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Flor yeast velum is a biofilm formed by certain yeast strains that distinguishes biologically aged wines such as Sherry wine from southern Spain from others. Although Saccharomyces cerevisiae is the most common species, 5.8 S-internal transcribed spacer (ITS) restriction fragment length polymorphism analyses have revealed the existence of non- Saccharomyces species. In order to uncover the flor microbiota diversity at a species level, we used ITS (internal transcribed spacer 1)-metabarcoding and matrix-assisted laser desorption/Ionization time of flight mass spectrometry techniques. Further, to enhance identification effectiveness, we performed an additional incubation stage in 1:1 wine:yeast extract peptone dextrose (YPD) before identification. Six species were identified: S. cerevisiae , Pichia manshurica , Pichia membranifaciens , Wickerhamomyces anomalus , Candida guillermondii , and Trichosporon asahii , two of which were discovered for the first time (C. guillermondii and Trichosporon ashaii) in Sherry wines. We analyzed wines where non- Saccharomyces yeasts were present or absent to see any potential link between the microbiota and the chemical profile. Only 2 significant volatile chemicals (out of 13 quantified), ethanol and ethyl lactate, and 2 enological parameters (out of 6 quantified), such as pH and titratable acidity, were found to differ in long-aged wines. Although results show a low impact where the non- Saccharomyces yeasts are present, these yeasts isolated from harsh environments (high ethanol and low nutrient availability) could have a potential industrial interest in fields such as food microbiology and biofuel production. [ABSTRACT FROM AUTHOR]

In this study, we evaluate the performance of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for rapid detection of carbapenemase activity in Enterobacterales in clinical microbiology laboratories during a multicenter networking validation study. The study was divided into three different stages: "software design," "intercenter evaluation," and "clinical validation." First, a standardized procedure with an online software for data analysis was designed. Carbapenem resistance was detected by measuring imipenem hydrolysis and the results were automatically interpreted using the Clover MS data analysis software (Clover BioSoft, Spain). Second, a series of 74 genotypically characterized Enterobacterales (46 carbapenemase-producers and 28 non carbapenemase-producers) were analyzed in 8 international centers to ensure the reproducibility of the method. Finally, the methodology was evaluated independently in all centers during a 2-month period and results were compared with the reference standard for carbapenemase detection used in each center. The overall agreement rate relative to the reference method for carbapenemase resistance detection in clinical samples was 92.5%. The sensitivity was 93.9% and the specificity, 100%. Results were obtained within 60 min and accuracy ranged from 83.3 to 100% among the different centers. Further, our results demonstrate that MALDI-TOF MS is an outstanding tool for rapid detection of carbapenemase activity in Enterobacterales in clinical microbiology laboratories. The use of a simple in-house procedure with online software allows routine screening of carbapenemases in diagnostics, thereby facilitating early and appropriate antimicrobial therapy. [ABSTRACT FROM AUTHOR]

The objectives of this study were to report on the antimicrobial susceptibility of 276 clinically significant anaerobic bacteria belonging to the major genera isolated between May 2017 and November 2018 in a tertiary hospital in Granada (Spain) and to describe key clinical features of the patients. Species identification was performed by MALDI-TOF MS. Antimicrobial susceptibility tests were performed against penicillin, amoxicillin-clavulanic, imipenem, meropenem, moxifloxacin, clindamycin, metronidazole, vancomycin, and piperacillin-tazobactam using the gradient diffusion technique and EUCAST breakpoints (except for moxifloxacin). The most frequent anaerobes were Bacteroides (29.7%; n = 82), Clostridioides difficile (15.9%, n = 44), Prevotella (10.8%, n = 30), and Propionibacterium (10.7%, n = 25). Metronidazole was not universally active against all genera tested, and some isolates showed resistance to this drug. Almost all tested anaerobes were susceptible to carbapenems and amoxicillin-clavulanate except for Clostridioides difficile (resistance rate of 94%) and Bacteroides (19%), respectively. High overall resistance rates to clindamycin were observed, especially for genera Finegoldia (54%), Bacteroides (49%), and Prevotella (40%). Resistance rates to carbapenems and amoxicillin-clavulanate were very low for the majority of tested genera but were high for Clostridioides difficile and Bacteroides spp., respectively. Resistance to clindamycin was very high, especially for Bacteroides , Finegoldia magna , Prevotella and Peptoniphilus. Routine antimicrobial susceptibility testing for anaerobes contributes information on the global situation and allows empirical therapies to be selected in accordance with local data on resistant strains. • Metronidazole was not universally active against all anaerobes. • Resistance rates to carbapenems and amoxicillin-clavulanate were very low. • Resistance rates of almost all genera to clindamycin were very high. • Routine antimicrobial susceptibility testing is highly recommended. [ABSTRACT FROM AUTHOR]

Bacteroides pyogenes is a Gram-negative obligate anaerobe, saccharolytic, non-motile, non-pigment-producing and non-spore-forming rod. Reports of human infections caused by B. pyogenes are scarce, with approximately 30 cases documented in scientific literature. The aim of this study was to describe the clinical characteristics of 8 different patients and to study the in vitro antibiotic susceptibility of the strains, as well as to evaluate the in vivo activity of the prescribed treatments. We conducted a descriptive retrospective study by searching all B. pyogenes isolates from January 2010 to March 2023 at the Basurto University Hospital. This included all cases, both in monomicrobial or polymicrobial cultures. Among the eight patients, three experienced severe infections such as bacteremia and osteomyelitis. All the strains were susceptible to amoxicillin/clavulanic acid, piperacillin/tazobactam, imipenem, meropenem, clindamycin, metronidazole and moxifloxacin. • B. pyogenes is a commensal of the oral cavity of cats and dogs. • In this work, half of the B. pyogenes infections were related to cat bites. • Identification of B. pyogenes isolates was succesfully performed by MALDI-TOF MS. • B. pyogenes isolates showed low MICs to all tested antibiotics. [ABSTRACT FROM AUTHOR]

Introduction: The correct identification of the species within the Candida parapsilosis complex has become relevant due to the resistance of Candida metapsilosis to antifungals. We describe the characteristics of the Candida parapsilosis complex isolates, with respect to antifungal resistance and biofilm formation., Methods: We perform a descriptive cross-sectional study in 30 strains, collected in a tertiary hospital. All strains, were identified by Vitek2, Vitek-MS™ systems and by ITS sequencing. The antifungal susceptibility profile was obtained with Sensititre™ panels, while biomass production and metabolic activity were quantified by means of crystal violet and XTT reduction assay, respectively., Results: There was a 100% correlation between Vitek-MS™ and ITS sequencing. All isolates were susceptible to the nine antifungals tested. The metabolic activity and biomass production tests did not show any difference among the subtypes., Conclusions: The Vitek-MS™ system provides acceptable identification. We did not find significant differences neither in azole resistance nor in biofilm formation., (Copyright © 2021 Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. Published by Elsevier España, S.L.U. All rights reserved.)

Abstract: Three ceftazidime-resistant strains isolated from the sewage water of a municipal hospital in Palma de Mallorca, Spain, were analysed phenotypically and genotypically to clarify their taxonomic positions. Sequence determinations and phylogenetic analyses of the 16S rRNA genes indicated that strains CS20.3T, CS39 and CS41 were affiliated with the species of the alphaproteobacterial genus Brevundimonas, most closely related to B. bullata, B. diminuta, B. naejangsanensis and B. terrae. Additional sequences analyses of the ITS1 region of the rRNA operon and the genes for the housekeeping enzymes DNA gyrase β-subunit and RNA polymerase β-subunit, genomic DNA–DNA hybridisation similarities, cell fatty acid profiles and physiological and biochemical characterizations supported the recognition of CS20.3T (CCUG 58127T =CECT 7729T) as a distinct and novel species, for which the name Brevundimonas faecalis sp. nov. is proposed. Strains CS39 and CS41 were ascribed to the species B. diminuta. [Copyright &y& Elsevier]

The objectives of this study were to report 10 episodes of clinically significant bacteremia caused by species of the genus Anaerococcus isolated between July 2018 and February 2021 from the microbiology laboratory of a tertiary hospital in Granada (Spain). None of the isolates were identified by MALDI-TOF MS, and the definitive species identification was performed by 16 S rRNA gene sequencing. No reference spectra of the Anaerococcus species were present in the MALDI-TOF MS database. Eight isolates were finally identified as A. octavius, one isolate as A. tetradius and the other as A. urinomassiliensis. The majority of these infections were seen in patients aged >70 years. Risk factors for anaerobic infection were observed in eight patients, especially diabetes mellitus, surgery, and the presence of cancer. Fever was present in all patients. Three patients died, but only one death was attributed to the infection. Mean detection time of positive blood cultures was 47.5 h (range 24-92 h). Antimicrobial susceptibility to penicillin, amoxicillin-clavulanate, imipenem, moxifloxacin, clindamycin, metronidazole, and piperacillin-tazobactam was tested using the gradient diffusion technique and EUCAST breakpoints (except for moxifloxacin). No resistance to amoxicillin-clavulanate, metronidazole, imipenem, or piperacillin-tazobactam was detected; however, the majority of isolates were resistant to clindamycin. When MALDI-TOF MS does not provide a correct identification at genus or species level, as in some isolates of Gram-positive anaerobic cocci, microbiologists should perform an additional confirmatory technique, such as gene sequencing analysis, to obtain a definitive diagnosis., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Several artisanal cheeses are elaborated in European countries, being commonly curdled with rennets of animal origin. However, in some Spanish regions some cheeses of type "Torta" are elaborated using Cynara cardunculus L. rennets. Two of these cheeses, "Torta del Casar" and "Torta de Trujillo", are elaborated in Cáceres province with ewe's raw milk and matured over at least 60 days without starters. In this work, we identified the lactic acid bacteria present in these cheeses using MALDI-TOF MS and pheS gene analyses, which showed they belong to the species Lactobacillus curvatus, Lactobacillus diolivorans, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactococcus lactis and Leuconostoc mesenteroides. The pheS gene analysis also allowed the identification of the subspecies La. plantarum subsp. plantarum, La. paracasei subsp. paracasei and Le. mesenteroides subsp. jonggajibkimchii. Low similarity values were found in this gene for some currently accepted subspecies of Lc. lactis and for the two subspecies of La. plantarum, and values near to 100% for the subspecies of Le. mesenteroides and La. paracasei. These results, which were confirmed by the calculated ANIb and dDDH values of their whole genomes, showed the need to revise the taxonomic status of these species and their subspecies. [ABSTRACT FROM AUTHOR]

The objectives of this study were to report those variables which are readily identifiable at the bedside and that are able to predict mortality in patients with bacteraemia caused by anaerobes. Patients with clinically significant anaerobic bacteraemias detected between January 2016 and December 2019 in a tertiary hospital in Granada (Spain) were retrospectively included. Species identification was performed by MALDI-TOF MS and/or molecular methods. Finally, 136 cases of anaerobic bacteraemia were included, being the most frequent anaerobes Bacteroides (45.5%; n = 62), Clostridium (24.2%, n = 33), and Gram-positive anaerobic cocci (16.1%, n = 22). Crude mortality was 25.7%, corresponding to 35 patients who died, with 82.8% of deaths directly attributable to bacteraemia. A multivariable logistic regression model with non-parametric bootstrap estimation identified three variables that were independently and significantly associated with an increased risk of death: 1) hospitalization in the intensive care unit; 2) septic shock; and 3) presence of any kind of cancer. These variables were as recorded at the time that the first positive blood culture was obtained. An index score, obtained from these variables, was calculated and divided patients into two groups with increasing likelihood of mortality resulting from anaerobic bacteraemia. The sensitivity and specificity of a prediction of death based on this model were 65.2% and 97%, respectively., Competing Interests: Declaration of competing interest Authors declare no conflict of interest., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Flea identification is a significant issue because some species are considered as important vectors of several human pathogens that have emerged or re-emerged recently, such as Bartonella henselae (Rhizobiales: Bartonellaceae) and Rickettsia felis (Rickettsiales: Rickettsiaceae). Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been evaluated in recent years for the identification of multicellular organisms, including arthropods. A preliminary study corroborated the usefulness of this technique for the rapid identification of fleas, creating a preliminary database containing the spectra of five species of flea. However, longterm flea preservation in ethanol did not appear to be an adequate method of storage in the context of specimen identification by MALDI-TOF MS profiling. The goal of the present work was to assess the performance of MALDI-TOF MS in the identification of seven flea species [Ctenocephalides felis (Siphonaptera: Pulicidae), Ctenocephalides canis, Pulex irritans (Siphonaptera: Pulicidae), Archaeopsylla erinacei (Siphonaptera: Pulicidae), Leptopsylla taschenbergi (Siphonaptera: Ceratophyllidae), Stenoponia tripectinata (Siphonaptera: Stenoponiidae) and Nosopsyllus fasciatus (Siphonaptera: Ceratophyllidae)] collected in the field and stored in ethanol for different periods of time. The results confirmed that MALDI-TOF MS can be used for the identification of wild fleas stored in ethanol. Furthermore, this technique was able to discriminate not only different flea genera, but also the two congeneric species C. felis and C. canis., (© 2018 The Royal Entomological Society.)

We describe for the first time a non-symbiotic species of the recently described genus Neorhizobium, lacking nodulation or nitrogen fixation genes. The strains were isolated from a dryland agricultural soil in southern Spain where no record of legume cultivation is available, thus we propose the name Neorhizobium tomejilense sp. nov. (type strain T17_20 T , LMG 30623 T and CECT 9621 T ). N. tomejilense exhibit a clear distinct lineage from the other Neorhizobium species, Neorhizobium galegae, Neorhizobium alkalisoli and Neorhizobium huautlense, based on polyphasic evidence. Phylogenetic marker analysis of 16S rDNA, atpD, glnII, recA, rpoB and thrC genes and genomic identity data derived from the draft genomic sequences showed that N. tomejilense strains clearly separated from the other Neorhizobium species and that N. galegae represents the closest species, with Average Nucleotide Identities (ANIb) ranging from 90% (for type strain HAMBI 540 T ) to just under 95.0% (for two N. galegae sv. officinalis strains). Genomes from N. galegae and N. tomejilense, however, clearly differed in important traits, such as the number of rRNA operon copies or the number of tRNAs. Phenotypic characterisation of N. tomejilense also displayed differences with the other Neorhizobium species. Whole-cell matrix-assisted laser-desorption time-of-flight mass spectrometry (WC MALDI-TOF-MS) fingerprint analysis and the dendrogram derived from the fingerprint profiles, showed a clearly distinct group formed by the three N. tomejilense isolates (T17_20 T , T20_22 and T11_12) from the other Neorhizobium especies., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

Genus Bradyrhizobium includes slow growing bacteria able to nodulate different legumes as well as species isolated from plant tumours. The slow growth presented by the members of this genus and the phylogenetic closeness of most of its species difficults their identification. In the present work we applied for the first time Matrix-Assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometry (MALDI-TOF MS) to the analysis of Bradyrhizobium species after the extension of MALDI Biotyper 2.0 database with the currently valid species of this genus. With this methodology it was possible to identify strains belonging to phylogenetically closely related species of genus Bradyrhizobium allowing the discrimination among species with rrs gene identities higher than 99%. The application of MALDI-TOF MS to strains isolated from nodules of different Lupinus species in diverse geographical locations allowed their correct identification when comparing with the results of rrs gene and ITS analyses. The nodulation of Lupinus gredensis, an endemic species of the west of Spain, by B. canariense supports the European origin of this species., (Copyright © 2013. Published by Elsevier GmbH.)