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.

Isolation of infectious microalga Prototheca wickerhamii from a carp (Cyprinus carpio) - a first confirmed case report of protothecosis in a fish.

Protothecosis is a rare infection caused by environmentally ubiquitous achlorophyllic microalgae of the genus Prototheca. Here, we describe a first case of protothecosis in a carp (Cyprinus carpio), which is at the same time the first case of protothecosis in a fish, confirmed by phenotype- and molecular-based methods, including PCR sequencing of the rDNA cluster and protein profiling using matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

A close-up on the epidemiology and transmission of multidrug-resistant tuberculosis in Poland.

Multidrug-resistant tuberculosis (MDR-TB) poses a serious challenge to the global control of the disease. The purpose of this study was to characterize MDR-TB patients from Poland and to determine the extent of MDR-TB disease attributable to recent transmission. The study included all 46 patients diagnosed with MDR-TB in Poland in 2004 and followed up for 6 years (until 2011). For each patient, sociodemographic and clinical characteristics, treatment outcomes, and bacteriological data were collected by the review of medical and laboratory records. Mycobacterium tuberculosis isolates from all patients were characterized using spoligotyping, mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) typing, IS6110 restriction fragment length polymorphism (RFLP) analysis, and sequencing analysis of drug resistance-associated loci (katG, mabA-inhA, rpoß, rpsL, and embB). The majority of patients were male (86.9%), 40-64 years of age (60.8%), with a history of TB treatment (84.8%), and producing smear-positive sputa (86.9%). Twenty-two (47.8%) patients suffered from concomitant diseases and 28 (60.8%) were alcohol abusers. Treatment outcome assessment revealed that 8 (17.4%) patients were cured or completed therapy, while 15 (32.6%) died of TB, 11 (23.9%) defaulted, 8 (17.4%) failed, and 1 (2.2%) was transferred and lost to follow-up. Upon genotyping, 10 (21.7%) isolates were allocated in four clusters. These were further subdivided by mutational profiling. Overall, in 6 (13%) patients, MDR-TB was a result of recent transmission. For 4 (8.7%) of these patients, a direct epidemiological link was established. The study shows that the transmission of MDR-TB occurs at a low rate in Poland. Of urgent need is the implementation of a policy of enforced treatment of MDR-TB patients in Poland.

Short communication: Antimicrobial susceptibility profiling and genotyping of Staphylococcus aureus isolates from bovine mastitis in Poland.

Staphylococcus aureus is the predominant causative agent of bovine mastitis, a disease that remains a major economic burden for the dairy industry worldwide. In this study, the antimicrobial resistance patterns and the genetic composition of 80 S. aureus mastitis isolates collected from 14 dairy farms in Eastern Poland were determined. Of the 10 antimicrobial agents evaluated, only testing for penicillin G produced drug resistance. As 41% of the S. aureus isolates were penicillin resistant, this drug along with other ß-lactamase-sensitive ß-lactams, should rather not be considered for the treatment of bovine mastitis caused by S. aureus. Upon genotyping, with a triplex PCR method, a total of 11 distinct PCR types were produced. The population structure of S. aureus isolates was highly clonal, with 1 predominant genotype circulating on each farm. The observed similarities in the genotype composition of S. aureus populations from geographically distant farms underscore the significance of interfarm transmission of S. aureus in Poland. This, in turn, argues for the establishment of a nationwide surveillance program for bovine mastitis due to this pathogen.

Chromoblastomycosis as an endemic disease in temperate Europe: first confirmed case and review of the literature.

This study reports a case of a 56-year-old white male, retired coal-miner, diagnosed with chromoblastomycosis lasting 20 years. The infection site was the burnt skin of the back. For many years the patient had not undertaken any treatment believing that the lesion had been a burn scar. A gradual increase in lesion size prompted the patient to start therapy. The diagnosis was made by histopathological examination and mycological culture. Identification of the causative agent at the species level was achieved by sequence analysis of the internal transcribed spacer (ITS) region and D1/D2 domains of the 26S rDNA. To our knowledge, this is the first documented case of chromoblastomycosis caused by Fonsecaea monophora in temperate Europe, outside the endemic area for the disease. This finding is highly significant for understanding the routes of infection of chromoblastomycosis and radically revises the traditional view of the natural ecology of the etiological agents of the disease.

Identification and differentiation of Trichophyton rubrum clinical isolates using PCR-RFLP and RAPD methods.

Trichophyton rubrum represents the most frequently isolated causative agent of superficial dermatophyte infections. Several genotyping methods have recently been introduced to improve the delineation between pathogenic fungi at both the species and the strain levels. The purpose of this study was to apply selected DNA fingerprinting methods to the identification and strain discrimination of T. rubrum clinical isolates. Fifty-seven isolates from as many tinea patients were subjected to species identification by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) analysis and strain differentiation using a randomly amplified polymorphic DNA (RAPD) method, with two primers designated 1 and 6. Using PCR-RFLP, 55 of the isolates studied were confirmed to be T. rubrum. Among those, a total of 40 and five distinct profiles were obtained by RAPD with primers 1 and 6, respectively. The combination of profiles from both RAPD assays resulted in 47 genotypes and an overall genotypic diversity rate of 85.4%. A dendrogram analysis performed on the profiles generated by RAPD with primer 1 showed most of the isolates (87.3%) to be genetically related. PCR-RFLP serves as a rapid and reliable method for the identification of T. rubrum species, while the RAPD analysis is rather a disadvantageous tool for T. rubrum strain typing.

Molecular characterization of Polish Prototheca zopfii mastitis isolates and first isolation of Prototheca blaschkeae in Poland.

Bovine mastitits caused by the colorless, yeast-like alga Prototheca zopfii is a serious and complex condition that results in heavy economic losses in the dairy industry, both through a substantial reduction in milk production and culling of infected animals. Based on the 18S rDNA sequence analysis, genotype-specific PCR assays have recently been developed to differentiate within the species P. zopfii three distinct P. zopfii genotypes (1-3), of which P. zopfii genotype 3 has been considered a new species P. blaschkeae sp. nov. The purpose of this study was to employ the newly-devised molecular approach for the detection of the two P. zopfii genotypes and P. blaschkeae sp. nov. among bovine mastitis isolates from Poland. This study is the first to provide molecular characterization of Polish P. zopfii mastitis isolates. It also gives the first description of bovine mammary protothecosis due to P. blaschkeae in Poland, as evidenced by genotypical, microbiological, and electron microscopy findings.

Molecular analysis of drug-resistant Mycobacterium tuberculosis isolates collected in central Poland.

In total, 48 Mycobacterium tuberculosis clinical isolates from 48 patients with drug-resistant pulmonary tuberculosis (TB) in central Poland during 2004 were analysed by spoligotyping and IS6110-Mtb1/Mtb2 PCR. Twelve (25%) isolates were clustered by spoligotyping in combination with IS6110-Mtb1/Mtb2 PCR. Patients from whom these isolates were obtained were assumed to have developed TB as a result of recent transmission. Spoligotyping, used alone, overestimated the number of clustered isolates. However, combined use of spoligotyping and IS6110-Mtb1/Mtb2 PCR was an efficient approach for revealing clonal relatedness among M. tuberculosis isolates.