Biofilm formation by <i>Prototheca zopfii</i> isolated from clinical and subclinical bovine mastitis in distinct growth conditions under different dyes

Ely, Valessa Lunkes; Gressler, Letícia Trevisan; Sutili, Fernando Jonas; Ribeiro, Márcio Garcia; Costa, Mateus Matiuzzi da; Vargas, Agueda Castagna de; Botton, Sônia de Avila

doi:10.1590/0103-8478cr20180574

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MICROBIOLOGY • Cienc. Rural 49 (2) • 2019 • https://doi.org/10.1590/0103-8478cr20180574 copy

Biofilm formation by Prototheca zopfii isolated from clinical and subclinical bovine mastitis in distinct growth conditions under different dyes

Formação de biofilme por Prototheca zopfii isolados de mastite bovina clínica e subclínica em distintas condições de crescimento sob diferentes corantes

Authorship SCIMAGO INSTITUTIONS RANKINGS

ABSTRACT:

Prototheca spp. have been reported as an emergent environmental mastitis pathogen in several countries. Biofilm formation is a significant factor associated with different degrees of virulence developed by many microorganisms, including Prototheca spp. The present study aimed to compare two growth conditions and two staining dyes to determine which combination was more appropriate to evaluate qualitatively and quantitatively the production of biofilm by P. zopfii. Biofilm formation was evaluated in polystyrene microplates under static and dynamic growth conditions and staining with crystal violet or cotton blue dye. All P. zopfii isolates from cows with mastitis were classified as biofilm-producers in all growth conditions and staining. The cotton blue dye proved to be more appropriate method to classify the intensity of P. zopfii biofilm production.

Key words:
Prototheca zopfii, dairy cattle; bovine mastitis, intramammary infections, microplate biofilm assay, virulence factor

Authorship

Valessa Lunkes Ely

Departamento de Medicina Veterinária Preventiva (DMVP), Programa de Pós-graduação em Medicina Veterinária (PPGMV), Programa de Pós-Graduação em Ciências Farmacêuticas (PPGCF), Centro de Ciências Rurais (CCR), Universidade Federal de Santa Maria (UFSM), 97105-900, Santa Maria, RS, Brasil. Universidade Federal de Santa Maria (UFSM)BrazilSanta Maria, RS, BrazilDepartamento de Medicina Veterinária Preventiva (DMVP), Programa de Pós-graduação em Medicina Veterinária (PPGMV), Programa de Pós-Graduação em Ciências Farmacêuticas (PPGCF), Centro de Ciências Rurais (CCR), Universidade Federal de Santa Maria (UFSM), 97105-900, Santa Maria, RS, Brasil.

http://orcid.org/0000-0003-4045-5213

Letícia Trevisan Gressler

Laboratório de Microbiologia e Doenças Infecciosas, Instituto Federal Farroupilha (IFFar), Frederico Westphalen, RS, Brasil.Instituto Federal de Educação, Ciência e Tecnologia Farroupilha (IFFar)BrazilFrederico Westphalen, RS, BrazilLaboratório de Microbiologia e Doenças Infecciosas, Instituto Federal Farroupilha (IFFar), Frederico Westphalen, RS, Brasil.

http://orcid.org/0000-0001-9141-7503

Fernando Jonas Sutili

Departamento de Fisiologia e Farmacologia, Universidade Federal de Santa Maria (UFSM), Santa Maria, RS, Brasil.Universidade Federal de Santa Maria (UFSM)BrazilSanta Maria, RS, BrazilDepartamento de Fisiologia e Farmacologia, Universidade Federal de Santa Maria (UFSM), Santa Maria, RS, Brasil.

http://orcid.org/0000-0002-0225-3357

Márcio Garcia Ribeiro

Departamento de Higiene Veterinária e Saúde Pública, Faculdade de Medicina Veterinária e Zootecnia, Universidade Estadual Paulista Julio Mesquita Filho (UNESP), Botucatu, SP, Brasil.Universidade Estadual Paulista Julio Mesquita Filho (UNESP)BrazilBotucatu, SP, BrazilDepartamento de Higiene Veterinária e Saúde Pública, Faculdade de Medicina Veterinária e Zootecnia, Universidade Estadual Paulista Julio Mesquita Filho (UNESP), Botucatu, SP, Brasil.

http://orcid.org/0000-0003-2682-9389

Mateus Matiuzzi da Costa

Universidade Federal do Vale do São Francisco (UNIVASF), Petrolina, PE, Brasil.Universidade Federal do Vale do São Francisco (UNIVASF)BrazilPetrolina, PE, BrazilUniversidade Federal do Vale do São Francisco (UNIVASF), Petrolina, PE, Brasil.

http://orcid.org/0000-0002-9884-2112

Agueda Castagna de Vargas

http://orcid.org/0000-0002-9637-4028

Sônia de Avila Botton ^*E-mail: sabott20@gmail.com. *Corresponding author.

http://orcid.org/0000-0002-8855-9217

E-mail: sabott20@gmail.com. ^*Corresponding author.

DECLARATION OF CONFLICTING INTERESTS

The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

AUTHORS’ CONTRIBUTIONS

The authors contributed equally to the manuscript.

SCIMAGO INSTITUTIONS RANKINGS

Tables

Tables (3)

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Table 1
Absorbance values of Prototheca zopfii stained with crystal violet and cotton blue and grown under static and dynamic conditions.

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Table 2
Classification of Prototheca zopfii biofilm-forming ability according to the growth and staining conditions.

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Table 3
Studies involving biofilm formation by Prototheca spp.

Table 1 Absorbance values of Prototheca zopfii stained with crystal violet and cotton blue and grown under static and dynamic conditions.

Isolate	---------------------Crystal violet-------------------		--------------------------------Cotton blue--------------------------
	Static	Dynamic	Static	Dynamic
2	3.25±0.09	3.32±0.05	0.17±0.09	0.17±0.16
4	3.16±0.12	3.28±0.04	0.18±0.20	0.48±0.13
5	3.41±0.04	1.96±0.80	0.28±0.01	0.09±0.06
6	2.28±0.08	2.95±0.64	0.17±0.03	0.07±0.07
7	2.97±0.18	3.05±0.18	0.11±0.04	0.02±0.02
8	3.27±0.03	3.30±0.03	0.14±0.05	0.24±0.06
9	2.49±0.68	3.05±0.37	0.10±0.001	0.02±0.01
10	3.24±0.09	3.34±0.03	0.12±0.04	0.03±0.02
11	2.94±0.35	2.78±0.45	0.26±0.07	0.13±0.06
12	1.87±0.17	2.68±0.63	0.07±0.03	0.19±0.02
13	2.68±0.52	2.83±0.40	0.05±0.02	0.13±0.02
14	2.43±0.75	3.08±0.09	0.09±0.01	0.16±0.003
15	2.91±0.07	2.98±0.39	0.09±0.01	0.18±0.007
16	2.66±0.21	1.90±0.27	0.15±0.03	0.03±0.006
17	3.20±0.07	2.57±0.37	0.26±0.06	0.10±0.03
18	2.78±0.24	3.34±0.15	0.13±0.03	0.19±0.05
19	3.20±0.16	3.34±0.08	0.09±0.03	0.26±0.07
20	2.37±0.69	2.69±0.43	0.19±0.04	0.36±0.16
21	3.34±0.15	3.31±0.02	0.033±0.09	0.11±0.003
166AD	2.91±0.36	4.34±1.44	0.15±0.06	0.19±0.09
172AD	3.37±0.02	3.15±0.18	0.25±0.12	0.07±0.03
172AE	2.54±0.60	2.98±0.34	0.12±0.01	0.20±0.03
192AE	1.92±0.15	1.64±0.13	0.10±0.02	0.05±0.02
267AD	1.66±0.92	1.96±0.42	0.14±0.07	0.13±0.05
267PE	2.47±0.21	2.73±0.34	0.11±0.04	0.10±0.02
287AD	1.74±0.34	2.96±0.29	0.022±0.01	0.016±0.01
287PE	3.31±0.03	1.96±1.00	0.18±0.06	0.12±0.03
288AD	3.00±0.28	3.37±0.05	0.32±0.18	1.00±0.05
289AD	1.85±0.40	3.19±0.28	0.19±0.04	0.23±0.10
304PD	3.35±0.04	3.34±0.05	0.22±0.02	0.15±0.05
308AD	3.22±0.03	1.00±0.24	0.14±0.05	0.17±0.12
SB119/12	3.31±0.10	2.89±0.41	0.17±0.02	0.12±0.04

Note: the controls (media only) absorbance values were subtracted from P. zopfii absorbance values. The results represent the mean±standard deviation (SD) for each triplicate.

Table 2 Classification of Prototheca zopfii biofilm-forming ability according to the growth and staining conditions.

Isolate	--------------------------Cotton blue dye--------------------------			--------------------------Crystal violet dye------------------------
	Static	Dynamic	Classification S/D^*	Static	Dynamic	Classification S/D
2	3.31	3.93	M/M	12.26	12.68	S/S
4	3.55	8.80	M/S	9.33	8.60	S/S
5	4.43	2.06	S/M	14.96	7.80	S/S
6	3.35	1.96	M/W	12.91	10.61	S/S
7	2.48	1.31	M/W	16.53	10.93	S/S
8	2.91	5.09	M/S	12.32	12.61	S/S
9	2.33	1.28	M/W	13.97	10.95	S/S
10	2.68	1.42	M/W	17.94	11.86	S/S
11	4.14	2.47	S/M	13.03	10.61	S/S
12	1.99	3.47	W/M	10.76	9.73	S/S
13	1.67	3.21	W/M	10.29	10.96	S/S
14	2.31	3.70	M/M	7.40	8.13	S/S
15	2.24	4.16	M/S	11.08	11.50	S/S
16	2.91	1.41	M/W	11.91	7.56	S/S
17	4.26	2.16	S/M	14.09	9.88	S/S
18	2.78	3.39	M/M	15.53	11.87	S/S
19	2.23	5.49	M/S	12.07	12.75	S/S
20	3.80	6.93	M/S	7.25	7.23	S/S
21	5.02	2.31	S/M	14.69	12.46	S/S
166 AD	3.05	4.27	M/S	11.07	16.28	S/S
172 AD	4.10	1.84	S/W	14.79	11.91	S/S
172 AE	2.80	4.26	M/S	7.69	7.91	S/S
192 AE	2.19	1.58	M/W	8.86	6.67	S/S
267 AD	3.07	3.11	M/M	5.38	5.54	S/S
267 PE	2.55	2.63	M/M	7.51	7.32	S/S
287 AD	1.29	1.20	W/W	10.08	10.64	S/S
287 PE	3.47	3.12	M/M	12.47	7.92	S/S
288 AD	5.31	2.26	S/M	16.69	11.99	S/S
289 AD	3.53	3.92	M/M	10.69	11.39	S/S
304 PD	4.09	3.57	S/M	12.61	12.77	S/S
308 AD	2.71	3.01	M/M	14.20	4.42	S/S
119/12	3.32	3.06	M/M	12.48	11.19	S/S

^*Correspond to static (S) or dynamic (D) growth condition. M-moderate, S-strong, W-weak.
Note: the values were obtained by the division of arithmetic mean of the absorbance values of each isolate by the mean of the control (culture medium only).

Table 3 Studies involving biofilm formation by Prototheca spp.

Study	Isolates/% positive	Tº	Mode of incubation	Dye/OD	Culture medium	Degree
Gonçalves et al. (2015GONÇALVES, J et al. Biofilm-producing ability and efficiency of sanitizing agents against Prototheca zopfii isolates from bovine subclinical mastitis. Journal of Dairy Science, 2015. v.98, n.6, p.3613-3621. Available from: <Available from: http://linkinghub.elsevier.com/retrieve/pii/S0022030215002283 >. Accessed Mar. 13, 2017. doi: 10.3168/jds.2014-9248. http://linkinghub.elsevier.com/retrieve/... )	10/100	37ºC/25ºC	48h/dynamic (156 rpm)	1% crystal violet/570nm	TSB+0.6% yeast extract	W, M or S
Farrag et al. (2015FARRAG, HA et al. Microbial colonization of irradiated pathogenic yeast to catheter surfaces: Relationship between adherence, cell surface hydrophobicity, biofilm formation and antifungal susceptibility. A scanning electron microscope analysis. International Journal of Radiation Biology, 2015. v.91, n.6, p.519-527. Available from: <Available from: https://www.tandfonline.com/doi/abs/10.3109/09553002.2015.1021959?journalCode=irab20 >. Accessed Mar. 11, 2017. doi: 10.3109/09553002.2015.1021959. https://www.tandfonline.com/doi/abs/10.3... )	1/100	37ºC	48h/static^*	1% crystal violet/570nm	YNB broth+0.9% glucose	-
Kwiecinski (2015KWIECINSKI, J. Biofilm formation by pathogenic Prototheca algae. Letters in Applied Microbiology, 2015. v.61, n.6, p.511-517. Available from: <Available from: https://onlinelibrary.wiley.com/doi/abs/10.1111/lam.12497 >. Accessed: Feb. 11, 2017. doi: 10.1111/lam.12497. https://onlinelibrary.wiley.com/doi/abs/... )	4/100	37ºC	24h/static^*	0.4% safranin/520nm	Minimal medium+10% human heparinized plasma or 10% skimmed milk	-
Morandi et al. (2016MORANDI, S et al. Molecular typing and differences in biofilm formation and antibiotic susceptibilities among Prototheca strains isolated in Italy and Brazil. Journal of Dairy Science, 2016. v.99, n.8, p.6436-6445. Available from: <Available from: http://linkinghub.elsevier.com/retrieve/pii/S0022030216302971 >. Accessed: Feb. 17, 2017. doi: 10.3168/jds.2016-10900. http://linkinghub.elsevier.com/retrieve/... )	46/100	37ºC	24h/static	0.4% safranin/450nm	Sabouraud broth + 6% glucose	W, M or S
Present study	32/100	37ºC	24h/static and dynamic (100rpm)	0.25% crystal violet or 0.05% cotton blue/550nm	TSB+1% glucose	W, M or S

TSB: Tryptic Soy Broth; YNB: Yeast Nitrogen Base; ^*: We assumed the mode as static once it is normally used and because the authors have not described the incubation as dynamic.
Degrees: W: Weak; M: Moderate; S: Strong.

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Biofilm formation by <i>Prototheca zopfii</i> isolated from clinical and subclinical bovine mastitis in distinct growth conditions under different dyes

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[1] E-mail: sabott20@gmail.com. ^*Corresponding author.