Uncovering the microbial diversity of Czech Republic archives: A study of metabolically active airborne microbes.

Despite the diligent efforts of libraries, archives, and similar institutions to preserve cultural monuments, biodeterioration continues to pose a significant threat to these objects. One of the main sources of microorganisms responsible for the biodeterioration process is the presence of airborne microorganisms. Therefore, this research aims to monitor and compare outcomes of both culture-dependent (utilising various cultivation strategies) and culture-independent approaches (RNA-based sequencing) to identifying metabolically active airborne microorganisms in archives in the Czech Republic. Through this study, several species that have the potential to pose risks to both cultural heritage objects and the health of institution employees were found. Additionally, the efficacy of different cultivation media was demonstrated to be varied across archive rooms, highlighting the necessity of employing multiple cultivation media for comprehensive analyses. Of noteworthy importance, the resuscitating-promoting factor (Rpf) proved to be a pivotal tool, increasing bacterial culturability by up to 30% when synergistically employed Reasoner's 2A agar (R2A) and R2A + Rpf media. Next, the study emphasises the importance of integrating both culture-dependent and culture-independent approaches. The overlap between genera identified by the culture-dependent approach and those identified also by the culture-independent approach varied from 33% to surpassing 94%, with the maximum alignment exceeding 94% in only one case. Our results highlight the importance of actively monitoring and assessing levels of microbial air contamination in archives to prevent further deterioration of cultural heritage objects and to promote improved conditions for employees in archives and similar institutions.

Prevalence of SARS-CoV-2 variants in Prague wastewater determined by nanopore-based sequencing.

The early detection of upcoming disease outbreaks is essential to avoid both health and economic damage. The last four years of COVID-19 pandemic have proven wastewater-based epidemiology is a reliable system for monitoring the spread of SARS-CoV-2, a causative agent of COVID-19, in an urban population. As this monitoring enables the identification of the prevalence of spreading variants of SARS-CoV-2, it could provide a critical tool in the fight against this viral disease. In this study, we evaluated the presence of variants and subvariants of SARS-CoV-2 in Prague wastewater using nanopore-based sequencing. During August 2021, the data clearly showed that the number of identified SARS-CoV-2 RNA copies increased in the wastewater earlier than in clinical samples indicating the upcoming wave of the Delta variant. New SARS-CoV-2 variants consistently prevailed in wastewater samples around a month after they already prevailed in clinical samples. We also analyzed wastewater samples from smaller sub-sewersheds of Prague and detected significant differences in SARS-CoV-2 lineage progression dynamics among individual localities studied, e.g., suggesting faster prevalence of new variants among the sites with highest population density and mobility.

Selective syngas fermentation to acetate under acidic and psychrophilic conditions using mixed anaerobic culture.

Syngas fermentation to acetate offers a promising solution for its valorisation, particularly when syngas contains a high N2 concentration, which otherwise impedes the utilisation of syngas biomethanation gaseous product in cogeneration or upgrading units. In this study, continuous lab-scale syngas fermentation assessing the effects of acidic pH and psychrophilic conditions (28 °C and 20 °C) on bioconversion efficiency and anaerobic consortium diversity was studied. The results showed that as temperature and pH decrease, acetate yield increases. The highest H2 and CO consumption rates were observed at 20 °C and pH 4.5, reaching 48.4 mmol/(L·d) and 31.5 mmol/(L·d), respectively, and methanogenic activity was not completely suppressed. The microbial community composition indicated an enhanced abundance of acetate-producing bacteria and hydrogenotrophic methanogens at 28 °C. The PICRUSt2 prediction of metabolic potential indicated that temperature and pH changes appear to have a more pronounced impact on acetotrophic methanogenesis genes than carbon dioxide-based methanogenesis genes.

Joining the bacterial conversation: increasing the cultivation efficiency of soil bacteria with acyl-homoserine lactones and cAMP.

IMPORTANCE: Microorganisms are a repository of interesting metabolites and functions. Therefore, accessing them is an important exercise for advancing not only basic questions about their physiology but also to advance technological applications. In this sense, increasing the culturability of environmental microorganisms remains an important endeavor for modern microbiology. Because microorganisms do not live in isolation in their environments, molecules can be added to the cultivation strategies to "inform them" that they are present in growth-permissive environmental conditions. Signaling molecules such as acyl-homoserine lactones and 3',5'-cyclic adenosine monophosphate belong to the plethora of molecules used by bacteria to communicate with each other in a phenomenon called quorum sensing. Therefore, including quorum sensing molecules can be an incentive for microorganisms, specifically soil bacteria, to increase their numbers on solid media.

Monitoring of monkeypox viral DNA in Prague wastewater.

Monkeypox virus (Mpxv) is a dsDNA virus that has become a global concern for human health in 2022. As both infected people and non-human hosts can shed the virus from their skin, faeces, urine and other body fluids, and the resulting sewage contains viral load representative of the whole population, it is highly promising to detect the spread of monkeypox virus in municipal wastewater. We established a methodology for sewage-based monitoring of Mpxv in Prague and analysed samples (n = 24) already early August-October of 2022 in a municipality with 1.4 million inhabitants that only reported 29 cumulative cases in this period. We isolated Mpxv DNA with the Wizard Enviro Total Nucleic Acid Kit, and thereafter detected Mpxv DNA using the EliGene® Monkeypox RT-PCR Kit. Prague wastewater was positive for Mpxv (in total 9 positive samples in periods with 1-9 new cases per week, coinciding with a weekly incidence of 0.07-0.64 per 100,000 inhabitants. The method for confirmation of wastewater positivity via semi-nested PCR and Sanger sequencing was successfully confirmed on positive controls including Mpxv particles and Mpxv-positive wastewater from the Netherlands. However, for Prague wastewater samples, amplification of Mpxv DNA via semi-semi-nested PCR was unsuccessful. This was probably due to extremely low case count, leading to the amplification of non-target bacterial DNA. Compared to other studies with much higher Mpxv prevalence, we show the outstanding sensitivity of our approach for monitoring the spread of monkeypox using wastewater.

Reaching unreachables: Obstacles and successes of microbial cultivation and their reasons.

In terms of the number and diversity of living units, the prokaryotic empire is the most represented form of life on Earth, and yet it is still to a significant degree shrouded in darkness. This microbial "dark matter" hides a great deal of potential in terms of phylogenetically or metabolically diverse microorganisms, and thus it is important to acquire them in pure culture. However, do we know what microorganisms really need for their growth, and what the obstacles are to the cultivation of previously unidentified taxa? Here we review common and sometimes unexpected requirements of environmental microorganisms, especially soil-harbored bacteria, needed for their replication and cultivation. These requirements include resuscitation stimuli, physical and chemical factors aiding cultivation, growth factors, and co-cultivation in a laboratory and natural microbial neighborhood.

Monitoring COVID-19 spread in selected Prague's schools based on the presence of SARS-CoV-2 RNA in wastewater.

The COVID-19 pandemic has demanded a broad range of techniques to better monitor its extent. Owing to its consistency, non-invasiveness, and cost effectiveness, wastewater-based epidemiology has emerged as a relevant approach to monitor the pandemic's course. In this work, we analyzed the extent of the COVID-19 pandemic in five primary schools in Prague, the Czech Republic, and how different preventive measures impact the presence of SARS-CoV-2 RNA copy numbers in wastewaters. Copy numbers were measured by reverse transcription-multiplex quantitative real-time PCR. These copy numbers were compared to the number of infected individuals in each school identified through regular clinical tests. Each school had a different monitoring regime and subsequent application of preventive measures to thwart the spread of COVID-19. The schools that constantly identified and swiftly quarantined infected individuals exhibited persistently low amounts of SARS-CoV-2 RNA copies in their wastewaters. In one school, a consistent monitoring of infected individuals, coupled with a delayed action to quarantine, allowed for the estimation of a linear model to predict the number of infected individuals based on the presence of SARS-CoV-2 RNA in the wastewater. The results show the importance of case detection and quarantining to stop the spread of the pandemic and its impact on the presence of SARS-CoV-2 RNA in wastewaters. This work also shows that wastewater-based epidemiological models can be reliably used even in small water catchments, but difficulties arise to fit models due to the nonconstant input of viral particles into the wastewater systems.

Solicola gregarius gen. nov., sp. nov., a soil actinobacterium isolated after enhanced cultivation with Micrococcus luteus culture supernatant.

An actinobacterial strain, designated A5X3R13T, was isolated from a compost soil suspension supplemented with extracellular material from a Micrococcus luteus-culture supernatant. The strain was cultured on tenfold-diluted reasoner's 2A agar. The cells were ovoid-to-rod shaped, non-motile, Gram-stain-positive, oxidase-negative, catalase-positive and had a width of 0.5 µm and a length of 0.8-1.2 µm. The results of both 16S rRNA-based phylogenetic and whole-genome analyses indicate that A5X3R13T forms a distinct lineage within the family Nocardioidaceae (order Propionibacteriales). On the basis of the 16S rRNA gene sequence, A5X3R13T was closely related to Aeromicrobium terrae CC-CFT486T (96.2 %), Nocardioides iriomotensis IR27-S3T (96.2 %), Nocardioides guangzhouensis 130T (95.6 %), Marmoricola caldifontis YIM 730233T (95.5 %), Aeromicrobium alkaliterrae KSL-107T (95.4 %), Aeromicrobium choanae 9H-4T (95.4 %), Aeromicrobium panaciterrae Gsoil 161T (95.3 %), and Nocardioides jensenii NBRC 14755T (95.2 %). The genome had a length of 4 915 757 bp, and its DNA G+C content was 68.5 mol %. The main fatty acids were 10-methyl C17 : 0, C16 : 0, C15 : 0, C18 : 0, C17 : 0 and iso-C16 : 0. The main polar lipids were phosphatidylglycerol, diphosphatidylglycerol, phosphatidylinositol and two unidentified phospholipids. MK-9(H4) was the predominant respiratory quinone. The peptidoglycan type was A3γ (A41.1) and contained alanine, glycine, glutamic acid and ll-diaminopimelic acid in a molar ratio of 1.2 : 0.9 : 1.0 : 0.8. On the basis of the results of the phylogenetic and phenotypic analyses and comparisons with other members of the family Nocardioidaceae, strain A5X3R13T is proposed to represent a novel species within a novel genus, for which the name Solicola gregarius gen. nov., sp. nov. is proposed. The type strain is A5X3R13T (=DSM 112953T=NCCB 100840T).

Pedomonas mirosovicensis gen. nov., sp. nov., a bacterium isolated from soil with the aid of Micrococcus luteus culture supernatant containing resuscitation-promoting factor.

An orange-golden iridescent culture, designated A1X5R2T, was isolated from a compost soil suspension which was amended with Micrococcus luteus NCTC 2665T culture supernatant. The cells were non-motile, Gram-stain-negative, 0.4-0.5 µm wide and 0.7-1.4 µm long. The 16S rRNA-based phylogenetic and whole-genome analyses revealed that strain A1X5R2T forms a distinct lineage within the family Sphingosinicellaceae and is closely related to members of the genus Sphingoaurantiacus (S. capsulatus, 93.04 % similarity, and S. polygranulatus, 92.77 %). The organism grew at 22-47 °C (optimal at 37 °C), salinity <3 % (optimal at 1.5 %) and at pH 7. The major respiratory quinone was ubiquinone-10, but a small quantity of ubiquinone-9 was also detected The major polyamine was homospermidine, but a small quantity of putrescine was also detected. The strain contained C18  :  1ω7c, C16 : 0, C16 : 1 ω7c and C18 : 0 as the major fatty acids. The main polar lipids were phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine, phosphatidylinositol, sphingoglycolipid, diphosphatidylglycerol, two unidentified phospholipids and three unidentified amino lipids. The DNA G+C content was 64.9 mol%. According to the results of phylogenetic and phylogenomic analyses, as well as its physiological characteristics, strain A2X5R2T represents the type species of a novel genus within the family Sphingosinicellaceae. The name Pedomonas mirosovicensis gen. nov., sp. nov. is proposed, with the type strain being A1X5R2T (=NCCB 100839T=DSM 112829T).