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

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

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

RESUMO:

Prototheca spp. tem sido relatado como um patógeno ambiental causador de mastite bovina em vários países. A formação de biofilme é um fator associado a diferentes graus de virulência desenvolvidos por muitos microrganismos, incluindo Prototheca zopfii. O presente estudo teve como objetivo comparar duas condições de crescimento e dois corantes para determinar a combinação mais adequada para avaliar qualitativa e quantitativamente a produção de biofilme por P. zopfii. A formação de biofilme foi avaliada em microplacas de poliestireno sob condições estáticas e dinâmicas de crescimento e coloração com cristal violeta ou azul de algodão. Todos os isolados de P. zopfii de vacas com mastite foram caracterizados como produtores de biofilme, independentemente das condições de crescimento e coloração. O corante azul de algodão demonstrou ser o método mais adequado para classificar a intensidade de produção de biofilme de P. zopfii.

Palavras-chave:
Prototheca zopfii; bovino de leite; mastite bovina; infecções intramamárias; ensaio de biofilme em microplaca; fator de virulência

INTRODUCION:

Prototheca spp. are unicellular achlorophyllous algae that reproduce asexually (SUDMAN & KAPLAN, 1973SUDMAN, MS; KAPLAN, W. Identification of the Prototheca species by immunofluorescence. Applied Microbiology, 1973. v.25, n.6, p.981-90. Available from: <Available from: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=380951&tool=pmcentrez&rendertype=abstract >. Accessed: Mar. 12, 2017.
http://www.pubmedcentral.nih.gov/article...
), and are widely distributed in the environment, especially in the presence of water and organic matter (PORE et al., 1983PORE, RS et al. Prototheca ecology. Mycopathologia, 1983. v.81, p.49-62. Available from: <Available from: https://link.springer.com/content/pdf/10.1007%2FBF00443909.pdf >. Accessed: Mar. 12, 2017. doi: 0301 486x/83/81/0049-14/$00.20/0.
https://link.springer.com/content/pdf/10...
). These microorganisms have also been isolated from diverse animal species, particularly from dairy cattle. The infections of mammary gland of cows by Prototheca spp., especially Prototheca zopfii, normally result in reduced milk production, early culling, as well as public health concerns (COSTA et al., 1997COSTA, EO et al. Epidemiologic study of environmental sources in a Prototheca zopfii outbreak of bovine mastitis. Mycopathologia, 1997. v.137, n.1, p.33-36. Available from: <Available from: https://www.ncbi.nlm.nih.gov/pubmed/9299756 >. Accessed Mar. 11, 2017. doi: 10.1023/A:1006871213521.
https://www.ncbi.nlm.nih.gov/pubmed/9299...
; VARGAS et al., 1998VARGAS, AC et al. Isolation of Prothoteca zopfii from a case of bovine mastitis in Brazil. Mycopathologia, 1998. v.142, n.3, p.135-137. Available from: <Available from: https://www.ncbi.nlm.nih.gov/pubmed/10052162 >. Accessed: Mar. 20, 2017.
https://www.ncbi.nlm.nih.gov/pubmed/1005...
; SALERNO et al., 2010SALERNO, T et al. In vitro algaecide effect of sodium hypochlorite and iodine based antiseptics on Prototheca zopfii strains isolated from bovine milk. Research in Veterinary Science, 2010. v.88, n.2, p.211-213. Available from: <Available from: https://www.sciencedirect.com/science/article/pii/S0034528809002082?via%3Dihub >. Accessed: Mar. 12, 2017. doi: 10.1016/j.rvsc.2009.08.001.
https://www.sciencedirect.com/science/ar...
). P. zopfii has been reported to be a causative agent of bovine mastitis across Europe (ROESLER et al., 2006ROESLER, U et al. Diversity within the current algal species Prototheca zopfii: A proposal for two Prototheca zopfii genotypes and description of a novel species, Prototheca blaschkeae sp. nov. International Journal of Systematic and Evolutionary Microbiology, 2006. v.56, n.6, p.1419-1425. Available from: <Available from: http://ijs.microbiologyresearch.org/content/journal/ijsem/10.1099/ijs.0.63892-0#tab2 >. Accessed: Mar. 20, 2017. doi: 10.1099/ijs.0.63892-0.
http://ijs.microbiologyresearch.org/cont...
), as well as in Japan (ONOZAKI et al., 2013ONOZAKI, M et al. Detection and identification of genotypes of Prototheca zopfii in clinical samples by quantitative PCR analysis. Japanese Journal of Infectious Diseases, 2013. v.66, n.5, p.383-390. Available from: <Available from: https://www.jstage.jst.go.jp/article/yoken/66/5/66_383/_article >. Accessed: Mar. 11, 2017. doi: 10.7883/yoken.66.383.
https://www.jstage.jst.go.jp/article/yok...
) and in Brazil (SALERNO et al., 2010SALERNO, T et al. In vitro algaecide effect of sodium hypochlorite and iodine based antiseptics on Prototheca zopfii strains isolated from bovine milk. Research in Veterinary Science, 2010. v.88, n.2, p.211-213. Available from: <Available from: https://www.sciencedirect.com/science/article/pii/S0034528809002082?via%3Dihub >. Accessed: Mar. 12, 2017. doi: 10.1016/j.rvsc.2009.08.001.
https://www.sciencedirect.com/science/ar...
). In Brazilian dairy cattle, protothecosis has been described as an important cause of environmental mastitis, occasioning great damage to the mammary gland and resulting in animal discard (SALERNO et al., 2010SALERNO, T et al. In vitro algaecide effect of sodium hypochlorite and iodine based antiseptics on Prototheca zopfii strains isolated from bovine milk. Research in Veterinary Science, 2010. v.88, n.2, p.211-213. Available from: <Available from: https://www.sciencedirect.com/science/article/pii/S0034528809002082?via%3Dihub >. Accessed: Mar. 12, 2017. doi: 10.1016/j.rvsc.2009.08.001.
https://www.sciencedirect.com/science/ar...
).

Mastitis diagnosis caused by P. zopfii may be performed through morphology and phenotypic methods (PORE, 1985PORE, RS. Prototheca taxonomy. Mycopathologia, 1985. v.90, n.3, p.129-139. Available from: <Available from: https://link.springer.com/content/pdf/10.1007%2FBF00436728.pdf >. Accessed: Feb. 15, 2017. doi: 10.1007/BF00436728.
https://link.springer.com/content/pdf/10...
; VARGAS et al., 1998VARGAS, AC et al. Isolation of Prothoteca zopfii from a case of bovine mastitis in Brazil. Mycopathologia, 1998. v.142, n.3, p.135-137. Available from: <Available from: https://www.ncbi.nlm.nih.gov/pubmed/10052162 >. Accessed: Mar. 20, 2017.
https://www.ncbi.nlm.nih.gov/pubmed/1005...
; ROESLER et al. 2006ROESLER, U et al. Diversity within the current algal species Prototheca zopfii: A proposal for two Prototheca zopfii genotypes and description of a novel species, Prototheca blaschkeae sp. nov. International Journal of Systematic and Evolutionary Microbiology, 2006. v.56, n.6, p.1419-1425. Available from: <Available from: http://ijs.microbiologyresearch.org/content/journal/ijsem/10.1099/ijs.0.63892-0#tab2 >. Accessed: Mar. 20, 2017. doi: 10.1099/ijs.0.63892-0.
http://ijs.microbiologyresearch.org/cont...
); although, molecular methods are necessary for genotyping (AOUAY et al., 2008AOUAY, A et al. Molecular characterization of Prototheca strains isolated from bovine mastitis. Journal de Mycologie Medicale, 2008. v.18, n.4, p.224-227. Available from: <https://www.sciencedirect.com/science/article/pii/S1156523308000954>. Accessed: Mar. 13, 2017. doi: 10.1016/j.mycmed.2008.10.002.
https://www.sciencedirect.com/science/ar...
). There is no effective therapeutic protocol for treatment of P. zopfii induced bovine mastitis due to its intrinsically high resistance to conventional antimicrobials, antifungals and antiseptic compounds (LASS-FLÖRL & MAYR, 2007LASS-FLÖRL, C; MAYR, A. Human protothecosis. Clinical Microbiology Reviews, 2007. v.20, n.2, p.230-242. Available from: <Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1865593/pdf/0032-06.pdf >. Accessed: Feb. 12, 2017. doi: 10.1128/CMR.00032-06.
https://www.ncbi.nlm.nih.gov/pmc/article...
). Although, the algae’s poor response to conventional therapy has long been known (COSTA et al., 2004COSTA, EO et al. Diagnosis of clinical bovine mastitis by fine needle aspiration followed by staining and scanning electron microscopy in a Prototheca zopfii outbreak. Mycopathologia, 2004. v.158, n.1, p.81-85. Available from: <Available from: https://www.ncbi.nlm.nih.gov/pubmed/15487325 >. Accessed Mar. 12, 2017. doi: 10.1023/B:MYCO.0000038423.49445.
https://www.ncbi.nlm.nih.gov/pubmed/1548...
), only recently it has been demonstrated that the biofilm formed by P. zopfii is associated with microorganism persistence in the mammary gland, and consequently to antimicrobial resistance (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/...
).

Biofilms are communities of microorganisms attached to a surface and surrounded by an extra cellular matrix (O’TOOLE et al., 2000O’TOOLE, G et al. Biofilm formation as microbial development. Annual Review of Microbiology, Oct. 2000. v.54, n.1, p.49-79. Available from: <Available from: http://www.annualreviews.org/doi/10.1146/annurev.micro.54.1.49 >. Accessed: Mar. 13, 2017. doi: 10.1146/annurev.micro.54.1.49.
http://www.annualreviews.org/doi/10.1146...
). These slime-embedded communities become highly resistant to both antimicrobials and host defenses (FUX et al, 2005FUX, CA et al. Survival strategies of infectious biofilms. Trends in Microbiology, 2005, v.13, n.1, p.34-40. Available from: <Available from: https://www.sciencedirect.com/science/article/pii/S0966842X04002641?via%3Dihub >. Accessed Dec. 17, 2018. doi: doi.org/10.1016/j.tim.2004.11.010.
https://www.sciencedirect.com/science/ar...
). Although, the formation of biofilms by P. zopfii had been reported, there is no agreement between the current characterization protocols (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/...
; 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/...
; 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/...
). Therefore, given the relevance of this issue, the aim was 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.

MATERIALS AND METHODS:

Thirty-two strains of P. zopfii isolated from cows with clinical (n=27) and subclinical (n=5) mastitis were used in this study. Clinical mastitis was characterized by abnormalities at visual observation and udder palpation, including udder swelling, hardness of the affected quarter, pain, watery milk, and reduced milk yield. Subclinical mastitis was considered when the visible inflammatory changes were absent in the milk or udder. All Prototheca spp.- positive milk samples were obtained from five medium-scale dairy farms, with 100 to 380 lactating cows, each producing 12-20 liters of milk daily and milked mechanically twice a day. All dairy farms were located in four Brazilian States from the South (Rio Grande do Sul and Paraná), Southeast (São Paulo) and Northeast (Pernambuco) regions. Milk samples were plated on defibrinated sheep blood agar (5%) and Sabouraud dextrose agar and incubated aerobically at 37°C for 72h. Yeast-like colonies were submitted for identification by phenotypic tests and carbohydrate fermentation (trehalose, fructose, galactose and glycerol) (PORE, 1985PORE, RS. Prototheca taxonomy. Mycopathologia, 1985. v.90, n.3, p.129-139. Available from: <Available from: https://link.springer.com/content/pdf/10.1007%2FBF00436728.pdf >. Accessed: Feb. 15, 2017. doi: 10.1007/BF00436728.
https://link.springer.com/content/pdf/10...
; VARGAS et al., 1998VARGAS, AC et al. Isolation of Prothoteca zopfii from a case of bovine mastitis in Brazil. Mycopathologia, 1998. v.142, n.3, p.135-137. Available from: <Available from: https://www.ncbi.nlm.nih.gov/pubmed/10052162 >. Accessed: Mar. 20, 2017.
https://www.ncbi.nlm.nih.gov/pubmed/1005...
; ZAROR et al., 2011ZAROR, L et al.,. Mastitis bovina por Prototheca zopfii: primer aislamiento en Chile. 2011. Archivos de Medicina Veterinária, v.43, n.2, p.173-176. Available from: <Available from: https://scielo.conicyt.cl/scielo.php?script=sci_arttext&pid=S0301-732X2011000200010&lng=es&nrm=iso >. Accessed: Dec. 17, 2018. doi: dx.doi.org/10.4067/S0301-732X2011000200010.
https://scielo.conicyt.cl/scielo.php?scr...
).

Biofilm assays were performed as previously described by RODRIGUES et al. (2010RODRIGUES, LB et al. Quantification of biofilm production on polystyrene by Listeria, Escherichia coli and Staphylococcus aureus isolated from a poultry slaughterhouse. Brazilian Journal of Microbiology, 2010. v.41, n.4, p.1082-1085. Available from: <Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3769765/ >. Accessed: May, 05, 2017. doi: 10.1590/S1517-838220100004000029.
https://www.ncbi.nlm.nih.gov/pmc/article...
) with few modifications. All isolates were grown in tryptone soya broth (TSB) with 1% glucose added, and incubated at 37°C for 48h. Next, an inoculum was prepared for all isolates and standardized at approximately 108 CFUmL-1 which corresponded to optical density (OD) of 0.24±0.02 by spectrophotometric observation at 600nm (OD600). Subsequently, 20µl of each P. zopfii suspension was added to 96-well polystyrene microplates containing 180µl/well TSB added 1% glucose. The microplates were incubated at 37°C for 24h under static or dynamic growth conditions (100 revolutions per minute, rpm). After incubation, the content in the wells was carefully aspirated, and each well was washed twice with 0.01M sterile saline phosphate buffer (PBS; pH 7.4) to remove all non-adherent cells. After drying of the plates at room temperature, the adherent cells were stained for 5min. with 100µl of 0.25% crystal violet (Synth®, Cat. No. 42535/42555) or 0.05% cotton blue (Newprov®, Cat. No. 771). Lastly, the content of the wells was aspirated, washed and dried as previously described. After, 200µl of a 60:40 alcohol/acetone solution was added to solubilize the stained biofilm. Biofilm was quantified by spectrophotometric observation at 550nm. To ensure quality control, the ATCC 25923 Staphylococcus aureus was used as positive control for biofilm formation (MARQUES et al., 2007MARQUES, SC et al. Formation of biofilms by Staphylococcus aureus on stainless steel and glass surfaces and its resistance to some selected chemical sanitizers. Brazilian Journal of Microbiology, 2007. v.38, n.3, p.538-543. Available from: <Available from: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1517-83822007000300029 >. Accessed: Mar. 20, 2017. doi: 10.1590/S1517-83822007000300029.
http://www.scielo.br/scielo.php?script=s...
), while the medium alone served as negative control. All tests were performed in triplicate. The arithmetic mean of the triplicates was calculated and the strains showing absorbance values greater than the negative control were considered positive for biofilm formation. Strains were classified regarding the biofilm-forming ability using the results of arithmetic mean, where ODct refers to the negative control and ODis refers to the strain analyzed. P. zopfii isolates were classified as weak (ODct<ODis< 2.ODct), moderate (2.ODct<ODis<4.ODct) or strong (4.ODct<ODis) biofilm-producers (STEPANOVIĆ et al., 2003STEPANOVIĆ, S et al. Influence of the incubation temperature, atmosphere and dynamic conditions on biofilm formation by Salmonella spp. Food Microbiology, 2003. v.20, n.3, p.339-343. Available from: <Available from: https://www.sciencedirect.com/science/article/pii/S0740002002001235?via%3Dihub >. Accessed: Oct. 02, 2018. doi: 10.1016/S0740-0020(02)00123-5.
https://www.sciencedirect.com/science/ar...
). Results of static and dynamic growth conditions, as well as the staining assays, were compared using the OD values averages of each triplicate by statistical methods. The homogeneity of variances among groups was assessed using the Levene’s test and the comparisons among those groups were carried out using one-way ANOVA and Tukey’s test (Statistica 7.0 Software). The minimum significance level was set at P≤0.05.

RESULTS AND DISCUSSION:

All P. zopfii isolates were classified as biofilm-producers in all growth conditions and staining methods analyzed. Absorbance values of P. zopfii stained with crystal violet and cotton blue grown under static or dynamic conditions are presented in Table 1. All isolates stained with crystal violet were classified as strong biofilm producers under static or dynamic growth conditions (Table 2). However, the microorganisms stained with cotton blue displayed different degrees of biofilm formation. Under static condition, 68.75% (22/32) were classified as moderate, 21.9% (7/32) as strong and 9.37% (3/32) as weak biofilm-producers (Table 2). P. zopfii isolates stained with cotton blue under dynamic conditions presented the same proportion of strong biofilm producers, while moderate isolates dropped to 53.1% (17/32), and the weak isolates proportion increased to 25.0% (8/32) (Table 2). However, statistical analyses did not reveal significant differences (P>0.05) with crystal violet and cotton blue grown under static or dynamic conditions.

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

Concerning the virulence factors and pathogenic aspects of this microorganism there is scarce information. Since the biofilm-formation has been reported as an important virulence factor of Prototheca spp., this study aimed to evaluate the biofilm formation by 32 P. zopfii isolates testing different methodologies employing two growth conditions, and two different dyes.

In the present study, all P. zopfii isolates were classified as biofilm producers. No significant difference in the biofilm-forming ability was detected with different staining methods (P>0.05), it was verified that the cotton blue dye was more appropriated to quantify biofilm formation by P. zopfii. According to the literature, crystal violet dye has been used mainly in biofilm assays with bacteria (O’TOOLE et al., 2000O’TOOLE, G et al. Biofilm formation as microbial development. Annual Review of Microbiology, Oct. 2000. v.54, n.1, p.49-79. Available from: <Available from: http://www.annualreviews.org/doi/10.1146/annurev.micro.54.1.49 >. Accessed: Mar. 13, 2017. doi: 10.1146/annurev.micro.54.1.49.
http://www.annualreviews.org/doi/10.1146...
), while cotton blue dye has been employed in fungal and yeast biofilm studies (JAYASINGHEARACHCHI & SENEVIRATNE, 2006JAYASINGHEARACHCHI, HS; SENEVIRATNE, G. Fungal solubilization of rock phosphate is enhanced by forming fungal-rhizobial biofilms. Soil Biology and Biochemistry, 2006. v.38, n.2, p.405-408. Available from: <Available from: https://www.sciencedirect.com/science/article/pii/S0038071705002439 >. Accessed: Mar. 11, 2017. doi: 10.1016/j.soilbio.2005.06.004.
https://www.sciencedirect.com/science/ar...
; SIQUEIRA et al., 2008SIQUEIRA, A et al. Protothecosis in companion animals and aspects of the disease in human. Ciência Rural, 2008. v.38, n.06, p.1794-1804. Available from: <Available from: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0103-84782008000600052&lng=en&nrm=iso&tlng=pt >. Accessed: May, 12, 2017.
http://www.scielo.br/scielo.php?script=s...
). In this study, the characterization of P. zopfii as weak, moderate or strong biofilm-producer was possible using the absorbance results from 0.05% cotton blue dye only. Several studies have demonstrated that microorganisms, even those from the same species, display variable biofilm-forming ability (O’TOOLE et al., 2000O’TOOLE, G et al. Biofilm formation as microbial development. Annual Review of Microbiology, Oct. 2000. v.54, n.1, p.49-79. Available from: <Available from: http://www.annualreviews.org/doi/10.1146/annurev.micro.54.1.49 >. Accessed: Mar. 13, 2017. doi: 10.1146/annurev.micro.54.1.49.
http://www.annualreviews.org/doi/10.1146...
), as observed using cotton blue staining. There is a hypothesis about the ability of P. zopfii strains to form biofilm depending on their genotypes which claims that the strains belonging to the genotype 1 are weak to moderate whilst those of the genotype 2 are strong biofilm producers (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/...
). However, without a standardized methodology this hypothesis cannot be tested appropriately.

The static or dynamic growth conditions had no significant influence in the production of biofilms by P. zopfii (P>0.05); however, it was possible to verify that the growth conditions (static and dynamic) had impact on the biofilm formation, which was noticeable when cotton blue was used (Table 2). Nevertheless, besides the technical particularities, the biofilm formation is also modulated by several growth conditions and nutrient sources, as well as interactions with different microorganisms and environmental conditions (COSTERTON et al., 1995COSTERTON, JW et al. Bacterial biofilms in nature and disease. Annual Reviews Microbiology, 1995. v.49, p.711-745. Available from: <Available from: https://www.annualreviews.org/doi/abs/10.1146/annurev.mi.41.100187.002251 >. Accessed Mar. 11, 2017. doi: 10.1146/annurev.mi.41.100187.002251.
https://www.annualreviews.org/doi/abs/10...
), making any comparison among microorganisms a challenge. Studies carried out on biofilm formation in isolates of Prototheca spp.; although restricted, present a large variability in their experimental design (Table 3) resulting from the lack of standardization.

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

In table 3 we summarized the current knowledge about biofilm formation by Prototheca spp. In 2015, the first study showing the biofilm formation by P. zopfii was reported (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/...
). The authors evaluated 10 subclinical isolates under different incubation temperatures (25°C and 37°C) and suggested some influence of this parameter on biofilm formation of Prototheca sp. The authors classified the isolates as weak, moderate, and strong biofilm-producers, and reported six weak and four moderate biofilm-producers employing a 48 h of incubation at 37°C. Later, another study testing 46 P. zopfii isolates identified 90% as strong biofilm-producers after a 24 h incubation at 37°C and by using safranin dye (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/...
). In the present study, employing a 24h incubation at 37°C, using crystal violet dye, as suggested by GONÇALVES et al. (2015)GONÇALVES, 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/...
, it was verified that 100% of the isolates were strong biofilm-producers. However, when cotton blue dye was used it was possible to classify the isolates as weak, moderate or strong biofilm-producers. Nevertheless, when in the static condition 90.65% of P. zopfii isolates were classified as moderate to strong and under dynamic condition there was a decrease in number of moderate and strong biofilm-forming isolates (75.00%) (Table 2).

Table 3
Studies involving biofilm formation by Prototheca spp.

The present research reinforces previous findings that indicate P. zopfii as a potent in vitro biofilm-producer, which may contribute to chronic bovine mammary mastitis, as well as the persistence of algae in milking machines or farm environments (OSUMI et al., 2008OSUMI, T et al. Prototheca zopfii genotypes isolated from cow barns and bovine mastitis in Japan. Veterinary Microbiology, 2008. v.131, n.3-4, p.419-423. Available from: <Available from: https://www.sciencedirect.com/science/article/pii/S0378113508001478?via%3Dihub > Accessed: May, 11, 2017. doi: 10.1016/j.vetmic.2008.04.012.
https://www.sciencedirect.com/science/ar...
). Based on these results, it is possible to propose that the most adequate conditions for testing the formation and classification of biofilm by P. zopfii would be static growth condition, a 24-hour incubation at 37°C and staining with 0.05% cotton blue. Nevertheless, we suggested that additional studies are necessary to characterize the mechanisms involved in biofilm production by P. zopfii to prevent and control the infections caused by this important pathogen.

ACKNOWLEDGEMENTS

The authors acknowledge the support received from development agencies to search Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) (finance code 001).

REFERENCES

  • 0
    CR-2018-0574.R2

Publication Dates

  • Publication in this collection
    2019

History

  • Received
    20 July 2018
  • Accepted
    02 Jan 2019
  • Reviewed
    08 Feb 2019
Universidade Federal de Santa Maria Universidade Federal de Santa Maria, Centro de Ciências Rurais , 97105-900 Santa Maria RS Brazil , Tel.: +55 55 3220-8698 , Fax: +55 55 3220-8695 - Santa Maria - RS - Brazil
E-mail: cienciarural@mail.ufsm.br