Rapid detection by MALDI-TOF MS of isolates from cystic fibrosis patients belonging to the epidemic clones Achromobacter xylosoxidans ST137 or Achromobacter ruhlandii DES.

Objective: Achromobacter spp. are increasingly reported among cystic fibrosis patients. Genotyping requires time consuming methods such as Multilocus-Sequence-Typing or Pulsed-Field-Gel-Electrophoresis. Therefore, data on the prevalence of the multiresistant epidemic clones, especially A. xylosoxidans ST137 (AxST137) and the Danish Epidemic Strain A. ruhlandii (DES) are lacking. We recently developed and published a database for Achromobacter species identification by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS, Bruker Daltonics). The aim of this study was to evaluate the ability of the MALDI-TOF MS to distinguish these multiresistant epidemic clones within Achromobacter species. Methods: All the spectra of A.xylosoxidans (n=1571) and A.ruhlandii (n=174) used to build the local database were analysed by ClinProTools™, MALDI Biotyper® PCA, MALDI Biotyper ® dendrogram and flexAnalysis™ softwares for biomarker peaks detection. Two-hundred-two isolates (including 48 isolates of AxST137 and 7 of DES) were tested. Results: Specific biomarker peaks were identified: absent peak at m/z 6651 for AxST137 isolates and present peak at m/z 9438 for DES isolates. All tested isolates were well typed by our local database and clustered within distinct groups (ST137 or non-ST137 and DES or non-DES) no matter the MALDI-TOF software or only by simple visual inspection of the spectra by any user. Conclusions: The use of MALDI-TOF MS allowed identifying isolates of A. xylosoxidans belonging to the AxST137 clone which spread in France and Belgium (the Belgian epidemic clone) and of A. ruhlandii belonging to the DES clone. This tool will help implementation of segregation measures to avoid inter-patient transmission of these resistant clones.

ЭТИОЛОГИЯ ВНЕБОЛЬНИЧНЫХ ПНЕВМОНИЙ В ПЕРИОД ЭПИДЕМИЧЕСКОГО РАСПРОСТРАНЕНИЯ COVID-19 В РОСТОВСКОЙ ОБЛАСТИ

Введение. Пандемия COVID-19 в 2020 году внесла свой вклад в эпидемиологию респираторных инфекций. Важным является своевременное проведение дифференциальной диагностики COVID-19 и сезонных острых респираторных заболеваний. У пациентов с новой коронавирусной инфекцией возрастает риск развития госпитальной пневмонии. Актуальным является анализ особенностей циркуляции резистентных к антибактериальным химиопрепаратам штаммов возбудителей внутрибольничных инфекций. Цель – изучение этиологической структуры внебольничных пневмоний в период эпидемического распространения COVID-19, оценка рисков присоединения возбудителей пневмоний, связанных с оказанием медицинской помощи. Материалы и методы: Исследовали биологический материал от 446 пациентов с диагнозом «внебольничная пневмония», находившихся на амбулаторном лечении или в стационарах г. Ростова-на-Дону. Верификация респираторных вирусов, включая РНК SARS-CoV-2, а также M. pneumoniae, C. pneumoniae, L. pneumophila выполнена методом полимеразной цепной реакции в мазках носоглотки. Бактериологический анализ мокроты проводили с использованием дифференциально-диагностических сред, идентификацию выделенных патогенов осуществляли с помощью времяпролетной масс-спектрометрии на приборе Autoflex (Bruker Daltonics) c программным обеспечением BioTyper 3,0. Результаты и обсуждение. В период распространения новой коронавирусной инфекции в Ростовской области доля положительных результатов на SARS-CoV-2 среди пациентов с диагнозом внебольничная пневмония составляет 35,6 %. Частота микстинфекций вирусной природы достоверно не отличалась среди пациентов с лабораторно подтвержденным диагнозом COVID-19 и пациентов с отрицательным результатом на SARS-CoV-2 (25,9 % и 26,2 %, соответственно). В структуре микробиоты превалировали грибы рода Candida и плазмокоагулирущие стафилококки. Достоверно чаще от пациентов с лабораторно подтвержденным COVID-19 изолировали культуры неферментирующих грамотрицательных бактерий. У 51,6% пациентов, проходивших лечение в стационаре, отмечено вторичное коинфицирование, вероятно связанное с объектами внешней среды или с передачей инфекции от персонала. Передача ИСМП между пациентами не установлена.

Features of Etiology of Community-Acquired Pneumonia Associated with COVID-19

Objective: comparative study of the etiological structure of community-acquired pneumonia in SARSCoV-2 “+”and SARS-CoV-2 “-“ patients who sought help from medical organizations in the Rostov Region.Materials and methods. Biological material from 508 patients diagnosed with community-acquired pneumonia who were on outpatient treatment or in hospitals in Rostov-on-Don was studied. Verification of respiratory viruses, including SARS-CoV-2 RNA, as well as M. pneumoniae, C. pneumoniae, and L. pneumophila was performed by polymerase chain reaction in nasopharyngeal smears. Bacteriological analysis of sputum was carried out using differential diagnostic media, identification of isolated pathogens was carried out using time-of-flight mass spectrometry on Autoflex (Bruker Daltonics) with BioTyper 3.0 software.Results and discussion. During the spread of a new coronavirus infection in the Rostov Region, the main etiological agent of community-acquired pneumonia is the new SARS-CoV-2 coronavirus. Specific character of pneumonia in patients with laboratory-confirmed COVID-19 is a higher incidence of mixed infection of both viral and bacterial etiology. Against the background of detection of a new coronavirus infection in patients with pneumonia, cases of detection of other types of coronaviruses have been registered (HKU-1,OC43, HL-63 and 229Е). The most common etiological agent of bacterial pneumonia in patients was Streptococcus spp., both in patients with COVID-19-associated pneumonia and in patients who tested negative for SARS-CoV-2. Coronavirus patients represent a high risk group for the development of mycotic lung lesions.

Evaluation of Rapid Sepsityper® protocol and specific MBT-Sepsityper module (Bruker Daltonics) for the rapid diagnosis of bacteremia and fungemia by MALDI-TOF-MS

During bloodstream infections, rapid adaptation of empirical treatment according to the microorganism identified is essential to decrease mortality. The aim of the present study was to assess the microbiological performances of a new rapid version of the Sepsityper® kit (Bruker Daltonics) allowing identification of bacteria and yeast by MALDI-TOF mass spectrometry directly from positive blood cultures in 10 min and of the specific MBT-Sepsityper module for spectra analysis, designed to increase identification performance. Identification rates were determined prospectively on 350 bacterial and 29 fungal positive blood cultures, and compared to conventional diagnostic method. Our rapid diagnosis strategy (Rapid Sepsityper® protocol: one spot with and one without formic acid extraction step) combined to MBT-Sepsityper module provided 65.4%, 78.9% and 62% reliable identification to the species level of monomicrobial positive blood cultures growing respectively Gram-positive, Gram-negative bacteria or yeast. Importantly, identification rates of Gram-positive bacteria were higher in anaerobic than in aerobic bottles (77.8% vs 22.2%; p = 0.004), if formic acid extraction step was performed (60.8% vs 39.2%; p = 1.8e−6) and if specific MBT-Sepsityper module was used (76.2% vs 61.9%, p = 0.041) while no significant differences were observed for Gram-negative bacteria. For yeasts identification, formic acid extraction step improved rapid identification rate by 37.9% while the specific MBT-Sepsityper module increased overall performances by 38%, providing up to 89.7% reliable identification if associated with the standard Sepsityper® protocol. These performances, associated with a reduce turnaround time, may help to implement a rapid identification strategy of bloodstream infections in the routine workflow of microbiology laboratories.

Identificación de aislamientos de Candida auris recuperados a través de la vigilancia por laboratorio en Colombia: un reto para el diagnóstico

Objetivo: Comparar los resultados obtenidos de diferentes sistemas de identificación de C. auris.Métodos: Análisis descriptivo con datos recopilados durante 2016-19 mediante la vigilancia nacional. Se evaluaron los resultados generados por los sistemas MicroScan, Phoenix BD, VITEK 2 y MALDI-TOF MS de instituciones hospitalarias de 843 aislamientos clínicos sospechosos de C. auris remitidos al INS y se compararon con los resultados generados de confirmación a través de MALDI- TOF MS (Bruker Daltonics) o PCR. Resultados: De los 843 aislamientos clínicos remitidos al INS, el 81,7% fueron confirmados como C. auris mediante MALDI- TOF MS o PCR en el INS y el resto, 18,3%, fueron identificados como otras especies de Candida spp. Las identificaciones correctas enviadas por los laboratorios representaron el 42,4%. MicroScan identificó C. auris principalmente como C. haemulonii, C. guilliermondii, C. albicans y C. famata; Phoenix BD, VITEK 2 y MALDI-TOF MS identificó C. auris como C. haemulonii. Discusión: Estudios señalan que C. auris exhibe una estrecha relación filogenética con C. haemulonii. Las identificaciones discrepantes pueden darse debido a que las bases de datos de los sistemas de diagnóstico son limitadas para este patógeno. Las deficiencias de los sistemas comerciales para la identificación de C. auris deben ser complementados con otros sistemas como MALDI-TOF MS o pruebas moleculares.

Isolation and Identification of Lactic Acid Bacteria in Wine Production by MALDI-TOF MS Biotyper

The aim of this study was to identify lactic acid bacteria (LAB) in grapes, must and wines. A total amount of 90 samples including grape (n = 30), must (no = 30) and wine (no = 30) were collected from vineyards in Slovakia. LAB were used cultured on MRS agar with subsequent confirmation with MALDI-TOF mass spectrometry (Bruker Daltonics). Altogether, 904 isolates were identified. Members of the family Lactobacillaeceae were the most abundant in grape (60%), must (46%) and wine (51%). Lactobacillus, Lactococcus, Leuconostoc, Pediococcus and Weissella genera and 27 species of LAB were isolated from the examined samples. Leuconostoc mesenteroides spp. mesenteroides was the most abundant species in grape, must and wine.

Identification of Yeasts with Mass Spectrometry during Wine Production

The aim of the present study was to identify yeasts in grape, new wine “federweisser” and unfiltered wine samples. A total amount of 30 grapes, 30 new wine samples and 30 wine samples (15 white and 15 red) were collected from August until September, 2018, from a local Slovak winemaker, including Green Veltliner (3), Mūller Thurgau (3), Palava (3), Rhein Riesling (3), Sauvignon Blanc (3), Alibernet (3), André (3), Blue Frankish (3), Cabernet Sauvignon (3), and Dornfelder (3) grapes; federweisser and unfiltered wine samples were also used in our study. Wort agar (WA), yeast extract peptone dextrose agar (YPDA), malt extract agar (MEA) and Sabouraud dextrose agar (SDA) were used for microbiological testing of yeasts. MALDI-TOF Mass Spectrometry (Microflex LT/SH) (Bruker Daltonics, Germany) was used for the identification of yeasts. A total of 1668 isolates were identified with mass spectrometry. The most isolated species from the grapes was Hanseniaspora uvarum, and from federweisser and the wine—Saccharomyces cerevisiae.

Integrated Analytical Tools for Accessing Acridones and Unrelated Phenylacrylamides from Swinglea glutinosa

In natural product studies, the purification of metabolites is an important challenge. To accelerate this step, alternatives such as integrated analytical tools should be employed. Based on this, the chemical study of Swinglea glutinosa (Rutaceae) was performed using two rapid dereplication strategies: Target Analysis (Bruker Daltonics®, Bremen, Germany) MS data analysis combined with MS/MS data obtained from the GNPS platform. Through UHPLC-HRMS data, the first approach allowed, from crude fractions, a quick and visual identification of compounds already reported in the Swinglea genus. Aside from this, by grouping compounds according to their fragmentation patterns, the second approach enabled the detection of eight molecular families, which presented matches for acridonic alkaloids, phenylacrylamides, and flavonoids. Unrelated compounds for S. glutinosa have been isolated and characterized by NMR experiments, Lansamide I, Lansiumamide B, Lansiumamide C, and N-(2-phenylethyl)cinnamamide.