Michael S. Gilmore

Virulence of enterococci.

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. Images

In vitro susceptibility studies of vancomycin-resistant Enterococcus faecalis.

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.

Relationships between Enterococcal Virulence and Antimicrobial Resistance

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 infections 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.

Modulation of virulence within a pathogenicity island in vancomycin-resistant Enterococcus faecalis

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.

Role of Enterococcus faecalis Surface Protein Esp in the Pathogenesis of Ascending Urinary Tract Infection

ABSTRACT Enterococcus faecalis bacteria isolated from patients with bacteremia, endocarditis, and urinary tract infections more frequently express the surface protein Esp than do fecal isolates. To assess the role of Esp in colonization and persistence of E. faecalis in an animal model of ascending urinary tract infection, we compared an Esp+strain of E. faecalis to its isogenic Esp-deficient mutant. Groups of CBA/J mice were challenged transurethrally with 108 CFU of either the parent or mutant strain, and bacteria in the urine, bladder, and kidneys were enumerated 5 days postinfection. Significantly higher numbers of bacteria were recovered from the bladder and urine of mice challenged with the parent strain than from the bladder and urine of mice challenged with the mutant. Colonization of the kidney, however, was not significantly different between the parent and mutant strains. Histopathological evaluations of kidney and bladder tissue done at 5 days postinfection did not show marked histopathological changes consistent with inflammation, mucosal hyperplasia, or apoptosis, and there was no observable difference between the mice challenged with the parent and those challenged with the mutant. We conclude that, while Esp does not influence histopathological changes associated with acute urinary tract infections, it contributes to colonization and persistence of E. faecalis at this site.

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.

Bacterial Endophthalmitis: Epidemiology, Therapeutics, and Bacterium-Host Interactions

SUMMARY Endophthalmitis is a severe inflammation of the interior of the eye caused by the introduction of contaminating microorganisms following trauma, surgery, or hematogenous spread from a distant infection site. Despite appropriate therapeutic intervention, bacterial endophthalmitis frequently results in visual loss, if not loss of the eye itself. Although the pathogenicity of bacterial endophthalmitis has historically been linked with toxin production during infection, a paucity of information exists as to the exact mechanisms of retinal toxicity and the triggers for induction of the intraocular immune response. Recently, research has begun to examine the bacterial and host molecular and cellular events that contribute to ocular damage during endophthalmitis. This review focuses on the causative agents and therapeutic challenges of bacterial endophthalmitis and provides current data from the analysis of the role of bacterial virulence factors and host inflammatory interactions in the pathogenesis of eye infections. Based on these and related studies, a hypothetical model for the molecular pathogenesis of bacterial endopthalmitis is proposed. Identifying and understanding the basic mechanisms of these bacterium-host interactions will provide the foundation for which novel, information-based therapeutic agents are developed in order to prevent vision loss during endophthalmitis.

Genetic Diversity among Enterococcus faecalis

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.

Multidrug-Resistant Enterococci Lack CRISPR-cas

ABSTRACT Clustered, regularly interspaced short palindromic repeats (CRISPR) provide bacteria and archaea with sequence-specific, acquired defense against plasmids and phage. Because mobile elements constitute up to 25% of the genome of multidrug-resistant (MDR) enterococci, it was of interest to examine the codistribution of CRISPR and acquired antibiotic resistance in enterococcal lineages. A database was built from 16 Enterococcus faecalis draft genome sequences to identify commonalities and polymorphisms in the location and content of CRISPR loci. With this data set, we were able to detect identities between CRISPR spacers and sequences from mobile elements, including pheromone-responsive plasmids and phage, suggesting that CRISPR regulates the flux of these elements through the E. faecalis species. Based on conserved locations of CRISPR and CRISPR-cas loci and the discovery of a new CRISPR locus with associated functional genes, CRISPR3-cas, we screened additional E. faecalis strains for CRISPR content, including isolates predating the use of antibiotics. We found a highly significant inverse correlation between the presence of a CRISPR-cas locus and acquired antibiotic resistance in E. faecalis, and examination of an additional eight E. faecium genomes yielded similar results for that species. A mechanism for CRISPR-cas loss in E. faecalis was identified. The inverse relationship between CRISPR-cas and antibiotic resistance suggests that antibiotic use inadvertently selects for enterococcal strains with compromised genome defense. IMPORTANCE For many bacteria, including the opportunistically pathogenic enterococci, antibiotic resistance is mediated by acquisition of new DNA and is frequently encoded on mobile DNA elements such as plasmids and transposons. Certain enterococcal lineages have recently emerged that are characterized by abundant mobile DNA, including numerous viruses (phage), and plasmids and transposons encoding multiple antibiotic resistances. These lineages cause hospital infection outbreaks around the world. The striking influx of mobile DNA into these lineages is in contrast to what would be expected if a self (genome)-defense system was present. Clustered, regularly interspaced short palindromic repeat (CRISPR) defense is a recently discovered mechanism of prokaryotic self-defense that provides a type of acquired immunity. Here, we find that antibiotic resistance and possession of complete CRISPR loci are inversely related and that members of recently emerged high-risk enterococcal lineages lack complete CRISPR loci. Our results suggest that antibiotic therapy inadvertently selects for enterococci with compromised genome defense. For many bacteria, including the opportunistically pathogenic enterococci, antibiotic resistance is mediated by acquisition of new DNA and is frequently encoded on mobile DNA elements such as plasmids and transposons. Certain enterococcal lineages have recently emerged that are characterized by abundant mobile DNA, including numerous viruses (phage), and plasmids and transposons encoding multiple antibiotic resistances. These lineages cause hospital infection outbreaks around the world. The striking influx of mobile DNA into these lineages is in contrast to what would be expected if a self (genome)-defense system was present. Clustered, regularly interspaced short palindromic repeat (CRISPR) defense is a recently discovered mechanism of prokaryotic self-defense that provides a type of acquired immunity. Here, we find that antibiotic resistance and possession of complete CRISPR loci are inversely related and that members of recently emerged high-risk enterococcal lineages lack complete CRISPR loci. Our results suggest that antibiotic therapy inadvertently selects for enterococci with compromised genome defense.

Comparative Genomics of Enterococci: Variation in Enterococcus faecalis, Clade Structure in E. faecium, and Defining Characteristics of E. gallinarum and E. casseliflavus

ABSTRACT The enterococci are Gram-positive lactic acid bacteria that inhabit the gastrointestinal tracts of diverse hosts. However, Enterococcus faecium and E. faecalis have emerged as leading causes of multidrug-resistant hospital-acquired infections. The mechanism by which a well-adapted commensal evolved into a hospital pathogen is poorly understood. In this study, we examined high-quality draft genome data for evidence of key events in the evolution of the leading causes of enterococcal infections, including E. faecalis, E. faecium, E. casseliflavus, and E. gallinarum. We characterized two clades within what is currently classified as E. faecium and identified traits characteristic of each, including variation in operons for cell wall carbohydrate and putative capsule biosynthesis. We examined the extent of recombination between the two E. faecium clades and identified two strains with mosaic genomes. We determined the underlying genetics for the defining characteristics of the motile enterococci E. casseliflavus and E. gallinarum. Further, we identified species-specific traits that could be used to advance the detection of medically relevant enterococci and their identification to the species level. IMPORTANCE The enterococci, in particular, vancomycin-resistant enterococci, have emerged as leading causes of multidrug-resistant hospital-acquired infections. In this study, we examined genome sequence data to define traits with the potential to influence host-microbe interactions and to identify sequences and biochemical functions that could form the basis for the rapid identification of enterococcal species or lineages of importance in clinical and environmental samples. The enterococci, in particular, vancomycin-resistant enterococci, have emerged as leading causes of multidrug-resistant hospital-acquired infections. In this study, we examined genome sequence data to define traits with the potential to influence host-microbe interactions and to identify sequences and biochemical functions that could form the basis for the rapid identification of enterococcal species or lineages of importance in clinical and environmental samples.