A strain of C. difficile that is highly resistant to clindamycin was responsible for large outbreaks of diarrhea in four hospitals in different states. The use of clindamycin is a specific risk factor for diarrhea due to this strain. Resistance to clindamycin further increases the risk of C. difficile-associated diarrhea, an established complication of antimicrobial use.

SummaryClostridium difficile is an emerging nosocomial pathogen of increasing importance and virulence but our ability to study the molecular mechanisms underlying the pathogenesis of C. difficile-associated disease has been limited because of a lack of tools for its genetic manipulation. We have now developed a reproducible method for the targeted insertional inactivation of chromosomal C. difficile genes. The approach relies on the observation that an Escherichia coli-Clostridium perfringens shuttle vector is unstable in C. difficile and can be used as a form of conditional lethal vector to deliver gene constructs to the chromosome. We have used this methodology to insertionally inactivate two putative response regulator genes, rgaR and rgbR, which encode proteins with similarity to the toxin gene regulator, VirR, from C. perfringens. Transcriptomic analysis demonstrated that the C. difficile RgaR protein positively regulated four genes, including a putative agrBD operon. The RgaR protein was also purified and shown to bind specifically to sites that contained two consensus VirR boxes located just upstream of the putative promoters of these genes. The development of this methodology will significantly enhance our ability to use molecular approaches to develop a greater understanding of the ability of C. difficile to cause disease.

Clostridium difficile is a nosocomial pathogen that causes a range of chronic intestinal diseases, usually as a result of antimicrobial therapy. Macrolidelincosamide-streptogramin B (MLS) resistance in C. difficile is encoded by the Erm B resistance determinant, which is thought to be located on a conjugative transposon, Tn5398. The 9630 bp Tn5398 element has been cloned and completely sequenced and its insertion site determined. Analysis of the resultant data reveals that Tn5398 is not a classical conjugative transposon but appears to be a mobilizable non-conjugative element. It does not carry any transposase or site-specific recombinase genes, nor any genes likely to be involved in conjugation. Furthermore, using PCR analysis it has been shown that isolates of C. difficile obtained from different geographical locations exhibit heterogeneity in the genetic arrangement of both Tn5398 and their Erm B determinants. These results indicate that genetic exchange and recombination between these determinants occurs in the clinical and natural environment.

Mobilisable transposons are transposable genetic elements that also encode mobilisation functions but are not in themselves conjugative. They rely on coresident conjugative elements to facilitate their transfer to recipient cells. Clostridial mobilisable transposons include Tn4451 and Tn4452 from Clostridium perfringens, and Tn4453a and Tn4453b from Clostridium difficile, all of which are closely related, and Tn5398 from C. difficile. The Tn4451 group of elements encodes resistance to chloramphenicol and is unusual in that transposition is dependent upon a large resolvase protein rather than a more conventional transposase or integrase. This group of elements also encodes the mobilisation protein TnpZ that, by acting at the RS(A) or oriT site located on the transposon, and in the presence of a coresident conjugative element, promotes the movement of the nonreplicating circular intermediate and of plasmids on which the transposon resides. The erythromycin resistance element Tn5398 is unique in that it encodes no readily identifiable transposition or mobilisation proteins. However, the element is still capable of intraspecific transfer between C. difficile isolates, by an unknown mechanism. The detailed analysis of these mobilisable clostridial elements provides evidence that the evolution and dissemination of antibiotic resistance genes is a complex process that may involve the interaction of genetic elements with very different properties.

The ErmB macrolide-lincosamide-streptogramin B (MLS) resistance determinant from Clostridium difficile 630 contains two copies of an erm(B) gene, separated by a 1.34-kb direct repeat also found in an Erm(B) determinant from Clostridium perfringens. In addition, both erm(B) genes are flanked by variants of the direct repeat sequence. This genetic arrangement is novel for an ErmB MLS resistance determinant.

Endolysin enzymes from bacteriophage cause bacterial lysis by degrading the peptidoglycan cell wall. The streptococcal C1 phage endolysin PlyC, is the most potent endolysin described to date and can rapidly lyse group A, C, and E streptococci. PlyC Primer name Sequence (5′-3′) General sequencing fw seq TTAGCGGATCCTACCTGACG This study rv seq TTTTATCAGACCGCTTCTGC This study Site saturation mutagenesis and GA ins fw CAGTGCCAAAGAAACTGCTAAATGTTTTAG This study ins rev GGTTAGTTTGATAATGACACCATTCTAAGTTATG This study vector fw CATAACTTAGAATGGTGTCATTATCAAACTAACC This study vector rv CTAAAACATTTAGCAGTTTCTTTGGCACTG This study R66 NNS fw GATGTAGAGGCTATCNNSAAGGCTATGAAAAAG This study R66 NNS rv CTTTTCATAGCCTTNNSGATAGCCTCTACATC This study K59 NNS fw GTCTATCAATATTAGTNNSTCTGATGTAGAGGC This study K59 NNS rv GCCTCTACATCAGANNSACTAATATTGATAGAC This study Y28 NNS fw GAAAAGAAAGTTACGGTNNSCGTGCTTTTATTAACG This study Y28 NNS rv CGTTAATAAAAGCACGNNSACCGTAACTTTCTTTTC This study E36 NNS fw CTTTTATTAACGGAGTTNNSATTGGTATTAAAGACATTG This study E36 NNS rv CAATGTCTTTAATACCAATNNSAACTCCGTTAATAAAAG This study Y26 NNS fw CATACCGATGGAAAAGAAAGTNNSGGTTATCGTGCTTTTATTAAC This study Y26 NNS rv GTTAATAAAAGCACGATAACCNNSACTTTCTTTTCCATCGGTATG This study PlyCB site-directed mutagenesis PlyCB R66A fw GTAAGTCTGATGTAGAGGCTATCGCAAAGGCTATGAA This study PlyCB R66A rv TTCATAGCCTTTGCGATAGCCTCTACATCAGACTTAC This study PlyCB R66K fw CTGATGTAGAGGCTATCAAAAAGGCTATGAA This study PlyCB R66K rv TTCATAGCCTTTTTGATAGCCTCTACATCAG This study PlyCB Y26A fw GATGGAAAAGAAAGTGCCGGTTATCGTGCTTT This study PlyCB Y26A rv AAAGCACGATAACCGGCACTTTCTTTTCCATC This study PlyCB Y26W fw CGATGGAAAAGAAAGTTGGGGTTATCGTGCTTT This study PlyCB Y26W rv AAAGCACGATAACCCCAACTTTCTTTTCCATCG This study PlyCB Y28H fw AAGAAAGTTACGGTCATCGTGCTTTTATTAAC This study PlyCB Y28H rv GTTAATAAAAGCACGATGACCGTAACTTTCTT This study Note: All plasmids except pSM85 have a pBAD24 vector backbone and an ampicillin resistance marker. pSM85 is based on pBAD33 with chloramphenicol resistance.

The Clostridium perfringens tetracycline resistance protein, TetA(P), is an inner-membrane protein that mediates the active efflux of tetracycline from the bacterial cell. This protein comprises 420 aa and is predicted to have 12 transmembrane domains (TMDs). Comparison of the TetA(P) amino acid sequence to that of several members of the major facilitator superfamily (MFS) identified a variant copy of the conserved Motif A. This region consists of the sequence E 59 xPxxxxxDxxxRK 72 and is located within the putative loop joining TMDs 2 and 3 in the predicted structural model of the TetA(P) protein. To study the functional importance of the conserved residues, site-directed mutagenesis was used to construct 17 point mutations that were then analysed for their effect on tetracycline resistance and their ability to produce an immunoreactive TetA(P) protein. Changes to the conserved Phe-58 residue were tolerated, whereas three independent substitutions of Pro-61 abolished tetracycline resistance. Examination of the basic residues showed that Arg-71 is required for function, whereas tetracycline resistance was retained when Lys-72 was substituted with arginine. These results confirm that the region encoding this motif is important for tetracycline resistance and represents a distant version of the Motif A region found in other efflux proteins and members of the MFS family. In addition, it was shown that Glu-117 of the TetA(P) protein, which is predicted to be located in TMD4, is important for resistance although a derivative with an aspartate residue at this position is also functional.

Macrolide-lincosamide-streptogramin B resistance is widespread, with the determinants encoding resistance to antibiotics such as erythromycin being detected in many bacterial pathogens. Resistance is most commonly mediated by the production of an Erm protein, a 23S rRNA methyltransferase. We have undertaken a mutational analysis of the Erm(B) protein from Clostridium perfringens with the objective of developing a greater understanding of the mechanism of action of this protein. A recombinant plasmid that carried the erm(B) gene was mutated by either in vitro hydroxylamine mutagenesis or passage through the mutator strain XL1-Red. Twenty-eight independently derived mutants were identified, nine of which had single point mutations in the erm(B) gene. These mutants produced stable but nonfunctional Erm(B) proteins, and all had amino acid changes within conserved methyltransferase motifs that were important for either substrate binding or catalysis. Modeling of the C. perfringens Erm(B) protein confirmed that the point mutations all involved residues important for the structure and/or function of this rRNA methyltransferase. These regions of the protein therefore represent potential targets for the rational development of methyltransferase inhibitors.Macrolide, lincosamide, and streptogramin B (MLS) antibiotics are a diverse group of antibacterial agents that are chemically distinct but that have similar modes of action. They act at the early stages of protein synthesis by blocking the growth of the nascent peptide chain (1), which then presumably causes the premature dissociation of the peptidyl-tRNA molecule from the ribosome (22). These antibiotics include erythromycin, clindamycin, and lincomycin and are active against various bacteria, including gram-positive cocci and rods and gramnegative cocci.Four mechanisms of bacterial resistance to MLS antibiotics have been detected; they involve enzymatic modification of the antibiotic, active efflux from the bacterial cell, mutation of the ribosomal target site, or, most commonly, enzyme-mediated chemical alteration of the rRNA target (18,19,33). The last mechanism is mediated by the synthesis of a 23S rRNA methyltransferase, which is responsible for the N 6 -dimethylation of a specific adenine residue in the 23S rRNA molecule (17).Methyltransferases are enzymes that methylate a wide variety of substrates; they use S-adenosyl-L-methionine (SAM) as the universal methyl donor and release S-adenosyl-L-homocysteine as the reaction product (4). Comparative analysis of over 40 DNA amino-methyltransferases (21) revealed the presence of nine conserved sequence motifs that are important in target sequence specificity, catalysis, and SAM binding (Fig. 1). Motif I is highly conserved and forms a secondary structure known as the G loop which binds to the methionine moiety of SAM.Motifs II and III are less conserved, with motif II containing a negatively charged amino acid that interacts with the ribose hydroxyls of SAM and a bulky hydrophobic side chain that makes contact with the...