Adaptation of key bacterial vaginosis-associated bacteria to a medium simulating genital tract secretions: a transcriptomic analysis

Frontiers in Genetics, Journal Year: 2025, Volume and Issue: 16

Published: March 26, 2025

Approximately 30% of reproductive-age women around the world are affected by bacterial vaginosis (BV), which is most common vaginal infection and main cause discharge (Peebles et al., 2019). BV a polymicrobial dysbiosis with substantial data supporting role sexual transmission BV-associated bacteria (BVAB) (Fethers 2008; Muzny 2022). In case BV, several strict facultative anaerobic increased in number microbiota replacing protective Lactobacillus species, major colonizers present healthy (Rosca 2020b; Chen 2021). These species recognized to be living biofilm adhered epithelial cells creating characteristic clue (Swidsinski 2024). Despite identification multiple cases (Ceccarani 2019), some may have greater impact on development than others (Gilbert 2019; Randis Ratner, Castro Previously, Gardnerella vaginalis, Fannyhessea vaginae, Prevotella bivia were detected, vivo specimens from early (Muzny 2018), further vitro experiments showed important synergisms between these suggesting an essential initial stages formation (Castro Having this mind, we recently investigated how three interact when growing nutrient-rich medium, allowed for growth. Notably, observed that hundreds genes differentially expressed comparing triple-species single-species biofilms (Sousa However, adaptation different growth media results alterations transcriptome consequent variations gene expression (Blair 2013; Smith 2018). As such, consideration culture medium suitable grow specific crucial depending will performed factors need analyzed. Although use allows better laboratory settings, especially fastidious microorganisms, using mimic conditions preferable trying evaluate microorganisms behave vivo-like (Pouget For study it useful simulates environment, simulating genital tract secretions (mGTS) contains amino acids, as well albumin, urea, mucin found fluid (Tietz Klein, 2018).Thus, investigate environment affects biofilm, aimed explore adaptations would compare mGTS, partially conditions.2Materials methodsBacterial strains conditionsG. vaginalis ATCC 14018, F. vaginae BAA-55, P. 29303 used study. The kept Brain Heart Infusion (Liofilchem, Roseto degli Abruzzi, Italy) supplemented 23% glycerol (98%, Panreac, Darmstadt, Germany) at -80ºC. Before each experiment, they grown Columbia Base Agar (Liofilchem) plates 5% defibrinated horse blood (CBA) (Thermo Fisher Scientific, Lenexa, KS) 48 h 37°C (AnaerocultTM A, Merck Millipore, Taufkirchen, Germany). New York City III (NYCIII) nutrient rich was prepared previously described 2020a). mGTS secretions, adapted previous studies (Liu 2011; Stingley 2014), purpose 2025).Biofilm Triple-species G. formed 24-well (Orange Braine-l'Alleud, Belgium), either NYCIII or competitive model 2022a). considers all initiation thus incubated same time form biofilm. suspension 24 h. since limited planktonic cultures biomass CBA day formation. Thereafter, both protocols, concentration adjusted 9107 CFU/mL reading optical density 620 nm suspensions then dispensed wells plate total volume 1 mL, final 1107 under conditions. characterization composition qPCR, removed washed once 0.9% NaCl (Sigma, Germany), followed mechanical detachment mL NaCl. content subsequently combined. Afterwards, genomic DNA extracted qPCR quantification primers 2022b). Using calibration curves designed (Lameira 2024), determined. RNA-seq experiments, removed, 1 phosphate-buffered saline (PBS) Scientific) suspended RNA protect Bacteria Reagent (Qiagen, MD, USA), diluted 2:1 PBS. samples centrifuged 5000 g 10 min room temperature. repeated least times. extractionTwelve pooled obtain enough analysis. extraction RNeasy Mini Kit (Qiagen), according manufacturer's instructions, minor modifications (França 2012). First, 600 μL lysis buffer RLT transferred tube 0.1 mm zirconium beads (Merck, Cells lysed BeadBug 6 Microtube Homogenizer (Benchmark NJ, USA) maximum speed 35 s. cycle four times ice 5 cycles. Then, centrifuged, supernatant recovered into new tube. Ethanol 70% added proportion (vol:vol) solution spin column. After washing steps, eluted RNase-free water (Grisp) treated Turbo DNase (Invitrogen, Waltham, Massachusetts, degrade following instructions rigorous protocols.cDNA library preparation sequencingRNA quality assessed Agilent 2100 Bioanalyzer (Agilent, CA, only indicators above 7 used. libraries Lexogen's CORALLTM Total kit (Lexogen, Vienna, Austria) 100 ng sample. RNA-seq, rRNA RiboCop (Mixed Bacterial Samples META) Depletion (Lexogen). Sequencing evaluated Fragment Analyzer System (Agilent) quantified QubitTM dsDNA HS Assay (Invitrogen). generated Illumina NextSeq 2000 single-end reads (SR100). FastQ files via bcl2fastq2 (v.2.17.1.14). individual sequences FastQC software after adapter trimming cutadapt (1.18). RNA-sequencing analysisFastQ analyzed CLC Genomics Workbench version 21.99). Quality trimming, including scores nucleotide ambiguity, genomics workbench default settings (Supplementary Table 1). Alignment species' NCTC10287 (NCBI reference sequence: NZ_LR134385.1), FDAARGOS_934 CP065631.1), DSM 20514 NZ_JH660658.1; NZ_JH660659.1; NZ_JH660660.1) genomes, also 2). Differential analysis Reads Per Kilobase per Million (RPKM) mapped fragments normalization strategy controls. Baggerley's test applied identify statistically significant alterations. Fold changes > 2 < -2 false discovery rate (FDR) p-value 0.05 considered bioinformatics analyses. Raw datasets deposited Gene Expression Omnibus database GSE279623.Functional annotationFunctional enrichment Search Tool Retrieval Interacting Genes/Proteins (STRING, 11.5) based Ontology (GO) Kyoto Encyclopedia Genes Genomes (KEGG) databases. Classes FDR-adjusted p-values enrichment. REVIGO (version 1.8.1) removing redundant GO terms. UniProt find homology hypothetical proteins.Statistical analysisPrincipal Component Analysis (PCA) graphs heatmaps created All other figures analyses GraphPad Prism 8.2 (La Jolla, USA). Statistical two-way ANOVA Tukey's comparisons test. differences p 0.05.3Data descriptionTo influence simulated interactions key BVAB, composed bivia, versus mimicking secretions. determine composition, revealing great majority shown Supplementary Figure 1. Regarding data, sequencing evaluating summary parameters mapping steps Tables 3 4, respectively. ranged 13137570 20716698. genome sequence revealed percentage 1.82% 75.76% 0.63% 67.48% medium. Overall, ones lowest percentages mapping.The PCA plots, depicted Figures 6, among triplicates condition. Triplicates more similar those bivia. contrast, condition vaginae. likely result biological heterogeneity 2014). clear separation indicating two caused species.The distribution 7. box display RPKM values, species. scatter represent correlation values conditions, upregulated (more displayed line), whereas downregulated below line). Finally, heat maps depicting normalized pattern across alteration 8 12). differential analysis, control. A 1315, 1202, 2184 MA volcano plots (Figure 1) has fold change -32 32 (-5 Log2 5). Moreover, wide range FDR comparison vaginalis. whose considered. This resulted 927, 492, 166 37, 104, 490 top ten their respective functions detailed To complete reported 13-15. categories processes, molecular functions, cellular components associated mainly metabolic processes. Among genes, namely active transmembrane transporter activity. terms category, process. anatomical entity process highest identified however, genes. category process, components. mostly within transport term KEGG pathways 16), enriched pathways, while ABC transporters identified. pathways. work highlights transcriptomic BVAB vivo-simulating compared It note that, influenced reflect shift abundance Additionally, particularly very stress responses lack capacity survive rather response functional Further investigation needed elucidate mechanisms involved contributions incident species.4Conflict InterestCAM reports receiving grants her institution BioNTech, Lupin, Abbott, Visby, Gilead Sciences, Inc. She honorarium and/or consulting fees Cepheid, biomerieux, Elsevier, Manuals, UpToDate, Roche. authors no disclosures. 5Author ContributionsCAM NC LS VP experiments. AF genomics. drafted manuscript. participated revising summary, contributed approved submitted manuscript.6FundingThis supported National Institute Allergy Infectious Diseases [R01AI146065-01A1 CAM]. funded Portuguese Foundation Science Technology (FCT), scope strategic funding unit [UIDB/04469/2020].7AcknowledgmentsLS acknowledges FCT financial support Grant [2020.04912.BD] acknowledge through program DL 57/2016 – Norma transitória (DL57/2016/CP1377/CT0032).

Language: Английский

ВОСПАЛИТЕЛЬНЫЕ ЗАБОЛЕВАНИЯ ОРГАНОВ МАЛОГО ТАЗА У ЖЕНЩИН СТАРШЕ 35 ЛЕТ

Bulletin of Osh State University, Journal Year: 2025, Volume and Issue: 1, P. 56 - 70

Published: March 25, 2025

Воспалительные заболевания органов малого таза (ВЗОМТ) представляют собой значимую медицинскую проблему, особенно у женщин старше 35 лет, так как в этой возрастной группе увеличивается риск развития хронических форм, бесплодия и спаечного процесса. В статье рассматриваются факторы риска, влияющие на течение заболевания, включая возрастные изменения, сопутствующие задержку обращения за медицинской помощью. Проведен анализ распространенности воспалительных заболеваний (специфических неспецифических возбудителей) среди указанной группы, их ненений ключевых этиологических факторов, Chlamydia trachomatis, Trichomania vaginalis, Gonorrhea Neisseria, Candida albicans, Gardnerella vaginalis. Полученные данные позволяют выделить приоритетные направления диагностике лечении что способствует снижению заболеваемости улучшению качества жизни пациенток.

Language: Русский