Comparative Analysis of Two Candida parapsilosis Isolates Originating from the Same Patient Harbouring the Y132F and R398I Mutations in the ERG11 Gene

This article belongs to the Special Issue Cellular and Molecular Mechanisms of Multiple Drug Resistance (MDR)This work presents a comparative analysis of two clinical isolates of C. parapsilosis, isolated from haemoculture (HC) and central venous catheter (CVC). Both strains harboured Y132F and R398I mutations in the gene ERG11 associated with resistance to fluconazole (FLC). Differences between the HC and CVC isolates were addressed in terms of virulence, resistance to FLC, and lipid distribution. Expression of the ERG6 and ERG9 genes, lipid analysis, fatty acid composition, and lipase activity were assessed via qPCR, thin-layer chromatography/high-performance liquid chromatography, gas chromatography, and spectrophotometry, respectively. Regulation of the ERG6 and ERG9 genes did not prove any impact on FLC resistance. Analysis of lipid metabolism showed a higher accumulation of lanosterol in both the isolates regardless of FLC presence. Additionally, a decreased level of triacylglycerols (TAG) with an impact on the composition of total fatty acids (FA) was observed for both isolates. The direct impact of the ERG11 mutations on lipid/FA analysis has not been confirmed. The higher lipase activity observed for C. parapsilosis HC isolate could be correlated with the significantly decreased level of TAG. The very close relatedness between both the isolates suggests that one isolate was derived from another after the initial infection of the host.This research was funded by the Slovak Research and Development Agency under contracts of SK-PT-18-0006 as part of the Bilateral Cooperation Program (2019–2022), APVV-21-0302 and grant VEGA 2/0036/22 from the Ministry of Education, Science, Research, and the Sport of the Slovak Republic.info:eu-repo/semantics/publishedVersio

Anti-biofilm Activity of Antibody Directed Against Surface Antigen Complement Receptor 3-Related Protein-Comparison of Candida Albicans and Candida Dubliniensis

Candida species (spp.) are a part of the normal human microbiota. Candida dubliniensis mostly colonizes the oral cavity and/or respiratory tract (Mahelová and Růžička 2017), especially in HIV-infected individuals (Coleman et al.1997; Sullivan et al.2004; Wahab et al.2014), while Candida albicans is a common inhabitant of the gastrointestinal tract, urogenital tract and oral cavity (Sardi et al.2013; Höfs, Mogavero and Hube 2016). Candidiasis is the most common global fungal infection (Sardi et al.2013). Candida albicans has been isolated in more than 50% of candidiasis; however, the number of non-albicans spp. able to cause serious candidiasis has increased in recent years (Yapar 2014; Pu et al.2015; Sandhu et al.2017). Although C. dubliniensis is phylogenetically very similar to C. albicans, it differs in some genes, especially those coding for virulence-associated proteins. Candida dubliniensis lacks more than 168 genes characteristic of its ‘yeast-cousin’ C. albicans (Jackson et al.2009), the majority of them encoding proteins related to the yeast-to-hyphae transition, tissue invasion or biofilm development (Moran et al.2004; Jackson et al.2009; Moran, Coleman and Sullivan 2012). Moreover, C. dubliniensis manifests a higher predisposition to develop resistance to fluconazole (Sullivan et al.1995; Moran, Coleman and Sullivan 2012; Jordan et al.2014). On the other hand, both C. albincans and C. dubliniensis are able to form a biofilm (Sullivan et al.2004; Borghi et al.2014). Adherence is the first and most crucial step in biofilm development, and various surface antigens participate in this process (Chaffin 2008; Gow and Hube 2012; Hebecker et al.2014). CR3-RP (complement receptor 3-related protein) is one of the cell surface antigens of Candida spp. with functional and structural similarity to the human complement receptor 3 (CR3) expressed on neutrophils, macrophages and monocytes. CR3-RP has been demonstrated to bind human complement fragment iC3b and to mediate leukocyte diapedesis (Heidenreich and Dierich 1985; Bujdáková et al.1997). Additionally, CR3-RP seems to be an important immunogenic mannoprotein participating in adhesion and biofilm development (Bujdáková et al.2008, 2010). A fragment of CR3-RP was sequenced (DINGGGATLPQ), and according to this sequence, CR3-RP was categorized into the DING protein family (named after DINGGG N termini) (Bujdáková et al.2008; Bernier 2013). Some other surface proteins contributing to biofilm development have been described, such as Eap protein, the Als protein family, the Hwp1 or MP65 proteins (Gomez et al.1996; Nailis et al.2010; Finkel and Mitchell 2011; Araújo, Henriques and Silva 2017). Additionally, antibodies generated after the immunization of animals with some of the above proteins seems to be promising in tools focused on fighting yeast infections (Fujibayashi et al.2009; Mishra, Ali and Shukla 2015; Torosantucci et al.2017). Recent studies showed that antibodies targeting Als3 (Coleman et al.2009), MP65 (De Bernardis et al.2007) or another 42.7 kDa unnamed surface antigen in the Candida cell wall (Mishra, Ali and Shukla 2015) decreased adhesion and biofilm formation

Effect of Quorum Sensing Molecule Farnesol on Mixed Biofilms of Candida albicans and Staphylococcus aureus

This article belongs to the Special Issue Microbial Biofilms, Antimicrobials, and Virulence Determinants.The natural bioactive molecule farnesol (FAR) is widely studied mainly for its antibiofilm and antimicrobial properties. In addition, it increases the effectiveness of some antimicrobial substances, which makes it interesting for the development of combined therapy. In the present work, the effect of FAR either alone or in combination with oxacillin (OXA) on mixed biofilms formed by clinically relevant pathogens, Candida albicans and Staphylococcus aureus, was studied. S. aureus isolates used for biofilm formation originated from blood cultures and central venous catheters (CVC) were characterized in terms of antimicrobial resistance. The minimal biofilm inhibitory concentration (MBIC50) for FAR of 48 h mixed biofilms formed by the C. albicans and methicillin-sensitive S. aureus (MSSA) was determined to be 125 M, and for the mixed biofilms with methicillin-resistant S. aureus (MRSA) was determined to be 250 M. Treatment of mixed biofilms with OXA (2 mg/mL) showed 4% inhibition; however, the combination of OXA (2 mg/mL) and FAR (300 M) resulted in 80% inhibition of biofilms. In addition, planktonic cells of S. aureus exhibited an increased susceptibility to OXA, cefoxitin and kanamycin in the presence of FAR (150 and 300 M). Scanning electron microscopy (SEM) micrographs confirmed patchy biofilm and lack of candidal hyphae in the samples treated with FAR and FAR/OXA in comparison to control and mixed biofilms treated only with OXA. Intriguingly, in a pilot experiment using fluorescence in situ hybridization (FISH), considerable differences in activity (as indicated by ribosome content) of staphylococcal cells were detected. While the activity rate of the staphylococci in mixed biofilms treated with FAR was high, no FISH-positive signal for staphylococcal cells was found in the biofilm treated with FAR/OXA.This research was funded by the Slovak Research and Development Agency under contracts of SK-PT-18-0006 as part of the Bilateral Cooperation Program (2019–2022), APVV-21-0302 and APVV-18-0075. This work was also supported by the EU Grant number 952398—CEMBO, Call: H2020-WIDESPREAD-05-2020—Twinning.info:eu-repo/semantics/publishedVersio

Development of novel apoferritin formulations for antitumour benzothiazoles

Background: The benzothiazole structure is important in medicinal chemistry, and 5‐fluoro‐2‐(3,4‐dimethoxyphenyl) benzothiazole (GW 610) is of particular interest as it shows outstanding anticancer activity in sensitive breast and colorectal carcinoma cell lines via generation of lethal DNA adducts in sensitive cancer cells. Despite promising activity, poor water solubility limits its applications. The apoferritin (AFt) protein cage has been proposed as a robust and biocompatible drug delivery vehicle. Aims: Here, we aim to enhance solubility of GW 610 by developing amino acid prodrug conjugates and utilizing the AFt capsule as drug delivery vessel. Methods and results: The potent experimental antitumour agent, GW 610, has been successfully encapsulated within AFt with more than 190 molecules per AFt cage. The AFt‐GW 610 complex exhibits dose‐dependent growth inhibition and is more potent than GW 610 alone in 5/7 cancer cell lines. To enhance both aqueous solubility and encapsulation efficiency, a series of amino acid esters of GW 608 prodrug were synthesized via N,N′‐dicyclohexylcarbodiimide ester coupling to produce molecules with different polarity. A dramatic increase in encapsulation efficiency was achieved, with more than 380 molecules of GW 608‐Lys molecules per AFt cage. Release studies show sustained release of the cargo over 12 hours at physiologically relevant pH. The AFt‐encapsulated amino acid modified GW 608 complexes are sequestered more rapidly and exhibit more potent anticancer activity than unencapsulated agent. Conclusion: These results indicate that AFt‐encapsulation of GW 610 prodrug provides a biocompatible delivery option for this potent, selective experimental antitumour agent and for amino acid‐modified GW 608. Of particular interest is the encapsulation efficiency and in vitro antitumour activity of AFt‐GW 608‐Lys, which warrants further preclinical evaluation

Antimicrobial Resistance and Biofilms Underlying Catheter-Related Bloodstream Coinfection by Enterobacter cloacae Complex and Candida parapsilosis

Biofilm-associated infections are a public health concern especially in the context of healthcare-associated infections such as catheter-related bloodstream infections (CRBSIs). We evaluated the biofilm formation and antimicrobials resistance (AMR) of Enterobacter cloacae complex and Candida parapsilosis co-isolated from a CRBSI patient. Antimicrobial susceptibility of central venous catheters (CVCs) and hemoculture (HC) isolates was evaluated, including whole genome sequencing (WGS) resistome analysis and evaluation of gene expression to obtain insight into their AMR determinants. Crystal violet assay was used to assess dual biofilm biomass and microscopy was used to elucidate a microorganism’s distribution within biofilms assembled on different materials. Bacteria were multidrug-resistant including resistance to colistin and beta-lactams, likely linked to the mcr-9-like phosphoethanolamine transferase and to an ACT family cephalosporin-hydrolyzing class C beta-lactamase, respectively. The R398I and Y132F mutations in the ERG11 gene and its differential expression might account for C. parapsilosis resistance to fluconazole. The phenotype of dual biofilms assembled on glass, polystyrene and polyurethane depends on the material and how biofilms were initiated by one or both pathogens. Biofilms assembled on polyurethane were denser and richer in the extracellular polymeric matrix, and microorganisms were differently distributed on the inner/outer surface of the CVC.publishedVersio

Profil osjetljivosti na kaspofungin i flukonazol i ekspresija gena Als1 i Als3 u stanicama biofilma te planktonskim stanicama vrste Candida albicans

The biofilm of Candida albicans has been implicated as a source of bloodstream infections. Dispersal cells, as the final biofilm stage, are responsible for its spread. The aim of this study was to compare the susceptibility of biofilm and dispersal cells vs. planktonic cells (overnight liquid culture) of C. albicans to caspofungin (CAS) and fluconazole (FLU) when the drugs were added: i) at the beginning of the experiment; ii) after 1.5 h (adherence stage); iii) after 24 h (early mature biofilm). The findings were evaluated after 48 h (mature biofilm) using the XTT reduction assay. Later administration of the drug increased biofi lm sessile minimal inhibitory concentration (SMIC80) of both FLU and CAS from 1 μg mL-1 to over 64 μg mL-1 and from 0.125 μg mL-1 to over 16 μg mL-1, respectively. Susceptibility of dispersal cells also decreased with time of administration. We also determined the expression of the Als1 and Als3 genes in 48-h sessile biofilm and dispersal cells of C. albicans SC5314 and compared it to planktonic cells. The expression was normalised to the standard Act1 gene in every condition tested. Quantitative real-time PCR revealed a strong up-regulation of the Als1 gene in the dispersal cells but not in biofilm and high expression of the Als3 gene in both biofilm and dispersal cells. High expression of both Als1 and Als3 genes supports the hypothesis that dispersal cells pose a high-risk of infection.Rasprostranjenju biofilma pridonose oslobođene, tzv. planktonske stanice, koje nastaju u posljednjoj fazi oblikovanja biofilma. U ovome istraživanju usporedili smo osjetljivost stanica biofilma i planktonskih stanica (prekonoćna bujonska kultura) C. albicans na kaspofungin (CAS) i fl ukonazol (FLU) u uvjetima kada su lijekovi dodavani: i) na početku pokusa; ii) nakon 1,5 h (faza priljepljivanja, adherencije); iii) nakon 24 h (rana zrelost biofilma). Nakon 48 h u fazi zrelog biofi lma provedeno je mjerenje primjenom testa redukcije XTT-a. Dobiveni rezultati potvrđuju da kasnija primjena lijeka povisuje sesilnu minimalnu inhibicijsku koncentraciju, SMIC80 (od engl. sessile minimal inhibitory concentration) i FLU i CAS (u rasponu od 1 μg mL-1 do ≥64 μg mL-1 te u rasponu od 0,125 μg mL-1 do ≥16 μg mL-1). Nadalje, uočili smo smanjenu osjetljivost planktonskih stanica na lijekove. U drugom dijelu pokusa usredotočili smo se na ekspresiju Als1 i Als3 gena u C. albicans SC5314, kako u sesilnim stanicama iz biofi lma starog 48 h tako i u stanicama koje su se izdvojile iz njega te smo ih usporedili s kulturom planktonskih stanica. Ekspresiju smo u svakome od ispitanih uvjeta testiranja normalizirali prema genu Act1 kao standardu. Primjenom kvantitativnog PCR-a u realnom vremenu (engl. Quantitative Real Time PCR) dokazali smo snažno pojačanu ekspresiju gena Als1 u zrelim stanicama koje se oslobađaju iz biofilma, ali ne i u sesilnom biofilmu, kao i visoku ekspresiju gena Als3 i u biofilmu i u stanicama koje su se izdvojile iz njega. Takvi rezultati upućuju na to da se osjetljivost stanica udruženih u biofilm te stanica oslobođenih iz njega na FLC i CAS smanjuje s kasnijim vremenom primjene lijeka, a, dodatno, visoka ekspresija gena Als1 i Als3 govori u prilog hipotezi da su stanice oslobođene iz biofi lma visoki čimbenik rizika i izvor infekcije

Impact of farnesol and Corsodyl® on Candida albicans forming dual biofilm with Streptococcus mutans

Objective: This work studied the biofilm formed by Candida albicans and Streptococcus mutans on a hydroxyapatite surface after exposure to the quorum-sensing molecule farnesol (200 µM) in comparison with the diluted mouthwash Corsodyl® (0.0001% chlorhexidine digluconate). Materials and Methods: The cytotoxicity of farnesol was evaluated by Galleria melonella surviving assay. The viability of biofilm cells after exposure to farnesol and Corsodyl® was determined by colony-forming units. The morphology and structure of a dual-species biofilm was evaluated by scanning electron microscopy. Results: Farnesol did not exhibit a toxic effect on larval survival. While 200 µM farnesol effectively reduced the yeast-to-hyphae transition in the dual biofilm, it did not affect the growth of S. mutans. Additionally, despite the presence of farnesol, many blastospores were observed. Corsodyl® reduced S. mutans in the dual biofilm, but did not influence C. albicans. Conclusion: This study showed that 200 µM farnesol modulated C. albicans in a dual-species biofilm with S. mutans, but did not exhibit antimicrobial activity against S. mutans. Moreover, it seems that S. mutans provides conditions that support the growth of the yeast form of C. albicans. The mouthwash Corsodyl® reduces S. mutans, but was not effective against C. albicans.We would like to thank COST Action TD1305 Improved Protection of Medical Devices against Infection (iPROMEDAI) for funding the Short-term scientific mission at the Instituto Nacional de Saúde Dr Ricardo Jorge, Departamento de Saúde Ambiental, Unidade de Investigação e Desenvolvimento-Lisboa, Avenida Padre Cruz, Lisboa, Portugal. This research was also funded by the Slovak Research and Development Agency under the contract No. [APVV-15-0347] and by the grant VEGA [1/0628/15] supported by the Ministry of Education, Science, Research and Sport of the Slovak Republic. We wish to thank to Isabel Nogueira from MicroLab–Instituto Superior Técnico in Lisbon, for her expert assistance with scanning electron microscopy.info:eu-repo/semantics/publishedVersio

Opportunist Coinfections by Nontuberculous Mycobacteria and Fungi in Immunocompromised Patients

ReviewNontuberculous mycobacteria (NTM) and many fungal species (spp.) are commonly associated with opportunistic infections (OPIs) in immunocompromised individuals. Moreover, occurrence of concomitant infection by NTM (mainly spp. of Mycobacterium avium complex and Mycobacterium abscessus complex) and fungal spp. (mainly, Aspergillus fumigatus, Histoplasma capsulatum and Cryptococcus neoformans) is very challenging and is associated with poor patient prognosis. The most frequent clinical symptoms for coinfection and infection by single agents (fungi or NTM) are similar. For this reason, the accurate identification of the aetiological agent(s) is crucial to select the best treatment approach. Despite the significance of this topic it has not been sufficiently addressed in the literature. This review aims at summarizing case reports and studies on NTM and fungi coinfection during the last 20 years. In addition, it briefly characterizes OPIs and coinfection, describes key features of opportunistic pathogens (e.g., NTM and fungi) and human host predisposing conditions to OPIs onset and outcome. The review could interest a wide spectrum of audiences, including medical doctors and scientists, to improve awareness of these infections, leading to early identification in clinical settings and increasing research in the field. Improved diagnosis and availability of therapeutic options might contribute to improve the prognosis of patients’ survival.This research was funded by Portuguese Fundação para a Ciência e a Tecnologia and by the Slovak Research and Development Agency under contract SK-PT-18-0006 as a part of the Bilateral Cooperation Program (2019-2021), APVV-15-0347,and by the grant VEGA 1/0537/19 from the Ministry of Education, Science, Research, and the Sport of the Slovak Republic.info:eu-repo/semantics/publishedVersio