Actinobacteriota (12.08–54.00%) and Proteobacteria (25.34–44.97%) dominated most of the samples. A total of 44 different species, out of 96 obtained isolates, were determined as part of the culturable bacteriobiota, with the predominance of species from the genus Bacillus. Bacillus simplex was the only isolated species simultaneously present in all investigated substrata within the church. The best antagonistic activity against 10 biodeteriogenic fungi was documented for Streptomyces anulatus, followed by Bacillus altitudinis, Chryseobacterium viscerum, and Streptomyces sp. with their highest PGI% values ranging of from 55.9% to 80.9%. These promising results indicate that characterized bacteria are excellent candidates for developing biocontrol strategies for suppressing deteriogenic fungi responsible for the deterioration of investigated fresco painting. Finally, isolate 11-11MM, characterized as Streptomyces sp., represents a new species for science prompting the need for further study.The principal aim of this study was to determine bacterial diversity within the Cave Church of Sts. Peter and Paul, via culturable and non-culturable approaches, and elucidate the antifungal potential of autochthonous antagonistic bacterial isolates against biodeteriogenic fungi. Furthermore, whole-genome sequencing of selected bacterial antagonists and the analysis of genes included in the synthesis of secondary metabolites were performed. With the highest RA values, determined in metabarcoding analysis, phyla  The Cave Church of Sts. Peter and Paul was built on Stara Planina Mountain, 22 km from the town of Pirot in the vicinity of the village Rsovci, the Republic of Serbia, in a limestone cave on Kalik hill, and is well known for its fresco painting of the “Bald-headed Jesus”. The fresco is also known by its other name, “the young Jesus” or “Jesus the Infant”. It is the only fresco painting present in the church and was painted on its northern wall. Serbian art historians emphasize that among fresco painters there are those who believe that the painting of the “Bald-headed Jesus” is the work of artists influenced by various denominations of Christianity, while others believe it to be the work of hermits from Sinai, who painted it during their stay in the cave on one of their pilgrimages. No accurate data exists on when it was painted. Based on the style of painting and the pigments used, it dates from the middle or second half of the 13th century, while others believe it belongs to a much older school of painting. At the time, hermit cells were not subject to church canon laws and rules on how to represent saints. Therefore, it is assumed that Jesus was painted in a manner that indicates that the painters were unaffected by the conventional painting techniques of that era, and accordingly, it is assumed that the painting very likely predates the 13th century. This fresco, unique among Orthodox fresco paintings, depicts Jesus as very young, bald-headed, dressed in Buddhist robes, with a divine halo, and an octagonal star, while three of his fingers are raised, signifying baptism . The opposite was noted for the iconostasis’ sample (sample 06). The genera Blastocatella (B. fastidiosa) and Crossiella. At the same time, for samples 09 and 10, Burkholderiales (Sulfurirhabdus sp.) was detected in a higher abundance. For samples 09 and 11, a greater dominance of Pyrinomonadaceae RB41 (Arenimicrobium sp.), Rubrobacter, and Nitrospira was noted. The taxa Rhodococcus (R. corynebacterioides), Geminicoccaceae (Arboricoccus sp.), and Alkanindiges were more frequent in sample 09. Sample 10, obtained in the proximity of the fresco itself, contained an abundance of Pseudonocardia, Sporichthya, Streptomyces, Thermoleophilia (Miltoncostaea sp.), Acidimicrobiia IMCC26256 (Aciditerrimonas sp.), Nocardia , Methylibium (M. petroleiphilum), Acidiferrobacteraceae (Acidiferrobacter sp.), Lysobacter, and Sphingorhabdus. Sample 11, also obtained from the proximity of the fresco, was characterized by Sphaerobacter sp., Tepidisphaera sp., Urbifossiella sp., Sphingomonas (S. rhizophila/sediminicola), Rhizobiales Incertae Sedis (Nordella sp.), Alphaproteobacteria (Chelativorans sp.), Sulfurirhabdus and Gemmatimonadaceae (Gemmatimonas sp.). Contrary to that, in sample 07 , communities of cyanobacteria such as Phormidium CYN64 (Timaviella sp.), Gloeocapsa PCC-7428 (Gloeocapsopsis crepidinum), Chroococcidiopsaceae (Gloeocapsopsis sp.) and Coleofasciculaceae (Symplocastrum sp.) were found to be the dominant ones, as well as Panacagrimonas (P. perspica), Methylotenera (Methylophilus methylotrophus), Methylophilaceae (Methylotenera sp.) and Variovorax (V. guangxiensis/soli). Obtained at an even greater distance from the fresco, sample 08 was dominated by communities of cyanobacteria such as Cyanobacterales (Loriellopsis sp.), Leptolyngbyaceae , as well as unidentified bacteria from the class Ktedonobacteria and the phylum Chloroflexi, with the sole presence of Chloroflexaceae (Chloroflexus sp.) and Chloronema.The most prevalent taxa linked to the various deposits on the walls  were Asanoa and Archangium, Nocardia, Pseudomonas, Gemmatimonas and Stenotrophobacter on the one hand and Ohtaekwangia, Asanoa, and Acidiphilium on the other. Moreover, a positive correlation was observed for Flavobacterium and Alkanindiges on the one hand and the unknown taxon from the class of cyanobacteria on the other. Furthermore, a positive correlation was noted between Phormidium, Gloeocapsa, and an unknown taxon from the family of Chroococcidiopsaceae, and unknown types from the classes of cyanobacteria and Thermoleophilia. A negative correlation was noted between the genera Methylobacterium-Methylorubrum and Nocardia on the one hand and Pseudomonas on the other. More negative correlations were found between Diplorickettsia and the genera Stenotrophobacter, Ohtaekwangia on the one hand and Gemmatimonas on the other, as well as between Fluviicola and Hansschlegelia on the one hand and Alkanindiges on the other. The relationships between taxonomic categories  were visualized based on the joint appearance of taxa , as depicted in The analysis of differential abundance was used to generate a matrix of the relative abundance of statistically significant species at all the taxonomic levels based on the origin of the samples . Asanoa and Plantactinospora) were present to a statistically significant extent. They were found only in the samples taken from the fresco and were therefore defined as core species. For all the remaining taxa, as stated in Based on the results, it was determined that only two of the known genera , Bacillus altitudinis/aerosphericus/stratosphericus (6-1 TSA), and Streptomyces lavenulae (11-11MM). Chryseobacterium ureilyticum (7-15/G14) was also selected since it was one of the rare isolates (in addition to the isolate 11-11 MM) which exhibited a good antifungal effect against P. album. Isolate 1-3 TSA, as the best antagonist in this study, statistically significantly inhibited the growth of P. citreonigrum, B. murorum, C. cladosporioides, A. aureulatus, and E. nigrum at the same time. All of the aforementioned antagonists were further analyzed using genome sequencing.Following the initial screening of 36 bacterial antagonists against 10 biodeteriogenic fungi of the same origin (previously isolated and molecularly identified to the species level), 16 antagonists were selected, whose results are shown in The whole genomes of the best antagonists were sequenced, and the results of the statistical analysis of the genomes with the closest reference strains and characteristics are shown in Streptomyces anulatus JCM 4721 with an ANI value greater than 97%. Moreover, it was concluded that 6-1 TSA belongs to the syntype strain  Bacillus altitudinis DSM 26896 (ANI 98.5%), and that 7-15/G14 belongs to the type strain Chryseobacterium viscerum 687B-08 (ANI > 95%). Unlike them, isolate 11-11MM was characterized only down to the genus level, as Streptomyces sp., since the ANI value was only confirmed to that taxonomic level and there are no similarities with any of the genomes deposited in the database  ranging from 7529 (1-3 TSA) and 8549 (11-11MM). However, in the case of isolate 6-1 TSA, that number was only 3923 CDS, which, at the same time, qualifies it as the smallest genome in this study, 3.8 Mb in size and with a low percentage of G+C content. The lowest G+C content was, however, noted for the 7-15/G14 isolate. Interestingly, as many as four types of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) were noted for isolate 1-3 TSA. Based on the taxonomic annotation  and a phylogenetic analysis (which included a comparison with numerous protein markers) it was determined that the 1-3 TSA isolate is closest to the type strain database . BearingS. anulatus was determined to have exceptional potential for the production  of AmfS class III lantipeptides, isorenieratene terpenes, warkmycin cs1/cs2 polyketide angucycline, ectoine, the non-ribosomal synthesized peptide (NRP) coelichelin, the polyketide alkylresorcinol, NRP+polyketide complex sgr ptms, phenazine cluster compounds , as well as siderophoric desferrioxamine b with a somewhat lower level of similarity (80%). However, the aforementioned isolate was also noted to produce melanin. Isolate 11-11 MM also had AmfS lantipeptides in common with 1-3 TSA, ectoine, and coelichelin, as well as the antibiotic azomycin, with an 83% overlap. In the case of the B. altitudinis, the presence of the gene for the antimicrobial peptides bacilysin, and lichenysin (for both 85%), as well as bacillibactin and fengycin was noted with a probability lower than 53%. Of the offered metabolites for C. viscerum, only the polyketide flexirubin had an overlap probability of 75%.The antiSMASH analysis of selected isolates showed potential for the production of different secondary metabolites. Thus, the Proteobacteria, Firmicutes, Bacteroidetes, and Actinobacteria [Actinobacteria [Pseudonocardia [Thioalkalivibrio, which belongs to the phylum Proteobacteria and is linked to the oxidation of sulfur, was dominant in the studied samples. A similar claim had previously also been proven [Sphingomonas, which was obtained from the damaged parts of the wall painting [Actinomycetospora, another representative of the dominant bacterial environment around the fresco and responsible for the formation of white deposits on the stone wall in our study, is believed to support the development of microbes on such a substrate [Rubrobacter and Nocardioides, it leads to the emergence of pink deposits on murals and wall paintings [Streptomyces species, which caused changes to the colors on the wall paintings in Egyptian tombs [Biodeterioration is conditioned by numerous factors such as the chemical makeup and nature of the material, exposure of the object itself, and its environment. At the same time, the frequency of cleaning and maintenance also significantly affects the further destruction of objects of cultural heritage. The cave microbiota, and especially stone colonizers, display exceptional biodiversity and complexity of types. It was previously determined that such cave ecosystems are dominated by the bacterial phyla bacteria , which wbacteria , others nocardia , which wn proven . In addipainting , confirmubstrate . Along waintings , which ian tombs , are thoSymplocastrum sp. from the family Coleofasciculaceae. Until recently, this genus of cyanobacteria was insufficiently studied and lacked a molecular definition, but lately, it was proven to colonize stone surfaces and rocks, as well as lakeshores and the surfaces of freshwater sponges in the Baikal lake [Timaviella, which was the most abundant in one of the samples of the cave walls in our study, is a recently described genus characteristic of a cave ecosystem, and the representatives of this genus (Timaviella circinata and Timaviella karstica) have previously been isolated from the Giant Cave lampenflora in Italy under typical cave conditions, characterized by high humidity and constant temperature [T. obliquedivisa, T. radians, Timaviella sp. WMT-WP7-NPA, and T. edaphica comb. nov. [Cyanobacteria were often considered pioneer colonizers in numerous harsh environments, primarily due to their ability to produce exopolymer substances which enabled them to adhere to a stone surface and very quickly develop communities [Leptolyngbya can bind significant amounts of calcium from a basic substrate such as the material underlying a painting or fresco, but this potential is still being studied. This genus often produces biofilms in cave habitats and in humid locations [Phormidium was found growing under black sulfate crusts which developed on limestone in Spain [Chroococcus spp. possess the ability to grow and actively penetrate the stone; that is, they show potential for endolithic growth, at the same time creating tunnels and inflicting damage, but are still important biodeteriogens of stone [In addition to real bacterial taxa, the metagenome data from our study also indicated the dominance of cyanobacterial communities, especially in samples of green and black wall patinas and deposits. The metagenome analysis confirmed the presence of many cyanobacterial strains including kal lake . The Timperature . Since tmb. nov. ,41, theimb. nov. . Even thmb. nov. , epilithmb. nov. . The envmunities . Numeroumunities . The resmunities . On the munities . Furthermunities  indicateocations . The endin Spain . Some Chof stone . OverallBacillus, Micrococcus, Streptomyces, Staphylococcus, Pseudomonas, Chryseobacterium, Paenibacillus, and Curtobacterium. Many of these species had previously been found on similar artifacts [Bacillus, the most dominant group of isolates, which is a very heterogeneous genus and includes hundreds of strains [Bacillus on the wooden iconostasis can be explained by the presence of extracellular enzymes such as cellulase or lactamase-like enzymes which dissolve lignin [Staphylococcus spp. indicates a highly likely anthropogenic or animal origin, which is also confirmed by the S. hominis and S. warneri isolates in our study, with the possibility of horizontal transfer of antibiotic resistance genes to other species [S. epidermidis also plays a role in biomineralization [Curtobacterium oceanosedimentum, another isolate in our study, has the ability to inhibit the growth of S. epidermidis [Massilia timonae, also has antifungal potential since it produces chitinases, as previously indicated [Paenibacillus is already known to possess the ability to form endospores, and to produce a broad spectrum of bioactive units and volatile organic compounds [Streptomyces destroys material by producing bio-pigments in conditions of high salinity, that is, disruptions in the natural environment. The pigments it produces are melanins, with variations in color ranging from brown to olive green, then carotenoids of red, purple, and pink color, and actinorhodin with a blue tint. These biopigments can paint colonized surfaces and are especially resilient to various external environmental conditions [Micrococcus is a genus with an exceptional ability to survive under stress and can also produce important metabolites and antimicrobial compounds. Previous studies described it as a genus with strong colonizing abilities on frescoes, able to damage both the glue and binding [M. luteus coexists with types of the genus Streptomyces due to M. luteus resilience to exoenzymes that are secreted by streptomycetes [P. stutzeri. Its efficiency was proven in the removal of salt crusts which are the end result of the accumulation of nitrate salts on the surface of wall paintings, and which are otherwise difficult to remove using standard methods of restoration [S. maltophilia was proven to be very effective in removing brown deposits of protein [Stenotrophomonas maltophilia, and the genus Rhodococcus, were both identified as heterotrophic bacterial communities which colonize speleothems or calcium carbonate cave deposits. Furthermore, it was determined that S. maltophilia can convert nitrates into nitrites and ammonia and that its calcium carbonate precipitation can contribute to the formation of cave speleothems [Klebsiella pneumoniae, which does so by using urease. It is believed that this species can successfully be used in the construction industry to repair ruptures in concrete by producing calcite crystals, as well as for the potential production of bioconcrete [Buttiauxella and Kluyvera were primarily isolated from unpolluted soil and water but were also found in abundance in the intestines of slugs and other mollusks [Following the isolation and identification of bacterial isolates, the presence of several species of different genera was confirmed, such as rtifacts , which p strains . Its cos strains . However strains . Furthere lignin . The pre species . In addilization . Curtobadermidis . Yet, anndicated . The genompounds ,61, whicompounds . It is wnditions . Microco binding . It was omycetes . On the toration . This ty protein . Stenotrleothems . In numeconcrete . The genmollusks . Thus, tStreptomyces anulatus 1-3 TSA was characterized as a strain that is potentially a prime candidate for the biocontrol of biodeteriogenic fungi. This species is already known for its production of secondary metabolites which are used in the pharmaceutical industry, agrochemical industry, and agriculture [Botrytis cinerea have been indicated [S. anulatus S37, including nigericin, which has the greatest antifungal effect, as well as piericidin A1 and streptochlorine, which have a somewhat lower effectiveness. In addition, this species is also known for the production of antibiotics and actinomycin D [B. altitudinis 6-1 TSA was most similar to an isolate originating from the Yerba mate plant from Argentine, which has a pronounced potential for the promotion of plant growth [Phytophthora sojae was proven for similar isolates by means of the production of reactive oxygen species and callose deposition on soybeans, as well as an increase in the expression of genes that react to salicylate [B. altitudinis was demonstrated against Fusarium verticillioides, Corynespora cassiicola, Fusarium oxysporum f.sp. pisi and Sclerotinia sclerotiorum [C. viscerum was initially isolated from dead or infected fish [In our study, four candidates were selected based on their strong antifungal activities for whole-genome sequencing. iculture , while indicated . The afoomycin D . What ist growth . The abilicylate . In addirotiorum . Even thted fish , recent ted fish . The surface of the fresco of Jesus the Infant, as well as the walls of the cave church interior, possesses an exceptional biodiversity of the microbiota. Some represent biodeteriogens, while others can successfully be used as a means of biocleaning, for removing altered and unwanted organic substances and other microorganisms from historical wall paintings and frescoes. A multidisciplinary approach is needed in the work of scientists, conservationists, and administrative bodies, with synchronized work protocols, for a step forward to be made in the field of cultural heritage protection. Over the past decades, bacterial biomineralization has been proposed as an ecologically acceptable technology for stone protection, and it is important to focus attention on that aspect as well . The mosThe Cave Church of the Sts. Peter and Paul, with a unique fresco painting, is located in the village of Rsovci (43°10′32″ N 22°46′35″ E), in the rocky massif of the Kalik hill, 22 km from Pirot, as we described previously . The samSterile Puritan™ HydraFlock™ swabs  were taken from all sampling points and stored in a DNA/RNA shield  during transport. The DNA extraction was performed using the Zymo BIOMICS DNA Mini Kit  from a minimum of two swabs collected from each sampling point, according to the manufacturer’s protocol. The concentration of isolated DNA was measured using a Qubit Fluorometric Quantitation device . Amplicon sequencing was performed using a 2 × 300 bp paired-end sequencer on a MiSeq sequencer according to the manufacturer’s instructions  at a commercial sequencing service . To identify bacterial communities, the V3-V4 region of the 16S rRNA gene was amplified with defined forward (5′-CCTAGCGGGNGGCWGCAG-3′) and reverse (5′-GACTACHVGGGTATCTAATCC-3′) primers .https://www.arb-silva.de/documentation/release-138/, accessed on 21 December 2021). RDP Naïve Bayesian Classifier [Sequence quality assessment was performed using the DADA2 R package . Primer assifier  with defP < 0.05. Statistical analysis was performed using the software program IBM SPSS Statistics v.23 . Alpha diversity was estimated using the Phyloseq R program  at all taxonomic levels up to ASV. Alpha diversity indices in terms of species richness ACE, Chao1, and a number of observed species (OBS) were used. Diversity is shown through the Shannon and Gini–Simpson indices. The obtained values for the alpha indices, supported by the Kruskal–Wallis test by ranks, were used for testing significance among the samples obtained from fresco (samples 01–05) and from the cave interior . Statistical significance applied in all tests was In the beginning, the diversity between different samples (beta diversity) was estimated at the ASV level (previously rarefied according to the sample with the lowest read count) and determined using Double Principle Coordinate Analysis—DPCoA . SequencP < 0.05) co-occurrence patterns were shown. Differential abundance analysis was estimated using the DESeq2 package [The relationships among taxonomic categories were visualized based on co-occurrence using the cooccur package . Only th package . Startin package  was used package  was used package .The collection of samples for the cultivation of bacteria was carried out on all sampling points using dry sterile swabs by dragging and rotating the swab tip onto the designated surface with present deterioration symptoms, whilst applying firm pressure to break through any biological growth present. After sampling, swabs were put on ice and transported to the laboratory under sterile conditions. In total, 750 µL of phosphate buffer (1 × PBS) was added to the swabs tubes, vortexed for a few minutes, and 100 µL of the suspension obtained from each sample was spread onto defined nutrient media (Luria-Bertani Agar (LA), Tryptic Soy Agar (TSA), Glucose Agar (GA), Minimal Medium (MM), and Cetrimide Agar (CA)), and growth was monitored for a minimum of 48 h, at 30 °C. Individual bacterial isolates to pure axenic cultures were obtained by multiple streeking onto media according to their growth requirements, and the final selection of isolates was obtained using LA medium.Total genomic DNA was isolated by the protocol described in Dimkić et al. . Molecul® 2100 bioanalyzer was used to assess the insert size. Quantitative real-time PCR (qPCR) was performed to detect the effective concentration of each library. Illumina PE150 technology was applied for sample sequencing on a NovaSeq 6000 sequencer according to the manufacturer’s instructions. For the whole-genome sequencing (WGS), four bacterial antagonists  were selected whose DNA ) was sent for commercial sequencing . A DNA library was prepared by fragmenting genomic DNA to a size of 350 bp, and the selected fragments were then polished, dA-tailed, and ligated with a full-length NEBNext adapter. The required fragments were PCR enriched by P5 and indexed P7 oligos. The Agilenthttp://ftp.ebi.ac.uk/pub/databases/Pfam/releases/Pfam34.0/, accessed on 1 October 2021) and hmmer 3.3.2 options [https://antismash.secondarymetabolites.org/#!/start, accessed on 7 October 2021). GO annotation was performed using the GO FEAT server via the following link http://computationalbiology.ufpa.br/gofeat, accessed on 7 October 2021 [Sequence filtering and adapter sequence trimming were started from untrimmed sequences using the trim galore 0.6.6, and additionally, 10 bp from the left side and 2–4 bp from the right side of the reads were removed, as well. Genome assembly was performed using the SPAdes genome assembler v3.15.2 program , using a options . The sec options  program ber 2021 . A multiber 2021 . For eacber 2021  with defber 2021 .Penicillium citreonigrum, Botryotrichum murorum, Cladosporium cladosporioides, Aspergillus aureulatus, Epicoccum nigrum, Parengyodontium album, Botrytis cinerea, Beaueria pseudobassiana, Mortierella alpine, and Trichoderma viridescens), previously isolated and molecularly identified [P < 0.05. Statistical analysis was conducted by the general procedures of IBM SPSS Statistics v.23 .The initial antifungal activity in vitro of selected bacterial isolates from the risk 1 group (36 isolates) was tested against 10 potentially biodeteriogenic fungi of the same origin (entified . In the entified . After tentified : (PGI)% The highest abundance of species was documented on the side walls of the church, while the lowest abundance was noted on the iconostasis.Samples from the fresco are mutually similar, as are the samples obtained from the side walls of the church.Actinobacteriota and Proteobacteria are dominant phyla in the majority of studied samples.Methylococcaceae, Blastocatellaceae, and Nocardioidaceae of Thermomicrobiales class.The dominant bacterial taxa belonged to families Blastocatella and Crossiella.The most prevalent genera linked to the various kinds of wall deposits were The number of positive correlations between bacterial genera far outnumbered the number of negative correlations.Bacillus. Bacillus simplex was the only isolated species simultaneously present in all investigated substrata within the church.A total of 44 bacteria were identified, out of 96 obtained isolates, with the dominance of species from genus Streptomyces anulatus (1-3 TSA), Bacillus altitudinis (6-1 TSA), Chryseobacterium viscerum (7-15/G14), and Streptomyces sp. (11-11MM).The most promising antagonistic bacteria, with the potential to suppress growth of deteriogenic fungi and which represent excellent candidates for developing biocontrol strategies, are Streptomyces sp. (11-11MM) represents a new species for science.Further research is necessary to design the most appropriate biocontrol formulation, taking into account the environment and sensitivity of the substrate, so that the desired effect of sustainable and long-term elimination of biodeteriogenic fungi can be achieved after in situ application.Complete diversity of the bacterial communities of the Cave Church of Sts. Peter and Paul was obtained via non-culturable and culturable analyses and bioconservation potential of best antagonistic bacterial isolates was assessed on the basis of which it was concluded:


Staphylococcus haemolyticus, Staphylococcus epidermidis, Staphylococcus hominis, Enterococcus faecalis, and Micrococcus luteus. When comparing the type of urine samples, bacteria were more common in samples from the first-void urine than from the midstream one. The absence of bacteria was found in 12.2% of samples from the first-void urine and in 24.7% from the midstream. There was no difference in the total incidence of species between streams (p = 0.85). Before fiducial implantation, the total number of detected bacterial species was significantly higher in comparison to the end of radiotherapy (p = 0.038), indicating that the administered therapy resulted in depleting the local microbiome. One month after radiotherapy, an increase in the number of isolated bacteria was observed. The number of bacterial species in urine did not correlate with blood parameters. The presence of leukocytes (p = 0.013) and proteins (p = 0.004) in urine was related to a greater variety of bacteria found in urine specimens. (4) Conclusions: We obtained a similar spectrum of bacteria from the initial and middle urine streams. We also showed that there is a change in bacteria species affected by the treatment of prostate cancer patients, with both antibiotics before gold fiducial implantation and radiotherapy.(1) Background: Little is known about the impact of urinary microflora, in particular, its effects on side effects after radiotherapy. The use of mass spectrometry identification method (MALDI) may bring a new look at the issue of the composition and significance of the urinary microbiome. This study aimed to use the mass spectrometry identification method (MALDI) to identify the microbiome of urine samples collected from 50 irradiated prostate cancer patients. (2) Methods: Blood and urine samples were collected before gold marker implantation, at the start and last day of radiotherapy, 1, 4 months after. Patients do not always collect the urine from the midstream; therefore, samples were collected from the first void and midstream in 12 patients for MALDI analysis; in the remaining 38 patients—from the midstream void for MALDI and biochemical analysis. (3) Results: Microorganisms were present in 140/181 urine samples. We found 33 different species 3G(−) and 30G(+). The most frequently isolated strains were:  Conducting radiotherapy in the pelvic area is accompanied by the occurrence of early and late complications. The type and severity of radiotherapy side effects depend on the dose, on the amount of healthy tissue that is exposed to the radiation, and they are patient specific. Newer technologies, such as intensity-modulated radiation therapy (IMRT), deliver the highest dose of radiation to the target while sparing the surrounding healthy tissue. This helps minimize the side effects of external beam radiation treatment. Still, some of the urinary tract side effects may significantly decrease patient quality of life, such as frequent, difficult, or painful urination, urinary leakage, blood in urine, abdominal cramping, nocturia.Radiobiological mechanisms of complications are known concerning cell killing by irradiation and tissue response to treatment . UnfortuNevertheless, there is still a lack of studies describing the importance of the urinary microbiome in radiation-induced changes in the present literature. The majority of the studies have been conducted to provide a relation between the urinary microbiome and urinary tract infections. In most studies, the identification of the microbiome was performed using the 16S rRNA gene sequencing technique, which is the one most commonly used to identify microorganisms in urine samples ,10. AddiThe present study aimed to use the MALDI technique to identify the microbiome and evaluate its changes in urine samples collected from patients treated for prostate cancer undergoing radiotherapy. The compositions of the microbiome were analyzed at various time points before fiducial implantation to see the original bacterial compositions in the urine (before antibiotics were used); then, at the beginning and end of radiotherapy; and during the follow-up, 1 month and 4 months after the end of irradiation. In addition, changes in the species composition of microorganisms at different stages of radiotherapy and the correlation of the obtained results with alteration in immune mediators  present in blood and urine samples were investigated.Patients qualified primarily or post-operatively for radical irradiation for prostate cancer at all stages, with or without hormonal treatment, with or without gold fiducial implantation. One to three gold fiducials are small gold coils, about the size of a grain of rice, which are placed into the prostate gland of patients treated primarily for prostate cancer.Prostate cancer radical radiation therapy and radiation treatment planning were conducted at the Maria Sklodowska-Curie National Research Institute of Oncology in accordance with the valid protocol. Patient characteristics are shown in The treatment decision was made according to disease stage, histopathological grade, and PSA level. Participation in the study did not affect the choice of method, irradiated volumes, and dose. Thirty patients were on hormonal treatment as analog LH-RH, with or without Flutamide. Radiotherapy was delivered on a linear accelerator (25 pts) or cyberknife (25 pts). The volumes included: prostate alone, prostate with a base of seminal vesicles, prostate with the seminal vesicles (primary treatment), irradiated to a total dose of 76–78 Gy delivered in 2 Gy/fraction standard fractionation, or hypofractionation of 36.25 Gy delivered in 7.25 Gy/fraction, using MV photons; prostate bed , irradiated to a total dose of 66–76 Gy delivered in 2 Gy/fraction using MV photons. One patient had a prostate boost of 15 Gy delivered with brachytherapy. If required, pelvic lymph nodes were irradiated to a total dose of 44–50 Gy delivered in 2 Gy/fraction using MV photons, with or without a boost to the involved lymph nodes to a total dose of 60–68 Gy, or delivered as a stereotactic boost of 16 Gy in 2 fractions. Appropriate regulatory approval, including ethical approval, was obtained in all jurisdictions. The study was approved by the NIO-PIB Ethics Committee KB/430-104/19 and conducted in accordance with the principles of Good Clinical Practice. All patients provided written informed consent.Urine and blood samples were prospectively collected from patients; a total of 181 urine samples were obtained from 50 patients undergoing radiotherapy of the pelvic area at different time points: t1—before gold fiducial implantation into the prostate gland (43), t2—before radiotherapy (49), t3—at the end of radiotherapy (42), at follow-up; t4—1 month after radiotherapy (36), t5—4 months after radiotherapy (11). At time point t1, patients had blood and urine collected at least 1 day before starting antibiotics and prior rectal debridement. Usually, Azithromycin 500 mg or Ciprofloxacin 250 mg was prescribed. An enema infusion the evening before and the morning of the day of gold marker implantation was usually recommended for cleaning the rectum before the procedure.It is recommended to collect urine from the midstream, but patients do not always do this correctly despite being instructed. Information for the patient midstream collection for microbiological studies is shown in For urine samples, general tests were performed, such as color, transparency, pH, specific gravity, and standard urine culture, performed obligatorily in each patient during radiotherapy; additionally, anaerobic media were used. In the case of blood samples, routine tests were performed at various time points. Tests included morphology, liver tests, lipids, creatinine, urea, electrolytes, glucose, C-reactive protein (CRP), interleukin 6, coagulation tests, PSA.−1 dilution. Subsequently, 100 μL of undiluted and diluted urine samples was plated onto 3 different types of culture media: Tryptic Soy Agar , Schaedler Agar , and CLED Agar , followed by incubation at 37 °C for 18–24 h. All culture media were in the form of ready-to-use powders. For obtaining pure cultures, single colonies demonstrating different morphologies were transferred onto new plates, streaked, and incubated at 37 °C for 18–24 h.Before microbiological examination, samples were defrosted at room temperature and then thoroughly vortexed. An amount of 0.5 mL of each urine sample was added to 4.5 mL sterile peptone water  to obtain the 10For the isolates’ identification, matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI TOF MS technique), MALDI Biotyper 3.0 platform , and bacterial protein extraction protocol according to Bruker’s guideline were used. Amounts of 300 µL of sterile deionized water and 1 inoculation loop (10 μL) of bacterial biomass were added to a 1.5 mL tube and mixed together using a vortex. Afterward, 900 μL of 96% ethyl alcohol was added, mixed again, and centrifuged . The supernatant was discarded, and the remaining cell pellet was dried using a vacuum centrifuge at room temperature. To extract proteins from bacterial cells, 10 µL of 70% formic acid and 10 µL acetonitrile were added to the cell pellet and mixed by pipetting. Then, the obtained extracts were centrifuged , and the supernatants were used for further analysis.Before identification, 1 µL of supernatant was transferred onto a MALDI MTP 384 ground steel target sample spot . After air drying, the samples were overlaid with 1 µL of MALDI matrix solution—10 mg/mL α-cyano-4-hydroxycinnamic acid  dissolved in standard solvent solution .Samples were analyzed using an ultrafleXtreme MALDI-TOF mass spectrometer  equipped with the smartbeam-II laser–positive mode. Spectra were collected manually through manufacturer software flexControl, using parameters described in a previous work . The colCategorical tests were carried out in an R environment . Chi-square test was performed using “chisq.test” R function to search for statistically significant differences between the expected and observed frequencies of bacteria isolated from urine samples obtained using different collection procedures and at different time points of the patient’s treatment course. In case the assessed expected frequencies were fewer than 5 for at least 20% of the variables, Fisher’s exact test was performed instead (“fisher.test” R function). Spearman correlation analysis (“corrplot” package) was conducted to observe possible relationships between the number of detected bacterial species, sampling time points, and biochemical parameters. The following analyses were conducted using IBM SPSS v.23 . The Kolmogorov–Smirnov test was used to assess the normality of data distribution. Mann–Whitney (non-parametric) and t or ANOVA (parametric) tests were carried out, aiming to point out bacteria that presented significant differences in their incidences depending on the sampling time point and to assess alterations in biochemical parameters between the studied sample cohorts.Microorganisms were present in 140/181 urine samples, using three different culture media obtained over 450 bacterial isolates. The best microbial growth was observed on TSA  and CLED ; the largest number of isolates was obtained in those media. MALDI-TOF MS identification revealed the presence of 33 different bacterial species—3 Gram-negative (G−) and 30 Gram-positive (G+) .Staphylococcaceae (39%), Corynebacteriaceae (15%), and Streptococcaceae (12%). Considering all the studied samples with bacteria, the most frequently isolated species were: Staphylococcus haemolyticus , Staphylococcus epidermidis , Staphylococcus hominis , Enterococcus faecalis , and Micrococcus luteus . The following combinations of species were most often isolated in one sample: S. epidermidis + S. haemolyticus (six cases), S. epidermidis + S. haemolyticus + S. hominis (six), E. faecalis + S. haemolyticus + S. hominis (five), and S. haemolyticus + S. hominis (five). In total, 61% of all the isolated bacterial species were present in at least two patients. When analyzing the samples collected from each patient separately, the following bacteria were most often isolated: S. epidermidis , S. haemolyticus , S. hominis , M. luteus , and E. faecalis . This means that they were isolated at least at one stage of the research from the same patient.The vast majority of the identified bacterial species were represented by the following families: p = 0.85). When evaluating the frequencies of individual species in both urine streams, Enterococcus faecalis and Corynebacterium accolens were the bacteria that presented the most expressive differential distribution between urinary fractions, both presenting greater-than expected-actual counts in the first-void urine . The frequency of some species was related to the urine stream. E. faecalis and S. hominis were more frequent in the first-void urine than in the midstream one. On the other hand, S. haemolyticus was more common in midstream urine. The incidences of S. epidermidis and M. luteus in both samples were comparable.Urine samples were collected from 12 patients from the first-void (40 samples) and middle (39 samples) streams. When comparing the type of urine samples, bacteria were more often found in the samples from the first-void urine than from the midstream one. The absence of bacteria was verified in 12.5% of samples from the first-void urine and in 35.9% of samples from the midstream one. More detailed information is presented in The prevalence of different bacteria in urine samples collected at different time points in the treatment course is shown in p = 0.19). The assessment of chi-square residuals , demonstrating that the variety of bacterial species tends to be inversely proportional to the progression of the treatment. As a consequence, time points 2 and 3 appear strongly positively correlated .The differential distribution of species’ variety according to the investigated sampling time point is suggested; however, it cannot be considered statistically relevant . Considering the incidence of individual bacterial genera at different time points, a greater alteration was indicated when comparing time points 1 and 3 for the Staphylococcus genus , whose incidence was significantly superior at the first sampling time point.The total number of detected bacterial species was significantly higher at time point 1 in comparison to time point 3  and lymphocytes (p = 0.012) in blood showed to have their values altered depending on the evaluated sampling times. The greatest difference was observed between t1 and t4, and the numbers of WBC  and lymphocytes  were significantly higher at the first sampling time point. Analysis of Spearman’s rank correlation coefficient (p = 9.20 × 10−5), as well as the registered levels of leukocytes  and proteins . Urine specific gravity also showed to be indirectly linked to sample pH . The number of bacterial species present in urine samples was also significantly correlated with sample coloration  and leukocytes concentration . The progression of treatment with time showed to be negatively connected—at moderate strength—with the WBC parameter  and other associated variables: the levels of neutrophils , lymphocytes , and monocytes . Such observations may denote a decrease in inflammatory response as the radiotherapy treatment advances. In addition to that, sample-matched PSA concentrations presented a negative and strong correlation with red blood cell count (RBC)  and hemoglobin amount . Total radiotherapy dose was inversely linked to the following biochemical parameters: WBC , neutrophils , and C-reactive protein levels .One-way ANOVA was performed, aiming to identify variables with statistically relevant alterations in their means across samples collected at different time points. The related parameters as white blood cell count (WBC) . Relative PSA levels (PSA at a referred time point/PSA at t1) were significantly lower in patients submitted to hormonotherapy for all the assessed treatment time points . In addition to that, such relative PSA concentrations across time were statistically inferior in the patient group treated using the linear accelerator as a radiotherapy device compared to Cyberknife . Within a short follow-up, this may be related to the frequent use of hormone therapy in the group of patients treated with the linear accelerator (only two patients were not on hormone therapy), in contrast to the patients treated with the Cyberknife, where 72% of patients were not on hormonal treatment.Additional statistical examination was performed on PSA data recorded for the following time periods: before gold fiducial implantation into the prostate gland (t = 1), 1 month (t4), 4 months (t5) after radiotherapy. It was demonstrated that PSA levels displayed a strong negative correlation with the elapsed time of treatment  were the most common in our research. Most of them are opportunistic pathogens, since their presence in urine does not always indicate an infection, but they can be associated with hospital-acquired infections, e.g., after catheters and bacteremia [S. haemolyticus is often part of the urine microbiome, especially among men [Research indicates that the urine microbiome has not yet been clearly defined, but among healthy people, it consists mainly of slow-growing and fastidious microorganisms, mainly belonging to five phyla: bacteria . Numeroubacteria ,17 and tbacteria . Among he cancer . Corynebular DNA . The useular DNA . The autcteremia . S. haemmong men . Studiesmong men . It is among men ,25. Our S. epidermidis [S. saprophyticus infections mostly cause UTI in young women, but in males, they have been associated with urethritis and prostatitis [S. hominis, M. luteus, and S. warneri) are considered to be potential probiotic strains. They produce peptides and enzymes, which give them antibacterial activities against pathogens, such as Escherichia coli or Staphylococcus aureus [E. coli and S. aureus) was observed in any of the samples with at least one of the probiotic strains mentioned above (over 46% of samples).However, urine samples can suffer from skin and perineal contamination if the periurethral area is not sterilized before urine collection . The prodermidis . Among istatitis . Three os aureus . In our E. faecalis, which was isolated in over one-third of the patients, is considered the second most important cause of urinary tract infections (UTI). This bacterium is part of the fecal microflora and can cause UTI through an endogenous route [Streptococcus and Staphylococcus species were more frequently isolated in middle urine. These types are indicated in other studies as most often isolated from urine samples of healthy men [us route . This balthy men . This obS. haemolyticus, S. epidermidis, S. hominis, E. faecalis, and M. luteus. It can be noticed that, only in the case of E. faecalis, no significant changes in the frequency of its occurrence were observed, which may indicate accidental contamination of samples by patients during their collection. In the case of the remaining strains, their presence in the urine at the end of RT was approximately 15% lower. The decreased number/species of bacteria was restored 1 month after irradiation. This suggests that our radiation treatment does not completely damage the urinary microflora, allowing regeneration. Four months after RT, S. hominis and S. haemolyticus were detected in approximately half of the collected samples. The remaining strains were found in a much smaller percentage. Moreover, studies show that differences in the number and diversity of bacteria are more important than individual types found in patients. This is because the composition of the microbiome is influenced by many different factors, such as genetic predisposition, environment, diet, and the use of antibiotics.There is a change in the number of bacteria species concerning the time of the sample collection. At the intermediary sampling time points (before radiotherapy and at the end of radiotherapy), generally, lower numbers of bacteria were detected in samples, indicating that the procedures before radiotherapy and radiotherapy itself result in depleting the local microbiome. At time point 1, before fiducial implantation, the samples represent the general urinary microbiome status in patients. Fiducial implantation requires the use of antibiotics that induce a decrease in the number/species of bacteria on the day of radiotherapy beginning. Antibiotics are the most popular treatment for urinary tract infections and are also used as a prophylactic treatment. However, the use of broad-spectrum antibiotics can negatively influence the beneficial microflora in the patient and consequential selective overgrowth of pathogenic bacteria. The exact effects of antibiotics on urinary microbiota are not known, but clinical trials are ongoing on this topic. Additional treatment with radiation also induces depletion of bacterial species if there is no co-infection associated with the occurrence of radiation side effects, such as difficulty in urinating and urine retention in the bladder. During the research, it was observed that the applied treatments, both RT and the hormone therapy, contributed to the overall decline in the diversity of the microbiome. Due to the small number of individual isolates, it is not possible to specify the changes in urine microbes resulting from the addition of androgen deprivation therapy (hormonotherapy). Including more patients in subsequent studies would make it much easier to find changes in the occurrence of individual strains in urine samples. In our group of patients, the most frequently isolated strains were Microbiome is extensively analyzed in the gut and its changes in variable situations, but little information is available on the microbiome and urinary tract in cancer patients. To our knowledge, this is the first study evaluating the differences in bacterial species in men with prostate cancer undergoing radiotherapy. Unfortunately, this study has its limitations due to an insufficient number of patients required to assess microbiome differences, taking into account different doses of radiotherapy or hormonal therapy usage, local advancement of the disease, and the Gleason score. We are aware that all of these factors influence the oncological response and, at the same time, can affect the microbiome. Here, we present the first result of an ongoing project in which we intend to include 300 patients. A larger number of patients will allow us to assess the differences between the variable groups of patients. For now, the obtained results emphasize the need for further research on changes in the microbiome in urine, both during and after oncological treatment, in order to correlate its changes with the early and late side effects of radiotherapy and the clinical outcome of the disease.Application of the MALDI identification appeared to be a suitable tool for the fast deciphering of differences in the microbial diversity of the urinary microbiota depending on the time point of radiotherapy, as well as between the type of urine specimens used—first-void and middle stream. The obtained results revealed a similar spectrum of bacteria from the initial and middle urine streams. In turn, the treatment of prostate cancer patients with both antibiotics before gold fiducial implantation and radiotherapy affected the bacterial species composition, namely depleting the urine microbiome, which recovered after ending the therapy. To our knowledge, this is the first study evaluating the differential bacterial species in men with prostate cancer undergoing radiotherapy. Further research should focus on the correlation of microbiome changes and their potential impact on radiation side effects in prostate cancer patients.


Chlorella sp. (Chlorophyceae), needs to be monitored because it has gamma radioresistance capabilities and can cause the corrosion of stainless steel.The water quality in the interim wet storage of spent fuel (ISSF) needs to be monitored due to its function as a radiation shield. Water in ISSF pools must be free from microorganisms such as microalgae that live in a radioactive environment. Moreover, particular microalgae are capable of causing corrosion to stainless steel, which is a component of ISSF. Therefore, this study aims to determine the diversity of microalgae in the ISSF and those living in a radioactive environment, which cause corrosion. The microalgae were detected using the diversity and Palmer indices. The sampling of microalgae water was carried out by vertical filtration method at eight sites of ISSF. The results show that the diversity of microalgae (H′) was low due to radiation exposure in pool water, hence, only specific species can survive. The evenness (J′) of the microalgae was low, causing a high dominance index (C) value. Furthermore, the dominating species, namely,  The interim wet storage of spent fuel (ISSF) in Serpong serves to receive and store spent fuel from the Multipurpose Reactor G.A. Siwabessy (RSG-GAS), and irradiated materials from radio metallurgy installation (RMI) and radioisotope installation (RI) . This faDemineralized water is used in the pool to function as a cooling system and radiation shield for spent fuel. The cooling system removes the decay heat generated by spent fuel through water circulation to maintain the temperature in the pool keep it under 35 °C . A waterHaematococcus pluvialis [Chlorella vulgaris causes an fourfold increase in the average corrosion rate [C. vulgaris to easily adhere to 316L stainless steel due to extracellular polymeric substances and cause corrosion after 21 days of incubation [C. vulgaris is an electrochemical process with anodic and cathodic reactions, namely, the dissolution of FeO and reduction of oxygen [Chlorella vulgaris attached to metal surfaces can form biofilms that cause corrosion due to the chloride ion content and oxygen produced by photosynthesis, which increases the concentration of dissolved oxygen (DO). Therefore, it accelerates the cathodic reaction and uptake of phosphate ions (P) for nutritional needs from C. vulgaris [Several investigations into microorganisms in nuclear facility pool water have been carried out in different locations. Microorganism detection in ISSF has found corrosion-causing bacteria with an estimated population of 90,000 CFU/mL . It was luvialis . Furtherluvialis . In mildion rate . Anothercubation . Corrosicubation . Meanwhif oxygen . Chlorelvulgaris ,8.Research to detect the presence of microalgae in ISSF in Serpong has not been carried out. Therefore, this study aims to understand the microalgae ecology in ISSF pool water to prevent downtime and minimize corrosion caused by microorganisms in the facility. This work was carried out using the diversity and Palmer indices to provide information on microalgae in a radioactive environment and the potential to corrode stainless steel materials as a parameter of water suitability in nuclear facilities. Furthermore, the chemical and physical parameters of the water are measured as data to support research on microalgae life in nuclear facilities.The ISSF facility is located in the Science and Technology Center, as part of the Radioactive Waste Technology Research Center, Serpong, Indonesia. Water sampling was carried out in December 2021 at 8 sites, where 3 are located in the pool  and 5 sites in the ISSF channel area based on the flow direction towards the main pool from RSG-GAS, PT. INUKI, and the channel junction, as presented in Good Practice for Water Quality Management of Research Reactor and Spent Fuel in Nuclear Facilities issued by the International Atomic Energy Agency (IAEA) world atomic agency [Water chemical–physical parameters were measured in situ. Light intensity was measured using a digital lux meter LX 1010B, while the pH, temperature, total dissolved solid (TDS), and electrical conductivity (EC) were estimated with a multiparameter instrument, Hanna Instruments 9811-5 . Subsequently, dissolved oxygen (DO) was evaluated with Hanna DO-5510, and water radioactivity using an STHF-R Water Proof High Dose Rate Probe by inserting the probe into the water. The maximum standard for water chemical–physical parameters for nuclear facilities refers to the book c agency .Samples were examined using a camera microscope  with a hemocytometer at 400× magnification. The samples were observed on the hemocytometer’s entire surface due to the microalgae’s low density. The genus of microalgae was identified based on the morphology, the shape of the flagellum, the number of flagella, the flagellum direction, the chloroplast location, and the shape of the chloroplast. Reference sources used for identifying microalgae are van Vuuren et al.  and Bell2), L = area of one field of view (mm2), P = number of chopped plankton, p = number of fields observed, v = volume of sample in under the cover glass (mL), V = volume of filtered water sample with plankton net (mL), w = volume of filtered microalgae sample (L). The Shannon–Wiener diversity index (H′) was calculated by the formula, The identified microalgae genus was processed quantitatively ecologically. The abundance of microalgae was calculated using Lackey’s drop (microtransect) method , with thiversity .The evenness of the microalgae was calculated using the Pielou evenness index (J′), with the formula, the same . The mic species .The identified microalgae were calculated by estimating algae pollution index based on Palmer  to deterChemical–physical parameters as data supporting the life of microalgae were calculated using the water quality index (WQI) with the formula,  Sevgili . The cri Sevgili , to deteChlorophyceae, consisting of Chlorella sp., Kirchneriella sp., and Chlamydomonas sp.; Cyanophyceae, with Chroococcus sp., Gloeocapsa sp., and Stephanodiscus sp.; Bacillariophyceae, including Nitzschia sp. and Navicula sp.; Zygnematophyceae, with Spirogyra sp.; and Trebouxiophyceae, with Dictyosphaerium sp. Chlorophyceae and Cyanophyceae have the highest percentage in ISSF, about 30%, while Bacillariophyceae have a percentage of 20%, and Zygnematophyceae and Treuboxiophyceae of 10%, as shown in The microalgae found in ISSF consist of 5 classes and 10 genera. The classes are Chlorella sp. was consistently found at all sampling sites, followed by Chroococcus sp., which was present at seven sites. The total abundance of microalgae in the ISSF was 10,867 Ind/L. Chlorella sp. had the highest abundance, namely, 8916 Ind/L, followed by Chroococcus sp., with a value of 957 Ind/L  value has a value of 0.76, and is classified as low diversity. The evenness index value (J′) has a value of 0.31, indicating low evenness. Meanwhile, the Simpsons dominance index (C) has a value of 0.68, indicating the dominance of microalgae in nuclear facilities .The maximum limits for the parameters of light intensity, DO, TDS, and radioactivity are not regulated by the International Atomic Energy Agency, IAEA. Meanwhile, the institutions have regulated other parameters such as temperature, pH, and electrical conductivity (EC). This section shows the results of measuring the chemical–physical parameters of water in pools and channels from the ISSF and the maximum limits set by the International Atomic Energy Agency (IAEA), as presented in The light intensity measured at the location is different due to the installation of lights only in the pool area , therefore, the channel area (K6 and K8) is 0, as shown in The DO values measured were 3.9–5.5 ppm, with the highest value at K3 (pool outlet), and the TDS at all sites in the ISSF is 0 ppm. Since the conductivity value of the ISSF is 1.02–1.9 µS/cm, it does not exceed the recommended limit of <10 µS/cm . The radChlorella sp., Chlamydomonas sp., Navicula sp., and Nitzschia sp. are pollution-resistant genera  and PT. INUKI. Moreover, microalgae can be sourced from both areas when the transfer of spent fuel takes place. When the transfer channel is closed, the water will only circulate in the channels and pools of the ISSF.In this study, chemical–physical parameters were used as supporting data for the diversity of microalgae in nuclear facilities. The measurement results show that the water’s temperature, pH, and electrical conductivity (EC) are still below the maximum limit. Based on the life support for microalgae in water, all parameters are appropriate except for light intensity and radioactivity. The light intensity measured at sites K6 and K8 has a value of 0 Lux, and some genera are still found. The radioactivity parameter in this study is a limiting factor for microalgae life; therefore, only specific genera can live in this environment. According to the water quality index (WQI), the value obtained is in a good category, with a score of 75 .Chlorophyceae [Chlorella sorokiniana can live in mixotrophic conditions and grow in autotrophic as well as heterotrophic conditions [Chlorophyceae group at site K8 may be caused by its ability to grow in mixotrophic conditions.Generally, high light intensity increases the number of ophyceae . Howeverophyceae . Chlorelnditions . The larChlorella sp. (Chlorophyta) and Chroococcus sp. (Cyanophyta) were the most abundant microalgae. Tolotti et al. [Chemical–physical parameters of water, such as DO, temperature, and pH, play an essential role in the distribution of these microorganisms . Phytopli et al.  stated tChlorella, which grows at pH 4–10 [Chlorella sp. It was also reported that the microalgae Geitlerinema amphibium (Cyanophyta) can grow and dominate in a radioactive environment with a pH of 8.4 [The degree of acidity significantly affect the growth of autotrophic microalgae such as  pH 4–10 . The pH H of 8.4 . FurtherH of 8.4 ,35.Chlorella sp., can trigger corrosion. There is a link between oxygen as the primary electron acceptor in accelerating the cathodic reaction, which will produce corrosion products [2O3 [The minimum DO level for the survival of aquatic organisms is 4 ppm . The meaproducts . The damcts [2O3 .The dissolved solids measured in the facility are 0 ppm, which indicates low organic matter in the channel and pool areas of the ISSF. When the TDS value is high, it shows the presence of organic matter that enters the water system. According to Rijaluddin et al. , an incrWater radioactivity is caused by piles of racks filled with spent nuclear fuel at the bottom of the pool . Since tChlorella sp. is almost always found at every site due to the radioactivity of water nuclear facilities. The effect of gamma irradiation on Chlorella sp. can cause a decrease in cell stability, size, chlorophyll content, and protein [Chlorella sp. showed a radioresistance response to low doses of gamma radiation at 10, 50, as well as 75 Gy, and formed radioprotective phytochemicals that will protect cells from radiation damage. Moreover, gamma radioresistance occurs by changing physiological and metabolic processes in cells to form primary defense mechanisms in microalgae species [Chlorella sp. to obtain mutants for increasing biomass and lipid production. This species had higher cell survival at doses of 100–600 Gy and lowered at doses of 800–900 Gy, and could also tolerate up to 900 Gy [ protein . Pradhan protein  stated t species ,42. Sent species  used Chlo 900 Gy . The effChlorella sp. as the genus with the highest abundance, namely, 8916 Ind/L, as shown in Planothidium frequentissimum and Crenotia angustio, in water sources with radioactive content and morphological deformities. Shuryak [Cyanophyceae group [Radioactivity in the water is one of the limiting factors for microalgae life that can impact species richness and abundance in nuclear facilities. In this study, the total abundance of microalgae is 10,867 Ind/L, with  Shuryak  conducteae group .Geitlerinema amphibium (Cyanophyta), Scenedesmus quadricauda (Chlorophyta), Aulacoseira ambigua (Bacillariophyta), and Pleurochloris imitans (Xantophyta). Geitlerinema amphibium predominates in this area. Duhovnaya et al. [The value of H′ in this study is less than 1, indicating that the diversity of microalgae is low, as shown in a et al.  stated tChlorella sp. The increasing number of species that dominate can cause sensitive species to be lost due to pollution [The evenness index value (J′) obtained is close to 0, which indicates low evenness or that the number of individuals of each genus has a very different distribution . When thollution . A high ollution .Chlorella sp. as pollution-resistant microalgae, based on the Palmer index, and its ability to cause the corrosion of stainless steel materials [The low algal index value is in line with the WQI value. Based on this result, the low input of organic matter will affect the water quality. This indicates that the quality of water in ISSF is still below the required standards. The water quality inspection process in the cooling system was previously only carried out chemically and physically ,52, whilaterials  is a warChlorella sp. (Chlorophyceae), needs to be monitored due to its gamma radioresistance capabilities, which causes corrosion in stainless steel. Apart from the existing chemical–physical parameters, the diversity and Palmer indices can be used for detecting microalgae, and as parameters of water suitability in nuclear facilities.Low diversity (H′) of microalgae is caused by radioactivity in the pool, therefore, only certain species can survive. Based on this study, the dominating genus,


This exploratory study aimed to investigate the resting-state electroencephalographic (rsEEG) correlates of the cognitive reserve from a life span perspective. Current source density (CSD) and lagged-linear connectivity (LLC) measures were assessed to this aim. We firstly explored the relationship between rsEEG measures for the different frequency bands and a socio-behavioral proxy of cognitive reserve, the Cognitive Reserve Index (CRI). Secondly, we applied moderation analyses to assess whether any of the correlated rsEEG measures showed a moderating role in the relationship between age and cognitive function. Moderate negative correlations were found between the CRI and occipital CSD of delta and beta 2. Moreover, inter- and intrahemispheric LLC measures were correlated with the CRI, showing a negative association with delta and positive associations with alpha 1, beta 1, and beta 2. Among those correlated measures, just two rsEEG variables were significant moderators of the relationship between age and cognition: occipital delta CSD and right hemispheric beta 2 LLC between occipital and limbic regions. The effect of age on cognitive performance was stronger for higher values of both measures. Therefore, lower values of occipital delta CSD and lower beta 2 LLC between right occipital and limbic regions might protect or compensate for the effects of age on cognition. Results of this exploratory study might be helpful to allocate more preventive efforts to curb the progression of cognitive decline in adults with less CR, possibly characterized by these rsEEG parameters at a neural level. However, given the exploratory nature of this study, more conclusive work on these rsEEG measures is needed to firmly establish their role in the cognition–age relationship, for example, verifying if these measures moderate the relationship between brain structure and cognition. Investigation of cognitively healthy older adults is also a fruitful avenue to provide information about the neural resilience mechanisms able to curb the onset or the progression of cognitive deficits as shown by the works of the Cambridge Centre for Ageing and Neuroscience (Cam-CAN) group . HoweverQuantifying CR is a matter of constant reviews and developments as it is challenging to assess it directly. Most measures are derived from socio-behavioral indexes used as indirect proxies, such as educational or occupational attainment, intelligence, leisure activities, and other lifetime experiences . Also, cIn this vein, brain imaging techniques, both structural (magnetic resonance imaging - MRI) and functional [functional MRI (fMRI) and positron emission tomography (PET)], have been used to investigate the potential neural correlates of CR along the age-related cognitive continuum. Structural MRI has been mainly employed to search for anatomical correlates of brain reserve, considered the more genetic and passive component of such reserve  and magnetoencephalography (MEG) have proven to be a promising, almost inexpensive method to study the CR neural correlates. (M)EEG is particularly useful, given its relatively non-invasive nature and high temporal resolution. Especially relevant in the context of FC, the oscillatory brain activity is thought to be a key index of the coordinated activity in long-range brain networks . Hence, Although the literature on the EEG correlates of CR in healthy aging is not as prolific as with other imaging techniques, several findings suggest that EEG is sensitive to the electrophysiological changes associated with various CR proxies . Some stConsidering all the results provided by both imaging and (M)EEG studies, it seems that CR can be reliably identified at a neural level. Moreover, The current perspectives regarding research on cognitive reserve have underlined the importance of studying aging following a life span approach to get reliable measures that reflect individual differences in brain structure and function built over the years . The reaTo achieve our main aim, rsEEG variables were derived from power spectral density and connectivity measures for the main EEG frequency bands in humans  since previous studies have found differences between high and low CR participants in some of these parameters ,2021. FuTherefore, following the conceptual research model proposed by Advertisements and informative talks were used to aid in recruiting young and middle-aged adults among University of Minho staff and students, as well as healthy older adults among users from different cultural associations and day-care centers in Portugal’s North region. Data were collected from 79 volunteers, of whom 56 participants met the inclusion criteria and composed the final sample. Selected participants (age range between 18 and 82 years), thus, met the following criteria: (1) intact or well-corrected sensory function (self-reported); (2) perform independently in instrumental activities of daily living according to the Portuguese version of the Lawton and Brody scale ; and, 3. Note thThe study protocol was approved by the Institutional Review Board of the University of Minho (CE.CVS 095/2018) and conformed with the principles embodied in the Declaration of Helsinki. Before data collection, all participants were informed about the study and signed the corresponding informed consent form.The CRIq  was admiRegarding the cognitive function assessment, several subtests of the CANTAB  were appPsychomotor speed: the Reaction Time test (RTI), where the participant must hold a button at the bottom of the screen and react as soon as possible to release this bottom and select one out of five circles presented above whenever a yellow dot appears in one of them. The outcome measure in this study was the median of the movement time.Memory: the Paired Associates Learning test (PAL) consists in learning the location of a series of patterns displayed behind boxes and, afterward, remembering where a specific pattern was initially located. The outcome measure used in this study was the total number of errors made by the participant. Also, the Spatial Span test (SSP), where a sequence of squares changing in color has to be remembered in the same order as they changed, with a progressive increase in the number of squares to be remembered. The outcome measure was the forward span length .Executive function: the Spatial Working Memory test (SWM) for which the participant must sequentially search for yellow “tokens” in each of several boxes. Planning and strategic thinking are needed as a box that had already contained a token cannot contain another one in the same trial. The selected outcome measure was the total number of errors . The Multitasking Test (MTT), where the participant has to indicate on which side of the screen an arrow appears or in which direction it is pointing according to a cue at the top of the screen (location vs. direction conditions). The task has single-task blocks, when the rule is consistent across trials, as well as multitasking blocks, when the cue changes from trial to trial in a randomized order. The outcome for this test was the median of the multitasking cost; that is, the difference between the median latency of response (from stimulus appearance to button press) during multitasking blocks and that of single-task blocks.In order to have a single measure of cognition, a CANTAB cognitive composite score was derived by computing the z scores for each of the five subtests and averaging across the five scores.At least 3 min of EEG data were recorded from each participant while sitting comfortably and relaxed with eyes closed using an ActiveTwo Biosemi system  with 64 active electrodes inserted in an elastic cap according to the international 10-10 system. A Common Mode Sense (CMS) and Driven Right Leg (DRL) montage of two electrodes located around the vertex was used as a reference. Electrode offset was kept below 30 mV. EEG data were filtered online between 0.01 and 100 Hz and digitized at a sampling rate of 512 Hz. Simultaneously with EEG recordings, ocular movements were recorded with two electrodes located supra and infra-orbitally to the left eye and another pair situated at the lateral canthi of each eye.1 An independent component analysis algorithm was then applied to extract independent components in the remaining data. ICLabel plug-in .eLORETA is a genuine inverse solution (not merely a linear imaging method) with exact, zero error localization in the presence of measurement and structured biological noise . ComputaIn addition, the eLORETA algorithm was also employed to obtain a measure of functional connectivity. Specifically, we calculated lagged-linear connectivity (LLC) as a measure of interdependence of rsEEG sources, given that it estimates linear inverse source connectivity while removing the artificially high zero-lag instantaneous interactions inherent to the low spatial resolution of the EEG tomography . AlthougBefore the main statistical session, an exploratory analysis of the data was carried out. There were no missing data for the demographic and health variables, the CRIq, or the EEG variables. However, for the CANTAB subtests, some missing values were present . These missing data were addressed by multiple imputations, creating 25 imputed datasets through a predictive mean matching procedure implemented in IBM SPSS  had a skewed distribution, and they were transformed to a logarithmic scale (log-10) . No outlr values. Given the exploratory nature of this study, instead of using the Bonferroni correction on associated p values to assess statistical significance, we have considered for further analyses only those correlations with at least a medium effect size . The correlation coefficients are reported as Pearson’s Secondly, to address whether rsEEG variables can be used as direct measures of CR, a moderation analysis using the regression-based approach in Hayes’ (2017) PROCESS macro (Version 3.5) for IBM SPSS was conducted  on Y (CANTAB composite score) at relatively low (16th percentile), moderate (50th percentile), and relatively high (84th percentile) values of M (rsEEG measures) were evaluated through this pick-a-point procedure . In addiModerate effect sizes were found for correlations between the CRI and several rsEEG CSD  for different frequency bands . NegativAs regard to the LLC  measures, moderate effect sizes were found for correlations between total CRI and inter- and intrahemispheric variables in different frequency bands . PositivA moderate negative correlation was found between CRI and delta band intrahemispheric LLC involving parietal and limbic regions in the right hemisphere . PositivFor those rsEEG variables with a moderate size correlation with total CRI, moderation analyses were performed. Just two of the 13 variables significantly interacted with age to moderate cognitive status: the CSD of the delta band in the occipital ROI and the LLC of the beta 2 band in the right hemisphere between occipital and limbic regions.R2 = 0.61, F = 19.62, p < 0.001. As expected, a negative effect of Age on CANTAB score was significant, b = −0.02, t(51) = −8.25, 95% BootLLCI = −0.028-BootULCI = −0.017; p < 0.001, and although the effect of delta CSD was not significant, b = −0.67, t(51) = −1.96, BootLLCI = −1.27-BootULCI = 0.025; p = 0.06, the age × delta CSD interaction was significant, b = −0.04, t(51) = −2.47, BootLLCI = −0.065-BootULCI = −0.009; p < 0.05; ΔR2 = 0.05, F = 6.59, indicating a moderator effect of delta CSD in the occipital region on the relationship between age and cognition. The covariate sex had no effect, b = 0.19, t(51) = 1.04, 95% BootLLCI = −0.25-BootULCI = 0.62; p = 0.30. The simple slope analysis indicated that the conditional effect of age on cognitive performance was statistically significant at any level of delta CSD in the occipital region  = 17.83, p < 0.001. The effect of age was significant, b = −0.02, t(51) = −7.88, BootLLCI = −0.03-BootULCI = −0.02, p < 0.001, but the effect of beta 2 intrahemispheric LLC was not, b = 0.05, t(51) = 0.21, BootLLCI = −0.41-BootULCI = 0.53, p = 0.83. The interaction term between age and beta 2 intrahemispheric LLC was significant, b = −0.03, t(51) = −2.63, BootLLCI = −0.04-BootULCI = −0.005, p < 0.05; ΔR2 = 0.06, F = 6.92. The covariate sex had no effect, b = 0.18, t(51) = 0.94, 95% BootLLCI = −0.19-BootULCI = 0.57; p = 0.35. Again, the conditional effect was significant for the three values of beta 2 LLC  as moderators failed to reach statistical significance for each of the possible interactions with age  as possible objective neural-based correlates of cognitive reserve following the model proposed by Numerous neuroimaging studies have employed the correlational methodology to build potential associations between CR and structural or functional brain measures. In line with our results, in resting-state fMRI studies, higher CR has been associated not only with increased brain functions  regions, greater local efficiency, and clustering in cuneus and occipital regions) but also with decreased activity  see  or positDespite the scarcity of rsEEG studies following the correlational approach, many studies have compared rsEEG variables between high and low CR groups. A negative correlation between the CRI and the CSD of beta 2 was also observed in the occipital region. In some studies, decreased brain activity in higher CR participants has been related to more efficient neural mechanisms, even in resting-state. For example, Inter- and intrahemispheric connectivity are considered measures of integration of the two cerebral hemispheres and hemispheric specialization, respectively. Further, they are paramount for good cognitive functioning . So, it Nevertheless, as we noted above, increased FC is not always associated with better network functioning, both in resting-state and task-related activity . Our resAlternatively, this complex pattern of positive and negative correlations between LLC and CRI may reflect differences in resting-state networks such as the DMN, dorsal attention network, and the frontoparietal network that have been previously shown to be anticorrelated in their activity . These rWe performed moderation analyses with the rsEEG variables that showed a moderate correlation with CRI as moderators of the age-related prediction on cognitive scores (CANTAB composite). Out of the thirteen variables tested, just delta CSD in the occipital region and right beta 2 LLC between occipital and limbic regions were significant moderators of this relationship. More specifically, the negative relationship between age and cognitive performance observed in our sample was attenuated among people with less occipital delta activity and lower levels of right beta 2 LLC between occipital and limbic regions. As a tentative hypothesis, this might mean that CR could be manifested as a more efficient network with weaker activation. People with lower CR probably showed intensified activations as an attempted, although unsuccessful, compensatory mechanism.As mentioned above, an increase in delta activity, especially in posterior areas, has been associated with cognitive impairment and reported in some pathological conditions such as AD , strokesA more paradoxical result was the moderation effect of the beta 2 LLC between occipital and limbic regions in the right hemisphere. The moderation analysis showed a more negative impact of age on the cognitive score as the beta 2 LLC increased, contrary to the positive correlation between occipital CSD in this frequency and CRI. This contradictory pattern seems to be due to the inclusion of the factor age and the interaction term in the moderation analyses. The effect of beta 2 LLC on CANTAB score was positive in the regression model , but when interacting with age, the effect turned negative. Although this relationship seemed the inverse at younger ages , as age increased, higher levels of beta 2 LLC seemed to lead to a worsened cognitive function. There are well-documented age-related differences in rsEEG variables in the literature, even in the field of CR. For example, A negative correlation between beta 2 activity and behavioral performance has been previously observed by Moderation analyses were also conducted using each of the five CANTAB subscales as dependent variables instead of the composite score. The obtained results were somehow confirmatory of the general results. However, the CANTAB subscales are highly correlated, so the results should be taken cautiously. In detail, the analyses for the Spatial Span test (SSP), a measure of recent memory, showed that occipital delta CSD moderated the relationship between age and SSP performance: as occipital delta CSD increases, the age-related decline for SSP performance also increases, just like the results observed for the CANTAB composite score. Besides this confirmatory result, the interhemispheric LLC for temporal beta 1 and the right intrahemispheric LLC between frontal and occipital regions for beta 1 were also significant moderators of this relationship, with greater connectivity associated with a lower age-related decline in cognition. These results, again, point to the potential existence of compensatory mechanisms that are reflected in increased connectivity in high- than low-CR individuals , a measure of executive function, moderation results were the same as for the composite CANTAB score. Therefore, a more negative effect of age on executive function was observed as the beta 2 intrahemispheric LLC increased. Taken together, results of the analyses with the different CANTAB subtests, may indicate the coexistence of compensatory mechanisms and increased neural efficiency processes in the resting-state brain electrical activity of high CR individuals, in line with the manuscript’s main results. Nevertheless, these results suggest that the performance in different cognitive domains is supported by distinct neural correlates of CR. In that vein, previous studies have shown that CR may be associated with specific cognitive domains while unrelated to other domains  dependinFinally, it is important to highlight the absence of moderation effects on the relationship between age and cognitive function by the CRI measures . To consider them reliable correlates of CR, they would be expected to moderate the age and cognitive function relationship. One reason that could explain this discrepancy is that these indices may reflect the amount of CR acquired during a person’s lifetime  but not In sum, many studies have proposed different neural measures as neural correlates of CR; however, as far as we know, none of them have tested the rsEEG variables as moderators in the relationship between age and cognitive status. Most of them have used the rsEEG measures as independent variables and split the sample into age and CR-based groups to perform ANOVAs. This approach presents limitations stemming from the use of an arbitrary median score to build the CR groups. Unlike previous studies, the strategy of the current work was not to compare age groups but instead to take the factor of age as a continuous measure. This life span approach, including participants from 18 to 82 years old, with higher ages indicating possible higher physiological neural deterioration, allows us to establish which rsEEG variables may be taken as probable protective or risk markers for accelerated cognitive decline as age increases. This, in turn, justifies to some extent the absence of a structural measure of brain integrity.Moreover, this study followed a correlational and regressional approach and considered CR as the ability to recruit brain networks more efficiently. In light of the reviewed evidence, CR might be reflected by increases or decreases in activity and functional connections across the brain. Indeed, current results showed that as age increases, higher levels of occipital delta CSD and stronger LLC between medial and posterior regions for beta 2 may indicate a less effective way to recruit networks in people with lower levels of CR.Although this results add valuable information to the research of aging and CR, they are not free from limitations and shortcomings. First, the large number of studied variables in this study increases the probability of a type I error, and, as such, some of our results could be false positives. However, given the study’s exploratory nature, which aims to generate hypotheses to be systematically and thoroughly evaluated in future studies, we adopted a rather lenient strategy regarding the problem of multiple comparisons. Second, there was a considerable amount of missing data for a dependent variable , which was subjected to a multiple imputation procedure. Although this is not the most desirable situation, a statistical comparison of CANTAB subtests’ score between the sample with imputed values and the sample without imputed values showed no significant differences, thus ensuring that the variable distributions were similar before and after the imputation procedure. Third, the lack of a measure of brain status did not allow us to align our results with the current research recommendations in the field of cognitive reserve . Hence, In this study, we aimed to find possible neural correlates of CR that can be used as objective measures of CR instead of indirect proxies, such as educational attainment or intelligence measures. To this end, we analyzed dozens of measures of rsEEG, including eLORETA solutions for cortical current density sources and connectivity, as moderators on age-related changes in cognitive function. Moderation analyses showed that lower delta activity in the occipital region and lower connectivity of beta 2 in the right hemisphere between occipital and limbic regions could index compensatory mechanisms and increase neural efficiency to maintain high cognitive performance during the aging process. Therefore, these patterns of rsEEG activity might be considered as putative neural correlates of the cognitive reserve to be deeply studied with more statistical control. As stated before, this study is highly exploratory with many variables under study, aiming to generate hypotheses to be refuted in future projects. This type of objective neural measure could be useful in the future to prevent the appearance of cognitive impairment, allocating more preventive efforts to those adults with rsEEG markers of low CR.https://osf.io/bdfyu/.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: Open Science Framework: This study involves human participants, so its protocols were reviewed and approved by the Institutional Review Board of the University of Minho (CE.CVS 095/2018). The patients/participants provided their written informed consent to participate in this study.AB, AS, and DP involved in the conception and design of the original study, as well as in the enrolment of the participants, and controlled the interpretation of the results. AB and DP carried out the neuropsychological assessments and EEG recordings and performed the data analysis in terms of database construction, EEG data processing, and statistical analyses. AB generated the first draft of the manuscript, and developed the final version of the manuscript. AS and DP critically reviewed the draft and modified it accordingly. All authors contributed to the article and approved the submitted version.