Identification of novel regulators of developmental hematopoiesis using Endoglin regulatory elements as molecular probes.

Enhancers are the primary determinants of cell identity, and specific promoter/enhancer combinations of Endoglin (ENG) have been shown to target blood and endothelium in the embryo. Here, we generated a series of embryonic stem cell lines, each targeted with reporter constructs driven by specific promoter/enhancer combinations of ENG, to evaluate their discriminative potential and value as molecular probes of the corresponding transcriptome. The Eng promoter (P) in combination with the -8/+7/+9-kb enhancers, targeted cells in FLK1 mesoderm that were enriched for blast colony forming potential, whereas the P/-8-kb enhancer targeted TIE2+/c-KIT+/CD41- endothelial cells that were enriched for hematopoietic potential. These fractions were isolated using reporter expression and their transcriptomes profiled by RNA-seq. There was high concordance between our signatures and those from embryos with defects at corresponding stages of hematopoiesis. Of the 6 genes that were upregulated in both hemogenic mesoderm and hemogenic endothelial fractions targeted by the reporters, LRP2, a multiligand receptor, was the only gene that had not previously been associated with hematopoiesis. We show that LRP2 is indeed involved in definitive hematopoiesis and by doing so validate the use of reporter gene-coupled enhancers as probes to gain insights into transcriptional changes that facilitate cell fate transitions.

Impaired functional capacity in potential liver transplant candidates predicts short-term mortality before transplantation.

Liver transplantation (LT) is a lifesaving treatment. Because of the shortage of donor organs, some patients will not survive long enough to receive a transplant. The identification of LT candidates at increased risk of short-term mortality without transplantation may affect listing decisions. Functional capacity, determined with cardiopulmonary exercise testing (CPET), is a measure of cardiorespiratory reserve and predicts perioperative outcomes. This study examined the association between functional capacity and short-term survival before LT and the potential for CPET to predict 90-day mortality without transplantation. A total of 176 patients who were assessed for nonacute LT underwent CPET. Ninety days after the assessment, 10 of the 164 patients who had not undergone transplantation were deceased (mortality rate = 6.1%). According to a comparison of survivors and nonsurvivors, the Model for End-Stage Liver Disease score, UK Model for End-Stage Liver Disease (UKELD) score, age, anaerobic threshold, and peak oxygen uptake (VO(2)) were significant univariate predictors of 90-day mortality without transplantation, but only the UKELD score and peak VO(2) retained significance in a multivariate analysis. The mean peak VO(2) was significantly lower for nonsurvivors versus survivors (15.2 ± 3.3 versus 21.2 ± 5.3 mL/minute/kg, P < 0.001). According to a receiver operating characteristic (ROC) curve analysis, peak VO(2) performed well as a diagnostic test (area under the ROC curve = 0.84, 95% confidence interval = 0.76-0.92, sensitivity = 0.90, specificity = 0.74, P < 0.001). The optimal cutoff value for predicting mortality was ≤17.6 mL/minute/kg. The positive predictive value of a peak VO(2) ≤ 17.6 mL/minute/kg for 90-day mortality was greatest for patients with high UKELD scores: 38% of the patients with a UKELD score ≥ 57 and a peak VO(2) ≤ 17.6 mL/minute/kg died, whereas only 6% of the patients with a UKELD score ≥ 57 and a peak VO(2) > 17.6 mL/minute/kg died (P = 0.03). In conclusion, patients assessed for LT with an impaired functional capacity have poorer short-term survival; this is particularly true for individuals with worse liver disease severity.

Metabolomic approaches reveal that cell wall modifications play a major role in ethylene-mediated resistance against Botrytis cinerea.

In Arabidopsis, resistance to the necrotrophic fungus Botrytis cinerea is conferred by ethylene via poorly understood mechanisms. Metabolomic approaches compared the responses of the wild-type, the ethylene-insensitive mutant etr1-1, which showed increased susceptibility, and the constitutively active ethylene mutants ctr1-1 and eto2 both exhibited decreased susceptibility to B. cinerea. Fourier transform-infrared (FT-IR) spectroscopy demonstrated reproducible biochemical differences between treatments and genotypes. To identify discriminatory mass-to-charge ratios (m/z) associated with resistance, discriminant function analysis was employed on spectra derived from direct injection electrospray ionisation-mass spectrometry on the derived principal components of these data. Ethylene-modulated m/z were mapped onto Arabidopsis biochemical pathways and many were associated with hydroxycinnamate and monolignol biosynthesis, both linked to cell wall modification. A high-resolution linear triple quadrupole-Orbitrap hybrid system confirmed the identity of key metabolites in these pathways. The contribution of these pathways to defence against B. cinerea was validated through the use of multiple Arabidopsis mutants. The FT-IR microspectroscopy indicated that spatial accumulation of hydroxycinnamates and monolignols at the cell wall to confine disease was linked ot ethylene. These data demonstrate the power of metabolomic approaches in elucidating novel biological phenomena, especially when coupled to validation steps exploiting relevant mutant genotypes.

Modeling reveals bistability and low-pass filtering in the network module determining blood stem cell fate.

Combinatorial regulation of gene expression is ubiquitous in eukaryotes with multiple inputs converging on regulatory control elements. The dynamic properties of these elements determine the functionality of genetic networks regulating differentiation and development. Here we propose a method to quantitatively characterize the regulatory output of distant enhancers with a biophysical approach that recursively determines free energies of protein-protein and protein-DNA interactions from experimental analysis of transcriptional reporter libraries. We apply this method to model the Scl-Gata2-Fli1 triad-a network module important for cell fate specification of hematopoietic stem cells. We show that this triad module is inherently bistable with irreversible transitions in response to physiologically relevant signals such as Notch, Bmp4 and Gata1 and we use the model to predict the sensitivity of the network to mutations. We also show that the triad acts as a low-pass filter by switching between steady states only in response to signals that persist for longer than a minimum duration threshold. We have found that the auto-regulation loops connecting the slow-degrading Scl to Gata2 and Fli1 are crucial for this low-pass filtering property. Taken together our analysis not only reveals new insights into hematopoietic stem cell regulatory network functionality but also provides a novel and widely applicable strategy to incorporate experimental measurements into dynamical network models.

Ontogeny of haematopoiesis: recent advances and open questions.

Unravelling the embryonic origins of the haematopoietic system has been the subject of sustained research for more than a century. Nevertheless, many important questions are still either unanswered or remain a matter of intense debate. Recent progress in mouse and embryonic stem cell model systems as well as imaging and post-genomic technologies has provided new insights into many of these open questions. Here we place into context recent reports on the anatomical site of blood stem cell emergence and, using red blood cells as an example, illustrate how the development of stem cells and the other blood lineages is both temporally and spatially decoupled. In addition, we outline how embryonic stem cell assays are increasingly used as a powerful surrogate for studying lineage relationships and developmental potential of early embryonic blood progenitors. Finally, we review how recent progress in the reconstruction of transcriptional regulatory networks is beginning to define the connectivity between key regulators that control early blood development. In light of these rapid recent advances, research into the embryonic origins of the haematopoietic system should remain one of the most vibrant disciplines within the wider field of haematology for the foreseeable future.

Endoglin expression in blood and endothelium is differentially regulated by modular assembly of the Ets/Gata hemangioblast code.

Endoglin is an accessory receptor for TGF-beta signaling and is required for normal hemangioblast, early hematopoietic, and vascular development. We have previously shown that an upstream enhancer, Eng -8, together with the promoter region, mediates robust endothelial expression yet is inactive in blood. To identify hematopoietic regulatory elements, we used array-based methods to determine chromatin accessibility across the entire locus. Subsequent transgenic analysis of candidate elements showed that an endothelial enhancer at Eng +9 when combined with an element at Eng +7 functions as a strong hemato-endothelial enhancer. Chromatin immunoprecipitation (ChIP)-chip analysis demonstrated specific binding of Ets factors to the promoter as well as to the -8, +7+9 enhancers in both blood and endothelial cells. By contrast Pu.1, an Ets factor specific to the blood lineage, and Gata2 binding was only detected in blood. Gata2 was bound only at +7 and GATA motifs were required for hematopoietic activity. This modular assembly of regulators gives blood and endothelial cells the regulatory freedom to independently fine-tune gene expression and emphasizes the role of regulatory divergence in driving functional divergence.

A proposed framework for the description of plant metabolomics experiments and their results.

The study of the metabolite complement of biological samples, known as metabolomics, is creating large amounts of data, and support for handling these data sets is required to facilitate meaningful analyses that will answer biological questions. We present a data model for plant metabolomics known as ArMet (architecture for metabolomics). It encompasses the entire experimental time line from experiment definition and description of biological source material, through sample growth and preparation to the results of chemical analysis. Such formal data descriptions, which specify the full experimental context, enable principled comparison of data sets, allow proper interpretation of experimental results, permit the repetition of experiments and provide a basis for the design of systems for data storage and transmission. The current design and example implementations are freely available (http://www.armet.org/). We seek to advance discussion and community adoption of a standard for metabolomics, which would promote principled collection, storage and transmission of experiment data.

Metabolic fingerprinting of salt-stressed tomatoes.

The aim of this study was to adopt the approach of metabolic fingerprinting through the use of Fourier transform infrared (FT-IR) spectroscopy and chemometrics to study the effect of salinity on tomato fruit. Two varieties of tomato were studied, Edkawy and Simge F1. Salinity treatment significantly reduced the relative growth rate of Simge F1 but had no significant effect on that of Edkawy. In both tomato varieties salt-treatment significantly reduced mean fruit fresh weight and size class but had no significant affect on total fruit number. Marketable yield was however reduced in both varieties due to the occurrence of blossom end rot in response to salinity. Whole fruit flesh extracts from control and salt-grown tomatoes were analysed using FT-IR spectroscopy. Each sample spectrum contained 882 variables, absorbance values at different wavenumbers, making visual analysis difficult and therefore machine learning methods were applied. The unsupervised clustering method, principal component analysis (PCA) showed no discrimination between the control and salt-treated fruit for either variety. The supervised method, discriminant function analysis (DFA) was able to classify control and salt-treated fruit in both varieties. Genetic algorithms (GA) were applied to identify discriminatory regions within the FT-IR spectra important for fruit classification. The GA models were able to classify control and salt-treated fruit with a typical error, when classifying the whole data set, of 9% in Edkawy and 5% in Simge F1. Key regions were identified within the spectra corresponding to nitrile containing compounds and amino radicals. The application of GA enabled the identification of functional groups of potential importance in relation to the response of tomato to salinity.

Investigating plant-plant interference by metabolic fingerprinting.

New analytical developments in post-genomic technologies are being introduced to the field of plant ecology. FT-IR fingerprinting coupled with chemometrics via cluster analysis is proposed as a tool for correlating global metabolic changes with abiotic or biotic perturbation and/or interactions. The current study concentrates on detecting chemical responses by inter-species competition between a monocotyledon Brachypodium distachyion and a dicotyledon Arabidopsis thaliana. Growth analysis of 42 days old plants showed differences in both species under competition. Clear changes in the FT-IR metabolic fingerprints of B. distachyion in competition with A. thaliana were observed, whilst there were no apparent chemical differences in the A. thaliana plant tissues. This study demonstrates the power of this approach in detecting changes in the global metabolic profiles of plants in response to biotic interactions, and we believe FT-IR is appropriate for rapid screening (10 s per sample) prior to targeted metabolite analyses.

Effect of a synbiotic on microbial community structure in a continuous culture model of the gastric microbiota in enteral nutrition patients.

Patients with dysphagia require long-term nutritional support. This can be delivered by the enteral route via a percutaneous endoscopic gastrostomy (PEG) tube. Enteral nutrition (EN) bypasses the body's innate defences that prevent the microbial colonization of the proximal gut, which predisposes to microbial overgrowth. A continuous culture model simulating the upper gastrointestinal tract microbiota of EN patients was used to investigate the effects of a synbiotic (Lactobacillus acidophilus DUN-311, Bifidobacterium bifidum BB-02, Bifidobacterium lactis BL-01, Synergy 1) on microbial community structure and metabolism. A PEG tube was inserted into the fermenters to study biofilm formation. The synbiotic delivered in sterile semi-skimmed milk (SSSM) was introduced either 48 h prior to or after PEG tube insertion. The synbiotic reduced biofilm formation on PEG tube surfaces, with suppression of Escherichia coli and Klebsiella pneumoniae when it was added subsequent to PEG insertion. When synbiotic feeding was commenced prior to PEG insertion, colonization by Staphylococcus aureus, Candida albicans and Candida famata was also inhibited. Lactate production increased in response the synbiotic or control (SSSM). These results indicate that the use of a synbiotic has the potential to reduce pathogen colonization on PEG tube surfaces in vivo, thereby reducing the incidence of biofilm-related infectious complications.

Integration of Elf-4 into stem/progenitor and erythroid regulatory networks through locus-wide chromatin studies coupled with in vivo functional validation.

The ETS transcription factor Elf-4 is an important regulator of hematopoietic stem cell (HSC) and T cell homeostasis. To gain insights into the transcriptional circuitry within which Elf-4 operates, we used comparative sequence analysis coupled with chromatin immunoprecipitation (ChIP) with microarray technology (ChIP-chip) assays for specific chromatin marks to identify three promoters and two enhancers active in hematopoietic and endothelial cell lines. Comprehensive functional validation of each of these regulatory regions in transgenic mouse embryos identified a tissue-specific enhancer (-10E) that displayed activity in fetal liver, dorsal aorta, vitelline vessels, yolk sac, and heart. Integration of a ChIP-sequencing (ChIP-Seq) data set for 10 key stem cell transcription factors showed Pu.1, Fli-1, and Erg were bound to the -10E element, and mutation of three highly conserved ETS sites within the enhancer abolished its activity. Finally, the transcriptional repressor Gfi1b was found to bind to and repress one of the Elf-4 promoters (-30P), and we show that this repression of Elf-4 is important for the maturation of primary fetal liver erythroid cells. Taken together, our results provide a comprehensive overview of the transcriptional control of Elf-4 within the hematopoietic system and, thus, integrate Elf-4 into the wider transcriptional regulatory networks that govern hematopoietic development.

Microbiological and immunological effects of enteral feeding on the upper gastrointestinal tract.

Enteral feeding via a percutaneous endoscopic gastrostomy tube is required for nutritional support in patients with dysphagia. Enteral tube feeding bypasses the innate defence mechanisms in the upper gastrointestinal tract. This study examined the surface-associated microbial populations and immune response in the gastric and duodenal mucosae of eight enteral nutrition (EN) patients and ten controls. Real-time PCR and fluorescence in situ hybridization were employed to assess microbiota composition and mucosal pro-inflammatory cytokine expression. The results showed that EN patients had significantly higher levels of bacterial DNA in mucosal biopsies from the stomach and duodenum (P<0.05) than the controls, and that enterobacteria were the predominant colonizing species on mucosal surfaces in these individuals. Expression of the pro-inflammatory cytokines interleukin (IL)-1α, IL-6 and tumour necrosis factor-α was significantly higher in gastric and small intestinal mucosae from patients fed normal diets in comparison with those receiving EN (P<0.05). These results indicate that EN can lead to significant bacterial overgrowth on upper gastrointestinal tract mucosae and a significantly diminished pro-inflammatory cytokine response.

LIF-independent JAK signalling to chromatin in embryonic stem cells uncovered from an adult stem cell disease.

Activating mutations in the tyrosine kinase Janus kinase 2 (JAK2) cause myeloproliferative neoplasms, clonal blood stem cell disorders with a propensity for leukaemic transformation. Leukaemia inhibitory factor (LIF) signalling through the JAK-signal transducer and activator of transcription (STAT) pathway enables self-renewal of embryonic stem (ES) cells. Here we show that mouse ES cells carrying the human JAK2V617F mutation were able to self-renew in chemically defined conditions without cytokines or small-molecule inhibitors, independently of JAK signalling through the STAT3 or phosphatidylinositol-3-OH kinase pathways. Phosphorylation of histone H3 tyrosine 41 (H3Y41) by JAK2 was recently shown to interfere with binding of heterochromatin protein 1α (HP1α). Levels of chromatin-bound HP1α were lower in JAK2V617F ES cells but increased following inhibition of JAK2, coincident with a global reduction in histone H3Y41 phosphorylation. JAK2 inhibition reduced levels of the pluripotency regulator Nanog, with a reduction in H3Y41 phosphorylation and concomitant increase in HP1α levels at the Nanog promoter. Furthermore, Nanog was required for factor independence of JAK2V617F ES cells. Taken together, these results uncover a previously unrecognized role for direct signalling to chromatin by JAK2 as an important mediator of ES cell self-renewal.

Acute liver injury associated with the use of herbal preparations containing glucosamine: three case studies.

The use of complementary and alternative medicines is becoming increasingly popular in Western society. As a result the number of reported adverse reactions is increasing. Glucosamine is a herbal remedy commonly used to ease joint pain in osteoarthritis, and only two previous reports of hepatotoxicity have been published in the scientific literature. The present report describes three patients who developed acute liver injury following exposure to glucosamine; one patient made a complete recovery on cessation of ingestion, the second developed chronic hepatitis and the third died following progression to fulminant hepatic failure. A diagnosis of glucosamine-induced hepatotoxicity was made based on the temporal relationship between onset of liver injury and glucosamine ingestion, exclusion of all other potential aetiologies and, in the two surviving cases, improvement in condition on withdrawal of the supplement.

Biphasic ethylene production during the hypersensitive response in Arabidopsis: a window into defense priming mechanisms?

The hypersensitive response (HR) is a cell death phenomenon associated with localized resistance to pathogens. Biphasic patterns in the generation of H(2)O(2), salicylic acid and ethylene have been observed in tobacco during the early stages of the HR. These biphasic models reflect an initial elicitation by pathogen-associated molecular patterns followed by a second phase, induced by pathogen-encoded avirulence gene products. The first phase has been proposed to potentiate the second, to increase the efficacy of plant resistance to disease. This potentiation is comparable to the "priming" of plant defenses which is seen when plants display systemic resistance to disease. The events regulating the generation of the biphasic wave, or priming, remains obscure, however recently we demonstrated a key role for nitric oxide in this process in a HR occurring in tobacco. Here we use laser photoacoustic detection to demonstrate that biphasic ethylene production also occurs during a HR occurring in Arabidopsis. We suggest that ethylene emanation during the HR represents a ready means of visualising biphasic events during the HR and that exploiting the genomic resources offered by this model species will facilitate the development of a mechanistic understanding of potentiating/priming processes.

Nitric oxide interacts with salicylate to regulate biphasic ethylene production during the hypersensitive response.

C(2)H(4) is associated with plant defense, but its role during the hypersensitive response (HR) remains largely uncharacterized. C(2)H(4) production in tobacco (Nicotiana tabacum) following inoculation with HR-eliciting Pseudomonas syringae pathovars measured by laser photoacoustic detection was biphasic. A first transient rise (C(2)H(4)-I) occurred 1 to 4 h following inoculation with HR-eliciting, disease-forming, and nonpathogenic strains and also with flagellin (flg22). A second (avirulence-dependent) rise, at approximately 6 h (C(2)H(4)-II), was only seen with HR-eliciting strains. Tobacco leaves treated with the C(2)H(4) biosynthesis inhibitor, aminoethoxyvinylglycine, suggested that C(2)H(4) influenced the kinetics of a HR. Challenging salicylate hydroxylase-expressing tobacco lines and tissues exhibiting systemic acquired resistance suggested that C(2)H(4) production was influenced by salicylic acid (SA). Disrupted expression of a C(2)H(4) biosynthesis gene in salicylate hydroxylase tobacco plants implicated transcriptional control as a mechanism through which SA regulates C(2)H(4) production. Treating leaves to increase oxidative stress or injecting with SA initiated monophasic C(2)H(4) generation, but the nitric oxide (NO) donor sodium nitroprusside initiated biphasic rises. To test whether NO influenced biphasic C(2)H(4) production during the HR, the NO synthase inhibitor N(G)-nitro-L-arginine methyl ester was coinoculated with the avirulent strain of P. syringae pv phaseolicola into tobacco leaves. The first transient C(2)H(4) rise appeared to be unaffected by N(G)-nitro-L-arginine methyl ester, but the second rise was reduced. These data suggest that NO and SA are required to generate the biphasic pattern of C(2)H(4) production during the HR and may influence the kinetics of HR formation.

A novel mode of enhancer evolution: the Tal1 stem cell enhancer recruited a MIR element to specifically boost its activity.

Altered cis-regulation is thought to underpin much of metazoan evolution, yet the underlying mechanisms remain largely obscure. The stem cell leukemia TAL1 (also known as SCL) transcription factor is essential for the normal development of blood stem cells and we have previously shown that the Tal1 +19 enhancer directs expression to hematopoietic stem cells, hematopoietic progenitors, and to endothelium. Here we demonstrate that an adjacent region 1 kb upstream (+18 element) is in an open chromatin configuration and carries active histone marks but does not function as an enhancer in transgenic mice. Instead, it boosts activity of the +19 enhancer both in stable transfection assays and during differentiation of embryonic stem (ES) cells carrying single-copy reporter constructs targeted to the Hprt locus. The +18 element contains a mammalian interspersed repeat (MIR) which is essential for the +18 function and which was transposed to the Tal1 locus approximately 160 million years ago at the time of the mammalian/marsupial branchpoint. Our data demonstrate a previously unrecognized mechanism whereby enhancer activity is modulated by a transposon exerting a "booster" function which would go undetected by conventional transgenic approaches.

Ulcerative colitis and autoimmunity induced by loss of myeloid alphav integrins.

The gastrointestinal tract is constantly challenged by foreign antigens and commensal bacteria but nonetheless is able to maintain a state of immunological quiescence. Recent advances have highlighted the importance of active suppression by regulatory lymphocytes and immunosuppressive cytokines in controlling mucosal immunity. Failures of these mechanisms contribute to the development of inflammatory bowel disease, but how these regulatory networks are established remains unclear. Here, we demonstrate key roles for alphav integrins in regulation of mucosal immunity. We report that deletion of alphav in the immune system causes severe colitis, autoimmunity, and cancer. Mice lacking immune cell alphav have fewer regulatory T (Treg) cells in the colon and corresponding increases in activated T cells and T cell cytokine production, leading to colitis. Using conditional gene targeting, we demonstrate that this is specifically attributable to loss of alphav from myeloid cells. Furthermore, we show that gut-associated macrophages and dendritic cells fail both to remove apoptotic cells efficiently and to induce Treg cells. Our results identify a vital role for myeloid alphav integrins in generating mucosal Treg cells and emphasize the importance of antigen-presenting cells in establishing immune tolerance.

Identifying gene regulatory elements by genomic microarray mapping of DNaseI hypersensitive sites.

The identification of cis-regulatory elements is central to understanding gene transcription. Hypersensitivity of cis-regulatory elements to digestion with DNaseI remains the gold-standard approach to locating such elements. Traditional methods used to identify DNaseI hypersensitive sites are cumbersome and can only be applied to short stretches of DNA at defined locations. Here we report the development of a novel genomic array-based approach to DNaseI hypersensitive site mapping (ADHM) that permits precise, large-scale identification of such sites from as few as 5 million cells. Using ADHM we identified all previously recognized hematopoietic regulatory elements across 200 kb of the mouse T-cell acute lymphocytic leukemia-1 (Tal1) locus, and, in addition, identified two novel elements within the locus, which show transcriptional regulatory activity. We further validated the ADHM protocol by mapping the DNaseI hypersensitive sites across 250 kb of the human TAL1 locus in CD34+ primary stem/progenitor cells and K562 cells and by mapping the previously known DNaseI hypersensitive sites across 240 kb of the human alpha-globin locus in K562 cells. ADHM provides a powerful approach to identifying DNaseI hypersensitive sites across large genomic regions.

Effect of pH and antibiotics on microbial overgrowth in the stomachs and duodena of patients undergoing percutaneous endoscopic gastrostomy feeding.

Enteral nutrition via a percutaneous endoscopic gastrostomy (PEG) tube is often part of management in patients with dysphagia due to neurological or oropharyngeal disease. Gastrostomy placement can affect normal innate defense mechanisms in the upper gut, resulting in bacterial overgrowth. In this study microbiological investigations were done with gastric and duodenal aspirates from 20 patients undergoing PEG tube placement and PEG tubes from 10 patients undergoing tube replacement. Aspirate and PEG tube microbiotas were assessed by using viable counts and selective solid media followed by aerobic and anaerobic incubation to assess cell viabilities. The antibiotic susceptibility profiles of the isolates were determined by the disk diffusion method, and gas chromatography was used to study the bacterial metabolic products in the aspirates. The aspirates and PEG tubes contained mainly streptococci, staphylococci, lactobacilli, yeasts, and enterobacteria. Enterococci were detected only in PEG tube biofilms and not in aspirates. Gastric pH affected the composition of the aspirate microbiotas but not the total microbial counts. Staphylococci, Escherichia coli, and Candida spp. were isolated only from antibiotic-treated patients, despite the sensitivities of the bacteria to the agents used. Antibiotic treatment had no effect on the incidence of infection or the length of hospital stay in these patients.