Candida albicans Isolates from the Gut of Critically Ill Patients Respond to Phosphate Limitation by Expressing Filaments and a Lethal Phenotype

Candida albicans is an opportunistic pathogen that proliferates in the intestinal tract of critically ill patients where it continues to be a major cause of infectious-related mortality. The precise cues that shift intestinal C. albicans from its ubiquitous indolent colonizing yeast form to an invasive and lethal filamentous form remain unknown. We have previously shown that severe phosphate depletion develops in the intestinal tract during extreme physiologic stress and plays a major role in shifting intestinal Pseudomonas aeruginosa to express a lethal phenotype via conserved phosphosensory-phosphoregulatory systems. Here we studied whether phosphate dependent virulence expression could be similarly demonstrated for C. albicans. C. albicans isolates from the stool of critically ill patients and laboratory prototype strains (SC5314, BWP17, SN152) were evaluated for morphotype transformation and lethality against C. elegans and mice during exposure to phosphate limitation. Isolates ICU1 and ICU12 were able to filament and kill C. elegans in a phosphate dependent manner. In a mouse model of intestinal phosphate depletion (30% hepatectomy), direct intestinal inoculation of C. albicans caused mortality that was prevented by oral phosphate supplementation. Prototype strains displayed limited responses to phosphate limitation; however, the pho4Δ mutant displayed extensive filamentation during low phosphate conditions compared to its isogenic parent strain SN152, suggesting that mutation in the transcriptional factor Pho4p may sensitize C. albicans to phosphate limitation. Extensive filamentation was also observed in strain ICU12 suggesting that this strain is also sensitized to phosphate limitation. Analysis of the sequence of PHO4 in strain ICU12, its transcriptional response to phosphate limitation, and phosphatase assays confirmed that ICU12 demonstrates a profound response to phosphate limitation. The emergence of strains of C. albicans with marked responsiveness to phosphate limitation may represent a fitness adaptation to the complex and nutrient scarce environment typical of the gut of a critically ill patient

Right sided upper extremity access for patients undergoing parallel graft placement during endovascular aortic repair is not associated with increased neurologic events when compared to left upper extremity access.

OBJECTIVE: The safety and efficacy of right axillary cannulation during complex aortic aneurysm repair for the deployment of chimney grafts is controversial, however there are few studies that compare right and left upper extremity access . We favor the right axillary approach because of the relative ease of access to the visceral branches and the ability of surgeons and nursing staff to work on the same side of the patient, while avoiding the left sided image intensifier. We aim to demonstrate that right sided access is equivalent or safer than left sided access in terms of technical success and complication rates, with a focus on neurologic outcomes. METHODS: This is a single institution retrospective study with a review of patients who underwent aortic intervention from January 2012 through December 2018. A total of 398 aortic interventions were performed, and 97 of these required brachial, axillary or subclavian arterial access for attempted ChEVAR or thoracic endovascular aortic repair with parallel chimney grafts. Primary endpoints that were analyzed were site or sites of upper extremity access, technical success, 30-day mortality, cerebrovascular events, and subclavian/axillary artery injury. The number of parallel grafts, age, mean hospital length of stay, prior aortic intervention, emergent or elective status were also analyzed RESULTS: 97 endovascular aortic operations required upper extremity access, with 67 using access from the right upper extremity, 26 using access from the left upper extremity, and 4 utilizing bilateral upper extremity access. A total of 68.0% of patients had undergone prior aortic surgery. Technical success was achieved in 85 cases (87.6%). 5 total patients suffered cerebrovascular accidents (CVA), with 2 occurring in left sided access (7.7%), 2 in right sided access (3.0%) , and 1 in bilateral access (25%). CONCLUSIONS: Right upper extremity access for patients undergoing parallel graft placement during endovascular aortic aneurysm repair is a safe and feasible approach that is not associated with an increased risk of stroke or neurological events as compared to left upper extremity access

<i>Candida albicans</i> Isolates from the Gut of Critically Ill Patients Respond to Phosphate Limitation by Expressing Filaments and a Lethal Phenotype

Candida albicans is an opportunistic pathogen that proliferates in the intestinal tract of critically ill patients where it continues to be a major cause of infectious-related mortality. The precise cues that shift intestinal C. albicans from its ubiquitous indolent colonizing yeast form to an invasive and lethal filamentous form remain unknown. We have previously shown that severe phosphate depletion develops in the intestinal tract during extreme physiologic stress and plays a major role in shifting intestinal Pseudomonas aeruginosa to express a lethal phenotype via conserved phosphosensory-phosphoregulatory systems. Here we studied whether phosphate dependent virulence expression could be similarly demonstrated for C. albicans. C. albicans isolates from the stool of critically ill patients and laboratory prototype strains (SC5314, BWP17, SN152) were evaluated for morphotype transformation and lethality against C. elegans and mice during exposure to phosphate limitation. Isolates ICU1 and ICU12 were able to filament and kill C. elegans in a phosphate dependent manner. In a mouse model of intestinal phosphate depletion (30% hepatectomy), direct intestinal inoculation of C. albicans caused mortality that was prevented by oral phosphate supplementation. Prototype strains displayed limited responses to phosphate limitation; however, the pho4Δ mutant displayed extensive filamentation during low phosphate conditions compared to its isogenic parent strain SN152, suggesting that mutation in the transcriptional factor Pho4p may sensitize C. albicans to phosphate limitation. Extensive filamentation was also observed in strain ICU12 suggesting that this strain is also sensitized to phosphate limitation. Analysis of the sequence of PHO4 in strain ICU12, its transcriptional response to phosphate limitation, and phosphatase assays confirmed that ICU12 demonstrates a profound response to phosphate limitation. The emergence of strains of C. albicans with marked responsiveness to phosphate limitation may represent a fitness adaptation to the complex and nutrient scarce environment typical of the gut of a critically ill patient.</p

Phosphate attenuates the formation of biofilm in <i>C. albicans</i> SC5314 <i>in vivo</i> and <i>in vitro</i>.

<p>(A) Scanning electron microscopy (SEM) of intestinal tissues from mice with cecal injection of <i>C. albicans</i> SC5314. Intestinal segments were prepared as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030119#s4" target="_blank">Materials and Methods</a> and viewed in Fei Nova Nano SEM200 at a distance of 5 µm. Biofilm formation is seen on the intestinal mucosa of mice subjected to hepatectomy (center panel) but not in sham operated mice (left panel) and mice subjected to hepatectomy and drinking phosphate solution (right panel). (B) Biofilm formation for SC5314 at high and low phosphate concentration. Biofilm was evaluated using XTT/menadione method as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030119#s4" target="_blank">Materials and Methods</a>, and normalized to cell density. n = 6, *p<0.01.</p

The effect of mutation in <i>CaGRF10</i> and <i>CaPHO4 on</i> the morphology and lethal effect of prototypic strains BWP17 and SN152.

<p>(A,B) Abundant filamentation is produced by <i>pho4</i>Δ on low phosphate solid medium (A) but not on high phosphate (B). (C) The percentage of filamented colonies in prototypic strains and its mutants <i>grf10</i>Δ and <i>pho4</i>Δ. n = 10 microscopy fields with 20-100 colonies/field, The number of filamented colonies on low Pi medium was significantly higher in SC5314, BWP17, and <i>pho4</i>Δ, *p<0.01, and in BWP17 low Pi as compared to <i>grf10</i>Δ low Pi, **p<0.01. On high phosphate medium, the number of filaments was significantly higher in <i>pho4</i>Δ compared to SN152, ***p<0.01. (D) Kaplan-Meyer survival curves in <i>C. elegans</i>. n = 70/variant, p<0.01 in between <i>pho4</i>Δ and SN152 induced mortality in low Pi medium. (E) Kaplan-Meyer survival curves in mice subjected hepatectomy and cecal injection of BWP17 or its derivative <i>grf10</i>Δ. n = 10/variant, *p<0.01.</p

Expression of <i>PHO4</i> and production of acid phosphatase in response to phosphate limitation.

<p>(A) QRT-PCR analysis demonstrating the fold expression of <i>PHO4</i> in (▪) ICU12 and (□) SN152 growing on low phosphate vs high phosphate solid PNMC medium. n = 3, p<0.01. (B-D) Acid phosphatase activity in (B) SN152, (C) <i>pho4</i>Δ, (D) ICU12 grown in (•) liquid PNMC, 25 µM Pi and in (○) liquid PNMC, 25 mM Pi. n = 3, *p<0.01.</p

Phosphate-dependent filamentation and lethality for <i>C. albicans</i> strains isolated from the stool of critically ill patients (ICU) compared to prototypic strains.

<p>(A, A’) morphological changes in (A) ICU and (A’) prototypic strains on high (25 mM) and low (<0.1 mM) phosphate-supplemented solid PNMC media. Filaments are indicated by arrows. (B, B’) <i>C. elegans</i> killing assay. Kaplan-Meyer survival curves of <i>C. elegans</i> demonstrating the effect of phosphate supplementation on the lethal effect of <i>C. albicans</i>. Dark dotted line, PNMC, 25 mM Pi; light dotted line, PNMC, <0.1mM Pi. (C, C’) Mortality of <i>C. elegans</i> due to avoidance behavior of <i>C. albicans</i>. ▪ PNMC, 25 mM Pi; □ PNMC, <0.1mM Pi. n = 5 worms/plate, 7 plates/experiment, 2-3 independent experiments for each <i>C. albicans</i> strain. Significant differences in survival (B) of <i>C. elegans</i> were demonstrated for <i>C. albicans</i> ICU1 (p<0.01), significant differences in mortality due to avoidance behavior (C) were demonstrated for <i>C. albicans</i> ICU1 and ICU12 (*p<0.01). (D, D’) Mouse model. Kaplan-Meyer survival curves demonstrating the effect of phosphate supplementation on the lethal effect of (D) ICU and (D’) prototypic strains of <i>C. albicans</i>. Black solid line, control (sham operated) mice; light dotted line, mice subjected to 30% hepatectomy; light solid line, mice subjected to 30% hepatectomy and drinking phosphate solution. n = 10 mice/group. p<0.01 for strains ICU1, ICU12, and SC5314.</p

Frequency of cultured microbial isolates.

<p>(A) Organisms from the stool of 7 healthy human volunteers and (B) Organisms from the stool of 15 critically ill patients confined to a care unit (ICU). 100% frequency of isolation indicates that that all patients tested were positive for a given species. Color bars reflect the species isolated at a frequency > 20%, each species has an unique color, and grey-scale bars reflect the species isolated at a frequency of <20%.</p