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Mycoses are accountable for millions of infections yearly worldwide. Invasive candidiasis is the most usual, presenting a high morbidity and mortality.

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.

[EN] 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. This research was funded by the Spanish Ministry of Science and Innovation (PID2020-117983RB-I00) and by the Consejería de Educación, Universidades e Investigación of Gobierno Vasco-Eusko Jaurlaritza (IT1607-22). A.P.-R. was funded by a Ph.D. grant from the University of the Basque Country (PIF17/167).

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. This research was supported by grants from the Spanish Ministry of Economy and Competitiveness (MINECO) [SAF2017-86188-P and PID2020-117983RB-I00] and from the Consejería de Educación, Universidades e Investigación of Gobierno Vasco-Eusko Jaurlaritza [GIC15/78 IT-990-16]. Ainara Hernando-Ortiz was funded by Ph.D. grants from the University of the Basque Country (PIF 16/39).

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.

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.

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. This work was supported by the Euskal Herriko Unibertsitatea [PIF 16/39]; Euskal Herriko Unibertsitatea [PIF17/167]; Eusko Jaurlaritza [GIC15/78 IT-990-16]; Ministerio de Economia y Competitividad [SAF2017-86188-P].

[EN]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. This work was supported by Gobierno Vasco-Eusko Jaurlaritza, Spain [grant number GIC15/78 IT-990-16, 2016] and Fundacion ONCE "Oportunidad al Talento", Spain and Fondo Social Europeo, Spain [CMA, 2018].

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. nivariensis. C. elegans proved to be a useful model system for assessing the pathogenicity of these closely related species.

AIM To analyse the prevalence, genetic diversity and antimicrobial susceptibility of Campylobacter spp. in northern Spain. METHODS AND RESULTS Campylobacter was isolated from 139 samples of broiler meat and faecal dropping of broiler and swine with a prevalence of 35·4, 62 and 42·8%, respectively. Campylobacter jejuni (n = 55) and Campylobacter coli (n = 31) were identified by multiplex-PCR in meat, faeces and human clinical samples while Campylobacter fetus (n = 3) was exclusively detected in the latter. Fingerprinting by flaA-RFLP and PFGE revealed 68 different genotypes from the 89 isolates with a Biodiversity Simpson's index of 0·98. The 86·5% of the isolates were resistant to ciprofloxacin, 85·4% to tetracycline and 49·4% to erythromycin; only three genotypes were susceptible to the three antimicrobial drugs. Multidrug resistance was detected in the 40·7% of the isolates. CONCLUSIONS Campylobacter remains prevalent in northern Spain with a high biodiversity degree. About 93·3% of the isolates were resistant to one or more drugs. SIGNIFICANCE AND IMPACT OF THE STUDY Although different measures are taken to control Campylobacter, the detection of isolates resistant to the drugs used in the treatment of campylobacteriosis is still high, including different species and genotypes. This evidences the need of additional strategies against this pathogen.

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.

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.

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.

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&reg, 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 &gt, 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.

EnglishCandidiasis is a mycotic infection caused by species of Candida genus. Candida albicans is the major aetiological agent, although other species of Candida, such as Candida glabrata, are considered emerging causes of this disease. The species, Candida bracarensis and Candida nivariensis, are phylogenetically similar to Candida glabrata and can be correctly differentiated by molecular techniques. These changes in the aetiology have serious implications for diagnosis, treatment and prognosis; considering that yeast pathogenesis or susceptibilities to current antifungal drugs may be different. Invertebrate models, such as the nematode Caenorhabditis elegans and the lepidopter Galleria mellonella, are an attractive alternative for the study of fungal pathogenesis and antifungal therapy. The aim of this research study was to evaluate the usefulness of these two non-convetional model to assess the in vivo virulence of the phylogenetically close-related species, Candida glabrata, Candida bracarensis and Candida nivariensis. euskaraKandidiasia Candida generoko espezieek eragindako infekzio mikotikoa da. Candida albicans agente etiologiko nagusia da baina gero eta gehiagotan Candida generoko beste espezie batzuk agertzen ari dira kandidiasiaren eragile bezala eta, hauen artean, Candida glabrata espeziea. Espezie honekin lotuta beste bi espezie daude, Candida bracarensis eta Candida nivariensis, teknika molekularrek ondo desberdindu ditzaketenak. Aldaketa etiologiko hauek ondorio larriak izan ditzakete kandidiasiaren diagnostikoan, tratamenduan edota pronostikoan, besteak beste, bere patogenia edo antifungikoekiko sentikortasuna oso desberdina izan daitezkeelako. Ordezko eredu esperimentalek erabiliz mikroorganismoek eragindako gaixotasunen patogenia eta terapia ezagutzeko ezinbesteko aukera ematen dizkigute, eta hauen artean, Caenorhabditis elegans nematodoa eta Galleria mellonella lepidopteroa ditugu. Ikerketa lan honetan konbentzionalak ez diren bi animalia eredu hauen erabilgarritasuna ebaluatu nahi izan da Candida glabrata, Candida bracarensis eta Candida nivariensis harreman filogenetiko estua duten hiru espezie hauen birulentzia in vivo aztertzeko.

Background Candidiasis is one of the most common opportunistic oral infections that presents different acute and chronic clinical presentations with diverse diagnostic and therapeutic approaches. The present study carries out a bibliographic review on the therapeutic tools available against oral candidiasis and their usefulness in each clinical situation. Material and Methods Recent studies on treatment of oral candidiasis were retrieved from PubMed and Cochrane Library. Results Nystatin and miconazole are the most commonly used topical antifungal drugs. Both antifungal drugs are very effective but need a long time of use to eradicate the infection. The pharmacological presentations of miconazole are more comfortable for patients but this drug may interact with other drugs and this fact should be assessed before use. Other topical alternatives for oral candidiasis, such as amphotericin B or clotrimazole, are not available in many countries. Oral fluconazole is effective in treating oral candidiasis that does not respond to topical treatment. Other systemic treatment alternatives, oral or intravenous, less used are itraconazole, voriconazole or posaconazole. Available novelties include echinocandins (anidulafungin, caspofungin) and isavuconazole. Echinocandins can only be used intravenously. Isavuconazole is available for oral and intravenous use. Other hopeful alternatives are new drugs, such as ibrexafungerp, or the use of antibodies, cytokines and antimicrobial peptides. Conclusions Nystatin, miconazole, and fluconazole are very effective for treating oral candidiasis. There are systemic alternatives for treating recalcitrant infections, such as the new triazoles, echinocandins, or lipidic presentations of amphotericin B. Key words:Oral candidiasis, antifungal treatment, azoles, echinocandins, fluconazole, miconazole, nystatin.

The oxidative metabolism of acetic acid bacteria (AAB) can be exploited for the production of several compounds, including D-gluconic acid. The production of D-gluconic acid in fermented beverages could be useful for the development of new products without glucose. In the present study, we analyzed nineteen strains belonging to eight different species of AAB to select those that could produce D-gluconic acid from D-glucose without consuming D-fructose. We tested their performance in three different media and analyzed the changes in the levels of D-glucose, D-fructose, D-gluconic acid and the derived gluconates. D-Glucose and D-fructose consumption and D-gluconic acid production were heavily dependent on the strain and the media. The most suitable strains for our purpose were Gluconobacter japonicus CECT 8443 and Gluconobacter oxydans Po5. The strawberry isolate Acetobacter malorum (CECT 7749) also produced D-gluconic acid; however, it further oxidized D-gluconic acid to keto-D-gluconates.

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.

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 CM-A was a recipient of a grant from Fundación Once (Oportunidad al Talento). EM, CM-A, EE, and GQ have received grant support from Consejería de Educación, Universidades e Investigación (GIC15 IT-990-16), Fondo de Investigación Sanitaria (FIS PI11/00203), and UPV/EHU (UFI 11/25) SI

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.

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.

Acetic acid bacteria (AAB) diversity from healthy, mould-infected and rot-affected grapes collected from three vineyards of Adelaide Hills (South Australia) was analyzed by molecular typing and identification methods. Nine different AAB species were identified from the 624 isolates recovered: Four species from Gluconobacter genus, two from Asaia and one from Acetobacter were identified by the analysis of 16S rRNA gene and 16S–23S rRNA gene internal transcribed spacer. However, the identification of other isolates that were assigned as Asaia sp. and Ameyamaea chiangmaiensis required more analysis for a correct species classification. The species of Gluconobacter cerinus was the main one identified; while one genotype of Asaia siamensis presented the highest number of isolates. The number of colonies recovered and genotypes identified was strongly affected by the infection status of the grapes; the rot-affected with the highest number. However, the species diversity was similar in all the cases. High AAB diversity was detected with a specific genotype distribution for each vineyard.

Highbush blueberries (Vaccinium corymbosum L.) are known to have positive health benefits. The production of blueberry vinegar is one method to preserve this seasonal fruit and allow extended consumption. In this study, blueberry wine acetification was performed with naturally occurring micro-organisms and with an inoculated Acetobacter cerevisiae strain. Acetifications were carried out in triplicate using the Schützenbach method. The successful spontaneous processes took up to 66% more time than the processes involving inoculation. The isolation of acetic acid bacteria (AAB) and the analysis of these AAB using molecular methods allowed the identification of the main genotypes responsible of the blueberry acetification. Although the Acet. cerevisiae strain was the predominant strain isolated from the inoculated process samples, Acetobacter pasteurianus was isolated from samples for both processes and was the only species present in the spontaneous acetification samples. To the best of our knowledge, this is the first report describing the identification and variability of AAB isolated during blueberry acetification. The isolated Acet. pasteurianus strains could be used for large-scale blueberry vinegar production or as a starter culture in studies of other vinegar production methods.

The aim of this work was to analyze the microbiota involved in the traditional vinegar elaboration of strawberry fruit during a spontaneous and inoculated process. In the spontaneous processes, low biodiversity was detected in both alcoholic fermentation (AF) and acetification. Nevertheless, a strain of Saccharomyces cerevisiae and of Acetobacter malorum were selected and tested as starter cultures in the inoculation study. The inoculated processes with these strains were compared with another spontaneous process, yielding a significant reduction in time for AF with a total imposition of the S. cerevisiae strain. The resulting strawberry wine was acetified in different containers (glass and wood) yielding an initial imposition of the A. malorum inoculated strain, although displacement by Gluconacetobacter species was observed in the wood barrels.

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. This work was supported by BMBF within the ChemBiofilm Network at the University of Hamburg and by the Grant GL2010-22152-C03-02 from the Spanish Ministry of Science and Innovation. Collaboration between the two groups was possible through the fellowship AP2009-0843 from the Spanish Ministry of Education, Culture and Sports for M.J. Valera.

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.

Persimmon ( Diospyros kaki ) is a seasonal fruit with important health benefits. In this study, persimmon use in wine and condiment production was investigated using molecular methods to identify the yeast and acetic acid bacteria (AAB) isolated from the alcoholic fermentation and acetification of the fruit. Alcoholic fermentation was allowed to occur either spontaneously, or by inoculation with a commercial Saccharomyces cerevisiae wine strain, while acetification was always spontaneous; all these processes were performed in triplicates. Non- Saccharomyces yeast species were particularly abundant during the initial and mid-alcoholic fermentation stages, but S. cerevisiae became dominant toward the end of these processes. During spontaneous fermentation, S. cerevisiae Sc1 was the predominant strain isolated throughout, while the commercial strain of S. cerevisiae was the most common strain isolated from the inoculated fermentations. The main non- Saccharomyces strains isolated included Pichia guilliermondii , Hanseniaspora uvarum , Zygosaccharomyces florentinus and Cryptococcus sp. A distinct succession of AAB was observed during the acetification process. Acetobacter malorun was abundant during the initial and mid-stages, while Gluconacetobacter saccharivorans was the main species during the final stages of these acetifications. Four additional AAB species, Acetobacter pasteurianus , Acetobacter syzygii , Gluconacetobacter intermedius and Gluconacetobacter europaeus , were also detected. We observed 28 different AAB genotypes, though only 6 of these were present in high numbers (between 25%–60%), resulting in a high biodiversity index.

Three molecular techniques [denaturing gradient gel electrophoresis (DGGE-PCR), restriction fragment length polymorphism of the 16S rRNA gene amplicon (RFLP-PCR 16S rRNA) and real-time PCR (RT-PCR) with SybrGreen and with specific TaqMan-Minor Groove Binder (MGB) probes] were used to identify and monitor acetic acid bacteria (AAB) species during a controlled acetification. This process was initiated by seeding a starter culture comprising a mixture of one strain each of Acetobacter pasteurianus, Gluconacetobacter europaeus and Gluconacetobacter hansenii. Analysis at the species level indicated that the population of A. pasteurianus increased quickly, subsequently acquiring a dominant position, whereas the other two species gradually disappeared. All three methods confirmed this result. When the total AAB population was estimated, the results obtained based on summing the three species by TaqMan-MGB RT-PCR, total AAB RT-PCR and the direct microscopic count method were similar. Using TaqMan-MGB RT-PCR we were able to detect species with populations 3 log units lower than that of the major species and which could not be detected by other methods.

Methyl tert-butyl ether (MTBE) is a widely used fuel ether, which has become a soil and water contaminant. In this study, 12 microbial strains were isolated from gasoline-contaminated soils and selected because of their capacity to grow in MTBE. The strains were identified by 16S/ITS rDNA gene sequencing and screened for their ability to consume MTBE aerobically in a simple mineral solution. Solid phase microoextraction and gas chromatography were used to detect MTBE degradation. High levels of MTBE biodegradation were obtained using resting cells of the bacteria Achromobacter xylosoxidans MCM1/1 (78%), Enterobacter cloacae MCM2/1 (50%), and Ochrobactrum anthropi MCM5/1 (52%) and the fungus Exophiala dermatitidis MCM3/4 (14%). Our phylogenetic analysis clearly shows that bacterial MTBE biodegraders belong to the clade of Proteobacteria. For further insight, MTBE-degrader strains were profiled by denaturing gel gradient electrophoresis (DGGE) of PCR-amplified 16S rRNA gene sequences. This approach could be used to analyse microbial community dynamics in bioremediation processes.

Claudio Hidalgo1, Estibaliz Mateo1, Ana Belen Cerezo2, Maria-Jesús Torija1, Albert Mas11Biotecnologia Enològica, Departament de Bioquimica i Biotecnologia, Facultat d’Enologia, Universitat Rovira i Virgili, Marcel-li Domingo, Tarragona; 2Área de Nutrición y Bromatología, Facultad de Farmacia, Universidad de Sevilla, Sevilla, SpainAbstract: Fruit surplus is common in intensive agriculture in many countries. This ecologic and economic problem requires alternative uses to be found for fruit. The aim of this study was to use surplus fruit to produce vinegar by traditional methods (alcoholic fermentation and acetification) from persimmon and strawberry. The process was performed with naturally occurring microorganisms and compared with inoculated commercial wine yeast for alcoholic fermentation. Alcoholic fermentation proceeded faster when inoculated due to the length of the lag phases observed in spontaneous fermentations. The alcoholic fermentations of strawberry mash were faster than those of persimmon mash. In contrast, acetifications were much faster in persimmon (30 days) than in strawberry (70 days), in the latter some acetifications were not finished. From the technologic point of view, to produce persimmon and strawberry wine and vinegar, it is better to avoid fruit pressing and perform the process with fruit mash. Inoculation is recommended for persimmon and is necessary for strawberry.Keywords: wine, vinegar, fruit seasonings, acetic acid bacteria

The traditional production of wine vinegar is a lengthy process with little or no microbiological control. The aim of this study was to shorten the acetification process via three different strategies: changes in wood type; barrel shape; and the inoculation of an Acetobacter pasteurianus pure culture. The barrel shape was modified by constructing two prototypes with higher liquid-air interface. We compared the changes in acetic acid bacteria (AAB) population dynamics in these barrels with those of a submerged method. The wood type had no effect on the acetification length, whereas the shape of the barrel resulted in a significant shortening of the acetification length. Although the selected AAB strain did not always take over, it reduced the biodiversity of the AAB. The inoculated strain was predominant in oak barrels, whereas in the highly aerated prototypes Gluconacetobacter species (Ga. intermedius and/or Ga. europaeus) displaced A. pasteurianus, as what occurs in the submerged method.

A crucial step in the production of high-quality wine vinegars is ageing in wood. This study aims to determine how wood barrels affect vinegar quality. For this purpose, vinegars were aged in barrels made from acacia, cherry, chestnut and oak wood. Two kinds of vinegars were studied: balsamic and red-wine vinegars. Analysis of samples included total phenol index (TPI), total monomeric anthocyanins (TA), dry extract, and phenolic and furanic compounds by liquid chromatography with photodiode array detection (LC–DAD) in a total of 98 samples. The concentration of HMF, 2-furfuraldehyde, protocatechualdehyde and vanillin significantly increased during ageing. Taxifolin is present in vinegars aged in cherry wood, confirmed by liquid chromatography–mass spectrometry/mass (LC–MS/MS). Hence, it can be a marker of vinegars aged in cherry wood. This is the first time that this compound has been identified in wine vinegars. On the other hand, ellagic acid is a characteristic compound for samples aged in chestnut and oak barrels. An unknown peak was found in all vinegar samples (groups F, T and M) which were aged in acacia wood. Linear discriminant analysis was used to classify samples as a function of the type of wood and ageing time, with phenolic compounds as variables. The linear functions obtained correctly classified – at 100% – red vinegars according to the type of wood. If ageing time (0–365 days) is considered, 62.5% and 100% of the red and balsamic vinegar samples, respectively, were well classified. The effect of wood on vinegar quality is obvious enough to be perceived by the senses in triangle tests. Woody, sweet and vanilla descriptors presented the highest scores for vinegars aged in oak wood. On the other hand, ageing in cherry wood increased markers for red-fruit attributes. These results prove that wood used in vinegar ageing determines both the chemical composition and sensorial properties.

Maria-Jesús Torija1, Estibaliz Mateo1, Carlos-Alfredo Vegas1, Carla Jara1, Angel González1, Montse Poblet1, Cristina Reguant1, Jóse-Manuel Guillamon2, Albert Mas11Biotecnología enológica. Departament de Bioquímica i Biotecnologia, Facultat d’enologia, Universitat Rovira i Virgili, Tarragona, Spain; 2Departamento de Biotecnología de los Alimentos, Instituto de Agroquímica y Tecnología de Alimentos (CSIC), Burjassot, València, SpainAbstract: Traditional vinegar production is a lengthy process which implies high operational risks and jeopardizes the organoleptic characteristics of the final product. In an effort to solve these problems without changing the traditional model, we modified the wood type and thickness of vinegar barrels. We acetified in triplicate in barrels made of acacia, cherry, chestnut, and oak and in three wood thicknesses (15, 20, and 25 mm) in two different vinegar plants. The operating volume was set at 60 L. Reducing wood thickness improved neither maximum acetification velocity or the total length of the process, and in some cases even worsened them. The process took longer in oak barrels than in other types of wood barrel in one of the vinegar plants. Therefore, the choice of wood is a parameter to be considered in the wine vinegar production. Keywords: acacia, cherry, chestnut, oak, acetic acid bacteria

The presence of phenolic compounds has been extensively studied in Sherry and Balsamic vinegars due to their impact on quality but little work has been done on red wine vinegars. Phenolic compounds were monitored during the acetification of red wine vinegars produced by surface culture in different wood barrels (oak, chestnut, acacia and cherry). A total of 166 samples were analysed for phenolic compounds using LC-DAD, the total phenol index (TPI) and the total monomeric anthocyanins (TA). Twelve phenolic compounds were identified corresponding to phenolic acids, flavanols and stilbens. Most phenolic acids did not significantly change their concentrations in the different acetifications. (+)-Catechin and resveratrol glycoside underwent significant decreases during acetification while gallic acid and gallic ethyl ester increased substantially for those vinegars produced in chestnut wood. The concentrations of phenolic compounds were used to build the functions for discriminant analysis. Samples belonging to two wine substrates (groups F and T) were correctly classified with 98.6% (group F) and 100% (group T) for the four types of wood barrels. During acetification a decrease (∼50%) in the content of total monomeric anthocyanins was observed. According to the results of triangle difference test the panel was able to distinguish most of the vinegars according to the different woods they were made in. The results of descriptive sensory analysis show that oak and cherry gave the maximum scores for most of the descriptors.

The present article reports the draft genome sequence of the strain Komagataeibacter europaeus CECT 8546, an acetic acid bacterium characterized by its ability to overproduce cellulose. This species is highly resistant to acetic acid and commonly found during vinegar elaboration.

Twenty Campylobacter jejuni and 16 Campylobacter coli strains isolated from humans and food/animals, including 17 isolates resistant to erythromycin, were analyzed. A combined mismatch amplification mutation assay-PCR technique was developed to detect the mutations A 2074 C and A 2075 G in the 23S rRNA gene associated with erythromycin resistance. All high-level erythromycin-resistant strains examined by DNA sequencing carried the transition mutation A 2075 G, whereas no isolate carried the A 2074 C mutation. No mutations were found among the susceptible and low-level erythromycin-resistant strains.

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. © 2014, Springer-Verlag Berlin Heidelberg.

10.1016/j.foodres.2012.12.055 The low recovery of acetic acid bacteria (AAB) on solid media is one of the biggest hurdles to studying the microbiota present in vinegar production. For this reason, we carried out a comparative analysis between culture-dependent and independent methods to evaluate if the main AAB species isolated by plating are representative of the microbiota present. 265 isolates from two different culture media were identified and characterized by the 16S rRNA gene PCR-RFLP, ERIC-PCR and (GTG)5-rep-PCR. Additionally, a phylogenetic analysis using the 16S rRNA gene was performed. Finally, qualitative and quantitative culture-independent techniques (DGGE-PCR, 16S rRNA gene PCR-RFLP and Real Time PCR) were used for detecting and quantifying the AAB directly from vinegar. In general, the main species recovered by culture-dependent method were the same as those detected by culture-independent methods, confirming that plating could provide a valid initial approach for determining the diversity of the microbiota. The species detected by both methods were Gluconacetobacter europaeus, Gluconacetobacter xylinus and Gluconacetobacter intermedius. Furthermore, plating allowed us to detect some putative new Gluconacetobacter species that would have remained undetected with culture-independent techniques.

A fragment of the gyrA gene was sequenced from 34 isolates of Campylobacter coli , including 23 isolates resistant to ciprofloxacin. All ciprofloxacin-resistant isolates examined by DNA sequencing carried a point mutation at position Thr-86 on the gyrA gene product, involving the replacement of Thr-86 by Ile. A combined PCR-restriction fragment length polymorphism technique using RsaI was developed to detect this mutation.