Esp Gene Research Articles

Changing trends in vancomycin-resistant enterococci in French hospitals, 2001-08

Unprecedented outbreaks of vancomycin-resistant enterococci (VRE) have occurred in French hospitals since 2004. The aim of this study was to provide a picture of the spread and control of VRE in France and to characterize the isolates. Notification of VRE cases to Institut de Veille Sanitaire has been mandatory since 2001. Isolates of VRE were sent to the National Reference Centre for species and vancomycin-resistance gene identification. Isolates were tested for antimicrobial susceptibility and typed by PFGE and multilocus sequence typing. Five hundred and four VRE notifications from 195 hospitals were recorded, corresponding to 2475 cases of infection (n=243) or colonization (n=2232) and 74 episodes of clustered cases. Outbreaks were controlled by implementation of infection control measures, although the number of new hospitals reporting isolation of VRE was increasing. The majority of 902 VRE isolated from 2006 to 2008 were Enterococcus faecium (94.8%) with the vanA or vanB gene. No isolate was resistant to linezolid, tigecycline or fusidic acid. PFGE analysis showed 161 different patterns. Generally a few predominant clones and several minor clones spread in a single hospital. In a subset of 46 representatives of PFGE clones, 13 different sequence types were characterized, all belonging to clonal complex CC17, while the esp and hyl genes were inconsistently detected. The national mandatory notification of unusual nosocomial events allowed rapid identification of VRE outbreaks and early implementation of control measures that have proved effective. However, VRE continue to emerge in a growing number of hospitals.

Virulence Factors in Enterococci from Partridges (Alectoris rufa) Representing a Food Safety Problem

Forty-three Enterococcus isolates recovered from fecal samples of partridges, during the partridge hunting season, were studied for gelatinase and β-hemolysis activities. The presence of fsr-gelE genes and the cyl operon was studied by polymerase chain reaction and correlated with gelatinase and β-hemolysis production, respectively. In addition, genes encoding additional virulence factors (cpd, hyl, agg, esp, and ace) was also analyzed in all enterococci. Most of our Enterococcus faecalis isolates showed gelatinase activity (10 of 15 isolates), and this activity was not present in the other enterococcal species. All enterococci that showed gelatinase activity harbored the gelE and fsr genes. A large proportion of our isolates harbored genes of the cyl operon (19 of 43 isolates), although only 1 isolate contained the five cyl tested genes (E. faecalis), being the only one that expressed β-hemolysis. From the additional virulence factors (cpd, hyl, agg, esp, and ace), at least one virulence gene was detected in 13 of the 15 E. faecalis isolates, with cpd being the most frequently detected gene (9 isolates), followed by agg (5 isolates) and ace (4 isolates) genes. These virulence genes were not detected in the other enterococcal species with the exception of one E. faecium and E. casseliflavus isolate that harbored the hyl and cpd genes, respectively. Moreover, the esp gene was not detected in any of our isolates. In conclusion, this study showed the presence of virulence factors in enterococci of partridges and the possible transmission to humans through the food chain.

Antimicrobial Resistance and Multilocus Sequence Typing of Vancomycin-ResistantEnterococcus faeciumIsolated from the Chungcheong Area

Background: Enterococcus faecium has emerged as an important nosocomial pathogen worldwide, and this trend has been associated with the dissemination of a genetic lineage designated clonal complex 17 (CC17). In the present study, characterization of the glycopeptide resistance mechanism, genetic relatedness, and pathogenicity in isolates of vancomycin-resistant E. faecium in the Chungcheong area were investigated. Methods: A total of 37 consecutive, non-duplicate, vancomycin-resistant E. faecium were isolated at three university hospitals in the Chungcheong area. The mechanism of glycopeptide resistance and pathogenicity factors were studied using PCR, and the genetic relatedness was determined via multilocus sequence type and esp repeat profile analysis. Additionally, the quinolone resistance-determining regions of parC and gyrA were sequenced to identify mutations involved in ciprofloxacin resistance. Results: Two genotypes of VRE were confirmed: VanAphenotype vanA genotype VRE (25 isolates) and VanB-phenotype vanA genotype VRE (12 isolates). MLST analysis revealed five sequence types. A significant result was that ST414 and CNS4 (4-11-1-1-1-1) were considered as belonging to CC17. The esp and hyl genes were found in 100% and 86.4% of the isolates, respectively. A total of 37 isolates showed genetic mutations in parC and gyrA. Conclusion: All isolated strains in the present study belonged to one of the CC17 genotypes including ST414 and CNS4 (4-1-1-1-1-1-1), which were not previously detected in Korea. The combination of MLST and the esp gene repeat profiles can be useful for genetic characterization of VREF isolates with regard to the evolutionary process and epidemiology of the clones. (Korean J Clin Microbiol 2011;14:6066)

Molecular Epidemiology of Vancomycin-ResistantEnterococcus faeciumBloodstream Infections Among Patients with Neutropenia Over a 6-Year Period in South Korea

Over a 6-year period (March 2000-February 2006), there were 60 vancomycin-resistant Enterococcus faecium (VREF) bloodstream infections (BSIs) in a hematology unit, accounting for 83.3% of all VREF BSIs in the hospital. We investigated 49 VREF isolates causing BSIs in patients with neutropenia to understand the molecular epidemiology at this unit. All isolates had the vanA genotype. Pulsed-field gel electrophoresis typing revealed high clonal diversity (23 types with nine clusters comprising 35 isolates) and 1 predominant type, type A (14/49, 28.6%), persisted at this unit throughout the study period, suggesting the clonal spread of this endemic strain by cross-contamination. Tn1546 types were less heterogeneous, with five main Tn1546 types, two of which (types I and IV) accounted for 67.4% of isolates. This indicates that in addition to clonal spread, the horizontal transfer of Tn1546 played a major role in the nosocomial dissemination of vancomycin resistance. The genetic diversity of VREF increased over time, implying an increasing influx of new strains into the unit and genetic changes, possibly attributable to the horizontal transfer of diverse Tn1546 types. Despite such diversity, all the isolates belonged to clonal complex 17, which is the epidemic clone worldwide, enriched with the esp (35/49, 71.4%) and hyl (24/49, 48.9%) virulence genes. This hospital-adapted clone has become endemic and is well suited to causing BSIs in patients with neutropenia in this unit.

Characterization of virulence factors and clonal diversity of Enterococcus faecalis isolates from treated dental root canals

The high prevalence of Enterococcus faecalis in root canal treated teeth with post-treatment disease, as evidenced by both molecular and traditional culturing methods, suggests that this species may be a key player in endodontic treatment failure. This study aimed to detect virulence factors by phenotypic and western blotting tests, and virulence genes by PCR from 20 clinical strains of E. faecalis isolated from treated root canals of teeth with (10) or without (10) apical periodontitis. Moreover, genomic diversity of these strains was assessed by pulsed-field gel electrophoresis (PFGE) and rep-PCR. All 20 strains presented the gelE gene (gelatinase), but 10 of them did not hydrolyze gelatin. Seven of the 10 gelatinase-producing isolates were recovered from root canals with lesions, which suggests a role for this virulence factor in the pathogenesis of post-treatment disease. The esp gene was expressed only in cases where gelatinase production was negative. The other virulence genes were found in 90% ( efaA and ace genes), 45% ( agg gene) and 95% ( cpd gene) of the E. faecalis isolates. As for PFGE and rep-PCR, no specific clonal type of E. faecalis was found in association with teeth with or without disease, revealing the interindividual clonal diversity of endodontic infections.

Comparative study among clinical and commensal isolates of Enterococcus faecalis for presence of esp gene and biofilm production

Because of increasing difficulty in treating enterococcal infections, effort is being devoted to understanding factors that are responsible for causing nosocomial infection, with a focus toward targeting these factors with new therapeutics. Evidence has emerged that the esp genemediates biofilm formation in vitro, which helps the organism colonize and cause infection. This study was conducted over a four-year period in a tertiary-care hospital. There were 200 clinical pathogenic strains isolated from nosocomial infections and 100 commensals from stool specimens of healthy individuals. The study compared the production of biofilm and detection of the esp gene among clinical and commensal isolates. Among 200 clinical isolates of Enterococcus faecalis 65 (32.5%) isolates were positive for biofilm production and 60 (30%) for the esp gene by PCR. Among 100 commensal isolates, 16(8%) and 14 (7%) were positive for biofilm formation and the esp gene, respectively. Five clinical and two commensal isolates produced biofilm without any amplification of the esp gene. The study shows a significant difference in production of biofilm and presence of the esp gene between clinical and commensal isolates (P < 0.002). Therefore, it can be concluded that biofilm production has an important role in causing nosocomial infection. Although detection of the esp gene correlates with biofilm production, it may not be the only factor determining the formation of biofilm since few isolates produced biofilm without the esp gene. Strains isolated from indwelling medical devices showed high production of biofilm and esp gene.

Investigation of human sewage pollution and pathogen analysis at Florida Gulf coast Beaches

Water quality at two Florida beaches was compared using faecal indicator bacteria measurements, microbial source tracking (MST) methods for detecting human source pollution and the assessment of pathogen presence. These values were also compared before and after remediation of wastewater infrastructure at one beach. Faecal coliforms, Escherichia coli and enterococci were enumerated in estuarine water and sediment samples. PCR assays for the human-associated esp gene of Enterococcus faecium and human polyomaviruses (HPyVs) were used to detect human sewage. Culturable Salmonella and enteric viruses were also analysed. MST identified human sewage contamination at one beach, leading to repair of a sewer main and relocation of portable restrooms. Exceedances of Florida recreational water regulatory standards were significantly reduced after remediation (by 52% for faecal coliforms and 39% for enterococci), and the frequency of detection of MST markers decreased. Coxsackie virus B4 and HPyVs were codetected following a major sewage spill, but Salmonella was not detected during the study. These data indicate that infrastructure remediation significantly reduced pollution from human sewage at the impacted beach. A comprehensive microbial water quality study that can identify contamination sources through the use of MST markers and close collaboration with local/and state agencies can result in tangible actions to improve recreational water quality and safety.

Biofilm formation and esp, gelE, ebpA genes and QS-fsr system in Enterococcus faecalis

Objective To investigate the association of esp, gelE, ebpA and QS-fsr system and biofilm formation in Enterococcus faecalis. Methods Totally 24 isolates of Enterococcus faecalis were collected from urine and catheter of clinical urine tract infection patients in Third Xiangya Hospital from Oct. 2007 to Jun. 2008, and were divided into biofilm group and non-biofilm group. The luminance ratios of esp, gelE, ebpA and fsrrB of Enterococcus faecalis in biofilm group and non-biofilm group were detected by RT-PCR. And the expression of esp, gelE, ebpA, fsrrB genes in different groups were detected by real-time PCR and were relatively quantitated through 2-△△Ct method. Moreover, the relevancies between that fourgenes and biofilm formation in Enterococcus faecalis were analyzed respectively. Results The expression of esp and ebpA in biofilm group were 298 times and 59 times more than the non-biofilm group. The expression level ofgelE and fsrB in biofilm group were 1/244 and 1/249 times less than the non-biofilm group, and the luminance ratios of esp, gelE, ebpA and fsrB were not significant between the two groups (rank sum was 92,79, 42 and 34 respectively,all P ＞ 0. 05 ). Conclusions The results showed that the biofilm formation in Enterococcus faecalis was promoted by esp and ebpA, and was inhabited by gelE and fsrB, which suggested that the expression of esp, ebpA and gelE genes was regulated by fsr system. Key words: Enterococcus faecalis; Bacterial proteins; Fimbriae,bacterial; Biofilms; Urinary tract infections

Prevalence of virulence factors and antibiotic resistance in vancomycin-resistant Enterococcus faecium isolated from sewage and clinical samples in Iran

Purpose: The purpose of the present study was to perform a molecular epidemiological survey by investigating the antibiotic resistance and the presence of known virulence factors in Enterococcus faecium isolates in Iran. The data collected from this study would allow us to control the spread and develop strategies for treatment of the enterococcal infections. Materials and Methods: In this study, 156 vancomycin-sensitive E. faecium (VSEF; 58) and vancomycin-resistant E. faecium (VREF; 98) samples were isolated from clinical specimen and sewage treatment plants (STPs). These isolates were screened for the presence of genes encoding for aggregation substance (asa1), cytolysin (cyl), enterococcal surface protein (esp), gelatinase (gelE) and hyaluronidase (hyl) by polymerase chain reaction (PCR). Results: Although significantly different, the results showed the presence of hyl and esp genes in both clinical (41 and 75%, respectively) and sewage (3.2 and 41%, respectively) isolates. Sensitivity of all isolates to seven antibiotics was examined. The results of the clinical isolates showed that the majority of esp positive isolates were also resistant to vancomycin, ciprofloxacin and erythromycin. Furthermore, cyl, gelE and asa1 were not found in either clinical or STP isolates. Finally, we determined the distinct types of isolates using Pulse Field Gel Electrophoresis (PFGE), which confirmed that most of the isolates were clonally unrelated. Conclusion: Our results demonstrated that higher number of the clinical E. faecium isolates carried virulence genes than the isolates from STP. Finally, the lack of the genes in clinical and STP isolates confirmed that these genes do not transfer horizontally.

An evolving integrative physiology: skeleton and energy metabolism

The adipocyte-derived hormone leptin regulates appetite and bone mass. Recent research demonstrates that reciprocally, osteoblasts have a role in controlling energy metabolism. Several genes expressed in osteoblasts are involved in this process, and one of them is the Esp gene. The remaining genes regulate Esp gene expression. OST-PTP, the protein name of Esp, regulates the carboxylation of osteocalcin secreted from osteoblasts, thus affecting insulin sensitivity and insulin secretion. This review provides evidence for a novel interpretation of the connection between bone and energy metabolism and expands our understanding of the novel physiology of bone beyond its classical functions.

Characterization of Vancomycin-Resistant Enterococci Isolated from Fecal Samples of Ostriches by Molecular Methods

The aim of this study was to estimate the occurrence of vancomycin-resistant enterococci (VRE) in fecal samples of ostriches from a farm of Southern Portugal, the mechanisms implicated, and the associated virulence factors, 13 years after the banning of the glycopeptide avoparcin as animal growth promoter in the European Union. Fifty-four fecal samples of ostriches were inoculated in Slanetz-Bartley supplemented with vancomycin (4 microg/mL) for VRE recovery. Susceptibility to 11 antibiotics was performed by disk-diffusion agar method in recovered VRE isolates. The mechanism of resistance to vancomycin and to other antibiotics and the presence of the esp and hyl virulence genes were determined by polymerase chain reaction and sequencing. VRE were detected in 7 of the 54 ostrich fecal samples (13%); Enterococcus durans isolates with the vanA genotype were found in 4 of the 54 fecal samples (7.4%), and Enterococcus gallinarum with the intrinsic vanC1 genotype in the remaining three VRE-positive samples. All vanA-containing E. durans isolates showed resistance to tetracycline and erythromycin, and one of them also to ciprofloxacin; they harbored the erm(B) and tet(M) genes, as well as the specific sequences of Tn916 and Tn5397 transposons, but not the esp or hyl virulence genes. Two of the three vanC1 isolates showed resistance to tetracycline [with the tet(M) gene] and one to erythromycin [with the erm(B) gene], and all three contained the hyl gene. Fecal samples of ostriches represent a reservoir of vanA-containing enterococci that could be transmitted to humans through the food chain.

Stored-product insects carry antibiotic-resistant and potentially virulent enterococci

A total of 154 enterococcal isolates from 95 stored-product insects collected from a feed mill, a grain storage silo, and a retail store were isolated and identified to the species level using PCR. Enterococcus casseliflavus represented 51% of the total isolates, followed by Enterococcus gallinarum (24%), Enterococcus faecium (14%), Enterococcus faecalis (7%), and Enterococcus hirae (5%). Many isolates were resistant to tetracycline (48%), followed by streptomycin (21%), erythromycin (14%), kanamycin (13%), ciprofloxacin (12%), ampicillin (4%), and chloramphenicol (<1%). Enterococci carried genes coding for virulence factors, including the gelatinase gene gelE (26% of isolates), an enterococcal surface protein gene esp (1%), and the cytolysin gene cylA (2%). An aggregation substance (asa1) gene was detected in six out of 10 E. faecalis isolates and five of these were positive for the aggregation substance. Enterococci were positive for hemolytic (57% of isolates) and gelatinolytic (23%) activity. The filter-mating assay showed that the tetracycline resistance gene, tetM, was transferable among E. faecalis by conjugation. These data demonstrated that stored-product insects can serve as potential vectors in disseminating antibiotic-resistant and potentially virulent enterococci.

Tn1546 structures and multilocus sequence typing of vanA-containing enterococci of animal, human and food origin

To characterize the Tn1546 structure and to perform the genetic typing of 51 PFGE-unrelated vanA-containing enterococci of different origin (clinical, food and faecal samples of healthy humans and healthy poultry). Tn1546 structure was characterized by a PCR primer walking strategy and sequencing. Multilocus sequence typing (MLST) was performed for Enterococcus faecium and Enterococcus faecalis strains, and esp and hyl genes were detected by PCR. Nine different Tn1546 structures were identified in the studied strains. Type I was the most prevalent structure (75%) (identical to GenBank M97297). Two new Tn1546 structures were identified (in three clinical and animal strains), containing two new insertion sequences (ISs; ISEfa11 disrupting vanS and ISEfa10 disrupting orf1). An additional new Tn1546 structure was found in one animal strain, containing ISEf1 interrupting vanY and IS1542 in the orf2-vanR region. A high diversity of sequence types (STs) was detected among clinical (6 ST/18 strains) and non-clinical E. faecium strains (18 ST/24 strains). STs associated with clonal complexes CC17 and CC9 were mainly detected among clinical and non-clinical E. faecium strains, respectively. Seven new STs were identified in non-clinical strains. The esp and hyl genes were only found among clinical E. faecium strains. A moderate variability in Tn1546 structure has been detected among unrelated vanA-containing enterococci of different origins, showing three new structures including two new ISs. A high diversity of STs was detected among E. faecium strains, especially among non-clinical strains, and new STs have been identified.

High prevalence of ST-78 infection-associated vancomycin-resistant Enterococcus faecium from hospitals in Asunción, Paraguay

Forty infection-associated VanA-type vancomycin-resistant Enterococcus faecium (VRE) strains obtained from five collaborating hospitals in Asunción, Paraguay were investigated. Genotyping using pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing revealed the presence of 17 cluster types and four STs, with 93% (37/40) of isolates comprising ST type 78. Other ST types included ST-132, ST-210 and one new ST type (ST-438). All but one isolate (ST-438) were associated with clonal complex 17 (CC17), and 97% of the total isolates carried the esp gene. Three Tn1546 variants were found, including a new lineage containing an ISEfa5 insertion in an existing IS1251 element.

Gene detection of vancomycin-resistant Enterococcus isolated from a teaching hospital in Shanghai

Objective To investigate the prevalence of VRE in Huashan hospital of Shanghai from 2007 to 2009, and to examine the molecular characteristics of the VRE isolates.Methods A total of 890 non-repetive clinical isolates of Enterococcus were screened by the agar screening method ( ADSP method).Broth dilution susceptibility test was performed to determine the antimicrobial susceptibility of Enterococcus isolates to vancomycin and teicoplanin.The resistant genes and virulent genes of VRE isolates were investigated by PCR and sequencing methods.VRE isolates were classified by MLST and six isolates of VRE from 2007 to 2008 were analyzed by PFGE.Results Thirteen VRE isolates were identified by ADSP method and broth dilution susceptibility test. Six of them were resistant to vancomycin but sensitive to teicoplanin ( vancomycin MICs were from 64 to 256 μg/ml).The sequencing data of PCR products indicated these isolates might harbor a potential novel vancomycin resistant gene, which was different from the one reported in previous studies. The rest 7 isolates harbored vanA gene. The MICs of these isolates to vancomycin and teicoplanin were 32 - 64 μg/ml and 16 - 32 μg/ml, respectively.MLST results revealed 4 STs were identified in 13 VRE isolates.Eleven isolates belonged to clonal complexes(CC) 17.The positive rates of esp gene and hyl gene were 69.2% and 30.8%, respectively.Conclusions This study suggests that the most common VRE clone in Huashan Hospital was CC17.A potentially novel vancomycin resistance gene was identified, and further work needs to be done to investgate the function and the location of this novel gene. Key words: vancomycin resistance; Enterococcus; Sequence homology; Bacterial typing techniques; Electrophoresis,gel, pulsed-field

Characterization of Enterococcus spp. from Human and Animal Feces Using 16S rRNA Sequences, theespGene, and PFGE for Microbial Source Tracking in Korea

Contamination from human and animal fecal waste is a primary cause of water pollution. Microbial source tracking (MST) may be a useful tool for high-quality environmental management and for assessing human health risks associated with water pollution. The goal of this study was to evaluate Enterococcus spp. as a target organism for MST. Thirty-four fecal samples were collected from five different sources (human, chicken, pig, cow, and goose) in South Korea. In total, 237 Enterococcus spp. were isolated from feces using membrane- Enterococcus indoxyl-beta-d-glucoside agar. The 16S rRNA gene and the whole genome were analyzed using nucleic acid sequencing and pulsed-field gel electrophoresis (PFGE), respectively. Both phylogenetic analysis and principal coordinate analysis using UniFrac were performed on the nucleic acid sequences of the 16S rRNA gene. According to P-tests from UniFrac, significant differences existed between Enterococcus spp. isolated from human feces and those from animal feces. In addition, we evaluated whether the esp gene of Enterococcus faecium could be a specific target for Enterococcus spp. isolated from human feces. Of 58 E. faecium isolates tested, only three were esp-positive. The specificity of the esp gene of E. faecium isolated from human feces was 100%, but the sensitivity was <10%. These results suggest that Enterococcus spp. have different molecular characteristics according to their fecal source and that these characteristics can be further identified by analyzing the esp gene and 16S rRNA sequences, whereas PFGE provides limited information on the fecal sources of Enterococcus spp.

Characterisation of vancomycin-resistant enterococci from hospitalised patients at a tertiary centre over a seven-year period

Vancomycin-resistant enterococci (VRE) were isolated from 41 patients at a medical centre in southern Taiwan from 1999 to 2005. This accounted for 0.55% of 7449 patients with clinical colonisation or infection due to Enterococcus species. Nine (22%) patients had clinical infections caused by VRE. Twenty-five (61%) VRE isolates were Enterococcus faecalis with VanA phenotype and vanA gene cluster, the remaining 16 (39%) isolates were Enterococcus faecium with VanB phenotype and vanB (15 isolates) or vanA (1 isolate) gene cluster. The esp gene cluster was found in 6 (24%) E. faecalis isolates and 14 (87.5%) E. faecium isolates, and hyl in 2 (8%) E. faecalis and 6 (37.5%) E. faecium isolates. Analysis of SmaI-digested DNA by pulsed-field gel electrophoresis revealed that most isolates of E. faecalis were distinct, indicating a limited person-to-person spread or sporadic nature of spread. By contrast, 10 (67%) of 15 VanB phenotype E. faecium isolates were identical, suggesting the presence of an occult and limited clonal spread at the hospital. The presence of the esp or hyl gene was not associated with the virulence or outbreak of VRE. In summary, epidemiological data and molecular typing suggest that there was occult clonal spread of VanB phenotype-vanB genotype E. faecium in the surgical ICU, and the limited person-to-person spread or sporadic nature of E. faecalis. Infection control measures are warranted due to the potential for spread of VRE.

Pyrosequencing-based comparative genome analysis of the nosocomial pathogen Enterococcus faecium and identification of a large transferable pathogenicity island

BackgroundThe Gram-positive bacterium Enterococcus faecium is an important cause of nosocomial infections in immunocompromized patients.ResultsWe present a pyrosequencing-based comparative genome analysis of seven E. faecium strains that were isolated from various sources. In the genomes of clinical isolates several antibiotic resistance genes were identified, including the vanA transposon that confers resistance to vancomycin in two strains. A functional comparison between E. faecium and the related opportunistic pathogen E. faecalis based on differences in the presence of protein families, revealed divergence in plant carbohydrate metabolic pathways and oxidative stress defense mechanisms. The E. faecium pan-genome was estimated to be essentially unlimited in size, indicating that E. faecium can efficiently acquire and incorporate exogenous DNA in its gene pool. One of the most prominent sources of genomic diversity consists of bacteriophages that have integrated in the genome. The CRISPR-Cas system, which contributes to immunity against bacteriophage infection in prokaryotes, is not present in the sequenced strains. Three sequenced isolates carry the esp gene, which is involved in urinary tract infections and biofilm formation. The esp gene is located on a large pathogenicity island (PAI), which is between 64 and 104 kb in size. Conjugation experiments showed that the entire esp PAI can be transferred horizontally and inserts in a site-specific manner.ConclusionsGenes involved in environmental persistence, colonization and virulence can easily be aquired by E. faecium. This will make the development of successful treatment strategies targeted against this organism a challenge for years to come.

Integrative Physiology: Defined Novel Metabolic Roles of Osteocalcin

The prevailing model of osteology is that bones constantly undergo a remodeling process, and that the differentiation and functions of osteoblasts are partially regulated by leptin through different central hypothalamic pathways. The finding that bone remodeling is regulated by leptin suggested possible endocrinal effects of bones on energy metabolism. Recently, a reciprocal relationship between bones and energy metabolism was determined whereby leptin influences osteoblast functions and, in turn, the osteoblast-derived protein osteocalcin influences energy metabolism. The metabolic effects of bones are caused by the release of osteocalcin into the circulation in an uncarboxylated form due to incomplete γ-carboxylation. In this regard, the Esp gene encoding osteotesticular protein tyrosine phosphatase is particularly interesting because it may regulate γ-carboxylation of osteocalcin. Novel metabolic roles of osteocalcin have been identified, including increased insulin secretion and sensitivity, increased energy expenditure, fat mass reduction, and mitochondrial proliferation and functional enhancement. To date, only a positive correlation between osteocalcin and energy metabolism in humans has been detected, leaving causal effects unresolved. Further research topics include: identification of the osteocalcin receptor; the nature of osteocalcin regulation in other pathways regulating metabolism; crosstalk between nutrition, osteocalcin, and energy metabolism; and potential applications in the treatment of metabolic diseases.

Molecular Epidemiologic Analysis ofEnterococcus faecalisIsolates in Cuba by Multilocus Sequence Typing

We carried out the first study of Enterococcus faecalis clinical isolates in Cuba by multilocus sequence typing linking the molecular typing data with the presence of virulence determinants and the antibiotic resistance genes. A total of 23 E. faecalis isolates recovered from several clinic sources and geographic areas of Cuba during a period between 2000 and 2005 were typed by multilocus sequence typing. Thirteen sequence types (STs) including five novel STs were identified, and the ST 64 (clonal complex [CC] 8), ST 6 (CC2), ST 21(CC21), and ST 16 (CC58) were found in more than one strain. Sixty-seven percent of STs corresponded to STs reported previously in Spain, Poland, and The Netherlands, and other STs (ST115, ST64, ST6, and ST40) were genetically close to those detected in the United States. Prevalence of both antimicrobial resistance genes [aac(6')-aph(2''), aph(3'), ant(6), ant(3'')(9), aph(2'')-Id, aph(2'')-Ic, erm(B), erm(A), erm(C), mef(A), tet(M), and tet(L)] and virulence genes (agg, gelE, cylA, esp, ccf, and efaAfs) were examined by polymerase chain reaction. Aminoglycoside resistance genes aac(6')-Ie-aph(2'')-Ia, aph(3'), ant(6), ant(3'')(9) were more frequently detected in ST6, ST16, ST23, ST64, and ST115. The multidrug resistance was distributed to all STs detected, except for ST117 and singleton ST225. The presence of cyl gene was specifically linked to the ST64 and ST16. Presence of the esp, gel, and agg genes was not specific to any particular ST. This research provided the first insight into the population structure of E. faecalis in Cuba, that is, most Cuban strains were related to European strains, whereas others to U.S. strains. The CC2, CC21, and CC8, three of the biggest CCs in the world, were evidently circulating in Cuba, associated with multidrug resistance and virulence traits.