Friend turned foe: evolution of enterococcal virulence and antibiotic resistance.

The enterococci are an ancient genus that evolved along with the tree of life. These intrinsically rugged bacteria are highly adapted members of the intestinal consortia of a range of hosts that spans the animal kingdom. Enterococci are also leading opportunistic hospital pathogens, causing infections that are often resistant to treatment with most antibiotics. Despite the importance of enterococci as hospital pathogens, the vast majority live outside of humans, and nearly all of their evolutionary history took place before the appearance of modern humans. Because hospital infections represent evolutionary end points, traits that exacerbate human infection are unlikely to have evolved for that purpose. However, clusters of traits have converged in specific lineages that are well adapted to colonize the antibiotic-perturbed gastrointestinal tracts of patients and that thrive in the hospital environment. Here we discuss these traits in an evolutionary context, as well as how comparative genomics is providing new insights into the evolution of the enterococci.

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title: Emergence and spread of vancomycin resistance among enterococci in Europe.
abstract: Nowadays, six types of acquired vancomycin resistance in enterococci are known; however, only VanA and to a lesser extent VanB are widely prevalent. Various genes encode acquired vancomycin resistance and these are typically associated with mobile genetic elements which allow resistance to spread clonally and laterally. The major reservoir of acquired vancomycin resistance is Enterococcus faecium; vancomycin-resistant Enterococcus faecalis are still rare. Population analysis of E. faecium has revealed a distinct subpopulation of hospital-acquired strain types, which can be differentiated by molecular typing methods (MLVA, MLST) from human commensal and animal strains. Hospital-acquired E. faecium have additional genomic content (accessory genome) including several factors known or supposed to be virulence-associated. Acquired ampicillin resistance is a major phenotypic marker of hospital-acquired E. faecium in Europe and experience has shown that it often precedes increasing rates of VRE with a delay of several years. Several factors are known to promote VRE colonisation and transmission; however, despite having populations with similar predispositions and preconditions, rates of VRE vary all over Europe.

title: Comparative Genomics of Vancomycin-Resistant Staphylococcus aureus Strains and Their Positions within the Clade Most Commonly Associated with Methicillin-Resistant S. aureus Hospital-Acquired Infection in the United States
abstract: Methicillin-resistant Staphylococcus aureus (MRSA) strains are leading causes of hospital-acquired infections in the United States, and clonal cluster 5 (CC5) is the predominant lineage responsible for these infections. Since 2002, there have been 12 cases of vancomycin-resistant S. aureus (VRSA) infection in the United States—all CC5 strains. To understand this genetic background and what distinguishes it from other lineages, we generated and analyzed high-quality draft genome sequences for all available VRSA strains. Sequence comparisons show unambiguously that each strain independently acquired Tn1546 and that all VRSA strains last shared a common ancestor over 50 years ago, well before the occurrence of vancomycin resistance in this species. In contrast to existing hypotheses on what predisposes this lineage to acquire Tn1546, the barrier posed by restriction systems appears to be intact in most VRSA strains. However, VRSA (and other CC5) strains were found to possess a constellation of traits that appears to be optimized for proliferation in precisely the types of polymicrobic infection where transfer could occur. They lack a bacteriocin operon that would be predicted to limit the occurrence of non-CC5 strains in mixed infection and harbor a cluster of unique superantigens and lipoproteins to confound host immunity. A frameshift in dprA, which in other microbes influences uptake of foreign DNA, may also make this lineage conducive to foreign DNA acquisition.  Invasive methicillin-resistant Staphylococcus aureus (MRSA) infection now ranks among the leading causes of death in the United States. Vancomycin is a key last-line bactericidal drug for treating these infections. However, since 2002, vancomycin resistance has entered this species. Of the now 12 cases of vancomycin-resistant S. aureus (VRSA), each was believed to represent a new acquisition of the vancomycin-resistant transposon Tn1546 from enterococcal donors. All acquisitions of Tn1546 so far have occurred in MRSA strains of the clonal cluster 5 genetic background, the most common hospital lineage causing hospital-acquired MRSA infection. To understand the nature of these strains, we determined and examined the nucleotide sequences of the genomes of all available VRSA. Genome comparison identified candidate features that position strains of this lineage well for acquiring resistance to antibiotics in mixed infection. Invasive methicillin-resistant Staphylococcus aureus (MRSA) infection now ranks among the leading causes of death in the United States. Vancomycin is a key last-line bactericidal drug for treating these infections. However, since 2002, vancomycin resistance has entered this species. Of the now 12 cases of vancomycin-resistant S. aureus (VRSA), each was believed to represent a new acquisition of the vancomycin-resistant transposon Tn1546 from enterococcal donors. All acquisitions of Tn1546 so far have occurred in MRSA strains of the clonal cluster 5 genetic background, the most common hospital lineage causing hospital-acquired MRSA infection. To understand the nature of these strains, we determined and examined the nucleotide sequences of the genomes of all available VRSA. Genome comparison identified candidate features that position strains of this lineage well for acquiring resistance to antibiotics in mixed infection.

title: Antibacterial resistance worldwide: causes, challenges and responses
abstract: The optimism of the early period of antimicrobial discovery has been tempered by the emergence of bacterial strains with resistance to these therapeutics. Today, clinically important bacteria are characterized not only by single drug resistance but also by multiple antibiotic resistance—the legacy of past decades of antimicrobial use and misuse. Drug resistance presents an ever-increasing global public health threat that involves all major microbial pathogens and antimicrobial drugs.

title: The phylogenetic position of Streptococcus and Enterococcus.
abstract: Streptococcus pyogenes, S. equinus, S. bovis, S. salivarius, S. sanguis, S. mutans, S. rattus, S. cricetus, S. lactis, S. raffinolactis and Enterococcus faecalis have been characterized by oligonucleotide cataloguing of their 16S ribosomal RNA. All the organisms form a loose but coherent group that is phylogenetically equivalent to those of lactobacilli, bacilli, the Brochothrix and Listeria group, and related taxa that constitute one of several sublines within the 'Clostridium' branch of Gram-positive eubacteria. Within the Steptococcus-Enterococcus group, organisms fall into three moderately related clusters defined by Enterococcus, the lactic acid streptococci and streptococci of the pyogenic and oral groups, respectively.

title: Transfer of Streptococcus faecalis and Streptococcus faecium to the Genus Enterococcus nom. rev. as Enterococcus faecalis comb. nov. and Enterococcus faecium comb. nov.
abstract: The results of deoxyribonucleic acid-deoxyribonucleic acid and deoxyribonucleic acid-ribosomal ribonucleic acid hybridization studies demonstrated that Streptococcus faecalis and Streptococcus faecium are distantly related to the non-enterococcal streptococci (Streptococcus bovis and Streptococcus equinus) of serological group D and to other streptococci. On the basis of our results and those of previous studies, we propose that S. faecalis and S. faecium be transferred to the genus Enterococcus (ex Thiercelin and Jouhaud) nom. rev. as Enterococcus faecalis (Andrewes and Horder) comb. nov. and Enterococcus faecium (Orla-Jensen) comb. nov., respectively. A description of the genus Enterococcus nom. rev. and emended descriptions of E. faecalis and E. faecium are given.

title: Census of the Bacterial Community of the Gypsy Moth Larval Midgut by Using Culturing and Culture-Independent Methods
abstract: Little is known about bacteria associated with Lepidoptera, the large group of mostly phytophagous insects comprising the moths and butterflies. We inventoried the larval midgut bacteria of a polyphagous foliivore, the gypsy moth (Lymantria dispar L.), whose gut is highly alkaline, by using traditional culturing and culture-independent methods. We also examined the effects of diet on microbial composition. Analysis of individual third-instar larvae revealed a high degree of similarity of microbial composition among insects fed on the same diet. DNA sequence analysis indicated that most of the PCR-amplified 16S rRNA genes belong to the γ-Proteobacteria and low G+C gram-positive divisions and that the cultured members represented more than half of the phylotypes identified. Less frequently detected taxa included members of the α-Proteobacterium, Actinobacterium, and Cytophaga/Flexibacter/Bacteroides divisions. The 16S rRNA gene sequences from 7 of the 15 cultured organisms and 8 of the 9 sequences identified by PCR amplification diverged from previously reported bacterial sequences. The microbial composition of midguts differed substantially among larvae feeding on a sterilized artificial diet, aspen, larch, white oak, or willow. 16S rRNA analysis of cultured isolates indicated that an Enterococcus species and culture-independent analysis indicated that an Entbacter sp. were both present in all larvae, regardless of the feeding substrate; the sequences of these two phylotypes varied less than 1% among individual insects. These results provide the first comprehensive description of the microbial diversity of a lepidopteran midgut and demonstrate that the plant species in the diet influences the composition of the gut bacterial community.

title: Native Microbial Colonization of Drosophila melanogaster and Its Use as a Model of Enterococcus faecalis Pathogenesis
abstract: Enterococci are commensal organisms of the gastrointestinal (GI) tracts of a broad range of mammalian and insect hosts, but they are also leading causes of nosocomial infection. Little is known about the ecological role of enterococci in the GI tract consortia. To develop a tractable model for studying the roles of these organisms as commensals and pathogens, we characterized the Drosophila melanogaster microflora and examined the occurrence of enterococci in the gastrointestinal consortium of Drosophila. In a survey of laboratory-reared Drosophila and wild-captured flies, we found that Drosophila was naturally colonized by representatives of five bacterial phyla. Among these organisms were several species of enterococci, including Enterococcus faecalis, Enterococcus faecium, Enterococcus gallinaraum, and Enterococcus durans, as well as a previously detected but uncultured Enterococcus species. Drosophila could be cured of enterococcal carriage by antibiotic treatment and could be reassociated with laboratory strains. High-level colonization by a well-characterized strain expressing the enterococcal cytolysin was found to be detrimental to Drosophila compared to the effect of an isogenic, noncytolytic control. The anatomical distribution of enterococci in the Drosophila GI tract was determined by immunohistochemical staining of thin sections of naturally colonized and reassociated flies.

title: Enterococci in insects.
abstract: Enterococci were obtained from 213 of 403 insects cultured during a 14-month period, in numbers from 10(3) to 3 x 10(7)/g of insect. Insects were taken only from nonurban, wild, and cultivated fields and woods. In species of insects carrying them, enterococci were not always present in every individual cultured, and often more than one species of enterococcus occurred within a species. Enterococci were obtained from certain insects taken in the field during the dormant season, suggesting their role as overwintering agents. They were generally present in species feeding on nectar, succulent plant parts, and on and ir forest litter, but not from insects feeding on less succulent leaves and stems. Streptococcus faecalis was recovered from 32%, Streptococcus faecium from 22.4%, and Streptococcus faecium var. casseliflavus from 43.5% of members of the 37 taxa of insects. S. faecalis and S. faecium var. casseliflavus exhibit a high percent of conformity to the properties published for them. The heterogeneity in properties of S. faecium is similar to that found for the species taken from plants. Many fail to grow in broth at 45 C or in broth containing 6.5% NaCl; 50% of the cultures ferment both melezitose and melibiose, and a few ferment neither sugar. The remainder ferment melibiose only. Failure to reduce methylene blue in milk by S. faecalis and S. faecium is correlated with the inability to ferment lactose. More than 93% of the cultures of S. faecalis digest casein in milk from the top downward, following the production of a soft, flowing curd. Because this property is not characteristic of S. faecalis taken from humans, the reaction in litmus milk is suggested as a means of differentiation between cultures of remote and innocent origin in nature and recent, human pollution.

title: From Commensal to Pathogen: Translocation of Enterococcus faecalis from the Midgut to the Hemocoel of Manduca sexta
abstract: A dynamic homeostasis is maintained between the host and native bacteria of the gastrointestinal tract in animals, but migration of bacteria from the gut to other organs can lead to disease or death. Enterococcus faecalis is a commensal of the gastrointestinal tract; however, Enterococcus spp. are increasingly frequent causes of nosocomial infections with a high mortality rate. We investigated the commensal-to-pathogen switch undergone by E. faecalis OG1RF in the lepidopteran model host Manduca sexta associated with its location in the host. E. faecalis persists in the harsh midgut environment of M. sexta larvae without causing apparent illness, but injection of E. faecalis directly into the larval hemocoel is followed by rapid death. Additionally, oral ingestion of E. faecalis in the presence of Bacillus thuringiensis insecticidal toxin, a pore-forming toxin that targets the midgut epithelium, induces an elevated mortality rate. We show that the loss of gut integrity due to B. thuringiensis toxin correlates with the translocation of E. faecalis from the gastrointestinal tract into the hemolymph. Upon gaining access to the hemolymph, E. faecalis induces an innate immune response, illustrated by hemocyte aggregation, in larvae prior to death. The degree of hemocyte aggregation is dependent upon the route of E. faecalis entry. Our data demonstrate the efficacy of the M. sexta larval model system in investigating E. faecalis-induced sepsis and clarifies controversies in the field regarding the events leading to larval death following B. thuringiensis toxin exposure.  This study advances our knowledge of Enterococcus faecalis-induced sepsis following translocation from the gut and provides a model for mammalian diseases in which the spatial distribution of bacteria determines disease outcomes. We demonstrate that E. faecalis is a commensal in the gut of Manduca sexta and a pathogen in the hemocoel, resulting in a robust immune response and rapid death, a process we refer to as the “commensal-to-pathogen” switch. While controversy remains regarding Bacillus thuringiensis toxin-induced killing, our laboratory previously found that under some conditions, the midgut microbiota is essential for B. thuringiensis toxin killing of Lymantria dispar (N. A. Broderick, K. F. Raffa, and J. Handelsman,  2006; B. Raymond, et al.,  2009; P. R. Johnston, and N. Crickmore,  2009). We and others have demonstrated that the role of the midgut microbiota in B. thuringiensis toxin killing is dependent upon the lepidopteran species and formulation of B. thuringiensis toxin (N. A. Broderick, K. F. Raffa, and J. Handelsman,  2006; N. A. Broderick, et al. 2009). This work reconciles much of the apparently contradictory previous data and reveals that the M. sexta-E. faecalis system provides a model for mammalian sepsis. This study advances our knowledge of Enterococcus faecalis-induced sepsis following translocation from the gut and provides a model for mammalian diseases in which the spatial distribution of bacteria determines disease outcomes. We demonstrate that E. faecalis is a commensal in the gut of Manduca sexta and a pathogen in the hemocoel, resulting in a robust immune response and rapid death, a process we refer to as the “commensal-to-pathogen” switch. While controversy remains regarding Bacillus thuringiensis toxin-induced killing, our laboratory previously found that under some conditions, the midgut microbiota is essential for B. thuringiensis toxin killing of Lymantria dispar (N. A. Broderick, K. F. Raffa, and J. Handelsman,  2006; B. Raymond, et al.,  2009; P. R. Johnston, and N. Crickmore,  2009). We and others have demonstrated that the role of the midgut microbiota in B. thuringiensis toxin killing is dependent upon the lepidopteran species and formulation of B. thuringiensis toxin (N. A. Broderick, K. F. Raffa, and J. Handelsman,  2006; N. A. Broderick, et al. 2009). This work reconciles much of the apparently contradictory previous data and reveals that the M. sexta-E. faecalis system provides a model for mammalian sepsis.

title: Occurrence of enterococci in animals in a wild environment.
abstract: Enterococci were obtained from the feces of 71% of 216 mammals, 86% of 70 reptiles, and 32% of 22 birds sampled in a truly wild environment, the Great Smoky Mountains National Park. Patterns of food dependence and also of species dependence were observed. Among the lower classes of the primarily herbivorous mammals, the enterococci occurred sporadically; however, of the six species of Sciuridae, the gray squirrel, and of four species of Cricetidae, the red-backed mouse, the enterococci appear to be natural hosts. The enterococci were not obtained from most specimens of moles, shrews, or rabbits but they were obtained from most specimens of bats and from the carnivorous mammals, such as fox, bear, raccon, skunk, and boar. Streptococcus faecalis was obtained from 12 reptiles, and a caseolytic variant was obtained from 37 specimens of the reptiles. The strongly reducing, tellurite-tolerant species, S. faecalis, its caseolytic variant, and S. faecalis var. zymogenes were isolated from 127 or 41% of 308 specimens cultured. S. faecium was recovered from 87 or 28% of the animals, chiefly from the wild boar (60 of 64 trials) and the black bear. S. zymogenes was obtained from 1 of 31 bats, 3 of 12 raccoons, and 1 of 3 owls.

title: Functionality of enterococci in dairy products.
abstract: Enterococci have important implications in the dairy industry. They occur as nonstarter lactic acid bacteria (NSLAB) in a variety of cheeses, especially artisan cheeses produced in southern Europe from raw or pasteurised milk, and in natural milk or whey starter cultures. They play an acknowledged role in the development of sensory characteristics during ripening of many cheeses and have been also used as components of cheese starter cultures. The positive influence of enterococci on cheese seems due to specific biochemical traits such as lipolytic activity, citrate utilisation, and production of aromatic volatile compounds. Some enterococci of dairy origin have also been reported to produce bacteriocins (enterocins) inhibitory against food spoilage or pathogenic bacteria, such as Listeria monocytogenes, Staphylococcus aureus, Vibrio cholerae, Clostridium spp., and Bacillus spp. The technological application of enterocins, shown to be produced during cheese manufacture, led to propose enterococci as adjunct starter or protective cultures in cheeses. There is evidence that enterococci, either added as adjunct starters or present as nonstarter NSLAB, could find potential application in the processing of some fermented dairy products. Literature suggest that the complex biochemical and ecological phenomena explaining the technological functionality of the enterococci in dairy products, are still to be fully understood. Clearly, the clinical research on enterococci underlines also that the safety of dairy products containing enterococci is an issue that the industry must carefully address before proceeding to their application.

title: Occurrence of enterococci on plants in a wild environment.
abstract: Enterococci were obtained from 14% of nearly 2,200 flowers, 3.4% of non-floral structures of angiosperms, and from 8.3% of samples of soil, water, and lesser plants of the Great Smoky Mountains National Park, an area little influenced by the presence of man. The enterococci were recovered from one or more flowers or flower clusters of 1,515 samples in 47 taxa, but not from flowers of 67 taxa with 654 samples. The per cent of recovery was influenced adversely by dense forest cover and by increase in elevation, as compared with recovery from flowers in sunny locations in the lower elevations. The per cent recovery increased directly with rising seasonal temperature, with the maximal per cent of recovery occurring in September. In no instance did all samples of a species of flower or plant yield enterococci on culture, and with only three genera, Cacalia, Delphinium, and Mitchella, were the bacteria obtained from more than 50% of the samples. Approximately 11% of the cultures isolated were identified as Streptococcus faecalis, 64% as the soft curd producing, caseolytic variant of S. faecalis, 4% as S. faecalis var. zymogenes, and 20% as S. faecium. The per cent distribution of these species on plants was reasonably similar to the distribution within wild animals in the same environment. It was concluded that the enterococci occurring on plants arise commonly from the wild animals, and that they do not represent plant-specific species or variants of the enterococci.

title: Intestinal colonization resistance
abstract: Dense, complex microbial communities, collectively termed the microbiota, occupy a diverse array of niches along the length of the mammalian intestinal tract. During health and in the absence of antibiotic exposure the microbiota can effectively inhibit colonization and overgrowth by invading microbes such as pathogens. This phenomenon is called ‘colonization resistance’ and is associated with a stable and diverse microbiota in tandem with a controlled lack of inflammation, and involves specific interactions between the mucosal immune system and the microbiota. Here we overview the microbial ecology of the healthy mammalian intestinal tract and highlight the microbe–microbe and microbe–host interactions that promote colonization resistance. Emerging themes highlight immunological (T helper type 17/regulatory T‐cell balance), microbiota (diverse and abundant) and metabolic (short‐chain fatty acid) signatures of intestinal health and colonization resistance. Intestinal pathogens use specific virulence factors or exploit antibiotic use to subvert colonization resistance for their own benefit by triggering inflammation to disrupt the harmony of the intestinal ecosystem. A holistic view that incorporates immunological and microbiological facets of the intestinal ecosystem should facilitate the development of immunomodulatory and microbe‐modulatory therapies that promote intestinal homeostasis and colonization resistance.

title: Colonization resistance
abstract: In 1987 we reviewed the literature on the concept of colonization resistance (CR) (12). In this concept, the indigenous anaerobic flora limits the concentration of potentially pathogenic (mostly aerobic) flora in the digestive tract. This implies that the risk of superinfections by aerobic flora would be eliminated by selecting those antimicrobial agents which spare the anaerobic flora. The concept of CR was based on experiments in animals and uncontrolled observations in patients. It was not validated conclusively. Since publication of our previous review (12), we have improved the design for studying the influence of antimicrobial agents on CR, especially by using human volunteers and analyzing the data in each volunteer separately with singlepatient statistics (57, 63). This methodology was applied in the study of several antimicrobial agents (57-61, 63, 64). The resulting data provide a better understanding of the impact of antimicrobial agents on the microbial flora of the bowel. At present, it seems to be the case that some former conclusions were made prematurely, especially those pertaining to the availability of antimicrobial agents that do not impair CR (12, 47). This review will be restricted to a discussion of CR of the bowel.

title: A simple model host for identifying Gram-positive virulence factors
abstract: We demonstrate the use of the nematode Caenorhabditis elegans as a facile and inexpensive model host for several Gram-positive human bacterial pathogens. Enterococcus faecalis, Streptococcus pneumoniae, and Staphylococcus aureus, but not Bacillus subtilis, Enterococcus faecium, or Streptococcus pyogenes, kill adult C. elegans. Focusing our studies on the enterococcal species, we found that both E. faecalis and E. faecium kill C. elegans eggs and hatchlings, although only E. faecalis kills the adults. In the case of adults, a low inoculum of E. faecalis grows to a high titer in the C. elegans intestine, resulting in a persistent infection that cannot be eradicated by prolonged feeding on E. faecium. Interestingly, a high titer of E. faecium also accumulates in the nematode gut, but does not affect the longevity of the worms. Two E. faecalis virulence-related factors that play an important role in mammalian models of infection, fsr, a putative quorum-sensing system, and cytolysin, are also important for nematode killing. We exploit the apparent parallels between Gram-positive infection in simple and more complex organisms by using the nematode to identify an E. faecalis virulence factor, ScrB, which is relevant to mammalian pathogenesis.

title: Nutrition of the Enterococci
abstract: In connection with the isolation of pantothenic acid from natural materials, Woolley and Hutchings (1940) reported the development of a chemically defined medium for a strain of Streptococcus zymogenes, a hemolytic group D streptococcus. In addition to glucose, inorganic salts, buffer, and a reducing substance, the medium contained the three vtins, riboflavin, pantothenic acid, and pyridoxine; and seven amino acids. Schuman and Farrell (1941) extended this work and showed that a similar medium, differing somewhat in amino acid content, would support the growth of a culture of Streptococcus fecalis, a non-hemolytic group D streptococcus. Later, Woolley (1941a) reported that a closely related Streptococcus zymogenes strain would not grow in the synthetic medium unless a larger variety of amino acids was used. Nicotinic acid also stimulated' the growth of this strain. In this laboratory, attempts to culture a number of enterococcus cultures in the above media have failed. A study was.therefore undertaken to determine the additional substances required by these organisms, and also to determine whether or not the four major varieties or "species" within the enterococcus group have similar nutritive requirements.

title: The Enterococci and Related Streptococci
abstract: Quite out of line with the usual "Presidential Address" I have chosen to speak to you briefly about some of our work of the past few months. These studies have concerned the extension of the Lancefield serological technique to those species or types of streptococci which are usually considered "enterococci," as well as to certain related forms. In doing this I must make use of material which belongs quite as much to others as to me. These investigations have been made possible during a comparatively brief span of time, amid many other duties, only by the untiring industry of my associates, Dr. Floyd R. Smith, now of the University of California, and Mr. Charles F. Niven.

title: Metagenomic Analysis of the Human Distal Gut Microbiome
abstract: The human intestinal microbiota is composed of 1013 to 1014 microorganisms whose collective genome (“microbiome”) contains at least 100 times as many genes as our own genome. We analyzed ∼78 million base pairs of unique DNA sequence and 2062 polymerase chain reaction–amplified 16S ribosomal DNA sequences obtained from the fecal DNAs of two healthy adults. Using metabolic function analyses of identified genes, we compared our human genome with the average content of previously sequenced microbial genomes. Our microbiome has significantly enriched metabolism of glycans, amino acids, and xenobiotics; methanogenesis; and 2-methyl-d-erythritol 4-phosphate pathway–mediated biosynthesis of vitamins and isoprenoids. Thus, humans are superorganisms whose metabolism represents an amalgamation of microbial and human attributes.

title: Prokaryotes: the unseen majority.
abstract: The number of prokaryotes and the total amount of their cellular carbon on earth are estimated to be 4-6 x 10(30) cells and 350-550 Pg of C (1 Pg = 10(15) g), respectively. Thus, the total amount of prokaryotic carbon is 60-100% of the estimated total carbon in plants, and inclusion of prokaryotic carbon in global models will almost double estimates of the amount of carbon stored in living organisms. In addition, the earth's prokaryotes contain 85-130 Pg of N and 9-14 Pg of P, or about 10-fold more of these nutrients than do plants, and represent the largest pool of these nutrients in living organisms. Most of the earth's prokaryotes occur in the open ocean, in soil, and in oceanic and terrestrial subsurfaces, where the numbers of cells are 1.2 x 10(29), 2.6 x 10(29), 3.5 x 10(30), and 0. 25-2.5 x 10(30), respectively. The numbers of heterotrophic prokaryotes in the upper 200 m of the open ocean, the ocean below 200 m, and soil are consistent with average turnover times of 6-25 days, 0.8 yr, and 2.5 yr, respectively. Although subject to a great deal of uncertainty, the estimate for the average turnover time of prokaryotes in the subsurface is on the order of 1-2 x 10(3) yr. The cellular production rate for all prokaryotes on earth is estimated at 1.7 x 10(30) cells/yr and is highest in the open ocean. The large population size and rapid growth of prokaryotes provides an enormous capacity for genetic diversity.

title: Enterococci in the Environment
abstract: Enterococci are common, commensal members of gut communities in mammals and birds, yet they are also opportunistic pathogens that cause millions of human and animal infections annually. Because they are shed in human and animal feces, are readily culturable, and predict human health risks from exposure to polluted recreational waters, they are used as surrogates for waterborne pathogens and as fecal indicator bacteria (FIB) in research and in water quality testing throughout the world. Evidence from several decades of research demonstrates, however, that enterococci may be present in high densities in the absence of obvious fecal sources and that environmental reservoirs of these FIB are important sources and sinks, with the potential to impact water quality. This review focuses on the distribution and microbial ecology of enterococci in environmental (secondary) habitats, including the effect of environmental stressors; an outline of their known and apparent sources, sinks, and fluxes; and an overview of the use of enterococci as FIB. Finally, the significance of emerging methodologies, such as microbial source tracking (MST) and empirical predictive models, as tools in water quality monitoring is addressed. The mounting evidence for widespread extraenteric sources and reservoirs of enterococci demonstrates the versatility of the genus Enterococcus and argues for the necessity of a better understanding of their ecology in natural environments, as well as their roles as opportunistic pathogens and indicators of human pathogens.

title: Sunlight Inactivation of Fecal Indicator Bacteria and Bacteriophages from Waste Stabilization Pond Effluent in Fresh and Saline Waters
abstract: Sunlight inactivation in fresh (river) water of fecal coliforms, enterococci, Escherichia coli, somatic coliphages, and F-RNA phages from waste stabilization pond (WSP) effluent was compared. Ten experiments were conducted outdoors in 300-liter chambers, held at 14°C (mean river water temperature). Sunlight inactivation (kS) rates, as a function of cumulative global solar radiation (insolation), were all more than 10 times higher than the corresponding dark inactivation (kD) rates in enclosed (control) chambers. The overall kS ranking (from greatest to least inactivation) was as follows: enterococci > fecal coliforms ≥ E. coli > somatic coliphages > F-RNA phages. In winter, fecal coliform and enterococci inactivation rates were similar but, in summer, enterococci were inactivated far more rapidly. In four experiments that included freshwater-raw sewage mixtures, enterococci survived longer than fecal coliforms (a pattern opposite to that observed with the WSP effluent), but there was little difference in phage inactivation between effluents. In two experiments which included simulated estuarine water and seawater, sunlight inactivation of all of the indicators increased with increasing salinity. Inactivation rates in freshwater, as seen under different optical filters, decreased with the increase in the spectral cutoff (50% light transmission) wavelength. The enterococci and F-RNA phages were inactivated by a wide range of wavelengths, suggesting photooxidative damage. Inactivation of fecal coliforms and somatic coliphages was mainly by shorter (UV-B) wavelengths, a result consistent with photobiological damage. Fecal coliform repair mechanisms appear to be activated in WSPs, and the surviving cells exhibit greater sunlight resistance in natural waters than those from raw sewage. In contrast, enterococci appear to suffer photooxidative damage in WSPs, rendering them susceptible to further photooxidative damage after discharge. This suggests that they are unsuitable as indicators of WSP effluent discharges to natural waters. Although somatic coliphages are more sunlight resistant than the other indicators in seawater, F-RNA phages are the most resistant in freshwater, where they may thus better represent enteric virus survival.

title: Survival of Indicator and Pathogenic Bacteria in Bovine Feces on Pasture
abstract: The survival of enteric bacteria was measured in bovine feces on pasture. In each season, 11 cow pats were prepared from a mixture of fresh dairy cattle feces and sampled for up to 150 days. Four pats were analyzed for Escherichia coli, fecal streptococci, and enterococci, and four inoculated pats were analyzed for Campylobacter jejuni and Salmonella enterica. Two pats were placed on drainage collectors, and another pat was fitted with a temperature probe. In the first 1 to 3 weeks, there were increases (up to 1.5 orders of magnitude) in the counts of enterococci (in four seasons), E. coli (three seasons), fecal streptococci (three seasons), and S. enterica (two seasons), but there was no increase in the counts of C. jejuni. Thereafter, the counts decreased, giving an average ranking of the times necessary for 90% inactivation of C. jejuni (6.2 days from deposition) < fecal streptococci (35 days) < S. enterica (38 days) < E. coli (48 days) < enterococci (56 days). The pat temperature probably influenced bacterial growth, but the pattern of increases and decreases was primarily determined by desiccation; growth occurred when the water content was greater than 80%, but at a water content of 70 to 75% counts decreased. E. coli and enterococcus regrowth appeared to result from pat rehydration. Of 20 monthly leaching losses of E. coli, 16 were <10% of the total counts in the pat, and 12 were <1%. Drainage losses of C. jejuni (generally <1%) were detected for only 1 to 2 months. Although enterococci exhibited the best survival rate, higher final counts suggested that E. coli is the more practical indicator of bovine fecal pollution.

title: Influence of Soil on Fecal Indicator Organisms in a Tidally Influenced Subtropical Environment
abstract: The potential regrowth of fecal indicator bacteria released into coastal environments in recreational water bodies has been of concern, especially in tropical and subtropical areas where the number of these bacteria can be artificially elevated beyond that from fecal impacts alone. The task of determining the factors that influence indicator bacterial regrowth was addressed though a series of field sampling and laboratory experiments using in situ densities of Escherichia coli, enterococci, and Clostridium perfringens in river water, sediment, and soil. Field sampling efforts included the collection of surface sediments along the cross section of a riverbank, a 20-cm-deep soil core, and additional surface soils from remote locations. In addition to field sampling, two types of laboratory experiments were conducted. The first experiment investigated the survival of bacteria already present in river water with the addition of sterile and unsterile sediment. The second experiment was designed to simulate the wetting and drying effects due to tidal cycles. The results from the sampling study found elevated numbers of E. coli and C. perfringens in surficial sediments along the riverbank near the edge of the water. C. perfringens was found in high numbers in the subsurface samples obtained from the soil core. Results from laboratory experiments revealed a significant amount of regrowth for enterococci and E. coli with the simulation of tides and addition of sterile sediment. Regrowth was not observed for C. perfringens. This study demonstrates the need to further evaluate the characteristics of indicator microbes within tropical and subtropical water systems where natural vegetation, soil embankments, and long-term sediment accumulation are present. In such areas, the use of traditional indicator microbes to regulate recreational uses of a water body may not be appropriate.

title: Growth of Enterococci in Unaltered, Unseeded Beach Sands Subjected to Tidal Wetting
abstract: Enterococci are indicator bacteria used to assess the risk of acquiring enteric disease from swimming in marine waters. Previous work identified beach sands as reservoirs of enterococci which can be transported from the sand to the sea, where they may instigate beach advisories. The present study establishes that naturally occurring enterococci can replicate in beach sands under environmentally relevant conditions. In unseeded, nonsterile microcosm experiments, it was shown that intermittent wetting of sands by seawater, like that which would occur at the high tide line, stimulates the transient replication of enterococci at rates of 0.20 to 0.63 per day (equivalent to doubling times of 1.1 to 3.5 days). Replication was not observed in control microcosms that were not subjected to wetting. Enterococci were enumerated using both culture-dependent (membrane filtration and mEI media) and culture-independent (quantitative PCR [QPCR], 23S rRNA gene based) techniques, which allowed tracking of both culturable and total enterococcus populations. Inhibition of QPCR and DNA extraction efficiencies were accounted for in the interpretation of the QPCR results. The results provide evidence that enterococci may not be an appropriate indicator of enteric disease risk at recreational beaches subject to nonpoint sources of pollution.

title: Diurnal Variation in Enterococcus Species Composition in Polluted Ocean Water and a Potential Role for the Enterococcal Carotenoid in Protection against Photoinactivation
abstract: Enterococcus species composition was determined each hour for 72 h at a polluted marine beach in Avalon, Santa Catalina Island, CA. Species composition during the day was significantly different from that at night, based on an analysis of similarity. Enterococcus faecium and E. faecalis were more prevalent at night than during the day, while E. hirae and other Enterococcus species were more prevalent during the day than the night. Enterococcus spp. containing a yellow pigment were more common during the day than the night, suggesting that the pigmented phenotype may offer a competitive advantage under sunlit conditions. A laboratory microcosm experiment established that the pigmented E. casseliflavus isolate and a pigmented E. faecalis isolate recovered from the field site decay slower than a nonpigmented E. faecalis isolate in a solar simulator in simulated, clear seawater. This further supports the idea that the yellow carotenoid pigment in Enterococcus provides protection under sunlit conditions. The findings are in accordance with previous work with other carotenoid-containing nonphotosynthetic and photosynthetic bacteria that suggests that the carotenoid is able to quench reactive oxygen species capable of causing photoinactivation and photostress. The results suggest that using enterococcal species composition as a microbial source tracking tool may be hindered by the differential environmental persistence of pigmented and nonpigmented enterococci.

title: Survival of Enterococcus faecalis in an Oligotrophic Microcosm: Changes in Morphology, Development of General Stress Resistance, and Analysis of Protein Synthesis
abstract: The ability of Enterococcus faecalis to metabolically adapt to an oligotrophic environment has been analyzed. E. faecalis is able to survive for prolonged periods under conditions of complete starvation established by incubation in tap water. During incubation in this microcosm, cells developed a rippled cell surface with irregular shapes. Exponentially growing cells survived to the same extent as cells starved for glucose prior to exposure to the multiple nutrient deficient stress. Chloramphenicol treatment during incubation in tap water led to a rapid decline in plate counts for exponentially growing cells but showed progressively reduced influence on stationary-phase cells harvested after different times of glucose starvation. During incubation in the oligotrophic environment, cells from the exponential-growth phase and early-stationary phase became progressively more resistant to other environmental stresses (heat [62°C], acid [pH 3.3], UV254 nm light [180 J/m2], and sodium hypochlorite [0.05%]) until they reached a maximum of survival characteristic for each treatment. In contrast, cells starved of glucose for 24 h did not become more resistant to the different treatments during incubation in tap water. Our combined data suggest that energy starvation induces a response similar to that triggered by oligotrophy. Analysis of protein synthesis by two-dimensional gel electrophoresis revealed the enhanced synthesis of 51 proteins which were induced in the oligotrophic environment. A comparison of these oligotrophy-inducible proteins with the 42 glucose starvation-induced polypeptides (J. C. Giard, A. Hartke, S. Flahaut, P. Boutibonnes, and Y. Auffray,  1997) showed that 16 are common between the two different starvation conditions. These proteins and the corresponding genes seem to play a key role in the observed phenomena of long-term survival and development of general stress resistance of starved cultures of E. faecalis.

title: Survival of Enterococcus faecalis in Seawater Microcosms Is Limited in the Presence of Bacterivorous Zooflagellates
abstract: The survival and persistence of growing and starved cells of Enterococcus faecalis in untreated and differentially filtered (20 μm, 5 μm, 3 μm, 1.2 μm, and 0.1 μm) seawater was analyzed in samples taken at different times over a 1-year period by plate counts and scanning electron microscopy. Whereas seawater filtered through a 0.1-μm mesh was not at all or only slightly bactericidal during incubation at 16°C in the dark, culturability of E. faecalis in the other systems decreased as a function of increasing pore size of the filters. Recovery of culturable, glucose pre-starved cells was always higher than that of cells harvested from the exponential growth phase. Electron microscopic analysis showed that the disappearance of enterococci appeared related to the presence and multiplication of various zooflagellates.

title: Bacteriophage lysis of Enterococcus host strains: a tool for microbial source tracking?
abstract: This paper describes the isolation of Enterococcus host strains, for potential use as simple bacteriophage (phage)-based microbial source tracking (MST) tools. Presumptive Enterococcus host strains were isolated from cattle feces, raw municipal wastewater, agricultural runoff, and waters impacted by farms or wastewater treatment works (WWTW) in southern England, United Kingdom (UK). All enterococcal host strains (n = 390) were first screened for their ability to detect phage in samples of raw municipal wastewater and fecal material from cattle, pigs, and sheep. Host strains that detected phage (n = 147) were ranked according to both their specificity to a particular fecal source and also the number of phages (expressed as plaque-forming units, PFU) that they detected per milliliter of sample. Host strains that demonstrated host specificity and which detected phages at levels greater than 100 PFU/mL (n = 29) were further tested using additional fecal samples of human and nonhuman origin. The specificity and sensitivity of the enterococcal host strains were found to vary, ranging from 44 to 100% and from 17 to 83%, respectively. Most notably, seven strains exhibited 100% specificity to either cattle, human, or pig samples. Isolates exhibiting specificity to cattle were identified as belonging to the species Enterococcus casseliflavus , Enterococcus mundtii , or Enterococcus gallinarum , while human and pig isolates were members of either Enterococcus faecium or Enterococcus faecalis . The high specificity of phages infecting Enterococcus hosts and the simplicity and relatively low cost of the approach collectively indicate a strong potential for using this method as a tool in MST.

title: ECOLOGICAL FACTORS IN SPECIATION
abstract: The past decades have seen the gradual formulation of the theory of geographical speciation, principally based on work in the fields of the systematics of mammals, birds, insects, and mollusks. This theory 'postulates that in bisexual animals a new species can develop only "if a population, which has become geographically isolated from its parental species, acquires during this period of isolation characters which prorhote or guarantee reproductive isolation when 'the external barriers break down" (Mayr, 1942). A number of workers in recent years have considered this statement an oversimplification. The omission of any reference in this description of the speciation process to the ecological factors-so vitally involved in the process of speciation-has also been objected to. Even though the importance of geographical speciation is universally admitted, there are a number of authors, particularly among the ichthyologists and entomologists, who believe that in addition to geographical speciation there is another process of speciation, variously referred to as ecological or sympatric speciation. Thorpe (1945), for example, is inclined to consider that in addition to geographical speciation there is a speciation process which is characterized by the fact "that local differences of habit may be the starting point for the evolution of new species." It appears to me that there is no real conflict between those authors who stress the ecological aspects of speciation, such as Stresemann (1943) and Thorpe (1945), and those who, like myself, have stressed the geographical aspects. This was pointed out by Timofeeff-Ressovsky (1943). The seeming differences are due partly to misunderstandings and partly to a different emphasis placed on various aspects of a single process of speciation. The field has suffered from looseness of thought and vagueness of expression, and it may therefore be ulseful to attempt a fresh analysis and possibly a synthesis of the two viewpoints. The principal obstacle blocking such synthesis up to now has been that those authors who have emphasized the role of ecological factors in speciation generally accept sympatric speciation as an integral part of ecological speciation. To remove this difficulty, a special section (pp. 269-285) has been devoted to a discussion of sympatric speciation. It is more important, however, to determine the respective roles of geographical and ecological factors in speciation, and to find out whether they operate consecutively or concomitantly. The field has suffered particularly from the lack of a clear-cut Fragestellung. The result has been that only few investigations have ever been undertaken which would give decisive answers to the unsolved questions. Most of the published studies sadly miss the crucial points. It is one additional object of this discussion to focus the attention of investigators on those aspects that are in particular need of further study.

title: The commensal microbiology of the gastrointestinal tract.
abstract: The gastrointestinal (GI) tract is a dynamic environment and therefore the stability of the commensal community, or microbiota, is under constant challenge. Microscopic observations have revealed that the majority of bacteria present in the GI tract are not detected using standard culturing techniques, however with the application of culture-independent techniques it has been estimated that between 500 to 1000 bacterial species inhabit the human GI tract. Numerically predominant organisms in the microbiota belong to two eubacterial divisions, the Cytophaga-Flavobacterium-Bacteroides (CFB) and the Firmicutes, and fall into three main groups; Clostridium rRNA subcluster XIVa, Clostridium rRNA subcluster IV and Bacteroides. The prevalence and diversity of bacteria in different areas of the GI tract is influenced by the different conditions at these sites and thus the microbiota of the stomach and jejunum varies with that of the large intestine. Additionally, host genotype, age and diet have all been shown to affect microbial diversity in the GI tract. The distal intestine harbours the highest bacterial cell densities for any known ecosystem. Characterizing the species composition of the healthy microbiota may be a key step in identifying bacterial or associated physiological conditions that are present or absent in an unhealthy microbiota.

title: Carriage of group D streptococci in the human bowel.
abstract: Sixty faecal samples, 39 from adults and 21 from neonates, were investigated by means of a simple scheme to isolate and identify any group D streptococci present. A number of differences were found between the two groups. Group D streptococci were absent from 43% of the neonates compared with only 13% of the adults; Streptococcus bovis was commoner in the neonates (23.8%) than in the adults (5%), and Streptococcus faecium was not isolated from any of the infant samples although it was common in the adult samples (25%). The viable counts of Strep. faecium were found to be, on average, 100-fold lower than those of Streptococcus faecalis. The methods and results are discussed with reference to the clinical significance of group D streptococci, especially in bacterial endocarditis and carcinoma of the colon.

title: Diet rapidly and reproducibly alters the human gut microbiome
abstract: Long-term dietary intake influences the structure and activity of the trillions of microorganisms residing in the human gut, but it remains unclear how rapidly and reproducibly the human gut microbiome responds to short-term macronutrient change. Here we show that the short-term consumption of diets composed entirely of animal or plant products alters microbial community structure and overwhelms inter-individual differences in microbial gene expression. The animal-based diet increased the abundance of bile-tolerant microorganisms (Alistipes, Bilophila and Bacteroides) and decreased the levels of Firmicutes that metabolize dietary plant polysaccharides (Roseburia, Eubacterium rectale and Ruminococcus bromii). Microbial activity mirrored differences between herbivorous and carnivorous mammals, reflecting trade-offs between carbohydrate and protein fermentation. Foodborne microbes from both diets transiently colonized the gut, including bacteria, fungi and even viruses. Finally, increases in the abundance and activity of Bilophila wadsworthia on the animal-based diet support a link between dietary fat, bile acids and the outgrowth of microorganisms capable of triggering inflammatory bowel disease. In concert, these results demonstrate that the gut microbiome can rapidly respond to altered diet, potentially facilitating the diversity of human dietary lifestyles.

title: Nonclinical and Clinical Enterococcus faecium Strains, but Not Enterococcus faecalis Strains, Have Distinct Structural and Functional Genomic Features
abstract: Certain strains of Enterococcus faecium and Enterococcus faecalis contribute beneficially to animal health and food production, while others are associated with nosocomial infections. To determine whether there are structural and functional genomic features that are distinct between nonclinical (NC) and clinical (CL) strains of those species, we analyzed the genomes of 31 E. faecium and 38 E. faecalis strains. Hierarchical clustering of 7,017 orthologs found in the E. faecium pangenome revealed that NC strains clustered into two clades and are distinct from CL strains. NC E. faecium genomes are significantly smaller than CL genomes, and this difference was partly explained by significantly fewer mobile genetic elements (ME), virulence factors (VF), and antibiotic resistance (AR) genes. E. faecium ortholog comparisons identified 68 and 153 genes that are enriched for NC and CL strains, respectively. Proximity analysis showed that CL-enriched loci, and not NC-enriched loci, are more frequently colocalized on the genome with ME. In CL genomes, AR genes are also colocalized with ME, and VF are more frequently associated with CL-enriched loci. Genes in 23 functional groups are also differentially enriched between NC and CL E. faecium genomes. In contrast, differences were not observed between NC and CL E. faecalis genomes despite their having larger genomes than E. faecium. Our findings show that unlike E. faecalis, NC and CL E. faecium strains are equipped with distinct structural and functional genomic features indicative of adaptation to different environments.

title: Genetic functions and cell–cell interactions in the pheromone‐inducible plasmid transfer system of Enterococcus faecalis
abstract: Pheromone‐inducible plasmid transfer is a novel form of bacterial conjugation which has, to date, been observed only in Enterococcus (Streptococcus) faecalis. This process includes several important stages of interaction between the donor and recipient cell. The initial interaction is the transmission of a chemical signal from the recipient to the donor cell. Recent evidence has shown that the signal is in the form of a small hydrophobic peptide, which is capable of inducing a complex mating response in the donor cell at concentrations as low as 1–5 molecules per responder cell. Most E. faecalis strains produce multiple pheromones, each of which induces a response only in cells carrying a particular plasmid (or member of a family of related plasmids). Genetic functions ascribed to the pheromone response include: (i) cell‐cell aggregation, which promotes initial close contact between mating cells; (ii) surface exclusion, which prevents plasmid transfer between aggregated donor cells; and (iii) highly efficient DNA transfer, which requires other unidentified functions in addition to aggregation. The first two processes appear to be mediated by proteinaceous surface antigens.

title: Pheromone-inducible conjugation in Enterococcus faecalis: interbacterial and host-parasite chemical communication
abstract: Conjugation is a major mechanism of horizontal genetic transfer in gram-positive cocci and the genes transferred often include antibiotic resistance and virulence determinants (9, 14, 15, 40). A number of enterococcal conjugative plasmids can transfer very efficiently (frequencies as high as 5 3 10 ) in liquid cultures. Expression of this highly efficient transfer system (which to date has been shown to be present only in the enterococci) in donor cells is induced by a peptide signal (pheromone) produced by recipient cells. Most work in this area has focused on two plasmids, the hemolysin-bacteriocin plasmid pAD1 (62), which has been described in detail in recent reviews by Clewell (10, 11) andWirth (69), and the tetracycline resistance plasmid pCF10 (16). These plasmids share many common features at the molecular and genetic levels and exhibit some very interesting differences. This minireview will focus on the cell surface receptors involved in mating pair formation and on the molecular mechanisms by which the pheromone acts as a chemical signal to induce expression of the transfer functions, with emphasis on pCF10. We will also briefly discuss possible roles in hostparasite interactions for the signalling systems and for the surface components involved in mating aggregate formation.

title: Mechanism of chromosomal transfer of Enterococcus faecalis pathogenicity island, capsule, antimicrobial resistance, and other traits
abstract: The Enterococcus faecalis pathogenicity island (PAI) encodes known virulence traits and >100 additional genes with unknown roles in enterococcal biology. Phage-related integration and excision genes, and direct repeats flanking the island, suggest it moves as an integrative conjugative element (ICE). However, transfer was observed not to require these genes. Transfer only occurred from donors possessing a pheromone responsive-type of conjugative plasmid, and was invariably accompanied by transfer of flanking donor chromosome sequences. Deletion of plasmid transfer functions, including the cis-acting origin of transfer (oriT), abolished movement. In addition to demonstrating PAI movement by a mechanism involving plasmid integration, we observed transfer of a selectable marker placed virtually anywhere on the chromosome. Transfer of this selectable marker was observed to be accompanied by chromosome-chromosome transfer of vancomycin resistance, MLST markers, and capsule genes as well. Plasmid mobilization therefore appears to be a major mechanism for horizontal gene transfer in the evolution of antibiotic resistant E. faecalis strains capable of causing human infection.

title: The Origins of C4 Grasslands: Integrating Evolutionary and Ecosystem Science
abstract: Grassland Emergence The evolution of the C4 photosynthetic pathway from the ancestral C3 pathway in grasses led to the establishment of grasslands in warm climates during the Late Miocene (8 to 3 million years ago). This was a major event in plant evolutionary history, and their high rates of foliage production sustained high levels of herbivore consumption. The past decade has seen significant advances in understanding C4 grassland ecosystem ecology, and now a wealth of data on the geological history of these ecosystems has accumulated and the phylogeny of grasses is much better known. Edwards et al. (p. 587) review this multidisciplinary research area and attempt to synthesize emerging knowledge about the evolution of grass species within the context of plant and ecosystem ecology. The evolution of grasses using C4 photosynthesis and their sudden rise to ecological dominance 3 to 8 million years ago is among the most dramatic examples of biome assembly in the geological record. A growing body of work suggests that the patterns and drivers of C4 grassland expansion were considerably more complex than originally assumed. Previous research has benefited substantially from dialog between geologists and ecologists, but current research must now integrate fully with phylogenetics. A synthesis of grass evolutionary biology with grassland ecosystem science will further our knowledge of the evolution of traits that promote dominance in grassland systems and will provide a new context in which to evaluate the relative importance of C4 photosynthesis in transforming ecosystems across large regions of Earth.

title: Host-pathogen interactions: the attributes of virulence.
abstract: Virulence is one of a number of possible outcomes of host-microbe interaction. As such, microbial virulence is dependent on host factors, as exemplified by the pathogenicity of avirulent microbes in immunocompromised hosts and the lack of pathogenicity of virulent pathogens in immune hosts. Pathogen-centered views of virulence assert that pathogens are distinguished from nonpathogens by their expression of virulence factors. Although this concept appears to apply to certain microbes that cause disease in normal hosts, it does not apply to most microbes that cause disease primarily in immunocompromised hosts. The study of virulence is fraught with the paradox that virulence, despite being a microbial characteristic, can only be expressed in a susceptible host. Thus, the question "What is a pathogen?" begs the question, "What is the outcome of the host-microbe interaction?" We propose that host damage provides a common denominator that translates into the different outcomes of host-microbe interaction.

title: Host resistance influences patterns of experimental viral adaptation and virulence evolution
abstract: Infectious diseases are major threats to all living systems, so understanding the forces of selection that limit the evolution of more virulent pathogens is of fundamental importance; this includes the practical application of identifying possible mitigation strategies for at-risk host populations. The evolution of more virulent pathogens has been classically understood to be limited by the tradeoff between within-host growth rate and transmissibility. Importantly, heterogeneity among hosts can influence both of these factors. However, despite our substantial understanding of how the immune system operates to control pathogen replication during infection, we have only a limited appreciation of how variability in intrinsic (i.e., genetically determined) levels of host resistance influences patterns of pathogen adaptation and virulence evolution. Here, we describe results from experimental evolution studies using a model host–pathogen (virus–mammal) system; we demonstrate that variability in intrinsic levels of resistance among host genotypes can have significant effects on patterns of pathogen adaptation and virulence evolution during serial passage. Both the magnitude of adaptive response as well as the degree of pathogen specialization was positively correlated with host resistance, while mean overall virulence of post-passage virus was negatively correlated with host resistance. These results are consistent with a model whereby resistant host genotypes impose stronger selection on adapting pathogen populations, which in turn leads to the evolution of more specialized pathogen variants whose overall (i.e., mean) virulence across host genotypes is reduced.

title: Evolution of Virulence in a Plant Host-Pathogen Metapopulation
abstract: In a wild plant–pathogen system, host resistance and pathogen virulence varied markedly among local populations. Broadly virulent pathogens occurred more frequently in highly resistant host populations, whereas avirulent pathogens dominated susceptible populations. Experimental inoculations indicated a negative trade-off between spore production and virulence. The nonrandom spatial distribution of pathogens, maintained through time despite high pathogen mobility, implies that selection favors virulent strains ofMelampsora lini in resistant Linum marginalepopulations and avirulent strains in susceptible populations. These results are consistent with gene-for-gene models of host-pathogen coevolution that require trade-offs to prevent pathogen virulence increasing until host resistance becomes selectively neutral.

title: Evidence supporting an intentional Neandertal burial at La Chapelle-aux-Saints
abstract: For several decades, scholars have questioned the existence of burial in Western Europe prior to the arrival of Anatomically Modern Humans. Therefore, an approach combining a global field recovery and the reexamination of the previously discovered Neandertal remains has been undertaken in the site of La Chapelle-aux-Saints (France), where the hypothesis of a Neandertal burial was raised for the first time. This project has concluded that the Neandertal of La Chapelle-aux-Saints was deposit in a pit dug by other members of its group and protected by a rapid covering from any disturbance. These discoveries attest the existence of West European Neandertal burial and of the Neandertal cognitive capacity to produce it. The bouffia Bonneval at La Chapelle-aux-Saints is well known for the discovery of the first secure Neandertal burial in the early 20th century. However, the intentionality of the burial remains an issue of some debate. Here, we present the results of a 12-y fieldwork project, along with a taphonomic analysis of the human remains, designed to assess the funerary context of the La Chapelle-aux-Saints Neandertal. We have established the anthropogenic nature of the burial pit and underlined the taphonomic evidence of a rapid burial of the body. These multiple lines of evidence support the hypothesis of an intentional burial. Finally, the discovery of skeletal elements belonging to the original La Chapelle aux Saints 1 individual, two additional young individuals, and a second adult in the bouffia Bonneval highlights a more complex site-formation history than previously proposed.

title: Evolution of virulence in opportunistic pathogens: generalism, plasticity, and control
abstract: Standard virulence evolution theory assumes that virulence factors are maintained because they aid parasitic exploitation, increasing growth within and/or transmission between hosts. An increasing number of studies now demonstrate that many opportunistic pathogens (OPs) do not conform to these assumptions, with virulence factors maintained instead because of advantages in non-parasitic contexts. Here we review virulence evolution theory in the context of OPs and highlight the importance of incorporating environments outside a focal virulence site. We illustrate that virulence selection is constrained by correlations between these external and focal settings and pinpoint drivers of key environmental correlations, with a focus on generalist strategies and phenotypic plasticity. We end with a summary of key theoretical and empirical challenges to be met for a fuller understanding of OPs.

title: Identification of high-risk enterococcal clonal complexes: global dispersion and antibiotic resistance.
abstract: Vancomycin-resistant Enterococcus faecium spread dramatically in hospital settings in the USA in the 1990s and reached endemicity at the turn of the century. Similarly, rising prevalence rates are currently observed in several European countries, with prevalence rates of greater than 10% reported in seven of these. On the basis of multilocus sequence typing (MLST), the population structure of E. faecium was elucidated and the existence of a distinct high-risk enterococcal clonal complex, designated clonal complex-17 (CC17), which is associated with the majority of hospital outbreaks and clinical infections in five continents, was revealed. This complex is correlated with ampicillin and quinolone resistance and with the presence of a putative pathogenicity island. Preliminary MLST data suggest that similar hospital-adapted complexes might also exist in E. faecalis.

title: Genetic Diversity among Enterococcus faecalis
abstract: Enterococcus faecalis, a ubiquitous member of mammalian gastrointestinal flora, is a leading cause of nosocomial infections and a growing public health concern. The enterococci responsible for these infections are often resistant to multiple antibiotics and have become notorious for their ability to acquire and disseminate antibiotic resistances. In the current study, we examined genetic relationships among 106 strains of E. faecalis isolated over the past 100 years, including strains identified for their diversity and used historically for serotyping, strains that have been adapted for laboratory use, and isolates from previously described E. faecalis infection outbreaks. This collection also includes isolates first characterized as having novel plasmids, virulence traits, antibiotic resistances, and pathogenicity island (PAI) components. We evaluated variation in factors contributing to pathogenicity, including toxin production, antibiotic resistance, polymorphism in the capsule (cps) operon, pathogenicity island (PAI) gene content, and other accessory factors. This information was correlated with multi-locus sequence typing (MLST) data, which was used to define genetic lineages. Our findings show that virulence and antibiotic resistance traits can be found within many diverse lineages of E. faecalis. However, lineages have emerged that have caused infection outbreaks globally, in which several new antibiotic resistances have entered the species, and in which virulence traits have converged. Comparing genomic hybridization profiles, using a microarray, of strains identified by MLST as spanning the diversity of the species, allowed us to identify the core E. faecalis genome as consisting of an estimated 2057 unique genes.

title: Relationships between enterococcal virulence and antimicrobial resistance.
abstract: Enterococci have become a vexing problem in clinical medicine because of their ability to infect patients who are typically receiving antibiotic therapy for unrelated underlying illness. Moreover, the infections have become extremely difficult to manage because of the accumulation of antibiotic resistances among enterococci. The ability of enterococci to cause disease is an intrinsic property of the organism or possibly subpopulations within enterococcal species. The probability of an infection's becoming established, however, is almost certainly in part a function of the enterococcal burden. By altering endogenous bacterial flora, antibiotic therapy promotes increased colonization by antibiotic-resistant organisms. Therefore, antibiotic resistance and intrinsic virulence both contribute to disease, but in separate and complementary ways. We review the virulence of enterococci, as distinct from the acquisition of antimicrobial resistance genes, and identify current gaps in our understanding of enterococcal virulence and the basis for disease.

title: Multilocus Sequence Typing Scheme for Enterococcus faecalis Reveals Hospital-Adapted Genetic Complexes in a Background of High Rates of Recombination
abstract: A multilocus sequence typing (MLST) scheme based on seven housekeeping genes was used to investigate the epidemiology and population structure of Enterococcus faecalis. MLST of 110 isolates from different sources and geographic locations revealed 55 different sequence types that grouped into four major clonal complexes (CC2, CC9, CC10, and CC21) by use of eBURST. Two of these clonal complexes, CC2 and CC9, are particularly fit in the hospital environment, as CC2 includes the previously described BVE clonal complex identified by an alternative MLST scheme and CC9 includes exclusively isolates from hospitalized patients. Identical alleles were found in genetically diverse isolates with no linkage disequilibrium, while the different MLST loci gave incongruent phylogenetic trees. This demonstrates that recombination is an important mechanism driving genetic variation in E. faecalis and suggests an epidemic population structure for E. faecalis. Our novel MLST scheme provides an excellent tool for investigating local and short-term epidemiology as well as global epidemiology, population structure, and genetic evolution of E. faecalis.

title: Comparative genomic analysis reveals significant enrichment of mobile genetic elements and genes encoding surface structure-proteins in hospital-associated clonal complex 2 Enterococcus faecalis
abstract: BackgroundEnterococci rank among the leading causes of nosocomial infections. The failure to identify pathogen-specific genes in Enterococcus faecalis has led to a hypothesis where the virulence of different strains may be linked to strain-specific genes, and where the combined endeavor of the different gene-sets result in the ability to cause infection. Population structure studies by multilocus sequence typing have defined distinct clonal complexes (CC) of E. faecalis enriched in hospitalized patients (CC2, CC9, CC28 and CC40).ResultsIn the present study, we have used a comparative genomic approach to investigate gene content in 63 E. faecalis strains, with a special focus on CC2. Statistical analysis using Fisher's exact test revealed 252 significantly enriched genes among CC2-strains. The majority of these genes were located within the previously defined mobile elements phage03 (n = 51), efaB5 (n = 34) and a vanB associated genomic island (n = 55). Moreover, a CC2-enriched genomic islet (EF3217 to -27), encoding a putative phage related element within the V583 genome, was identified. From the draft genomes of CC2-strains HH22 and TX0104, we also identified a CC2-enriched non-V583 locus associated with the E. faecalis pathogenicity island (PAI). Interestingly, surface related structures (including MSCRAMMs, internalin-like and WxL protein-coding genes) implicated in virulence were significantly overrepresented (9.1%; p = 0.036, Fisher's exact test) among the CC2-enriched genes.ConclusionIn conclusion, we have identified a set of genes with potential roles in adaptation or persistence in the hospital environment, and that might contribute to the ability of CC2 E. faecalis isolates to cause disease.

title: Emergence of Epidemic Multidrug-Resistant Enterococcus faecium from Animal and Commensal Strains
abstract: Enterococcus faecium, natively a gut commensal organism, emerged as a leading cause of multidrug-resistant hospital-acquired infection in the 1980s. As the living record of its adaptation to changes in habitat, we sequenced the genomes of 51 strains, isolated from various ecological environments, to understand how E. faecium emerged as a leading hospital pathogen. Because of the scale and diversity of the sampled strains, we were able to resolve the lineage responsible for epidemic, multidrug-resistant human infection from other strains and to measure the evolutionary distances between groups. We found that the epidemic hospital-adapted lineage is rapidly evolving and emerged approximately 75 years ago, concomitant with the introduction of antibiotics, from a population that included the majority of animal strains, and not from human commensal lines. We further found that the lineage that included most strains of animal origin diverged from the main human commensal line approximately 3,000 years ago, a time that corresponds to increasing urbanization of humans, development of hygienic practices, and domestication of animals, which we speculate contributed to their ecological separation. Each bifurcation was accompanied by the acquisition of new metabolic capabilities and colonization traits on mobile elements and the loss of function and genome remodeling associated with mobile element insertion and movement. As a result, diversity within the species, in terms of sequence divergence as well as gene content, spans a range usually associated with speciation.  Enterococci, in particular vancomycin-resistant Enterococcus faecium, recently emerged as a leading cause of hospital-acquired infection worldwide. In this study, we examined genome sequence data to understand the bacterial adaptations that accompanied this transformation from microbes that existed for eons as members of host microbiota. We observed changes in the genomes that paralleled changes in human behavior. An initial bifurcation within the species appears to have occurred at a time that corresponds to the urbanization of humans and domestication of animals, and a more recent bifurcation parallels the introduction of antibiotics in medicine and agriculture. In response to the opportunity to fill niches associated with changes in human activity, a rapidly evolving lineage emerged, a lineage responsible for the vast majority of multidrug-resistant E. faecium infections. Enterococci, in particular vancomycin-resistant Enterococcus faecium, recently emerged as a leading cause of hospital-acquired infection worldwide. In this study, we examined genome sequence data to understand the bacterial adaptations that accompanied this transformation from microbes that existed for eons as members of host microbiota. We observed changes in the genomes that paralleled changes in human behavior. An initial bifurcation within the species appears to have occurred at a time that corresponds to the urbanization of humans and domestication of animals, and a more recent bifurcation parallels the introduction of antibiotics in medicine and agriculture. In response to the opportunity to fill niches associated with changes in human activity, a rapidly evolving lineage emerged, a lineage responsible for the vast majority of multidrug-resistant E. faecium infections.

title: The rise of the Enterococcus: beyond vancomycin resistance
abstract: The genus Enterococcus includes some of the most important nosocomial multidrug-resistant organisms, and these pathogens usually affect patients who are debilitated by other, concurrent illnesses and undergoing prolonged hospitalization. This Review discusses the factors involved in the changing epidemiology of enterococcal infections, with an emphasis on Enterococcus faecium as an emergent and challenging nosocomial problem. The effects of antibiotics on the gut microbiota and on colonization with vancomycin-resistant enterococci are highlighted, including how enterococci benefit from the antibiotic-mediated eradication of Gram-negative members of the gut microbiota. Analyses of enterococcal genomes indicate that there are certain genetic lineages, including an E. faecium clade of ancient origin, with the ability to succeed in the hospital environment, and the possible virulence determinants that are found in these genetic lineages are discussed. Finally, we review the most important mechanisms of resistance to the antibiotics that are used to treat vancomycin-resistant enterococci.

title: Modulation of virulence within a pathogenicity island in vancomycin-resistant Enterococcus faecalis
abstract: Enterococci are members of the healthy human intestinal flora, but are also leading causes of highly antibiotic-resistant, hospital-acquired infection. We examined the genomes of a strain of Enterococcus faecalis that caused an infectious outbreak in a hospital ward in the mid-1980s (ref. 2), and a strain that was identified as the first vancomycin-resistant isolate in the United States, and found that virulence determinants were clustered on a large pathogenicity island, a genetic element previously unknown in this genus. The pathogenicity island, which varies only subtly between strains, is approximately 150 kilobases in size, has a lower G + C content than the rest of the genome, and is flanked by terminal repeats. Here we show that subtle variations within the structure of the pathogenicity island enable strains harbouring the element to modulate virulence, and that these variations occur at high frequency. Moreover, the enterococcal pathogenicity island, in addition to coding for most known auxiliary traits that enhance virulence of the organism, includes a number of additional, previously unstudied genes that are rare in non-infection-derived isolates, identifying a class of new targets associated with disease which are not essential for the commensal behaviour of the organism.

title: Multiple Functional Domains of Enterococcus faecalis Aggregation Substance Asc10 Contribute to Endocarditis Virulence
abstract: Aggregation substance proteins encoded by sex pheromone plasmids increase the virulence of Enterococcus faecalis in experimental pathogenesis models, including infectious endocarditis models. These large surface proteins may contain multiple functional domains involved in various interactions with other bacterial cells and with the mammalian host. Aggregation substance Asc10, encoded by plasmid pCF10, is induced during growth in the mammalian bloodstream, and pCF10 carriage gives E. faecalis a significant selective advantage in this environment. We employed a rabbit model to investigate the role of various functional domains of Asc10 in endocarditis. The data suggested that the bacterial load of the infected tissue was the best indicator of virulence. Isogenic strains carrying either no plasmid, wild-type pCF10, a pCF10 derivative with an in-frame deletion of the prgB gene encoding Asc10, or pCF10 derivatives expressing other alleles of prgB were examined in this model. Previously identified aggregation domains contributed to the virulence associated with the wild-type protein, and a strain expressing an Asc10 derivative in which glycine residues in two RGD motifs were changed to alanine residues showed the greatest reduction in virulence. Remarkably, this strain and the strain carrying the pCF10 derivative with the in-frame deletion of prgB were both significantly less virulent than an isogenic plasmid-free strain. The data demonstrate that multiple functional domains are important in Asc10-mediated interactions with the host during the course of experimental endocarditis and that in the absence of a functional prgB gene, pCF10 carriage is actually disadvantageous in vivo.

title: The ecology, epidemiology and virulence of Enterococcus.
abstract: Enterococci are Gram-positive, catalase-negative, non-spore-forming, facultative anaerobic bacteria, which usually inhabit the alimentary tract of humans in addition to being isolated from environmental and animal sources. They are able to survive a range of stresses and hostile environments, including those of extreme temperature (5-65 degrees C), pH (4.5-10.0) and high NaCl concentration, enabling them to colonize a wide range of niches. Virulence factors of enterococci include the extracellular protein Esp and aggregation substances (Agg), both of which aid in colonization of the host. The nosocomial pathogenicity of enterococci has emerged in recent years, as well as increasing resistance to glycopeptide antibiotics. Understanding the ecology, epidemiology and virulence of Enterococcus species is important for limiting urinary tract infections, hepatobiliary sepsis, endocarditis, surgical wound infection, bacteraemia and neonatal sepsis, and also stemming the further development of antibiotic resistance.

title: Aggregation Substance Promotes Adherence, Phagocytosis, and Intracellular Survival of Enterococcus faecalis within Human Macrophages and Suppresses Respiratory Burst
abstract: The aggregation substance (AS) of Enterococcus faecalis, encoded on sex pheromone plasmids, is a surface-bound glycoprotein that mediates aggregation between bacteria thereby facilitating plasmid transfer. Sequencing of the pAD1-encoded Asa1 revealed that this surface protein contains two RGD motifs which are known to ligate integrins. Therefore, we investigated the influence of AS on the interaction of E. faecalis with human monocyte-derived macrophages which constitutively express β2 integrins (e.g., CD18). AS was found to cause a greater-than-fivefold increase in enterococcal adherence to macrophages and a greater-than-sevenfold increase in phagocytosis. Adherence was mediated by an interaction between the RGD motif and the integrin CD11b/CD18 (complement receptor type 3) as demonstrated by inhibition studies with monoclonal antibodies and RGD peptide. AS-bearing enterococci were significantly more resistant to macrophage killing during the first 3 h postinfection, probably due to inhibition of the respiratory burst as indicated by reduced concentrations of superoxide anion.

title: Aggregation substance of Enterococcus faecalis mediates adhesion to cultured renal tubular cells
abstract: The sex pheromone system of Enterococcus faecalis is a unique, highly efficient plasmid collection mechanism for this species. A crucial role in this system is played by an adhesin called aggregation substance which enables the cell-cell contact between donor and recipient strains. The existence of the amino acid motif Arg-Gly-Asp-Ser in the adhesin prompted us to look for a possible binding of E. faecalis cells expressing aggregation substance to eucaryotic cells. We were able to show that the adhesin mediated binding to cultured renal tubular cells (porcine cell line LLC-PK1) via light microscopic, electron microscopic, and enzyme-linked immunosorbent assay-based studies. Synthesis of the adhesin was induced by some component(s) of serum. These data are interpreted to mean that aggregation substance is an adhesin mediating not only cell-cell contact between different E. faecalis strains but also binding of E. faecalis to eucaryotic cells, and therefore it might contribute to virulence.

title: A plasmid-encoded surface protein on Enterococcus faecalis augments its internalization by cultured intestinal epithelial cells.
abstract: Aggregation substance, a plasmid-encoded Enterococcus faecalis surface protein, plays a role in mediating the formation of mating aggregates, resulting in plasmid transfer. The role of aggregation substance in the internalization of E. faecalis by cultured intestinal epithelial cells, namely HT-29 cells, was analyzed. It was associated with a significant increase in endocytosis of E. faecalis by HT-29 cells: Numbers of internalized enterococci were fewer than of an invasive strain of Listeria monocytogenes, similar to Salmonella typhimurium and another L. monocytogenes strain, and greater than relatively noninvasive strains of E. faecalis, Proteus mirabilis, and Escherichia coli. Electron microscopy confirmed aggregation substance on the surface of strains interacting with the enterocyte microvillous surface, and intracellular enterococci were localized within membrane-bound vacuoles in the enterocyte cytoplasm. Thus, aggregation substance may facilitate E. faecalis internalization by host epithelial cells.

title: Plasmid-associated hemolysin and aggregation substance production contribute to virulence in experimental enterococcal endocarditis
abstract: A rabbit endocarditis model was utilized to evaluate the virulence conferred by the conjugative plasmid pAD1 with the following strains: Enterococcus faecalis plasmid-free FA2-2 and FA2-2 containing plasmids pAD1 (hemolysin and aggregation substance positive), pAM9058 (insertional inactivation of hemolysin), and pAM944 or pAM947 (insertional inactivation of aggregation substance). All isolates were similar in ability to produce endocarditis. Mean vegetation weight was greater in animals inoculated with strains that produced aggregation substance (P < 0.01). Mortality was significantly increased in animals given FA2-2 containing pAD1 compared with those given all other strains (P < 0.01). These results suggest that the combination of hemolysin and aggregation substance is associated with increased mortality and that vegetation weight is associated with production of aggregation substance in experimental E. faecalis endocarditis.

title: Inducible Expression of Enterococcus faecalis Aggregation Substance Surface Protein Facilitates Bacterial Internalization by Cultured Enterocytes
abstract: Aggregation substance (AS) is an Enterococcus faecalissurface protein that may contribute to virulence. Using a recently described system for controlled expression of AS in E. faecalis and the heterologous host Lactococcus lactis, experiments were designed to assess the effect of AS on bacterial internalization by HT-29 and Caco-2 enterocytes. AS expression was associated with increased internalization of E. faecalis by HT-29 enterocytes and of L. lactis by HT-29 and Caco-2 enterocytes. Compared to enterocytes cultivated under standard conditions, either cultivation in hypoxia or 1-h pretreatment of enterocytes with calcium-free medium resulted in increased internalization of both E. faecalis and L. lactis (with and without AS expression). Also, AS expression augmented these increases when E. faecalis was incubated with pretreated HT-29 enterocytes and when L. lactis was incubated with pretreated Caco-2 and HT-29 enterocytes. These data indicated that AS might facilitate E. faecalisinternalization by cultured enterocytes.

title: Sex pheromones and plasmid transfer in Enterococcus faecalis.
abstract: Plasmid-free Enterococcus faecalis excrete peptides (sex pheromones) which specifically induce a mating response in strains harboring certain conjugative plasmids. The response is characterized by the synthesis of a "fuzzy" surface material, visible by electron microscopy, which is believed to facilitate the aggregation of donors and recipients. Transconjugants which receive a specific plasmid shut down the production of endogenous pheromone; however, they continue to produce pheromones specific for donors harboring different classes of plasmids. In this review, we summarize what is known about the biochemistry and genetics of this phenomenon. Some emphasis is given to the hemolysin plasmid pAD1 and the regulation of its conjugal transfer.

title: The antisense RNA of the par locus of pAD1 regulates the expression of a 33‐amino‐acid toxic peptide by an unusual mechanism
abstract: The par stability determinant of the Enterococcus faecalis plasmid pAD1 is the first antisense RNA‐regulated post‐segregational killing system (PSK) identified in a Gram‐positive organism. Par encodes two small, convergently transcribed RNAs, designated RNA I and RNA II, which are the toxin and antidote of the par PSK system respectively. RNA I encodes an open reading frame of 33 codons designated fst. The results presented here demonstrate that the peptide encoded by fst is the par toxin. The fst sequence was shown to be sufficient for cell killing, and removal of the final codon inactivated the toxin. In vitro translation reactions of purified RNA I transcript produced a product of the expected size for the fst‐encoded peptide. This product was not produced when purified RNA II transcript was added to the translation reaction. Toeprint analysis demonstrated that purified RNA II was able to inhibit ribosome binding to RNA I. These data suggest that fst expression is regulated by RNA II via an antisense RNA mechanism. In vitro translation studies and toeprint analyses also indicated that fst expression is internally regulated by a stem–loop structure at the 5′ end of RNA I. Removal of this structure resulted in better ribosome binding to RNA I and a 300‐fold increase in production of the fst‐encoded peptide. Finally, RNA II was shown to be less stable than RNA I in vivo, providing a basis for the selective expression of fst in plasmid‐free cells.

title: Structure, Function, and Biology of the Enterococcus faecalis Cytolysin
abstract: Enterococcus faecalis is a Gram-positive commensal member of the gut microbiota of a wide range of organisms. With the advent of antibiotic therapy, it has emerged as a multidrug resistant, hospital-acquired pathogen. Highly virulent strains of E. faecalis express a pore-forming exotoxin, called cytolysin, which lyses both bacterial and eukaryotic cells in response to quorum signals. Originally described in the 1930s, the cytolysin is a member of a large class of lanthionine-containing bacteriocins produced by Gram-positive bacteria. While the cytolysin shares some core features with other lantibiotics, it possesses unique characteristics as well. The current understanding of cytolysin biosynthesis, structure/function relationships, and contribution to the biology of E. faecalis are reviewed, and opportunities for using emerging technologies to advance this understanding are discussed.

title: Lantibiotics: peptides of diverse structure and function.
abstract: The current need for antibiotics with novel target molecules has coincided with advances in technical approaches for the structural and functional analysis of the lantibiotics, which are ribosomally synthesized peptides produced by gram-positive bacteria. These peptides have antibiotic or morphogenetic activity and are structurally defined by the presence of unusual amino acids introduced by posttranslational modification. Lantibiotics are complex polycyclic molecules formed by the dehydration of select Ser and Thr residues and the intramolecular addition of Cys thiols to the resulting unsaturated amino acids to form lanthionine and methyllanthionine bridges, respectively. Importantly, the structural and functional diversity of the lantibiotics is much broader than previously imagined. Here we discuss this growing collection of molecules and introduce some recently discovered peptides, review advances in enzymology and protein engineering, and discuss the regulatory networks that govern the synthesis of the lantibiotics by the producing organisms.

title: The Sequence of the Enterococcal Cytolysin Imparts Unusual Lanthionine Stereochemistry
abstract: The enterococcal cytolysin is a two-component lantibiotic of unknown structure with hemolytic activity that is important for virulence. We prepared cytolysin by co-expression of each precursor peptide with the synthetase CylM in E. coli, and characterized its structure. Surprisingly, cytolysin is the first example of a lantibiotic containing lanthionine and methyllanthionine structures with different stereochemistries in the same peptide, which is determined by the sequence of the substrate peptide.

title: Enterococcal cytolysin: a novel two component peptide system that serves as a bacterial defense against eukaryotic and prokaryotic cells.
abstract: The cytolysin is a novel, two-peptide lytic toxin produced by some strains of Enterococcus faecalis. It is toxic in animal models of enterococcal infection, and associated with acutely terminal outcome in human infection. The cytolysin exerts activity against a broad spectrum of cell types including a wide range of gram positive bacteria, eukaryotic cells such as human, bovine and horse erythrocytes, retinal cells, polymorphonuclear leukocytes, and human intestinal epithelial cells. The cytolysin likely originated as a bacteriocin involved with niche control in the complex microbial ecologies associated with eukaryotic hosts. However, additional anti-eukaryotic activities may have been selected for as enterococci adapted to eukaryotic cell predation in water or soil ecologies. Cytolytic activity requires two unique peptides that possess modifications characteristic of the lantibiotic bacteriocins, and these peptides are broadly similar in size to most cationic eukaryotic defensins. Expression of the cytolysin is tightly controlled by a novel mode of gene regulation in which the smaller peptide signals high-level expression of the cytolysin gene cluster. This complex regulation of cytolysin expression may have evolved to balance defense against eukaryotic predators with stealth.

title: Hemolysin of Streptococcus faecalis subspecies zymogenes contributes to virulence in mice
abstract: The conjugative plasmid pAD1 (56.7 kilobases) in Streptococcus faecalis confers hemolysin-bacteriocin (Hly-Bcn) expression and a mating response to the sex pheromone cAD1 excreted by recipient cells. We examined the contribution of hemolysin to pathogenicity in intraperitoneally infected mice by using Tn916 and Tn917 insertion mutants altered in hemolysin expression. Strains exhibiting the normal hemolysin phenotype were significantly more virulent than the nonhemolytic insertion mutants. A mutant plasmid with an increased copy number which gave rise to a larger-than-normal zone of hemolysis on blood agar rendered host strains more virulent than the wild-type streptococci in mice.

title: Contribution of the pAD1-encoded cytolysin to the severity of experimental Enterococcus faecalis endophthalmitis
abstract: The contribution of the pAD1-encoded cytolysin to Enterococcus faecalis virulence in an experimental endophthalmitis model was studied by using isogenic strains differing only in the location of transposon Tn917. The course of experimental endophthalmitis in New Zealand White rabbits was evaluated by postoperative reduction in retinal neuroresponsiveness, thin-section histopathology, and transmission electron microscopy. Infections caused by cytolytic E. faecalis resulted in 99% loss of retinal function at postoperative day 3, while isogenic, noncytolytic strains produced reductions of only 74.2%. Light microscopy revealed near-total destruction of retinal architecture at 24 h postinfection with cytolytic E. faecalis, while noncytolytic strains produced few or no destructive changes. Transmission electron microscopy revealed tissue destruction in retinal layers as early as 6 h postinfection with cytolytic E. faecalis. In vivo and in vitro growth rates of cytolytic and noncytolytic E. faecalis showed similar kinetics. These data demonstrate the contribution of the pAD1-encoded cytolysin to both the course and the severity of experimental E. faecalis endophthalmitis.

title: Genetic analysis of the pAD1 pheromone response in Streptococcus faecalis, using transposon Tn917 as an insertional mutagen
abstract: The conjugative plasmid pAD1 (56.7 kilobases) in Streptococcus faecalis has been shown to confer a mating response to the sex pheromone cAD1 excreted by recipient strains. The response is characterized by the synthesis of a proteinaceous adhesin which coats the surface of the pAD1 -containing donor cell and facilitates the formation of mating aggregates. Donors exposed to cAD1 -containing filtrates of recipients undergo self-aggregation (clumping), an event believed to be associated with an interaction between the adhesin and a binding substance always present on the surface of both recipients and donors. To analyze the molecular processes involved in the mating response, mutants were generated by the erythromycin resistance transposon Tn917 . Transpositions to pAD1 in S. faecalis DS16 gave rise to a number of derivatives that exhibited "constitutive clumping" and the ability to transfer at high frequencies in short (10-min) matings. These mutants fell into two subclasses, which exhibited colony morphologies that were "dry" or "normal". The Tn917 insertions were mapped by restriction enzyme analysis to two separate clusters, designated traA and traB. The dry colony subclass corresponded to traA and represented a span of 1.5 kilobases, whereas the normal subclass corresponded to traB and spanned 1.3 kilobases. The two clusters were separated by 1.7 kilobases in which insertions of Tn917 did not affect the ability to respond normally to cAD1 . Neither type of constitutive clumper produced cAD1 . Another series of insertions exhibited reduced donor potential. In two cases, the reduction in transfer was three to four orders of magnitude; these mapped in traA . In two other cases, the reduction was one to two orders of magnitude. These mapped outside of traA and traB, and one was associated with an increase in plasmid copy number.

title: SURVEY OF THE BACTERIOCINES OF ENTEROCOCCI
abstract: Brock, Thomas D. (Indiana University, Bloomington), Barbara Peacher, and Deborah Pierson. A survey of the bacteriocines of enterococci.  1963.—A survey has been made of bacteriocine production by a wide variety of well-characterized strains of group D streptococci. On the basis of spectrums and sensitivity to chloroform, heat, and proteolytic enzymes, five distinct bacteriocines can be defined. Type 1 is produced by all Streptococcus zymogenes (S. faecalis var. zymogenes) strains, is active against a wide variety of gram-positive bacteria, and is also a hemolysin. Type 2 is produced by some S. liquefaciens (S. faecalis var. liquefaciens) strains, and acts on many enterococci as well as on certain other lactic acid bacteria. Type 3 is produced by certain strains of both S. faecalis and S. faecium, and inhibits a wide variety of group D streptococci, but is inactive against all other lactic acid bacteria tested except Leuconostoc citrovorum. Type 4 is produced by certain S. faecium strains and resembles in certain ways the type 3 activity, but differs from it in other ways. Type 5 has been found to be produced by only one proteolytic strain of S. zymogenes, and this bacteriocine has a very narrow spectrum. The strain that produces this bacteriocine also produces type 1 activity. No strain is sensitive to a bacteriocine of the type it produces.

title: High incidence of hemolysin production by Enterococcus (Streptococcus) faecalis strains associated with human parenteral infections
abstract: Hemolysin production, clumping (pheromone) response, transferability of the hemolytic trait, and drug resistance were examined in 97 clinical isolates of Enterococcus (Streptococcus) faecalis. The isolates were derived from various sources (i.e., urine, pus, vagina, sputum, bile, and blood), and approximately 60% were found to be hemolytic. About 85% of the hemolytic strains exhibited a clumping response, compared with about 49% of the nonhemolytic strains. Over 50% of the hemolytic strains carried transferable hemolysin determinants, and in no case were drug resistance genes linked. The hemolytic strains exhibited multiple drug resistance more frequently than did the nonhemolytic strains. In contrast to the high frequency of hemolysin producers among parenteral isolates, strains derived from fecal specimens of healthy individuals exhibited a low (17%) incidence of hemolysin production.

title: Microbial telesensing: probing the environment for friends, foes, and food.
abstract: Bacterial-sensing circuits may be triggered by molecules originating from the environment (e.g., nutrients and chemoattractants). Bacteria also actively probe the environment for information by releasing molecular probes to measure conditions beyond the cell surface: a process known as telesensing. Perceiving the environment beyond is achieved by sensing environmentally induced changes in those probes, as occurs when a siderophore chelates an iron atom or a quorum-sensing signal is inactivated by a specific enzyme or adsorbent. This information, captured by chemical and physical changes induced in specifically produced molecules transiting through the environment, enables bacteria to mount a contextually appropriate response.

title: Infection-Derived Enterococcus faecalisStrains Are Enriched in esp, a Gene Encoding a Novel Surface Protein
abstract: We report the identification of a new cell wall-associated protein of Enterococcus faecalis. Studies on the distribution of the gene encoding this novel surface protein, Esp, reveal a significant (P < 0.001) enrichment in infection-derived E. faecalis isolates. Interestingly, the esp gene was not identified in any of 34 clinical E. faecium isolates or in 4 other less pathogenic enterococcal species tested. Analysis of the structural gene among various E. faecalis isolates reveals the existence of alternate forms of expression of the Esp protein. The deduced primary structure of the Esp protein from strain MMH594, inferred to be 1,873 amino acids (aa) with a predicted mass of ∼202 kDa, reveals a core region consisting of repeat units that make up 50% of the protein. Esp bears global organizational similarity to the Rib and C alpha proteins of group B streptococci. Identity among Esp, Rib, and C alpha proteins is strikingly localized to a stretch of 13 aa within repeats of similar length. The high degree of conservation of this 13-residue sequence suggests that it plays an important role in the natural selection for this trait among infection-derived E. faecalis and group B streptococcal isolates.

title: Variant esp gene as a marker of a distinct genetic lineage of vancomycinresistant Enterococcus faecium spreading in hospitals
abstract: In the USA, vancomycin-resistant Enterococcus faecium (VREF) is endemic in hospitals, despite lack of carriage among healthy individuals. In Europe, however, hospital outbreaks are rare, but VREF carriage among healthy individuals and livestock is common. We used amplified fragment-length polymorphism analysis to genotype 120 VREF isolates associated with hospital outbreaks and 45 non-epidemic isolates from the USA, Europe, and Australia. We also looked for the esp virulence gene in these isolates and in 98 VREF from animals. A specific E. faecium subpopulation genetically distinct from non-epidemic VREF isolates was found to be the cause of the hospital epidemics in all three continents. This subpopulation contained a variant of the esp gene that was absent in all non-epidemic and animal isolates. Identification of the variant esp gene will be important in guiding infection-control strategies, and the Esp protein could be a new target for antibacterial therapy.

title: Identification of a Staphylococcus aureus extracellular matrix-binding protein with broad specificity
abstract: A staphylococal surface protein capable of binding several extracellular matrix glycoproteins was purified as a result of our attempts to identify a receptor(s) for bone sialoprotein (BSP) on Staphylococcus aureus cells. Proteins from different staphylococcal strains were solubilized in sodium lauryl sulfate, separated by polyacrylamide gel electrophoresis, blotted onto Immobilon P membranes, and probed with 125I-BSP. Several bacterial proteins bound the radiolabeled ligand, and various strains expressed different repertoirs of BSP-binding proteins. Major BSP-binding proteins with apparent M(r)s of 72,000 or 60,000 were present on most strains, and these proteins were further studied. The 72- and 60-kDa proteins were preferentially expressed when bacteria were cultured in Luria broth compared with when they were cultured on tryptic soy broth, and the abundance of the proteins could be correlated to an increased 125I-BSP binding. Both the 72-kDa and the 60-kDa proteins were solubilized by extraction of cells with 1 M LiCl and were purified by cation-exchange chromatography. Amino acid composition analysis of the purified 72-kDa protein indicated a high content of lysine (11.9%) and hydrophobic amino acids (28.0% combined). In Western ligand blotting (immunoblotting) experiments, the 72-kDa protein bound not only BSP but also radiolabeled fibronectin, fibrinogen, vitronectin, thrombospondin, and, to some extent, collagen. Addition of the purified 60-kDa protein to S. aureus cells did not inhibit binding of the different ligands but in some cases resulted in an augmentation of the binding of 125I-ligand. Purified 60-kDa protein could hemagglutinate sheep erythrocytes at a concentration of 61 micrograms/ml. The agglutination reaction was inhibited by high concentrations of fucose, mannose, or melibiose. These data suggest that the purified proteins may serve as bacterial receptors with broad specificity for matrix glycoproteins and that the proteins may act as carbohydrate-binding proteins.

title: The Enterococcal Surface Protein, Esp, Is Involved in Enterococcus faecalis Biofilm Formation
abstract: The enterococcal surface protein, Esp, is a high-molecular-weight surface protein of unknown function whose frequency is significantly increased among infection-derived Enterococcus faecalisisolates. In this work, a global structural similarity was found between Bap, a biofilm-associated protein of Staphylococcus aureus, and Esp. Analysis of the relationship between the presence of the Esp-encoding gene (esp) and the biofilm formation capacity in E. faecalis demonstrated that the presence of the esp gene is highly associated (P < 0.0001) with the capacity of E. faecalis to form a biofilm on a polystyrene surface, since 93.5% of the E. faecalis esp-positive isolates were capable of forming a biofilm. Moreover, none of the E. faecalis esp-deficient isolates were biofilm producers. Depending on theE. faecalis isolate, insertional mutagenesis ofesp caused either a complete loss of the biofilm formation phenotype or no apparent phenotypic defect. Complementation studies revealed that Esp expression in an E. faecalis esp-deficient strain promoted primary attachment and biofilm formation on polystyrene and polyvinyl chloride plastic from urine collection bags. Together, these results demonstrate that (i) biofilm formation capacity is widespread among clinical E. faecalis isolates, (ii) the biofilm formation capacity is restricted to the E. faecalis strains harboringesp, and (iii) Esp promotes primary attachment and biofilm formation of E. faecalis on abiotic surfaces.

title: A bone sialoprotein-binding protein from Staphylococcus aureus: a member of the staphylococcal Sdr family.
abstract: Staphylococcus aureus bacteria, isolated from bone and joint infections, specifically interact with bone sialoprotein (BSP), a glycoprotein of bone and dentine extracellular matrix, via a cell-surface protein of M(r) 97000 [Yacoub, Lindahl, Rubin, Wendel, Heinegârd and Rydén, (1994) ]. Amino acid sequences of seven trypsin fragments from the 97000-M(r) BSP-binding protein were determined. A gene encoding a protein encompassing all seven peptide sequences was identified from chromosomal DNA isolated from S. aureus strain O24. This gene encodes a protein with 1171 amino acids, called BSP-binding protein (Bbp), which displays similarity to recently described proteins of the Sdr family from S. aureus. SdrC, SdrD and SdrE encode putative cell-surface proteins with no described ligand specificity. Bbp also shows similarity to a fibrinogen-binding protein from S. epidermidis called Fbe. A serine-aspartic acid repeat sequence was found close to the cell-wall-anchoring Leu-Pro-Xaa-Thr-Gly sequence in the C-terminal end of the protein. Escherichia coli cells were transformed with an expression vector containing a major part of the bbp gene fused to the gene for glutathione S-transferase. The affinity-purified fusion protein bound radiolabelled native BSP, and inhibited the binding of radiolabelled BSP to staphylococcal cells. Serum from patients suffering from bone and joint infection contained antibodies that reacted with the fusion protein of the BSP-binding protein, indicating that the protein is expressed during an infection and is immunogenic. The S. aureus Bbp protein may be important in the localization of bacteria to bone tissue, and thus might be of relevance in the pathogenicity of osteomyelitis.

title: The capsular polysaccharide of Enterococcus faecalis and its relationship to other polysaccharides in the cell wall
abstract: With the goal of identifying and characterizing traits of Enterococcus faecalis that play key roles in human disease, we identified an operon specifying synthesis of a capsular carbohydrate of the type most commonly expressed by clinical isolates. This surface-exposed carbohydrate consists of glycerol phosphate, glucose, and galactose residues, and its biosynthesis is encoded by a determinant that includes 11 ORFs. Insertional inactivation of genes in this pathway yielded mutants with enhanced susceptibility to phagocytic killing in vitro and compromised in the ability to persist in regional lymph nodes in vivo.

title: Survey for virulence determinants among Enterococcus faecalis isolated from different sources.
abstract: A collection of Enterococcus faecalis strains from clinical isolates, healthy individuals and the environment was screened for the presence of virulence factor genes, such as those for collagen-binding protein (ace), endocarditis antigen (efaA), haemolysin activator (cylA), gelatinase (gelE), aggregation substances (asa1 and asa373), a surface protein (esp) and two novel putative surface antigens (EF0591 and EF3314). Apart from some genes that were present in all strains (ace, efaA and EF3314), the gelE gene was the most common factor, although its presence did not correlate with its expression. The genes that encode Esp and CylA were never detected in endocarditis isolates, whereas an association was noted between the esp gene and isolates from urinary tract infection (UTI) and bacteraemia. An aggregation substance gene was always present in commensal strains. As for gelatinase, the presence of the cylA and asa genes did not correlate completely with their phenotypic expression. Generally, isolates from endocarditis, biliary stents and the environment were equipped with fewer virulence factors than isolates from other sources. UTI strains possessed the highest number of factors.

title: Diversity of ace, a Gene Encoding a Microbial Surface Component Recognizing Adhesive Matrix Molecules, from Different Strains of Enterococcus faecalis and Evidence for Production of Ace during Human Infections
abstract: Our previous work reported that most Enterococcus faecalis strains adhered to the extracellular matrix proteins collagen types I and IV and laminin after growth at 46°C, but not 37°C, and we subsequently identified an E. faecalissequence, ace, that encodes a bacterial adhesin similar to the collagen binding protein Cna of Staphylococcus aureus. In this study, we examined the diversity of E. faecalis-specific ace gene sequences among different isolates obtained from various geographic regions as well as from various clinical sources. A comparison of nucleotide and deduced amino acid sequences of Ace from nine E. faecalis strains identified a highly conserved N-terminal A domain, followed by a variable B domain which contains two to five repeats of 47 amino acids in tandem array, preceded by a 20-amino-acid partial repeat. Using 17 other strains collected worldwide, the 5′ region of acethat encodes the A domain was sequenced, and these sequences showed ≥97.5% identity. Among the previously reported five amino acids critical for collagen binding by Cna of S. aureus, four were found to be identical in Ace from all strains tested. Polyclonal immune rabbit serum prepared against recombinant Ace A derived fromE. faecalis strain OG1RF detected Ace in mutanolysin extracts of seven of nine E. faecalis strains after growth at 46°C; Ace was detected in four different molecular sizes that correspond to the variation in the B repeat region. To determine if there was any evidence to indicate that Ace might be produced under physiological conditions, we quantitatively assayed sera collected from patients with enterococcal infections for the presence of anti-Ace A antibodies. Ninety percent of sera (19 of 21) from patients withE. faecalis endocarditis showed reactivity with titers from 1:32 to >1:1,024; the only 2 sera which lacked antibodies to Ace A had considerably lower titers of antibodies to other E. faecalis antigens as well. Human-derived, anti-Ace A immunoglobulins G purified from an E. faecalis endocarditis patient serum inhibited adherence of 46°C-grown E. faecalis OG1RF to collagen types I and IV and laminin. In conclusion, these results show that ace is highly conserved among isolates of E. faecalis, with at least four variants related to the differences in the B domain, is expressed by different strains during infection in humans, and human-derived antibodies can block adherence to these extracellular matrix proteins.

title: Importance of the Collagen Adhesin Ace in Pathogenesis and Protection against Enterococcus faecalis Experimental Endocarditis
abstract: Ace is an adhesin to collagen from Enterococcus faecalis expressed conditionally after growth in serum or in the presence of collagen. Here, we generated an ace deletion mutant and showed that it was significantly attenuated versus wild-type OG1RF in a mixed infection rat endocarditis model (P<0.0001), while no differences were observed in a peritonitis model. Complemented OG1RFΔace (pAT392::ace) enhanced early (4 h) heart valve colonization versus OG1RFΔace (pAT392) (P = 0.0418), suggesting that Ace expression is important for early attachment. By flow cytometry using specific anti-recombinant Ace (rAce) immunoglobulins (Igs), we showed in vivo expression of Ace by OG1RF cells obtained directly from infected vegetations, consistent with our previous finding of anti-Ace antibodies in E. faecalis endocarditis patient sera. Finally, rats actively immunized against rAce were less susceptible to infection by OG1RF than non-immunized (P = 0.0004) or sham-immunized (P = 0.0475) by CFU counts. Similarly, animals given specific anti-rAce Igs were less likely to develop E. faecalis endocarditis (P = 0.0001) and showed fewer CFU in vegetations (P = 0.0146). In conclusion, we have shown for the first time that Ace is involved in pathogenesis of, and is useful for protection against, E. faecalis experimental endocarditis.

title: Purification and substrate specificity of a strongly hydrophobic extracellular metalloendopeptidase ("gelatinase") from Streptococcus faecalis (strain 0G1-10).
abstract: An extracellular Zn-endopeptidase was purified to homogeneity from the culture filtrates of Streptococcus faecalis (human oral strain 0G1-10) by a procedure that comprised concentration in an Amicon Hollow Fiber System, ammonium sulfate precipitation, gel permeation chromatography, hydrophobic interaction chromatography (batch operation on phenyl-sepharose Cl-4B), followed by fast protein liquid chromatography (FPLC) on a phenyl-Superose HR 5/5 column, and finally FPLC on a Superose 12 HR 10/30 column. The enzyme is a 31.5-kDa strongly hydrophobic protein with an isoelectric point of 4.6 and a broad pH optimum of 6 to 8. The substrate specificity of the enzyme is similar to that of the mammalian membrane endopeptidase-24.11 and Streptococcus thermophilus thermolysin (EC 3.4.24.4) in hydrolyzing preferentially the Phe24-Phe25 bond in insulin B-chain, followed by cleavage of the His5-Leu6 bond. The enzyme was especially active on Azocoll and gelatin; soluble and insoluble collagens were hydrolyzed at a lower rate. S. faecalis sex pheromone-related peptides and several mammalian bioactive peptides were cleaved at sites involving pronounced hydrophobicity. The enzyme did not hydrolyze small synthetic peptide derivatives (phenylazobenzyloxycarbonyl-L-Pro-L-Leu-Gly-L-Pro-D-Arg and 2-furylacryloyl-L-Leu-Gly-L-Ala) that are typically attacked by "true" bacterial collagenases. Chemical modification indicated the importance of histidyl, carboxyl, and tyrosyl groups in enzyme activity, suggesting that this enzyme may thus be classified as a metalloprotease II (EC 3.4.24.4). The enzyme is strongly inhibited by a 720-kDa factor present in rat inflammatory exudate. The pronounced ability of the enzyme to attack collagenous materials and certain bioactive peptides suggests its participation in inflammatory processes involving the presence of S. faecalis.

title: Ace Is a Collagen-binding MSCRAMM from Enterococcus faecalis *
abstract: A putative collagen-binding MSCRAMM, Ace, of Enterococcus faecalis was identified by searching bacterial genome data bases for proteins containing domains homologous to the ligand-binding region of Cna, the collagen-binding MSCRAMM fromStaphylococcus aureus. Ace was predicted to have a molecular mass of 71 kDa and contains features characteristic of cell surface proteins on Gram-positive bacteria, including a LPXTG motif for cross-linking to the cell wall. The N-terminal region of Ace contained a region (residues 174–319) in which 56% of the residues are identical or similar when compared with the minimal ligand-binding region of Cna (Cna 151–318); the remainder of the Ace A domain has 46% similarity with the corresponding region of the Cna A domain. Antibodies raised against recombinant Ace A domain were used to verify the cell surface expression of Ace on E. faecalis. These antibodies also effectively inhibited the adhesion of enterococcal cells to a collagen substrate, suggesting that Ace is a functional collagen-binding MSCRAMM. Structural modeling of the conserved region in Ace (residues 174–319) suggested a structure very similar to that reported for residues 151–318 of the Cna collagen-binding domain in which the ligand-binding site was identified as a trench transversing a β-sheet face (Symersky, J., Patti, J. M., Carson, M., House-Pompeo, K., Teale, M., Moore, D., Jin, L., DeLucas, L. J., Höök, M., and Narayana, S. V. L. (1997) . Biochemical analyses of recombinant Ace and Cna A domains supported the modeling data in that the secondary structures were similar as determined by CD spectroscopy and both proteins bound at multiple sites in type I collagen with micromolar affinities, but with different apparent kinetics. We conclude that Ace is a collagen-binding MSCRAMM on enterococci and is structurally and functionally related to the staphylococcal Cna protein.

title: The Fsr Quorum-Sensing System of Enterococcus faecalisModulates Surface Display of the Collagen-Binding MSCRAMM Ace through Regulation of gelE
abstract: Ace, a known virulence factor and the first identified microbial surface component recognizing adhesive matrix molecule (MSCRAMM) of Enterococcus faecalisis associated with host cell adherence and endocarditis. The Fsr quorum-sensing system of E. faecalis, a two-component signal transduction system, has also been repeatedly linked to virulence in E. faecalis, due in part to the transcriptional induction of an extracellular metalloprotease, gelatinase (GelE). In this study, we discovered that disruption of the Fsr pathway significantly increased the levels of Ace on the cell surface in the latter phases of growth. Furthermore, we observed that, in addition to fsrBmutants, other strains identified as deficient in GelE activity also demonstrated a similar phenotype. Additional experiments demonstrated the GelE-dependent cleavage of Ace from the surface of E. faecalis, confirming that GelE specifically reduces Ace cell surface display. In addition, disruption of the Fsr system or GelE expression significantly improved the ability of E. faecalisto adhere to collagen, which is consistent with higher levels of Ace on the E. faecalissurface. These results demonstrate that the display of Ace is mediated by quorum sensing through the action of GelE, providing insight into the complicated world of Gram-positive pathogen adhesion and colonization.

title: What are antibiotics? Archaic functions for modern activities
abstract: Secondary metabolites are proposed to have played important roles in the evolution of the reactions of living forms on earth, in effecting and modulating reactions during biochemical evolution by chemical and structural interaction with ‘receptor’ sites in primitive macromolecular templates. For example, in the evolution of the translation system, as the polymerizing reactions became more complex and proteins became involved, the low molecular‐weight effectors were functionally replaced by polypeptides, but retained their ability to interact with receptor sites in nucleic acids and proteins. Many of these low molecular‐weight effectors now play a different role, that of antagonists, by interacting with the original receptor sites in contemporary activity as antibiotics.

title: Origins and Evolution of Antibiotic Resistance
abstract: Antibiotics have always been considered one of the wonder discoveries of the 20th century. This is true, but the real wonder is the rise of antibiotic resistance in hospitals, communities, and the environment concomitant with their use. The extraordinary genetic capacities of microbes have benefitted from man's overuse of antibiotics to exploit every source of resistance genes and every means of horizontal gene transmission to develop multiple mechanisms of resistance for each and every antibiotic introduced into practice clinically, agriculturally, or otherwise. This review presents the salient aspects of antibiotic resistance development over the past half-century, with the oft-restated conclusion that it is time to act. To achieve complete restitution of therapeutic applications of antibiotics, there is a need for more information on the role of environmental microbiomes in the rise of antibiotic resistance. In particular, creative approaches to the discovery of novel antibiotics and their expedited and controlled introduction to therapy are obligatory.

title: The antibiotic paradox : how the misuse of antibiotics destroys their curative powers
abstract: In this totally revised and updated edition of the classic primer on the misuse of antibiotics, Dr. Stuart Levy reveals how our cavalier and naive attitude about the power of antibiotics can have -and already has had-dire consequences. He explains that we are currently witnessing a massive evolutionary change in bacteria. This build-up of new antibiotic-resistant bacteria in individuals and the environment, mixed with our overzealous use of antibiotic soaps and the unregulated dispensing of antibiotics worldwide, is leading us into a dangerous territory where our "miracle" drugs will no longer help.

title: Antibiotic resistance : implications for global health and novel intervention strategies : workshop summary
abstract: Years of using, misusing, and overusing antibiotics and other antimicrobial drugs has led to the emergence of multidrug-resistant 'superbugs.' The IOM's Forum on Microbial Threats held a public workshop April 6-7 to discuss the nature and sources of drug-resistant pathogens, the implications for global health, and the strategies to lessen the current and future impact of these superbugs.

title: Horizontal gene transfer and the genomics of enterococcal antibiotic resistance.
abstract: Enterococci are Gram-positive bacteria that normally colonize gastrointestinal tracts of humans and animals. They are of growing concern because of their ability to cause antibiotic resistant hospital infections. Antibiotic resistance has been acquired, and has disseminated throughout enterococci, via horizontal transfer of mobile genetic elements. This transmission has been mediated mainly by conjugative plasmids of the pheromone-responsive and broad host range incompatibility group 18 type. Genome sequencing is revealing the extent of diversity of these and other mobile elements in enterococci, as well as the extent of recombination and rearrangement resulting in new phenotypes. Pheromone-responsive plasmids were recently shown to promote genome plasticity in antibiotic resistant Enterococcus faecalis, and their involvement has been implicated in E. faecium as well. Further, incompatibility group 18 plasmids have recently played an important role in mediating transfer of vancomycin resistance from enterococci to methicillin-resistant strains of S. aureus.

title: Virulence of enterococci
abstract: Enterococci are commensal organisms well suited to survival in intestinal and vaginal tracts and the oral cavity. However, as for most bacteria described as causing human disease, enterococci also possess properties that can be ascribed roles in pathogenesis. The natural ability of enterococci to readily acquire, accumulate, and share extrachromosomal elements encoding virulence traits or antibiotic resistance genes lends advantages to their survival under unusual environmental stresses and in part explains their increasing importance as nosocomial pathogens. This review discusses the current understanding of enterococcal virulence relating to (i) adherence to host tissues, (ii) invasion and abscess formation, (iii) factors potentially relevant to modulation of host inflammatory responses, and (iv) potentially toxic secreted products. Aggregation substance, surface carbohydrates, or fibronectin-binding moieties may facilitate adherence to host tissues. Enterococcus faecalis appears to have the capacity to translocate across intact intestinal mucosa in models of antibiotic-induced superinfection. Extracellular toxins such as cytolysin can induce tissue damage as shown in an endophthalmitis model, increase mortality in combination with aggregation substance in an endocarditis model, and cause systemic toxicity in a murine peritonitis model. Finally, lipoteichoic acid, superoxide production, or pheromones and corresponding peptide inhibitors each may modulate local inflammatory reactions.

title: Diversity among multidrug-resistant enterococci.
abstract: Enterococci are associated with both community- and hospital-acquired infections. Even though they do not cause severe systemic inflammatory responses, such as septic shock, enterococci present a therapeutic challenge because of their resistance to a vast array of antimicrobial drugs, including cell-wall active agents, all commercially available aminoglycosides, penicillin and ampicillin, and vancomycin. The combination of the latter two occurs disproportionately in strains resistant to many other antimicrobial drugs. The propensity of enterococci to acquire resistance may relate to their ability to participate in various forms of conjugation, which can result in the spread of genes as part of conjugative transposons, pheromone-responsive plasmids, or broad host-range plasmids. Enterococcal hardiness likely adds to resistance by facilitating survival in the environment (and thus enhancing potential spread from person to person) of a multidrug-resistant clone. The combination of these attributes within the genus Enterococcus suggests that these bacteria and their resistance to antimicrobial drugs will continue to pose a challenge.

title: Streptomyces as symbionts: an emerging and widespread theme?
abstract: Streptomyces bacteria are ubiquitous in soil, conferring the characteristic earthy smell, and they have an important ecological role in the turnover of organic material. More recently, a new picture has begun to emerge in which streptomycetes are not in all cases simply free-living soil bacteria but have also evolved to live in symbiosis with plants, fungi and animals. Furthermore, much of the chemical diversity of secondary metabolites produced by Streptomyces species has most likely evolved as a direct result of their interactions with other organisms. Here we review what is currently known about the role of streptomycetes as symbionts with fungi, plants and animals. These interactions can be parasitic, as is the case for scab-causing streptomycetes, which infect plants, and the Streptomyces species Streptomyces somaliensis and Streptomyces sudanensis that infect humans. However, in most cases they are beneficial and growth promoting, as is the case with many insects, plants and marine animals that use streptomycete-produced antibiotics to protect themselves against infection. This is an exciting and newly emerging field of research that will become increasingly important as the search for new antibiotics switches to unusual and under-explored environments.

title: Aminoglycoside antibiotic-inactivating enzymes in actinomycetes similar to those present in clinical isolates of antibiotic-resistant bacteria.
abstract: Various species of Streptomyces possess aminoglycoside-modifying enzymes. Streptomyces kanamyceticus contains an enzyme that acetylates the 6'-amino group of kanamycin A and B, gentamicin C(1a), and neomycin. Streptomyces spectabilis produces an enzyme that acetylates the 2'-amino group of the hexose ring of gentamicin C(1a). These enzymes catalyze reactions identical to those catalyzed by enzymes found in gram-negative bacteria containing R(antibiotic resistance)-factors. The discovery of these enzymes suggests the possibility of an evolutionary relationship between the aminoglycosideinactivating enzymes (produced by resistance determinants) in bacteria containing R-factors and similar enzymes found in the actinomycetes.

title: Sampling the Antibiotic Resistome
abstract: Microbial resistance to antibiotics currently spans all known classes of natural and synthetic compounds. It has not only hindered our treatment of infections but also dramatically reshaped drug discovery, yet its origins have not been systematically studied. Soil-dwelling bacteria produce and encounter a myriad of antibiotics, evolving corresponding sensing and evading strategies. They are a reservoir of resistance determinants that can be mobilized into the microbial community. Study of this reservoir could provide an early warning system for future clinically relevant antibiotic resistance mechanisms.

title: In vitro susceptibility studies of vancomycin-resistant Enterococcus faecalis
abstract: Vancomycin resistance exhibited by Enterococcus faecalis isolates V583, V586, and V587 is described. The vancomycin MICs ranged from 32 to 64 micrograms/ml. Although resistant to vancomycin, the isolates were susceptible to teicoplanin (MIC, less than or equal to 0.5 micrograms/ml). Such a glycopeptide susceptibility profile has not been previously described for E. faecalis. Time kill studies showed that vancomycin resistance adversely affected the synergistic activity that vancomycin and aminoglycoside combinations usually demonstrate against enterococci. However, the ability to detect vancomycin resistance varied with the susceptibility testing method used. Whereas broth microdilution, broth macrodilution, and agar dilution methods detected resistance, disk-agar diffusion and the AutoMicrobic system Gram-Positive GPS-A susceptibility card (Vitek Systems Inc., Hazelwood, Mo.) did not. To detect vancomycin resistance reliably and establish the incidence of such E. faecalis isolates, adjustments in some susceptibility testing methods may be necessary.

title: Plasmid-mediated resistance to vancomycin and teicoplanin in Enterococcus faecium.
abstract: THE glycopeptide antibiotic agents vancomycin and teicoplanin are useful in the treatment of severe infections due to gram-positive bacteria.1 2 3 Staphylococci, streptococci, and enterococci are a...

title: Twenty-five years of shared life with vancomycin-resistant enterococci: is it time to divorce?
abstract: Twenty-five years ago, isolation of vancomycin-resistant Enterococcus faecium (VREm) was reported both in the UK and in France. Since then, VREm has spread worldwide in hospitals. Hospital outbreaks appeared to be related to the evolution since the end of 1980s of a subpopulation of E. faecium highly resistant to ampicillin and fluoroquinolones (the so-called clonal complex CC17) that later acquired resistance to vancomycin. CC17 isolates are presumably better adapted than other E. faecium isolates to the constraints of the hospital environment and most contain mobile genetic elements, phage genes, genes encoding membrane proteins, regulatory genes, a putative pathogenicity island and megaplasmids. Colonization and persistence are major features of VREm. Inherent characteristics of E. faecium including a remarkable genome plasticity, in part due to acquisition of IS elements, in particular IS16, have facilitated niche adaptation of this distinct E. faecium subpopulation that is multiply resistant to antibiotics. Quinupristin/dalfopristin and linezolid are licensed for the treatment of VREm infections, with linezolid often used as a first-line treatment. However, the emergence of plasmid-mediated resistance to linezolid by production of a Cfr methyltransferase in Enterococcus faecalis is worrying. Daptomycin has not been extensively evaluated for the treatment of VREm infections and resistant mutants have been selected under daptomycin therapy. Although control of VRE is challenging, a laissez-faire policy would result in an increased number of infections and would create an irreversible situation. Although so far unsuccessful, dissemination of glycopeptide-resistant Staphylococcus aureus with van genes acquired from resistant enterococci cannot be ruled out.

title: Antibiotic resistance is ancient
abstract: The discovery of antibiotics more than 70 years ago initiated a period of drug innovation and implementation in human and animal health and agriculture. These discoveries were tempered in all cases by the emergence of resistant microbes. This history has been interpreted to mean that antibiotic resistance in pathogenic bacteria is a modern phenomenon; this view is reinforced by the fact that collections of microbes that predate the antibiotic era are highly susceptible to antibiotics. Here we report targeted metagenomic analyses of rigorously authenticated ancient DNA from 30,000-year-old Beringian permafrost sediments and the identification of a highly diverse collection of genes encoding resistance to β-lactam, tetracycline and glycopeptide antibiotics. Structure and function studies on the complete vancomycin resistance element VanA confirmed its similarity to modern variants. These results show conclusively that antibiotic resistance is a natural phenomenon that predates the modern selective pressure of clinical antibiotic use.

title: DNA sequence resembling vanA and vanB in the vancomycin-resistant biopesticide Bacillus popilliae.
abstract: The origin of high-level vancomycin resistance in enterococci is unknown. Biopesticidal powders containing spores of Bacillus popilliae, which is vancomycin-resistant, have been used for >50 years in the United States for suppression of Japanese beetle populations. Using a polymerase chain reaction assay designed to amplify the vanB gene in enterococci, an amplicon in B. popilliae was identified and sequenced. The putative ligase gene in B. popilliae had 76.8% and 68.4%-68.9% nucleotide identity to the sequences of the vanA and vanB genes, respectively. There was 75.3% and 69.3%-69.9% identity between the translation of the putative ligase gene in B. popilliae and the translation of the vanA and vanB genes, respectively. We have identified a gene resembling vanA and vanB in B. popilliae. The gene in B. popilliae may have been a precursor to or have had an ancestral gene in common with vancomycin resistance genes in enterococci.

title: Vancomycin-resistant enterococci from nosocomial, community, and animal sources in the United States
abstract: The presence of vancomycin-resistant enterococci (VRE) was looked for in fecal samples from 104 healthy volunteers (3 with hospital exposure), 100 selected hospitalized patients, and various environmental sources (44 commercial chickens, 5 farm-raised chickens, 3 turkeys, and 2 chicken farm lagoon slurries). Five probiotic preparations were also studied. No VRE with vanA or vanB genes were isolated from the healthy volunteers without hospital exposure, environmental sources, or probiotic preparations. VRE with vanB were found in the stools of 16% of the high-risk hospitalized patients and in one volunteer with hospital contact. All VRE examined could be classified into one of two clones by pulsed-field gel electrophoresis. VRE from 11 of the colonized patients were quantified and ranged from 10(3) to 10(6) CFU/g of stool. This study, in contrast to findings in Europe, failed to find evidence of VanA- or VanB-type VRE in the community or environmental sources in Houston, Texas, and suggests that these settings are not a likely source of VRE in hospitals in this geographic area.

title: Epidemiologic Evaluation of Antimicrobial Resistance in Community-Acquired Enterococci
abstract: Fecal samples from 200 consecutive patients admitted to a community hospital yielded 107 enterococci. High-level gentamicin resistance occurred in 10 (14%) of the Enterococcus faecalisisolates. Ampicillin resistance occurred in two (3%) of the E. faecalis isolates and six (23%) of the Enterococcus faecium isolates. There were no vancomycin-resistant enterococci. Risk factors for enterococci with high-level aminoglycoside (gentamicin) or ampicillin resistance included prior hospitalization and previous antibiotic use.

title: Characterization of antimicrobial resistance in enterococci of animal origin
abstract: Among 97 enterococci cultured from animals, gentamicin MICs were > or = 2,000 micrograms/ml for 9 isolates and between 250 and 1,024 micrograms/ml for 6 isolates. For two isolates tested (gentamicin MICs, 256 and 512 micrograms/ml, respectively), there was no in vitro synergy with penicillin plus gentamicin, resistance was transferable, and there was no hybridization with a probe specific for 6'-aminoglycoside acetyltransferase-2"-aminoglycoside phosphotransferase. The results of the study indicate the presence of a unique gentamicin resistance genotype in enterococci of animal origin.

title: Antimicrobial Resistance in Enterococci Isolated from Turkey Flocks Fed Virginiamycin
abstract: From 125 separate cloacal cultures from three turkey flocks fed virginiamycin, 104 Enterococcus faecium and 186Enterococcus faecalis isolates were obtained. As the turkeys aged, there was a higher percentage of quinupristin-dalfopristin-resistant E. faecium isolates, with isolates from the oldest flock being 100% resistant. There were no vancomycin-resistant enterococci. Results of pulsed-field gel electrophoresis (PFGE) indicated there were 11 PFGE types of E. faecalis and 7 PFGE types of E. faecium that were in more than one group of flock cultures.

title: Inducible resistance to vancomycin in Enterococcus faecium D366.
abstract: Strain D366, a clinical isolate of Enterococcus faecium, is resistant (minimum inhibitory concentration [MIC] 32 mg/L) to vancomycin. When exponential-phase cultures were exposed to half the MIC of vancomycin, a lag of 3-4 h occurred before growth resumed. Cells preexposed to 1/2 MICs of vancomycin did not show any lag. Pregrowth of D366 with vancomycin caused resistance to all glycopeptides tested. Pregrowth in vancomycin resulted in synthesis of a 3.95-kDa cytoplasmic-membrane-associated protein. This protein was correlated with resistance in mutants with high-level resistance, in the presence of NaCl, which inhibited the activity of vancomycin, and when several glycopeptides with varying activities were tested. Vancomycin-grown cells appeared abnormal and lysed at a much slower rate than did normal cells. We conclude that (1) vancomycin resistance in D366 is inducible; (2) resistance is correlated with the synthesis of 39.5-kDa cytoplasmic membrane protein; and (3) this protein play an additional role in the inhibition of normal lytic functions.

title: Emergence of vancomycin-resistant enterococci in New York City
abstract: Enterococci, a common cause of nosocomial infection, are intrinsically resistant to most antimicrobials and readily acquire additional resistance. Vancomycin-resistant enterococci (VRE) have caused clusters of nosocomial infections since 1988. In April, 1991, the New York City Department of Health asked all city laboratories to submit suspected VRE isolates for confirmation. Clinical and epidemiological characteristics of the first 100 patients with VRE were identified, and antimicrobial susceptibility testing, restriction enzyme analysis, and DNA-DNA hybridisation with the vanA gene probe were done. From September, 1989, to October, 1991, 361 patients with VRE were identified at 38 hospitals. The number of hospitals reporting VRE increased from 1 in 1989 to 38 by October, 1991. 98% of 100 VRE infections were nosocomially acquired and 83% patients had received vancomycin and/or a cephalosporin in the 30 days before isolation of VRE. Of 23 isolates from 21 of the first 100 patients, 19 (83%) were resistant to all available antimicrobials. Four vanA probing patterns were noted, and restriction enzyme analysis of the 23 isolates revealed 14 strains. VRE have emerged rapidly in New York City. Molecular analyses suggest that a highly mobile genetic element--eg, a transposon--is responsible for the rapid spread of vancomycin resistance.

title: Historical Yearly Usage of Glycopeptides for Animals and Humans: The American-European Paradox Revisited
abstract: Kirst et al. (5) in their letter on clinical use of vancomycin in the United States and major European countries state that “although it is well recognized that vancomycin resistance is more prevalent in the United States than in Europe, it has not been explained why avoparcin usage fails to correlate with the different epidemiologies of resistance between the two continents; avoparcin was never approved for use in animals in the United States, in contrast to its broad use as a growth-promoting agent in Europe.” 
 
I do not agree that vancomycin resistance is more prevalent in the United States than in Europe. In Europe, vancomycin-resistant Enterococcus faecium (VRE) is highly prevalent in the community (livestock, food, and humans) (8). In contrast, a zero prevalence has been found in healthy humans and in livestock in the United States (3, 7). 
 
The frequent occurrence of VRE in the community correlates well with the use of large amounts of the glycopeptide avoparcin as a growth-promoting agent in livestock in Europe. A direct relationship between avoparcin usage and occurrence of VRE in livestock has been shown (1, 2). In Denmark, with a population of 5.2 million inhabitants, 24 kg of active vancomycin was used for humans in 1994. In comparison, 24,000 kg of active avoparcin was used for swine and broilers the same year. Thus, the amounts of active avoparcin used for livestock in Denmark in 1994 exceeded the total clinical use of vancomycin in the United States and in Europe in 1994 (Table ​(Table1)!1)! Data on total use of avoparcin in Europe is unfortunately not available. 
 
 
 
TABLE 1 
 
Yearly use of glycopeptides for animals and humans in the United States and some European countries 
 
 
 
Avoparcin was never approved for animals in the United States due to its carcinogenic effect (6). This correlates well with the absence of VRE in healthy humans and livestock in the United States. 
 
The incidence of human infections with VRE is higher in the United States relative to Europe. This correlates with a high usage of vancomycin in hospitals in the United States and a lower usage in Europe (5). 
 
The most likely sources of VRE in European hospitals are community sources (6, 8). The primary sources of VRE in hospitals in the United States are not known. I propose that likely sources of VRE in the United States are travel, tourists, and imported food. With a high consumption of vancomycin in hospitals, even rare introductions of VRE may lead to a high incidence of infection because of the extreme tenacity of this organism and its capability of causing nosocomial outbreaks (4, 6). 
 
Considering the huge community reservoir, the incidence of VRE infection in Europe would probably surpass that in the United States were the use vancomycin to reach similar levels. Using last-resort antibiotics as growth promoters appears to present an unacceptable public health risk. 
 
We need data on antibiotic consumption to understand the epidemiology of antibiotic-resistant bacteria. I therefore welcome the initiative of Kirst et al. (5). To my mind, monitoring of antibiotic resistance in bacteria and consumption of antibiotics must go hand in hand.

title: Genetic Analysis of a High-Level Vancomycin-Resistant Isolate of Staphylococcus aureus
abstract: Vancomycin is usually reserved for treatment of serious infections, including those caused by multidrug-resistant Staphylococcus aureus. A clinical isolate of S. aureus with high-level resistance to vancomycin (minimal inhibitory concentration = 1024 μg/ml) was isolated in June 2002. This isolate harbored a 57.9-kilobase multiresistance conjugative plasmid within which Tn1546 (vanA) was integrated. Additional elements on the plasmid encoded resistance to trimethoprim (dfrA), β-lactams (blaZ), aminoglycosides (aacA-aphD), and disinfectants (qacC). Genetic analyses suggest that the long-anticipated transfer of vancomycin resistance to a methicillin-resistant S. aureus occurred in vivo by interspecies transfer of Tn1546 from a co-isolate of Enterococcus faecalis.

title: Fitness Cost of VanA-Type Vancomycin Resistance in Methicillin-Resistant Staphylococcus aureus
abstract: We have quantified the biological cost of VanA-type glycopeptide resistance due to the acquisition of the resistance operon by methicillin-resistant Staphylococcus aureus (MRSA) from Enterococcus sp. Exponential growths of recipient strain HIP11713, its transconjugant VRSA-1, VRSA-5, and VRSA-6 were compared in the absence or, except for HIP11713, in the presence of vancomycin. Induction of resistance was performed by adding vancomycin in both the preculture and the culture or the culture at only 1/50 the MIC. In the absence of vancomycin, the growth rates of the vancomycin-resistant S. aureus (VRSA) strains were similar to that of susceptible MRSA strain HIP11713. When resistance was induced, and under both conditions, there was a significant reduction of the growth rate of the VRSA strains relative to that of HIP11713 and to those of their noninduced counterparts, corresponding to a ca. 20% to 38% reduction in fitness. Competition experiments between isogenic VRSA-1 and HIP11713 mixed at a 1:1, 1:100, or 100:1 ratio revealed a competitive disadvantage of 0.4% to 3% per 10 generations of the transconjugant versus the recipient. This slight fitness burden can be attributed to the basal level of expression of the van genes in the absence of induction combined with a gene dosage effect due to the presence of the van operon on multicopy plasmids. These data indicate that VanA-type resistance, when induced, is highly costly for the MRSA host, whereas in the absence of induction, its biological cost is minimal. Thus, the potential for the dissemination of VRSA clinical isolates should not be underestimated.

title: Identification of a genetic determinant in clinical Enterococcus faecium strains that contributes to intestinal colonization during antibiotic treatment.
abstract: Intestinal colonization by antibiotic-resistant Enterococcus faecium is the first step in a process that can lead to infections in hospitalized patients. By comparative genome analysis and subsequent polymerase chain reaction screening, we identified a locus that encodes a putative phosphotransferase system (PTS). The PTS locus was widespread in isolates from hospital outbreaks of infection (84.2%) and nonoutbreak clinical infections (66.0%) but absent from human commensal isolates. Deletion of pstD, which is predicted to encode the enzyme IID subunit of this PTS, significantly impaired the ability of E. faecium to colonize the murine intestinal tract during antibiotic treatment. This is the first description of a determinant that contributes to intestinal colonization in clinical E. faecium strains.