Drug Susceptibility Profiling of Prototheca Species Isolated from Cases of Human Protothecosis.

Prototheca are unicellular, achlorophyllous, yeast-like microalgae that occur in a wide range of natural habitats. At least five species have been implicated as the causative agents of opportunistic infections of men. Human protothecosis typically manifests as cutaneous, articular, or systemic disease. Treatment is largely empirical with poorly predictable and often unsuccessful outcomes. This is largely due to the frequently observed resistance of Prototheca species to conventional antimicrobial agents. This work is the first to perform drug susceptibility profiling exclusively on isolates from human cases of protothecosis. A total of 23 such isolates were tested against amphotericin B and 9 azoles, including efinaconazole and luliconazole, whose activities against Prototheca have never been studied before. Efinaconazole was the most active, with median minimum inhibitory concentration (MIC) and minimum algicidal concentration (MAC) values of 0.031 mg/L and 0.063 mg/L, respectively. Fluconazole and luliconazole had the lowest activity, with median MIC and MAC values of 128 mg/L. To conclude, amphotericin B and most of the azoles showed in vitro activity, with an algicidal rather than algistatic effect, against Prototheca. Still, the activity of individual drugs differed significantly between the species and even between strains of the same species. These differences can be attributed to a species-specific potential for acquiring drug resistance, which, in turn, might be linked to the treatment history of the patient from whom the strain was recovered. The results of this study underscore the potential clinical utility of efinaconazole as a promising therapeutic agent for the treatment of human protothecosis.

Occurrence of Prototheca Microalgae in Aquatic Ecosystems with a Description of Three New Species, Prototheca fontanea, Prototheca lentecrescens, and Prototheca vistulensis.

Prototheca species are unicellular, nonphotosynthetic, saprophytic, and occasionally pathogenic, microalgae, with an extensive environmental reservoir. This study explores, for the first time, the occurrence of Prototheca in aquatic ecosystems by using a molecular profiling approach. A total of 362 samples were collected from 80 natural and artificial waterbodies at 88 sampling sites in 26 localities across Poland during a 1.5-year period. The overall isolation rate of Prototheca from water environments was 14.1%. Prototheca were most prevalent in rivers of urbanized areas, indicating that the algae are primarily adapted to lotic ecosystems with a high input of organic matter. Interestingly, it is not the amount of organic matter per se but its quality that seems to shape the habitat potential of the protothecae. The two most frequently isolated species were P. wickerhamii and P. pringsheimii, representing a third and a fourth of the strains, respectively. Additionally, three novel species were described, namely, P. fontanea, P. lentecrescens, and P. vistulensis. The high species diversity of the genus Prototheca may reflect the complexity of water ecosystems along with ecological and functional adaptations of the algae to such environments. For further investigations, the study provides a revised scheme for identification of all 18 Prototheca species currently recognized. IMPORTANCE The study investigates the occurrence of very rare and poorly studied microalgae of the genus Prototheca, potentially pathogenic to humans and animals, in different water environments. Given the potential hazard to human and animal health from exposure to water-inhabiting protothecae, the prevalence of the algae in aquatic habitats deserves an insightful examination. The study is the first since the 1980s to explore the aquatic habitat of Prototheca spp. and the first ever performed to do this by molecular methods. Although the Prototheca isolation rate was low, a high species diversity was observed. The algae appear to represent allochthonous microflora, brought into waterbodies from various anthropogenic sources. Large rivers of urbanized areas were the most Prototheca-abundant. The study provides a description of three new Prototheca species, namely, P. fontanea, P. lentecrescens, and P. vistulensis. The study also delivers a new identification scheme for all Prototheca species currently recognized.

Tryptophan, Kynurenine and Kynurenic Acid Concentrations in Milk and Serum of Dairy Cows with Prototheca Mastitis.

The aim of this work was to investigate serum and milk levels of tryptophan (TRP), kynurenine (KYN), and kynurenic acid (KYNA), as well as the activity of indoleamine 2,3-dioxygenase (IDO) in cows with mastitis due to Prototheca algae. The study was prompted by previous research showing a link between the KYN pathway of TRP metabolism and bovine mastitis of bacterial etiology. The study was carried out over a 2-year period (2018-2019) and included quarter milk and serum samples collected from six dairy herds in Poland. The samples were obtained from healthy cows and cows with Prototheca mastitis of either clinical and subclinical manifestation, as determined upon direct measurement of the somatic cell count or indirectly by performing a California Mastitis Test on suspected quarters. Both TRP and KYN concentrations were significantly lower in milk of mastitic cows compared to healthy animals (0.8 vs. 8.72 µM, p = 0.001; 0.07 vs. 0.32 µM, p = 0.001, respectively). The difference in TRP and KYN concentrations in the sera of the two animal groups was much less pronounced (25.55 vs. 27.57 µM, 3.03 vs. 3.56 nM, respectively). The concentration of KYNA was almost at the same level in milk (1.73 vs. 1.70 nM) and in serum (80.47 vs. 75.48 nM) of both mastitic and healthy cows. The data showed that the level of TRP and its metabolites in serum was conspicuously higher compared to milk in all cows under the study. The activity of IDO was significantly higher in milk of cows with Prototheca mastitis compared to healthy animals (71.4 vs. 40.86, p < 0.05), while in serum it was pretty much the same (135.94 vs. 124.98, p > 0.05). The IDO activity differed significantly between serum and milk both for mastitic (135.94 vs. 71.4, p < 0.05) and healthy cows (124.98 vs. 40.86, p < 0.001). In conclusion, low values of TRP and KYN concentrations or elevated IDO activity in milk samples might be used as markers of mastitis due to infectious causes, including Prototheca spp.

Prototheca-ID: a web-based application for molecular identification of Prototheca species.

The genus Prototheca houses unicellular, achlorophyllous, yeast-like algae, widely distributed in the environment. Protothecae are the only known plants that have repeatedly been reported to infect vertebrates, including humans. Although rare, protothecosis can be clinically demanding, with an unpredictable and treatment-resistant behavior. Accurate identification of Prototheca species relies upon DNA sequence-based typing of the mitochondrially encoded CYTB gene. However, no bioinformatic tool for the processing and analyzing of protothecal sequence data exists. Moreover, currently available sequence databases suffer from a limited number of records and lack of or flawed sequence annotations, making Prototheca identification challenging and often inconclusive. This report introduces the Prototheca-ID, a user-friendly, web-based application providing fast and reliable speciation of Prototheca isolates. In addition, the application offers the users the possibility of depositing their sequences and associated metadata in a fully open Prototheca-ID database, developed to enhance research integrity and quality in the field of Protothecae and protothecosis. Database URL: The Prototheca-ID application is available at https://prototheca-id.org.

The first outbreak of methicillin-resistant Staphylococcus aureus in dairy cattle in Poland with evidence of on-farm and intrahousehold transmission.

Staphylococcus aureus is a widely recognized pathogen responsible for many serious diseases in both humans and animals. It is also one of the major causative agents of bovine mastitis. Methicillin-resistant S. aureus (MRSA), although relatively rare in this pathology, has been increasingly reported in livestock animals, mainly in pigs, but also cattle, sheep, and poultry. The recent emergence of livestock-associated (LA-)MRSA is cause for an immediate public health concern due to the risk of zoonotic transmission to humans, and is of particular concern for people who work in animal husbandry or have prolonged contact with livestock animals. This study reports on the first LA-MRSA outbreak in dairy cattle and the first probable case of MRSA transmission between humans and cows in Poland. A single dairy farm located in Eastern Poland was monitored on a regular basis for the occurrence of mastitis. Over a 1-yr study period, 717 quarter-milk samples from 583 cows were collected and examined microbiologically. A total of 5 MRSA isolates from as many cows with subclinical mastitis were cultured. They all belonged to the same outbreak, given a 2-mo time window in which they were identified. During the outbreak, 24 oral and nasal swabs were voluntarily taken from 6 people: a milker, a veterinarian, and 4 members of the veterinarian's family. Eight swabs from a milker, veterinarian, and 2 family members yielded positive MRSA cultures. All MRSA isolates were genotyped with a combination of multiple-locus variable number tandem repeat analysis, multilocus sequence typing, and staphylococcal protein A gene (spa) typing. Eleven bovine (n = 5; 5 cases) and human (n = 6; 4 cases) isolates showed an identical drug-susceptibility profile and were indistinguishable upon multiple-locus variable number tandem repeat analysis (pattern A), multilocus sequence typing (ST398) and spa (t034) typing. The results of this study provide the evidence of transmission of MRSA between humans and cows, and between humans in the family setting. This work, despite being a preliminary investigation, underscores the risk of intra- and interspecies transmission of LA-MRSA and urges enhancement of the existing biosecurity measures aimed at preventing MRSA (and other milk pathogens) spread at both the farm- and household levels.