Carbapenem-resistant Enterobacteriaceae colonization (CRE) and subsequent risk of infection and 90-day mortality in critically ill patients, an observational study.

BACKGROUND:Carbapenem-resistant Enterobacteriaceae (CRE) have emerged as an urgent public health threat. Intestinal colonization with CRE has been identified as a risk factor for the development of systemic CRE infection, but has not been compared to colonization with third and/or fourth generation cephalosporin-resistant (Ceph-R) Enterobacteriaceae. Moreover, the risk conferred by colonization on adverse outcomes is less clear, particularly in critically ill patients admitted to the intensive care unit (ICU). METHODS:We carried out a cohort study of consecutive adult patients screened for rectal colonization with CRE or Ceph-R upon ICU entry between April and July 2013. We identified clinical variables and assessed the relationship between CRE or Ceph-R colonization and subsequent systemic CRE infection within 30 days (primary outcome) and all-cause mortality within 90 days (secondary outcome). RESULTS:Among 338 ICU patients, 94 (28%) were colonized with either Ceph-R or CRE. 26 patients developed CRE infection within 30 days of swab collection; 47% (N = 17/36) of CRE-colonized and 3% (N = 2/58) of Ceph-R colonized patients. 36% (N = 13/36) of CRE-colonized patients died within 90 days compared to 31% (N = 18/58) of Ceph-R-colonized and 15% (N = 37/244) of non-colonized patients. In a multivariable analysis, CRE colonization independently predicted development of a systemic CRE infection at 30 days (aOR 10.8, 95% CI2.8-41.9, p = 0.0006); Ceph-R colonization did not (aOR 0.5, 95% CI0.1-3.3, p = 0.5). CRE colonization was associated with increased 90-day mortality in a univariable analysis (p-value 0.001), in a multivariable model, previous hospitalization and medical ICU admission were independent predictors of 90-day mortality whereas CRE colonization approached significance (aOR 2.3, 95% CI1.0-5.3, p = 0.056). CONCLUSIONS:Our study highlights the increased risk of CRE infection and mortality in patients with CRE colonization at the time of ICU admission. Future studies are needed to assess how CRE colonization can guide empiric antibiotic choices and to develop novel decolonization strategies

Characterization of Metabolically Quiescent <i>Leishmania</i> Parasites in Murine Lesions Using Heavy Water Labeling

<div><p>Information on the growth rate and metabolism of microbial pathogens that cause long-term chronic infections is limited, reflecting the absence of suitable tools for measuring these parameters <i>in vivo</i>. Here, we have measured the replication and physiological state of <i>Leishmania mexicana</i> parasites in murine inflammatory lesions using ²H₂O labeling. Infected BALB/c mice were labeled with ²H₂O for up to 4 months, and the turnover of parasite DNA, RNA, protein and membrane lipids estimated from the rate of deuterium enrichment in constituent pentose sugars, amino acids, and fatty acids, respectively. We show that the replication rate of parasite stages in these tissues is very slow (doubling time of ~12 days), but remarkably constant throughout lesion development. Lesion parasites also exhibit markedly lower rates of RNA synthesis, protein turnover and membrane lipid synthesis than parasite stages isolated from <i>ex vivo</i> infected macrophages or cultured <i>in vitro</i>, suggesting that formation of lesions induces parasites to enter a semi-quiescent physiological state. Significantly, the determined parasite growth rate accounts for the overall increase in parasite burden indicating that parasite death and turnover of infected host cells in these lesions is minimal. We propose that the <i>Leishmania</i> response to lesion formation is an important adaptive strategy that minimizes macrophage activation, providing a permissive environment that supports progressive expansion of parasite burden. This labeling approach can be used to measure the dynamics of other host-microbe interactions <i>in situ</i>.</p></div

Stage-specific changes in <i>Leishmania</i> growth rates.

<p><b>A</b>. Schematic overview of ²H₂O labeling protocol. Parasite stages were cultivated axenically in the presence 5% ²H₂O, or isolated from infected macrophages or BALB/c lesion incubated or infused with ²H₂O (final concentration 5%). Parasite stages were harvested at multiple time points and extracts containing total DNA/RNA, or total proteins and lipids generated from purified parasite fraction. Levels of deuterium enrichment in constituent dRib/Rib, amino acids and fatty acids were subsequently quantitated by GC-MS. <b>B</b>. Kinetics of ²H-labeling of DNA dRib in cultured promastigotes (Pro^log, Pro^stat) and amastigotes (Ama^axenic), and in amastigotes isolated from macrophages (Ama^Mø) and murine lesions (Ama^lesion). The fraction of new cells (Y-axis) was calculated from the level of ²H-enrichment in dRib relative to maximum labeling observed in each parasite stage after long term (equilibrium) labeling. Calculated doubling times for each stage are shown in inset boxes. <b>C</b>. Comparative growth rates of different <i>Leishmania</i> stages, calculated from ²H-enrichment in dRib. <b>D</b>. Section of stained cutaneous lesion (with detail in insert) and calculated range of parasite numbers/phagolysosome. Abbreviations: dRib; deoxyribose, Rib; ribose.</p

Lesion amastigotes utilize both salvage and <i>de novo</i> biosynthetic pathways to supply their fatty acid needs.

<p><b>A</b>. The maximum level of ²H-enrichment (EM1, %) in major cellular fatty acids of Pro^log and Ama^lesion were determined after labeling for 7 days and >6 weeks, respectively. ²H-enrichment in the total plasma lipid of infected mice was also measured to determine the potential contribution of labeled host fatty acids to the parasite labeling. Note that while saturated and unsaturated C18 fatty acids are predominant fatty acids in both stages, the fatty acid composition of Ama^lesion differs from cultured promastigotes in containing elevated levels of C20:4 n-6, and polyunsaturated very long chain fatty acids (C22:4 n-6, C22:6 n-3) (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004683#ppat.1004683.s008" target="_blank">S8 Fig</a>.). The C:D nomenclature refers to overall chain length and number of double bonds in each fatty acid, respectively. n-3 and n-6 refers to the two major biosynthetic pathways involved in unsaturated fatty acid biosynthesis (where-3 and-6 refer to the position of double bond relative to the methyl carbon). <b>B</b>. Stage-specific differences in the levels of ²H-enrichment in fatty acid pools can be used to infer the contributions of <i>de novo</i> biosynthesis and salvage pathways. In particular, levels of ²H-enrichment in the major Ama^lesion C18 fatty acids, C18:1 (oleic acid) and C18:2 (linoleic acid), were appreciably higher than in host plasma, indicating that these stages are dependent on <i>de novo</i> biosynthesis. Conversely, the elevated levels of ²H-enrichment in C20:4 n-6 and C22:6n-3 compared to C18:1 precursor (comparable to plasma pools) indicate that these very long chain polyunsaturated fatty acids are primarily scavenged from the host cell.</p

Rates of protein turnover in cultured and intracellular <i>Leishmania</i> stages.

<p>Parasite stages were ²H₂O-labeled in culture or <i>in situ</i> in infected BALB/c mice and harvested at the indicated time points. Kinetics of ²H-labeling of proteinogenic alanine in (<b>A</b>) cultured parasite stages (Pro^log, Pro^stat, Ama^axenic) and (<b>B</b>) amastigotes isolated from BALB/c lesion (Ama^lesion). The fraction of new molecules (Y-axis) was calculated from the level of ²H-enrichment in alanine relative to maximum labeling observed in each parasite stage after long term labeling. Inset boxes in A and B show turnover (t_1/2) in days. <b>C</b>. Comparative rates of protein turnover in different <i>Leishmania</i> developmental stages. Note that similar estimates of protein turnover were obtained by measuring deuterium incorporation into other proteinogenic amino acids (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004683#ppat.1004683.s006" target="_blank">S6 Fig</a>.).</p

Rates of RNA turnover in cultured and intracellular <i>Leishmania</i> stages.

<p>Kinetics of ²H-labeling of RNA ribose in (<b>A</b>) cultured parasite stages (Pro^log, Pro^stat, Ama^axenic) (<b>B</b>) amastigotes isolated from infected J774 macrophages (Ama^Mø) and (<b>C</b>) amastigotes isolated from BALB/c lesions (Ama^lesion). The fraction of new molecules (Y-axis) was calculated from the level of ²H-enrichment in Rib relative to maximum labeling observed in each parasite stage after long term labeling. Inset boxes shows estimated RNA turnover (t_1/2 in days) in each stage. <b>D</b>. Comparative rates of RNA turnover in different <i>Leishmania</i> developmental stages.</p