Characterization of a new erm-related macrolide resistance gene present in probiotic strains of Bacillus clausii

Abstract

The mechanism of resistance to macrolides, lincosamides, and streptogramins B was studied in four Bacillus clausii strains that are mixed in a probiotic administered to humans for prevention of gastrointestinal side effects due to oral antibiotic chemotherapy and in three reference strains of B. clausii, DSM8716, ATCC 21536, and ATCC 21537. An 846-bp gene called erm(34), which is related to the erm genes conferring resistance to these antibiotics by ribosomal methylation, was cloned from total DNA of B. clausii DSM8716 into Escherichia coli. The deduced amino acid sequence presented 61% identity with that of Erm(D) from B. licheniformis, B. halodurans, and B. anthracis. Pulsed-field gel electrophoresis of total DNA digested by I-CeuI, followed by hybridization with an erm(34)-specific probe, indicated a chromosomal location of the gene in all B. clausii strains. Repeated attempts to transfer resistance to macrolides by conjugation from B. clausii strains to Enterococcus faecalis JH2-2, E. faecium HM1070, and B. subtilis UCN19 were unsuccessful.

FIG. 1.

erm(34) DNA sequence and deduced amino acid sequence. The nucleotide sequence of erm(34) is shown together with the deduced amino acid sequence of Erm34 methylase and its leader peptide. Putative ribosome-binding sites are underlined.

FIG. 2.

Localization of the erm(34) gene in B. clausii NR, OC, SIN, T, and DSM8716. (Left) Total DNAs from B. clausii strains NR (lane 1), OC (lane 2), SIN (lane 3), T (lane 4), and DSM8716 (lane 5) were digested with the I-CeuI restriction enzyme and submitted to pulsed-field gel electrophoresis. M, molecular size standard (Saccharomyces cerevisiae chromosomal DNA). (Right) DNA was transferred to a nylon membrane and hybridized with an erm(34) probe labeled with digoxigenin.

FIG. 3.

Phylogenetic relationships among Erm methylases. The tree was constructed by using the neighbor-joining method. Sequences are from S. aureus [Erm(A) (accession no. X03216) and Erm(C) (accession no. J01755)], Streptococcus pneumoniae [Erm(B) (accession no. X52632)], B. licheniformis [Erm(D) (accession no. M29832)], Saccharopolyspora erythraea [Erm(E) (accession no. X51891)], Bacteroides fragilis [Erm(F) (accession no. M14730)], B. sphaericus [Erm(G) (accession no. M15332)], Streptomyces thermotolerans [Erm(H) (accession no. P13079)], Streptomyces fradiae [Erm(N) (accession no. X97721) and [Erm(S)], Streptomyces lividans [Erm(O) (accession no. M74717)], Clostridium perfringens [Erm(R) (accession no. L22689)], Arthrobacter sp. [Erm(R) (accession no. M11276)], Lactobacillus reuteri [Erm(T) (accession no. M64090)], Streptomyces lincolnensis [Erm(U) (accession no. X62867)], Corynebacterium diphtheriae [Erm(X) (accession no. M36726)], M. griseorubida [Erm(W) (accession no. D14532)], and B. clausii [Erm34].