Robb M. Garni

Comparative virulence of Candida albicans yeast and filamentous forms in orally and intravenously inoculated mice.

ObjectiveCandida albicans, a dimorphic fungus that switches from yeast to filamentous forms, is a major cause of complicating systemic infection in intensive care patients. The aim of this study was to compare the pathogenic potential of C. albicans yeast and filamentous forms. DesignSeparate groups of mice were inoculated either intravenously or orally with C. albicans CAF2 (wild type), HLC54 (yeast forms defective in filament formation), or BCa2-10 (constitutively filamentous). Mice were killed 1, 7, 14, and 21 days after intravenous C. albicans and kidneys and liver were quantitatively cultured; cohort groups were observed for mortality. Mice were pretreated with antibiotics for 3 days before oral inoculation with C. albicans, and killed 3 days later with dexamethasone administered for the latter 3 days; at sacrifice, the mesenteric lymph nodes and kidneys were cultured to monitor extraintestinal dissemination of C. albicans. SettingUniversity teaching hospital research laboratory. SubjectsFemale, Swiss Webster, adult mice. Measurements and Main ResultsIn intravenously inoculated mice, mortality was highest with wild-type C. albicans CAF2 (92%), intermediate with HLC54 (56%), and not detected with constitutively filamentous BCa2-10 (0%); BCa2-10 was cleared from the kidney and liver, but CAF2 and HLC54 were recovered at approximately 105–7/g kidney and 104–5/g liver. There was only occasional mortality in orally inoculated mice and the numbers of cecal C. albicans CAF2 and HLC54 were similarly high (approximately 10/g), whereas numbers of cecal BCa2-10 were at least 100-fold lower. Extraintestinal dissemination was greatest with HLC54, intermediate with CAF2, and undetectable with BCa2-10. ConclusionsOf the three C. albicans strains studied, wild-type CAF2 was most virulent in intravenously inoculated mice and HLC54 (defective in filament formation) was most virulent in orally inoculated mice. The constitutively filamentous BCa2-10 was avirulent in both models, suggesting that filamentous forms by themselves might not be critically important for C. albicans virulence.

Cecal Colonization and Systemic Spread of Candida albicans in Mice Treated with Antibiotics and Dexamethasone

Infections with Candida albicans have become a significant problem among very low birth weight infants in the neonatal intensive care unit. Risk factors are multiple and include administration of antibiotics and glucocorticoids, such as dexamethasone. Experiments were designed to study the combined effect of oral broad-spectrum antibiotics and parenteral dexamethasone on cecal colonization and extraintestinal dissemination of C. albicans in separate groups of mice that were orally inoculated with one of four C. albicans strains that were either wild-type INT1/INT1 or had one or more disruptions of the INT1 gene. Intestinal colonization was monitored by quantitative culture of the mouse cecum, and extraintestinal invasion was monitored by quantitative culture of the draining mesenteric lymph nodes and kidneys. At sacrifice, the average numbers of cecal C. albicans differed from 7.7 log10/g to 6.7 log10/g (p < 0.01) in mice orally inoculated with C. albicans containing two functional copies of INT1 and no functional copies of INT1, respectively. The incidence of extraintestinal dissemination to mesenteric lymph nodes and kidneys correspondingly varied from 57 to 13% (p < 0.01) and 83 to 4% (p < 0.01) in mice inoculated with these two C. albicans strains. Mice orally inoculated with C. albicans containing one functional copy of INT1 had intermediate levels of cecal colonization and extraintestinal dissemination. Thus, cecal colonization and extraintestinal dissemination of C. albicans was facilitated in antibiotic-treated mice given dexamethasone. In addition, the presence of two functional copies of the INT1 gene was associated with the greatest levels of cecal colonization and extraintestinal dissemination of C. albicans.

Gastrointestinal colonization by Candida albicans mutant strains in antibiotic-treated mice.

ABSTRACT Antibiotic-treated mice orally inoculated with one of threeCandida albicans strains (including two mutant strains) or indigenous Candida pelliculosa showed levels of candidal gastrointestinal colonization that were strain specific. However, regardless of strain, the numbers of viable candida were intermediate to high in the stomach, were consistently lowest in the upper small intestine, and increased progressively down the intestinal tract.

Role of heparan sulfate in interactions of Listeria monocytogenes with enterocytes

Abstract. Heparan sulfate is known to participate in binding a wide variety of microbes to mammalian cells, but few studies have focused on the enterocyte. Normal human colonic and small intestinal enterocytes, and cultured HT-29 (but not Caco-2) enterocytes, reacted prominently with antibodies specific for heparan sulfate and for the core protein of syndecan-1 (a heparan sulfate proteoglycan). The heparan sulfate analog, heparin, inhibited interactions of Listeria monocytogenes (adherence and internalization) with HT-29, but not Caco-2, enterocytes. Internalization of L. monocytogenes by HT-29 enterocytes was inhibited by heparan sulfate and to a lesser extent by chondroitin sulfate, but not by the non-sulfated glycosaminoglycan hyaluronic acid. Compared to plasmid control ARH-77 cells, adherence of L. monocytogenes, was increased using ARH-77 cells transfected with syndecan-1 cDNA. Heparin binding protein(s) on L. monocytogenes were confirmed using biotinylated heparin. To determine if these in vitro observations might have in vivo relevance, L. monocytogenes was preincubated with heparin and then orally inoculated into mice. Compared to L. monocytogenes not pretreated with heparin, L. monocytogenes pretreated with heparin was associated with decreased extraintestinal dissemination to the mesenteric lymph nodes and liver of orally inoculated mice. Thus, heparan sulfate (possibly as the heparan sulfate proteoglycan syndecan-1) appears to participate in interactions of L. monocytogenes with enterocytes.

Hypoxia and extraintestinal dissemination of Candida albicans yeast forms.

Candida albicans is a pleomorphic fungus with budding yeast and filamentous forms, and is a frequent cause of complicating infections in patients who are postsurgical, in shock, and have trauma. Many cases of systemic candidiasis are thought to originate from the intestine, but it is unclear if the filament or the yeast is the more invasive form. Because C. albicans is relatively noninvasive and because mesenteric ischemia is thought to facilitate extraintestinal microbial dissemination, wild-type C. albicans CAF2 and mutant HLC54 (defective in filament formation) were orally inoculated into antibiotic-treated mice that were housed exclusively in room air, or were intermittently exposed to 10% oxygen for 1-h intervals. Both strains of C. albicans colonized the cecum in similar numbers (approximately 106.7/g). C. albicans translocation to the draining mesenteric lymph nodes was not detected in mice inoculated with CAF2 (normoxic or hypoxic) or in normoxic mice inoculated with HLC54, but was detected in 33% (P < 0.01) of hypoxic mice inoculated with HLC54. Using Caco-2 and HT-29 enterocytes cultivated on plastic dishes and pretreated for 48 h in 10% oxygen, adherence of C. albicans HLC54 was decreased compared with wild-type CAF2, and hypoxia had no noticeable effect on adherence of either CAF2 or HLC54. Using enterocytes cultivated on permeable 8-&mgr;m filters, transepithelial migration of C. albicans CAF2 and HLC54 appeared similar. Thus, C. albicans HLC54 (defective in filament formation) was more invasive in hypoxic mice compared with wild-type CAF2, and host factors (e.g., mesenteric ischemia) rather than an innate ability to interact with enterocytes might play a more important role in extraintestinal dissemination of C. albicans yeast forms.

Effect of lipopolysaccharide on virulence of intestinal candida albicans1

BACKGROUND Candida albicans is a polymorphic fungus that frequently causes systemic infection in postsurgical and trauma patients. Others have reported that Escherichia coli lipopolysaccharide (LPS) acts as a copathogen to enhance the virulence of parenteral C. albicans. Experiments were designed to clarify the effect of parenteral LPS on systemic candidiasis initiated via the oral route. MATERIALS AND METHODS Antibiotic-treated mice were orally inoculated with C. albicans CAF2 (wild-type) or mutant HLC54 (defective in filament formation), and were given 100 microg parenteral LPS 16 h before sacrifice. Separate groups of mice were additionally exposed to intermittent hypoxia prior to LPS. At sacrifice, cecal flora and microbial translocation to the mesenteric lymph nodes were quantified. C. albicans adherence to cultured HT-29 and Caco-2 enterocytes (pretreated with LPS, or calcium-free medium to expose the enterocyte lateral surface, or both) was quantified by enzyme-linked immunoabsorbent assay. RESULTS All mice had high numbers of cecal C. albicans, and LPS was associated with an additional increase in cecal concentrations of HLC54 but not CAF2. Translocation of HLC54, but not CAF2, appeared facilitated by hypoxia, but LPS did not facilitate translocation in any treatment group. Exposure of the lateral surface of cultured enterocytes had no effect on C. albicans adherence, although LPS consistently decreased adherence of both C. albicans strains. CONCLUSIONS In contrast to experiments where systemic candidiasis was initiated by the parenteral route, parenteral LPS did not act as a copathogen in mice with systemic candidiasis initiated by the oral route, and these results might be related to LPS-induced alterations in C. albicans adherence to host enterocytes.

Escherichia coli modulates extraintestinal spread of Staphylococcus aureus

Staphylococcus aureus remains one of the most frequent causes of life-threatening systemic infection in surgical and trauma patients. It is understood that S. aureus colonization predisposes to complicating infection, but extraintestinal dissemination of S. aureus from the intestinal lumen to the draining mesenteric lymph nodes has not been systematically studied. After oral inoculation with high numbers of S. aureus, otherwise normal mice had low levels of cecal S. aureus (6.7 log10/g) and the incidence of extraintestinal dissemination was 30%. As expected, parenteral Escherichia coli lipopolysaccharide (LPS) was associated with increased numbers of cecal S. aureus, but the incidence of translocation remained unchanged. Purified LPS had no effect on S. aureus internalization by cultured HT-29 enterocytes and no effect on S. aureus transmigration through confluent enterocytes. To begin to clarify the effect of alterations in cecal bacteria on S. aureus translocation, mice were orally inoculated with E. coli and S. aureus. Compared with mice inoculated with S. aureus alone, these mice had increased numbers of cecal E. coli and S. aureus, and the incidence of S. aureus translocation nearly doubled from 46% to 88%. Experiments with HT-29 enterocytes indicated that viable E. coli had no effect on S. aureus internalization, but viable E. coli was at least 40 times more potent in inducing S. aureus transmigration across confluent enterocytes compared with a corresponding amount of purified LPS. Thus, S. aureus disseminated from the intestinal tract of normal mice by a mechanism that could involve paracellular migration across the intestinal epithelial barrier.

Comparative abilities of Candida glabrata and Candida albicans to colonize and translocate from the intestinal tract of antibiotic-treated mice

Candida albicans is the primary cause of candidemia in hospitalized patients, and the intestinal tract is considered the source of most systemic infections. C. glabrata has emerged as the second or third most frequent cause of candidemia, but little is known about its epidemiology and pathogenesis. Our goal was to compare the intestinal colonization and extra-intestinal dissemination of C. glabrata and C. albicans (wild type and filamentation-defective mutant). Mice were pretreated with antibacterial agents to alter their resident microflora, and then orally inoculated with C. glabrata and/or C. albicans. Elimination of detectable cecal bacteria facilitated colonization with both Candida species. Selective elimination of aerobic/facultative gram-negative bacilli did not noticeably affect Candida colonization, but Escherichia coli overgrowth inhibited colonization. In all situations, C. glabrata colonized the cecum equally well or better than C. albicans, and the ability of C. albicans to form filaments did not facilitate colonization. In vitro generation times had little relevance to the resulting cecal population levels of C. glabrata and C. albicans, and neither species readily disseminated to mesenteric lymph nodes. Thus, like C. albicans, the intestinal tract may be an epidemiological reservoir for C. glabrata and antibiotic-induced alterations in intestinal bacteria may facilitate colonization.

103 COMPARATIVE VIRULENCE OF CANDIDA ALBICANS AND CANDIDA PARAPSILOSIS IN A MOUSE MODEL OF SYSTEMIC INFECTION.

Background Candida species are important nosocomial pathogens in the neonatal intensive care unit (NICU), resulting in a relatively high rate of morbidity and mortality. While Candida albicans is the most frequently isolated species and considered most pathogenic, Candida parapsilosis accounts for approximately 25% of nosocomially acquired candidiasis on the NICU—a situation unique to the neonate. Unlike C. albicans, the epidemiology and virulence of C. parapsilosis have not been systematically evaluated. Objective To compare the virulence of C. parapsilosis and C. albicans, as well as to evaluate the pattern of organ involvement, following systemic infection in the mouse. Design/Methods Separate groups of mice were inoculated intravenously (IV) with 108 C. parapsilosis, 108 C. albicans, or 105 C. albicans and monitored for mortality and morbidity. Mortality was determined by observation for 14 days following inoculation (n = 12/treatment, in each of two experiments). To determine morbidity, a separate series of mice were sacrificed on days 1, 7, and 14 following intravenous inoculation, with liver and kidneys cultured for viable microbes. (n = 4-5 mice/treatment/day, in each of three experiments). Results Mortality was highest following IV inoculation with 108 C. albicans (> 50% by day 14). Significantly fewer mice died following injection with 108 C. parapsilosis and 105 C. albicans (≈8% by day 14, p < .05). The inoculated Candida species was recovered from the liver in ≈65% of each treatment group on day 1, with clearance of the yeast by day 14 in the surviving mice (p < .01). On day 1 following inoculation, respective organisms were recovered from the kidneys of ≈65% of mice inoculated with 108 C. parapsilosis or 108 C. albicans and 43% of mice inoculated with 105 C. albicans. Approximately 40% of surviving mice inoculated with 108 C. parapsilosis or 105 C. albicans showed persistence of the yeast in the kidney by day 14 (104.4-4.9/g tissue). Surviving mice inoculated with 108 C. albicans showed persistence of the organism in the kidneys to day 7 (105 /g tissue); however, no mice survived to sacrifice on day 14 following this inoculation. Conclusions C. albicans is more virulent than C. parapsilosis when the outcome variable is death. However, the two organisms are equally virulent with respect to the ability to cause persistent infection in the kidney following IV inoculation.