
], for commensal bacteria in the human gut.