Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease (Mpro) autocatalytically releases itself out of the viral polyprotein to form a fully active mature dimer in a manner that is not fully understood. Here, we introduce several tools to help elucidate differences between cis (intramolecular) and trans (intermolecular) proteolytic processing and to evaluate inhibition of precursor Mpro. We found that many mutations at the P1 position of the N-terminal autoprocessing site do not block cis autoprocessing but do inhibit trans processing. Notably, substituting the WT glutamine at the P1 position with isoleucine retains Mpro in an unprocessed precursor form that can be purified and further studied. We also developed a cell-based reporter assay suitable for compound library screening and evaluation in HEK293T cells. This assay can detect both overall Mpro inhibition and the fraction of uncleaved precursor form of Mpro through separable fluorescent signals. We observed that inhibitory compounds preferentially block mature Mpro. Bofutrelvir and a novel compound designed in-house showed the lowest selectivity between precursor and mature Mpro, indicating that inhibition of both forms may be possible. Additionally, we observed positive modulation of precursor activity at low concentrations of inhibitors. Our findings help expand understanding of the SARS-CoV-2 viral life cycle and may facilitate development of strategies to target precursor form of Mpro for inhibition or premature activation of Mpro.

• MeSH
• antivirové látky * farmakologie   chemie   MeSH
• farmakoterapie COVID-19 MeSH
• HEK293 buňky MeSH
• inhibitory proteas farmakologie   chemie   MeSH
• koronavirové proteasy 3C * metabolismus   antagonisté a inhibitory   chemie   genetika   MeSH
• lidé MeSH
• mutace MeSH
• objevování léků * metody   MeSH
• proteolýza MeSH
• SARS-CoV-2 * enzymologie   účinky léků   metabolismus   genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

The aim of this study is to evaluate opportunistic pathogenic bacteria of the genus Pseudomonas in anthropogenically impacted bathing waters, primarily focusing on bathing ponds. The findings include the detection of these bacteria, their susceptibility to selected antibiotics, and the determination of the Exotoxin A (exoA) gene using PCR method. P. aeruginosa was present in most samples, albeit in low concentrations (1-14 CFU/100 mL). The presence of P. otitidis, which is associated with ear infection, in this type of bathing water, was not rare (up to 90 CFU/100 mL). This species would not be detected by the standard methods, including tests on acetamid medium, used for P. aeruginosa in water. The isolated strains of P. otitidis lack the exoA gene and exhibited higher resistance to meropenem compared to P. aeruginosa.

• MeSH
• ADP-ribosatransferasy genetika   MeSH
• antibakteriální látky * farmakologie   MeSH
• bakteriální léková rezistence MeSH
• bakteriální proteiny genetika   MeSH
• bakteriální toxiny genetika   MeSH
• exotoxin A z Pseudomonas aeruginosa MeSH
• exotoxiny genetika   MeSH
• faktory virulence genetika   MeSH
• mikrobiální testy citlivosti * MeSH
• mikrobiologie vody * MeSH
• polymerázová řetězová reakce MeSH
• Pseudomonas * genetika   izolace a purifikace   klasifikace   účinky léků   MeSH
• rybníky * mikrobiologie   MeSH
• Publikační typ
• časopisecké články MeSH

Laboratorní diagnostika infekčního agens je založena na metodách přímého průkazu (kultivace, mikroskopie, PCR, antigenní test) a na metodách nepřímého průkazu (detekce protilátek). Sérologie zůstává důležitou součástí laboratorní diagnostiky infekčních onemocnění, přestože význam některých sérologických testů klesá s rozvojem molekulárně biologických metod. Pro pochopení, jakou roli hrají protilátky v procesu imunitní odpovědi na infekční agens, je nutné znát obecné mechanismy, které jsou s tvorbou protilátek spojené. Interpretace sérologických výsledků je složitá a závisí na fázi imunitní odpovědi, antigenních vlastnostech patogenu, použité metodě, senzitivitě a specificitě testů a klinickém kontextu konkrétního pacienta. Správná interpretace sérologických testů vyžaduje hlubokou znalost patogeneze infekce (specifická reakce na bakterie, viry nebo prvoky), metodických limitací a klinických souvislostí, což je zásadní pro efektivní diagnostiku a léčbu pacientů v klinické praxi.

Martinek J, Lochmanová A, Maďar R. Interpretation of serological results in the diagnosis of infectious diseases Laboratory diagnosis of infectious agents is based on direct detection methods (culture, microscopy, PCR, antigen test) and indirect detection methods (antibody detection). Serology remains an important part of the laboratory diagnosis of infectious diseases, although the importance of some serological tests is declining with the development of molecular biological methods. To understand the role of antibodies in the immune response to infectious agents, it is necessary to know the general mechanisms involved in antibody production. Interpretation of serological results is complex and depends on the phase of the immune response, the antigenic properties of the pathogen, the method used, the sensitivity and specificity of the tests, and the clinical context of the individual patient. Correct interpretation of serological tests requires an in-depth knowledge of the pathogenesis of infection (specific response to bacteria, viruses or protozoa), methodological limitations and clinical context, which is essential for effective diagnosis and treatment of patients in clinical practice.

BackgroundDuring the COVID-19 pandemic, non-pharmaceutical interventions (NPIs) such as social distancing, lockdowns and enhanced hygiene led to a decrease in respiratory pathogens. However, as NPIs were relaxed, a resurgence in several respiratory pathogens was observed including one local Chlamydia pneumoniae outbreak in Switzerland, prompting the need for a better understanding of C. pneumoniae epidemiology.AimTo assess temporal and geographical variations in C. pneumoniae detection before, during and after the COVID-19 pandemic.MethodsData on C. pneumoniae PCR detection ratios (number of positive tests/ total number of tests) across pre-pandemic (2018-2019), pandemic (2020-2022) and post-pandemic (2023) periods were collected via a global survey disseminated through various professional networks.ResultsC. pneumoniae detection ratios were analysed across 28 sites (27 in Europe, one in Taiwan) in 2023 (Dataset A, n = 172,223 tests) and 20 sites from 2018 to 2023 (Dataset B, n = 693,106 tests). Twenty-seven sites were laboratories (hospital or clinical) and one a surveillance system (Denmark). A significant decrease in detection ratios was observed during the pandemic period (from 1.05% to 0.23%, p < 0.001). In 2023, detection ratios increased to 0.28% (p < 0.002). Notable regional variations were found, with statistically significant increases in detection ratios at six sites located in Switzerland and Slovenia, where ratios ranged from 0.52% to 3.25%.DiscussionThe study highlights how NPIs influenced C. pneumoniae epidemiology, with reduced detection during the pandemic and partial resurgence afterwards. Regional variations suggest differing NPI impacts and underscore the need for continued surveillance.

• MeSH
• Chlamydophila pneumoniae * izolace a purifikace   genetika   MeSH
• COVID-19 * epidemiologie   MeSH
• infekce bakteriemi rodu Chlamydophila * epidemiologie   diagnóza   MeSH
• lidé MeSH
• pandemie MeSH
• polymerázová řetězová reakce MeSH
• SARS-CoV-2 MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Evropa MeSH
• Taiwan MeSH

BackgroundOn 29 January 2024, the European Centre for Disease Prevention and Control distributed an alert about a metronidazole-resistant Clostridioides difficile outbreak of PCR ribotype (RT) 955 in England.AimWe aimed to investigate the presence of RT955 in Czech, Slovak and Polish C. difficile isolates and evaluate different culture media for detecting its metronidazole resistance.MethodsIsolates with binary toxin genes identified as 'unknown' by the WEBRIBO PCR ribotyping database up to 2023 were re-analysed after adding the RT955 profile to the database. The RT955 isolates were characterised by whole genome sequencing and tested for susceptibility to 15 antimicrobials.ResultsWe did not find RT955 in Czech (n = 6,661, 2012-2023) and Slovak (n = 776, 2015-2023) isolates, but identified 13 RT955 cases (n = 303, 2021-2023) in three hospitals in Poland. By whole genome multilocus sequence typing, 10 isolates clustered into one clonal complex including a sequence of United Kingdom strain ERR12670107, and shared similar antimicrobial resistance genes/mutations. All 13 isolates were resistant to ciprofloxacin/moxifloxacin, erythromycin/clindamycin and ceftazidime. All isolates had a mutation in the nimB gene promoter and in NimB (Tyr130Ser and Leu155Ile). The metronidazole resistance was detected in all isolates using brain-heart-infusion agar supplemented with haemin and Chocolate agar. Results were discrepant with the European Committee on Antimicrobial Susceptibility Testing-recommended Fastidious anaerobe agar and Brucella blood agar.ConclusionThe identification of clonally related haem-dependent metronidazole-resistant C. difficile RT955 in multiple hospitals indicates a need for prospective surveillance to estimate its prevalence in Europe.

• MeSH
• antibakteriální látky * farmakologie   MeSH
• bakteriální léková rezistence * genetika   MeSH
• Clostridioides difficile * genetika   účinky léků   izolace a purifikace   klasifikace   MeSH
• epidemický výskyt choroby MeSH
• klostridiové infekce * epidemiologie   mikrobiologie   farmakoterapie   MeSH
• lidé MeSH
• metronidazol * farmakologie   MeSH
• mikrobiální testy citlivosti MeSH
• multilokusová sekvenční typizace MeSH
• polymerázová řetězová reakce MeSH
• ribotypizace * MeSH
• sekvenování celého genomu MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Česká republika MeSH
• Polsko MeSH
• Slovenská republika MeSH

Current antibiotics and chemotherapeutics are becoming ineffective because pathogenic bacteria and tumor cells have developed multiple drug resistance. Therefore, it is necessary to find new substances that can be used in treatment, either alone or as sensitizing molecules in combination with existing drugs. Peptaibols are bioactive, membrane-active peptides of non-ribosomal origin, mainly produced by filamentous fungi such as Trichoderma spp. This study focused on producing peptaibol-rich extracts from Trichoderma atroviride O1, cultivated on malt extract agar (MA) under circadian and constant darkness conditions for 13 days. Peptaibol production was detected by MALDI-TOF mass spectrometry after six days of cultivation. The extracts demonstrated antibacterial activity against Staphylococcus aureus strains, particularly the methicillin-resistant variant, but not against the Gram-negative Pseudomonas aeruginosa. Quorum sensing interference revealed that a peptaibol-rich extract suppressed Vibrio campbellii BAA-1119's AI-2 signaling system to a degree comparable with gentamycin. Beyond antibacterial properties, the extracts exhibited notable antiproliferative activity against human ovarian cancer cells and their adriamycin-resistant subline in both 2D and 3D models. Specifically, MA-derived extracts reduced ovarian cancer cell viability by 70% at 50 μg/mL, especially under light/dark regime of cultivation. Compared to previously published results for PDA-based extracts, MA cultivation shifted the biological effects of peptaibol-containing extracts toward anticancer potential. These findings support the idea that modifying fungal cultivation parameters, the bioactivity of secondary metabolite mixtures can be tailored for specific therapeutic applications.

• MeSH
• agar * chemie   MeSH
• antibakteriální látky * farmakologie   metabolismus   MeSH
• Hypocreales MeSH
• kultivační média chemie   MeSH
• lidé MeSH
• mikrobiální testy citlivosti MeSH
• nádorové buněčné linie MeSH
• peptaiboly * farmakologie   metabolismus   biosyntéza   chemie   MeSH
• proliferace buněk účinky léků   MeSH
• protinádorové látky * farmakologie   metabolismus   MeSH
• Pseudomonas aeruginosa účinky léků   MeSH
• Staphylococcus aureus účinky léků   MeSH
• Trichoderma * metabolismus   růst a vývoj   chemie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

BACKGROUND: Tick-borne encephalitis virus (TBEV) is a significant threat to human health. The virus causes potentially fatal disease of the central nervous system (CNS), for which no treatments are available. TBEV infected individuals display a wide spectrum of neuronal disease, the determinants of which are undefined. Changes to host metabolism and virus-induced immunity have been postulated to contribute to the neuronal damage observed in infected individuals. In this study, we evaluated the cytokine, chemokine, and metabolic alterations in the cerebrospinal fluid (CSF) of symptomatic patients infected with TBEV presenting with meningitis or encephalitis. Our aim was to investigate the host immune and metabolic responses associated with specific TBEV infectious outcomes. METHODS: CSF samples of patients with meningitis (n = 27) or encephalitis (n = 25) were obtained upon consent from individuals hospitalised with confirmed TBEV infection in Brno. CSF from uninfected control patients was also collected for comparison (n = 12). A multiplex bead-based system was used to measure the levels of pro-inflammatory cytokines and chemokines. Untargeted metabolomics followed by bioinformatics and integrative omics were used to profile the levels of metabolites in the CSF. Human motor neurons (hMNs) were differentiated from induced pluripotent stem cells (iPSCs) and infected with the highly pathogenic TBEV-Hypr strain to profile the role(s) of identified metabolites during the virus lifecycle. Virus infection was quantified via plaque assay. RESULTS: Significant differences in proinflammatory cytokines (IFN-α2, TSLP, IL-1α, IL-1β, GM-CSF, IL-12p40, IL-15, and IL-18) and chemokines (IL-8, CCL20, and CXCL11) were detected between neurological-TBEV and control patients. A total of 32 CSF metabolites differed in TBE patients with meningitis and encephalitis. CSF S-Adenosylmethionine (SAM), Fructose 1,6-bisphosphate (FBP1) and Phosphoenolpyruvic acid (PEP) levels were 2.4-fold (range ≥ 2.3-≥3.2) higher in encephalitis patients compared to the meningitis group. CSF urocanic acid levels were significantly lower in patients with encephalitis compared to those with meningitis (p = 0.012209). Follow-up analyses showed fluctuations in the levels of O-phosphoethanolamine, succinic acid, and L-proline in the encephalitis group, and pyruvic acid in the meningitis group. TBEV-infection of hMNs increased the production of SAM, FBP1 and PEP in a time-dependent manner. Depletion of the metabolites with characterised pharmacological inhibitors led to a concentration-dependent attenuation of virus growth, validating the identified changes as key mediators of TBEV infection. CONCLUSIONS: Our findings reveal that the neurological disease outcome of TBEV infection is associated with specific and dynamic metabolic signatures in the cerebrospinal fluid. We describe a new in vitro model for in-depth studies of TBEV-induced neuropathogenesis, in which the depletion of identified metabolites limits virus infection. Collectively, this reveals new biomarkers that can differentiate and predict TBEV-associated neurological disease. Additionally, we have identified novel therapeutic targets with the potential to significantly improve patient outcomes and deepen our understanding of TBEV pathogenesis.

• MeSH
• cytokiny mozkomíšní mok   MeSH
• dospělí MeSH
• klíšťová encefalitida * mozkomíšní mok   metabolismus   MeSH
• kultivované buňky MeSH
• lidé středního věku MeSH
• lidé MeSH
• metabolom * fyziologie   MeSH
• metabolomika MeSH
• mladý dospělý MeSH
• neurony * metabolismus   virologie   MeSH
• senioři MeSH
• viry klíšťové encefalitidy * MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH

Bats are the natural reservoirs for a variety of emerging and re-emerging viruses. Among them, rabies virus (genus Lyssavirus, family Rhabdoviridae) is one of the first and most emblematic described in these animals. Since its first description, several new bat lyssaviruses have been regularly identified. In addition to lyssaviruses, other bat rhabdoviruses have also been discovered, including members of the genera Vesiculovirus, Ledantevirus and, more recently, Alphanemrhavirus and Tupavirus. However, the family Rhabdoviridae is one of the most abundant and diverse viral families, with 434 officially recognized species, divided into 5 subfamilies and 56 different genera. The number of rhabdoviruses associated with bats is therefore probably higher than that currently available. In this study, we first developed and validated a combined nested RT-qPCR technique (pan-rhabdo RT-nqPCR) dedicated to the broad detection of animal rhabdoviruses. After validation, this technique was used for a large retrospective screening of archival bat samples (n = 1962), including blood (n = 816), brain (n = 723) and oral swab (n = 423). These samples were collected from various bat species over a 12-year period (2007-2019) in 9 different countries in Europe and Africa. A total of 23 samples (1.2%) from bat species Miniopterus schreibersii, Rhinolophus euryale and Rhinolophus ferrumequinum tested positive for rhabdovirus infection, including 17 (2.1%) blood and 6 (1.4%) oral swab samples, all collected from bats originating from the Mediterranean region. Complete virus genome sequences were obtained by next-generation sequencing for most of the positive samples. Molecular and phylogenetic analysis of these sequences demonstrated that the virus isolates, named Mediterranean bat virus (MBV), were closely related and represented a new species, Mediterranean vesiculovirus, within the genus Vesiculovirus. MBV was more specifically related to other bat vesiculoviruses previously described from China and North America, together clustering into a distinct group of bat viruses within this genus. Interestingly, our results suggest that MBV is widespread, at least in the western part of the Mediterranean region, where it circulates in the blood of several bat species. These results expand the host range and viral diversity of bat vesiculoviruses, and pave the way for further studies to determine the transmission route and dissemination dynamics of these viruses in bat colonies, as well as to assess their potential threat to public health.

• MeSH
• Chiroptera * virologie   MeSH
• fylogeneze MeSH
• genom virový MeSH
• infekce viry z čeledi Rhabdoviridae * veterinární   epidemiologie   virologie   MeSH
• kvantitativní polymerázová řetězová reakce MeSH
• Vesiculovirus * genetika   izolace a purifikace   klasifikace   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Středomoří MeSH

BACKGROUND: Since the incidence of vancomycin-resistant enterococci (VRE) is increasing and treatment options remain limited, we aimed to investigate the epidemiology of vancomycin- and tigecycline-resistant enterococci in a university hospital using whole genome sequencing (WGS). METHODS: Between April and December 2021, 102 VRE isolates were collected from a single tertiary care hospital in the Czech Republic. Forty selected isolates underwent antimicrobial susceptibility testing and WGS (Illumina short reads and long reads with MinION in selected isolates). RESULTS: All Enterococcus faecium isolates were resistant to ampicillin, carrying the PBP5_Met485Ala, PBP5_Glu629Val, and fluoroquinolones carrying the GyrA_Ser83Ile and ParC_Ser80Ile substitutions. The vanA operon was found on pELF2-like plasmids and plasmids carrying rep17 and/or rep18b genes. The novel Tn1546 structural variants were identified in vanA-carrying isolates. The vanB operon was located on the chromosome within a Tn1549 structural variant. Linezolid resistance was detected in one isolate carrying the 23S rDNA_G2576T substitution. Twenty-two isolates were resistant to tigecycline (tet(L), tet(M) and rpsJ_del 155-166 or RpsJ_Lys57Arg). Discrepancies between phenotypic and genotypic resistance profiles were observed for daptomycin (RpoB_Ser491Phe), trimethoprim/sulfamethoxazole (dfrG gene), nitrofurantoin (NmrA_Gln48Lys substitution without the EF0404 and EF0648 genes) and tetracycline (truncated TetM). The two multilocus sequence typing (MLST) schemes identified different numbers of STs: 5 STs, with ST117 as the predominant one (n = 32, 80%), versus 10 STs, with ST138 (27.5%), ST136 (25%), and ST1067 (20%) being the most frequent, respectively. The whole genome MLST revealed clonal clustering (0-7 allele differences) among isolates of the same ST. When comparing ST117 isolates from our study with 2,204 ST117 isolates from 15 countries, only one Czech isolate clustered closely with strains from Germany and the Netherlands, differing by just 16 alleles. CONCLUSIONS: The spread of E. faecium isolates ST117 resistant to vancomycin and tigecycline was identified. The discrepancies between resistance genotypes and phenotypes highlight the importance of combining molecular and phenotypic surveillance in antimicrobial resistance monitoring.

• MeSH
• antibakteriální látky * farmakologie   MeSH
• bakteriální proteiny genetika   MeSH
• Enterococcus faecium * genetika   účinky léků   izolace a purifikace   klasifikace   MeSH
• enterokoky rezistentní vůči vankomycinu * genetika   účinky léků   izolace a purifikace   MeSH
• genom bakteriální MeSH
• grampozitivní bakteriální infekce * mikrobiologie   epidemiologie   MeSH
• lidé MeSH
• mikrobiální testy citlivosti MeSH
• mnohočetná bakteriální léková rezistence genetika   MeSH
• multilokusová sekvenční typizace MeSH
• rezistence na vankomycin genetika   MeSH
• sekvenování celého genomu MeSH
• tigecyklin * farmakologie   MeSH
• vankomycin * farmakologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Česká republika MeSH

Molecular surveillance was widely used during the COVID-19 pandemic to detect rapidly emerging variants and monitor the transmission of SARS-CoV-2 within communities. In 2021, the Czech COVID-19 Genomics Consortium (COG-CZ) was set up to coordinate a new SARS-CoV-2 molecular surveillance network. In the Czech Republic, molecular surveillance employed whole genome sequencing (WGS) and variant discrimination polymerase chain reaction (VD-PCR) on samples collected through passive, active and sentinel surveillance. All WGS data was uploaded to GISAID and the PANGO lineages used by GISAID were compared to the main variants determined by VD-PCR. To assess the effectiveness and reliability of the gathered data in adapting pandemic responses, the capabilities and turnaround times of the molecular surveillance methods are evaluated. VD-PCR results were available within 48 h of sample collection for 81.5% of cases during the Delta/Omicron transition. WGS enabled the detection of low-frequency novel variants in infection clusters. WGS surveillance showed there was community spread of AY.20.1, a variant that gained novel mutations within the Czech Republic. Molecular surveillance informed the implementation of public health measures; temporal comparisons of restrictions and outcomes are described. Further areas for improvement have been identified for monitoring and managing future pandemics.

• MeSH
• COVID-19 * epidemiologie   virologie   MeSH
• genom virový MeSH
• lidé MeSH
• pandemie MeSH
• SARS-CoV-2 * genetika   izolace a purifikace   MeSH
• sekvenování celého genomu MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Česká republika MeSH