Susan E. Flannagan

Unconstrained bacterial promiscuity: the Tn916–Tn1545 family of conjugative transposons

Conjugative transposons are highly ubiquitous elements found throughout the bacterial world. Members of the Tn916-Tn1545 family carry the widely disseminated tetracycline-resistance determinant Tet M, as well as additional resistance genes. They have been found naturally in, or been introduced into, over 50 different species and 24 genera of bacteria. Recent investigations have led to insights into the molecular basis of movement of these interesting mobile elements.

Plasmid Content of a Vancomycin-Resistant Enterococcus faecalis Isolate from a Patient Also Colonized by Staphylococcus aureus with a VanA Phenotype

ABSTRACT Vancomycin-resistant Enterococcus faecalis coisolated with vancomycin-resistant (VanA) Staphylococcus aureus was found to contain two plasmids, designated pAM830 (45 kb) and pAM831 (95 kb). pAM830, found to be conjugative and closely related to the Inc18 family of broad-host-range conjugative plasmids, encodes resistances to vancomycin (via a Tn1546-like element) and erythromycin; pAM831 encodes resistances to gentamicin, streptomycin, and erythromycin.

Enterococcal plasmid transfer: sex pheromones, transfer origins, relaxases, and the Staphylococcus aureus issue.

Certain conjugative plasmids in Enterococcus faecalis encode a mating response to peptide sex pheromones encoded on the chromosome of potential recipient (plasmid-free) strains. The pheromone precursors correspond to the precursors of surface lipoproteins with the mature peptides coming from the last 7-8 residues of the related signal sequences. Processing that gives rise to the pAD1-related peptide involves a chromosome-encoded metalloprotease (Eep) that is believed to operate within the cytoplasmic membrane. Mutations in the determinants for cAD1 and cAM373, cad and camE, respectively, do not affect cell viability; and when the related plasmid is present, the pheromone response is normal. A cAM373-like activity is produce by Staphylococcus aureus, but the corresponding lipoprotein determinant (camS) is unrelated to the enterococcal determinant (camE). pAD1 has two origins of transfer, oriT1 and oriT2 and encodes a relaxase (TraX), which has been shown to specifically nick in oriT2. pAM373 has a site, oriT, that is similar to oriT2 of pAD1. Both sites (oriT2 of pAD1 and oriT of pAM373) have a series of short direct repeats (5-6 bp with 5-6 bp-spacings) adjacent to a long inverted repeat (140 bp). The direct repeats differ significantly and confer specificity to the two systems. pAD1 and pAM373 are both able to mobilize the nonconjugative plasmid pAMalpha1, which encodes two relaxases that are involved in transfer. Relevant information concerning the possible movement of vancomycin resistance from E. faecalis to S. aureus in a clinical environment is discussed.

The Conjugative Transposons of Gram-Positive Bacteria

Conjugative transposons are characterized by their ability to move from one bacterial cell to another by a process requiring cell-to-cell contact. Evidence for such elements became apparent about 13 years ago from studies of Enterococcus (formerly Streptococcus) faecalis strain DS16, a clinical isolate obtained in 1975 from St. Joseph’s Mercy Hospital in Ann Arbor, Michigan (38, 39). DS16 was of interest at that time because of its multiple antibiotic resistance and hemolytic properties. It was found to harbor a hemolysin/bacteriocin plasmid, pAD1, and a resistance plasmid, pAD2 (26, 112). pAD1 was conjugative and conferred a mating response to a peptide sex pheromone secreted by potential recipient (pAD1-free) cells, whereas pAD2 conferred resistance to erythromycin, streptomycin, and kanamycin and was nonconjugative. The erythromycin resistance determinant (erm) of pAD2 was associated with a transposon designated Tn917 (111). Derivatives of DS16 cured of both pAD1 and pAD2 maintained a resistance to tetracycline (Tc), indicating that a Tc-resistance determinant (tet) was located on the bacterial chromosome. A series of filter membrane mating experiments designed to examine transfer of the various resistance determinants of DS16 showed that tet could be mobilized at frequencies of 10−5 per donor (38). The majority of transconjugants (about 90%) harbored pAD1 and had tet on the chromosome. Among most of the remaining transconjugants, tet was linked with pAD1, and this correlated with insertion of a 16-kb segment of DNA. A surprising result arose when certain “control” experiments were performed using a plasmid-free (cured) derivative of DS16 as a donor. As reported by Franke and Clewell (38), tet was able to transfer from such strains at a frequency of about 10−8 per donor, and transconjugants could pass on the trait in subsequent matings. Intercellular transfer was DNase resistant and was not affected by the presence of a Rec−allele in either the donor or the recipient. In addition, donor filtrates did not transfer tet to recipients, nor did donor cells exposed to chloroform prior to mating. Because cell contact appeared necessary, the term “conjugative transposon,” was adopted, and the element was designated Tn916. Additional studies showing interspecies transfer added strong support that the transposon encoded its own fertility functions.

Antibiotic resistance gene transfer between Streptococcus gordonii and Enterococcus faecalis in root canals of teeth ex vivo.

Multiple bacterial species coexisting in infected root canals might interact, but evidence for interspecies gene transfer is lacking. This study tested the hypothesis that horizontal exchange of antibiotic resistance can occur between different bacterial species in root canals. Transfer of the conjugative plasmid pAM81 carrying erythromycin resistance between 2 endodontic infection-associated species, Streptococcus gordonii and Enterococcus faecalis, was investigated in an ex vivo tooth model. Equal numbers of each species (one with pAM81 and the other plasmid-free) were combined in prepared root canals of sterilized teeth and incubated at 37 degrees C. At 24 and 72 hours, bidirectional interspecies antibiotic resistance gene transfer was evident in microorganisms recovered from teeth; average transfer frequencies from S. gordonii to E. faecalis were 10(-3) transconjugants per donor and from E. faecalis to S. gordonii were 10(-6) and 10(-7) transconjugants per donor at 24 and 72 hours, respectively. Microbial accumulations were observed on root canal walls with scanning electron microscopy. Horizontal genetic exchange in endodontic infections might facilitate adoption of an optimal genetic profile for survival.

Identification and characterization of genes encoding sex pheromone cAM373 activity in Enterococcus faecalis and Staphylococcus aureus

The sex pheromone cAM373 of Enterococcus faecalis and the related staph‐cAM373 of Staphylococcus aureus were found to correspond to heptapeptides located within the C‐termini of the signal sequences of putative prelipoproteins. The deduced mature forms of the lipoproteins share no detectable homology and presumably serve unrelated functions in the cells. The chromosomally encoded genetic determinants for production of the pheromones have been identified and designated camE (encoding cAM373) and camS (encoding staph‐cAM373). Truncated and full‐length clones of camE were generated in Escherichia coli, in which cAM373 activity was expressed. In E. faecalis, insertional inactivation in the middle of camE had no detectable phenotypic effects on the pheromone system. Establishment of an in frame translation stop codon within the signal sequence resulted in reduction of cAM373 activity to 3% of normal levels. The camS determinant has homologues in Staphylococcus epidermidis, Bacillus subtilis and Listeria monocytogenes; however, corresponding heptapeptides present within those sequences do not resemble staph‐cAM373 closely. The particular significance of staph‐cAM373 as a potential intergeneric inducer of transfer‐proficient genetic elements is discussed.

Indication of Transposition of a Mobile DNA Element Containing the vat(D) and erm(B) Genes in Enterococcus faecium

ABSTRACT The vat(D) and erm(B) genes encoding streptogramin resistance in Enterococcus faeciumtransferred together, and a direct physical link betweenerm(B) and vat(D) was detected. Both thevat(D) and erm(B) probes hybridized to fragments of different sizes in the donor and transconjugants, which indicated a transposition event.

A Genetic Determinant in Streptococcus gordonii Challis Encodes a Peptide with Activity Similar to That of Enterococcal Sex Pheromone cAM373, Which Facilitates Intergeneric DNA Transfer

Enterococcus faecalis strains secrete multiple peptides representing different sex pheromones that induce mating responses by bacteria carrying specific conjugative plasmids. The pheromone cAM373, which induces a response by the enterococcal plasmid pAM373, has been of interest because a similar activity is also secreted by Streptococcus gordonii and Staphylococcus aureus. The potential to facilitate intergeneric DNA transfer from E. faecalis is of concern because of extensive multiple antibiotic resistance, including vancomycin resistance, that has emerged among enterococci in recent years. Here, we characterize the related pheromone determinant in S. gordonii and show that the peptide it encodes, gordonii-cAM373, does indeed induce transfer of plasmid DNA from E. faecalis into S. gordonii. The streptococcal determinant camG encodes a lipoprotein with a leader sequence, the last 7 residues of which represent the gordonii-cAM373 heptapeptide SVFILAA. Synthetic forms of the peptide had activity similar to that of the enterococcal cAM373 AIFILAS. The lipoprotein moiety bore no resemblance to the lipoprotein encoded by E. faecalis. We also identified determinants in S. gordonii encoding a signal peptidase and an Eep-like zinc metalloprotease (lspA and eep, respectively) similar to those involved in processing certain pheromone precursors in E. faecalis. Mutations generated in camG, lspA, and eep each resulted in the ablation of gordonii-cAM373 activity in culture supernatants. This is the first genetic analysis of a potential sex pheromone system in a commensal oral streptococcal species, which may have implications for intergeneric gene acquisition in oral biofilms.

Bacteriocin-Related Siblicide in Clinical Isolates of Enterococci

Siblicide is a phenomenon defined in the present context as an Enterococcus strain that, while growing as a colony on solid media, exhibits an inhibitory effect on a lawn composed of the identical strain. It was shown to occur in seven clinical isolates of enterococci (one E. faecalis and six E. faecium). Four involve inhibitory anti-listerial activities consistent with class II bacteriocins, two of which appear to be up-regulated by extracellular autoinducers.

Fluoride Levels in Unstimulated Whole Saliva following Clinical Application of Different 5% NaF Varnishes

The aim of this study was to evaluate the fluoride release from differently formulated 5% NaF varnishes into unstimulated whole saliva in vivo. The fluoride concentration in unstimulated whole saliva was determined after the application of 3 different 5% NaF varnishes (5% NaF, 5% NaF + tricalcium phosphate [TCP], and 5% NaF + amorphous calcium phosphate [ACP]) or a placebo. Fifteen subjects were recruited and enrolled following Institutional Review Board approval based upon the inclusion/exclusion criteria of this study. A cross-over study design was used for the application of either one of the 5% NaF varnishes or a placebo. Unstimulated whole saliva was collected at baseline and at 1, 4, 6, 26, and 50 h following application and analyzed for supernatant ionic fluoride and whole fluoride by microdiffusion. Linear mixed-effects models (5% significance level) were used to determine the effects of varnish and time on the salivary fluoride concentration. The highest amount of fluoride in saliva was found 1 h after application of the fluoride varnishes, with no significant differences among the treatment varnishes with respect to whole fluoride but with lower levels for 5% NaF + ACP in the saliva supernatant. Salivary fluoride levels at 4, 6, and 26 h decreased at each time point and were generally significantly higher for 5% NaF and 5% NaF + TCP. After 50 h, fluoride levels in saliva for all groups were at or below baseline levels. In conclusion, the formulation of other ingredients in fluoride varnishes can affect the fluoride concentration in saliva. The reasons for this phenomenon warrant further investigation since it might affect efficacy of the treatment. This trial is registered at ClinicalTrials.gov (NCT01629290).