Comparison of sample preparation methods for the identification of Staphylococcus Aureus by MALDI-FOF MS

Coagulase-positive staphylococci include 3 species, Staphylococcus aureus, S. hyicus and S. intermedius. Of these three species, S. aureus is the most well-known human pathogen. S. aureus is part of the human and animal normal microbiota, however, it is capable of producing several staphylococcal enterotoxins (SEs) that cause intoxication symptoms of varying intensity in humans after consuming contaminated food. Selective media which are used for the determination of coagulase-positive staphylococci from foods are not able to identify isolates at a species. With the MALDI-TOF MS technique, we can identify S. aureus cheaper and faster than by using molecular methods. This paper describes the results of the study of the presence of coagulase-positive staphylococci and S. aureus in many food products, and the application of three sample preparation methods: direct sample preparation, formic acid suspension and ethanol extraction

Investigation of the antibiotic resistance of staphylococcus species isolated from foods

The presence of methicillin-resistant Staphylococcus aureus (MRSA) strains in the food chain has been confirmed by several studies in the European Union, but there are only limited data available in Hungary. The objective of the present study was to investigate the antibiotic resistance of Staphylococcus strains isolated from foods, using classical microbiological, molecular biological methods and the MALDI-TOF-MS technique, as well as the multi-locus sequence typing (MLST) of antibiotic resistant strains. During the study, 47 coagulase-positive (CPS) and 30 coagulase-negative (CNS) Staphylococcus isolates were collected. In the course of the MALDI-TOF-MS investigations, all CPS isolates (n=47) were found to be S. aureus species, while 8 different species were identified in the case of the CNS strains. Methicillin resistance was confirmed in two S. aureus strains, one of which had a sequence type not yet known, while the other MRSA strain was type ST398, which is the most common type of MRSA strain isolated from farm animals in the EU/EEA

Applicability of diatom metabarcoding in the ecological status assessment of Hungarian lotic and soda pan habitats

Diatoms, eukaryotic algae with silica cell wall have been proved to be reliable bioindicators and are applied in ecological status assessment of aquatic ecosystems using indices calculated from pollution sensitivity and indicator values of species. Traditional method of identification of diatom species is based on morphological features of frustule that requires in-depth taxonomical knowledge and expertise. Metabarcoding that combines barcodes and high-throughput sequencing offers a promising alternative. In this pilot study we tested the applicability of metabarcoding of benthic diatom assemblages for ecological status assessment of water bodies in Hungary comparing its performance to that of morphology-based identification of species. Samples from 78 rivers and streams arranged along a trophic gradient and 14 soda pans representing unique types of habitats were investigated. Sequences of the barcode region of rbcL gene suggested by Vasselon et al. (2017) were acquired revealing 1135 diatom amplicon sequence variants (ASVs) of which 54% could be assigned at species level using Diat.barcode (Rimet et al. 2019) as reference database. Morphological investigation detected more species and intraspecific taxa in the lotic and lentic samples (413 and 78, respectively) than metabarcoding (190 and 75). Correspondence between taxa detected based on morphology and DNA sequences was relatively low (on average 24% in lotic and 26% in lentic samples) but considerably increased (on average 66% and 56%, respectively) when taking into account only the taxa reaching higher than 5% relative abundance in microscopy counting. The differences mainly derived from that a considerable portion (59% and 32%, respectively) of the morphologically identified taxa were not recorded in the reference database. Community structure of samples from both running and standing waters based on microscopy and DNA sequence analysis showed significant correlation as revealed using Mantel test. For rivers and streams diatom index IPS (Specific Pollution Sensitivity Index, (Coste in Cemagref, 1982) for soda pans indices H (Ziemann et al., 2001) and IBD (Indice Biologique Diatomées, Lenoir and Coste, 1996) were calculated from the results of the two aspects. Strong correlation was found between morphology- and sequence-based indices and in about half of the samples, the ecological status class obtained with the two methods coincided. Our results suggest that metabarcoding inheres a great opportunity and could be successfully applied in benthic diatom-based ecological status assessment of Hungarian waters after the reference database is supplemented with taxa frequently occurring in these habitats. ReferencesCemagref, 1982. Etude des méthodes biologiques quantitativesd'appréciation de la qualité des eaux, Rapport Division Qualité des Eaux Lyon. Agence financière de Bassin Rhône-Méditerranée-Corse, Pierre-Bénite, France.Lenoir, A., Coste, M., 1996. Development of a practical diatom index of overall water quality applicable to the French National Water Board Network. Presented at the International symposium, Volksbildungsheim Grilhof Vill, AUT, 17-19 September 1995, Universität Innsbruck, pp. 29–43.Rimet, F., Gusev, E., Kahlert, M., Kelly, M.G., Kulikovskiy, M., Maltsev, Y., Mann, D.G., Pfannkuchen, M., Trobajo, R., Vasselon, V., Zimmermann, J., Bouchez, A., 2019. Diat.barcode, an open-access curated barcode library for diatoms. Sci. Rep. 9, 15116.Vasselon, V., Rimet, F., Tapolczai, K., Bouchez, A., 2017. Assessing ecological status with diatoms DNA metabarcoding: Scaling-up on a WFD monitoring network (Mayotte island, France). Ecol. Indic. 82, 1–12.Ziemann, H., Kies, L., Schulz, C.-J., 2001. Desalinization of running waters: III. Changes in the structure of diatom assemblages caused by a decreasing salt load and changing ion spectra in the river Wipper (Thuringia, Germany). Limnologica 31, 257–280

Élelmiszerekből izolált staphylococcus fajok antibiotikum rezisztencia vizsgálata

A methicillin-rezisztens Staphylococcus aureus (MRSA) törzsek élelmiszerláncban előforduló jelenlétét számos tanulmány igazolta az Európai Unióban, azonban Magyarországon kevés adat áll rendelkezésünkre ezzel kapcsolatban. Jelen vizsgálat célja az élelmiszerekből izolált Staphylococcus törzsek antibiotikum rezisztenciájának vizsgálata klasszikus mikrobiológiai, molekuláris biológiai módszerekkel és MALDI-TOF-MS technikával, továbbá az antibiotikum rezisztens törzsek multilókusz szekvencia tipizálása (MLST). A vizsgálat során 47 koaguláz-pozitív (CPS) és 30 koaguláz-negatív Staphylococcus (CNS) izolátumot gyűjtöttünk. A MALDI-TOF-MS vizsgálat során minden CPS izolátum (n=47) S. aureus fajnak bizonyult, míg a CNS törzsek esetében 8 különböző fajt azonosítottunk. Két S. aureus törzs esetében állapítottunk meg methicillin-rezisztenciát, amelyek közül az egyik izolátum eddig még nem ismert szekvencia típusba, míg a másik MRSA törzs az ST398 típusba tartozott, amely a mezőgazdasági haszonállatokból izolált MRSA törzsek leggyakoribb típusa az EU/EGT területén

Wastewater based epidemiology in countries with poor wastewater treatment - SARS-CoV-2 RNA in surface waters

Background Wastewater-based epidemiology surveillance of COVID-19 and other outbreaks in the future is a challenge for developing countries, as the majority of households are not connected to sewerage systems. In December 2019, we have detected SARS-CoV-2 RNA in the Danube River at a site that is severely affected by wastewater of Belgrade. Considering that rivers are much more complex systems in comparison to wastewater, additional efforts are needed to address all the factors which might influence the adoption of WBE as an alternative of targeting raw wastewater. Objectives The major objective of this study was to provide a more detailed insight in the potential of SARS-CoV-2 surveillance in Serbian surface waters under consideration of epidemiological, microbiological, physico-chemical and hydro-morphological parameters for epidemiological purposes. Methodology Water samples were collected at 12 sites at Sava and Danube Rivers in the Belgrade city area during the fourth COVID-19 wave in Serbia that started in late February 2021. SARS-CoV-2 RNA was quantified using RT-qPCR with primer sets targeting nucleocapsid (N1 and N2) and envelope (E) protein genes. Microbiological (standard fecal indicator bacteria and microbial faecal source tracking markers), epidemiological, physico-chemical and hydro-morphological parameters were analysed in parallel. Results Out of 44 samples analyzed, 31 were positive for at least one of the target regions of SARS-CoV-2. The results indicated that surveillance of SARS-CoV-2 RNA in surface waters in context with the large amount of epidemiological and environmental metadata can be used as epidemiological early-warning tool in countries with poor wastewater treatment

Making Waves:Collaboration in the time of SARS-CoV-2 - rapid development of an international co-operation and wastewater surveillance database to support public health decision-making

The presence of SARS-CoV-2 RNA in wastewater was first reported in March 2020. Over the subsequent months, the potential for wastewater surveillance to contribute to COVID-19 mitigation programmes has been the focus of intense national and international research activities, gaining the attention of policy makers and the public. As a new application of an established methodology, focused collaboration between public health practitioners and wastewater researchers is essential to developing a common understanding on how, when and where the outputs of this non-invasive community-level approach can deliver actionable outcomes for public health authorities. Within this context, the NORMAN SCORE “SARS-CoV-2 in sewage” database provides a platform for rapid, open access data sharing, validated by the uploading of 276 data sets from nine countries to-date. Through offering direct access to underpinning meta-data sets (and describing its use in data interpretation), the NORMAN SCORE database is a resource for the development of recommendations on minimum data requirements for wastewater pathogen surveillance. It is also a tool to engage public health practitioners in discussions on use of the approach, providing an opportunity to build mutual understanding of the demand and supply for data and facilitate the translation of this promising research application into public health practice.</p

Making Waves : Collaboration in the time of SARS-CoV-2-rapid development of an international co-operation and wastewater surveillance database to support public health decision-making

The presence of SARS-CoV-2 RNA in wastewater was first reported in March 2020. Over the subsequent months, the potential for wastewater surveillance to contribute to COVID-19 mitigation programmes has been the focus of intense national and international research activities, gaining the attention of policy makers and the public. As a new application of an established methodology, focused collaboration between public health practitioners and wastewater researchers is essential to developing a common understanding on how, when and where the outputs of this non-invasive community-level approach can deliver actionable outcomes for public health authorities. Within this context, the NORMAN SCORE "SARS-CoV-2 in sewage" database provides a platform for rapid, open access data sharing, validated by the uploading of 276 data sets from nine countries to-date. Through offering direct access to underpinning meta-data sets (and describing its use in data interpretation), the NORMAN SCORE database is a resource for the development of recommendations on minimum data requirements for wastewater pathogen surveillance. It is also a tool to engage public health practitioners in discussions on use of the approach, providing an opportunity to build mutual understanding of the demand and supply for data and facilitate the translation of this promising research application into public health practice. (C) 2021 Elsevier Ltd. All rights reserved.Peer reviewe

Examination of the usability of the MALDI-TOF method in sunflower genetic identity analyses

Economically, one of our most important crop is the sunflower. Regarding its production it is essential to use top quality seeds. As the conditions of seed certification and the certification parameters are regulated by laws and ordinances, it is highly important to farmers and seed producers to detect seeds of low quality or dubious origins.Nowadays, the examination of the cultivar homogenity of sunflower is based on a reference method of the International Seed Testing Association Rules International (ISTA), Chapter 8. This standard reference method uses ultrathin poliakrilamid isoelectric focusing gel electrophoresis (IEF-UTL, or simply IEF). With this work we have set out to compare the results of MALDI-TOF to the test results – used as reference results – achieved by this reference method. The aim is to develop a new, quick, cheap and reliable method. In this article I summarized the results of some of the experiments that will provide basis for my further work.During our previous experiments we have concluded that out of four extracting agents we can get the most protein markers using NaCl acid buffer, 1 propanol buffer