Biofilm analyses and exoproduct release by clinical and environmental isolates of Burkholderia pseudomallei from Brazil

Abstract

Objective: To characterize biofilm production by clinical (n=21) and environmental (n=11) isolates of Burkholderia pseudomallei and evaluate the production of proteases, hemolysins and siderophores. Methods: Initially, the 32 strains were evaluated for biofilm production in Müller-Hinton broth-1% glucose (MH-1% glucose) and BHI broth-1% glucose, using the crystal violet staining technique. Subsequently, growing (48 h) and mature (72 h) biofilms were evaluated by confocal microscopy. Finally, the production of proteases, hemolysins and siderophores by planktonic aggregates, growing biofilms and mature biofilms was evaluated. Results: All isolates produced biofilms, but clinical isolates had significantly higher biomass in both MH-1% glucose (P<0.001) and BHI-glucose 1% (P=0.005). The structural analyses by confocal microscopy showed thick biofilms, composed of multiple layers of cells, homogeneously arranged, with mature biofilms of clinical isolates presenting higher biomass (P=0.019) and thickness of the entire area (P=0.029), and lower roughness coefficient (P=0.007) than those of environmental isolates. Protease production by growing biofilms was significantly greater than that of planktonic (P<0.001) and mature biofilms (P<0.001). Hemolysin release by planktonic aggregates was higher than that of biofilms (P<0.001). Regarding siderophores, mature biofilms presented higher production than growing biofilms (P<0.001) and planktonic aggregates (P<0.001). Conclusions: Clinical isolates have higher production of biofilms than their environmental counterparts; protease and siderophores seem important for growth and maintenance of Burkholderia pseudomallei biofilms.

Keywords

Mangosteen / Burkholderia pseudomallei / Biofilms / Proteases / Hemolysins / Siderophores

References

[1] Limmathurotsakul D, Golding N, Dance DA, Messina JP, Pigott DM, Moyes CL, et al. Predicted global distribution of Burkholderia pseudomallei and burden of melioidosis. Nat Microbiol 2016; 1:5008.

[2] Dunachie SJ, Jenjaroen K, Reynolds CJ, Quigley KJ, Sergeant R, Sumonwiriya M, et al. Infection with Burkholderia pseudomallei-immune correlates of survival in acute melioidosis. Sci Rep 2017; 7(1):1-14.

[3] Rolim DB, Lima RXR, Ribeiro AKC, Colares RM, Lima LDQ, Rodríguez-Morales AJ, et al. Melioidosis in South America. Trop Med Infect Dis 2018; 3(2):60.

[4] Samy RP, Stiles BG, Sethi G, Lim LHK. Melioidosis: Clinical impact and public health threat in the tropics. PLoS Negl Trop Dis 2017; 11(5):e0004738.

[5] Limmathurotsakul D, Paeyao A, Wongratanacheewin S, Saiprom N, Takpho N, Thaipadungpanit J, et al. Role of Burkholderia pseudomallei biofilm formation and lipopolysaccharide in relapse of melioidosis. Clin Microbiol Infect 2017; 20(11):854-856.

[6] Ahmad L, Hung TL, Akhir NAM, Mohamed R, Nathan S, Firdaus-Raih M. Characterization of Burkholderia pseudomallei protein BPSL1375 validates the putative hemolytic activity of the COG3176 N-acyltransferase family. BMC Microbiol 2015; 15(270):1-12.

[7] Lee MA, Liu Y. Sequencing and characterization of a novel serine metalloprotease from Burkholderia pseudomallei. FEMS Microbiol Lett 2000; 192(1):67-72.

[8] Stone JK, DeShazer D, Brett PJ, Burtnick MN. Melioidosis: Molecular aspects of pathogenesis. Expert Rev Anti Infect Ther 2014; 12(12):1487-1499.

[9] Ramírez-Larrota JS, Eckhard U. An Introduction to bacterial biofilms and their proteases, and their roles in host infection and immune evasion. Biomolecules 2022; 12(2):306.

[10] Butt AT, Thomas MS. Iron acquisition mechanisms and their role in the virulence of Burkholderia species. Front Cell Infect Microbiol 2014; 7:460.

[11] Bandeira TJPG, Moreira CA, Brilhante RSN, Castelo-Branco DSCM, Neto MPA, Cordeiro RA, et al. In vitro activities of amoxicillin-clavulanate, doxycycline, ceftazidime, imipenem, and trimethoprim-sulfamethoxazole against biofilm of brazilian strains of Burkholderia pseudomallei. Antimicrob Agents Chemother 2013; 57(11):5771-5773.

[12] Stepanović S, Vuković D, Hola V, Bonaventura GD, Djukić S, Cirković I, et al. Quantification of biofilm in microtiter plates: Overview of testing conditions and practical recommendations for assessment of biofilm production by staphylococci. APMIS 2007; 115(8):891-899.

[13] Sidrim JJC, Vasconcelos DC, Riello GB, Guedes GMM, Serpa R, Bandeira TJPG, et al. Promethazine improves antibiotic efficacy and disrupts biofilms of Burkholderia pseudomallei. Biofouling 2017; 33(1):88-97.

[14] Heydorn A, Nielsen AT, Hentzer M, Sternberg C, Givskov M, Ersboll K, et al. Quantification of biofilm structures by the novel computer program COMSTAT. Microbiology 2000; 146(10):2395-2407.

[15] Sidrim JJC, Ocadaque CJ, Amando BR, Guedes GMM, Costa CL, Brilhante RSN, et al. Rhamnolipid enhances Burkholderia pseudomallei biofilm susceptibility, disassembly and production of virulence factors. Future Microbiol 2020; 15:1109-1121.

[16] Charney J, Tomarelli RM. A colorimetric method for the determination of the proteolytic activity of duodenal juice. J Biol Chem 1947; 171(2):501-505.

[17] Tang F, Li L, Meng XM, Bing L, Wang CQ, Wang SQ, et al. Inhibition of alpha-hemolysin expression by resveratrol attenuates Staphylococcus aureus virulence. Microb Pathog 2019; 127:85-90.

[18] Phansri K, Sarnthima R, Thammasirirak S, Boonchalee P, Khammuang S. Antibacterial activity of Bauhinia acuminata L. seed protein extract with low hemolytic activity against human erythrocytes. Chiang Mai J Sci 2011; 38(2):242-251.

[19] Ali SS, Vidhale NN. Evaluation of siderophore produced by different clinical isolate Pseudomonas aeruginosa. Int J Microbiol Res 2011; 3(3):131-135.

[20] Wongtrakoongate P, Tumapa S, Tungpradabkul S. Regulation of a quorum sensing system by stationary phase sigma factor RpoS and their co-regulation of target genes in Burkholderia pseudomallei. Microbiol Immunol 2012; 56(5):281-294.

[21] Procop GW, Church DL, Hall GS, Janda WM, Koneman EW, Schreckenberger PC, et al, editors. Microbiological diagnosis. Edition 7. Philadelphia: Wolters Kluwer Health; 2017.

[22] Kamjumphol W, Chareonsudjai S, Chareonsudjai P, Wongratanacheewin S, Taweechaisupapong S. Environmental factors affecting Burkholderia pseudomallei biofilm formation Southeast Asian. J Trop Med Public Health 2013; 44(1):72-81.

[23] Pakkulnan R, Anutrakunchai C, Kanthawong S, Taweechaisupapong S, Chareonsudjai P, Chareonsudjai S. Extracellular DNA facilitates bacterial adhesion during Burkholderia pseudomallei biofilm formation. PLoS One 2019; 14(3):e0213288.

[24] Trunk T, Khalil HS, Leo JC. Bacterial autoaggregation. AIM Microbiol 2018; 4(1):140-164.

[25] Mangalea MR, Borlee GI, Borlee BR. The current status of extracellular polymeric substances produced by Burkholderia pseudomallei. Curr Trop Med Rep 2017; 4:117-126.

[26] Bottagisio M, Soggiu A, Piras C, Bidossi A, Grego V, Pieroni L, et al. Proteomic analysis reveals a biofilm-like behavior of planktonic aggregates of Staphylococcus epidermidis grown under environmental pressure/stress. Front Microbiol 2019; 10:1909.

[27] Haaber J, Cohn MT, Frees D, Andersen TJ, Ingmer H. Planktonic aggregates of Staphylococcus aureus protect against common antibiotics. PLoS One 2012; 7:e41075.

[28] Esoda CN, Kuehn MJ. Pseudomonas aeruginosa leucine aminopeptidase influences early biofilm composition and structure via vesicle-associated antibiofilm activity. MBio 2019; 10(6):e02548.

[29] Vellasamy KM, Vasu C, Puthucheary SD, Vadivelu J. Comparative analysis of extracellular enzymes and virulence exhibited by Burkholderia pseudomallei from different sources. Microb Pathog 2009; 47(3):111-117.

[30] Liu Q, Sun S, Piao M, Yang JY. Purification and characterization of a protease produced by a Planomicrobium sp. L-2 from gut of Octopus vulgaris. Prev Nutr Food Sci 2013; 18(4):273-279.

[31] Yadav V, Harjai K, Kaur R, Joshi K, Sharma S. Urovirulence of Pseudomonas aeruginosa: Planktonic cells vs. biofilm cells. Folia Microbiol 2004; 49(4):465-470.

[32] Banerjee M, Moulick S, Bhattacharya KK, Parai D, Chattopadhyay S, Mukherjee SK. Attenuation of Pseudomonas aeruginosa quorum sensing, virulence and biofilm formation by extracts of Andrographis paniculate. Microb Pathog 2017; 113:85-93.

[33] Milivojevic D, Šumonja N, Medić S, Pavic A, Moric I, Vasiljevic B, et al. Biofilm-forming ability and infection potential of Pseudomonas aeruginosa strains isolated from animals and humans. Pathog Dis 2018; 76(4). doi: 10.1093/femspd/fty041.

[34] Thomson EL, Dennis JJ. A Burkholderia cepacia complex non-ribosomal peptide-synthesized toxin is hemolytic and required for full virulence. Virulence 2012; 3(3):286-298.

[35] Wilson BR, Bogdan AR, Miyazawa M, Hashimoto K, Tsuji Y. Siderophores in iron metabolism: From mechanism to therapy potential. Trends Mol Med 2016; 22(12):1077-1090.

[36] Kamjumphol W, Chareonsudjai S, Chareonsudjai P, Wongratanacheewin S, Taweechaisupapong S. Environmental factors affecting Burkholderia pseudomallei biofilm formation. Southeast Asian J Trop Med Public Health 2013; 44(1):72-81.

[37] Yu S, Wei Q, Zhao T, Guo Y, Ma LZ. A survival strategy for Pseudomonas aeruginosa that uses exopolysaccharides to sequester and store iron to stimulate psl-dependent biofilm formation. Appl Environ Microbiol 2016; 82(21):6403-6413.