Fever-like temperature impacts on Staphylococcus aureus and Pseudomonas aeruginosa interaction, physiology, and virulence both in vitro and in vivo.
Interaction
Pseudomonas aeruginosa
Staphylococcus aureus
Temperature
Virulence
Journal
BMC biology
ISSN: 1741-7007
Titre abrégé: BMC Biol
Pays: England
ID NLM: 101190720
Informations de publication
Date de publication:
05 Feb 2024
05 Feb 2024
Historique:
received:
31
07
2023
accepted:
18
01
2024
medline:
6
2
2024
pubmed:
6
2
2024
entrez:
5
2
2024
Statut:
epublish
Résumé
Staphylococcus aureus (SA) and Pseudomonas aeruginosa (PA) cause a wide variety of bacterial infections and coinfections, showing a complex interaction that involves the production of different metabolites and metabolic changes. Temperature is a key factor for bacterial survival and virulence and within the host, bacteria could be exposed to an increment in temperature during fever development. We analyzed the previously unexplored effect of fever-like temperatures (39 °C) on S. aureus USA300 and P. aeruginosa PAO1 microaerobic mono- and co-cultures compared with 37 °C, by using RNAseq and physiological assays including in vivo experiments. In general terms both temperature and co-culturing had a strong impact on both PA and SA with the exception of the temperature response of monocultured PA. We studied metabolic and virulence changes in both species. Altered metabolic features at 39 °C included arginine biosynthesis and the periplasmic glucose oxidation in S. aureus and P. aeruginosa monocultures respectively. When PA co-cultures were exposed at 39 °C, they upregulated ethanol oxidation-related genes along with an increment in organic acid accumulation. Regarding virulence factors, monocultured SA showed an increase in the mRNA expression of the agr operon and hld, pmsα, and pmsβ genes at 39 °C. Supported by mRNA data, we performed physiological experiments and detected and increment in hemolysis, staphyloxantin production, and a decrease in biofilm formation at 39 °C. On the side of PA monocultures, we observed an increase in extracellular lipase and protease and biofilm formation at 39 °C along with a decrease in the motility in correlation with changes observed at mRNA abundance. Additionally, we assessed host-pathogen interaction both in vitro and in vivo. S. aureus monocultured at 39 Our results highlight a relevant change in the virulence of bacterial opportunistic pathogens exposed to fever-like temperatures in presence of competitors, opening new questions related to bacteria-bacteria and host-pathogen interactions and coevolution.
Sections du résumé
BACKGROUND
BACKGROUND
Staphylococcus aureus (SA) and Pseudomonas aeruginosa (PA) cause a wide variety of bacterial infections and coinfections, showing a complex interaction that involves the production of different metabolites and metabolic changes. Temperature is a key factor for bacterial survival and virulence and within the host, bacteria could be exposed to an increment in temperature during fever development. We analyzed the previously unexplored effect of fever-like temperatures (39 °C) on S. aureus USA300 and P. aeruginosa PAO1 microaerobic mono- and co-cultures compared with 37 °C, by using RNAseq and physiological assays including in vivo experiments.
RESULTS
RESULTS
In general terms both temperature and co-culturing had a strong impact on both PA and SA with the exception of the temperature response of monocultured PA. We studied metabolic and virulence changes in both species. Altered metabolic features at 39 °C included arginine biosynthesis and the periplasmic glucose oxidation in S. aureus and P. aeruginosa monocultures respectively. When PA co-cultures were exposed at 39 °C, they upregulated ethanol oxidation-related genes along with an increment in organic acid accumulation. Regarding virulence factors, monocultured SA showed an increase in the mRNA expression of the agr operon and hld, pmsα, and pmsβ genes at 39 °C. Supported by mRNA data, we performed physiological experiments and detected and increment in hemolysis, staphyloxantin production, and a decrease in biofilm formation at 39 °C. On the side of PA monocultures, we observed an increase in extracellular lipase and protease and biofilm formation at 39 °C along with a decrease in the motility in correlation with changes observed at mRNA abundance. Additionally, we assessed host-pathogen interaction both in vitro and in vivo. S. aureus monocultured at 39
CONCLUSIONS
CONCLUSIONS
Our results highlight a relevant change in the virulence of bacterial opportunistic pathogens exposed to fever-like temperatures in presence of competitors, opening new questions related to bacteria-bacteria and host-pathogen interactions and coevolution.
Identifiants
pubmed: 38317219
doi: 10.1186/s12915-024-01830-3
pii: 10.1186/s12915-024-01830-3
doi:
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
27Subventions
Organisme : Agencia Nacional de Promoción Científica y Tecnológica
ID : PICT2018-2017
Organisme : Alexander von Humboldt-Stiftung
ID : Retourn Fellowship
Organisme : Exzellenzclusters Entzündungsforschung
ID : Cluster of Excellence EXC 2124 - Controlling Microbes to Fight Infections - 390838134
Organisme : Foundation for the National Institutes of Health
ID : SC3GM125556
Informations de copyright
© 2024. The Author(s).
Références
Wang M-G, Liu Z-Y, Liao X-P, Sun R-Y, Li R-B, Liu Y, et al. Retrospective data insight into the global distribution of carbapenemase-producing Pseudomonas aeruginosa. Antibiotics. 2021;10:548.
pubmed: 34065054
pmcid: 8151531
doi: 10.3390/antibiotics10050548
Yebra G, Harling-Lee JD, Lycett S, Aarestrup FM, Larsen G, Cavaco LM, et al. Multiclonal human origin and global expansion of an endemic bacterial pathogen of livestock. Proc Natl Acad Sci U S A. 2022;119:e2211217119.
pubmed: 36469788
pmcid: 9897428
doi: 10.1073/pnas.2211217119
Cheung GYC, Bae JS, Otto M. Pathogenicity and virulence of Staphylococcus aureus. Virulence. 2021;12:547–69.
pubmed: 33522395
pmcid: 7872022
doi: 10.1080/21505594.2021.1878688
Reynolds D, Kollef M. The epidemiology and pathogenesis and treatment of Pseudomonas aeruginosa infections: an update. Drugs. 2021;81:2117–31.
pubmed: 34743315
pmcid: 8572145
doi: 10.1007/s40265-021-01635-6
Biswas L, Götz F. Molecular mechanisms of Staphylococcus and Pseudomonas interactions in cystic fibrosis. Front Cell Infect Microbiol. 2022;11:1383.
Mashburn LM, Jett AM, Akins DR, Whiteley M. Staphylococcus aureus serves as an iron source for Pseudomonas aeruginosa during in vivo coculture. J Bacteriol. 2005;187:554–66.
pubmed: 15629927
pmcid: 543556
doi: 10.1128/JB.187.2.554-566.2005
Voggu L, Schlag S, Biswas R, Rosenstein R, Rausch C, Götz F. Microevolution of cytochrome bd oxidase in Staphylococci and its implication in resistance to respiratory toxins released by Pseudomonas. J Bacteriol. 2006;188:8079.
pubmed: 17108291
pmcid: 1698191
doi: 10.1128/JB.00858-06
Biswas L, Biswas R, Schlag M, Bertram R, Götz F. Small-colony variant selection as a survival strategy for Staphylococcus aureus in the presence of Pseudomonas aeruginosa. Appl Environ Microbiol. 2009;75:6910–2.
pubmed: 19717621
pmcid: 2772425
doi: 10.1128/AEM.01211-09
Szamosvári D, Böttcher T. An unsaturated quinolone N-oxide of Pseudomonas aeruginosa modulates growth and virulence of Staphylococcus aureus. Angew Chem Int Ed. 2017;56:7271–5.
doi: 10.1002/anie.201702944
Camus L, Briaud P, Bastien S, Elsen S, Doléans-Jordheim A, Vandenesch F, et al. Trophic cooperation promotes bacterial survival of Staphylococcus aureus and Pseudomonas aeruginosa. ISME J. 2020;14:3093–105.
pubmed: 32814867
pmcid: 7784975
doi: 10.1038/s41396-020-00741-9
Millette G, Langlois JP, Brouillette E, Frost EH, Cantin AM, Malouin F. Despite antagonism in vitro, Pseudomonas aeruginosa enhances Staphylococcus aureus colonization in a murine lung infection model. Front Microbiol. 2019;10:2880.
pubmed: 31921058
pmcid: 6923662
doi: 10.3389/fmicb.2019.02880
Monteiro R, Magalhães AP, Pereira MO, Sousa AM. Long-term coexistence of Pseudomonas aeruginosa and Staphylococcus aureus using an in vitro cystic fibrosis model. 2021;16:879–93. https://doi.org/10.2217/fmb-2021-0025 .
Worlitzsch D, Tarran R, Ulrich M, Schwab U, Cekici A, Meyer KC, et al. Effects of reduced mucus oxygen concentration in airway Pseudomonas infections of cystic fibrosis patients. J Clin Invest. 2002;109:317–25.
pubmed: 11827991
pmcid: 150856
doi: 10.1172/JCI0213870
Pallett R, Leslie LJ, Lambert PA, Milic I, Devitt A, Marshall LJ. Anaerobiosis influences virulence properties of Pseudomonas aeruginosa cystic fibrosis isolates and the interaction with Staphylococcus aureus. Scientific Reports. 2019;9:1–18.
doi: 10.1038/s41598-019-42952-x
Klinkert B, Narberhaus F. Microbial thermosensors. Cell Mol Life Sci. 2009;66:2661–76.
pubmed: 19554260
doi: 10.1007/s00018-009-0041-3
Knapp BD, Huang KC. The effects of temperature on cellular physiology. Annu Rev Biophys. 2022;51:499–526.
pubmed: 35534014
doi: 10.1146/annurev-biophys-112221-074832
Yu KW, Xue P, Fu Y, Yang L. T6ss mediated stress responses for bacterial environmental survival and host adaptation. Int J Mol Sci. 2021;22:1–13.
Loh E, Righetti F, Eichner H, Twittenhoff C, Narberhaus F. RNA thermometers in bacterial pathogens. Microbiol Spectr. 2018;6:55–73.
Barbier M, Damron FH, Bielecki P, Suárez-Diez M, Puchałka J, Albertí S, et al. From the environment to the host: re-wiring of the transcriptome of Pseudomonas aeruginosa from 22°C to 37°C. PLoS ONE. 2014;9:e89941.
pubmed: 24587139
pmcid: 3933690
doi: 10.1371/journal.pone.0089941
Bastock RA, Marino EC, Wiemels RE, Holzschu DL, Keogh RA, Zapf RL, et al. Staphylococcus aureus responds to physiologically relevant temperature changes by altering its global transcript and protein profile. mSphere. 2021;6:10–128.
Brewer SM, Twittenhoff C, Kortmann J, Brubaker SW, Honeycutt J, Massis LM, et al. A Salmonella Typhi RNA thermosensor regulates virulence factors and innate immune evasion in response to host temperature. PLoS Pathog. 2021;17:e1009345.
pubmed: 33651854
pmcid: 7954313
doi: 10.1371/journal.ppat.1009345
Pienkoß S, Javadi S, Chaoprasid P, Holler M, Roßmanith J, Dersch P, et al. RNA thermometer-coordinated assembly of the Yersinia injectisome. J Mol Biol. 2022;434:167667.
pubmed: 35667470
doi: 10.1016/j.jmb.2022.167667
Tribelli PM, López NI. Insights into the temperature responses of Pseudomonas species in beneficial and pathogenic host interactions. Appl Microbiol Biotechnol. 2022;106:7699–709.
pubmed: 36271255
doi: 10.1007/s00253-022-12243-z
Diard M, Hardt W-D. Evolution of bacterial virulence. FEMS Microbiol Rev. 2017;41:679–97.
pubmed: 28531298
doi: 10.1093/femsre/fux023
Ribet D, Cossart P. How bacterial pathogens colonize their hosts and invade deeper tissues. Microbes Infect. 2015;17:173–83.
pubmed: 25637951
doi: 10.1016/j.micinf.2015.01.004
Abu Kwaik Y, Bumann D. Microbial quest for food in vivo : ‘Nutritional virulence’ as an emerging paradigm. Cell Microbiol. 2013;15:882–90.
pubmed: 23490329
doi: 10.1111/cmi.12138
Lam O, Wheeler J, Tang CM. Thermal control of virulence factors in bacteria: a hot topic. Virulence. 2014;5:852.
pubmed: 25494856
pmcid: 4601195
doi: 10.4161/21505594.2014.970949
Balli S, Shumway KR, Sharan S. Physiology, Fever. 2022.
Islam MA, Kundu S, Alam SS, Hossan T, Kamal MA, Hassan R. Prevalence and characteristics of fever in adult and paediatric patients with coronavirus disease 2019 (COVID-19): a systematic review and meta-analysis of 17515 patients. PLoS ONE. 2021;16:e0249788.
pubmed: 33822812
pmcid: 8023501
doi: 10.1371/journal.pone.0249788
González Plaza JJ, Hulak N, Zhumadilov Z, Akilzhanova A. Fever as an important resource for infectious diseases research. Intractable Rare Dis Res. 2016;5:97–102.
pubmed: 27195192
pmcid: 4869589
doi: 10.5582/irdr.2016.01009
Saladin K. Major themes on anatomy and physiology. In: Anatomy & Physiology: The Unity of Form and Function. 6th ed. NY, USA: McGraw-Hill; 2011. p. 1–27.
Elhadad D, McClelland M, Rahav G, Gal-Mor O. Feverlike temperature is a virulence regulatory cue controlling the motility and host cell entry of typhoidal Salmonella. J Infect Dis. 2015;212:147–56.
pubmed: 25492917
doi: 10.1093/infdis/jiu663
Schlag S, Fuchs S, Nerz C, Gaupp R, Engelmann S, Liebeke M, et al. Characterization of the oxygen-responsive NreABC regulon of Staphylococcus aureus. J Bacteriol. 2008;190:7847–58.
pubmed: 18820014
pmcid: 2583599
doi: 10.1128/JB.00905-08
Polack B, Dacheux D, Delic-Attree I, Toussaint B, Vignais PM. The Pseudomonas aeruginosa fumC and sodA genes belong to an iron-responsive operon. Biochem Biophys Res Commun. 1996;226:555–60.
pubmed: 8806672
doi: 10.1006/bbrc.1996.1393
Hempel N, Görisch H, Mern DS. Gene ercA, encoding a putative iron-containing alcohol dehydrogenase, is involved in regulation of ethanol utilization in Pseudomonas aeruginosa. J Bacteriol. 2013;195:3925–32.
pubmed: 23813731
pmcid: 3754586
doi: 10.1128/JB.00531-13
Clauditz A, Resch A, Wieland KP, Peschel A, Götz F. Staphyloxanthin plays a role in the fitness of Staphylococcus aureus and its ability to cope with oxidative stress. Infect Immun. 2006;74:4950–3.
pubmed: 16861688
pmcid: 1539600
doi: 10.1128/IAI.00204-06
Pelz A, Wieland KP, Putzbach K, Hentschel P, Albert K, Götz F. Structure and biosynthesis of Staphyloxanthin from Staphylococcus aureus. J Biol Chem. 2005;280:32493–8.
pubmed: 16020541
doi: 10.1074/jbc.M505070200
Moormeier DE, Bayles KW. Staphylococcus aureus biofilm: a complex developmental organism. Mol Microbiol. 2017;104:365–76.
pubmed: 28142193
pmcid: 5397344
doi: 10.1111/mmi.13634
Quinn B, Rodman N, Jara E, Fernandez JS, Martinez J, Traglia GM, et al. Human serum albumin alters specific genes that can play a role in survival and persistence in Acinetobacter baumannii. Sci Rep. 2018;8:1–16.
doi: 10.1038/s41598-018-33072-z
Egesten A, Frick IM, Mörgelin M, Olin AI, Björck L. Binding of albumin promotes bacterial survival at the epithelial surface. J Biol Chem. 2011;286:2469–76.
pubmed: 21098039
doi: 10.1074/jbc.M110.148171
Earn DJD, Andrews PW, Bolker BM. Population-level effects of suppressing fever. Proceedings of the Royal Society B: Biological Sciences. 2014;281:20132570.
pmcid: 3906934
doi: 10.1098/rspb.2013.2570
Evans SS, Repasky EA, Fisher DT. Fever and the thermal regulation of immunity: the immune system feels the heat. Nat Rev Immunol. 2015;15:335–49.
pubmed: 25976513
pmcid: 4786079
doi: 10.1038/nri3843
Wurtzel O, Yoder-Himes DR, Han K, Dandekar AA, Edelheit S, Greenberg EP, et al. The single-nucleotide resolution transcriptome of Pseudomonas aeruginosa grown in body temperature. PLoS Pathog. 2012;8:e1002945.
pubmed: 23028334
pmcid: 3460626
doi: 10.1371/journal.ppat.1002945
Hamamoto H, Panthee S, Paudel A, Ohgi S, Suzuki Y, Makimura K, et al. Transcriptome change of Staphylococcus aureus in infected mouse liver. Commun Biol. 2022;5:721.
pubmed: 35859002
pmcid: 9300722
doi: 10.1038/s42003-022-03674-5
Reslane I, Halsey CR, Stastny A, Cabrera BJ, Ahn J, Shinde D, et al. Catabolic Ornithine Carbamoyltransferase activity facilitates growth of Staphylococcus aureus in defined medium lacking glucose and arginine. mBio. 2022;13:e00395–22.
Palela M, Giol ED, Amzuta A, Ologu OG, Stan RC. Fever temperatures impair hemolysis caused by strains of Escherichia coli and Staphylococcus aureus. Heliyon. 2022;8:e08958.
pubmed: 35243078
pmcid: 8859000
doi: 10.1016/j.heliyon.2022.e08958
Wrotek S, Legrand EK, Dzialuk A, Alcock J. Let fever do its job The meaning of fever in the pandemic era. https://doi.org/10.1093/emph/eoaa044 .
Sabat AJ, Pantano D, Akkerboom V, Bathoorn E, Friedrich AW. Pseudomonas aeruginosa and Staphylococcus aureus virulence factors as biomarkers of infection. Biol Chem. 2021;402:1565–73.
pubmed: 34505460
doi: 10.1515/hsz-2021-0243
Laakso HA, Marolda CL, Pinter TB, Stillman MJ, Heinrichs DE. A heme-responsive regulator controls synthesis of Staphyloferrin B in Staphylococcus aureus. J Biol Chem. 2016;291:29.
pubmed: 26534960
doi: 10.1074/jbc.M115.696625
Lin Y-C, Cornell WC, Jo J, Price-Whelan A, Dietrich LEP. The Pseudomonas aeruginosa complement of lactate dehydrogenases enables use of d- and l-lactate and metabolic cross-feeding. mBio. 2018;9:10–128.
Chekabab SM, Silverman RJ, Lafayette SL, Luo Y, Rousseau S, Nguyen D. Staphylococcus aureus inhibits IL-8 responses induced by Pseudomonas aeruginosa in airway epithelial cells. PLoS ONE. 2015;10:e0137753.
pubmed: 26360879
pmcid: 4567135
doi: 10.1371/journal.pone.0137753
Alves PM, Al-Badi E, Withycombe C, Jones PM, Purdy KJ, Maddocks SE. Interaction between Staphylococcus aureus and Pseudomonas aeruginosa is beneficial for colonisation and pathogenicity in a mixed biofilm. Pathog Dis. 2018;76:fty003.
Oka T, Oka K, Kobayashi T, Sugimoto Y, Ichikawa A, Ushikubi F, et al. Characteristics of thermoregulatory and febrile responses in mice deficient in prostaglandin EP1 and EP3 receptors. J Physiol. 2003;551:945–54.
pubmed: 12837930
pmcid: 2343282
doi: 10.1113/jphysiol.2003.048140
Shiraki C, Horikawa R, Oe Y, Fujimoto M, Okamoto K, Kurganov E, et al. Role of TRPM8 in switching between fever and hypothermia in adult mice during endotoxin-induced inflammation. Brain Behav Immun Health. 2021;16:100291.
pubmed: 34589786
pmcid: 8474285
doi: 10.1016/j.bbih.2021.100291
Eskilsson A, Shionoya K, Engblom D, Blomqvist A. Fever during localized inflammation in mice is elicited by a humoral pathway and depends on brain endothelial interleukin-1 and interleukin-6 signaling and central EP
pubmed: 33941650
pmcid: 8211540
doi: 10.1523/JNEUROSCI.0313-21.2021
Catalan-Moreno A, Cela M, Menendez-Gil P, Irurzun N, Caballero CJ, Caldelari I, et al. RNA thermoswitches modulate Staphylococcus aureus adaptation to ambient temperatures. Nucleic Acids Res. 2021;49:3409–26.
pubmed: 33660769
pmcid: 8034633
doi: 10.1093/nar/gkab117
Hussein H, Fris ME, Salem AH, Wiemels RE, Bastock RA, Righetti F, et al. An unconventional RNA-based thermosensor within the 5’ UTR of Staphylococcus aureus cidA. PLoS One. 2019;14:e0214521.
Grosso-Becerra MV, Croda-García G, Merino E, Servín-González L, Mojica-Espinosa R, Soberón-Chávez G. Regulation of Pseudomonas aeruginosa virulence factors by two novel RNA thermometers. Proc Natl Acad Sci. 2014;111:15562–7.
pubmed: 25313031
pmcid: 4217398
doi: 10.1073/pnas.1402536111
Bolger AM, Lohse M, Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 2014;30:2114–20.
pubmed: 24695404
pmcid: 4103590
doi: 10.1093/bioinformatics/btu170
Tjaden B. A computational system for identifying operons based on RNA-seq data. Methods. 2020;176:62–70.
pubmed: 30953757
doi: 10.1016/j.ymeth.2019.03.026
Ogata H, Goto S, Sato K, Fujibuchi W, Bono H, Kanehisa M. KEGG: Kyoto Encyclopedia of Genes and Genomes. Nucleic Acids Res. 1999;27:29–34.
pubmed: 9847135
pmcid: 148090
doi: 10.1093/nar/27.1.29
Caspi R, Billington R, Ferrer L, Foerster H, Fulcher CA, Keseler IM, et al. The MetaCyc database of metabolic pathways and enzymes and the BioCyc collection of pathway/genome databases. Nucleic Acids Res. 2016;44:D471-480.
pubmed: 26527732
doi: 10.1093/nar/gkv1164
Szklarczyk D, Gable AL, Lyon D, Junge A, Wyder S, Huerta-Cepas J, et al. STRING v11: protein–protein association networks with increased coverage, supporting functional discovery in genome-wide experimental datasets. Nucleic Acids Res. 2019;47:D607–13.
pubmed: 30476243
doi: 10.1093/nar/gky1131
Vitko NP, Richardson AR. Laboratory maintenance of methicillin‐resistant Staphylococcus aureus (MRSA). Curr Protoc Microbiol. 2013;28:9C–2.
Valliammai A, Selvaraj A, Muthuramalingam P, Priya A, Ramesh M, Pandian SK. Staphyloxanthin inhibitory potential of thymol impairs antioxidant fitness, enhances neutrophil mediated killing and alters membrane fluidity of methicillin resistant Staphylococcus aureus. Biomed Pharmacother. 2021;141:111933.
pubmed: 34328107
doi: 10.1016/j.biopha.2021.111933
Schneider CA, Rasband WS, Eliceiri KW. NIH Image to ImageJ: 25 years of image analysis. Nat Methods. 2012;9:671–5.
pubmed: 22930834
pmcid: 5554542
doi: 10.1038/nmeth.2089
Tribelli PM, Nikel PI, Oppezzo OJ, López NI. Anr, the anaerobic global regulator, modulates the redox state and oxidative stress resistance in Pseudomonas extremaustralis. Microbiology. 2013;159(Pt 2):259–68.
pubmed: 23223440
doi: 10.1099/mic.0.061085-0
O’Toole GA, Kolter R. Flagellar and twitching motility are necessary for Pseudomonas aeruginosa biofilm development. Mol Microbiol. 1998;30:295–304.
pubmed: 9791175
doi: 10.1046/j.1365-2958.1998.01062.x
O’May C, Tufenkji N. The swarming motility of Pseudomonas aeruginosa is blocked by cranberry proanthocyanidins and other tannin-containing materials. Appl Environ Microbiol. 2011;77:3061–7.
pubmed: 21378043
pmcid: 3126419
doi: 10.1128/AEM.02677-10
R Core Team. R: A language and environment for statistical computing. 2022.
Solar Venero E, Tribelli PM, Ricardi M. RNAseq of Pseudomonas aeruginosa PAO1 and Staphylococcus aureus USA300 in monocultures and co-cultivated at 37 and 39°C in microaerobiosis. Supplementary Datasets. BioStudies accession: E-MTAB-12581 https://www.ebi.ac.uk/biostudies/arrayexpress/studies/E-MTAB-12581?key=6e13e32b-cae0-4154-9e08-1ccfa6fd46c8 . 2023.