David E. Nelson

Outcomes of patients with do-not-intubate orders treated with noninvasive ventilation

Objective:To determine whether diagnosis and bedside observations predict outcomes of patients who have declined intubation but accept noninvasive positive pressure ventilation (NPPV) to treat their respiratory failure. Design:Prospective multiple-center cohort trial. Setting:Two teaching hospitals and two community-based hospitals in southeastern New England from January through October 1999. Patients:All patients receiving NPPV for acute respiratory failure were screened and enrolled if they had a written do-not-intubate (DNI) order. Interventions:Patients were begun on NPPV with mean inspiratory and expiratory pressures of 13.4 ± 0.3 and 5.0 ± 1 cm H2O, respectively. Respiratory therapists recorded demographic information, blood gases, and ventilator type and settings, and they made bedside assessments of cough strength, presence of airway secretions, awake state, and agitation. Patients were followed until discharge for duration of NPPV, survival status, and disposition. Measurements and Main Results:Of 1,211 screened patients, 114 had a DNI status and were enrolled into the study. Of these, 49 (43%) survived to discharge. Age, gender, location in a community vs. teaching hospital, and initial pH and Pao2 did not affect survival, but a higher baseline Paco2 was associated with a favorable odds ratios for survival to discharge. Diagnosis was an important determinant of survival, with congestive heart failure patients having significantly better survival rates than those with chronic obstructive pulmonary disease, cancer, pneumonia, or other diagnoses. A stronger cough and being awake were also associated with increased probability of survival. Conclusion:Patients with respiratory failure and a DNI status have a high overall mortality rate when treated with NPPV, but those with diagnoses such as congestive heart failure or chronic obstructive pulmonary disease, who have a strong cough, or who are awake have better prognoses. These data should be useful when counseling DNI patients and their families on use of NPPV.

Biogeography of the ecosystems of the healthy human body

BackgroundCharacterizing the biogeography of the microbiome of healthy humans is essential for understanding microbial associated diseases. Previous studies mainly focused on a single body habitat from a limited set of subjects. Here, we analyzed one of the largest microbiome datasets to date and generated a biogeographical map that annotates the biodiversity, spatial relationships, and temporal stability of 22 habitats from 279 healthy humans.ResultsWe identified 929 genera from more than 24 million 16S rRNA gene sequences of 22 habitats, and we provide a baseline of inter-subject variation for healthy adults. The oral habitat has the most stable microbiota with the highest alpha diversity, while the skin and vaginal microbiota are less stable and show lower alpha diversity. The level of biodiversity in one habitat is independent of the biodiversity of other habitats in the same individual. The abundances of a given genus at a body site in which it dominates do not correlate with the abundances at body sites where it is not dominant. Additionally, we observed the human microbiota exhibit both cosmopolitan and endemic features. Finally, comparing datasets of different projects revealed a project-based clustering pattern, emphasizing the significance of standardization of metagenomic studies.ConclusionsThe data presented here extend the definition of the human microbiome by providing a more complete and accurate picture of human microbiome biogeography, addressing questions best answered by a large dataset of subjects and body sites that are deeply sampled by sequencing.

Evidence of Uncultivated Bacteria in the Adult Female Bladder

ABSTRACT Clinical urine specimens are usually considered to be sterile when they do not yield uropathogens using standard clinical cultivation procedures. Our aim was to test if the adult female bladder might contain bacteria that are not identified by these routine procedures. An additional aim was to identify and recommend the appropriate urine collection method for the study of bacterial communities in the female bladder. Consenting participants who were free of known urinary tract infection provided urine samples by voided, transurethral, and/or suprapubic collection methods. The presence of bacteria in these samples was assessed by bacterial culture, light microscopy, and 16S rRNA gene sequencing. Bacteria that are not or cannot be routinely cultivated (hereinafter called uncultivated bacteria) were common in voided urine, urine collected by transurethral catheter (TUC), and urine collected by suprapubic aspirate (SPA), regardless of whether the subjects had urinary symptoms. Voided urine samples contained mixtures of urinary and genital tract bacteria. Communities identified in parallel urine samples collected by TUC and SPA were similar. Uncultivated bacteria are clearly present in the bladders of some women. It remains unclear if these bacteria are viable and/or if their presence is relevant to idiopathic urinary tract conditions.

Penicillin Binding Protein 5 Affects Cell Diameter, Contour, and Morphology of Escherichia coli

Although general physiological functions have been ascribed to the high-molecular-weight penicillin binding proteins (PBPs) of Escherichia coli, the low-molecular-weight PBPs have no well-defined biological roles. When we examined the morphology of a set of E. coli mutants lacking multiple PBPs, we observed that strains expressing active PBP 5 produced cells of normal shape, while mutants lacking PBP 5 produced cells with altered diameters, contours, and topological features. These morphological effects were visible in untreated cells, but the defects were exacerbated in cells forced to filament by inactivation of PBP 3 or FtsZ. After filamentation, cellular diameter varied erratically along the length of individual filaments and many filaments exhibited extensive branching. Also, in general, the mean diameter of cells lacking PBP 5 was significantly increased compared to that of cells from isogenic strains expressing active PBP 5. Expression of cloned PBP 5 reversed the effects observed in DeltadacA mutants. Although deletion of PBP 5 was required for these phenotypes, the absence of additional PBPs magnified the effects. The greatest morphological alterations required that at least three PBPs in addition to PBP 5 be deleted from a single strain. In the extreme cases in which six or seven PBPs were deleted from a single mutant, cells and cell filaments expressing PBP 5 retained a normal morphology but cells and filaments lacking PBP 5 were aberrant. In no case did mutation of another PBP produce the same drastic morphological effects. We conclude that among the low-molecular-weight PBPs, PBP 5 plays a principle role in determining cell diameter, surface uniformity, and overall topology of the peptidoglycan sacculus.

Contributions of PBP 5 and DD-carboxypeptidase penicillin binding proteins to maintenance of cell shape in Escherichia coli.

Escherichia coli has 12 recognized penicillin binding proteins (PBPs), four of which (PBPs 4, 5, and 6 and DacD) have DD-carboxypeptidase activity. Although the enzymology of the DD-carboxypeptidases has been studied extensively, the in vivo functions of these proteins are poorly understood. To explain why E. coli maintains four independent loci encoding enzymes of considerable sequence identity and comparable in vitro activity, it has been proposed that the DD-carboxypeptidases may substitute for one another in vivo. We tested the validity of this equivalent substitution hypothesis by investigating the effects of these proteins on the aberrant morphology of DeltadacA mutants, which produce no PBP 5. Although cloned PBP 5 complemented the morphological phenotype of a DeltadacA mutant lacking a total of seven PBPs, controlled expression of PBP 4, PBP 6, or DacD did not. Also, a truncated PBP 5 protein lacking its amphipathic C-terminal membrane binding sequence did not reverse the morphological defects and was lethal at low levels of expression, implying that membrane anchoring is essential for the proper functioning of PBP 5. By examining a set of mutants from which multiple PBP genes were deleted, we found that significant morphological aberrations required the absence of at least three different PBPs. The greatest defects were observed in cells lacking, at minimum, PBPs 5 and 6 and one of the endopeptidases (either PBP 4 or PBP 7). The results further differentiate the roles of the low-molecular-weight PBPs, suggest a functional significance for the amphipathic membrane anchor of PBP 5 and, when combined with the recently determined crystal structure of PBP 5, suggest possible mechanisms by which these PBPs may contribute to maintenance of a uniform cell shape in E. coli.

Generation of targeted Chlamydia trachomatis null mutants.

Chlamydia trachomatis is an obligate intracellular bacterial pathogen that infects hundreds of millions of individuals globally, causing blinding trachoma and sexually transmitted disease. More effective chlamydial control measures are needed, but progress toward this end has been severely hampered by the lack of a tenable chlamydial genetic system. Here, we describe a reverse-genetic approach to create isogenic C. trachomatis mutants. C. trachomatis was subjected to low-level ethyl methanesulfonate mutagenesis to generate chlamydiae that contained less then one mutation per genome. Mutagenized organisms were expanded in small subpopulations that were screened for mutations by digesting denatured and reannealed PCR amplicons of the target gene with the mismatch specific endonuclease CEL I. Subpopulations with mutations were then sequenced for the target region and plaque-cloned if the desired mutation was detected. We demonstrate the utility of this approach by isolating a tryptophan synthase gene (trpB) null mutant that was otherwise isogenic to its parental clone as shown by de novo genome sequencing. The mutant was incapable of avoiding the anti-microbial effect of IFN-γ–induced tryptophan starvation. The ability to genetically manipulate chlamydiae is a major advancement that will enhance our understanding of chlamydial pathogenesis and accelerate the development of new anti-chlamydial therapeutic control measures. Additionally, this strategy could be applied to other medically important bacterial pathogens with no or difficult genetic systems.

Characteristic Male Urine Microbiomes Associate with Asymptomatic Sexually Transmitted Infection

Background The microbiome of the male urogenital tract is poorly described but it has been suggested that bacterial colonization of the male urethra might impact risk of sexually transmitted infection (STI). Previous cultivation-dependent studies showed that a variety of non-pathogenic bacteria colonize the urethra but did not thoroughly characterize these microbiomes or establish links between the compositions of urethral microbiomes and STI. Methodology/Findings Here, we used 16S rRNA PCR and sequencing to identify bacteria in urine specimens collected from men who lacked symptoms of urethral inflammation but who differed in status for STI. All of the urine samples contained multiple bacterial genera and many contained taxa that colonize the human vagina. Uncultivated bacteria associated with female genital tract pathology were abundant in specimens from men who had STI. Conclusions Urine microbiomes from men with STI were dominated by fastidious, anaerobic and uncultivated bacteria. The same taxa were rare in STI negative individuals. Our findings suggest that the composition of male urine microbiomes is related to STI.

Predisposition, insult/infection, response, and organ dysfunction: A new model for staging severe sepsis.

Objective: To generate and validate an initial version of the predisposition, insult/infection, response, and organ dysfunction (PIRO) staging model for risk stratification in severe sepsis. The goal was to create distinct levels of mortality risk within each of the four categories (P, I, R, and O), and that these risk levels would be meaningful in terms of prediction independent of the other categories. Design: Retrospective analysis using a statistical model utilizing two large, global databases of patients with severe sepsis. Setting and Patients: Database #1: Placebo-treated patients from a phase III clinical trial of patients with severe sepsis (PROtein C Worldwide Evaluation in Severe Sepsis [PROWESS], 840 patients). Database #2: Global severe sepsis registry performed in 276 intensive care units in 37 countries (PROmoting Global Research Excellence in Severe Sepsis [PROGRESS], 10,610 patients). Interventions: None. Methods: Classification and regression trees were used to classify patients and derive a scoring system from the PROWESS and PROGRESS databases with internal validation. Regression tree parameters included Chi-square tests and a minimum of five patients per node. The risk levels were done in a stepwise manner, adjusting for the previous categories. Initially, the predisposition scoring was developed, and subsequently, the infection scoring was then developed after adjusting for the predisposition levels, and so on. Logistic regression analyses, odds ratios, and area under the receiver operator characteristic curve were used to evaluate the scoring systems. Measurements and Main Results: Each of the four PIRO components had similar odds ratios in multivariable logistic regressions. In PROWESS, the correlation of the PIRO total score and in-hospital mortality rates was 0.974 (p < 0.0001), and in PROGRESS, the correlation of the PIRO total score and hospital mortality rates was 0.998 (p < 0.0001). Conclusions: PIRO can develop into an effective model for staging severe sepsis, seems to be predictive of mortality, and may be useful in future sepsis research.

Physiological functions of D-alanine carboxypeptidases in Escherichia coli

Bacterial cell shape is, in part, mediated by the peptidoglycan (murein) sacculus. Penicillin-binding proteins (PBPs) catalyze the final stages of murein biogenesis and are the targets of beta-lactam antibiotics. Several low molecular mass PBPs including PBP4, PBP5, PBP6 and DacD seem to possess DD-carboxypeptidase (DD-CPase) activity, but these proteins are dispensable for survival in laboratory culture. The physiological functions of DD-CPases in vivo are unresolved and it is unclear why bacteria retain these seemingly non-essential and enzymatically redundant enzymes. However, PBP5 clearly contributes to maintenance of cell shape in some PBP mutant backgrounds. In this review, we focus on recent findings concerning the physiological functions of the DD-CPases in vivo, identify gaps in the current knowledge of these proteins and suggest some possible courses for future study that might help reconcile current models of bacterial cell morphology.

Pathogenic Diversity among Chlamydia trachomatis Ocular Strains in Nonhuman Primates Is Affected by Subtle Genomic Variations

Chlamydia trachomatis is the etiological agent of trachoma, the leading cause of preventable blindness. Trachoma presents distinct clinical syndromes ranging from mild and self-limiting to severe inflammatory disease. The underlying host and pathogen factors responsible for these diverse clinical outcomes are unclear. To assess the role played by pathogen variation in disease outcome, we analyzed the genomes of 4 trachoma strains representative of the 3 major trachoma serotypes, using microarray-based comparative genome sequencing. Outside of ompA, trachoma strains differed primarily in a very small subset of genes (n = 22). These subtle genetic variations were manifested in profound differences in virulence as measured by in vitro growth rate, burst size, plaque morphology, and interferon-gamma sensitivity but most importantly in virulence as shown by ocular infection of nonhuman primates. Our findings are the first to identify genes that correlate with differences in pathogenicity among trachoma strains.