Pan-Genome-Wide Association Study reveals a key role of the salmochelin receptor IroN in the biofilm formation of Salmonella Typhimurium and its monophasic variant 4,[5],12:i:.

Salmonella enterica subsp. enterica serovar Typhimurium variant 4,[5],12:i:- (so called S. 4,[5],12:i:-) has rapidly become one of the most prevalent serovars in humans in Europe, with clinical cases associated with foodborne from pork products. The mechanisms, genetic basis and biofilms relevance by which S. 4,[5],12:i:- maintains and spreads its presence in pigs remain unclear. In this study, we examined the genetic basis of biofilm production in 78 strains of S. 4,[5],12:i:- (n = 57) and S. Typhimurium (n = 21), from human gastroenteritis, food products and asymptomatic pigs. The former showed a lower Specific Biofilm Formation index (SBF) and distant phylogenetic clades, suggesting that the ability to form biofilms is not a crucial adaptation for the S. 4,[5],12:i:- emerging success in pigs. However, using a pan-Genome-Wide Association Study (pan-GWAS) we identified genetic determinants of biofilm formation, revealing 167 common orthologous groups and genes associated with the SBF. The analysis of annotated sequences highlighted specific genetic deletions in three chromosomal regions of S. 4,[5],12:i:- correlating with SBF values: i) the complete fimbrial operon stbABCDE widely recognized as the most critical factor involved in Salmonella adherence; ii) the hxlA, hlxB, and pgiA genes, which expression in S. Typhimurium is induced in the tonsils during swine infection, and iii) the entire iroA locus related to the characteristic deletion of the second-phase flagellar genomic region in S. 4,[5],12:i:-. Consequently, we further investigated the role of the iro-genes on biofilm by constructing S. Typhimurium deletion mutants in iroBCDE and iroN. While iroBCDE showed no significant impact, iroN clearly contributed to S. Typhimurium biofilm formation. In conclusion, the pan-GWAS approach allowed us to uncover complex interactions between genetic and phenotypic factors influencing biofilm formation in S. 4,[5],12:i:- and S. Typhimurium.

Characterization of a monoclonal antibody directed against Salmonella enterica serovar Typhimurium and serovar [4,5,12:i:-].

Flagellar extracts of Salmonella enterica serovars expressing phase 2 H1 antigenic complex (H:1,2, H:1,5, H:1,6, and H:1,7) and a mutant flagellin obtained by site-directed mutagenesis of the fljB gene from serovar Typhimurium at codon 218, transforming threonine to alanine, expressed in Escherichia coli (fljB218(A)) were used to analyze the H1 antigenic complex. Cross-reactions were detected by Western blotting and dot blotting using commercial polyclonal antibodies against the different wild-type extracts and mutant FljB218(A). Therefore, we produced a monoclonal antibody (MAb), 23D4, isotyped as immunoglobulin M, against H:1,2 S. enterica serovar Typhimurium flagellin. The mutant flagellin was not recognized by this MAb. When a large number of phase 1 and phase 2 flagellin antigens of different serovars were used to characterize the 23D4 MAb, only extracts of serovars Typhimurium and [4,5,12:i:-] reacted. The protein composition of phase 1 and phase 2 extracts and highly purified H:1,2 flagellin from serovar Typhimurium strain LT2 and extract of strain 286 (serovar [4,5,12:i:-]), which reacted with the MAb, was studied. Phase 2 flagellin (FljB(H:1,2)) was detected in phase 1 and phase 2 flagellar heat extracts of serovar Typhimurium and was the single protein identified in all spots of purified H:1,2 flagellin. FliC, FlgK, and other proteins were detected in some immunoreactive spots and in the flagellar extract of serovar [4,5,12:i:-]. Immunoelectron microscopy of complete bacteria with 23D4 showed MAb attachment at the base of flagella, although the MAb failed to recognize the filament of flagella. Nevertheless, the results obtained by the other immunological tests (enzyme-linked immunosorbent assay, Western blotting, and dot blotting) indicate a reaction against flagellins. The epitopes could also be shared by other proteins on spots where FljB is not present, such as aminopeptidase B, isocitrate lyase, InvE, EF-TuA, enolase, DnaK, and others. In conclusion, MAb 23D4 can be useful for detection and diagnostic purposes of S. enterica serovar Typhimurium and serovar [4,5,12:i:-] and could be also helpful for epitope characterization of flagellum-associated antigens.

Serological differentiation of experimentally induced Candida dubliniensis and Candida albicans infections.

Using a rabbit model of systemic infection, we show that it is possible to differentiate infections caused by Candida dubliniensis and other Candida species by detecting the antibody response mounted by the infected animals. These results confirm our previous observation in a patient with C. dubliniensis candidemia and suggest that detection of C. dubliniensis-specific antibodies is useful in the diagnosis of invasive candidiasis caused by this yeast.

Influence of environmental pH on the reactivity of Candida albicans with salivary IgA.

Salivary secretory IgA reacts with a group of heat-shock mannoproteins preferentially expressed on Candida albicans yeast cells and germ tubes grown at 37 degrees C. Since other environmental factors can also modulate the expression of those antigens, we have investigated the influence of the pH of the culture medium on the expression of the antigens reacting with human salivary IgA by C. albicans. By indirect immunofluorescence, yeast cells grown in Sabouraud glucose broth at 37 degrees C showed a statistically significant increase in reactivity with salivary IgA (p < 0.0001) when compared with cells grown at 25 degrees C at the 4 pH values studied (3.3, 5.9, 7.5, and 9.5), the highest reactivity and the major heat-shock effect being observed at pH 5.9. The decrease in reactivity with salivary IgA observed in C. albicans cells grown at pH values of 3.3 and 9.5 was confirmed by Western blotting. Salivary IgA reacted with polydispersed materials from the cell walls of molecular masses > 55 kDa, which were more expressed at neutral pH than at acidic or alkaline pH values. A similar reactivity was observed when the antigenic extracts were stained with an antiserum directed against oligosaccharides present in antigen 6 of C. albicans serotype A. The differences in reactivity presented by salivary IgA may be related to a decrease in the expression of polysaccharides present on the surfaces of the yeast cells of C. albicans grown at acidic or alkaline pH values. The low reactivity of salivary IgA with C. albicans cells grown at acidic pH values may help to explain the association between acidic saliva and the carriage of Candida in the oral cavity, as well as with oral candidiasis.

Cell surface proteins of Candida albicans: preparation of extracts and improved detection of proteins.

We have reexamined the detection of the components in a beta-mercaptoethanol and ammonium carbonate buffer extract of surface proteins of Candida albicans and the effects of postextraction manipulation of the extract on recovery of extract components. Following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), preferential staining of some moieties was observed when bands detected by a commercial silver staining method or a Coomassie Brilliant Blue (CBB) staining method were compared. Additional protein bands that were either not detected or poorly detected by a single method alone were readily observed by a combined silver-CBB staining method. This method also detected alterations in the profile of extracted proteins from organisms grown in the presence of galactose or hemoglobin rather than glucose. Two-dimensional electrophoresis (2-DE) gel analysis by double stain showed better detection of several acidic and basic protein spots. Less than 10% of the extract as determined by a dye-binding assay was lost following either or both lyophilization and dialysis. These manipulations of the extract did not change the protein profile following SDS-PAGE as determined by the combined staining or Western blot analysis of a 70 kDa protein. These observations suggest that soluble cell wall proteins are not unusually sensitive to procedures routinely used in protein purification. In addition, these studies suggest that a modified staining method that combines both silver stain and CBB stain provides improved detection of cell wall proteins compared to either method alone.

Interactions between Candida albicans and the human extracellular matrix component tenascin-C.

Tenascins are large multimeric proteins that contain repeated structural motifs that include epidermal growth factor (EGF)-like repeats, fibronectin type III repeats and a globular fibrinogen-like domain, and are involved in tissue and organ morphogenesis, as well as in adhesion and migration of cells. C. albicans germ-tubes, but not blastospores, were able to bind to soluble human tenascin-C as revealed by an indirect immunofluorescence assay. However, materials present in cell wall extracts from both morphologies attached to tenascin-C immobilized in wells of a microtiter plate. The binding specificity was demonstrated by the inhibitory effect of antibodies against C. albicans cell wall components and an anti-tenascin antibody, but not anti-laminin antibody. Fibronectin, but not fibrinogen, inhibited binding, thus indicating a role of the fibronectin type III repeats in the interaction between the fungus and tenascin-C. Binding of C. albicans cell wall materials to tenascin was RGD- and divalent cation-independent.

Rapid identification of Candida dubliniensis by indirect immunofluorescence based on differential localization of antigens on C. dubliniensis blastospores and Candida albicans germ tubes.

There is a clear need for the development of a rapid and reliable test for the identification of Candida dubliniensis and for the discrimination of this species from Candida albicans. In the present study we have investigated the potential use of C. dubliniensis-specific antigens as a basis for its identification. We produced an anti-C. dubliniensis serum which, after adsorption with C. albicans blastospores, was found to differentially label C. dubliniensis isolates in an indirect immunofluorescence test. In this test, the antiserum reacted with blastospores and germ tubes of C. dubliniensis and with blastospores of Candida krusei and Rhodotorula rubra but did not react with blastospores of several other Candida species including C. albicans. The antiserum also reacted with C. albicans germ tubes. The anti-C. dubliniensis adsorbed serum reacted with specific components of 25, 28, 37, 40, 52, and 62 kDa in the C. dubliniensis extract and with a variety of antigens from other yeast species. The antigens from non-C. dubliniensis yeasts showing reactivity with the anti-C. dubliniensis adsorbed serum are mostly expressed within the cell walls of these yeast species, and this reactivity does not interfere with the use of the anti-C. dubliniensis adsorbed serum in an indirect immunofluorescence test for the rapid identification of C. dubliniensis.

Detection of antibodies to Candida albicans germ tubes during experimental infections by different Candida species.

Identification and characterization of Candida albicans germ tube-specific antigens may be of relevance for the serodiagnosis of invasive candidiasis since they could be the basis for the development of new diagnostic tests. In this study, we have identified two antigens of 180 and >200 kDa in the cell wall of C. albicans germ tubes which are responsible for the induction of antibodies to C. albicans germ tubes. Antigens of similar molecular masses have been demonstrated in the cell walls of the Candida species C. stellatoidea, C. parapsilosis, C. guilliermondii, C. tropicalis, and C. krusei, but not C. glabrata. The kinetics of the antibody responses to C. albicans germ tubes were studied in rabbits infected with different Candida species. Although these antibodies were detected in rabbits infected with all Candida species except C. glabrata, the kinetics of the antibody responses to C. albicans germ tubes induced by the Candida species studied were different. Both the highest titer and the earliest response of antibodies to C. albicans germ tubes were observed in rabbits infected with either of the two serotypes of C. albicans used. However, the time needed to elicit the antibodies to C. albicans germ tubes can be reduced as the result of an anamnestic antibody response. The results presented in this study show that a test designed to detect antibodies against C. albicans germ tube antigens may be suitable for the diagnosis of infections caused by most of the medically important Candida species.

Comparison of morphotypic and genotypic methods for strain delineation in Candida.

We compared two phenotypic methods, colony morphotyping on Sabouraudtripheniltetrazolium agar (STTZ) and serotyping, with two genotypic methods, karyotyping and Random Amplified Polymorphic DNA bands obtained by PCR amplification (RAPD-PCR), for strain delineation in 33 Candida clinical isolates and two C. albicans strains from culture collections. Analysis of isolates on STTZ showed 11 different morphotypes. In two patients there was a switch in the morphotype coincidential with a change in the susceptibility of the isolates to azole antifungals. C. albicans isolates were divided into two serotypes. Sixteen and 18 different patterns were identified among the Candida isolates by karyotyping and RAPD-PCR, respectively. No relationship was found between any of the typing methods used and the source of the isolates. The combination of karyotyping and morphotyping on STTZ yielded useful epidemiological information, since it allowed the differentiation among the Candida species studied and the discrimination of clusters within C. albicans as well as to check the capacity of a strain to generate variants with different susceptibility to some antifungals.

[Utility of Fluoroplate Candida for the rapid identification of Candida albicans]. / Utilidad de las placas de agar Fluoroplate Candida para la identificación rápida de Candida albicans.

BACKGROUND: Candida albicans infections are frequent in immunocompromised patients and a prompt diagnosis could favor an early and proper antifungal treatment. The rapid identification of clinical yeast isolates facilitate this diagnosis. METHODS: The utility of Fluoroplate Candida ready-to-use plates for Candida albicans rapid identification was evaluated with 653 clinical isolates from 23 yeast species, including 307 C. albicans plated onto Fluoroplate Candida agar (Merck, Germany). Rapid identification of C. albicans was based on the hydrolysis of 4-methylumbelliferyl-N-acetyl-beta-D-galactosaminide by the galactosaminidase activity of C. albicans producing white fluorescent colonies under ultraviolet light. Identification on Fluoroplate Candida was confirmed by germ tube, chlamydoconidia formation and API-ATB ID 32C assays. RESULTS: Three hundred and five of 306 isolates showing fluorescent colonies were C. albicans and one was Candida glabrata (false positive). The rest of the isolates showed colonies without fluorescence and with the exception of two false negatives, these isolates were identified as non-C. albicans by other methods. CONCLUSIONS: Fluoroplate Candida allows a rapid and excellent identification of C. albicans showing a sensitivity and specificity of 99.3 and 99.7%, respectively.

Reactivity of Candida albicans germ tubes with salivary secretory IgA.

Salivary secretory IgA (sIgA) has been shown to react with a group of heat shock mannoproteins preferentially expressed on yeast cells grown at 37 degrees C. Since at this temperature C. albicans can induce germ tubes, we explored the role of germ tube induction on human salivary sIgA reactivity in both germinative and agerminative C. albicans strains, in an attempt to investigate whether the germ tube expressed the heat shock mannoproteins reactive with sIgA. The reactivity with sIgA of the agerminative strain, grown at 25 and 37 degrees C for different times, was measured spectrofluorometrically and was fairly constant with time. Yeast cells grown at 37 degrees C tended to be more reactive than those grown at 25 degrees C. In contrast, when compared with the yeast cells of the germinative strain grown at 25 degrees C, there was a statistically significant decrease in reactivity with sIgA during germ tube formation. Serum IgA and IgG did not show statistically significant changes in reactivity with C. albicans during germination, suggesting differences in reactivity with C. albicans cell wall antigens between mucosal and systemic humoral responses. Cell wall mannoproteins of molecular masses > 60 kDa were characterized by Western blotting as responsible for the decrease in sIgA reactivity observed in the germ tube, and the fall in sIgA reactivity was related to the release of cell wall mannoproteins into the culture medium. The release of these mannoproteins may be a mechanism whereby C. albicans avoids the action of sIgA, and it may play an important role in the post-parasite relationship in oral candidiasis.

Candida albicans stress mannoprotein, SMP200, enhances tumour necrosis factor secretion in the murine macrophage cell line ANA-1.

Tumour necrosis factor (TNF) secretory activity has been studied in the murine macrophage cell line ANA-1 following in vitro exposure to Candida albicans 200 kDa stress mannoprotein (SMP200). Treatment of ANA-1 murine macrophages with 200 kDa stress mannoprotein results in increased TNF secretion. The phenomenon is (i) dose- and time-dependent, (ii) abrogated by 200 kDa stress mannoprotein preincubation with a specific monoclonal antibody, and (iii) dependent on intact murine macrophage Ca2+/calmodulin-dependent protein kinase function.

A new method of antibiotyping yeasts for subspecies discrimination and distribution in human clinical specimens.

A study of the antibiotypes of 764 isolates of the genera Candida and Torulopsis from different clinical specimens is reported. The typing method was based on the susceptibility results obtained by the standardized and partially automated kit ATB-Fungus (API-bioMérieux), giving to each strain a code of six figures, according to these criteria: susceptibility to 5-fluorocytosine, amphotericin B, nystatin, miconazole, econazole, and ketoconazole. Candida albicans serotypes were determined by the Candida Check test (Iatron, Japan). Twenty-six antibiotypes were found in C. albicans (482 isolates), 21 types in serotype A, and 15 in serotype B strains. Candida parapsilosis (115 isolates) was divided into 11 antibiotypes, Torulopsis glabrata (53 isolates) into five, Candida guilliermondii (36 isolates) into 10 and Candida tropicalis (31 isolates) into eight. Depending on the sample origin, 000000 (susceptibility to all the antifungals tested) was the predominant C. albicans antibiotype (92.9% of blood isolates, 41.2% of vaginal isolates, 33.3% of respiratory isolates, isolates, 31.01% or oral and digestive tract isolates, and 25.0% of nail and skin isolates). No predominant antibiotypes were found in strains from respiratory tract, skin ad nails. A reproducibility close to 99% was found with the test. Simplicity and standardization could make this method useful for typing Candida and Torulopsis isolates.

Variability in expression of antigens responsible for serotype specificity in Candida albicans.

The monoclonal antibody (mAb) B9E, which reacts with a cell wall surface determinant of Candida albicans serotype A, and a polyclonal monospecific antiserum against the antigen 6 (IF6) were used to investigate the expression of the antigens responsible for the serotype specificity in C. albicans under different growth conditions. By indirect immunofluorescence, both antibodies reacted with the cell wall surface of serotype A yeast cells and germ tubes grown in vitro but no reactivity was observed with serotype B yeast cells. In some cases, only a weak reactivity restricted to a zone close to the parent yeast cell was observed in serotype B germ tubes stained with mAb B9E. Both antibodies reacted strongly with yeast cells and germ tubes present in kidney abscesses from rabbits infected with both serotypes, but only serotype A yeast cells and germ tubes present in smears from patients with vulvovaginal candidiasis reacted with B9E and IF6 antibodies. The expression of antigens reactive with both antibodies was modulated by the pH of the environment in which the fungus was grown. Both antibodies showed a similar pattern of reactivity when studied with a spectrofluorometer. Serotype A yeast cells showed maximum reactivity when cells were grown on Sabouraud dextrose broth supplemented with yeast extract at pH 4.6. The lowest reactivity was observed in cells grown at pH 2.0. Conversely, the reactivity of serotype B yeast cells increased at alkaline pH values, the highest being in cells grown at pH values of 7.2 and 9.5.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of antigens reacting with anti-Candida albicans germ tube antibodies.

Anti-Candida albicans germ tube antibodies can be induced in rabbits immunized with different C. albicans extracts. Antigens responsible for the induction of those antibodies have molecular weights of approximately 230-250, 62, 43 and 41 kDa. These antigens are present in the cell wall of both C. albicans morphological forms, although their location seems to be different.