RESEARCH ARTICLE Open Access Analysis of a Clostridium difficile PCR ribotype 078 100 kilobase island reveals the presence of a novel transposon, Tn 6164 Jeroen Corver 1,4* , Dennis Bakker 1 , Michael S M Brouwer 2 , Céline Harmanus 1 , Marjolein P Hensgens 1 , Adam P Roberts 2 , Len J A Lipman 3 , Ed J Kuijper 1 and Hans C van Leeuwen 1 Abstract Background: Clostridium difficile is the main cause of antibiotic associated diarrhea. In the past decade, the number of C. difficile patients has increased dramatically, coinciding with the emergence of two PCR ribotypes 027 and 078. PCR ribotype 078 is also frequently found during C. difficile outbreaks in pigfarms. Previously, the genome of the PCR ribotype 078 strain M120, a human isolate, was described to contain a unique insert of 100 kilobases. Results: Analysis of this insert revealed over 90 open reading frames, encoding proteins originating from transposons, phages and plasmids. The insert was shown to be a transposon (Tn 6164 ), as evidenced by the presence of an excised and circularised molecule, containing the ligated 5 ’ and 3 ’ ends of the insert. Transfer of the element could not be shown through filter-mating experiments. Whole genome sequencing of PCR ribotype 078 strain 31618, isolated from a diarrheic piglet, showed that Tn 6164 was not present in this strain. To test the prevalence of Tn 6164 , a collection of 231 Clostridium difficile PCR ribotype 078 isolates from human (n = 173) and porcine (n = 58) origin was tested for the presence of this element by PCR. The transposon was present in 9 human, tetracycline resistant isolates, originating from various countries in Europe, and none of the pig strains. Nine other strains, also tetracycline resistant human isolates, contained half of the transposon, suggesting multiple insertion steps yielding the full Tn 6164 . Other PCR ribotypes (n = 66) were all negative for the presence of the transposon. Multi locus variable tandem repeat analysis revealed genetic relatedness among transposon containing isolates. Although the element contained several potential antibiotic resistance genes, it did not yield a readily distinguishable phenotype. Conclusions: Tn 6164 is a newly described transposon, occurring sporadically in C. difficile PCR ribotype 078 strains. Although no transfer of the element could be shown, we hypothesize that the element could serve as a reservoir of antibiotic resistance genes for other bacteria. Further research is needed to investigate the transfer capabilities of the element and to substantiate the possible role of Tn 6164 as a source of antibiotic resistance genes for other gut pathogens. Keywords: Clostridium difficile , Transposable element, Phage, Antimicrobial resistance, Virulence * Correspondence: [email protected] 1 Department of Medical Microbiology, Section Experimental Microbiology, Center of Infectious Diseases, Leiden University Medical Center, Leiden, The Netherlands 4 LUMC, Medical Microbiology, E4P, Postbus 9600, 2300 RC Leiden, The Netherlands Full list of author information is available at the end of the article © 2012 Corver et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Corver et al. BMC Microbiology 2012, 12 :130 http://www.biomedcentral.com/1471-2180/12/130 Background Over the past decade, Clostridium difficile has emerged as an important gut pathogen, causing hospital- and community-acquired diarrhea. The number of patients and the severity of disease have increased dramatically, due to the emergence of two hypervirulent PCR ribo- type, 027 [1] and 078 [2,3]. Traditionally, PCR ribotype 027 has been linked to nosocomial outbreaks. In con- trast, PCR ribotype 078 has been detected frequently in farming animals, especially pigs [2,4], and is found more during community acquired infection. The increase in C. difficile infections (CDI) of humans has boosted interest in C. difficile biology, diagnostics and pathogenesis. In the past few years, multiple genome sequences of several PCR ribotypes have been determined [5-8]. The analyses of the genomes, aided by comparative genomics of DNA-DNA microarrays [9,10] has shown that the genomes of C. difficile are highly variable with inserts of mobile DNA from phage, plasmid or transposon origin. These mobile DNA elements are actively moving within C. difficile genomes and are frequently passed on to neighboring bacteria, harboring mosaic genomes [7,11]. It is unclear what role the mobile elements play in the virulence of C. difficile. Some virulence linked genes, for example the holin-like tcdE , have a phage origin [12]. In fact, it has been suggested that the whole pathogenicity locus (PaLoc), encoding the major C. difficile virulence factors TcdA and TcdB, is of phage origin [13,14]. Recently, phages have been shown to upregulate toxin production in C. difficile , thereby increasing the viru- lence [15]. C. difficile transposons have been shown to contain antibiotic resistance genes [5,7,16,17], and there- fore acquiring such an element could increase the viru- lence and/or colonization potential of a particular strain. Mobile elements play an important role in the diversi- fication of bacterial genomes. One important group of mobile genetic elements is the Tn 916 family of conjugative transposons (also known as integrative and conjugative elements [ICEs]) [18]. These conjugative transposons usually code for tetracycline resistance and are found primarily in the Firmicutes . Numerous trans- posons have been described to be present in C. difficile genomes [5,7,11,17,19]. Several elements closely related to Tn 916 are present in diverse C. difficile strains, in- cluding Tn 5397 which confers tetracycline resistance [20,21]. Other transposons have been described to con- fer resistance to chloramphenicol and erythromycin [5]. Recently, the first full length genome of a PCR ribo- type 078 strain was published [5]. This M120 strain has been isolated from an Irish diarrheic patient. It was shown that PCR ribotype 078 is highly divergent from PCR ribotype 027, 001, 017 and 012. In addition, this PCR ribotype 078 strain was described to contain a unique 100 kb insert that showed 80% similarity to sequences of Thermoanaerobacter species and Strepto- coccus pneumoniae [5]. In this paper we show that the 100 kb insert is a mobile element that is only sporadic- ally present in PCR ribotype 078 strains. Furthermore, we show that the 100 kb consists of at least two inde- pendent mobile elements that were fused during evolution. Results Previously, an insert, unique for C. difficile ,was described in the genome of strain M120, a PCR ribotype (see Additional file 1). In addition, within the insert the different modules could be distinguished by their G + C contents. The G + C content of module A, B, C, D and E was 31%, 41%, 35%, 28% and 31%, respectively. The 100 kb insert is a transposon Based on the bioinformatic comparison of the insert described above, the possible excision of 3 (independent) elements was predicted. Primers were designed (primers 14 – 20, see Table 3) to amplify the circular intermediates of the complete insert (primers 14 and 15) , the putative Thermoanaerobacter sp. phage (module B, primers 15 and 16) and the C. fetus pathogenicity island (module D, primers 17 and 18) of the element. PCR confirmed only the excision and circularisation of the entire insert (results not shown). It is expected that the serine recom- binase at the 3 ’ end of the element is responsible for excision (see Table 1). Sequencing of the circular inter- mediate was used to determine the precise ends of the element, showing the element is flanked by a TG di- nucleotide; serine recombinases prefer a 2 bp crossover site identical in the target site and joint of the circular intermediate [27]. In silico extraction of this sequence from the genome confirms that the element is present in the homologous target site of CTn 2 in strain 630 [7]. The precise size of the element is 106,711 bp and it runs from bp 418,525-525,236 (including the TG dinucleotide at both ends) in the M120 genomic sequence (GenBank accession no. FN665653). Upon our request, the trans- poson number Tn 6164 was provided by the Transposon registry [28] (http://www.ucl.ac.uk/eastman/tn/index.php). To test the conjugative transfer of the element, filter mating assays were performed, selecting for the possible tetracycline resistance by means of the tet (44) gene. How- ever, M120 contains also a copy of tet (M) present on a conjugative transposon with 97% sequence identity to Tn 916 [16], which we have designated Tn 6190 .Thiselem- ent has inserted intragenically in the homologue of C. diffi- cile strain 630 ORF CD2015. Tn 6190 contains homologues to all Tn 916 ORFs except orf 12 which is involved in regu- lation of tet (M) through transcriptional attenuation [29]. During filter mating experiments with M120 as a donor strain and CD37 as a recipient, all putative trans- conjugants were identified as the recipient strain. In total 70 transconjugants were tested by PCR, using pri- mers Lok1, Lok3 [13],19,20, Tn 916 Fw, and Tn 916 Rev [30]. However, none contained Tn 6164, all contained only Tn 6190 (results not shown). Tn 6164 is sporadically present in PCR ribotype 078 Simultaneously with the publication of the M120 se- quence, we obtained Illumina sequence reads of the C. difficile strain 31618, which was isolated from a diar- rheic piglet from a pig farm in the Netherlands [16]. Comparative genomic analysis of 31618 to M120 revealed an almost complete overlap of the two genomes. How- ever, reference assembly of the 31618 reads to M120 showed that Tn Table 1 Open reading frames encoded by Tn 6164 Gene Position on Tn 6164 Module Sequence identity to Annotation Gene Position on Tn 6164 Module Sequence identity to Annotation Orf1 650-1930 A - putative modification methylase Orf25 26793-27122 B - conserved hypothetical protein Orf2 1915-3186 A - putative modification methylase Orf26 27189-28451 B Thermoanaerobacter sp. HK97 family phage portal protein Orf3 3252-3962 A - hypothetical protein Orf27 28448-29128 B Thermoanaerobacter sp. Peptidase S14, ClpP Orf4 3952-5031 A - ATPase associated with various cellular activities Orf28 29140-30339 B Thermoanaerobacter sp. HK97 family phage major capsid protein Orf5 5047-6312 A - LlaJI restriction endonuclease Orf29 30585-30899 B Thermoanaerobacter sp. uncharacterized phage protein Orf6 C 7557-6361 A - Protein with unknown function, contains a C-terminal CGNR Zinc finger motif Orf30 30903-31238 B Thermoanaerobacter sp. phage head-tail adaptor, putative Orf7 8000-8494 B Thermoanaerobacter sp. ECF RNA polymerase sigma-24 factor Orf31 31252-31662 B Thermoanaerobacter sp. HK97 family phage protein Orf8 8809-9126 B Thermoanaerobacter sp. rRNA biogenesis protein rrp5, putative Orf32 31659-32012 B Thermoanaerobacter sp. Protein of unknown function (DUF806); Orf9 9123-10250 B Thermoanaerobacter sp. Table 1 Open reading frames encoded by Tn 6164 (Continued) Orf24 25462-26685 B Thermoanaerobacter sp. phage terminase Orf48 51251-51979 C E. faecalis pEF418 putative spectinomycin/streptomycin adenyltransferase Orf49 52403-53176 E S. pneumoniae phage protein/replication initiator Orf71 77648-79216 E S. pneumoniae putative surface protein Orf50 53176-54000 E S. pneumoniae DNA replication protein Orf72 79231-80088 E S. pneumoniae putative bacteriocin Orf51 53993-54478 E S. pneumoniae DUF 3801 Orf73 80162-80773 E S. pneumoniae Predicted transcriptional regulator Orf52 54475-55209 E S. pneumoniae phage antirepressor protein Orf74 80766-81749 E S. pneumoniae Protein with unknown function Orf53 55202-56890 E S. pneumoniae TraG/TraD family protein Orf75 82268-82621 E S. pneumoniae transcriptional regulator, ArsR family Orf54 57454-58486 E - DUF 318 Predicted Permease (HHPred) Orf76 82696-83940 E S. pneumoniae major facilitator superfamily MFS_1 Orf55 59048-59398 D C. fetus glyoxalase family protein Orf77 83927-84403 E S. pneumoniae toxin-antitoxin system, toxin component, GNAT domain protein Orf56 59411-59938 D C. fetus transcriptional regulator Orf78 84758-86491 E S. pneumoniae DNA topoisomerase III Orf57 59988-61910 D C. fetus tetracycline resistance protein Orf79 86484-87449 E S. pneumoniae possible DNA (cytosine-5-)-methyltransferase Orf58 62225-63082 D