ORIGINAL ARTICLE A conjugative macrolide resistance gene, mef(A), in environmental Clostridium perfringens carrying multiple macrolide and ⁄ or tetracycline resistance genes O.O. Soge, L.D. Tivoli, J.S. Meschke and M.C. Roberts Department of Environmental and Occupational Health Sciences, University of Washington, Seattle, WA, USA Introduction Clostridium perfringens is a gram-positive spore-forming anaerobe, which is a normal inhabitant of the gastrointes- tinal tract and causes a wide range of diarrhoeal diseases in man and animals (Bruggemann 2005). Clostridium per- fringens is widely distributed in soil, sewage and food (Rood and Cole 1991). Tetracycline-, macrolide- and ⁄ or chloramphenicol-resistant human and animal-associated C. perfringens isolates have been identified since the 1970s (Brefort et al. 1977; Rood et al. 1978). The chlorampheni- col [cat(P), cat(Q)], macrolide-lincosamide-streptogramin B (MLS B )[erm(B), erm(Q)], and tetracycline [tetA(P), tetB(P), tet(M) and tet(Q)] genes have since been identi- fied in C. perfringens (Abraham et al. 1985; Berryman et al. 1994; Berryman and Rood 1995; Lyras and Rood 1996; Martel et al. 2004). However, the number of differ- ent antibiotic resistance genes carried, their distribution and their association with mobile elements has varied by study and geographic location of the isolates. The tetA(P) gene has been the most frequently identi- fied tetracycline (tet) resistance gene in C. perfringens with 100% of tetracycline-resistant (Tc r ) strains carrying the tetA(P) gene in one study (Lyras and Rood 1996). The tetB(P) gene overlaps the tetA(P) gene and has always been associated with the tetA(P) gene. In one study, 50% of the Tc r strains carried the tetA(P) and tetB(P) genes with 16% of the isolates carrying these genes on conjuga- tive plasmids. An additional 40% carried the tetA(P) and an unlinked tet(M) gene (Lyras and Rood 1996). Two different MLS B genes, erm(B) and erm(Q), have been identified in human and animal C. perfringens (Berryman Keywords C. perfringens, ecology, macrolide, mef(A), multidrug, tetracycline. Correspondence Marilyn C. Roberts, Department of Environmental and Occupational Health Sciences, Box 357234, School of Public Health and Community Medicine, University of Washington, Seattle 98195, WA, USA. E-mail: marilynr@u.washington.edu 2008 ⁄ 0693: received 23 April 2008, revised 28 May 2008 and accepted 9 June 2008 doi:10.1111/j.1365-2672.2008.03960.x Abstract Aims: To determine if environmental Clostridium perfringens carry antibiotic resistance genes and if the genes are mobile. Methods and Results: Clostridium perfringens from water, soil and sewage (2003–2006) were screened for the tetracycline and macrolide resistance genes previously described in animal and human C. perfringens [erm(B), erm(Q), tetA(P), tetB(P) and tet(M) genes] and the macrolide resistance mef(A) gene. Of the 160 isolates, 108 (67Æ5%) carried ‡1 of the six antibiotic resistance gene(s). The tetA(P), tetB(P) and tet(M) genes were in 53%, 22% and 8%, and the erm(B), erm(Q) and mef(A) genes in 26%, 1% and 18% of the isolates, respectively. The mef(A) gene and flanking regions were sequenced. The tet(M), erm(B), erm(Q) and mef(A) genes transfer independently from C. perfringens donors to the Enterococcus faecalis recipient. Conclusions: Six resistance genes were found in the environmental C. perfrin- gens with the most common being the tetA(P) gene and the erm(Q) gene the least common. Significance and Impact of the Study: This is the first time conjugal transfer of macrolide resistance genes and ⁄ or the tet(M) gene from C. perfringens has been demonstrated. The data presented supports the hypothesis that antibiotic- resistant environmental C. perfringens are capable of acting as reservoirs for these antibiotic resistance genes. Journal of Applied Microbiology ISSN 1364-5072 34 Journal compilation ª 2008 The Society for Applied Microbiology, Journal of Applied Microbiology 106 (2009) 34–40 ª 2008 The Authors