Antimicrobial Peptides with Anti-Candida Activity.

Mycoses are accountable for millions of infections yearly worldwide. Invasive candidiasis is the most usual, presenting a high morbidity and mortality. Candida albicans remains the prevalent etiologic agent, but the incidence of other species such as Candida parapsilosis, Candida glabrata and Candida auris keeps increasing. These pathogens frequently show a reduced susceptibility to commonly used antifungal drugs, including polyenes, triazoles and echinocandins, and the incidence of emerging multi-drug-resistant strains of these species continues to increase. Therefore, the need to search for new molecules that target these pathogenic species in a different manner is now more urgent than ever. Nature is an almost endless source of interesting new molecules that could meet this need. Among these molecules, antimicrobial peptides, present in different sources in nature, possess some advantages over conventional antifungal agents, even with their own drawbacks, and are considered as a promising pharmacological option against a wide range of microbial infections. In this review, we describe 20 antimicrobial peptides from different origins that possess an activity against Candida.

Isolation, host specificity and genetic characterization of Campylobacter specific bacteriophages from poultry and swine sources.

The isolation and characterization of 304 Campylobacter specific bacteriophage isolates from broiler and swine sources is reported in this study. Genome size characterization determined by PFGE classified these isolates,called CAM1-CAM304, within the campylophages group II (n = 18) and group III (n = 286). Host range analyses showed a high host specificity and similar lytic spectrum among isolates of the same group. Campylophages of group II infected C. jejuni, C. coli and even a C. fetus strain whereas those of group III only infected C. jejuni strains. The most promising 59 campylophage candidates were selected according to their lytic activity and their genetic diversity was analyzed by RFLP using SmiI and HhaI endonucleases for group II and III campylophages, respectively. Moreover, RAPD-PCR technique was for the first time assessed in the genetic characterization of campylophages and it was shown to be effective only for those of group II. Bacteriophage isolates grouped in a same genotype displayed different host ranges, therefore, 13 campylophages of group II and eight of group III were differentiated considering all the approaches assayed. An in-depth analysis of these bacteriophages will be performed to confirm their promising potential for the biocontrol of Campylobacter within the farm to fork process.

Caenorhabditis elegans as a Model System To Assess Candida glabrata, Candida nivariensis, and Candida bracarensis Virulence and Antifungal Efficacy.

Although Candida albicans remains the major etiological agent of invasive candidiasis, Candida glabrata and other emerging species of Candida are increasingly isolated. This species is the second most prevalent cause of candidiasis in many regions of the world. However, clinical isolates of Candida nivariensis and Candida bracarensis can be misidentified and are underdiagnosed due to phenotypic traits shared with C. glabrata Little is known about the two cryptic species. Therefore, pathogenesis studies are needed to understand their virulence traits and their susceptibility to antifungal drugs. The susceptibility of Caenorhabditis elegans to different Candida species makes this nematode an excellent model for assessing host-fungus interactions. We evaluated the usefulness of C. elegans as a nonconventional host model to analyze the virulence of C. glabrata, C. nivariensis, and C. bracarensis The three species caused candidiasis, and the highest virulence of C. glabrata was confirmed. Furthermore, we determined the efficacy of current antifungal drugs against the infection caused by these species in the C. elegans model. Amphotericin B and azoles showed the highest activity against C. glabrata and C. bracarensis infections, while echinocandins were more active for treating those caused by C. nivariensisC. elegans proved to be a useful model system for assessing the pathogenicity of these closely related species.

Utility of two PCR-RFLP-based techniques for identification of Candida parapsilosis complex blood isolates.

BACKGROUND: Candida parapsilosis is the second or third most frequently isolated Candida species related to nosocomial infections, even overtaking Candida albicans in some hospitals. C. parapsilosis constitutes a complex of closely related species: Candida parapsilosis sensu stricto, Candida orthopsilosis and Candida metapsilosis. Accurate detection of these species is of importance, as the incidence of C. orthopsilosis has been reported to surpass that of Candida krusei. OBJECTIVE: To evaluate the diagnostic utility of two PCR-RFLP methods targeting the SADH and FKS1 genes and to determine the prevalence of cryptic species in 96 bloodstream isolates of C. parapsilosis from 93 patients. METHODS: Restriction patterns of the SADH and FKS1 genes were analysed, and sequencing of the D1/D2 regions of the ribosomal RNA was used to evaluate the reliability of both PCR-RFLP methods. RESULTS: In our study, 77 C. parapsilosis sensu stricto, 13 C. orthopsilosis and five C. metapsilosis were identified by sequencing. Both PCR-RFLP methods demonstrated strong agreement with D1/D2 sequencing in the identification of C. parapsilosis and C. orthopsilosis, while both methods were unable to identify the C. metapsilosis isolates. Moreover, unexpected restriction patterns were observed for two isolates on SADH PCR-RFLP and for four isolates on FKS1 PCR-RFLP. Mixed bloodstream infections of C. parapsilosis sensu stricto and C. orthopsilosis were detected for three patients, for which differential growth characteristics were observed. CONCLUSION: The molecular method chosen for identification could have an impact on determination of the real prevalence of C. metapsilosis in candidaemia, and mixed fungaemias can remain undetected.

Efficient isolation of Campylobacter bacteriophages from chicken skin, analysis of several isolation protocols.

The application of Campylobacter specific bacteriophages appears as a promising food safety tool for the biocontrol of this pathogen in the poultry meat production chain. However, their isolation is a complicated challenge since their occurrence appears to be low. This work assessed the efficiency of seven protocols for recovering Campylobacter phages from chicken skin samples inoculated at phage loads from 5.0 × 101 to 5.0 × 106 PFU/g. The enrichment of chicken skin in selective Bolton broth containing target isolates was the most efficient procedure, showing a low detection limit of 5.0 × 101 PFU/g and high recovery rates of up to 560%. This method's effectiveness increased as phage concentration decreased, showing its suitability for phage isolation. When this method was applied to isolate new Campylobacter phages from retail chicken skin, a total of 280 phages were recovered achieving an isolation success rate of 257%. From the 109 samples 68 resulted phage positive (62%). Chicken skin could be, therefore, considered a rich source in Campylobacter phages. This method is a simple, reproducible and efficient approach for the successful isolation of both group II and III Campylobacter specific bacteriophages, which could be helpful for the enhancement of food safety by reducing this pathogen contamination in broiler meat.

GqqA, a novel protein in Komagataeibacter europaeus involved in bacterial quorum quenching and cellulose formation.

BACKGROUND: We report on the functional screening and identification of an active quorum quenching (QQ) gene in the Komagataeibacter europaeus strain CECT 8546, which is a member of the acetic acid bacteria (AAB). RESULTS: Using a previously published screening protocol (Schipper et al., in Appl Environ Microbiol 75:224-233, 2009. doi: 10.1128/AEM.01389-08 ) for QQ genes, we identified a single gene, designated gqqA, that interfered strongly with bacterial quorum sensing (QS) in various reporter strains. It encodes for a 281-amino acid protein with a molecular mass of 30 kDa. Although the GqqA protein is similar to predicted prephenate dehydratases, it does not complement Escherichia coli mutants of the pheA gene, thus indicating a potentially different function. Recombinant GqqA protein attenuated QS-dependent pyocyanin production and swarming motility in the Pseudomonas aeruginosa strain PAO1. Moreover, GqqA quenched the QS response of the Agrobacterium tumefaciens NTL4 and the Chromobacterium violaceum CV026 reporter strains. Interestingly, the addition of recombinant GqqA protein to growing cultures of the Komagataeibacter europaeus strain CECT 8546 altered the cellulose production phenotype of CECT 8546 and other AAB strains. In the presence of GqqA protein, cells were planktonic, and no visible cellulose biofilms formed. The addition of low levels of N-acylhomoserine lactones maintained the biofilm formation phenotype. CONCLUSIONS: Our data provide evidence for an interconnection between QS and AAB cellulose biofilm formation as well as QQ activity of the GqqA protein.

Cellulose production and cellulose synthase gene detection in acetic acid bacteria.

The ability of acetic acid bacteria (AAB) to produce cellulose has gained much industrial interest due to the physical and chemical characteristics of bacterial cellulose. The production of cellulose occurs in the presence of oxygen and in a glucose-containing medium, but it can also occur during vinegar elaboration by the traditional method. The vinegar biofilm produced by AAB on the air-liquid interface is primarily composed of cellulose and maintains the cells in close contact with oxygen. In this study, we screened for the ability of AAB to produce cellulose using different carbon sources in the presence or absence of ethanol. The presence of cellulose in biofilms was confirmed using the fluorochrome Calcofluor by microscopy. Moreover, the process of biofilm formation was monitored under epifluorescence microscopy using the Live/Dead BacLight Kit. A total of 77 AAB strains belonging to 35 species of Acetobacter, Komagataeibacter, Gluconacetobacter, and Gluconobacter were analysed, and 30 strains were able to produce a cellulose biofilm in at least one condition. This cellulose production was correlated with the PCR amplification of the bcsA gene that encodes cellulose synthase. A total of eight degenerated primers were designed, resulting in one primer pair that was able to detect the presence of this gene in 27 AAB strains, 26 of which formed cellulose.

Acetic acid bacteria from biofilm of strawberry vinegar visualized by microscopy and detected by complementing culture-dependent and culture-independent techniques.

Acetic acid bacteria (AAB) usually develop biofilm on the air-liquid interface of the vinegar elaborated by traditional method. This is the first study in which the AAB microbiota present in a biofilm of vinegar obtained by traditional method was detected by pyrosequencing. Direct genomic DNA extraction from biofilm was set up to obtain suitable quality of DNA to apply in culture-independent molecular techniques. The set of primers and TaqMan--MGB probe designed in this study to enumerate the total AAB population by Real Time--PCR detected between 8 × 10(5) and 1.2 × 10(6) cells/g in the biofilm. Pyrosequencing approach reached up to 10 AAB genera identification. The combination of culture-dependent and culture-independent molecular techniques provided a broader view of AAB microbiota from the strawberry biofilm, which was dominated by Ameyamaea, Gluconacetobacter, and Komagataeibacter genera. Culture-dependent techniques allowed isolating only one genotype, which was assigned into the Ameyamaea genus and which required more analysis for a correct species identification. Furthermore, biofilm visualization by laser confocal microscope and scanning electronic microscope showed different dispositions and cell morphologies in the strawberry vinegar biofilm compared with a grape vinegar biofilm.

Efficacy of the combination of amphotericin B and echinocandins against Candida auris in vitro and in the Caenorhabditis elegans host model.

IMPORTANCE: Multidrug resistance is a rising problem among non-Candida albicans species, such as Candida auris. This therapeutic problem has been very important during the COVID-19 pandemic. The World Health Organization has included C. auris in its global priority list of health-threatening fungi, to study this emerging multidrug-resistant species and to develop effective alternative therapies. In the present study, the synergistic effect of the combination of amphotericin B and echinocandins has been demonstrated against blood isolates of C. auris. Different susceptibility responses were also observed between aggregative and non-aggregative phenotypes. The antifungal activity of these drug combinations against C. auris was also demonstrated in the Caenorhabditis elegans host model of candidiasis, confirming the suitability and usefulness of this model in the search for solutions to antimicrobial resistance.

Acetobacter malorum and Acetobacter cerevisiae identification and quantification by Real-Time PCR with TaqMan-MGB probes.

The identification and quantification of Acetobacter malorum and Acetobacter cerevisiae in wine and vinegar were performed using the Real-Time PCR (RT-PCR) with two TaqMan-MGB probes designed to amplify the internal transcribed spacer (ITS) region between the 16S-23S rRNA genes. The primers and probes were highly specific, with a detection limit of 10² cells/ml for both species, and the efficiency of the technique was >80%. The RT-PCR technique with these two new TaqMan-MGB probes, together with the five (Acetobacter aceti, Acetobacter pasteurianus, Gluconobacter oxydans, Gluconacetobacter hansenii and Gluconacetobacter europaeus) that are already available (Torija et al., 2010), were validated on known concentrations of Acetic Acid Bacteria (AAB) grown in glucose medium (GY) and in inoculated matrices of wine and vinegar. Furthermore, this technique was applied to evaluate the AAB population in real wine samples collected in the Canary Islands. PCR enrichment performed prior to RT-PCR increased the accuracy of quantification and produced results similar to those detected with SYBR-Green. In real wine samples, the total AAB enumeration ranged from 9 × 10² to 106 cells/ml, and the seven AAB species tested were detected in more than one sample. However, AAB recovery on plates was poor; the isolates obtained on plates were A. malorum, G. oxydans, A. cerevisiae and A. pasteurianus species. RT-PCR with TaqMan-MGB probes is an accurate, specific and fast method for the identification and quantification of AAB species commonly found in wine and vinegar.

In vitro and in vivo anti-Candida activity of citral in combination with fluconazole.

BACKGROUND: The ability of Candida to develop biofilms on inert surfaces or living tissues favors recalcitrant and chronic candidiasis associated, in many instances, with resistance to current antifungal therapy. AIM: The aim of this study was to evaluate the antifungal activity of citral, a phytocompound present in lemongrass essential oil, in monotherapy and combined with fluconazole against azole-resistant Candida planktonic cells and biofilms. The effect of citral combined with fluconazole was also analysed with regard to the expression of fluconazole resistance-associated genes in Candida albicans and the effectiveness of the combination therapy in a Caenorhabditis elegans model of candidiasis. RESULTS: Citral reduced biofilm formation at initial stages and the metabolic activity of the mature biofilm. The combination of citral with fluconazole was synergistic, with a significant increase in the survival of C. elegans infected with Candida. RNA analysis revealed a reduction of the expression of the efflux pump encoded by MDR1, leading to a greater effect of fluconazole. CONCLUSION: Citral in monotherapy and in combination with fluconazole could represent an interesting therapy to treat recalcitrant Candida infections associated to biofilms.

Candidiasis by Candida glabrata, Candida nivariensis and Candida bracarensis in Galleria mellonella: Virulence and Therapeutic Responses to Echinocandins.

Candida albicans is the major etiological agent of invasive candidiasis but the increasing prevalence of emerging species of Candida, such as Candida glabrata and phylogenetically closely related species, Candida nivariensis and Candida bracarensis, requires special attention. Differences in virulence among these species and their therapeutic responses using in vivo non-mammalian models are scarcely analysed. The aim of this study was analyse the survival of G. mellonella and host-pathogen interactions during infection by C. glabrata, C. nivariensis and C. bracarensis. Moreover, therapeutic responses to echinocandins were also assessed in the G. mellonella model of candidiasis. These three species produced lethal infection in G. mellonella; C. glabrata was the most virulent species and C. bracarensis the less. Haemocytes of G. mellonella phagocytised C. bracarensis cells more effectively than those of the other two species. Treatment with caspofungin and micafungin was most effective to protect larvae during C. glabrata and C. nivariensis infections while anidulafungin was during C. bracarensis infection. The model of candidiasis in G. mellonella is simple and appropriate to assess the virulence and therapeutic response of these emerging Candida species. Moreover, it successfully allows for detecting differences in the immune system of the host depending on the virulence of pathogens.

In vitro activities of carvacrol, cinnamaldehyde and thymol against Candida biofilms.

Oral candidiasis is frequently associated with Candida biofilms. Biofilms are microbial communities related to persistent, recalcitrant and difficult to-treat infections. Conventional treatments are not sufficient to overcome biofilm-associated candidiasis; thus, the search of new antifungal compounds is necessary. In the current study, we have evaluated the effect of three phytocompounds, carvacrol, cinnamaldehyde and thymol, against Candida planktonic and sessile cells. Reduction in biofilm biomass and metabolic activity was assessed during adhesion and mature biofilm phases. Candida albicans was the most biofilm-producing Candida species. All phytocompounds tested were fungicidal against Candida planktonic cells. Cinnamaldehyde was the most active in inhibiting biofilm adhesion, but carvacrol and thymol significantly reduced both mature biofilm biomass and metabolic activity. These results highlight the role of cinnamaldehyde, carvacrol and thymol as promising alternatives for the treatment of candidiasis due to their antibiofilm capacities, and stress the necessity to continue studies on their safety, toxicity and pharmacodynamics and pharmacokinetics.

Virulence of Candida auris from different clinical origins in Caenorhabditis elegans and Galleria mellonella host models.

Candida auris is an emerging multidrug-resistant fungal pathogen responsible for nosocomial outbreaks of invasive candidiasis. Although several studies on the pathogenicity of this species have been reported, the knowledge on C. auris virulence is still limited. This study aims to analyze the pathogenicity of C. auris, using one aggregating isolate and eleven non-aggregating isolates from different clinical origins (blood, urine and oropharyngeal specimens) in two alternative host models of candidiasis: Caenorhabditis elegans and Galleria mellonella. Furthermore, possible associations between virulence, aggregation, biofilm-forming capacity, and clinical origin were assessed. The aggregating phenotype isolate was less virulent in both in vivo invertebrate infection models than non-aggregating isolates but showed higher capacity to form biofilms. Blood isolates were significantly more virulent than those isolated from urine and respiratory specimens in the G. mellonella model of candidiasis. We conclude that both models of candidiasis present pros and cons but prove useful to evaluate the virulence of C. auris in vivo. Both models also evidence the heterogeneity in virulence that this species can develop, which may be influenced by the aggregative phenotype and clinical origin.

Disinfectant Activity of A Portable Ultraviolet C Equipment.

Healthcare-associated infections (HAIs) can be caused by microorganisms present in common practice instruments generating major health problems in the hospital environment. The aim of this work was to evaluate the disinfection capacity of a portable ultraviolet C equipment (UV Sanitizer Corvent® -UVSC-) developed to disinfect different objects. For this purpose, six pathogens causing HAIs: Acinetobacter baumannii, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Candida albicans, were inoculated on slides and discs of different biomaterials (borosilicate, polycarbonate, polyurethane, silicone, Teflon and titanium) and exposed to ultraviolet C radiation. UVSC disinfection was compared with ethanol and chlorhexidine antimicrobial activities following the standards EN14561 and EN14562. Disinfection, established as a reduction of five logarithms from the initial inoculum, was achieved with the UVSC at 120 s of exposure time, with and without the presence of organic matter. The disinfectant effect was observed against S. aureus, P. aeruginosa, E. coli, B. subtilis and C. albicans (reduction >99.999%). Disinfection was also achieved with 70% ethanol and 2% chlorhexidine. As conclusion, UVSC was effective disinfecting the most contaminated surfaces assayed, being a promising alternative for disinfecting hospital materials and inanimate objects that cannot be immersed in liquid biocides, reducing the risk of pathogen transmission.

Corrigendum: Antifungal Activity of the Human Uterine Cervical Stem Cells Conditioned Medium (hUCESC-CM) Against Candida albicans and Other Medically Relevant Species of Candida.

[This corrects the article DOI: 10.3389/fmicb.2018.02818.].

Project-based learning methodology in the area of microbiology applied to undergraduate medical research.

In the recent years, there has been a decrease in the number of medical professionals dedicated to a research career. There is evidence that students with a research experience during their training acquire knowledge and skills that increase the probability of getting involved in research more successfully. In the Degree of Medicine (University of the Basque Country) the annual core subject 'Research Project' introduces students to research. The aim of this work was to implement a project-based learning methodology, with the students working on microbiology, and to analyse its result along time. Given an initial scenario, the students had to come up with a research idea related to medical microbiology and to carry out a research project, including writing a funding proposal, developing the experimental assays and analyzing and presenting their results to a congress organized by the University. Summative assessment was performed by both students and teachers. A satisfaction survey was carried out to gather the students' opinion. The overall results regarding to the classroom dynamics, learning results and motivation after the implementation were favourable. Students referred a greater interest about research than they had before. They would choose the project based methodology versus the traditional one.

The continuous changes in the aetiology and epidemiology of invasive candidiasis: from familiar Candida albicans to multiresistant Candida auris.

Recent changes in the aetiology and epidemiology of invasive candidiasis have serious implications for current and future diagnosis, treatment and prognosis. The aim of the current review was to discuss the epidemiology of invasive candidiasis, the distribution of Candida species in different regions of the world, the medical concerns of the changing aetiology and the emergence of antifungal resistance. Overall burden of invasive candidiasis remains high, especially in vulnerable persons, such as the elderly, immunosuppressed or debilitated patients. Moreover, there is a progressive shift in the aetiology of invasive candidiasis from Candida albicans to other species of Candida, probably related to the increased use of azole drugs with a clear trend towards increased antifungal resistance. Finally, the emergence and rise of multiresistant species, such as Candida auris or Candida glabrata, is a major threat making necessary invasive candidiasis worldwide surveillances. These changes have serious implications for the diagnosis, treatment and prognosis of invasive candidiasis. Updated knowledge of the current local epidemiology of invasive candidiasis is critical for the clinical management.

Prevalence and antifungal susceptibility profiles of Candida glabrata, Candida parapsilosis and their close-related species in oral candidiasis.

OBJECTIVE: To evaluate the importance of Candida glabrata, Candida parapsilosis and their close-related species, Candida bracarensis, Candida nivariensis, Candida metapsilosis and Candida orthopsilosis in patients with oral candidiasis and, to determine the in vitro activities of antifungal drugs currently used for the treatment. METHODS: One hundred fourteen isolates of C. glabrata and 97 of C. parapsilosis, previously identified by conventional mycological methods, were analysed by molecular techniques. In vitro antifungal susceptibility to fluconazole, itraconazole, miconazole, and nystatin was evaluated by CLSI M44-A2 disk diffusion test, and by CLSI M27-A3 microdilution for fluconazole. RESULTS: All C. glabrata isolates were identified as C. glabrata sensu stricto, 93 out of 97 C. parapsilosis isolates as C. parapsilosis sensu stricto, three as C. orthopsilosis and one as C. metapsilosis. Candida glabrata was mainly isolated in mixed cultures but C. parapsilosis complex was more frequent in pure culture. Candida metapsilosis and C. orthopsilosis were isolated as pure culture and both species were susceptible to all antifungal agents tested. Most C. glabrata isolates were susceptible to miconazole and nystatin, but resistant to fluconazole and itraconazole. Azole cross resistance was also observed. Candida parapsilosis isolates were susceptible to fluconazole although azole cross resistance to miconazole and itraconazole was observed. CONCLUSION: This study highlights the importance of accurate identification and antifungal susceptibility testing of oral Candida isolates in order to have an in-depth understanding of the role of C. glabrata and C. parapsilosis in oral candidiasis.

Antifungal Activity of the Human Uterine Cervical Stem Cells Conditioned Medium (hUCESC-CM) Against Candida albicans and Other Medically Relevant Species of Candida.

Background: Candidiasis is a major cause of human morbidity and mortality. Human uterine cervical stem cells conditioned medium (hUCESC-CM) is obtained from stromal stem cells of the cervical transformation zone, which are in permanent contact with a wide array of potential vaginal pathogens. In previous reports we have found that hUCESC-CM has antitumor and antibacterial potential. Since Candida is the most prevalent yeast in the human vagina, it seems plausible that hUCESC-CM might also show activity against it. Methods: In a preliminary step, to evaluate if hUCESC-CM showed any activity at all on Candida growth, in vitro activities of hUCESC-CM against fluconazole-susceptible reference strains of Candida albicans, Candida glabrata, Candida krusei, and Candida parapsilosis were studied with a microdilution method on RPMI 1640, using the BioScreen C microbiological incubator. Each measurement was repeated five times. The same methodology was used subsequently on fluconazole-susceptible and fluconazole-resistant Candida isolates from blood and vagina of those species corresponding to the reference strains of Candida against which activity had been detected in the previous study. Moreover, two fluconazole-resistant clinical isolates of Candida auris from blood and urine were also included. Findings: In vitro inhibitory activity of hUCESC-CM ranged from 57.5 to 96.6% growth-reduction against fluconazole-susceptible reference strains of Candida albicans, Candida glabrata, and Candida parapsilosis. hUCESC-CM also reduced the growth of all fluconazole-susceptible tested vaginal isolates by more than 50%. For fluconazole-resistant isolates, growth-reduction was higher than 67% for Candida albicans, regardless of its origin (vagina or blood). The isolate of Candida auris from urine with a MIC > 128 µ/ml for fluconazole was also significantly inhibited. However, hUCESC-CM was almost inactive against any of the fluconazole-resistant blood isolates of Candida glabrata, Candida parapsilosis, and Candida auris tested. Interpretation: This is the first report about the growth-inhibiting properties of conditioned medium from human stromal stem cells against different species of Candida. Antifungal activity of stromal stem cells depends on their site of origin, being most effective against Candida species most prevalent at that particular location. If confirmed in further studies, these findings might result in a completely new therapeutic approach against superficial and invasive candidiasis.