SHOCK, Vol. 49, No. 1, pp. 62–70, 2018
GASTROINTESTINAL COLONIZATION OF CANDIDA ALBICANS INCREASES SERUM (1!3)-B-D-GLUCAN, WITHOUT CANDIDEMIA, AND WORSENS CECAL LIGATION AND PUNCTURE SEPSIS IN MURINE MODEL Wimonrat Panpetch, * † Naraporn Somboonna, †‡ Dewi Embong Bulan, § Jiraphorn Issara-Amphorn, † Navaporn Worasilchai, † Malcolm Finkelman, jj Ariya Chindamporn, † Tanapat Palaga, ‡ Somying Tumwasorn, † and Asada Leelahavanichkul †ô ** *Interdisciplinary
Program of Medical Microbiology, Graduate School, Chulalongkorn University, Bangkok, Thailand; † Department of Microbiology, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand; ‡ Omics Sciences and Bioinformatics Center, Faculty of Science, Chulalongkorn University, Bangkok, Thailand; §Department of Water Resources Management, Faculty of Fisheries and Marine Science, Mulawarman University, Samarinda, Indonesia; jjAssociates of Cape Cod, Inc, East Falmouth, Massachusetts; ôCenter of Excellence in Immunology and Immune-mediated Diseases, Department of Microbiology, Faculty of Medicine, Bangkok, Thailand; and **STAR on Craniofacial and Skeleton Disorders, Faculty of Dentistry, Chulalongkorn University, Bangkok, Thailand Received 9 Feb 2017; first review completed 8 Mar 2017; accepted in final form 3 May 2017
ABSTRACT—The role of intestinal Candida albicans in bacterial sepsis, in the absence of candidemia, was investigated in murine models. Live C albicans or normal saline solution (NSS) was administered orally once, followed by 5 days of daily oral antibiotic-mixtures (ATB). Cecal ligation and puncture (CLP) was then performed to induce sepsis. Fecal Candida was detected by culture only in models with Candida administration. Oral Candida administration with/without ATB enhanced gutpathogenic bacteria as determined by microbiome analysis. Despite negative candidemia, serum (1!3)-b-D-glucan (BG) was higher in CLP with Candida preconditioning models than in CLP-controls (NSS-preconditioning) at 6 and/or 18 h postCLP. Blood bacterial burdens were not increased with Candida administration. Additionally, CLP with high-dose Candida (106 colony forming units) induced higher levels of fecal Candida, serum BG, serum IL-6, and mortality than the lowest dose (100 colony forming units). Interestingly, fluconazole attenuated fecal Candida and improved survival in mice with liveCandida administration, but not in the CLP-controls. Heat-killed Candida preparations or their supernatants reduced bone marrow-derived macrophage killing activity in vitro but enhanced cytokine production. In conclusion, intestinal abundance of fungi and/or fungal-molecules was associated with increased bacterial sepsis severity, perhaps through cytokine storm induction and/or decreased macrophage killing activity. These observations suggest that further investigation of the potential role of intestinal fungal burdens in sepsis is warranted. KEYWORDS—Cecal ligation and puncture, gastrointestinal Candida, sepsis, serum (1!3)-b-D-glucan
INTRODUCTION
molecules results in vigorous systemic inflammation (4). With this background, it is telling that gut permeability barrier failure is reported in sepsis (5, 6). While the significance of guttranslocation of bacterial molecules is appreciated (7), the impact of fungal molecules in bacterial sepsis is unknown. (1!3)-b-D-glucan (BG), a key component of the cell wall in most fungi, has been used as a biomarker of fungal infection. Fungal BG, released from fungi during fungal growth and the tissue invasion process (8), activates immune responses through several receptors (9, 10). Interestingly, higher serum BG from gut-translocation in bacterial sepsis, without fungemia, is associated with greater sepsis severity (6). However, the importance of intestinal fungi in bacterial sepsis, in the absence of fungemia, has not been adequately explored. As C albicans is the predominant fungal species in human intestine but not in mouse (11), we investigated this using murine models in which C albicans was orally administered prior to sepsis induction by cecal ligation and puncture (CLP). The hypotheses investigated included that intestinal colonization with C albicans enhances bacterial sepsis severity through the gut translocation of BG in the absence of candidemia and that intestinal fungi suppression, by fluconazole, attenuates serum BG, mortality, and severity of inflammation as judged by serum IL-6.
A complex balance of host pro- and anti-inflammatory responses to pathogens has been established (1). Sepsis is a syndrome of dysregulated host responses to systemic infection, independent of the organisms, resulting in organ dysfunction (2). Sepsis is the most common cause of death among patients in intensive care unit (3) and is a critically important worldwide health-care problem. Intestinal bacteria are the main sources of pathogen-associated molecular patterns (PAMPs) capable of immune activation, are essential for host immunity development, and gut-translocation of viable bacteria or bacterial Address reprint requests to Asada Leelahavanichkul, MD, PhD, Department of Microbiology, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand. E-mail:
[email protected] AL worked under Center of Excellence in Immunology and Immune-mediated Diseases, Department of Microbiology, Faculty of Medicine, Bangkok, Thailand. WP, NS, ST, and AL contributed equally to this work. This study was funded by grants from Chulalongkorn University including Ratchadapiseksompotch Fund, Faculty of Medicine (RA60/035); Grant for Development of New Faculty Staff and Thailand Government Fund. WP was supported by Thailand Research Fund through the Royal Golden Jubilee PhD Program (PHD/ 0316/2552). BG assays were partly supported by Associates of Cape Cod, Inc The authors report no conflicts of interest. DOI: 10.1097/SHK.0000000000000896 Copyright ß 2017 by the Shock Society
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SHOCK JANUARY 2018
GASTROINTESTINAL CANDIDA WORSEN BACTERIAL SEPSIS
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Cecal ligation and puncture at 5 days postoral administration of Candida albicans (CLP with 5 day Candida colonization model) All patients with sepsis receive antibiotics, an intervention that may enhance intestinal C albicans colonization (13). Oral administration of Candida, with antibiotics, but not antibiotic administration alone, has been shown to lead to significant intestinal Candida overgrowth in mice (14). Hence, an animal model of Candida colonization with antibiotic-administration should more closely resemble the septic patient. We administered a single dose of C albicans at 1 106, 1 104, or 1 102 CFU together with daily antibiotic-cocktails containing gentamicin (3.5 mg/kg), colistin (4.2 mg/kg), metronidazole (21.5 mg/kg), and vancomycin (4.5 mg/kg) (separately purchased from Sigma-Aldrich, St Louis, Mo), by twice-daily gavage, for 5 days prior to CLP surgery. To attempt to influence serum (1!3)-b-D-glucan (BG) burdens, due to growth of Candida, fluconazole, (Sigma-Aldrich, St Louis, Mo) at 10 mg/kg in 0.5 mL of NSS or NSS alone was administered orally at 3 and 0.5 h preoperation and at 6 h postCLP. Of note, fluconazole at lower doses and exposure frequency did not reduce fecal Candida in our pilot experiments (data not shown). A high dose of fluconazole was shown to be necessary for the reduction of fecal fungi in a previously described Candida-administered mouse model (15). FIG. 1. Survival analysis of mice with cecal ligation and puncture (CLP) at 3 h after different oral administration doses of Candida albicans (n ¼ 7/group). Fungemia was demonstrated only with Candida administration at 11010 CFU. (*, P < 0.001 vs. NSS alone; (), % of mice with fungemia at 18 h of CLP). NSS indicates normal saline solution.
METHODS Candida albicans preparation Candida albicans ATCC 90028 (Fisher Scientific, Waltham, Mass), a fluconazole susceptible strain (minimal inhibitory concentration 0.25 mg/mL to 1 mg/mL) was used. C albicans was cultured overnight on Sabouraud dextrose broth (Thermo Scientific, Hampshire, UK) and counted in a hemocytometer (Bright-Line, Denver, Colo) before use. Heat-killed C albicans was prepared by immersion in a water bath at 608C for 1 h. For the in vitro experiments, C albicans lysate was prepared by vigorous sonication of heat-killed cells (Sonics Vibra Cell, VCX 750, Sonics and Materials Inc, Newtown, Conn) until a homogenous solution was formed. Both well-mixed complete homogenate and the supernatant after centrifugation of the preparation were used as heat-killed C albicans and lysate, respectively. BG titers >523 pg/mL were established for both the heat-killed C albicans and the cell lysate, demonstrating that administration of these preparations also included measurable BG.
Animals and animal models The US National Institutes of Health animal care and use protocols (#85-23, revised 1985) were followed. Male, 8-week-old, ICR mice (National Laboratory Animal Center, Nakhornpathom, Thailand) were used. Only male mice were selected due to the gender difference in sepsis severity (12). The animal protocols were approved by the Institutional Animal Care and Use Committee of the Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand.
Cecal ligation and puncture at 3 h after oral administration of Candida albicans The oral administration of high-dose Candida might induce candidemia after CLP surgery. To see the effect of intestinal Candida, in the absence of candidemia, upon bacterial sepsis, several oral dose levels of live C albicans, 1 104, 1 106, and 1 1010 colony forming units (CFU), were administered to each group of mice at 3 h prior to CLP surgery. CLP procedures were slightly modified from the previous publication (6). Briefly, cecum was ligated at 10 mm from cecal tip with silk 2-0 and then punctured twice with a 21-gauge needle through an abdominal incision under isoflurane anesthesia. Postoperative fentanyl, consisting of 0.03 mg/kg in 0.5 mL of normal saline solution (NSS), was administered subcutaneously for analgesia and fluid replacement immediately after CLP, and repeated 6 h later. Antibiotic [imipenem/cilastatin, 14 mg/kg in 0.3 mL of NSS] was administered subcutaneously at 6 h post-surgery. Subsequent to these experiments, live C albicans oral administration at 1 106 CFU was selected for further investigation due to the absence, at this dosage, of concomitant candidemia after CLP surgery (Fig. 1).
Mouse blood sample analysis Blood (50 mL) was collected through tail vein nicking at the indicated timepoint and with cardiac puncture under isoflurane anesthesia at sacrifice. For quantitative bacterial analysis of blood, 25 mL of blood was spread directly onto blood agar plates (Oxoid, Hampshire, UK), kept at 378C under aerobic conditions, and bacterial colonies were enumerated at 48 h. The colonies were speciated by standard biochemical tests. The rest of the blood was centrifuged to separate serum and kept at 808C until analysis. Serum creatinine (Scr) and alanine transaminase (ALT) was measured with QuantiChrom Creatinine Assay kit (DICT-500, Bioassay, Hayward, Calif) and EnzyChrom Alanine Transaminase Assay kit (EALT-100, BioAssay, Hayward, Calif), respectively. Serum cytokines (TNF-a, IL-6, and IL-10) were measured with ELISA (ReproTech, NJ). BG was analyzed with Fungitell (Associates of Cape Cod Inc, East Falmouth, Mass). BG values at 523.4 pg/mL (beyond the lower and upper ranges of the standard curve) were recorded as 0 pg/mL and 523 pg/mL, respectively. All assays were performed according to the manufacturer’s protocol.
Culture of fecal fungi Feces were collected by placing an individual mouse in an empty cage for 0.5 to 1 h before CLP for the 0 h time-point. At 6 and 18 h post CLP, mice were sacrificed and feces from descending colon and/or rectum were collected. Feces were well-mixed with phosphate buffer solution (PBS) in a ratio of 1 mg/ 1 mL before plating directly in sabouraud dextrose agar with 0.1% chloramphenicol (Thermo Scientific) kept at 358C for 72 h for fungal colony enumeration.
Fecal microbiome The microbiota analysis protocol was performed as previously reported (16). In short, feces from individual mice were used for metagenomic DNA extractions, with three independent extractions of 0.25 g performed per sample. The DNA Isolation Kit (MoBio, Carlsbad, Calif) was used to extract total nucleic acid. Metagenomic DNA quality was assessed by agarose gel electrophoresis and nanodrop spectrophotometry. Universal prokaryotic 515F (forward; (50 GTGCCAGCMGCCGCGGTAA-30 ) and 806R (reverse; 50 -GGACTACHVGGGTWTCTAAT-30 ), with appended 50 Illumina adapter and 30 Golay barcode sequences, were used for 16S rRNA gene V4 library construction (16). Independent triplicate PCR experiments were performed and pooled to prevent stochastic PCR bias. The 16S rDNA amplicons of 381 base pairs (bp) were purified by the GenepHlow Gel Extraction Kit (Geneaid Biotech Ltd, New Taipei City, Taiwan), and quantified with Picogreen (Invitrogen, Eugene, Ore). Samples, at 240 ng, were pooled for sequencing by Miseq300 platform (Illumina, San Diego, Calif) (17). Raw sequences were quality screened by Mothur’s MiSeq platform procedures (18). Quality screening steps including the removal of reads that have ambiguous bases, >1 mismatch in the reverse primer sequence, >10 homopolymers, a minimum quality score of 3)-beta-D-glucans in health promotion and disease alleviation. Crit Rev Biotechnol 25(4):205–230, 2005. 28. Jang HR, Gandolfo MT, Ko GJ, Satpute S, Racusen L, Rabb H: Early exposure to germs modifies kidney damage and inflammation after experimental ischemiareperfusion injury. Am J Physiol Renal Physiol 297(5):F1457–F1465, 2009. 29. Wen K, Li G, Bui T, Liu F, Li Y, Kocher J, Lin L, Yang X, Yuan L: High dose and low dose Lactobacillus acidophilus exerted differential immune modulating effects on T cell immune responses induced by an oral human rotavirus vaccine in gnotobiotic pigs. Vaccine 30(6):1198–1207, 2012. 30. Samonis G, Gikas A, Toloudis P, Maraki S, Vrentzos G, Tselentis Y, Tsaparas N, Bodey G: Prospective study of the impact of broad-spectrum antibiotics on the yeast flora of the human gut. Eur J Clin Microbiol Infect Dis 13(8):665–667, 1994. 31. Eggimann P, Pittet D: Candida colonization index and subsequent infection in critically ill surgical patients: 20 years later. Intensive Care Med 40(10):1429– 1448, 2014. 32. Hoffman OA, Olson EJ, Limper AH: Fungal beta-glucans modulate macrophage release of tumor necrosis factor-alpha in response to bacterial lipopolysaccharide. Immunol Lett 37(1):19–25, 1993. 33. Seong SK, Kim HW: Potentiation of innate immunity by beta-Glucans. Mycobiology 38(2):144–148, 2010. 34. Geijtenbeek TB, Gringhuis SI: Signalling through C-type lectin receptors: shaping immune responses. Nat Rev Immunol 9(7):465–479, 2009. 35. Kikkert R, Bulder I, de Groot ER, Aarden LA, Finkelman MA: Potentiation of Toll-like receptor-induced cytokine production by (1–>3)-beta-D-glucans: implications for the monocyte activation test. J Endotoxin Res 13(3):140– 149, 2007.
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