The presence of bacteria and yeast like fungi in specimens from surgical patients

The aim of this study was to determine the incidence of Candida spp. strains in specimens obtained from surgically treated patients as well as to analyze the accompanying bacterial flora, both aerobic and anaerobic. The material came from two groups of patients. In the first group consisting of patients operated for colon and rectum carcinoma, the samples included peritoneal fluid, colon or rectum bioptates, pus, blood, and wound swabs. In the other group, biopsy material and smears from post operation wounds were taken from patients who underwent a surgical treatment of larynx carcinoma. Altogether, 282 various clinical specimens from 165 patients were analysed, and 41 Candida spp. strains were isolated: 39 strains of C. albicans and 2 strains of C. tropicalis. In 20 out of 41 specimens infected with Candida spp. (48.8%) the co-infection with bacterial aerobic flora was found. In 10 cases (24.4%), the fungi were isolated together with aerobic and anaerobic bacterial flora, whereas in 2 specimens (4.9%) the anaerobes and Candida albicans were diagnosed. The remaining 9 samples showed only the presence of Candida spp. (21.9%). From among aerobic bacterial flora Enterococcus spp. strains (n = 17) and Gram negative rods from Enterobacteriaceae family (n = 13) were the most frequently isolated. The bacterial strains of Streptococcus spp. (n = 5), Pseudomonas spp. (n = 3), Staphylococcus spp. and Corynebacterium spp. (2 strains, both) were identified more rarely. Bacteroides spp. were the most frequent members of bacterial anaerobic flora (n = 10). Other isolated anaerobic bacteria were classified as Fusobacterium spp. or Peptostreptococcus spp. (1 strain each). E. coli and Enterococcus spp. strains of aerobic bacterial flora were more frequently isolated together with Candida spp. CONCLUSIONS: (i) Mixed bacterial flora was found to predominate in the clinical material from the patients after surgery. (ii) Candida spp. were most frequently found together with aerobic bacterial flora.     

院内患者深部真菌感染的流行病学调查

目的 了解长海医院院内患者深部真菌感染的流行病学情况。方法 对2000-2004年间在长海医院住院治疗患者的深部真菌感染进行调查和分析。结果 5年间院内深部真菌感染的发病率上升明显,院内深部真菌感染的主要部位是呼吸道和泌尿道,主要的致病菌为念珠菌。结论 院内深部真菌感染病例近年呈上升趋势。白念珠菌仍是院内深部真菌感染的主要病原菌。     

Candida albicans proteinases and host/pathogen interactions

Candida infections are common, debilitating and often recurring fungal diseases and a problem of significant clinical importance. Candida albicans, the most virulent of the Candida spp., can cause severe mucosal and life-threatening systemic infections in immunocompromised hosts. Attributes that contribute to C. albicans virulence include adhesion, hyphal formation, phenotypic switching and extracellular hydrolytic enzyme production. The extracellular hydrolytic enzymes, especially the secreted aspartyl proteinases (Saps), are one of few gene products that have been shown to directly contribute to C. albicans pathogenicity. Because C. albicans is able to colonize and infect almost every tissue in the human host, it may be crucial for the fungus to possess a number of similar but independently regulated and functionally distinct secreted proteinases to provide sufficient flexibility in order to survive and promote infection at different niche sites. The aim of this review is to explore the functional roles of the C. albicans proteinases and how they may contribute to the host/pathogen interaction in vivo.     

Yeasts from Macrobrachium amazonicum: a focus on antifungal susceptibility and virulence factors of Candida spp

In the present study, it was sought to compare yeast microbiota of wild and captive Macrobrachium amazonicum and evaluate the antifungal susceptibility and production of virulence factors by the recovered isolates of Candida spp. Additionally, cultivation water was monitored for the presence of fungi. Overall, 26 yeast isolates belonging to three genera and seven species were obtained, out of which 24 were Candida spp., with Candida famata as the most prevalent species for both wild and captive prawns. From cultivation water, 28 isolates of filamentous fungi were obtained, with Penicillium spp., Cladosporium spp. and Aspergillus spp. as the most frequent genera. Eight out of 24 Candida spp. isolates were resistant to azole derivatives, out of which four were recovered from wild-harvested prawns. As for production of virulence factors, three (12.5%) and eight (33.3%) isolates presented phospholipase and protease activity, respectively. This is the first comparative study between wild and captive prawns and the first report on yeast microbiota of M. amazonicum. The most relevant finding was the high percentage of resistant Candida spp., including from wild individuals, which suggests the occurrence of an environmental imbalance in the area where these prawns were captured.     

Occurrence ofCandida strains in cases of paronychia

A total of 43 patients, 11 males and 32 females, with paronychia of the fingernails were examined for the presence of Candida spp. The yeast species isolated were identified using standard laboratory methods, including germ-tube production, morphology on rice agar with Tween 80, and mainly fermentation and assimilation of saccharides. In the male group, two Candida species were detected: C. albicans as the dominant species in 9 patients and C. parapsilosis in 2 cases. Similarly, C. albicans was the prevalent species also in females (n = 17); other Candida species detected were C. parapsilosis (n = 7), C. tropicalis (5) and C. krusei (3). In addition to the genus Candida, the following anaerobic and aerobic microorganisms were isolated from patients of both groups: Fusobacterium spp., Bacteroides spp., Staphylococcus aureus, alpha-hemolytic streptococci, group A beta-hemolytic streptococci, Klebsiella pneumoniae, Neisseria spp. and Pseudomonas aeruginosa.     

院内深部真菌感染92例临床分析

目的探讨院内深部真菌感染的临床表现、感染相关因素及诊断治疗措施.方法对1993年1月至2002年12月间经微生物检查证实的92例院内深部真菌感染的病例临床资料进行回顾性分析.结果在92例患者中,肺部疾病26例,糖尿病19例,血液病及肿瘤19例,外科手术后13例,消化系统疾病6例,慢性肾脏病6例,风湿性疾病3例.主要感染真菌为白色念珠菌和酵母菌.感染相关因素:长时间应用广谱抗生素病人65例,应用糖皮质激素22例,应用免疫抑制剂18例,应用气管切开或气管内插管以及中心静脉留置导管17例,进行血液净化治疗的9例.治疗痊愈59例(64.1%),好转10例(10.9%),死亡23例(25%).结论白色念珠菌是院内深部真菌感染的主要致病菌株,有逐年增加趋势,病死率高.与长时间应用广谱抗生素及免疫抑制剂等有相关性.     

The effects of macrophage source on the mechanism of phagocytosis and intracellular survival of Leishmania

Leishmania spp. protozoa are obligate intracellular parasites that replicate in macrophages during mammalian infection. Efficient phagocytosis and survival in macrophages are important determinants of parasite virulence. Macrophage lines differ dramatically in their ability to sustain intracellular Leishmania infantum chagasi (Lic). We report that the U937 monocytic cell line supported the intracellular replication and cell-to-cell spread of Lic during 72?h after parasite addition, whereas primary human monocyte-derived macrophages (MDMs) did not. Electron microscopy and live cell imaging illustrated that Lic promastigotes anchored to MDMs via their anterior ends and were engulfed through symmetrical pseudopods. In contrast, U937 cells bound Lic in diverse orientations, and extended membrane lamellae to reorient and internalize parasites through coiling phagocytosis. Lic associated tightly with the parasitophorous vacuole (PV) membrane in both cell types. PVs fused with LAMP-1-expressing compartments 24?h after phagocytosis by MDMs, whereas U937 cell PVs remained LAMP-1 negative. The expression of one phagocytic receptor (CR3) was higher in MDMs than U937 cells, leading us to speculate that parasite uptake proceeds through dissimilar pathways between these cells. We hypothesize that the mechanism of phagocytosis differs between primary versus immortalized human macrophage cells, with corresponding differences in the subsequent intracellular fate of the parasite.     

HIV-1 and its transmembrane protein gp41 bind to different Candida species modulating adhesion

Oral candidiasis in HIV-1-infected individuals is widely believed to be triggered by the acquired T-lymphocyte immunodeficiency. Recently, binding of the HIV-1 envelope protein gp160 and its subunit gp41, and also of the whole virus itself, to Candida albicans has been shown. The present study shows that, in addition to C. albicans, HIV-1 gp41 also binds to yeast and hyphal forms of Candida dubliniensis, a species which is closely related to C. albicans, and to Candida tropicalis but not to Candida krusei, Candida glabrata or Saccharomyces cerevisiae. The previous finding that gp41 binding to C. albicans augments fungal virulence in vitro is supported by the observation that the yeast showed an enhanced adhesion to HIV-infected H9 cells in comparison to uninfected cells. In line with these results soluble gp41 itself reduced binding of C. albicans to both endothelial and epithelial cell lines, confirming a dominant role of the gp41 binding moiety on the surface of Candida for adhesion. Surface-associated secreted aspartic proteinases (Saps) play an important role in candidial adhesion, but are not likely to be involved in the interaction as gp41 binding to the C. albicans parental wild-type strain was comparable to that of three different isogenic Sap deletion mutants. Furthermore, gp41 binding to the yeast killer toxin-susceptible C. albicans strain 10S was not inhibitable by an anti-YKT receptor antibody. In conclusion, HIV-1 interacts with different clinically important Candida spp., and may thereby affect the outcome of the respective fungal infection.     

Human Pathogenic Fungi in China??Emerging Trends from Ongoing National Survey for 1986, 1996, and 2006

This epidemiological survey was a retrospective study on three nodes during the past three decades on fungal infections representing the China, including Taiwan. Owing to rare publications reporting on dynamic epidemiological trends in the pathogen epidemiology in China, we surveyed the isolation rates and pathogenic fungi from 8 representative districts in China using uniform identification with uniform methodology. The pathogenic fungi isolation rates and species obtained from 1986 (n=9,096), 1996 (n=19,009), and 2006 (n=33,022) suggested that Trichophyton rubrum was the commonest organism cultured in 1980s (45.4%) and 1990s (34.5%), but Candida albicans increased significantly and reaching to its peak (26.9%) in 2006s' survey, and has become the most common isolate of fungal infections in China currently. In addition, Candida glabrata became the most common non-albicans species of Candida in 2006s' survey. At the same time, the incidence of molds also gradually increased. According to comparative analysis of the results of these three surveys, we found apparent differences in the isolation rates of different pathogenic fungi and the forefront 10 species in China varied significantly, and the dermatophytes decreased markedly, while yeasts, especially the Candida species and the molds, increased gradually during the past three decades. Less dermatophytic infections may suggest better access to healthcare or increase in Candida species indicated higher incidence of hospital acquired infections.     

Significance of Molecular Identification and Antifungal Susceptibility of Clinically Significant Yeasts and Moulds in a Global Antifungal Surveillance Programme

The increasing diversity of opportunistic fungi causing serious invasive fungal infections (IFI) has been documented. Accurate identification (ID) is important in guiding therapy, determining prognosis for IFIs and in epidemiological surveys. We assessed the utility of PCR-based methods for the ID of yeasts and moulds that either were uncommon, failed conventional ID, or represented unusual biochemical or phenotypic profiles of common species. Among 1,790 viable fungal clinical isolates received during the SENTRY Program in 2010, 322 strains from 40 study sites had ID confirmed by molecular methods. Isolates were previously identified in participant institutions. Yeasts that were not confirmed by morphology on CHROMagar, growth at 45?°C (Candida albicans/dubliniensis), or assimilation of trehalose (C. glabrata) as well as non-Candida yeasts and all moulds were amplified and sequenced using primers amplifying one or more of the following genes: ITS, 28S, β-tubulin (Aspergillus spp.), TEF (Fusarium spp.), IGS (Trichosporon spp.). The isolates selected for molecular ID included 149 isolates of Candida species, 77 of Aspergillus species, 73 non-Candida yeasts, and 23 other moulds (a total of 41 different species). Overall, the ID determined by the submitting site was confirmed for 189 isolates (58.7?%): Aspergillus spp. (64.1?% correct); Candida spp. (60.1?% correct); non-Candida yeasts (58.9?% correct); non-Aspergillus moulds (30.4?% correct). Species with high levels of concordance between conventional and molecular ID included A. fumigatus (95.0 %), C. lusitaniae (100?%), C. dubliniensis (92.3?%), C. kefyr (100?%), and C. neoformans (90.2?%). Only 50.0?% of isolates of C. albicans and 59.1?% of C. glabrata selected due to unusual phenotypic or biochemical features were found to be correctly identified by the submitting site. Molecular methods for the identification of fungal pathogens are an important adjunct to the conventional identification of many less common clinically relevant yeasts and moulds including species of Candida with unusual or erroneous phenotypic or biochemical profiles. Molecular confirmation of fungal identification is essential in epidemiological surveys such as SENTRY.     

眼部真菌感染菌种分布特征

目的分析眼部真菌感染的致病情况和菌种分布特征。方法统计山东省眼科研究所2000～2005年间所有真菌感染病例的培养结果,按照感染部位分类并分析不同菌种的致病情况。结果6年间共有970份真菌培养资料,阳性结果691份,阳性率为71.2%(691/970)。其中角膜溃疡阳性率为86.2%(669/776),镰刀菌占73.2%(490/669);眼内炎阳性率为10.4%(19/182),曲霉菌占68.4%(13/19);泪囊炎阳性率为25.0%(3/12),念珠菌占100.0%(3/3)。结论不同的感染部位常见的真菌菌种不同,镰刀菌是真菌性角膜溃疡的主要致病菌,曲霉菌是真菌性眼内炎的主要致病菌,而念珠菌则是泪囊炎的主要致病菌。     

Airborne Fungal Colony-Forming Units in Outdoor and Indoor Environments in Yokohama,Japan

The fungal concentration and flora in indoor and outdoor air in Yokohama, Japan were analyzed with a Reuter centrifugal air sampler and dichloran 18% glycerol agar (DG18), and compared with the levels assessed with potato dextrose agar (PDA). The number of fungal colony-forming units (CFU) in outdoor air was < 13–2750/m³; Cladosporium spp. predominated, followed by Alternaria spp. and Penicillium spp. The fungal concentration in outdoor air peaked in September. The concentrations of fungi in outdoor air (n = 288) were significantly correlated with the maximum temperature of the day, minimum temperature of the day, average temperature of the day, average velocity of wind of the day, average temperature of the month, average relative humidity of the month and precipitation of the month. In indoor air, the fungal CFU was < 13–3750/m³. Cladosporium spp. predominated, followed by the xerophilic fungi such as the Aspergillus restrictus group, Wallemia sebi, the A. glaucus group, and Penicillium spp. The fungal concentration in indoor air peaked in October. The concentrations of fungi in indoor air (n = 288) were significantly correlated with the indoor temperature, indoor relative humidity and the outdoor climatic factors mentioned above, except for the average velocity of wind of the day. This revised version was published online in June 2006 with corrections to the Cover Date.     

Active and passive immunization with rHyr1p-N protects mice against hematogenously disseminated candidiasis

We previously reported that Candida albicans cell surface protein Hyr1 encodes a phagocyte killing resistance factor and active vaccination with a recombinant N-terminus of Hyr1 protein (rHyr1p-N), significantly protects immunocompetent mice from disseminated candidiasis. Here we report the marked efficacy of rHyr1p-N vaccine on improving the survival and reducing the fungal burden of disseminated candidiasis in both immunocompetent and immunocompromised mice using the FDA-approved adjuvant, alum. Importantly, we also show that pooled rabbit anti-Hyr1p polyclonal antibodies raised against 8 different peptide regions of rHyr1p-N protected mice in a hematogenously disseminated candidiasis model, raising the possibility of developing a successful passive immunotherapy strategy to treat this disease. Our data suggest that the rabbit anti-Hyr1p antibodies directly neutralized the Hyr1p virulence function, rather than enhanced opsonophagocytosis for subsequent killing by neutrophil in vitro. Finally, the rHyr1p-N vaccine was protective against non-albicans Candida spp. These preclinical data demonstrate that rHyr1p-N is likely to be a novel target for developing both active and passive immunization strategies against Candida infections.     

Virulence phenotypes result from interactions between pathogen ploidy and genetic background

Studying fungal virulence is often challenging and frequently depends on many contexts, including host immune status and pathogen genetic background. However, the role of ploidy has often been overlooked when studying virulence in eukaryotic pathogens. Since fungal pathogens, including the human opportunistic pathogen Candida albicans, can display extensive ploidy variation, assessing how ploidy impacts virulence has important clinical relevance. As an opportunistic pathogen, C. albicans causes nonlethal, superficial infections in healthy individuals, but life‐threatening bloodstream infections in individuals with compromised immune function. Here, we determined how both ploidy and genetic background of C. albicans impacts virulence phenotypes in healthy and immunocompromised nematode hosts by characterizing virulence phenotypes in four near‐isogenic diploid and tetraploid pairs of strains, which included both laboratory and clinical genetic backgrounds. We found that C. albicans infections decreased host survival and negatively impacted host reproduction, and we leveraged these two measures to survey both lethal and nonlethal virulence phenotypes across the multiple C. albicans strains. In this study, we found that regardless of pathogen ploidy or genetic background, immunocompromised hosts were susceptible to fungal infection compared to healthy hosts. Furthermore, for each host context, we found a significant interaction between C. albicans genetic background and ploidy on virulence phenotypes, but no global differences between diploid and tetraploid pathogens were observed.     

In vitro antifungal and anti-elastase activity of some aliphatic aldehydes from Olea europaea L. fruit

Olea europaea preparations are traditionally employed in a variety of troubles, including skin infections. Olive extracts and some of their pure compounds have shown antimicrobial activity in vitro. The present study deals with the antifungal activity of some aliphatic aldehydes from olive fruit [hexanal, nonanal, (E)-2-hexenal, (E)-2-heptenal, (E)-2-octenal, (E)-2-nonenal] against Tricophyton mentagrophytes (6 strains), Microsporum canis (1 strains) and Candida spp. (7 strains). The capability of these substances to inhibit elastase, a virulence factor essential for the dermatophytes colonization, and their cytotoxicity on cultures of reconstructed human epidermis, are also described. Aldehydes tested, inhibited the growth of T. mentagrophytes and M. canis in the range of concentration between <1.9 and 125 microg/ml; the unsaturated aldehydes showed the most broad spectrum of activity in that inhibited all strains tested. None of the aldehydes exhibited activity against Candida spp. strains. (E)-2-octenal and (E)-2-nonenal inhibited the elastase activity in a concentration-dependent manner; the anti-elastase activity suggests an additional target of the antimicrobial activity of these compounds. Aldehydes were devoid of cytotoxicity on cultures of human reconstructed epidermis. The antifungal activity of the aldehydes from olive fruit here reported, substantiates the use of olive and olive oil in skin diseases and suggests that these natural compounds could be useful agents in the topical treatment of fungal cutaneous infections.     

Adaptation of Candida albicans to the host environment: the role of morphogenesis in virulence and survival in mammalian hosts

Although morphological flexibility could be a key contributor to fungal virulence, no molecular data has unambiguously established fungal morphogenesis as a virulence factor for Candida albicans, nor can specific forms of Candida be regarded as absolutely indicative of saprophytism or infection at a given site on the host. The fitness of the fungus in vivo probably reflects its adaptation to the variety of microenvironments in which this opportunist must survive.     

Development of an in vitro model for the multi-parametric quantification of the cellular interactions between Candida yeasts and phagocytes

We developed a new in vitro model for a multi-parameter characterization of the time course interaction of Candida fungal cells with J774 murine macrophages and human neutrophils, based on the use of combined microscopy, fluorometry, flow cytometry and viability assays. Using fluorochromes specific to phagocytes and yeasts, we could accurately quantify various parameters simultaneously in a single infection experiment: at the individual cell level, we measured the association of phagocytes to fungal cells and phagocyte survival, and monitored in parallel the overall phagocytosis process by measuring the part of ingested fungal cells among the total fungal biomass that changed over time. Candida albicans, C. glabrata, and C. lusitaniae were used as a proof of concept: they exhibited species-specific differences in their association rate with phagocytes. The fungal biomass uptaken by the phagocytes differed significantly according to the Candida species. The measure of the survival of fungal and immune cells during the interaction showed that C. albicans was the more aggressive yeast in vitro, destroying the vast majority of the phagocytes within five hours. All three species of Candida were able to survive and to escape macrophage phagocytosis either by the intraphagocytic yeast-to-hyphae transition (C. albicans) and the fungal cell multiplication until phagocytes burst (C. glabrata, C. lusitaniae), or by the avoidance of phagocytosis (C. lusitaniae). We demonstrated that our model was sensitive enough to quantify small variations of the parameters of the interaction. The method has been conceived to be amenable to the high-throughput screening of mutants in order to unravel the molecular mechanisms involved in the interaction between yeasts and host phagocytes.     

Candida albicans secreted aspartyl proteinases in virulence and pathogenesis.

Candida albicans is the most common fungal pathogen of humans and has developed an extensive repertoire of putative virulence mechanisms that allows successful colonization and infection of the host under suitable predisposing conditions. Extracellular proteolytic activity plays a central role in Candida pathogenicity and is produced by a family of 10 secreted aspartyl proteinases (Sap proteins). Although the consequences of proteinase secretion during human infections is not precisely known, in vitro, animal, and human studies have implicated the proteinases in C. albicans virulence in one of the following seven ways: (i) correlation between Sap production in vitro and Candida virulence, (ii) degradation of human proteins and structural analysis in determining Sap substrate specificity, (iii) association of Sap production with other virulence processes of C. albicans, (iv) Sap protein production and Sap immune responses in animal and human infections, (v) SAP gene expression during Candida infections, (vi) modulation of C. albicans virulence by aspartyl proteinase inhibitors, and (vii) the use of SAP-disrupted mutants to analyze C. albicans virulence. Sap proteins fulfill a number of specialized functions during the infective process, which include the simple role of digesting molecules for nutrient acquisition, digesting or distorting host cell membranes to facilitate adhesion and tissue invasion, and digesting cells and molecules of the host immune system to avoid or resist antimicrobial attack by the host. We have critically discussed the data relevant to each of these seven criteria, with specific emphasis on how this proteinase family could contribute to Candida virulence and pathogenesis.     

Discovery of novel indazole-linked triazoles as antifungal agents

The in vitro and in vivo activities of a series of (2R,3R)-2-(2,4-difluorophenyl)-3-(substituted indazol-1-yl)-1-(1H-1,2,4-triazol-1-yl)butan-2-ol as potential antifungal agents are described. In particular, the analog 12j having 5-bromo substitution on the indazole ring exhibited significant antifungal activity against a variety of fungal cultures (Candida spp. and Aspergillus spp.). In addition, oral administration of 12j showed its excellent efficacy against Candida albicans in a murine infection model and the significantly improved survival rates of the infected mice.     

Differential susceptibility of mitogen-activated protein kinase pathway mutants to oxidative-mediated killing by phagocytes in the fungal pathogen Candida albicans

The role of four mitogen-activated protein (MAP) kinase pathways in the survival of Candida albicans following infection of human phagocytes has been addressed through the analysis of mutants defective in their respective MAP kinase. While the contribution of the cell integrity (Mkc1-mediated) or mating (Cek2-mediated) pathways is relatively minor to survival, clear and opposite effects were observed for cek1 and hog1 mutants, despite the fact that these two MAP kinases are important virulence determinants in the mouse model of experimental infection. The Cek1-mediated pathway is involved in sensitivity to phagocyte-mediated killing, while the HOG pathway contributes to the survival of the fungal cells in this interaction. Furthermore, reporter genes have been developed to quantify oxidative and nitrosative stress. hog1 mutants show an oxidative and nitrosative stress response augmented - albeit non-protective - when challenged with oxidants and NO donors in vitro or phagocytic cells (macrophages, neutrophils and the myelomonocytic cell line HL-60), suggesting this as the cause of their reduced virulence in the murine model of infection. These data have important consequences for the development of novel antifungal therapies to combat against fungal infection.