Characterization of Prototheca CYP51/ERG11 as a possible target for therapeutic drugs.

Prototheca spp. are achlorophyllous algae, ubiquitous in nature. An increasing number of human and animal cases of Prototheca infection (protothecosis) are reported, and antifungal azoles, which inhibit sterol 14α-demethylase (CYP51/ERG11) involved in ergosterol biosynthesis, have empirically been used for the treatment of protothecosis. Although Prototheca, like fungi, has ergosterol in the cell membrane, efficacy of the antifungal azoles in the treatment of protothecosis is controversial. For investigating the interaction of azole drugs with Prototheca CYP51/ERG11, the CYP51/ERG11 genomic genes of four strains of P. wickerhamii and one strain each of P. cutis and P. miyajii were isolated and characterized in this study. Compared with the CYP51/ERG11 gene of chlorophyllous Auxenochlorella Protothecoides, it is possible that ProtothecaCYP51/ERG11 gene, whose exon-intron structure appeared to be species-specific, lost introns associated with the loss of photosynthetic activity. Analysis of the deduced amino acid sequences revealed that Prototheca CYP51/ERG11 and fungal CYP51/ERG11 are phylogenetically distant from each other although their overall structures are similar. Our basic in silico studies predicted that antifungal azoles could bind to the catalytic pocket of Prototheca CYP51/ERG11. It was also suggested that amino acid residues away from the catalytic pocket might affect the drug susceptibility. The results of this study may provide useful insights into the phylogenetic taxonomy of Prototheca spp. in relationship to the CYP51/ERG11 structure and development of novel therapeutic drugs for the treatment of protothecosis. LAY SUMMARY: Cases of infection by microalgae of Prototheca species are increasing. However, effective treatment has not been established yet. In this study, gene and structure of Prototheca's CYP51/ERG11, an enzyme which might serve as a target for therapeutic drugs, were characterized for the first time.

Prototheca from bovine milk and associated minimal algaecide concentration of chlorhexidine and povidone-iodine in Taiwan.

OBJECTIVE: Protothecal mastitis has emerged and affected the dairy industries globally in last decades; however, little is known about the occurrence of Prototheca in Taiwan. The present study identified the Prototheca isolated from bovine milk and revealed the algaecide efficacy of 2 disinfectants. MATERIAL AND METHODS: From 2011 to 2014, a total of 176 Prototheca isolates were obtained from 10 625 milk samples collected from dairy cows in 24 Taiwanese dairy farms. Of these, 149 isolates were identified by biochemical tests and genotype differentiation with a specific PCR protocol. Minimal algaecide concentration (MAC) of chlorhexidine and povidone-iodine were obtained from 16 isolates by microdilution method. RESULTS: Almost all (n = 147) isolates were P. zopfii, and remaining 2 isolates were P. wickerhamii and unidentified, respectively. Based on molecular genotyping, all P. zopfii isolates belonged to genotype 2. The MAC of 2 commonly used disinfectants, chlorhexidine and povidone-iodine, against 16 P. zopfii isolates was 1.56-3.13 and 48.83-390.63 µg/ml, respectively. CONCLUSION: This report revealed for the first time the prevalence and the predominant P. zopfii genotype 2 of bovine protothecal infection in Taiwan. In vitro algaecide efficacy indicated that both tested disinfectants were effective against P. zopfii.

Multiple cutaneous infections caused by Prototheca wickerhamii.

BACKGROUND: Prototheca species are a group of organisms ubiquitously existing in nature but have become a pathogenic threat to public health, which has aroused wide attention. Species identification and antifungal susceptibility have essential and valuable meanings to clinical diagnosis and treatment. METHODS: A case of an 84-year-old patient who had suffered from multiple cutaneous infections was reported. Tissue samples of the damaged skin were collected from the patient and used for microscopic examination and tissue culture. Staining methods, the VITEK system with YSD card and the molecular identification method based on partial mitochondrion-encoded cytochrome b (cytb) gene amplification and sequencing were used for species identification. Antifungal susceptibility testing was completed by using YeastOne plate. RESULTS: The patient had type II diabetes mellitus. Round, grape-like, and scattered morula forms were observed under the microscope in bright blue with lactophenol cotton blue staining and in green fluorescence with fungus fluorescence staining. Yeast-like colonies were grown on both the blood plates and the Sabouraud agar. P wichehamii was identified and presented resistance to three echinocandins, fluconazole, and 5-fluorocytosine, while was susceptible to amphotericin B, posaconazole, itraconazole, and voriconazole. CONCLUSION: Our result revealed that an old patient with diabetes mellitus might be a dangerous population of cutaneous protothecosis. It also highlighted the contribution to microbial methodology on the diagnosis and treatment of such rare fungus infection.

In vitro algicidal effect of polypyrrole on Prototheca species isolates from bovine mastitisAlgicidal activity of polypyrrole on Prototheca spp.

Algae of the genus Prototheca are microorganisms involved in the occurrence of diseases in humans and animals. In bovine species, Prototheca spp. cause environmental mastitis, productive losses in dairy herds, mainly leading to the discard of infected cows. Currently, there are no effective anti-Prototheca spp. drugs to combat this infection. Thus, the search for an efficacious therapy for Prototheca spp. infections have become essential. Highly soluble polypyrrole (Ppy) is a molecule with known antimicrobial activity. This study aimed to characterize Prototheca spp. isolates from bovine mastitis as well as to evaluate the susceptibility profile and to verify the morphological alterations on Prototheca spp. isolates treated with Ppy. In this research, 36 Brazilian isolates of Prototheca spp. were characterized by restriction fragment length polymorphism polymerase chain reaction (RFLP-PCR) assay for the mitochondrial cytB gene. Additionally, Ppy algicidal activity against these isolates of Prototheca spp. was assessed by minimal microbicidal concentration method in microplates. Further, scanning electron microscopy (SEM) was performed in order to verify the morphological alterations on Prototheca spp. isolates in response to Ppy. The isolates were characterized as belonging to Prototheca zopfii genotype 2 (35/36) and Prototheca blaschkeae (1/36). Ppy had an algicidal effect on all isolates tested at concentrations ranging from 15.625 µg ml-1 to 62.5 µg ml-1. SEM showed changes on planktonic and sessile P. zopfii, including a decrease of the number of cells with the presence of an amorphous substance involving the cells. The algicidal activity of Ppy suggests the therapeutic potential of this molecule in the prevention and treatment of Prototheca spp. in bovine mastitis.

Preliminary Results, Perspectives, and Proposal for a Screening Method of In Vitro Susceptibility of Prototheca Species to Antimicrotubular Agents.

Microorganisms belonging to the genus Prototheca are achlorophyllous microalgae, occasionally behaving as environmental pathogens that cause severe mastitis in milk cows, as well as localized or systemic infections in humans and animals. Among the different species belonging to the genus, Prototheca zopfii genotype 2 (recently reclassified as P. bovis) and P. blaschkeae are most commonly associated with bovine mastitis. To date, no pharmacological treatment is available to cure protothecal mastitis, and infected animals must be quarantined to avoid spreading the infection. The few antibiotic and antifungal drugs effective in vitro against Prototheca give poor results in vivo This failure is likely due to the lack of specificity of such drugs. As microalgae are more closely related to plants than to bacteria or fungi, an alternative possibility is to test molecules with herbicidal properties, in particular, antimicrotubular herbicides, for which plant rather than animal tubulin is the selective target. Once a suitable test protocol was set up, a panel of 11 antimicrotubular agents belonging to different chemical classes and selective for plant tubulin were tested for the ability to inhibit growth of Prototheca cells in vitro Two dinitroanilines, dinitramine and chloralin, showed strong inhibitory effects on P. blaschkeae at low micromolar concentrations, with half-maximal inhibitory concentrations (IC50) of 4.5 and 3 µM, respectively, while both P. zopfii genotype 1 (now reclassified as P. ciferrii) and P. bovis showed susceptibility to dinitramine only, to different degrees. Suitable screening protocols for antimitotic agents are suggested.

In vitro algaecid effect of itraconazole and ravuconazole on Prototheca species.

Protothecosis is a rare infection in humans, dogs, and cats, and its causative agent is Prototheca species, which consists of achlorophyllic algae that are ubiquitous in natural environments and hosts intestinal flora. Ravuconazole (RVZ) is a new available human azole drug in Japan since 2018 and broad-spectrum antifungal agent. In the present study, the in vitro susceptibility of clinical and environmental isolates of P. wickerhamii, P. zopfii, and P. blaschkeae to itraconazole (ITZ) voriconazole (VRZ), posaconazole (PCZ), and RVZ. RVZ was more potent than the other azoles against Protheca species and has considerable potential for use as a therapeutic agent for human and animal protothecosis.

Algicidal effect of blue light on pathogenic Prototheca species.

Prototheca spp. are pathogenic algae with important zoonotic potential. Most importantly, these algae often infect dairy cattle. Since there is no effective therapy against the algae, the standard recommendation is the disposal or culling of infected cows to avoid outbreaks. This study investigated the ability of blue light to inactivate pathogenic Prototheca species. Blue LED light (λ = 410 nm) was used to inactivate in vitro suspensions of P. zopfii genotypes 1 and 2, and P. blaschkeae. Our results showed that blue light irradiation induced a strain-specific dose-dependent algicidal effect against all tested strains. P. zopfii genotype 1, was more sensitive than genotype 2 and P. blaschkeae was the most tolerant. Even though we observed different inactivation kinetics, all strains presented significant photoinactivation levels within feasible procedure periods. Therefore, we conclude that blue light irradiation offers promising potential for the development of novel technologies that control contaminations and infections caused by Prototheca spp.

In vitro aminolevulinic acid mediated-antimicrobial photodynamic therapy inactivates growth of Prototheca wickerhamii but does not change antibacterial and antifungal drug susceptibiltity profile.

BACKGROUND: Antimicrobial photodynamic therapy(aPDT) has been used to treat localized cutaneous fungal infections that have an enhanced antifungal susceptibility profile. The aim of this study was to evaluate the effect of ALA aPDT on both the growth and the antimicrobial and antifungal susceptibility of Prototheca wickerhamii. METHODS: Six isolates of P. wickerhamii were used in the present study. The inocula in sterile 6-well microtiter plates were irradiated with narrow band LED (633 ± 10 nm) at the light intensity of 100 mW/cm2 and at a distance of 1 cm for 900 s. The ALA was tested at concentrations of 1, 5, and 10 mmol/l, while 10-µl aliquots of suspensions from each group were inoculated on Sabouraud dextrose agar to test the photoinactivation. Antibiotic susceptibility was investigated by the disc-diffusion method. RESULTS: Our study shows ALA aPDT induced 46% ± 24.23% reduction of the growth of all tested P. wickerhamii strains in T1 group. ALA aPDT induced 50.39% ± 19.88% reduction of the growth of all tested P. wickerhamii strains in T2 group. ALA aPDT induced 52.68 ± 20.22% reduction of the growth of all tested P. wickerhamii strains inT3 group. Single ALA aPDT induced 32.97% ± 1.6% growith reduction of three tested strains(O23d, O23e and 62,207), while repeated ALA aPDT induced 51.65 ± 2.91% reduction of the growth(P value = 0.000). There were no significant difference of the inhibitory zone diameter of both antibacterial and antifungal agents before and after ALA aPDT. CONCLUSIONS: ALA aPDT can inactivate the growth of P. wickerhamii, and repeated aPDT has more photoinactivation of P. wickerhamii. ALA aPDT does not change antibacterial agents and antifungal drugs susceptibility profile of P. wickerhamii.

The activity of silver nanoparticles against microalgae of the Prototheca genus.

AIM: To investigate the in vitro activity of silver NPs (AgNPs) against pathogenic microalgae of the Prototheca genus. MATERIALS & METHODS: The antialgal potential of AgNPs against Prototheca species of both clinical and environmental origin was assessed from minimum inhibitory (algistatic) and algicidal concentrations. The in vitro cytotoxicity of AgNPs against bovine mammary epithelial cell line was evaluated by means of the standard MTT assay. RESULTS: AgNPs showed a strong killing activity toward Prototheca algae, as the minimal algicidal concentration (MAC) values matched perfectly the corresponding minimum inhibitory concentration (MIC) values for all species (MAC = MIC, 1-4 mg/l), except P. stagnora (MIC > 8 mg/l). The concentrations inhibitory to pathogenic Prototheca spp. (MIC, 1-4 mg/l) were below the concentrations at which any toxicity in epithelial cells could be observed (CC20 > 6 mg/l). CONCLUSION: The study emphasizes the potential of AgNPs as a new therapeutic tool for the management of Prototheca infections.

The in vitro efficacy of SunSmile® Fruit & Vegetable Rinse against pathogenic strains of Prototheca algae that cause mastitis in cows.

The research concerns algae of the genus Prototheca. They are found in the natural environment and they can cause a disease in animals and humans called protothecosis. The aim of the study was to evaluate the in vitro activity of the fruit and vegetable rinse agent SunSmile® Fruit & Vegetable Rinse (Sunrider International) against P. zopfii isolates. The materials consisted of ten P. zopfii strains isolated from the milk of cows with mastitis. The following antifungal chemotherapeutic agents were also used in the study for comparison: nystatin, ketoconazole, amphothericin B, miconazole, clotrimazole, econazole, fluconazole, and flucytosine. The tube dilution method were used to evaluate the effect of a fruit and vegetable rinse agent and the disc-diffusion method to evaluate the effect of antifungal chemotherapeutic agents on P. zopfii strains. All tested strains of P. zopfii were susceptible to the action of the SunSmile® agent. The MMC was in the range of 0.0024-0.0190%. The SunSmile® Fruit & Vegetable Rinse can be used in prevention of mastitis in cows and in human protothecosis due to its safe, natural composition and efficacy.

The Effect of Some Natural Essential Oils Against Bovine Mastitis Caused by Prototheca zopfii Isolates In Vitro.

The aim of the study was to evaluate the effect of essential oils obtained from Thymus vulgaris L., Origanum vulgare L., Origanum majerana L., Mentha × piperita L. and Allium ursinum L. against Prototheca zopfii strains that cause inflammation of the udder (mastitis) in cows. The study was conducted on ten strains derived from milk samples. The microdilution method was used to determine the sensitivity of P. zopfii strains to the studied essential oils, and the disk diffusion method was used to determine the sensitivity to antifungal chemotherapeutics. The plates were incubated for 48 h at 37 °C under aerobic conditions. All strains of algae were sensitive to the essential oils marjoram, thyme and oregano and resistant to mint and garlic oils. MIC values ranged from 0.25 to 1 µl/ml. Marjoram oil demonstrated the greatest activity, and oregano oil the weakest. Among the antifungal agents tested, 90% of strains showed sensitivity to nystatin. One of the tested strains (71/IV) was resistant to all investigated antifungal agents. The tested essential oils are known to have anti-algae activity and can be used as natural agents for prophylaxis in animals, particularly in mastitis-affected cows.

Short communication: Cold atmospheric plasma inactivation of Prototheca zopfii isolated from bovine milk.

Mastitis is a serious bovine diseases that can be caused by Prototheca zopfii, yeast-like algae belonging to the family Chlorellaceae. The substantial economic losses and health damage associated with bovine mastitis emphasize the need to develop effective strategies aimed at control of the infection. Unfortunately, P. zopfii is highly resistant to most common antibacterial and antifungal agents, as well as to heat treatment. We report here the first attempt to use cold atmospheric plasma to inactivate this pathogen. We studied 20 strains of P. zopfii isolated from milk samples taken from cows with clinical or subclinical mastitis. The studies confirmed the high level of resistance of P. zopfii to typical antifungal agents, such as voriconazole, fluconazole, amphotericin B, caspofungin, anidulafungin, and micafungin. In contrast, each of the strains revealed high susceptibility to cold atmospheric plasma, >2-fold higher compared with a reference strain of Candida albicans. The obtained results are promising and open up a new approach in the fight against P. zopfii.

A comparative study of the in vitro activity of iodopropynyl butylcarbamate and amphotericin B against Prototheca spp. isolates from European dairy herds.

The objective of this study was to assess the in vitro effect of iodopropynyl butylcarbamate (IPBC) and amphotericin B (AMB) on Prototheca zopfii genotype 2 and Prototheca blaschkeae isolates recovered from dairy herds of Belgium, France, Italy, Germany, and Poland. The combination of IPBC with AMB on Prototheca isolates and toxicity of IPBC to the bovine mammary epithelial cells were also evaluated. The in vitro activity of IPBC and AMB against 96 isolates of P. zopfii genotype 2 and 42 isolates of P. blaschkeae was performed. Minimum inhibitory concentrations (MIC) and minimum algicidal concentrations (MAC) of IPBC and AMB were determined. To determine any synergistic, additive, or antagonistic effect of the combination of IPBC and AMB, 2-dimensional checkerboard combination tests were also performed to calculate fractional inhibitory concentrations. Cytotoxicity analysis of IPBC to the bovine mammary epithelial cell line was performed using a 3-(4,5-dimethyl-2-thiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The MIC for 50 and 90% of isolates (MIC50 and MIC90, respectively) for IPBC were 4 and 8 mg/L versus 0.5 and 1 mg/L for AMB, respectively. The MIC profiles differed between P. zopfii genotype 2 and P. blaschkeae, with the latter species being more susceptible to both compounds. The MIC50 and MIC90 of IPBC were 4 and 8 mg/L for P. zopfii genotype 2 and 1 and 2 mg/L for P. blaschkeae, respectively. The MIC50 and MIC90 of AMB were both 1 mg/L for P. zopfii genotype 2 and 0.25 and 1 mg/L for P. blaschkeae, respectively. Both IPBC and AMB exhibited the ability to kill Prototheca spp. The MAC for 90% of isolates of IPBC was twice the MIC90, whereas an 8-fold increase of the MIC90 was algicidal in the case of AMB. Overall, the combined use of IPBC and AMB exhibited an increased algicidal effect, albeit the fractional inhibitory concentration index showed synergistic activity only against 3 P. zopfii genotype 2 isolates. For all the remaining isolates (87.5%), this combination produced only an additive effect. The MTT assay results showed both IPBC and AMB, at the concentrations employed in the study, to be nontoxic to the epithelial mammary gland cells (cell viability >90%). Notably, only IPBC at the highest concentration (i.e., 8 mg/L) exerted a slight cytotoxic effect on the cell line tested (mean cell viability: 88.54 ± 3.88 and 90.66 ± 3.0, after 2 and 4 h of MTT treatment, respectively). The anti-Prototheca activity of IPBC was here demonstrated for the first time. In addition, the combined use of IPBC with AMB enhanced each other's effect, creating an additive rather than synergistic interaction. Both agents, used at concentrations corresponding to MIC values against Prototheca spp., showed no toxic effect for the mammary epithelial cells. In conclusion, IPBC, used either alone or in combination with AMB, can be considered a promising option in the treatment armamentarium for protothecal mastitis in dairy cows.

In vitro algicidal effect of guanidine on Prototheca zopfii genotype 2 strains isolated from clinical and subclinical bovine mastitis.

Prototheca species have increasingly been reported to be opportunistic pathogens that cause mastitis in dairy herds, and it poses an emergent problem because at present, there are no effective therapies for the treatment of protothecal mastitis. This study investigated the in vitro algicidal effect of guanidine on 75 Prototheca zopfii genotype 2 strains isolated from 75 cases of clinical and subclinical bovine mastitis. All strains were susceptible to guanidine in vitro with minimal algaecide concentrations ranging from 0·001 to 0·035%. Guanidine is known to have a high microbicidal effect and is considered to be a new generation microbicidal compound. It is not toxic to human mucous membranes and conjunctivas at low concentrations and has been used as a disinfectant in swimming pools and as an antiseptic for human wounds. The algicidal action of guanidine at low concentrations indicates that it could be an alternative disinfectant or antiseptic for cleaning of the dairy environment and milking equipment, in pre- and postdipping solutions, in the chemical dry therapy of bovine teats and even in the intramammary therapy of P. zopfii infections. This is the first report of the in vitro algicidal effect of guanidine on P. zopfii strains of animal origin. SIGNIFICANCE AND IMPACT OF THE STUDY: Prototheca zopfii genotype 2 is an opportunistic pathogen of bovine mastitis. To date, no effective therapies against protothecal mastitis have been developed. The in vitro algicidal effect of guanidine on 75 P. zopfii genotype 2 strains isolated from cows revealed that all of the isolates were susceptible to the compound at low concentrations, which indicates that guanidine may be used as an antiseptic/disinfectant for dairy milking equipment, in pre- and postdipping solutions, and as a chemical dry therapy or an intramammary therapy. This study describes the in vitro algicidal effect of guanidine on P. zopfii for the first time.

The In Vitro Efficacy of Essential Oils and Antifungal Drugs Against Prototheca zopfii.

BACKGROUND: The algae of the genus Prototheca are environmental pathogens whose main reservoir is the habitat of cows. They can cause protothecosis in domestic and wild animals, as well as human beings, with the main etiological agents being Prototheca zopfii in animals and Prototheca wickerhamii in humans. AIM: The aim of the study was to evaluate the in vitro activity of selected essential oils and antifungal antibiotics against P. zopfii isolates. MATERIALS AND METHODS: The material consisted of nine P. zopfii strains isolated from the milk of cows suffering from mastitis. Eight essential oils produced by POLLENA-AROMA, Poland, and nine antifungal agents were tested. The effects of essential oils on P. zopfii were evaluated by microdilution with liquid Sabouraud dextrose broth, and susceptibility to antifungal agents was tested using the disk-diffusion method. RESULTS: All used essential oils inhibited the activity of P. zopfii isolates, with MIC values ranging from 0.2 to 10.5 µl/ml. Cinnamon, clove, and thyme demonstrated the highest activity against the tested P. zopfii strains at concentrations ranging from 0.6 to 1.0 µl/ml. Of the antifungal agents, the tested strains were the most sensitive to nystatin (100 %). CONCLUSIONS: The tested essential oils can be used to complement protothecosis therapy in animals and human beings.

In vitro photoinactivation of bovine mastitis related pathogens.

BACKGROUND: Bovine mastitis is considered the most important disease of worldwide dairy industry. Treatment of this disease is based on the application intramammary antibiotic, which favors an increase in the number of resistant bacteria in the last decade. Photodynamic inactivation (PDI) has been investigated in different areas of Health Sciences, and has shown great potential for inactivating different pathogens, without any selection of resistant microorganisms. The objective of this study was to investigate the efficacy of PDI in the inactivation of pathogens associated with bovine mastitis. METHODS: We tested the effectiveness of PDI against antibiotic resistant strains, isolated from bovine mastitis, from the following species: Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, Corynebacterium bovis, and the alga Prototheca zopfii. Nine experimental groups were evaluated: control, no treatment; light only, irradiation of a red light-emitting diode (λ=662 (20) nm) for 180 s; exposure to 50 µM methylene blue alone for 5 min; and PDI for 5, 10, 30, 60, 120 and 180 s. RESULTS: S. dysgalactiae, S. aureus, and C. bovis were inactivated after 30s of irradiation, whereas S. agalactiae was inactivated after 120 s and P. zopfii at 180 s of irradiation. CONCLUSION: These results show that PDI can be an interesting tool for inactivating pathogens for bovine mastitis.

Characterization of Prototheca zopfii Genotypes Isolated from Cases of Bovine Mastitis and Cow Barns in China.

Protothecal mastitis, caused mostly by Prototheca zopfii (P. zopfii), is increasing in dairy herds and is being reported globally. The present study was aimed at studying the epidemiology of mastitis and at molecular characterization of P. zopfii isolates from dairy herds and their surroundings in three provinces of China using microbiological, biochemical and molecular methods, and antibiotic susceptibility tests. Samples from milk (n = 620) of mastitic cows and their barns sources (n = 410) including feces, feed, bedding materials and drinking water were analyzed. Among other pathogens recovered from mastitic milk, 84 (13.5%) of the isolates were identified as P. zopfii. All of the P. zopfii isolates recovered from milk were recognized as genotype 2, whereas 58 (73.4%) and 21 (26.6%) isolates from environmental sources were found to be P. zopfii genotypes 1 and 2, respectively. The isolates were susceptible to some antibiotics and antifungal agents, including amikacin (78.1%), streptomycin (58.5%), gentamicin (17.8%), amphotericin B (68.6%) and nystatin (64.4%). Additionally, the two genotypes displayed versatile patterns of susceptibility to different antimicrobials agents. Phylogeny of the genotypes on the basis of 18S SSU rDNA and 28S SSU rDNA was also investigated. The isolates of the two genotypes separated into different clades, and no interrelationship was observed among these as shown by phylogenetic analysis. The genotype 1 isolates from cow barn sources were non-pathogenic and may not present any risk of mastitis. We conclude that P. zopfii genotype 2 might play an important role in bovine mastitis in China.

Biofilm formation by pathogenic Prototheca algae.

UNLABELLED: Prototheca microalgae are the only plants known to cause infections in humans and animals. The mechanisms of Prototheca infections are poorly understood, and no good treatments are available. Biofilms-surface-attached, three-dimensional microbial communities contributing to chronic infections-are formed by many pathogenic bacteria and fungi, but it is not known if Prototheca algae also have this ability. This study shows that various Prototheca species form biofilms composed of surface-attached cells in all growth phases, linked together by matrix containing DNA and polysaccharides. Biofilm formation was modulated by the presence of host plasma or milk. Compared to planktonic cells, Prototheca biofilms caused decreased release of IL-6 by mononuclear immune cells and responded differently to treatment with antimicrobials. Prototheca biofilms possibly contribute to chronic and hard-to-treat character of those algal infections. SIGNIFICANCE AND IMPACT OF THE STUDY: Prototheca algae are the only existing pathogenic plants. Almost nothing is known about mechanisms of Prototheca infections. This study identifies that, similar to pathogenic bacteria and fungi, Prototheca algae can form biofilms. These biofilms induce reduced immune cell activation relative to planktonic cells, and are also less susceptible to antimicrobials. Biofilm formation by Prototheca could be the first in vitro correlate of pathogenicity, opening a new research field for this pathogen.

Biofilm-producing ability and efficiency of sanitizing agents against Prototheca zopfii isolates from bovine subclinical mastitis.

The objectives of the present study were to evaluate (1) the capacity of the microalga Prototheca zopfii isolated from subclinical bovine mastitis cases to form biofilms; and (2) the resistance of these isolates to sanitizing agents. Ten isolates of P. zopfii from cows with subclinical mastitis (somatic cell count>200×10(3) cells/mL), distributed in 5 dairy farms, were evaluated for their capacity to form biofilms in polystyrene microplate assays and stainless steel coupons, at 25°C and 37°C±1°C. Prototheca zopfii were isolated from milk samples via microbiological culture and analyzed by 18S rRNA gene sequencing. Biofilm formation on the coupons was observed by scanning electron microscopy. The resistance to sanitizing agents was assessed using the biofilm-forming P. zopfii isolates in stainless steel coupon assays, which were subjected to 3 sanitizers: peracetic acid, sodium hypochlorite, and iodine solution. To evaluate resistance to the sanitizers, the minimum inhibitory concentration (MIC) technique was performed using decreasing concentrations of the sanitizing agents (20, 10, 5, 2.5, 1.25, 0.625, 0.312, 0.156, 0.078, 0.039, and 0.019 g/L). After inoculating the isolates, all concentrations were evaluated at 3 distinct incubation periods (24, 48, and 72 h) to assess the effect of incubation time on the MIC. Using the polystyrene microplate assays, 1 isolate showed weak biofilm production, 5 moderate, and 4 strong, when incubated at 25°C±1. For isolates incubated at 37°C±1, 6 showed weak biofilm production and 4 moderate. All P. zopfii isolates (n=10) had the capacity to form biofilms on stainless steel coupons. The longer the incubation period of the P. zopfii isolates at different dilutions, the greater the concentrations of sanitizer needed to prevent growth of the microalgae under the tested conditions. We detected a significant effect of sanitizer and time of incubation (24, 48, and 72 h) on MIC values against P. zopfii isolates. The isolates were sensitive in vitro to peracetic acid (MIC90≥0.019 g/L), sodium hypochlorite (MIC90≥0.312 g/L), and iodine solution (MIC90≥0.625 g/L), after 24 h of incubation (where MIC90=concentration needed to inhibit 90% of isolates). Of the tested sanitizers, peracetic acid had the greatest efficiency against P. zopfii. We conclude that P. zopfii isolates are capable of biofilm production, which may contribute to their persistence in a milking and dairy environment.