In a survey of 640 human subjects, a subgroup of 356 persons without recent exposure to antibiotics demonstrated that those with a high prevalence of Hg resistance in their intestinal floras were significantly more likely to also have resistance to two or more antibiotics. This observation led us to consider the possibility that mercury released from amalgam ("silver") dental restorations might be a selective agent for both mercuryand antibiotic-resistant bacteria in the oral and intestinal floras of primates. Resistances to mercury and to several antibiotics were examined in the oral and intestinal floras of six adult monkeys prior to the installation of amalgam fillings, during the time they were in place, and after replacement of the amalgam fillings with glass ionomer fillings (in four of the monkeys). The monkeys were fed an antibiotic-free diet, and fecal mercury concentrations were monitored. There was a statistically significant increase in the incidence of mercuryresistant bacteria during the 5 weeks following installation of the amalgam fillings and during the 5 weeks immediately following their replacement with glass ionomer fillings. These peaks in incidence of mercuryresistant bacteria correlated with peaks of Hg elimination (as high as 1 mM in the feces) immediately following amalgam placement and immediately after replacement of the amalgam fillings. Representative mercuryresistant isolates of three selected bacterial families (oral streptococci, members of the family Enterobacteriaceae, and enterococci) were also resistant to one or more antibiotics, including ampicillin, tetracycline, streptomycin, kanamycin, and chloramphenicol. While such mercury-and antibiotic-resistant isolates among the staphylococci, the enterococci, and members of the family Enterobacteriaceae have been described, this is the first report of mercury resistance in the oral streptococci. Many of the enterobacterial strains were able to transfer mercury and antibiotic resistances together to laboratory bacterial recipients, suggesting that the loci for these resistances are genetically linked. Our findings indicate that mercury released from amalgam fillings can cause an enrichment of mercury resistance plasmids in the normal bacterial floras of primates. Many of these plasmids also carry antibiotic resistance, implicating the exposure to mercury from dental amalgams in an increased incidence of multiple antibiotic resistance plasmids in the normal floras of nonmedicated subjects.

Staphylococci are increasingly aggressive human pathogens suggesting that active evolution is spreading novel virulence and resistance phenotypes. Large staphylococcal plasmids commonly carry antibiotic resistances and virulence loci, but relatively few have been completely sequenced. We determined the plasmid content of 280 staphylococci isolated in diverse geographical regions from the 1940s to the 2000s and found that 79% of strains carried at least one large plasmid >20 kb and that 75% of these large plasmids were 20–30 kb. Using restriction fragment length polymorphism (RFLP) analysis, we grouped 43% of all large plasmids into three major families, showing remarkably conserved intercontinental spread of multiresistant staphylococcal plasmids over seven decades. In total, we sequenced 93 complete and 57 partial staphylococcal plasmids ranging in size from 1.3 kb to 64.9 kb, tripling the number of complete sequences for staphylococcal plasmids >20 kb in the NCBI RefSeq database. These plasmids typically carried multiple antimicrobial and metal resistances and virulence genes, transposases and recombinases. Remarkably, plasmids within each of the three main families were >98% identical, apart from insertions and deletions, despite being isolated from strains decades apart and on different continents. This suggests enormous selective pressure has optimized the content of certain plasmids despite their large size and complex organization.

Nine polymorphic mer loci carried by 185 gram-negative fecal bacterial strains from humans and nonhuman primates are described. The loci were characterized with specific intragenic and intergenic PCR primers to amplify distinct regions covering approximately 80% of the typical gram-negative mer locus. These loci were grouped phylogenetically with respect to each other and with respect to seven previously sequenced mer operons from gram-negative bacteria (the latter designated loci 1, 2, 3, 6, 7, 8, and ⌬8 by us here for the purpose of this analysis). Six of the mer loci recovered from primates are similar either to these previously sequenced mer loci or to another locus recently observed in environmental isolates (locus 4), and three are novel (loci 5, 9, and 10). We have observed merC, or a merC-like gene, or merF on the 5 side of merA in all of the loci except that of Tn501 (here designated mer locus 6). The merB gene was observed occasionally, always on the 3 side of merA. Unlike the initial example of a merB-containing mer locus carried by plasmid pDU1358 (locus 8), all the natural primate loci carrying merB also had large deletions of the central region of the operon (and were therefore designated locus ⌬8). Four of the loci we describe (loci 2, 5, 9, and 10) have no region of homology to merB from pDU1358 and yet strains carrying them were phenylmercury resistant. Two of these loci (loci 5 and 10) also lacked merD, the putative secondary regulator of operon expression. Phylogenetic comparison of character states derived from PCR product data grouped those loci which have merC into one clade; these are locus 1 (including Tn21), locus 3, and locus 4. The mer loci which lack merC grouped into a second clade: locus 6 (including Tn501) and locus 2. Outlying groups lacked merD or possessed merB. While these mer operons are characterized by considerable polymorphism, our ability to discern coherent clades suggests that recombination is not entirely random and indeed may be focused on the immediate 5 and 3 proximal regions of merA. Our observations confirm and extend the idea that the mer operon is a genetic mosaic and has a predominance of insertions and/or deletions of functional genes immediately before and after the merA gene. chi sites are found in several of the sequenced operons and may be involved in the abundant reassortments we observe for mer genes.

Gram-negative fecal bacteria from three longitudinal Hg exposure experiments and from two independent survey collections were examined for their carriage of the mercury resistance (mer) locus. The occurrence of antibiotic resistance was also assessed in both mercury-resistant (Hg r) and mercury-susceptible (Hg s) isolates from the same collections. The longitudinal studies involved exposure of the intestinal flora to Hg released from amalgam "silver" dental restorations in six monkeys. Hg r strains were recovered before the installation of amalgams, and frequently these became the dominant strains while amalgams were installed. Such persistent Hg r strains always carried the same mer locus throughout the experiments. In both the longitudinal and survey collections, certain mer loci were preferentially associated with one genus, whereas other mer loci were recovered from many genera. In general, strains with any mer locus were more likely to be multiresistant than were strains without mer loci; this clustering tendency was also seen for antibiotic resistance genes. However, the association of antibiotic multiresistance with mer loci was not random; regardless of source, certain mer loci occurred in highly multiresistant strains (with as many as seven antibiotic resistances), whereas other mer loci were found in strains without any antibiotic resistance. The majority of highly multiresistant Hg r strains also carried genes characteristic of an integron, a novel genetic element which enables the formation of tandem arrays of antibiotic resistance genes. Hg r strains lacking antibiotic resistance showed no evidence of integron components.

The mercury resistance operon, mer, of the transposon Tn21 is transcribed from two overlapping divergent promoters: PR for the regulatory gene, merR, and PTPCAD for the structural genes, merTPCAD. Transcription of merTPCAD is repressed in the absence of Hg(II) and activated in the presence of Hg(II) by the regulatory protein, MerR. In addition, MerR represses its own expression regardless of the presence of Hg(II). MerR binds as a dimer to a single region of dyad symmetry lying between the -35 and -10 hexamers of PTPCAD. Analysis of the expression of transcriptional fusions to hydroxylamine- and oligonucleotide-generated mutants of this divergent operator-promoter region identified key bases involved in MerR-dependent repression of PTPCAD and of PR and in activation of PTPCAD. Six of the seven mutants affecting the palindromic region were altered in their ability to bind the MerR protein in vitro as measured by fragment retardation assays. These differences in in vitro MerR binding correlated well with the in vivo measurements of repression or of activation. Bases identified as functionally relevant by this genetic analysis coincide extensively with those previously identified as relevant via in vivo footprinting. Four major points emerge from this analysis: (i) transition and transversion mutations within the spacer between the -10 and -35 hexamers of PTPCAD generally have little effect on the MerR-independent (i.e., unregulated) expression of either promoter; (ii) alteration of certain bases in the MerR-binding dyad affects repression of PTPCAD differently than repression of PR; (iii) certain dyad changes can impair activation of PTPCAD more severely than repression of this promoter; and (iv) mutations in the -10 hexamer of PTPCAD which also effect PR expression define one of two potential -10 hexamers in PR as actually functional in vivo.