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Enterococcus faecium has recently emerged as a multidrug-resistant nosocomial pathogen involved in outbreaks worldwide. A high rate of resistance to different antibiotics has been associated with virulent clonal complex 17 isolates carrying the esp and hyl genes and the purK1 allele.
Twelve clinical vancomycin-resistant Enterococcus faecium (VREF) isolates were obtained from pediatric patients at the Hospital Infantil de México Federico Gómez (HIMFG). Among these VREF isolates, 58.3% (7/12) were recovered from urine, while 41.7% (5/12) were recovered from the bloodstream. The VREF isolates showed a 100% rate of resistance to ampicillin, amoxicillin-clavulanate, ciprofloxacin, clindamycin, chloramphenicol, streptomycin, gentamicin, rifampicin, erythromycin and teicoplanin. In addition, 16.7% (2/12) of the isolates were resistant to linezolid, and 66.7% (8/12) were resistant to tetracycline and doxycycline. PCR analysis revealed the presence of the vanA gene in all 12 VREF isolates, esp in 83.3% (10/12) of the isolates and hyl in 50% (6/12) of the isolates. Phylogenetic analysis via molecular typing was performed using pulsed-field gel electrophoresis (PFGE) and demonstrated 44% similarity among the VREF isolates. MLST analysis identified four different sequence types (ST412, ST757, ST203 and ST612).
This study provides the first report of multidrug-resistant VREF isolates belonging to clonal complex 17 from a tertiary care center in Mexico City. Multidrug resistance and genetic determinants of virulence confer advantages among VREF in the colonization of their host. Therefore, the prevention and control of the spread of nosocomial infections caused by VREF is crucial for identifying new emergent subclones that could be challenging to treat in subsequent years.
Keywords:Enterococcus faecium; Multidrug-resistant; Clonal complex; Pulsotypes; Virulence
Enterococci are opportunistic pathogens of the normal intestinal microbiota of humans and animals [1,2]. The most common species of Enterococcus involved in nosocomial infections is Enterococcus faecium (E. faecium) [1,2]. This pathogen is associated with hospital-acquired infections such as UTIs (urinary tract infections), wounds, bacteremia, endocarditis and meningitis [1,2].
In recent years, the emergence of multidrug-resistant E. faecium has increased [3-5]. The recommended treatment for Enterococcus infections has been penicillin alone or combined with aminoglycosides. However, due to increased resistance to aminoglycosides, vancomycin is currently the antibiotic employed to treat these infections. In the last several decades, the number of vancomycin-resistant enterococci (VRE) has increased. The first VRE isolates were reported in the United Kingdom in the late 1980s [6]. In the United States, more than 80% of E. faecium isolates from hospitals are now resistant to vancomycin, and virtually all of them (>90%) exhibit ampicillin resistance [7]. Vancomycin-resistant Enterococcus faecium (VREF) has been associated with outbreaks in hospitals worldwide [2]. The rates of VREF colonization and infection have risen steadily, with most cases being caused by strains displaying glycopeptide resistance to VanA and VanB [8-11].
In addition to multidrug resistance, E. faecium produces diverse factors that contribute to its pathogenesis, including virulence molecules such as secreted antigen SagA [12], cell wall-anchored collagen adhesin (Acm) [13], hyaluronidase (Hyl) [14] and enterococcal surface protein (Esp) [15]. However, the traits that contribute to the transition of E. faecium from a commensal to a nosocomial pathogen have not been identified [16].
Molecular typing methods are essential for identifying hospital-associated outbreaks of E. faecium. Multilocus sequence typing (MLST) has revealed the existence of host-specific genogroups, including a specific genetic lineage designated clonal complex 17, associated with hospital-related isolates [1,17]. MLST of E. faecium is based on identifying alleles from DNA sequences in internal fragments of housekeeping genes (atpA, ddl, gdh, purK, gyd, pstS and adk), resulting in a numeric allelic profile, with each profile then being assigned a sequence type (ST) [17].
Complex 17 most likely evolved from the primary E. faecium ancestor ST-22, while ST-17 represents an important secondary founder with additional linages designated to complex 17 [18]. Clonal complex 17 is characterized by ampicillin and quinolone resistance and the presence of a putative pathogenicity island that includes the esp and/or hyl genes in the majority of isolates [1,18-20]. Various STs belonging to clonal complex 17, such as ST16, ST17, ST18, ST203 and ST412, are currently being disseminated worldwide [21,22]. Interestingly, half of the STs within the clonal complex 17 polyclonal subpopulation have also been identified in samples obtained from healthy humans, swine, poultry and pets [16].
In Mexico, there is little available information about the prevalence of VREF isolates, and no study related to clonal complex 17 has been performed in pediatric patients. The aim of this study was to genotypically and phenotypically characterize VREF clinical isolates from 12 immunocompromised pediatric patients at the Hospital Infantil de México Federico Gómez (HIMFG). This study involved amplification of the resistance genes vanA and vanB and two virulence genes (esp and hyl) and molecular typing via pulsed-field gel electrophoresis (PFGE) and MLST.
Bacterial isolates
Twelve E. faecium isolates of clinical importance were obtained from 12 patients with nosocomial infections in the PICU (Pediatric Intensive Care Unit), oncology, gastroenterology and transplant wards of HIMFG during the period from July 2009 to April 2011. The isolates were maintained at −70°C in skim milk (Becton Dickinson, New Jersey, USA) and cultured on 5% sheep blood agar plates (Becton Dickinson, New Jersey, USA) at 37°C under 5% CO2 for 24 h. The E. faecalis ATCC® 29212, E. faecalis ATCC® 51299 and E. faecium ATCC® 51559 strains (American Type Culture Collection Manassas, VA, USA) were used as controls.
Biochemical tests
Bacteria were grown on blood agar, and identification was performed using manual methods. All colonies were grown in brain heart infusion broth (BHI) (Becton Dickinson, New Jersey, USA) with 6.5% NaCl and on bile esculin agar (Oxoid Sunnyvale, California, USA) to determine their hydrolysis grade. Disks impregnated with the substrate L-pyrrolidonyl-beta-naphthylamide were used to perform pyrrolidonase tests (Oxoid Biochemical Identification System, Oxoid LTD., Basingstoke, Hampshire, England). Reduction of tellurite (Merck, Darmstadt, Germany) was evaluated via growing the bacteria on 0.04% potassium tellurite.
Antibiotic susceptibility
The antibiotic susceptibility profiles of the 12 VREF isolates were determined via the minimum inhibitory concentration (MIC) technique by means of the microdilution method using Mueller-Hinton broth (MHB), as recommended by the Clinical and Laboratory Standards Institute. MIC tests were performed for vancomycin (MP Biomedicals, Solon, Ohio, USA), teicoplanin (Sigma-Aldrich, St. oLouis, Missouri, USA), chloramphenicol (MP Biomedicals, Solon, Ohio, USA), ciprofloxacin (MP Biomedicals, Solon, Ohio, USA), streptomycin (Alexis Biochemical, San Diego California, USA), linezolid (Sigma-Aldrich, St. Louis, Missouri, USA), rifampicin (MP, Biomedicals, Ohio, USA), nitrofurantoin (MP Biomedicals, Solon, Ohio, USA), tetracycline (MP Biomedicals, Solon, Ohio, USA), doxycycline (Sigma-Aldrich, St. Louis, Missouri, USA), erythromycin (MP Biomedicals, Solon, Ohio, USA), tigecycline (Sigma-Aldrich, St. Louis, Missouri, USA), gentamicin (MP Biomedicals, Solon, Ohio, USA) and amoxicillin-clavulanate (Glaxo-Smith-Kline, Philadelphia, Pennsylvania, USA). Several concentrations (256–0.625 μg/ml) of the antibiotics were tested in Mueller Hinton broth, with 100 μl of those dilutions being loaded into each well of a microplate. For each dilution, 100 μl of a bacterial suspension (1.5x108 CFU/ml) was inoculated and grown overnight at 37°C under a CO2 atmosphere. After bacterial growth was detected, the MIC for each isolate of E. faecium was reported as the highest concentration (μg/ml) of antibiotics in which no growth was observed. The E. faecalis ATCC® 29212 strain (American Type Culture Collection Manassas, VA, USA) was used as a control. These isolates were also evaluated for high-level aminoglycoside resistance (HLAR) to streptomycin (1,000 μg/ml) and gentamicin (500 μg/ml).
Detection of the glycopeptide resistance genes vanA and vanB
PCR was performed to detect the glycopeptide resistance genes vanA and vanB in the 12 E. faecium clinical isolates using specific primers (Table 1) [23]. Briefly, genomic DNA was purified using the Wizard Genomic DNA Purification Kit (Promega Madison, Wisconsin, USA) from a bacterial culture grown in BHI broth incubated at 37°C for 24 h. The amplification reactions were prepared in a final volume of 50 μl, as follows: 25 μl of amplification mix (22 mM Tris/HCl, pH 8.4; 55 mM KCl; 1.65 mM MgCl2; 25 μM each dNTP; 0.6 U recombinant Taq DNA polymerase/ml), 100 ng/μl of bacterial DNA, 10 μl of H2O and 5 μl of primer solution (10 pg/μl). A Perkin Elmer 9600 thermocycler was programmed to run for 30 cycles with the following parameters: denaturing at 94°C for 3 m, annealing at 55°C for 45 s and extension at 72°C for 1 m, with a final extension at 72°C for 2 m. The samples were analyzed via electrophoresis in 1% agarose gels (Agarose LE, Promega) using a 100 bp DNA ladder (Gibco/BRL Life Technologies, Breda, The Netherlands). E. faecium strain ATCC 51559 (vanA+) and E. faecalis strain ATCC® 51299 (vanB+) were used as controls in the PCR experiments [24].
Table 1. Primers sequences used in this study
PCR screening for the esp and hyl genes
DNA from bacterial cultures was extracted and amplified via PCR using primers for the espEfm and hylEfm genes (Table 1), generating bands of 954 bp and 661 bp, respectively [14,25].
Molecular typing of VREF
PFGE of the 12 VREF clinical isolates was carried out following the protocols of Morrison et al. [26,27]. Briefly, the samples were digested with 50 U of SmaI (New England Biolab, Ipswich, MA, USA) for 4 h at 25°C. The digested plugs were separated via electrophoresis in 1% agarose gels (BioRad, Hercules, California, USA) using ultra-pure DNA agarose (BioRad, Hercules, California, USA), with 0.5X TBE as the running buffer in the CHEF MAPPER system (BioRad Laboratories, Hercules, California, USA), run at 6 V/cm at 14°C under two different linear ramped pulse times: 1 to 10 s for 16 h and 10 to 40 s for 22 h. A PFGE lambda ladder (New England Biolabs, Hertfordshire, England, UK) was used as a molecular weight marker, and the gels were stained for 40 m with 0.5 mg/ml of ethidium bromide for visualization under UV light. The obtained banding patterns were initially interpreted via visual inspection according to the criteria specified by Tenover et al. [28]. Cluster analysis was performed with BioNumerics (Applied Maths, Inc., Austin, TX, USA) using the DICE correlation coefficient and the unweighted pair group mathematical average algorithm (UPGMA) as the grouping method [29].
The PFGE pulsotypes of the 12 VREF clinical isolates were also genotyped through multilocus sequence typing (MLST) according to a standard protocol described by Homan et al. [17]. Fragments of seven housekeeping genes (atpA, ddl, gdh, purK, gyd, pstS and adk) were sequenced using a 3730xl DNA Analyzer (Applied Biosystems, Foster City, California, USA), thus obtaining their allelic profiles, and the STs for each unique allelic profile were designated on the basis of information from the MLST website (http://efaecium.mlst.net webcite).
Origin of the strains
A total of 12 VREF clinical isolates obtained during the period from July 2009 to April 2011 were included in this study. The risk factors of the 12 patients were characterized by a minimum hospital stay of 4 days, assistance in the PICU and treatment with vancomycin. During their stay, the 12 patients were subjected to surgical procedures and received a central venous catheter, steroids and immunosuppressive treatment. Among the VREF isolates, 58.3% (7/12) were obtained from urine, while 41.6% (5/12) were obtained from the bloodstream. The VREF isolates were obtained from patients with different pathologies (Table 2).
Table 2. Characteristics of the 12 VREF isolates related to the patients' clinical diagnosis, source of clinical samples, ward, PFGE, sequence type and clonal complex
Detection of susceptibility patterns and glycopeptide resistance in the VREF isolates
The results obtained for the 12 VREF clinical isolates showed a 100% rate of resistance to ampicillin, amoxicillin-clavulanate, ciprofloxacin, clindamycin, chloramphenicol, streptomycin, gentamicin, rifampicin, erythromycin and teicoplanin. The MIC values for each VREF isolate are presented in Table 3. In addition, 16.7% (2/12) of the VREF clinical isolates were resistant to linezolid, and 67% (8/12) were resistant to tetracycline and doxycycline (Table 3). However, all of the VREF isolates were susceptible to nitrofurantoin and tigecycline (Table 3). The HLAR values for gentamicin (500 μg/ml), streptomycin (1,000 μg/ml) and gentamicin/streptomycin (500/1,000 μg/ml) were determined with to 50% (6/12), 25% (3/12) and 25% (3/12), respectively.
Table 3. Minimum Inhibitory Concentration (MIC) to 12 clinical isolates of multidrug-resistant Enterococcus faecium
The vanA and vanB genes of the 12 VREF clinical isolates were amplified via PCR. Interestingly, only the vanA gene was detected in all the VREF clinical isolates, as a 1,030 bp amplicon (data not shown), whereas the vanB gene, with a length of 433 bp, was not identified in the isolates (data not shown). The E. faecium ATCC® 51559 (vanA+) and E. faecalis ATCC® 51299 (vanB+) strains were used as positive controls in the PCR assays [24].
Prevalence of the esp and hyl virulence genes in the VREF isolates
The esp and hyl virulence genes, which are associated with a clonal subcluster known as clonal complex 17 in VREF clinical isolates, were detected via PCR. The esp and hyl genes were highly prevalent in the isolates. The esp virulence gene was detected in 83.3% (10/12) of the isolates, and the hyl virulence gene was present in 50% (6/12) of them. Therefore, three genotypes were determined for the VREF clinical isolates: esp+/hyl-, esp+/hyl+ and esp-/hyl+, at prevalence rates of 50% (6/12), 33.3% (4/12) and 16.7% (2/12), respectively.
Molecular typing analysis of the E. faecium isolates via PFGE and MLST
The VREF isolates were analyzed via PFGE following SmaI digestion of genomic DNA. Data obtained through PFGE were analyzed using a dendrogram profile, which included the PFGE pulsotypes obtained from VREF (Figure 1). A total of four clusters (I-IV) with five DNA pulsotypes were identified, showing patterns consisting of 12 to 20 DNA fragments ranging in size from 48.5 to 339.5 Kb (Figure 1). Interestingly, 25% (3/12) of the VREF clinical isolates observed via PFGE were categorized as pulsotype B and 16.7% (2/12) as pulsotype B1, with 92% genetic similarity being observed among these isolates (Figure 1). Meanwhile, 25% (3/12) of the VREF isolates were classified as pulsotype A, showing a different pattern from pulsotypes B, C and D (Figure 1). However, 16.7% (2/12) of the VREF isolates were classified as pulsotypes C and D, which displayed 50% genetic similarity. In addition, a maximum of 44% similarity was observed among all clusters of VREF isolates.
Figure 1. PFGE analysis of 12 VREF isolates recovered at HIMFG and detection of the virulence factors esp and hyl, sequence type, isolation ward and type of sample. Phylogenetic analysis was performed using the DICE coefficient in association with the UPGMA algorithm as the grouping method. The dendrogram was evaluated by obtaining the cophenetic correlation coefficient using the Mantel test, which yielded an r value of 0.97769.
In this study, 12 VREF clinical isolates were subjected to MLST genotyping. Six of the 12 VREF isolates (50%) belonged to ST412, three to ST757, two to ST203 and one to ST612 (Table 2). eBURST analysis of the VREF isolates revealed four different STs (ST412, ST612, ST757 and ST203), three of which belonged to clonal complex 17; ST757 was not related to this clonal complex (Figure 2).
Figure 2. Clustering of MLST profiles using the eBURST database algorithm. Our profiles showed that ST412, ST612 and ST203, but not ST757, belong to clonal complex 17.
E. faecium is a highly resistant nosocomial pathogen and has recently emerged as an important threat in hospitals worldwide [2]. In this study, the 12 examined VREF isolates exhibited multidrug resistance to ampicillin, amoxicillin-clavulanate, ciprofloxacin, clindamycin, chloramphenicol, streptomycin, gentamicin, rifampicin, erythromycin and teicoplanin. At HIMFG, several types of enterococcal infections in pediatric patients are commonly treated with a combination of drugs (aminoglycoside-β-lactams, such as gentamicin/ampicillin) as the first choice, while vancomycin is the second choice; vancomycin-aminoglycoside or linezolid is the third choice; and tigecycline is the fourth choice. Interestingly, 16.7% (2/12) of the VREF clinical isolates were also resistant to linezolid, and 67% (8/12) were resistant to both tetracycline and doxycycline. The emergence of high levels of resistance to the most common anti-enterococcal antibiotics (vancomycin) might constitute a real challenge in the treatment of these infections. In the present study, 100% (12/12) of the examined VREF isolates were susceptible to tigecycline and nitrofurantoin. The VREF resistance patterns observed in this study are in agreement with the findings of other authors [30,31]. However, these authors observed VREF isolates that were susceptible to linezolid and nitrofurantoin, in contrast to our data, which showed that two of the VREF isolates were resistant to linezolid. Nevertheless, the low resistance to linezolid observed in the VREF clinical isolates is in accord with data reported in other countries [11,32].
Few instances of the isolation of HLAR E. faecium have been documented worldwide [22,33,34]. In this study, the examined VREF clinical isolates showed HLAR to gentamicin (500 μg/ml), streptomycin (1,000 μg/ml) and gentamicin/streptomycin (500/1,000 μg/ml), displaying resistance values of 50%, 25% and 25%, respectively. Treatment of severe enterococcal infection requires combined therapy to achieve a synergistic bactericidal effect [35]. However, the results obtained in cases of severe infections associated with enterococci have shown that HLAR should not be treated with combined therapy (gentamicin/ampicillin) [35]. Therefore, the treatment of HLAR E. faecium is restricted [36].
The enterococcal surface protein Esp, which is encoded by genes that appear to have been acquired and localized within a pathogenicity island, is commonly found in clinical isolates and anchors to the cell wall. This protein also affects biofilm formation and plays a role in experimental UTI and/or endocarditis models [2]. The presence of the esp gene has been associated with hospital outbreaks, although this gene is not exclusively found in epidemic strains [19,30,37,38]. The esp gene was detected in 83.3% of our VREF clinical isolates. In addition, the majority of esp+ strains of E. faecium isolates were multidrug-resistant to more than three antibiotics, in accord with data reported by other researchers [39-41].
On the other hand, the hyl gene was found in 50% of the VREF clinical isolates and displayed a higher prevalence compared to the prevalences of 29.8% (29/131) reported in isolates of E. faecium in the Picardy Region of France, 38% (83/220) in isolates from the US and 3% in European clinical isolates. However, in the United Kingdom, a hyl gene prevalence of 71% (20/28) was observed in E. faecium isolates [14,42,43]. We believe that the differences observed in the detection rates of the hyl gene are due to the region in which the samples were isolated. The rates of the occurrence of esp+/hyl-, esp+/hyl+ and esp-/hyl+ isolates were found to be 50% (6/12), 33.3% (4/12) and 16.7% (2/12), respectively, which is in accord with the findings of Vankerckhoven et al. and Rice et al. [14,42,44]. The VREF clinical isolates of Mexican origin in which the esp and/or hyl gene was amplified (alone or together), were resistant to more than three antibiotics; in contrast, other studies have shown a significant correlation between the presence of the esp gene and resistance to ampicillin, imipenem and ciprofloxacin [40,41].
PFGE and MLST analyses have been proposed as alternative methods for the molecular characterization of clinical isolates of E. faecium[45]. According to our PFGE analysis, the 12 VREF isolates showed a heterogeneous pattern associated with a profile of multidrug resistance to different antibiotics and the presence of the vanA gene. The data obtained through PFGE revealed four clusters (I-IV), with a low similarity of 44% being detected among the VREF isolates and therefore high diversity. Furthermore, the VREF isolates within clusters I, II-B and III showed an identical banding profile in the PFGE analysis. However, the MLST data indicated different STs due to changes in the nucleotide sequences of the analyzed housekeeping genes; these data are consistent with the findings of Poh et al. [46]. In addition, the VREF isolates within clusters II-B1 and IV displayed identical PFGE and MLST profiles, in agreement with other authors [22,33]. Nevertheless, pulsotypes from different wards showed similar multidrug resistance profiles, possibly due to horizontal genetic transference between these isolates.
MLST is an important tool for studying the molecular epidemiology of outbreaks of E. faecium and microbial population biology [44]. MLST analysis of VREF clinical isolates revealed four STs: ST203, ST412, ST612 and ST757. As previously reported, clonal complex 17 harbors various STs that have been involved in hospital outbreaks. Our results revealed two allelic profiles, ST203 and ST412, belonging to clonal complex 17 STs involved in hospital outbreaks. However, clonal complex 17 has been resolved into two different subgroups, one of which harbors ST17 and ST18, while the second harbors ST78 [47]. ST17, ST18 and ST203 are the major groups in the genetic lineage of E. faecium; they are distributed worldwide and have been associated with outbreaks [18,48]. ST412 was the most frequent sequence type found in the VREF isolates from HIMFG and was genetically linked to the ST78 lineage. Interestingly, ST412 has been identified worldwide and associated with outbreaks [49]. According to the eBURST analysis, ST612 showed characteristics of the STs belonging to the 18 lineage. ST757 has not been characterized within clonal complex 17. In addition, ST757 displayed resistance markers (ampicillin and quinolones), virulence genes (esp+ and/or hyl+) and the purK1 allele; however, it has not been associated with outbreaks. Nevertheless, this community of multidrug-resistant strains is able to infect humans and might contribute to the spreading of these bacteria in the hospital, highlighting the importance of molecular typing via MLST to identify STs involved in nosocomial outbreaks.
Recently, it was shown that MLST analysis of typified E. faecium based on selected alleles may generate misleading results due to the recombination of five alleles (atpA, ddl, gdh, gyd and pstS). As only the purk and adk alleles are located in regions where there is no predicted recombination, the results must be interpreted with care [50]. The genome of E. faecium is highly plastic due to the few existing barriers to the acquisition of foreign genetic elements [51,52]. Recent studies have provided evidence of high levels of recombination through comparative genomics analyses [51-54]. Whole-genome sequencing platforms are superior to conventional typing methods, providing an excellent tool for determining phylogenies and regions of recombination and for accurately discriminating between outbreak- and non-outbreak-causing VREF isolates [50,55]. Thus, whole-genome sequence information, rather than data on just one or a few genes, could be used to distinguish between closely related strains.
In this study, MLST and PFGE analysis were applied for the molecular characterization of clinical VREF isolates to identify different clonal complexes with different pulsotypes that were not related to outbreaks. However, according to the results obtained through PFGE, four multidrug-resistant clones of VREF were identified at HIMFG; in addition, these VREF isolates were identified at different periods. Therefore, these data suggest that these clones have circulated endemically at HIMFG.
In the case of cluster II, the clones have evolved from cluster II-B to cluster II-B1 due to the high similarity (> 90%) observed via PFGE analysis and based on the acquisition of three bands for B1, suggesting a mechanism of horizontal gene transfer. The results obtained in this study highlight the importance of monitoring circulating VREF isolates in different wards of this institution to efficiently control multidrug resistance and prevent outbreaks of these clones.
Little is known about VREF isolates in Mexican hospitals. In this study, the detected virulence genes (esp and hyl), multidrug profiles and allelic patterns were associated with clonal complex 17 VREF clinical isolates obtained from pediatric patients at HIMFG. To our knowledge, this is the first report describing clonal complex 17 VREF isolates in a tertiary care center in Mexico City.
Multidrug resistance and genetic determinants of virulence confer advantages in VREF in the colonization of their hosts. The genome of E. faecium is highly plastic, showing an ability to readily acquire genes involved in environmental persistence, colonization and virulence, favoring the selection of specific clonal complexes in a hospital environment. Therefore, the prevention and control of the propagation of nosocomial infections caused by VREF is crucial for identifying new emergent subclones that could be challenging to treat in subsequent years.
Ethics statement
The study was reviewed and approved by the Research (Dr. Onofre Muñoz Hernández), Ethics (Dr. Amparo Faure Fontenla) and Biosecurity (Dr. Herlinda Vera Hermosillo) Committee of HIMFG, under permit numbers HIM/2011/019. After looking at the medical history of each patient, E. faecium isolates were recovered from clinical samples, and the patients were asked by the physicians in the Infectology Department of HIMFG for their permission for their samples be used in this study. Analyses of E. faecium isolates obtained from clinical samples are not considered routine studies. Informed consent was obtained from the patient for the publication of this report and any accompanying images.
Am: Ampicillin; Amc: Amoxacillin/clavulanate; CIP: Ciprofloxacin; CC: Clindamycin; C: Chloramphenicol; GM: Gentamicin; S: Streptomycin; RA: Rifampin; E: Erythromycin; Va: Vancomycin; TEI: Teicoplanin; Te: Tetracycline; D: Doxycycline; LZN: Linezolid; F/M: Nitrofurantoin; TGC: Tigecycline; Hyl: E. faecium hyaluronidase; Esp: Enterococcal surface protein.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
SAO, LBD and GE performed the susceptibility pattern analysis, molecular genetics experiments and PFGE and MLST assays. SAO, ZS and ACC participated in editing the manuscript and the data analysis. VCD, CAE, BLM, RHC and GAJ conducted the diagnoses of the patients, interpreted data, collaborated in the collection of samples and revised the manuscript. JXC is the principal investigator and conceived the study, designed the experiments, performed data analysis and wrote the manuscript. All authors read and approved the final version.
We thank Ma. del Carmen Castellanos Cruz and Ana Karina Espinosa Mazariego for their technical assistance in this study. This work was partially supported by grants HIM/2011/019 from Federal Funds from the Hospital Infantil de México Federico Gómez, CONACyT 133451 and the Fondo Sectorial de Investigación en Salud y Seguridad Social, FOSSIS-CONACYT 2012-01-182651.
Top J, Willems R, Bonten M: Emergence of CC17 Enterococcus faecium: from commensal to hospital-adapted pathogen.
FEMS Immunol Med Microbiol 2008, 52(3):297-308. PubMed Abstract | Publisher Full Text
Arias CA, Murray BE: The rise of the Enterococcus: beyond vancomycin resistance.
Nat Rev Microbiol 2012, 10(4):266-278. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Grayson ML, Eliopoulos GM, Wennersten CB, Ruoff KL, De Girolami PC, Ferraro MJ, Moellering RC Jr: Increasing resistance to beta-lactam antibiotics among clinical isolates of Enterococcus faecium: a 22-year review at one institution.
Antimicrob Agents Chemother 1991, 35(11):2180-2184. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Jones RN, Sader HS, Erwin ME, Anderson SC: Emerging multiply resistant enterococci among clinical isolates. I. Prevalence data from 97 medical center surveillance study in the United States. Enterococcus Study Group.
Diagn Microbiol Infect Dis 1995, 21(2):85-93. PubMed Abstract | Publisher Full Text
Rice LB: Emergence of vancomycin-resistant enterococci.
Emerg Infect Dis 2001, 7(2):183-187. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Uttley AH, Collins CH, Naidoo J, George RC: Vancomycin-resistant enterococci.
Hidron AI, Edwards JR, Patel J, Horan TC, Sievert DM, Pollock DA, Fridkin SK: NHSN annual update: antimicrobial-resistant pathogens associated with healthcare-associated infections: annual summary of data reported to the national healthcare safety network at the centers for disease control and prevention, 2006–2007.
Infect Control Hosp Epidemiol 2008, 29(11):996-1011. PubMed Abstract | Publisher Full Text
Coque TM, Tomayko JF, Ricke SC, Okhyusen PC, Murray BE: Vancomycin-resistant enterococci from nosocomial, community, and animal sources in the United States.
Antimicrob Agents Chemother 1996, 40(11):2605-2609. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Rice LB: Antimicrobial resistance in gram-positive bacteria.
Johnson AP, Uttley AH, Woodford N, George RC: Resistance to vancomycin and teicoplanin: an emerging clinical problem.
Clin Microbiol Rev 1990, 3(3):280-291. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Deshpande LM, Fritsche TR, Moet GJ, Biedenbach DJ, Jones RN: Antimicrobial resistance and molecular epidemiology of vancomycin-resistant enterococci from North America and Europe: a report from the SENTRY antimicrobial surveillance program.
Diagn Microbiol Infect Dis 2007, 58(2):163-170. PubMed Abstract | Publisher Full Text
Teng F, Kawalec M, Weinstock GM, Hryniewicz W, Murray BE: An Enterococcus faecium secreted antigen, SagA, exhibits broad-spectrum binding to extracellular matrix proteins and appears essential for E. faecium growth.
Infect Immun 2003, 71(9):5033-5041. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Nallapareddy SR, Singh KV, Murray BE: Contribution of the collagen adhesin Acm to pathogenesis of Enterococcus faecium in experimental endocarditis.
Infect Immun 2008, 76(9):4120-4128. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Rice LB, Carias L, Rudin S, Vael C, Goossens H, Konstabel C, Klare I, Nallapareddy SR, Huang W, Murray BE: A potential virulence gene, hylEfm, predominates in Enterococcus faecium of clinical origin.
J Infect Dis 2003, 187(3):508-512. PubMed Abstract | Publisher Full Text
Heikens E, Bonten MJ, Willems RJ: Enterococcal surface protein Esp is important for biofilm formation of Enterococcus faecium E1162.
J Bacteriol 2007, 189(22):8233-8240. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Willems RJ, Van Schaik W: Transition of Enterococcus faecium from commensal organism to nosocomial pathogen.
Future Microbiol 2009, 4(9):1125-1135. PubMed Abstract | Publisher Full Text
Homan WL, Tribe D, Poznanski S, Li M, Hogg G, Spalburg E, Van Embden JD, Willems RJ: Multilocus sequence typing scheme for Enterococcus faecium.
J Clin Microbiol 2002, 40(6):1963-1971. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Willems RJ, Top J, Van Santen M, Robinson DA, Coque TM, Baquero F, Grundmann H, Bonten MJ: Global spread of vancomycin-resistant Enterococcus faecium from distinct nosocomial genetic complex.
Emerg Infect Dis 2005, 11(6):821-828. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Leavis H, Top J, Shankar N, Borgen K, Bonten M, Van Embden J, Willems RJ: A novel putative enterococcal pathogenicity island linked to the esp virulence gene of Enterococcus faecium and associated with epidemicity.
J Bacteriol 2004, 186(3):672-682. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Bonten MJ, Willems R, Weinstein RA: Vancomycin-resistant enterococci: why are they here, and where do they come from?
Lancet Infect Dis 2001, 1(5):314-325. PubMed Abstract | Publisher Full Text
Damani A, Klapsa D, Panopoulou M, Spiliopoulou I, Pantelidi K, Malli E, Kolonitsiou F, Grapsa S, Alepopoulou E, Frantzidou F, et al.: A newly described vancomycin-resistant ST412 Enterococcus faecium predominant in Greek hospitals.
Eur J Clin Microbiol Infect Dis 2010, 29(3):329-331. PubMed Abstract | Publisher Full Text
Panesso D, Reyes J, Rincon S, Diaz L, Galloway-Pena J, Zurita J, Carrillo C, Merentes A, Guzman M, Adachi JA, et al.: Molecular epidemiology of vancomycin-resistant Enterococcus faecium: a prospective, multicenter study in South American hospitals.
J Clin Microbiol 2010, 48(5):1562-1569. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Clark NC, Cooksey RC, Hill BC, Swenson JM, Tenover FC: Characterization of glycopeptide-resistant enterococci from U.S. hospitals.
Antimicrob Agents Chemother 1993, 37(11):2311-2317. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Kariyama R, Mitsuhata R, Chow JW, Clewell DB, Kumon H: Simple and reliable multiplex PCR assay for surveillance isolates of vancomycin-resistant enterococci.
J Clin Microbiol 2000, 38(8):3092-3095. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Shankar V, Baghdayan AS, Huycke MM, Lindahl G, Gilmore MS: Infection-derived Enterococcus faecalis strains are enriched in esp, a gene encoding a novel surface protein.
Infect Immun 1999, 67(1):193-200. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Morrison D, Woodford N, Barrett SP, Sisson P, Cookson BD: DNA banding pattern polymorphism in vancomycin-resistant Enterococcus faecium and criteria for defining strains.
J Clin Microbiol 1999, 37(4):1084-1091. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Turabelidze D, Kotetishvili M, Kreger A, Morris JG Jr, Sulakvelidze A: Improved pulsed-field gel electrophoresis for typing vancomycin-resistant enterococci.
J Clin Microbiol 2000, 38(11):4242-4245. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Tenover FC, Arbeit RD, Goering RV, Mickelsen PA, Murray BE, Persing DH, Swaminathan B: Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing.
J Clin Microbiol 1995, 33(9):2233-2239. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Mullane NR, Whyte P, Wall PG, Quinn T, Fanning S: Application of pulsed-field gel electrophoresis to characterise and trace the prevalence of Enterobacter sakazakii in an infant formula processing facility.
Int J Food Microbiol 2007, 116(1):73-81. PubMed Abstract | Publisher Full Text
Torres E, Perez S, Vindel A, Rodriguez-Bano J, Camba V, Villanueva R, Coque TM, Bou G: Glycopeptide-resistant Enterococcus faecium in a hospital in northern Spain. Molecular characterization and clinical epidemiology.
Enferm Infecc Microbiol Clin 2009, 27(9):511-517. PubMed Abstract | Publisher Full Text
Pourakbari B, Aghdam MK, Mahmoudi S, Ashtiani MT, Sabouni F, Movahedi Z, Alyari AE, Sadeghi RH, Mamishi S: High frequency of vancomycin-resistant Enterococcus faecalis in an Iranian referral children medical hospital.
Maedica 2012, 7(3):201-204. PubMed Abstract | PubMed Central Full Text
Werner G, Klare I, Fleige C, Witte W: Increasing rates of vancomycin resistance among Enterococcus faecium isolated from German hospitals between 2004 and 2006 are due to wide clonal dissemination of vancomycin-resistant enterococci and horizontal spread of vanA clusters.
Int J Med Microbiol 2008, 298(5–6):515-527. PubMed Abstract | Publisher Full Text
Weng PL, Ramli R, Shamsudin MN, Cheah YK, Hamat RA: High Genetic Diversity of Enterococcus faecium and Enterococcus faecalis Clinical Isolates by Pulsed-Field Gel Electrophoresis and Multilocus Sequence Typing from a Hospital in Malaysia.
Biomed Res Int 2013, 2013:938937. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Araoka H, Kimura M, Yoneyama A: A surveillance of high-level gentamicin-resistant enterococcal bacteremia.
J Infect Chemother 2011, 17(3):433-434. PubMed Abstract | Publisher Full Text
Murray BE: Vancomycin-resistant enterococcal infections.
N Engl J Med 2000, 342(10):710-721. PubMed Abstract | Publisher Full Text
Watanabe S, Kobayashi N, Quinones D, Nagashima S, Uehara N, Watanabe N: Genetic diversity of enterococci harboring the high-level gentamicin resistance gene aac(6′)-Ie-aph(2″)-Ia or aph(2″)-Ie in a Japanese hospital.
Microb Drug Resist 2009, 15(3):185-194. PubMed Abstract | Publisher Full Text
Leavis HL, Willems RJ, Top J, Spalburg E, Mascini EM, Fluit AC, Hoepelman A, De Neeling AJ, Bonten MJ: Epidemic and nonepidemic multidrug-resistant Enterococcus faecium.
Emerg Infect Dis 2003, 9(9):1108-1115. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Coque TM, Willems R, Canton R, Del Campo R, Baquero F: High occurrence of esp among ampicillin-resistant and vancomycin-susceptible Enterococcus faecium clones from hospitalized patients.
J Antimicrob Chemother 2002, 50(6):1035-1038. PubMed Abstract | Publisher Full Text
Eaton TJ, Gasson MJ: A variant enterococcal surface protein Esp(fm) in Enterococcus faecium; distribution among food, commensal, medical, and environmental isolates.
FEMS Microbiol Lett 2002, 216(2):269-275. PubMed Abstract | Publisher Full Text
Dupre I, Zanetti S, Schito AM, Fadda G, Sechi LA: Incidence of virulence determinants in clinical Enterococcus faecium and Enterococcus faecalis isolates collected in Sardinia (Italy).
J Med Microbiol 2003, 52(Pt 6):491-498. PubMed Abstract | Publisher Full Text
Billstrom H, Lund B, Sullivan A, Nord CE: Virulence and antimicrobial resistance in clinical Enterococcus faecium.
Int J Antimicrob Agents 2008, 32(5):374-377. PubMed Abstract | Publisher Full Text
Vankerckhoven V, Van Autgaerden T, Vael C, Lammens C, Chapelle S, Rossi R, Jabes D, Goossens H: Development of a multiplex PCR for the detection of asa1, gelE, cylA, esp, and hyl genes in enterococci and survey for virulence determinants among European hospital isolates of Enterococcus faecium.
J Clin Microbiol 2004, 42(10):4473-4479. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Biendo M, Adjide C, Castelain S, Belmekki M, Rousseau F, Slama M, Ganry O, Schmit JL, Eb F: Molecular characterization of glycopeptide-resistant enterococci from hospitals of the picardy region (france).
Int J Microbiol 2010, 2010:150464. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Cha JO, Jung YH, Lee HR, Yoo JI, Lee YS: Comparison of genetic epidemiology of vancomycin-resistant Enterococcus faecium isolates from humans and poultry.
J Med Microbiol 2012, 61(Pt 8):1121-1128. PubMed Abstract
Kuriyama T, Williams DW, Patel M, Lewis MA, Jenkins LE, Hill DW, Hosein IK: Molecular characterization of clinical and environmental isolates of vancomycin-resistant Enterococcus faecium and Enterococcus faecalis from a teaching hospital in Wales.
J Med Microbiol 2003, 52(Pt 9):821-827. PubMed Abstract | Publisher Full Text
Poh LW, Rukman AW, Cheah YK, Norital Z, Nazri AM, Mariana NS: Vancomycin-resistant Enterococcus faecium of multi locus sequence type 18 in Malaysia.
Med J Malaysia 2012, 67(6):639-640. PubMed Abstract | Publisher Full Text
Willems RJ, Top J, Van Schaik W, Leavis H, Bonten M, Siren J, Hanage WP, Corander J: Restricted gene flow among hospital subpopulations of Enterococcus faecium.
MBio 2012, 3(4):e00151-00112. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Werner G, Coque TM, Hammerum AM, Hope R, Hryniewicz W, Johnson A, Klare I, Kristinsson KG, Leclercq R, Lester CH, et al.: Emergence and spread of vancomycin resistance among enterococci in Europe.
Freitas AR, Novais C, Ruiz-Garbajosa P, Coque TM, Peixe L: Dispersion of multidrug-resistant Enterococcus faecium isolates belonging to major clonal complexes in different Portuguese settings.
Appl Environ Microbiol 2009, 75(14):4904-4908. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Howden BP, Holt KE, Lam MM, Seemann T, Ballard S, Coombs GW, Tong SY, Grayson ML, Johnson PD, Stinear TP: Genomic insights to control the emergence of vancomycin-resistant enterococci.
Galloway-Pena J, Roh JH, Latorre M, Qin X, Murray BE: Genomic and SNP analyses demonstrate a distant separation of the hospital and community-associated clades of Enterococcus faecium.
PLoS One 2012, 7(1):e30187. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Palmer KL, Godfrey P, Griggs A, Kos VN, Zucker J, Desjardins C, Cerqueira G, Gevers D, Walker S, Wortman J, et al.: Comparative genomics of enterococci: variation in Enterococcus faecalis, clade structure in E. faecium, and defining characteristics of E. gallinarum and E. casseliflavus.
MBio 2012, 3(1):e00318-00311. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
De Been M, Van Schaik W, Cheng L, Corander J, Willems RJ: Recent recombination events in the core genome are associated with adaptive evolution in Enterococcus faecium.
Genome Biol Evol 2013, 5(8):1524-1535. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Van Schaik W, Top J, Riley DR, Boekhorst J, Vrijenhoek JE, Schapendonk CM, Hendrickx AP, Nijman IJ, Bonten MJ, Tettelin H, et al.: Pyrosequencing-based comparative genome analysis of the nosocomial pathogen Enterococcus faecium and identification of a large transferable pathogenicity island.
BMC Genomics 2010, 11:239. PubMed Abstract | BioMed Central Full Text | PubMed Central Full Text
Reuter S, Ellington MJ, Cartwright EJ, Koser CU, Torok ME, Gouliouris T, Harris SR, Brown NM, Holden MT, Quail M, et al.: Rapid bacterial whole-genome sequencing to enhance diagnostic and public health microbiology.
JAMA Intern Med 2013, 173(15):1397-1404. PubMed Abstract | Publisher Full Text


text:
Diversified Financial Services
Company Overview of Gulf Investment Corporation
BOARD MEMBERS AFFILIATED WITH Turki Almalik *
Director, Member of Audit Committee, and Member of the Remuneration and Human Resources Committee
*Data is at least as current as the most recent Definitive Proxy.
MOST CONNECTED PEOPLE TO Turki Almalik
|Hussain Ali Al-Abdulah
|65 Relationships
|Darwish bin Ismail bin Ali Al-Bulushi
|53 Relationships
|Khalid Abdulla Al-Bassam
|37 Relationships
|Saeed Rashed AlYateem
|22 Relationships
|Abdul Kader Ahmed Askalan
|21 Relationships
Sponsored Financial Commentaries
Sponsored Links
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subset: commoncrawl


text:
Gmail adds drag and drop attachment saving
Google has added a feature to its Gmail service whereby you don't need to click to save an attachment, and then select a destination - you can now simply drag and drop your email attachments to your desktop or a designated folder.
The process, which only currently works with Google's Chrome browser, lets you select the icon of any attached files and drag them to where you want them. If you hover over the icon you get a text prompt telling you that you can do this.
You can still have your browser in full screen to make use of this function. In Windows 7, for example, you can drag the file to your taskbar explorer icon and select a folder to drop in that way, or drag the file to the "show desktop" button in the bottom right corner to make use of it that way.
This new feature completes a drag and drop hat-trick for Gmail. Users can also drag and drop attachments into an email, as well as dragging images straight into the body of the email as well.
It's all very Mac-like, but it makes life a bit easier.
In other Gmail news, the ability to sign in to multiple accounts is also being rolled out. The feature is not showing up for all users as of yet, but when it does, you will see the option for multiple sign-in on your account settings.
If you click to "change" the settings you get a message from Google saying that this is a feature that will work with Gmail, Google Calendar, Google Sites, Google Reader, Google Voice, App Engine and Google Code.
The Google help centre says:
"If you use multiple sign-in, the first account you use to sign in during that browser session will be your default account for the rest of that session. If you visit other Google products that don't support multiple accounts after you've signed in, you will automatically sign in to your default account for that product. If you sign out of any Google product while signed in to any account, you will be signed out of all your Google Accounts at once".
Again, this is a nice little addition from the G-meister, that will make your online life a little bit easier.
Are you a Gmail user? If so, what do you think of the new features? Give us a shout below.
Twitter Moments explained: The best of Twitter - all under one tab
Master HTML5 and CSS3 web design for 81 per cent off
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text:
Tale of DespereauxDirector: Sam Fell, Rob Stevenhagen
Cast: Sam Fell, Rob Stevenhagen, Matthew Broderick, Robbie Coltrane
View All Available Formats & Editions
A diminutive mouse strives to make a mammoth change in his troubled kingdom in this adaptation of author Kate DiCamillo's Newbery Medal-winning children's book. A long time ago, in the distant kingdom of Dor, a horrible accident broke the heart of the king, left a beautiful princess crestfallen, and filled the townspeople with despair. As the sun disappeared from the… See more details below
A diminutive mouse strives to make a mammoth change in his troubled kingdom in this adaptation of author Kate DiCamillo's Newbery Medal-winning children's book. A long time ago, in the distant kingdom of Dor, a horrible accident broke the heart of the king, left a beautiful princess crestfallen, and filled the townspeople with despair. As the sun disappeared from the sky and the flowers were drained of color, the laughter slowly ceased in this once-magical land. It was into this darkened world that a tiny mouse named Despereaux Tilling was born, and while this virtuous little rodent may have been short in stature, his bravery was ultimately too big for such a small world to contain. An unlikely hero with oversized ears and a discernible wheeze, Despereaux was taken with tales of chivalry, and longed to one day become a noble figure among his people. Sometimes in order to realize their true destiny, heroes must first experience great hardship, however, and when Despereaux fails to adhere to the rigid rules of his society, he is banished from Dor. Later, while exploring the outer realms, the lonely Despereaux is joined by a kindhearted rat named Roscuro. Roscuro, too, has been excommunicated from his homeland, and still cannot stand to look into a mirror lest he be reminded of his painful past. Later, as Despereaux embarks on a treacherous quest to rescue the endangered Princess Pea, Roscuro is set down the path of self-destruction after receiving a painful rejection from his highness. Now, on their way to realizing their extraordinary destinies, these two unlikely heroes will face off against the malevolent leader of the sewer rats, who is plotting revenge on humans from his subterranean kingdom; befriend a common serving girl who longs to become princess; and meet a whole host of colorful characters that they will never forget. Matthew Broderick, Dustin Hoffman, Emma Watson, and Tracey Ullman lead a powerhouse cast in this animated feature produced by Gary Ross and Allison Thomas. Also lending voices to the project are Sigourney Weaver, Kevin Kline, Stanley Tucci, William H. Macy, and Christopher Lloyd.
Editorial Reviews
Product Details
- Release Date:
- 04/07/2009
- 0025195053051
- Original Release:
- 2008
- Rating:
- Source:
- Universal Studios
- Presentation:
- [Wide Screen]
- Sound:
- [DTS 5.1-Channel Surround Sound, Dolby AC-3 Surround Sound]
- Time:
- 1:34:00
- Sales rank:
- 14,993
Cast & Crew
|Matthew Broderick
|Robbie Coltrane
|Gregory,The Jailer
|Frances Conroy
|Antoinette,Voice Only
|Tony Hale
|Furlough,Voice Only
|Emma Watson
|Pea,Princess Pea
|Ciarán Hinds
|Botticelli,Voice Only
|Tracey Ullman
|Mig,Miggery Sow
|Dustin Hoffman
|Kevin Kline
|Voice Only,Andre
|Richard Jenkins
|Voice Only,Principal
|Frank Langella
|Voice Only,Mayor
|William H. Macy
|Voice Only,Lester
|Christopher Lloyd
|Hovis,Voice Only
|James Nesbitt
|Voice Only
|Sigourney Weaver
|Voice Only,Narrator
|Charles Shaughnessy
|Stanley Tucci
|Voice Only,Boldo
|Justin Long
|Voice Only
Technical Credits
|Sam Fell
|Rob Stevenhagen
|Glen Ballard
|Robin Bissell
|Executive Producer
|Brad Blackbourn
|Ryan Kavanaugh
|Executive Producer
|David Lipman
|Executive Producer
|Will McRobb
|Original Story
|Kathy Nelson
|Musical Direction/Supervision
|Gary Ross
|William Ross
|Score Composer
|William Sargent
|Executive Producer
|Tracy Shaw
|Mark Solomon
|Dave Stewart
|Allison Thomas
|Chris Viscardi
|Original Story
|Debra Zane
Scene Index
Customer Reviews
Average Review:
and post it to your social network
Most Helpful Customer Reviews
See all customer reviews >


text:
Tri Tri Tri Again
Thursday - October 15
Wednesday - October 14
Tuesday - October 13
Monday - October 12
I've been a slacker blogging in the last couple of day. The day was long, the course was rough, the wind was tremendous. I promise the world's longest race report in the next week or so.
THANK YOU FOR ALL OF YOU WHO HAVE SHARED THIS JOURNEY WITH ME. Your support has meant more to me during the dark times than you will ever know.
Sunday - October 11
Saturday - October 10
- RACE DAY: Ford Ironman World Championship
Friday - October 9
Danette rented a GPS to track me during the Ironman tomorrow.
Web Browser and iPhone use: www.mapmyathlete.com/vemap.aspx?name=052311
For PDA/Blackberry/Palm use: www.mapmyathlete.com/pda.aspx?name=052311
Thanks for all of the well wishes.
I packed everything up and checked the bike in. I almost left my running shoes at home. Yeah--might need them.
wieczynski's Training Log
Actual vs Planned
Upcoming races
- September's totals
- August's totals
- 2015 totals
- 2014 totals
Run: 17h 43m 26s - 94.38 Mi
Device Upload: 35m
Stretching: 05m
Walking: 10m
Run: 21h 33m 32s - 110.15 Mi
Walking: 15m
Swim: 00m 12s - 1.08 Yd
Run: 120h 26m 51s - 603.42 Mi
Strength: 2h 05m
Core Training: 1h 15m
Device Upload: 14h 58m
Stretching: 1h 15m
Walking: 2h 35m
Swim: 2h 04m 39s - 7964.16 Yd
Bike: 10h 22m 17s - 192.67 Mi
Run: 183h 58m 08s - 930.18 Mi
Strength: 1h 25m
Bocce: 1h 00m
Hiking: 1h 52m
Home Improvement: 5h 00m
Kayaking: 2h 40m
Stretching: 6h 10m
Table Tennis: 30m
Walking: 9h 45m