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A large diarrhea outbreak due to enteroinvasive Escherichia coli (EIEC) serogroup O143 occurring in Houston, Tex., provided the opportunity to investigate aspects of the molecular epidemiology of this and related organisms. This was done by comparing the plasmid patterns and the chromosomal restriction endonuclease digestion patterns by pulsed-field gel electrophoresis (PFGE) of EIEC from the outbreak, other E. coli from the same serogoup (O143), and EIEC isolated from other patients with diarrhea. Among the isolates studied, there was marked restriction fragment length polymorphism. All 3 non-O143 EIEC isolates had very different restriction endonuclease digestion patterns, as did 5 of 5 O143 non-EIEC isolates and 6 of 15 O143 EIEC isolates. Four Houston outbreak O143 EIEC isolates had the same restriction pattern as an O143 EIEC strain isolated 2 months before in Mexico and was nearly identical to another two O143 EIEC Mexican isolates. These related strains also had the same plasmid pattern; however, the presence of only a few plasmid bands, versus the 21 to 30 chromosomal bands seen with PFGE, suggests that plasmid patterns could be a less specific way to distinguish different strains. These results demonstrate that PFGE can distinguish between different E. coli strains of the same serogroup and phenotype. This technique can also identify relatedness within O143 EIEC, and our data suggest the spread of a strain of EIEC from Mexico to Houston, where it caused a large outbreak. PFGE may be useful to study the epidemiology of EIEC.

Members of the Streptococcus bovis group are important causes of endocarditis. However, factors associated with their pathogenicity, such as adhesins, remain uncharacterized. We recently demonstrated that endocarditis-derived Streptococcus gallolyticus subsp. gallolyticus isolates frequently adhere to extracellular matrix (ECM) proteins. Here, we generated a draft genome sequence of an ECM protein-adherent S. gallolyticus subsp. gallolyticus strain and found, by genome-wide analyses, 11 predicted LPXTG-type cell wall-anchored proteins with characteristics of MSCRAMMs, including a modular architecture of domains predicted to adopt immunoglobulin (Ig)-like folding. A recombinant segment of one of these, Acb, showed high-affinity binding to immobilized collagen, and cell surface expression of Acb correlated with the presence of acb and collagen adherence of isolates. Three of the 11 proteins have similarities to major pilus subunits and are organized in separate clusters, each including a second Ig-fold-containing MSCRAMM and a class C sortase, suggesting that the sequenced strain encodes three distinct types of pili. Reverse transcription-PCR demonstrated that all three genes of one cluster, acb - sbs7 - srtC1 , are cotranscribed, consistent with pilus operons of other gram-positive bacteria. Further analysis detected expression of all 11 genes in cells grown to mid to late exponential growth phases. Wide distribution of 9 of the 11 genes was observed among S. gallolyticus subsp. gallolyticus isolates with fewer genes present in other S. bovis group species/subspecies. The high prevalence of genes encoding putative MSCRAMMs and pili, including a collagen-binding MSCRAMM, among S. gallolyticus subsp. gallolyticus isolates may play an important role in the predominance of this subspecies in S. bovis endocarditis.

En este estudio, presentamos un esquema de tipificación de secuencia trilocus (TLST) basado en regiones intragénicas de dos genes antigénicos, ace y salA (que codifican una adhesina de colágeno / laminina y un antígeno asociado a la pared celular, respectivamente), y un gen asociado con antibiótico resistencia, lsa (que codifica un transportador ABC putativo), para la diferenciación de subespecies de Enterococcus faecalis. Cada uno de los alelos se analizó utilizando 50 aislamientos de E. faecalis que representan 42 tipos de secuencias multilocus diversos (ST M; basado en siete genes de mantenimiento) y cuatro grupos de aislados unidos clonalmente (por electroforesis en gel de campo pulsado [PFGE]). Los perfiles alélicos y / o secuencias concatenadas de los tres genes coincidieron con los resultados de la tipificación de secuencias multilocus (MLST) para la tipificación de 49 de los 50 aislamientos; Además de la única excepción, se encontró que dos aislamientos tenían tipos de TLST idénticos pero eran variantes de un solo locus (que se diferenciaban por un solo nucleótido) según MLST y, por lo tanto, también se clasificaron como relacionados clonalmente por MLST. TLST también fue comparable a PFGE para establecer relaciones epidemiológicas a corto plazo, tipificando todos los aislados clasificados como relacionados clonalmente por PFGE con el mismo tipo. A continuación, se aplicó TLST a aislamientos representativos (de cada subtipo de PFGE y año de aislamiento) de una colección de 48 aislamientos hospitalarios y se demostraron las mismas relaciones entre los aislamientos de una cepa del brote que los encontrados por MLST y PFGE. En conclusión, el esquema TLST descrito aquí demostró ser exitoso para investigar la epidemiología a corto plazo en un entorno hospitalario y puede proporcionar una alternativa a MLST para discriminar aislamientos. In this study, we present a trilocus sequence typing (TLST) scheme based on intragenic regions of two antigenic genes, ace and salA (encoding a collagen/laminin adhesin and a cell wall-associated antigen, respectively), and a gene associated with antibiotic resistance, lsa (encoding a putative ABC transporter), for subspecies differentiation of Enterococcus faecalis. Each of the alleles was analyzed using 50 E. faecalis isolates representing 42 diverse multilocus sequence types (STM; based on seven housekeeping genes) and four groups of clonally linked (by pulsed-field gel electrophoresis [PFGE]) isolates. The allelic profiles and/or concatenated sequences of the three genes agreed with multilocus sequence typing (MLST) results for typing of 49 of the 50 isolates; in addition to the one exception, two isolates were found to have identical TLST types but were single-locus variants (differing by a single nucleotide) by MLST and were therefore also classified as clonally related by MLST. TLST was also comparable to PFGE for establishing short-term epidemiological relationships, typing all isolates classified as clonally related by PFGE with the same type. TLST was then applied to representative isolates (of each PFGE subtype and isolation year) of a collection of 48 hospital isolates and demonstrated the same relationships between isolates of an outbreak strain as those found by MLST and PFGE. In conclusion, the TLST scheme described here was shown to be successful for investigating short-term epidemiology in a hospital setting and may provide an alternative to MLST for discriminating isolates.

Previously, TX5179, a disruption mutant of the enterococcal polysaccharide antigen (epa) gene cluster of Enterococcus faecalis strain OG1RF was shown to be attenuated in translocation, biofilm, mouse peritonitis and was more susceptible to polymorphonuclear leukocyte phagocytic killing. Here, wild-type E. faecalis OG1RF and TX5179 strains were tested in a mixed-infection (inoculum, approximately 1:1) mouse urinary tract infection model. Wild-type OG1RF outnumbered TX5179 in the kidneys (P < .001) and bladder (P < .001). In conclusion, the epa locus of E. faecalis OG1RF contributes to murine urinary tract infection and is the first such enterococcal polysaccharide locus shown to be important in this site.

Pathogenic streptococci and enterococci primarily rely on the conserved secretory (Sec) pathway for the translocation and secretion of virulence factors out of the cell. Since many secreted virulence factors in gram-positive organisms are subsequently attached to the bacterial cell surface via sortase enzymes, we sought to investigate the spatial relationship between secretion and cell wall attachment in Enterococcus faecalis . We discovered that sortase A (SrtA) and sortase C (SrtC) are colocalized with SecA at single foci in the enterococcus. The SrtA-processed substrate aggregation substance accumulated in single foci when SrtA was deleted, implying a single site of secretion for these proteins. Furthermore, in the absence of the pilus-polymerizing SrtC, pilin subunits also accumulate in single foci. Proteins that localized to single foci in E. faecalis were found to share a positively charged domain flanking a transmembrane helix. Mutation or deletion of this domain in SrtC abolished both its retention at single foci and its function in efficient pilus assembly. We conclude that this positively charged domain can act as a localization retention signal for the focal compartmentalization of membrane proteins.

Drs. Cesar Arias and Barbara Murray write that we have arrived at a point as frightening as the preantibiotic era: for patients infected with multidrug-resistant bacteria, there is no magic bullet.

Antecedentes. Staphylococus aureus resistente a la meticilina (MRSA) es un patógeno nosocomial y asociado a la comunidad (CA) importante. Recientemente, una variante del clon MRSA USA300 surgió y se diseminó en América del Sur, causando importantes problemas clínicos. Métodos. Se recolectaron prospectivamente aislamientos de S. aureus (2006-2008) de 32 hospitales terciarios en Colombia, Ecuador, Perú y Venezuela. Los aislados de MRSA se sometieron a pruebas de susceptibilidad a los antimicrobianos y electroforesis en gel de campo pulsado y se clasificaron como clones similares a los de la atención médica (HA) o CA según las características genotípicas y la detección de genes que codifican la leucocidina Panton-Valentine y el cromosoma del casete de estafilococos ( SCC) mec IV. Además, se realizó la tipificación de secuencias multilocus de aislados representativos de cada pulsotipo CAMRSA principal, y se investigó la presencia de toxinas asociadas con USA300 y el gen arcA para todos los aislamientos categorizados como CA-MRSA. Resultados. Se incluyó un total de 1570 S. aureus; 651 eran MRSA (41%) - con la tasa más alta de aislamiento de MRSA en Perú (62%) y la más baja en Venezuela (26%) - y el 71%, 27% y 2% se clasificaron como HA-like, CA- clones similares y no similares a CA / HA, respectivamente. Se confirmó que solo 9 aislados de MRSA tenían una susceptibilidad reducida a los glicopéptidos (fenotipo de S. aureus intermedio con glicopéptidos). El pulsotipo más común (designado ComA) entre las cepas de MRSA similares a CA se encontró en el 96% de los aislados, y la mayoría (81%) tiene una diferencia de banda ≤ S6 con la cepa USA300-0114. Los aislados representativos de este clon fueron el tipo de secuencia 8; sin embargo, a diferencia de la cepa USA300-0114, albergaban un subtipo diferente de SCCmec IV y carecían de arcA (un indicador del elemento móvil catabólico de arginina). Conclusión. Se ha establecido una variante del clon CA-MRSA USA300 en América del Sur y, en algunos países, es endémica en entornos hospitalarios. Background. Methicillin-resistant Staphylococus aureus (MRSA) is an important nosocomial and communityassociated (CA) pathogen. Recently, a variant of the MRSA USA300 clone emerged and disseminated in South America, causing important clinical problems. Methods. S. aureus isolates were prospectively collected (2006-2008) from 32 tertiary hospitals in Colombia, Ecuador, Peru, and Venezuela. MRSA isolates were subjected to antimicrobial susceptibility testing and pulsedfield gel electrophoresis and were categorized as health care-associated (HA)-like or CA-like clones on the basis of genotypic characteristics and detection of genes encoding Panton-Valentine leukocidin and staphylococcal cassette chromosome (SCC) mec IV. In addition, multilocus sequence typing of representative isolates of each major CAMRSA pulsotype was performed, and the presence of USA300-associated toxins and the arcA gene was investigated for all isolates categorized as CA-MRSA. Results. A total of 1570 S. aureus were included; 651 were MRSA (41%)-with the highest rate of MRSA isolation in Peru (62%) and the lowest in Venezuela (26%)-and 71%, 27%, and 2% were classified as HA-like, CA-like, and non-CA/HA-like clones, respectively. Only 9 MRSA isolates were confirmed to have reduced susceptibility to glycopeptides (glycopeptide-intermediate S. aureus phenotype). The most common pulsotype (designated ComA) among the CA-like MRSA strains was found in 96% of isolates, with the majority (81%) having a ≤S6-band difference with the USA300-0114 strain. Representative isolates of this clone were sequence type 8; however, unlike the USA300-0114 strain, they harbored a different SCCmec IV subtype and lacked arcA (an indicator of the arginine catabolic mobile element). Conclusion. A variant CA-MRSA USA300 clone has become established in South America and, in some countries, is endemic in hospital settings.

Pulsed-field gel electrophoresis was used to compare 34 isolates of Enterococcus faecium from six different geographic locations. This procedure generated an average of 13 discernible fragment bands per isolate (range, 10 to 19 fragment bands) of 34 to 485 kb. The resulting restriction endonuclease digestion patterns were quite heterogeneous and were able to differentiate 27 of 34 isolates from each other, as defined by one or more mismatched fragment bands. Five patterns were shared by two or more isolates, and each set of isolates with matching patterns (shared pattern) originated in the same medical center, suggesting a common epidemiologic background, including highly penicillin resistant isolates in Richmond and Philadelphia. We conclude that pulsed-field gel electrophoresis of DNA digested with low-frequency-cleavage restriction enzymes offers a relatively simple method of comparing E. faecium for the purpose of epidemiologic study.

Three previously reported beta-lactamase-producing (Bla+) enterococci with distinct but related antibiotic resistance phenotypes, plasmid profiles, and plasmid restriction endonuclease digestion patterns were isolated at the West Haven Veterans Administration Medical Center in Connecticut (WH245, WH257, WH571) in July 1986 and March 1987. In this study, we analyzed the whole-cell DNA of these isolates by using pulsed-field gel electrophoresis of large chromosomal fragments generated by SmaI digestion. The three West Haven isolates showed very similar chromosomal restriction endonuclease digestion patterns; these patterns were distinct from those of Bla+ and Bla- enterococci from other geographic areas and from those of other West Haven enterococci, suggesting an endemic Bla+ strain at this institution. Clinical information regarding these isolates suggests that exposure to the genitourinary clinic, obstructive urinary tract disease, and frequent antibiotic therapy may have been common risk factors for acquisition of this endemic strain.

Emergence of resistance to antimicrobial agents among previously susceptible organisms continues to be an important obstacle to the successful treatment of bacterial infections. In hospitals, plasmid-mediated resistance to third-generation cephalosporins and monobactams has recently appeared in gram-negative bacilli, due primarily to mutations in TEM- and SHV-type enzymes. Among nosocomial enterococci, vancomycin resistance, beta-lactamase production, and high-level resistance to all aminoglycosides have recently been added to this organism's already formidable armamentarium of resistance properties. Also, resistance to fluoroquinolones and rifampin has been emerging in methicillin-resistant Staphylococcus aureus. In the community, organisms in which resistance plays a particularly important role are shigellae, Haemophilus influenzae, gonococci, and pneumococci, particularly in developing countries. beta-lactamase-producing meningococci have been reported for the first time. The selective pressure generated by the use of antimicrobial agents, together with the ability of bacteria to acquire and spread resistance and the capacity of humans to transmit bacteria, suggest that antimicrobial resistance will continue to be a problem for the foreseeable future.

In Enterococcus faecalis, the genetic determinant encoding gentamicin resistance (Gmr) on the conjugative plasmid pBEM10 previously has been shown to be on a mobile element. In the current study, this element, termed Tn5281, was shown to relocate in the absence of homologous recombination in E. faecalis UV202. On the basis of restriction endonuclease analysis and DNA-DNA hybridization studies, Tn5281 was shown to be similar, if not identical, to the Gmr transposons Tn4001 found in Australian isolates of Staphylococcus aureus and Tn4031 found in U.S. isolates of Staphylococcus epidermidis, since all three of these transposons have symmetrically located HindIII (2.5 kb apart), ClaI (slightly more than 2.5 kb apart), and HaeIII (3.9 kb apart) sites. Restriction endonuclease digestion patterns of Tn5281 generated with HincII, ScaI, and AluI were also consistent with Tn4001 and Tn4031. By using a probe specific for the external portion of the terminal inverted repeat of Tn4031, it was determined that each terminus of Tn5281 contained a 0.35-kb HaeIII fragment and a 0.7-kb HindIII-HaeIII fragment. The sizes of these fragments are identical to those found in the staphylococcal transposons, which is a further indication that inverted repeats like IS256 are present in Tn5281. A 1-kb HaeIII fragment in pBEM10 also hybridized with this probe, which indicates that Tn5281 in pBEM10 contains a double copy of the inverted repeat at one end.

Beta-lactamase-producing (Bla+) enterococci have been reported in three state and two countries. Pulsed-field gel electrophoresis was used to compare 14 Bla+ Enterococcus (Streptococcus) faecalis isolated from hospitalized patients in seven states and three continents. The restriction endonuclease digestion patterns of isolates from Connecticut, Massachusetts, Lebanon, and Argentina were all markedly different, indicating that these were different strains. However, isolates from Delaware, Texas, Pennsylvania (Philadelphia and Pittsburgh), Florida, and Virginia were similar, indicating that these isolates were derivatives of a single strain. This conclusion was supported by hybridization using individual fragments as probes. Spread of Bla+ enterococci within the hospital setting was also demonstrated. These findings illustrate the value of pulsed-field gel electrophoresis for epidemiologic analyses and support the importance of identifying and containing organisms with new resistance properties in an effort to decrease their transmission to and from, as well as within, hospitals.

Members of the Streptococcus bovis group are frequent colonizers of the intestinal tract, which can also cause endocarditis. However, their ability to adhere to and colonize host tissues and the factors associated with pathogenicity are largely unknown. Here, we assessed 17 endocarditis-derived human isolates [identified here as 15 Streptococcus gallolyticus ssp. gallolyticus (S. bovis biotype I), one S. gallolyticus ssp. pasteurianus (biotype II/2) and one Streptococcus infantarius ssp. coli (biotype II/1)] for their in vitro adherence to components of the extracellular matrix (ECM). Adherence to collagen type I was found to be the most common phenotype exhibited by 76% of isolates, followed by collagen type IV (53%), fibrinogen (47%), collagen type V (35%) and fibronectin (35%). Pulsed-field gel electrophoresis analyses showed that >50% of endocarditis-derived S. gallolyticus ssp. gallolyticus isolates are genetically diverse, although two clusters of two and four isolates were observed. The diversity of strains and differences observed in adherence characteristics to distinct host ECM proteins suggest that isolates of S. gallolyticus ssp. gallolyticus produce different surface components, similar to other gram-positive pathogens, to colonize the host and cause infection.

The treatment of multidrug-resistant enterococcal infections continues to be a challenge for clinicians. Glycopeptide and beta-lactam resistance is now a common feature of the majority of Enterococcus faecium hospital isolates, and resistance to aminoglycosides, quinupristin-dalfopristin, linezolid and daptomycin further complicates the problem. New antibiotics, such as tigecycline, lipoglycopeptides (dalbavancin, oritavancin and telavancin) and cephalosporins with activity against Enterococcus faecalis (ceftobiprole and ceftaroline), may have potential activity against certain resistant enterococcal strains in specific clinical settings, as may some older antibiotics, such as ampicillin, chloramphenicol, doxycycline, minocycline and nitrofurantoin. However, the treatment of endovascular infections (particularly endocarditis, where bactericidal therapy is important for optimal cure rates) caused by resistant enterococci continues to be an immense challenge even with the availability of new agents. The optimal therapy for these infections is not well established and clinical data are usually limited to case reports with conflicting results. Therefore, treatment decisions may have to be based on animal models and sporadic experiences and the best approach is for the physician to consider carefully each patient on a case by case manner and gather all the clinical and microbiological information possible regarding species identification and susceptibilities in order to choose a therapeutic regimen that would appear to be active.

Attention has recently been drawn to Enterococcus faecium because of an increasing number of nosocomial infections caused by this species and its resistance to multiple antibacterial agents. However, relatively little is known about the pathogenic determinants of this organism. We have previously identified a cell-wall-anchored collagen adhesin, Acm, produced by some isolates of E. faecium, and a secreted antigen, SagA, exhibiting broad-spectrum binding to extracellular matrix proteins. Here, we analysed the draft genome of strain TX0016 for potential microbial surface components recognizing adhesive matrix molecules (MSCRAMMs). Genome-based bioinformatics identified 22 predicted cell-wall-anchored E. faecium surface proteins (Fms), of which 15 (including Acm) had characteristics typical of MSCRAMMs, including predicted folding into a modular architecture with multiple immunoglobulin-like domains. Functional characterization of one [Fms10; redesignated second collagen adhesin of E. faecium (Scm)] revealed that recombinant Scm(65) (A- and B-domains) and Scm(36) (A-domain) bound to collagen type V efficiently in a concentration-dependent manner, bound considerably less to collagen type I and fibrinogen, and differed from Acm in their binding specificities to collagen types IV and V. Results from far-UV circular dichroism measurements of recombinant Scm(36) and of Acm(37) indicated that these proteins were rich in beta-sheets, supporting our folding predictions. Whole-cell ELISA and FACS analyses unambiguously demonstrated surface expression of Scm in most E. faecium isolates. Strikingly, 11 of the 15 predicted MSCRAMMs clustered in four loci, each with a class C sortase gene; nine of these showed similarity to Enterococcus faecalis Ebp pilus subunits and also contained motifs essential for pilus assembly. Antibodies against one of the predicted major pilus proteins, Fms9 (redesignated EbpC(fm)), detected a 'ladder' pattern of high-molecular-mass protein bands in a Western blot analysis of cell surface extracts from E. faecium, suggesting that EbpC(fm) is polymerized into a pilus structure. Further analysis of the transcripts of the corresponding gene cluster indicated that fms1 (ebpA(fm)), fms5 (ebpB(fm)) and ebpC(fm) are co-transcribed, a result consistent with those for pilus-encoding gene clusters of other Gram-positive bacteria. All 15 genes occurred frequently in 30 clinically derived diverse E. faecium isolates tested. The common occurrence of MSCRAMM- and pilus-encoding genes and the presence of a second collagen-binding protein may have important implications for our understanding of this emerging pathogen.

Enterococcus faecium recently evolved from a generally avirulent commensal into a multidrug-resistant health care-associated pathogen causing difficult-to-treat infections, but little is known about the factors responsible for this change. We previously showed that some E. faecium strains express a cell wall-anchored collagen adhesin, Acm. Here we analyzed 90 E. faecium isolates (99% acm + ) and found that the Acm protein was detected predominantly in clinically derived isolates, while the acm gene was present as a transposon-interrupted pseudogene in 12 of 47 isolates of nonclinical origin. A highly significant association between clinical (versus fecal or food) origin and collagen adherence ( P ≤ 0.0003) was also demonstrated, and levels of adherence were highly correlated ( r = 0.879) with the amount of cell surface Acm detected by whole-cell enzyme-linked immunosorbent assay and flow cytometry. Thirty-seven of 41 sera from patients with E. faecium infections showed reactivity with recombinant Acm, while only 4 of 30 community and hospitalized patient control group sera reacted ( P ≤ 0.0003); importantly, antibodies to Acm were present in all 14 E. faecium endocarditis patient sera. Although pulsed-field gel electrophoresis indicated that multiple strains expressed collagen adherence, multilocus sequence typing demonstrated that the majority of collagen-adhering isolates, as well as 16 of 17 endocarditis isolates, are part of the hospital-associated E. faecium genogroup referred to as clonal complex 17 (CC17), which has emerged globally. Taken together, our findings support the hypothesis that Acm has contributed to the emergence of E. faecium and CC17 in nosocomial infections.

Background. Most previous studies have found that Enterococcus faecalis isolates do not show significant adherence to fibronectin and fibrinogen. Methods. The influence of various conditions on E. faecalis adherence to extracellular matrix (ECM) proteins was evaluated using a radiolabeled-cell adherence assay. Results. Among the conditions studied, growth in 40% horse serum (a biological cue with potential clinical relevance) elicited adherence of all 46 E. faecalis strains tested to fibronectin and fibrinogen but not to elastin; adherence levels were independent of strain source, and adherence was eliminated by treating cells with trypsin. As previously reported, serum also elicited adherence to collagen. Although prolonged exposure to serum during growth was needed for enhancement of adherence to fibrinogen, brief exposure (

The cfr (chloramphenicol-florfenicol resistance) gene encodes a 23S rRNA methyltransferase that confers resistance to linezolid. Detection of linezolid resistance was evaluated in the first cfr -carrying human hospital isolate of linezolid and methicillin-resistant Staphylococcus aureus (designated MRSA CM-05) by dilution and diffusion methods (including Etest). The presence of cfr was investigated in isolates of staphylococci colonizing the patient's household contacts and clinical isolates recovered from patients in the same unit where MRSA CM-05 was isolated. Additionally, 68 chloramphenicol-resistant Colombian MRSA isolates recovered from hospitals between 2001 and 2004 were screened for the presence of the cfr gene. In addition to erm (B), the erm (A) gene was also detected in CM-05. The isolate belonged to sequence type 5 and carried staphylococcal chromosomal cassette mec type I. We were unable to detect the cfr gene in any of the human staphylococci screened (either clinical or colonizing isolates). Agar and broth dilution methods detected linezolid resistance in CM-05. However, the Etest and disk diffusion methods failed to detect resistance after 24 h of incubation. Oxazolidinone resistance mediated by the cfr gene is rare, and acquisition by a human isolate appears to be a recent event in Colombia. The detection of cfr -mediated linezolid resistance might be compromised by the use of the disk diffusion or Etest method.

An unusual increase in infections caused by vancomycin-resistant Enterococcus gallinarum (VREG) was identified in May 2004, in a Colombian tertiary care teaching hospital. A case-control study was subsequently designed to identify risk factors associated with the development of infections due to these organisms. All VREG isolates were subjected to antimicrobial susceptibility testing, vancomycin resistance gene detection and pulsed-field gel electrophoresis (PFGE) typing. Additionally, the presence of genes associated with an acquired pathogenicity island of E. faecalis and a hyl-like gene of E. faecium was assessed by hybridisation assays. Eleven cases of VREG were identified between May through June 2004. VREG was isolated from blood (N=4), surgical secretions (N=4), paranasal sinus secretion (N=1), lung abscess (N=1) and urine (N=1). Infections with VREG were associated with mucositis, hospitalisation in the haematology ward and surgical unit, length of hospital stay prior to culture and invasive procedures within 30 days prior to the culture. Logistic regression found that female sex and hospitalisation in the surgical unit were independent factors for VREG infection. All isolates were identified as E. gallinarum, harboured the vanC1 gene and exhibited indistinguishable restriction patterns by PFGE. Virulence-associated genes were not detected. This is the first documented hospital-wide outbreak of VREG and highlights the fact that uncommon species of enterococci are capable of nosocomial dissemination.

SUMMARYShigellosis is endemic in Central Australia and the infections are predominantly due toShigella flexneri6,Shigella flexneri2a andShigella sonnei. Plasmid profiles of isolates collected from 1985–9, suggested that infections caused byShigella flexneri6 were predominantly due to a single clone, whereas those caused byShigella flexneri2a andShigella sonneiwere due to several genetically diverse strains, although strains with identical plasmid profiles were found in widely separated geographical areas and in different years.

In a previous study, we found fecal colonization with multiresistant Escherichia coli exhibiting high-level trimethoprim resistance in 19% of diapered children attending six day-care centers in Houston, Tex. To examine the potential risk factors associated with this finding, we conducted cross-sectional studies among 203 children attending 12 day-care centers, 51 children attending a well-child clinic (controls), and 64 medical students. The prevalence of fecal colonization with trimethoprim-resistant E. coli among children attending day-care centers (30%) was higher (P less than 0.001) than among control children (6%) or medical students (8%). The prevalence of colonization among the children attending the 12 centers ranged from 0 to 59% and was correlated with the number of diapered children enrolled (r = 0.73; P less than 0.01). In a case control study among the day-care center children, significant risk factors were an age of less than 12 months and attendance at a center with an enrollment of over 40 diapered children (odds ratios of 2.2 and 3.5, respectively); ethnicity, duration of attendance, and prior antibiotic administration were not associated with colonization. Plasmid analysis of 60 of the day-care center strains revealed 22 profiles, each of which was unique to a given day-care center. Transmission and carriage of trimethoprim-resistant strains for as long as 6 months was documented in one center studied on three occasions. Given the documented transmission of enteric pathogens among diapered children attending day-care centers and their spread into family members, it is likely that day-care centers are an important community reservoir of plasmid-associated antibiotic-resistant E. coli.

The neurologic symptoms in human shigellosis have often been attributed to Shiga toxin, although its exact role has not been determined. By use of a [3H] thymidine-labeled HeLa cell assay, cytotoxic activity was demonstrated in stool but not cerebrospinal fluid or serum from five patients with shigellosis presenting with seizures or encephalopathy. Bacterial isolates produced 16.0-88.2 CD50 (50% cytotoxic dose) of cytotoxin/mg of protein. The toxin activity in stool and the cytotoxic activity of the isolates were not neutralized by antiserum to purified Shiga toxin. DNA hybridization studies showed that Shigella isolates from these patients lacked the structural genes for Shiga toxin. The cytotoxin produced was also distinct from Shiga-like toxins I and II. Sonicates of the Shigella strains injected intraperitoneally into mice caused lethargy and lethality. The toxin activity was heat-labile and sensitive to trypsin, indicating that its active component is protein. Ultrafiltration and gel filtration chromatography showed a molecular mass of 100-125 kDa. Thus Shiga toxin production is not essential for the development of neurologic manifestations of shigellosis; other toxic products may play a role.

Enterococci are important human pathogens that are increasingly resistant to antimicrobial agents. These organisms were previously considered part of the genus Streptococcus but have recently been reclassified into their own genus, called Enterococcus. To date, 12 species pathogenic for humans have been described, including the most common human isolates, Enterococcus faecalis and E. faecium. Enterococci cause between 5 and 15% of cases of endocarditis, which is best treated by the combination of a cell wall-active agent (such as penicillin or vancomycin, neither of which alone is usually bactericidal) and an aminoglycoside to which the organism is not highly resistant; this characteristically results in a synergistic bactericidal effect. High-level resistance (MIC, greater than or equal to 2,000 micrograms/ml) to the aminoglycoside eliminates the expected bactericidal effect, and such resistance has now been described for all aminoglycosides. Enterococci can also cause urinary tract infections; intraabdominal, pelvic, and wound infections; superinfections (particularly in patients receiving expanded-spectrum cephalosporins); and bacteremias (often together with other organisms). They are now the third most common organism seen in nosocomial infections. For most of these infections, single-drug therapy, most often with penicillin, ampicillin, or vancomycin, is adequate. Enterococci have a large number of both inherent and acquired resistance traits, including resistance to cephalosporins, clindamycin, tetracycline, and penicillinase-resistant penicillins such as oxacillin, among others. The most recent resistance traits reported are penicillinase resistance (apparently acquired from staphylococci) and vancomycin resistance, both of which can be transferred to other enterococci. It appears likely that we will soon be faced with increasing numbers of enterococci for which there is no adequate therapy.

Invasive disease due to Acinetobacter baumannii is an increasing problem in health care settings worldwide. Whether certain clones of A. baumannii are more likely to cause invasive disease in hospitalized patients is unknown. We studied all patients at a public teaching hospital in Houston, Texas, from whom the Acinetobacter calcoaceticus-Acinetobacter baumannii complex was isolated over a 14-month period in 2005 to 2006. One hundred seven unique patient isolates were identified, with 87 of the strains classified as being A. baumannii , the majority of which were multidrug resistant. The A. baumannii isolates were comprised of 18 unique pulsed-field types, with strains of clone A and clone B accounting for 66 of the 87 isolates. Epidemiologic analysis showed the predominance of the two A. baumannii clones at distinct time periods, with the remainder of the A. baumannii and non- A. baumannii strains being evenly distributed. Patients from whom clone A strains were isolated were more likely to be bacteremic than were patients with other A. baumannii isolates. Conversely, clone B strains were more likely to be isolated from patients with tertiary peritonitis. Patients from whom clone A was isolated had a significantly higher rate of mortality. Multilocus sequence typing demonstrated that clones A and B are related to each other and to A. baumannii strains previously isolated in Western Europe, sharing five of seven alleles. Taken together, we conclude that the outbreak of the A. calcoaceticus-A. baumannii complex in our institution was due to two distinct A. baumannii clones that were associated with significantly different patient outcomes.

Deletion mutants of the two sortase genes of Enterococcus faecalis OG1RF were constructed. srtC (renamed here bps for biofilm and pilus-associated sortase) was previously shown to be necessary for the production of Ebp pili and important for biofilm formation and endocarditis. Here, we report that a srtA deletion mutant showed a small (5%) yet significant (P = 0.037) reduction in biofilm relative to OG1RF, while a DeltasrtA Deltabps double mutant showed a much greater reduction (74% versus OG1RF and 44% versus the Deltabps mutant). In a murine urinary tract infection (UTI), the 50% infective doses of both the DeltasrtA Deltabps and Deltabps mutants were approximately 2 log10 greater than that of OG1RF or the DeltasrtA mutant. Similarly, approximately 2 log10 fewer bacteria were recovered from the kidneys after infection with the Deltabps mutant (P = 0.017) and the DeltasrtA Deltabps double mutant (P = 0.022) compared to wild-type strain OG1RF. In a competition UTI, the Deltabps mutant was slightly, but not significantly, less attenuated than the DeltasrtA Deltabps double mutant. Fluorescence-activated cell sorter analysis with Ebp-specific antibodies confirmed that a minority of OG1RF cells express Ebp pili on their surface in vitro and that Bps has a major role in Ebp pilus biogenesis but also indicated a function for SrtA in surface localization of the pilus subunit protein EbpA. In conclusion, deletion of bps had a major effect on virulence in murine UTIs, as well as biofilm; deletion of srtA from OG1RF had little effect on these phenotypes, but its deletion from a bps mutant had a pronounced effect on biofilm, suggesting that Bps and/or the proteins it anchors may compensate for the loss of some SrtA function(s).

We identify ef1090 (renamed ebpR ) and show its importance for the transcriptional regulation of expression of the Enterococcus faecalis pilus operon, ebpABC . An ebpR deletion (Δ ebpR ) mutant was found to have reduced ebpABC expression with loss of pilus production and a defect in primary adherence with, as a consequence, reduced biofilm formation.

Strains of Enterococcus faecium express a cell wall-anchored protein, Acm, which mediates adherence to collagen. Here, we (i) identify the minimal and high-affinity binding subsegments of Acm and (ii) show that anti-Acm immunoglobulin Gs (IgGs) purified against these subsegments reduced E. faecium TX2535 strain collagen adherence up to 73 and 50%, respectively, significantly more than the total IgGs against the full-length Acm A domain (28%) ( P < 0.0001). Blocking Acm adherence with functional subsegment-specific antibodies raises the possibility of their use as therapeutic or prophylactic agents.

A patient with native valve endocarditis caused by a vancomycin "heteroresistant" strain of Enterococcus faecium experienced failure of daptomycin monotherapy without evidence of daptomycin resistance. The infecting organism exhibited in vivo emergence of a vancomycin-susceptible subpopulation lacking vanA. Treatment with a combination of high-dose daptomycin, gentamicin, and high-dose ampicillin cleared the infection.

Ceftobiprole is a novel broad-spectrum cephalosporin with good in vitro activity against methicillin-resistant Staphylococcus aureus and Enterococcus faecalis. The objective of this study was to assess the in vivo activity of ceftobiprole against four strains of E. faecalis, including β-lactamase- producing (Bla+) and vancomycin-resistant strains. Mice were infected intraperitoneally with strains of E. faecalis: (i) the Bla+ strain HH22; (ii) two vancomycin-resistant strains (TX2484 and V583); and (iii) OG1RF (a laboratory strain), using 10 × the LD50 for each strain. Ceftobiprole doses of 25, 12.5 and 6.25 mg/kg (single doses) and ampicillin 50, 25, 12.5 and 6.25 mg/kg (single and double doses) were administered subcutaneously immediately after bacterial challenge and mice were monitored for 96 h. All four E. faecalis had ceftobiprole MICs ≤0.5 mg/L. Despite being susceptible in vitro at the standard inoculum, ampicillin (single and double doses) did not protect mice against intraperitoneal challenge with Bla+ E. faecalis HH22, with a 50% protective dose (PD50) of >100 mg/kg, whereas ceftobiprole was protective (PD50 of 2 mg/kg). Ceftobiprole PD50s for vancomycin-resistant isolates TX2484 and V583 were 7.7 and 5.2 mg/kg, respectively, similar to those of single dose ampicillin (12.5 and 16.4 mg/kg, respectively). For OG1RF, both ampicillin and ceftobiprole protected all mice at doses of 25 and 12.5 mg/kg, respectively, with a PD50 of 4.2 and 8 mg/kg for ceftobiprole and ampicillin, respectively. Ceftobiprole had comparable in vivo activity to that of ampicillin against vancomycin-resistant and ampicillin-susceptible strains of E. faecalis in the mouse peritonitis model. Ceftobiprole was superior in vivo to ampicillin against the Bla+ strain HH22. Our data support the further study of ceftobiprole as a therapeutic agent in humans infected with E. faecalis.

Ceftobiprole (BAL9141) is an investigational cephalosporin with broad in vitro activity against gram-positive cocci, including enterococci. Ceftobiprole MICs were determined for 93 isolates of Enterococcus faecalis (including 16 β-lactamase [Bla] producers and 17 vancomycin-resistant isolates) by an agar dilution method following the Clinical and Laboratory Standards Institute recommendations. Ceftobiprole MICs were also determined with a high inoculum concentration (10 7 CFU/ml) for a subset of five Bla producers belonging to different previously characterized clones by a broth dilution method. Time-kill and synergism studies (with either streptomycin or gentamicin) were performed with two β-lactamase-producing isolates (TX0630 and TX5070) and two vancomycin-resistant isolates (TX2484 [VanB] and TX2784 [VanA]). The MICs of ceftobiprole for 50 and 90% of the isolates tested were 0.25 and 1 μg/ml, respectively. All Bla producers and vancomycin-resistant isolates were inhibited by concentrations of ≤1 and ≤4 μg/ml, respectively, at the standard inoculum concentration. Ceftobiprole MICs at a high inoculum concentration for a subset of five Bla + E. faecalis isolates were ≤1 μg/ml. Bactericidal activity was observed against four isolates tested at concentrations as low as 1 μg/ml regardless of the production of β-lactamase or vancomycin resistance. A combination of ceftobiprole (0.5 μg/ml) and streptomycin (25 μg/ml) was synergistic against Bla + TX0630 and TX5070. Ceftobiprole (0.5 μg/ml) plus gentamicin (10 μg/ml) was synergistic against VanB isolate TX2484 and showed enhanced killing, but not synergism, against TX2784 (VanA), despite the absence of high-level resistance to gentamicin. In conclusion, ceftobiprole exhibited good in vitro activity against E. faecalis , including Bla + and vancomycin-resistant strains, and exhibited synergism with aminoglycosides against selected isolates.

The commensal Enterococcus faecalis is among the most common causes of nosocomial infections. Recent findings regarding increased abundance of enterococci in the intestinal microbiota of patients with inflammatory bowel diseases and induction of colitis in IL-10-deficient (IL-10-/-) mice put a new perspective on the contribution of E. faecalis to chronic intestinal inflammation. Based on the expression of virulence-related genes in the inflammatory milieu of IL-10-/- mice using RNA-sequencing analysis, we characterized the colitogenic role of two bacterial structures that substantially impact on E. faecalis virulence by different mechanisms: the enterococcal polysaccharide antigen and cell surface-associated lipoproteins. Germ-free wild type and IL-10-/- mice were monoassociated with E. faecalis wild type OG1RF or the respective isogenic mutants for 16 weeks. Intestinal tissue and mesenteric lymph nodes (MLN) were collected to characterize tissue pathology, loss of intestinal barrier function, bacterial adhesion to intestinal epithelium and immune cell activation. Bone marrow-derived dendritic cells (BMDC) were stimulated with bacterial lysates and E. faecalis virulence was additionally investigated in three invertebrate models. Colitogenic activity of wild type E. faecalis (OG1RF score: 7.2±1.2) in monoassociated IL-10-/- mice was partially impaired in E. faecalis lacking enterococcal polysaccharide antigen (ΔepaB score: 4.7±2.3; p, Author Summary Enterococcus faecalis is a commensal of the human intestinal core microbiota harboring several putative virulence factors, which highlight its role as opportunistic pathogen. This dualistic character is supported by recent evidence linking Enterococcus spp. to the pathogenesis of inflammatory bowel diseases (IBD). Although several studies suggest a crucial role for opportunistic pathogens in IBD pathogenesis targeting genetically susceptible individuals, the dynamic relationship between disease-relevant host compartments and specific bacterial structures able to trigger intestinal inflammation remain unclear. Here, we report that cell surface-associated lipoproteins and the enterococcal polysaccharide antigen, which are relevant for E. faecalis virulence in invertebrate infection models, but whose expression is minimally affected by the intestinal inflammatory milieu, exhibit colitogenic activity in a mouse model susceptible for chronic colitis. Bacterial lipoproteins trigger innate immune cell activation and are a critical prerequisite for E. faecalis-induced colitis. The enterococcal polysaccharide antigen mediates bacterial mucus penetration and adhesion to mucosal surfaces, promotes the formation of biofilm and contributes to E. faecalis colitogenic activity. Using E. faecalis as a model organism, we demonstrate that colitogenic activity of opportunistic pathogens can be assigned to specific bacterial structures, a finding that may help to identify the most essential steps in IBD-related microbe-host interactions.

In the present study, the commensal and pathogenic host-microbe interaction of Enterococcus faecalis was explored using a Caenorhabditis elegans model system. The virulence of 28 E. faecalis isolates representing 24 multilocus sequence types (MLSTs), including human commensal and clinical isolates as well as isolates from animals and of insect origin, was investigated using C. elegans strain glp-4 ( bn2ts ); sek-1 ( km4 ). This revealed that 6 E. faecalis isolates behaved in a commensal manner with no nematocidal effect, while the remaining strains showed a time to 50% lethality ranging from 47 to 120 h. Principal component analysis showed that the difference in nematocidal activity explained 94% of the variance in the data. Assessment of known virulence traits revealed that gelatinase and cytolysin production accounted for 40.8% and 36.5% of the observed pathogenicity, respectively. However, coproduction of gelatinase and cytolysin did not increase virulence additively, accounting for 50.6% of the pathogenicity and therefore indicating a significant (26.7%) saturation effect. We employed a comparative genomic analysis approach using the 28 isolates comprising a collection of 82,356 annotated coding sequences (CDS) to identify 2,325 patterns of presence or absence among the investigated strains. Univariate statistical analysis of variance (ANOVA) established that individual patterns positively correlated ( n = 61) with virulence. The patterns were investigated to identify potential new virulence traits, among which we found five patterns consisting of the phage03-like gene clusters. Strains harboring phage03 showed, on average, 17% higher killing of C. elegans ( P = 4.4e −6 ). The phage03 gene cluster was also present in gelatinase-and-cytolysin-negative strain E. faecalis JH2-2. Deletion of this phage element from the JH2-2 clinical strain rendered the mutant apathogenic in C. elegans , and a similar mutant of the nosocomial V583 isolate showed significantly attenuated virulence. Bioinformatics investigation indicated that, unlike other E. faecalis virulence traits, phage03-like elements were found at a higher frequency among nosocomial isolates. In conclusion, our report provides a valuable virulence map that explains enhancement in E. faecalis virulence and contributes to a deeper comprehension of the genetic mechanism leading to the transition from commensalism to a pathogenic lifestyle.

Hybrid total knee arthroplasty in which the femoral component is cementless and the tibial component is cemented remains controversial. We report on the long-term results of 65 total knee arthroplasties followed for an average of 15 years. There were 18 revisions (27%); of these, 11 were femoral component revisions. The overall implant survival with revision as the end point was estimated as 64% at 15 years. The femoral component survivorship at 15 years was 72%. Because of the high failure rate, hybrid fixation on the femoral side with this design has been abandoned at our institution; but hybrid fixation with a trabecular metal tibia is currently under investigation.

Background. We recently demonstrated that the ubiquitous Enterococcus faecalis ebp (endocarditis-and biofilmassociated pilus) operon is important for biofilm formation and experimental endocarditis. Here, we assess its role in murine urinary tract infection (UTI) by use of wild-type E. faecalis OG1RF and its nonpiliated, ebpA allelic replacement mutant (TX5475).Methods. OG1RF and TX5475 were administered transurethrally either at an ∼1:1 ratio (competition assay) or individually (monoinfection). Kidney pairs and urinary bladders were cultured 48 h after infection. These strains were also tested in a peritonitis model.Results. No differences were observed in the peritonitis model. In mixed UTIs, OG1RF significantly outnumbered TX5475 in kidneys (P = .0033 ) and bladders (P ⩽ .0001 ). More OG1RF colony-forming units were also recovered from the kidneys of monoinfected mice at the 4 inocula tested (P = .015 to P = .049), and 50% infective doses of OG1RF for kidneys and bladder ( 9.1 × 10 1 and 3.5 × 10 3 cfu, respectively) were 2–3 log 10 lower than those of TX5475. Increased tropism for the kidney relative to the bladder was observed for both OG1RF and TX5475.Conclusion. The ebp locus, part of the core genome of E. faecalis, contributes to infection in an ascending UTI model and is the first such enterococcal locus shown to be important in this site.

During an evaluation of PCR for identification of isolates of Enterococcus hirae , a homologue with 82% identity to E. hirae mur-2 was identified in Enterococcus durans and was named mur-2 ed . PCR using primers for two genes ( copY and murG ) of E. hirae strains showed amplification with E. hirae strains only. PCR (under high-stringency conditions) with primers for the mur-2 ed gene gave the expected amplification product only with E. durans strains. A combination of murG and mur-2 ed primers in a multiplex PCR assay differentiated E. hirae from E. durans in all cases. PCR using these primers appears to be a rapid alternative for identification of E. hirae and E. durans isolates.

Inactivation by allelic exchange in clinical isolates of the emerging nosocomial pathogen Enterococcus faecium has been hindered by lack of efficient tools, and, in this study, transformation of clinical isolates was found to be particularly problematic. For this reason, a vector for allelic replacement (pTEX5500ts) was constructed that includes (i) the pWV01-based gram-positive repAts replication region, which is known to confer a high degree of temperature intolerance, (ii) Escherichia coli oriR from pUC18, (iii) two extended multiple-cloning sites located upstream and downstream of one of the marker genes for efficient cloning of flanking regions for double-crossover mutagenesis, (iv) transcriptional terminator sites to terminate undesired readthrough, and (v) a synthetic extended promoter region containing the cat gene for allelic exchange and a high-level gentamicin resistance gene, aph ( 2 ′′) -Id , to distinguish double-crossover recombination, both of which are functional in gram-positive and gram-negative backgrounds. To demonstrate the functionality of this vector, the vector was used to construct an acm (encoding an adhesin to collagen from E. faecium ) deletion mutant of a poorly transformable multidrug-resistant E. faecium endocarditis isolate, TX0082. The acm -deleted strain, TX6051 (TX0082Δ acm ), was shown to lack Acm on its surface, which resulted in the abolishment of the collagen adherence phenotype observed in TX0082. A mobilizable derivative (pTEX5501ts) that contains oriT of Tn 916 to facilitate conjugative transfer from the transformable E. faecalis strain JH2Sm::Tn 916 to E. faecium was also constructed. Using this vector, the acm gene of a nonelectroporable E. faecium wound isolate was successfully interrupted. Thus, pTEX5500ts and its mobilizable derivative demonstrated their roles as important tools by helping to create the first reported allelic replacement in E. faecium ; the constructed this acm deletion mutant will be useful for assessing the role of acm in E. faecium pathogenesis using animal models.

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 .

In the current study, the gls24 disruption mutant TX10100, previously shown to be more sensitive to bile salts and attenuated in a mouse peritonitis model, showed an approximately fivefold higher 50% infective dose than wild-type OG1RF in a rat endocarditis model. When administered as a mixture, TX10100, unlike a downstream glsB mutant, was significantly outnumbered by OG1RF in vegetations, organs, and blood, despite being inoculated in greater numbers. These results indicate that gls24 is important in the pathogenesis of enterococcal endocarditis.

Enterococci, although part of the normal flora of the human gastrointestinal tract, have been recognized as an important cause of nosocomial infection for over two decades ([8][1], [11][2]) and are commonly implicated in urinary tract infections, bacteremia, intra-abdominal and surgical wound

Previously, in our laboratory, we established a two-chamber system to study translocation of Enterococcus faecalis across monolayers of polarized human colon carcinoma-derived T84 cells. By using the same system in the present study, we now show that disruption of gelE of strain OG1RF, which also has a polar effect on the cotranscribed sprE , as well as disruption of its regulatory system ( fsrA , fsrB , and fsrC ) resulted in a loss of detectable translocation by E. faecalis OG1RF; these mutants lost gelatinase (GelE) and serine protease (SprE) production by standard assay. A gelE deletion mutant of OG1RF (GelE − SprE + ) also showed that significantly reduced translocation and complementation with the gelE gene (pTEX5438) in trans restored gelatinase and translocation, demonstrating that gelatinase is important for E. faecalis translocation. Complementation of fsrA , fsrB , and fsrC mutants with all three fsr genes also resulted in production of gelatinase and translocation. Furthermore, introduction of fsr genes into two non-gelatinase-producing E. faecalis isolates, the well-characterized laboratory strain JH2-2 and a human-derived fecal isolate, TX1322 (both of which have gelE but not fsrA or fsrB , are gelatinase negative, and do not translocate), resulted in gelatinase production by these strains and restored translocation across T84 monolayers, while transformation with pTEX5438 ( gelE ) showed little or no translocation and no detectable gelatinase, confirming the importance of both fsr and gelatinase for E. faecalis translocation. The importance of gelatinase production was also corroborated among 20 E. faecalis human isolates (7 fecal, 7 endocarditis, and 6 urine isolates), which showed translocation by all gelatinase-positive isolates but little to no translocation for gelatinase nonproducers. These results indicate that gelatinase is important for the successful in vitro translocation of E. faecalis across human enterocyte-like T84 cells.

We have previously shown that the Enterococcus faecalis lsa gene, encoding the putative ABC protein Lsa, influences resistance to quinupristin-dalfopristin (Q-D) and clindamycin (CLI). We have now found that, while cloned lsa from E. faecalis strain V583 ( lsa V ) fully restored resistance to Q-D, CLI, and dalfopristin (DAL) lost by the OG1 lsa disruption mutant TX5332 and also caused increased MICs for Lactococcus lactis LM2301, cloned lsa from OG1 ( lsa OG ) did not cause any increase in MICs for either species. Sequencing of ca. 2 kb of these two lsa alleles found differences between lsa OG and lsa V in the upstream region as well as in the 5′ and 3′ halves of the lsa gene. To investigate the reason for the phenotypic differences expressed by the two cloned loci, 5′ half plus 3′ half hybrid constructs were created. When introduced into both TX5332 and L. lactis , cloned lsa V5 ′ OG3 ′ conferred increases in MICs of Q-D, CLI, and DAL similar to those of cloned lsa V while cloned lsa OG5 ′ V3 ′ showed a moderate increase in MICs relative to those of lsa OG , indicating that both halves of the locus can influence resistance expression. After site-directed mutagenesis of the cloned lsa alleles at positions −131 and −133 (relative to the putative Lsa start codon ATG), which converted two A's of lsa V to the G and T of lsa OG and vice versa, MIC testing showed that mutagenized lsa OG ( lsa OG-M ) was strongly influenced by these changes in terms of conferring increased MICs of Q-D, CLI, and DAL relative to lsa OG while the phenotype of mutagenized lsa V ( lsa V-M ) was less influenced, with moderately decreased MICs, primarily to CLI, relative to lsa V . In conclusion, this study found that changes in different regions of the E. faecalis lsa locus influence the ability of cloned lsa to confer resistance to Q-D, CLI, and DAL.

A previously identified gene sprE of Enterococcus faecalis strain OG1 was shown to encode an extracellular serine protease that appears to belong to the glutamyl endopeptidase I staphylococcal group. A single form of SprE with a molecular mass of 25 kDa and a pH optimum between 7.0 and 7.5 was isolated from culture supernatant of wild-type E. faecalis strain OG1RF (TX4002); this form was apparently generated by cleavage of the Ser −1 -Leu 1 and Arg 230 -Leu 231 peptide bonds of the secreted zymogen. In contrast, the culture supernatant of the gelatinase-null mutant, TX5264, with a nonpolar deletion of gelE which encodes the E. faecalis gelatinase, was found to contain several forms of SprE proteolytically processed on both the N and C termini; in addition to a full-length zymogen and a truncated zymogen, three mature forms of the SprE proteinase, Leu 1 -Ala 237 , Ser −1 -Glu 227 , and Leu 1 -Glu 227 , were identified. As with the V8 proteinase of Staphylococcus aureus , the closest homologue of SprE, all of the active forms cleaved specifically Glu-Xaa peptide bonds but with substantially different efficiencies, while none was able to hydrolyze peptide bonds with Asp in the P1 position. The most active of all these enzyme forms against several substrates, including human fibrinogen and β-chain insulin, was the Ser −1 -Glu 227 ( −1 S-SprE) isolated from TX5264; −1 S-SprE, in contrast to other forms of SprE, was unstable at 37°C, apparently due to autodegradation. In conclusion, our results demonstrate that sprE encodes a highly specific serine-type glutamyl endopeptidase, the maturation of which is dependent on the presence of gelatinase. In the absence of gelatinase activity, the aberrant processing of pro-SprE results in the appearance of a “superactive” form of the enzyme, −1 S-SprE.

The recently published Enterococcus faecalis genome [ Paulsen, I. T., Banerjei, L., Myers, G. S. & 29 other authors (2003). Science 299, 2071–2074) ] was examined and 41 putative cell-wall-anchored proteins were identified. Seventeen of these proteins are predicted to contain tandemly repeated immunoglobulin-like folds characteristic of the structural organization of staphylococcal adhesins of the MSCRAMM (microbial surface component recognizing adhesive matrix molecules) type. Two of the nine proteins selected for further study appear to represent cell-wall-anchored enzymes. It is proposed that the remaining seven proteins constitute a family of structurally related proteins potentially interacting with proteins of the host. This family includes the previously identified collagen/laminin-binding MSCRAMM ACE [ Rich, R. L., Kreikemeyer, B., Owens, R. T., LaBrenz, S., Narayana, S. V., Weinstock, G. M., Murray, B. E. & Hook, M. (1999). J Biol Chem 274, 26939–26945 ]. It is further demonstrated that genes encoding the seven putative MSCRAMMs are present in all E. faecalis strains tested and these proteins appear to be expressed during infection in humans, since sera from infected individuals contain antibodies reacting with recombinant versions of the enterococcal proteins.

We examined 215 Enterococcus faecalis isolates and found that neither the two-component regulatory locus fsr ( E. faecalis regulator) nor gelatinase production was more common in disease-associated isolates than in isolates colonizing healthy individuals (ca. 60 to 65%). The majority of gelatinase-negative isolates, including 14 endocarditis isolates (of 80 isolates tested), contained the previously described 23.9-kb deletion and lacked fsrA and fsrB . While these findings indicate that neither fsr nor gelatinase is required for E. faecalis to cause infection, this study did not address whether fsr or gelatinase affects the severity of disease, as it does in animal models.

We used a two-chamber system to study transcytosis of Enterococcus faecalis across monolayers of human colon carcinoma-derived T84 cells, which show structural resemblance to the native intestine. Among 16 E. faecalis isolates from different sources, the well-characterized strain OG1RF and 8 other isolates (2 endocarditis isolates, 1 urine isolate, and all 5 fecal isolates) showed translocation in this assay, while 6 clinical isolates (3 endocarditis and 3 urine isolates), the recipient strain JH2-2, and the control, Escherichia coli DH5α, had no detectable translocation. Of two OG1RF mutants involving the previously studied epa (enterococcal polysaccharide antigen) gene cluster, known to be needed for virulence and resistance to killing by polymorphonuclear leukocytes, one epa mutant (TX5179) was unable to translocate, while TX5180, with an epa disruption farther downstream, showed a moderate decrease in translocation relative to that of the wild-type strain OG1RF ( P < 0.01), indicating that the epa gene cluster is important for translocation across a T84 monolayer. This observation was confirmed by complementation of the epa mutant (TX5179) with epa genes and restoration of its translocation ability. In conclusion, we have demonstrated translocation of at least some strains of E. faecalis across T84 monolayers, although strains differ considerably in this ability, and we have demonstrated that epa mutations can cause marked changes in successful translocation. These results suggest that this model may be a useful in vitro system for studying the process of translocation from the intestinal tract.

The article by Edmond et al. [1] in this issue illustrates many of the features common to infection or colonization by vancomycin-resistant enterococci (VRE), such as the more frequent occurrence of VRE in teaching hospitals. VRE are also more common in larger hospitals, as was shown in a 1993 survey that found vancomycin resistance in 3.6% of enterococci in hospitals with >500 beds (the hospital in the report by Edmond et al. had 542 beds), in 1.8% of enterococci in hospitals with 200-500 beds, and none in hospitals with