Spore Adhesion Research Articles

The Multifaceted Roles of Fungal Cutinases during Infection.

Cuticles cover the aerial epidermis cells of terrestrial plants and thus represent the first line of defence against invading pathogens, which must overcome this hydrophobic barrier to colonise the inner cells of the host plant. The cuticle is largely built from the cutin polymer, which consists of C16 and C18 fatty acids attached to a glycerol backbone that are further modified with terminal and mid-chain hydroxyl, epoxy, and carboxy groups, all cross-linked by ester bonds. To breach the cuticle barrier, pathogenic fungal species employ cutinases—extracellular secreted enzymes with the capacity to hydrolyse the ester linkages between cutin monomers. Herein, we explore the multifaceted roles that fungal cutinases play during the major four stages of infection: (i) spore landing and adhesion to the host plant cuticle; (ii) spore germination on the host plant cuticle; (iii) spore germ tube elongation and the formation of penetrating structures; and (iv) penetration of the host plant cuticle and inner tissue colonisation. Using previous evidence from the literature and a comprehensive molecular phylogenetic tree of cutinases, we discuss the notion whether the lifestyle of a given fungal species can predict the activity nature of its cutinases.

Pathogenicity of Beauveria bassiana strains against Drosophila suzukii (Diptera: Drosophilidae)

The biological control (BC) is an efficient tool in helping to reduce pests in crops of economic interest. In addition to the efficiency, there are advantages in using biological control agents, such as human safety and other non-target organisms and decreasing pesticide residues. The general aim of this work was to evaluate in vitro the pathogenicity power of fifteen Beauveria bassiana strains aiming the biological control of Drosophila suzukii (Matsumura, 1931). Successful infection of entomopathogenic fungi depends primarily on the adherence and penetration ability of a fungus to the host integuments. In addition, scanning electron microscopy was realized. To verify the spore adherence after the spraying tests, insects were collected throughout the bioassays in order to confirm spore evolution on the insect cuticle. In this work, D. suzukii adults were not susceptible to the aqueous suspensions of conidia at concentrations of 108 spores·mL−1 of these strains. There wasn’t death confirmed by fungus extrusion after maintenance in humid chamber.

Using a ligate intestinal loop mouse model to investigate Clostridioides difficile adherence to the intestinal mucosa in aged mice.

Interaction of Clostridioides difficile spores with the intestinal mucosa contributes to the persistence and recurrence of the infection. Advanced age is one of the main risk factors for C. difficile infection and recurrence of the disease. However, interaction of C. difficile spores with the intestinal mucosa during aging has not been evaluated. In the present work, using intestinal ligated loop technique in a mouse model, we analyzed C. difficile spore adherence and internalization to the ileum and colonic mucosa during aging. Additionally, we provide visual documentation of the critical steps of the procedure. Consequently, our data suggest that spore internalization in the ileum and colonic mucosa is higher in elderly mice rather than adults or young mice. Also, our data suggest that spore adherence to the ileum and colonic mucosa decreases with aging.

The role of Glabridin in antifungal and anti-inflammation effects in Aspergillus fumigatus keratitis

PurposeTo investigate the effect of Glabridin (GLD) in Aspergillus fumigatus keratitis and its associated mechanisms. MethodsAspergillus fumigatus (A. fumigatus) conidia was inoculated in 96-well plate, and minimal inhibitory concentration (MIC) and biofilm formation ability were evaluated after GLD treatment. Spore adhesion ability was evaluated in conidia infected human corneal epithelial cells (HCECs). Keratitis mouse model was created by corneal intrastromal injection with A. fumigatus conidia, and GLD treatment started at the day after infection. The number of fungal colonies was calculated by plate count, and degree of corneal inflammation was assessed by clinical score. Flow cytometry, myeloperoxidase (MPO), and immunofluorescence staining (IFS) experiments were used to assess neutrophil infiltrations. PCR, ELISA and Western blot were conducted to determine levels of TLR4, Dectin-1 as well as downstream inflammatory factors. ResultsGLD treatment suppressed the proliferation, biofilm formation abilities and adhesive capability of A. fumigatus. In mice upon A. fumigatus infection, treatment of GLD showed significantly decreased severity of corneal inflammation, reduced number of A. fumigatus in cornea, and suppressed neutrophil infiltration in cornea. GLD treatment obviously inhibited mRNA and protein levels of Dectin-1, TLR4 and proinflammatory mediators such as IL-1β, HMGB1, and TNF-α in mice corneas compared to the control group. ConclusionGLD has antifungal and anti-inflammatory effects in fungal keratitis through suppressing A. fumigatus proliferation and alleviating neutrophil infiltration, and repressing the expression of TLR4, Dectin-1 and proinflammatory mediators.

Pathogenicity of Cordyceps javanica (Hypocreales: Cordycipitaceae) to Diaphorina citri (Hemiptera: Liviidae) Adults, with Ultrastructural Observations on the Fungal Infection Process

Entomopathogenic fungi are proposed biological control agents against the Asian citrus psyllid (Diaphorina citri). We quantified the pathogenicity of Cordyceps javanica strain Apopka 97 strain (Cja Apopka 97) (blastospores and conidia) against D. citri using the spray technique. We also used light and scanning electron microscopy to observe the Cja Apopka 97, infection process against D. citri adults at different stages pre- and post-mortem. Our findings demonstrated that psyllid mortality in the blastospore treatment ranged from 8 to 25% within 24–48 h of exposure, compared to 0% in the conidial and control treatments. However, psyllid mortality rate had reached 100% by 7 days after exposure at a concentration of 107 spores/mL, under both fungal treatments compared to the controls (0%). SEM and light microscopy revealed several stages in in the Cja Apopka 97 infection process of D. citri, including spore adherence and germ tube formation within 24 h post-inoculation, penetration pegs and mycelia growth on wings after 72 h, rupturing of cuticle after 96 h and mycelial mass colonizing host body after 144 h. Our study findings provide basic information on the interaction of entomopathogenic fungi with D. citri which will assist in the understanding of the infection process and the potential roles of entomopathogenic fungi in its management.

Enigmatic Pilus-Like Endospore Appendages of Bacillus cereus Group Species.

The endospores (spores) of many Bacillus cereus sensu lato species are decorated with multiple hair/pilus-like appendages. Although they have been observed for more than 50 years, all efforts to characterize these fibers in detail have failed until now, largely due to their extraordinary resilience to proteolytic digestion and chemical solubilization. A recent structural analysis of B. cereus endospore appendages (Enas) using cryo-electron microscopy has revealed the structure of two distinct fiber morphologies: the longer and more abundant “Staggered-type” (S-Ena) and the shorter “Ladder-like” type (L-Ena), which further enabled the identification of the genes encoding the S-Ena. Ena homologs are widely and uniquely distributed among B. cereus sensu lato species, suggesting that appendages play important functional roles in these species. The discovery of ena genes is expected to facilitate functional studies involving Ena-depleted mutant spores to explore the role of Enas in the interaction between spores and their environment. Given the importance of B. cereus spores for the food industry and in medicine, there is a need for a better understanding of their biological functions and physicochemical properties. In this review, we discuss the current understanding of the Ena structure and the potential roles these remarkable fibers may play in the adhesion of spores to biotic and abiotic surfaces, aggregation, and biofilm formation.

Infection of the Western Flower Thrips, Frankliniella occidentalis, by the Insect Pathogenic Fungus Beauveria bassiana

The western flower thrips, Frankliniella occidentalis, is an aggressive agricultural insect pest causing significant damage to a wide range of fruit, vegetable, and ornamental crops. Beauveria bassiana is a broad-host-range entomopathogenic fungus capable of infecting and killing F. occidentalis. Infection of thrips by B. bassiana strain BbYT12 using a concentration of 1 × 108 conidia/mL resulted in 81.48% mortality in adults in 6 d (LT50 = 90 ± 15.1 h). Scanning electron microscopy of the infection process revealed preferential adhesion and germination of fungal spores to inter-segmental folds or grooves on the insect body surface with penetrating germlings and extended hyphae visualized during the initial stages of infection (6–24 h). Histological analyses showed the appearance of in vivo hyphal bodies in sagittal sections and the fat body as early as 24 h post-infection. Within 72 h, hyphal bodies and hyphae could be found throughout the infected organism including in the midgut, Malphigian tubules, alimentary canal, ovarioles (in females), and an extended hyphal network could be seen on insect cadavers (>72 h post-infection). Real-time RT-PCR analyses of the expression of select genes implicated in virulence including the Pr1 protease, beauvericin synthase, involved in the production of the secondary metabolite beauvericin, two cytochrome P450 monooxygenases implicated in cuticular hydrocarbon degradation, two multidrug efflux proteins, a perilipin involved in lipid storage, and the Hog1 MAP kinase and protein kinase A signaling factors revealed discrete patterns of infection-time dependent expression. These data provide basic insights into the process of B. bassiana infection of F. occidentalis.

Adhesion of Penicillium italicum and Penicillium digitatum spores to materials commonly used in the citrus packaging chain

PurposeThe purpose of this study was to investigate the adhesion of Penicillium italicum and Penicillium digitatum spores on four materials commonly used in the citrus packaging chain (plastic, PVC, stainless steel, 316L and wood).Research methodsThe physicochemical characterization of spores and material surfaces was carried out using the contact angle method. The number of adhered spores was estimated after being detached from supports in an ultrasonic bath. The results showed that all citrus materials processes were classified as hydrophobic except for the wood packaging. Surface spores of P. digitatum presented a relatively hydrophobic character, and surface spores of P. italicum presented a hydrophilic character. Both of the spores and all materials presented high electron donor/acceptor characters.FindingsThe results showed that P. digitatum and P. italicum sporescould adhere to all the studied substrates. Furthermore, the highest adhesion was observed by P. italicum and P. digitatum spores on wood packaging (58 I 106 CFU/cm2) and (45 I 106 CFU/cm2), respectively. The wood packaging was the least hygienic material concerning the adhesion ability of P. digitatum and P. italicum spores, followed by plastic packaging, PVC, and 316 L stainless steel. A correlation between substratum physicochemical properties and spore adhesion was also examined, while a good correlation was observed between spore adhesion and donor electron character.Research limitations: There were no limitations to this study.Originality/value: This research studied the adhesion of spores on materials commonly used in the citrus packaging chain.

An insight of anopheline larvicidal mechanism of Trichoderma asperellum (TaspSKGN2)

Anopheline larvicidal property of T. asperellum has been found recently in medical science. The mechanism of actions exhibited by T. asperellum to infect mosquito larvae is the pivotal context of our present study. To infect an insect, entomopathogens must undergo some events of pathogenesis. We performed some experiments to find out the mechanisms of action of T. asperellum against anopheline larvae and compared its actions with other two well recognized entomopathogens like Metarhizium anisopliae and Beauveria bassiana. The methodology adopted for this includes Compound light and SE Microscopic study of host–pathogen interaction, detection of fungal spore adhesion on larval surface (Mucilage assay), detection of cuticle degrading enzymes (Spore bound pr1, chitinase and protease) by spectro-photometric method, Quantitative estimation of chitinase and protease enzymes, and determination of nuclear degeneration of hemocyte cells of ME (methanolic extract) treated larvae by T. asperellum under fluorescence microscope. Compound light microscopic studies showed spore attachment, appressorium and germ tube formation, invasion and proliferated hyphal growth of T. asperellum on epicuticle and inside of dead larvae. SEM study also supported them. After 3 h of interaction, spores were found to be attached on larval surface exhibiting pink colored outer layer at the site of attachment indicating the presence of mucilage surrounding the attached spores. The enzymatic cleavage of the 4-nitroanilide substrate yields 4-nitroaniline which indicates the presence of spore-bound PR1 protein (Pathogenecity Related 1 Protein) and it was highest (absorbance 1.298 ± 0.002) for T. asperellum in comparison with control and other two entomopathogens. T. asperellum exhibited highest enzymatic index values for both chitinase (5.20) and protease (2.77) among three entomopathogens. Quantitative experiment showed that chitinase enzyme concentration of T. asperellum (245 µg mL−1) was better than other two M. anisopliae (134.59 µg mL−1) and B. bassiana (128.65 µg mL−1). Similarly protease enzyme concentration of this fungus was best (298.652 µg mL−1) among three entomopathogens. Here we have detected and estimated fragmentized nuclei of hemocyte cells by fluorescence microscopy in treated larvae with different ME doses of T. asperellum, and also observed that mosquito larvae exposed to 0.1 mg mL−1 dose of ME showed maximum (100%) nuclear fragmentations of hemocytes and while 20, 45, 70 and 85% of nuclear deformities were recorded at 0.02, 0.04, 0.06 and 0.08 mg mL−1 concentrations of ME. The knowledge of this work certainly will help in understanding of mechanism of action of T. asperellum for anopheline larval killing and consequently in eradication of malaria vector.

Unc-119 mutants have an increased fungal spore adhesion that is not rescued by Cb-unc-119.

If the cuticle acts as a protective barrier against environmental insults, several pathogens have developed strategies that use it as a way to infect C. elegans. The fungus Drechmeria coniospora produces spores that attach to the cuticle, before hyphae invade the body. Mutants with an altered surface coat, the outermost layer of the cuticle, including bus-2, bus-4, bus-12 and bus-17 show increased adhesion of fungal spores (Rouger et al, 2014; Zugasti et al, 2016). We unexpectedly found that D. coniospora spores attach unusually densely around the mouth of unc-119 mutants. Interestingly, this phenotype is not rescued by the C. briggsae unc-119 construct that is conventionally used to rescue neuronal unc-119 phenotypes.

Corticosteroids alter alveolar macrophage control of Lichtheimia corymbifera spores in an ex vivo mouse model.

The aim of this study was to describe the impact of corticosteroids on alveolar macrophage (AM) capacities to control Mucorales growth in a new murine ex vivo model. Corticosteroids enhanced fungal growth of L. corymbifera through AM phagocytosis inhibition and burst oxidative decrease.

Antagonistic effects of native strains of the soil fungus Paecilomyces against gastrointestinal nematode and protozoan parasites of pigs in Panama.

A variety of gastrointestinal parasites naturally infect domestic pigs in Panama which may also occur as zoonotic infections in humans. Anthelmintic drug treatment, including mass drug administration, can lead to drug resistance, reflecting a need for alternatives. The objectives of this exploratory and observational study were: (1) to isolate and cultivate natives species of Paecilomyces from natural soils in Panama, and (2) to evaluate isolated strains for their capacity to parasitize endemic gastrointestinal nematode and protozoan parasites recovered from naturally infected domestic pigs by observing cultures for spore adhesion and hyphae penetration phases. Using microcultivation and inoculation techniques, four strains of Paecilomyces were isolated from three locations in Panama, out of which three successfully adhered to and penetrated free-living stages (eggs, cysts and oocysts) of Balantidium suis, coccidia, Trichuris suis and hookworm. To our knowledge, this is the first published report of a nematophagous fungus such as Paecilomyces successfully infecting this range of gastrointestinal parasites, particularly protozoan parasites.

Description of hyperparasitism of microsporidia in nematode (Ascaridia galli) parasitic in fowl, Gallus gallus domesticus

Microsporidia are intracellular, obligate parasites. They have wide infection range from diverse phyla of invertebrates to vertebrates and also these eukaryotic pathogens parasitize those hosts which themselves are parasites, thus revealing hyperparasitism. The present study describe intracellular pathogen microsporidia which is isolated from a nematode parasite, Ascaridia sp. which itself is a parasite of fowl, Gallus gallus domesticus. The initial detection by optical microscopy represented numerous refractive oval spores in the homogenate samples of nematodes with mean spore length and width of 4.32±0.32μm×1.57±0.26μm (N=10). Giemsa staining exhibited demarcating stages of spore development. Spores developing in sporophorous vesicle, mature spores of considerable size and shape, dividing sporonts and sporoblasts, were clearly noticeable. However, the phases of proliferation (merogony) were indistinguishable. The ultrastructural study by Scanning electron microscopy illustrated intracellular development of microspor-idian spore inside nematode tissues. Xenoma formation as well as spore adherence to the epithelial wall most likely indicated the degradation and auto-infection by the microsporidian spores in the host tissue. A spore infecting host tissue by ejecting its polar tubule was also captured. Thus, the overall study focuses on developing microsporidia as hyperparasite in nematodes, although further classification for determining the genus has been left to be explored.

The sps Genes Encode an Original Legionaminic Acid Pathway Required for Crust Assembly in Bacillus subtilis.

The crust is the outermost spore layer of most Bacillus strains devoid of an exosporium. This outermost layer, composed of both proteins and carbohydrates, plays a major role in the adhesion and spreading of spores into the environment. Recent studies have identified several crust proteins and have provided insights about their organization at the spore surface. However, although carbohydrates are known to participate in adhesion, little is known about their composition, structure, and localization. In this study, we showed that the spore surface of Bacillus subtilis is covered with legionaminic acid (Leg), a nine-carbon backbone nonulosonic acid known to decorate the flagellin of the human pathogens Helicobacter pylori and Campylobacter jejuni We demonstrated that the spsC, spsD, spsE, spsG, and spsM genes of Bacillus subtilis are required for Leg biosynthesis during sporulation, while the spsF gene is required for Leg transfer from the mother cell to the surface of the forespore. We also characterized the activity of SpsM and highlighted an original Leg biosynthesis pathway in B. subtilis Finally, we demonstrated that Leg is required for the assembly of the crust around the spores, and we showed that in the absence of Leg, spores were more adherent to stainless steel probably because of their reduced hydrophilicity and charge.IMPORTANCEBacillus species are a major economic and food safety concern of the food industry because of their food spoilage-causing capability and persistence. Their persistence is mainly due to their ability to form highly resistant spores adhering to the surfaces of industrial equipment. Spores of the Bacillus subtilis group are surrounded by the crust, a superficial layer which plays a key role in their adhesion properties. However, knowledge of the composition and structure of this layer remains incomplete. Here, for the first time, we identified a nonulosonic acid (Leg) at the surfaces of bacterial spores (B. subtilis). We uncovered a novel Leg biosynthesis pathway, and we demonstrated that Leg is required for proper crust assembly. This work contributes to the description of the structure and composition of Bacillus spores which has been under way for decades, and it provides keys to understanding the importance of carbohydrates in Bacillus adhesion and persistence in the food industry.

Colonization of Vitis vinifera L. by the Endophyte Trichoderma sp. Strain T154: Biocontrol Activity Against Phaeoacremonium minimum.

Trichoderma strains used in biological control products usually exhibit high efficiency in the control of plant diseases. However, their behavior under field conditions is difficult to predict. In addition, the potential of indigenous strains has been poorly assayed as well as their possible behavior as endophytes. Hence, niche colonization is a key feature for an effective protection. In this study, we aimed to: (i) explore the possibility of using a new Trichoderma strain isolated from vine to control pathogens, (ii) study the in planta interaction with the pathogen Phaeoacremonium minimum W. Gams, Crous, M.J. Wingf. & L. Mugnai (formerly Phaeoacremonium aleophilum), a pioneer fungus involved in Grapevine Trunk Diseases (GTDs) such as esca. For this purpose, fluorescently tagged Trichoderma sp. T154 and a P. minimum strain were used for scanning electron microscopy and confocal scanning laser microscopy analyses. Data showed that the Trichoderma strain is able to colonize plants up to 12 weeks post inoculation and is located in xylem, fibers, as well as in parenchymatic tissues inside the wood. The beneficial fungus reduced colonization of the esca-related pathogen colonizing the same niches. The main observed mechanism involved in biocontrol of Trichoderma against the esca pathogen was spore adhesion, niche exclusion and only few typical hypha coiling was found between Trichoderma and the pathogen. These results suggest that the Trichoderma strain has potential for reducing the colonization of Phaeoacremonium minimum and thus, an inoculation of this biological control agent can protect the plant by limiting the development of GTD, and the strain can behave as an endophyte.

Control of Post-harvest Citrus Green Mold using Ulva lactuca Extracts as a Source of Active Substances

Penicillium digitatum, the causal agent of citrus green mold, is responsible for 90% of post-harvest losses. Chemical fungicides are responsible for damage to human health and the environment. The exploitation of green algae as a source of pigments and carbohydrate polymers deserves a special attention. In this study, we evaluated the ability of Ulva lactuca extracts collected from Lebanon and China, to develop products for the biocontrol of post-harvest citrus green mold. Seaweed aqueous extracts (SAE), organic extracts (SOE) and chlorophylls and Ulvans fractions of Ulva lactuca are analyzed by chromatography and spectroscopy methods. The chemical compositions in functional molecules (pigments, organic acids, and monosaccharides) from extracts are compared for the samples. It was found that Lebanese Ulva lactuca is particularly rich in ulvans and other polysaccharides, while Food grade Ulva lactuca has the highest content in lipophilic molecules. Four approaches were used to evaluate their antifungal properties: inhibition of the conidia germination of P. digitatum, fungus viability, adhesion of spores to epidermal orange cells, and the disk diffusion method. For the different seaweed liquid extracts (SLEs), inhibitions of spore germination were observed between 70 and 87%. Moreover, the samples exhibited inhibitory activities towards the adhesion of P. digitatum spores. More particularly, SOE showed inhibitions of spore multiplication between 85 and 97%. According zone diamaters, Ulvan fractions showed maximum growth spore inhibitions. These findings are interesting to select extracts for safe and effective formulations dedicated to citrus fruit post harvesting protection.

Anti-Biofouling Features of Eco-Friendly Oleamide-PDMS Copolymers.

The biofouling of marine organisms on a surface induces serious economic damage. One of the conventional anti-biofouling strategies is the use of toxic chemicals. In this study, a new eco-friendly oleamide–PDMS copolymer (OPC) is proposed for sustainable anti-biofouling and effective drag reduction. The anti-biofouling characteristics of the OPC are investigated using algal spores and mussels. The proposed OPC is found to inhibit the adhesion of algal spores and mussels. The slippery features of the fabricated OPC surfaces are examined by direct measurement of pressure drops in channel flows. The proposed OPC surface would be utilized in various industrial applications including marine vehicles and biomedical devices.

Fungal spore adhesion on glycidoxypropyltrimethoxy silane modified silica nanoparticle surfaces as revealed by single cell force spectroscopy.

Thin film coatings prepared from commercially available glycidoxypropyltrimethoxysilane (GPS) modified silica nanoparticles (SiNPs) (Bindzil® CC301 and Bindzil® CC302) have previously shown excellent antifouling performance against a broad range of microbes [Molino et al., "Hydration layer structure of biofouling-resistant nanoparticles," ACS Nano 12, 11610 (2018)]. In this work, single cell force spectroscopy (SCFS) was used to measure the biological interactions between Epicoccum nigrum fungal spores and the same silica nanoparticle-based surfaces used in the aforementioned study, including a: glass coverslip, unmodified SiNP coatings, and both low (Bindzil® CC301) and high density (CC302) GPS functionalized SiNP coatings as a function of NaCl concentration. From the SCFS curves, the spore adhesion to the surface was greatest on the glass coverslip (20-80 nN) followed by the unmodified SiNP (3-5 nN) across all salt concentrations. Upon approach to both surfaces, the spores showed a long-range attraction generally with a profile characteristic of biointeractions and likely those of the outer cell wall structures or biological constituents. The attractive force allowed the spores to initially adhere to the surface and was found to be linearly proportional to the spore adhesion. In comparison, both high and low density GPS-SINP significantly reduced the spore adhesion (0.5-0.9 nN). In addition, the spore adhesion on high density GPS-SiNP occurred in only 14%-27% of SCFS curves (40%-48% for low density GPS-SiNP) compared to 83%-97% for the unmodified SiNP, indicating that in most cases the GPS functionalization completely prevented spore adhesion. The GPS-SiNP surfaces conversely showed a long-range electrostatic repulsion at low 1mM NaCl that was replaced by short-range repulsion at the higher salt concentrations. From the findings, it is proposed that the attractive force is a critical step in initial adhesion processes of the spore. The effective antifouling properties of the GPS are attributed to the ability to negate the attractive forces, either through electrostatic repulsion in low salt conditions and primarily from short-range repulsion correlating to the previously reported combined steric-hydration effect of the GPS functionalization on SiNP coatings.

Calcineurin signaling pathway influences Aspergillus niger biofilm formation by affecting hydrophobicity and cell wall integrity

BackgroundBiofilms, as a kind of fixed-cell community, can greatly improve industrial fermentation efficiency in immobilized fermentation, but the regulation process is still unclear, which restricts their application. Ca2+ was reported to be a key factor affecting biofilm formation. However, the effect of Ca2+ on biofilm structure and microbiology was yet only studied in bacteria. How Ca2+-mediated calcineurin signaling pathway (CSP) alters biofilm formation in bacteria and fungi has rarely been reported. On this basis, we investigated the regulation of CSP on the formation of biofilm in Aspergillus niger.ResultsDeletion of the key genes MidA, CchA, CrzA or CnaA in the CSP lowered the Ca2+ concentration in the mycelium to a different extent, inhibited the formation of A. niger biofilm, reduced the hydrophobicity and adhesion of spores, destroyed the cell wall integrity of hyphae, and reduced the flocculation ability of hyphae. qRT-PCR results showed that the expression of spore hydrophobic protein RodA, galactosaminogalactan (GAG) biosynthesis genes (uge3, uge5, agd3, gtb3), and α-1,3-glucan biosynthesis genes (ags1, ags3) in the ∆MidA, ∆CchA, ∆CrzA, ∆CnaA strains were significantly down-regulated compared with those of the wild type (WT). In addition, the transcription levels of the chitin synthesis gene (chsB, chsD) and β-1,3-glucan synthesis gene (FksA) were consistent with the change in chitin and β-1,3-glucan contents in mutant strains.ConclusionThese results indicated that CSP affected the hydrophobicity and adhesion of spores, the integrity of mycelial cell walls and flocculation by affecting Ca2+ levels in mycelium, which in turn affected biofilm formation. This work provides a possible explanation for how CSP changes the formation of A. niger biofilm, and reveals a pathway for controlling biofilm formation in industrial immobilized fermentation.

Biocide Resistance and Transmission of Clostridium difficile Spores Spiked onto Clinical Surfaces from an American Health Care Facility.

Clostridium difficile is the primary cause of antibiotic-associated diarrhea globally. In unfavorable environments, the organism produces highly resistant spores which can survive microbicidal insult. Our previous research determined the ability of C. difficile spores to adhere to clinical surfaces, finding that spores had markedly different hydrophobic properties and adherence abilities. Investigation into the effect of the microbicide sodium dichloroisocyanurate on C. difficile spore transmission revealed that sublethal concentrations increased spore adherence without reducing viability. The present study examined the ability of spores to transmit across clinical surfaces and their response to an in-use disinfection concentration of 1,000 ppm of chlorine-releasing agent sodium dichloroisocyanurate. In an effort to understand if these surfaces contribute to nosocomial spore transmission, surgical isolation gowns, hospital-grade stainless steel, and floor vinyl were spiked with 1 × 106 spores/ml of two types of C. difficile spore preparations: crude spores and purified spores. The hydrophobicity of each spore type versus clinical surface was examined via plate transfer assay and scanning electron microscopy. The experiment was repeated, and spiked clinical surfaces were exposed to 1,000 ppm sodium dichloroisocyanurate at the recommended 10-min contact time. Results revealed that the hydrophobicity and structure of clinical surfaces can influence spore transmission and that outer spore surface structures may play a part in spore adhesion. Spores remained viable on clinical surfaces after microbicide exposure at the recommended disinfection concentration, demonstrating ineffectual sporicidal action. This study showed that C. difficile spores can transmit and survive between various clinical surfaces despite appropriate use of microbicides.IMPORTANCEClostridium difficile is a health care-acquired organism and the causative agent of antibiotic-associated diarrhea. Its spores are implicated in fecal to oral transmission from contaminated surfaces in the health care environment due to their adherent nature. Contaminated surfaces are cleaned using high-strength chemicals to remove and kill the spores; however, despite appropriate infection control measures, there is still high incidence of C. difficile infection in patients in the United States. Our research examined the effect of a high-strength biocide on spores of C. difficile which had been spiked onto a range of clinically relevant surfaces, including isolation gowns, stainless steel, and floor vinyl. This study found that C. difficile spores were able to survive exposure to appropriate concentrations of biocide, highlighting the need to examine the effectiveness of infection control measures to prevent spore transmission and to consider the prevalence of biocide resistance when decontaminating health care surfaces.