A global view on fungal infections in humans and animals: infections caused by dimorphic fungi and dermatophytoses.

Fungal infections are still underappreciated and their prevalence is underestimated, which renders them a serious public health problem. Realistic discussions about their distribution, symptoms, and control can improve management and diagnosis and contribute to refinement of preventive actions using currently available tools. This article represents an overview of dermatophytes and endemic fungi that cause infections in humans and animals. In addition, the impact of climate change on the fungal spread is discussed. The endemic fungal infections characterized in this article include coccidioidomycosis, histoplasmosis, blastomycosis, lobomycosis, emergomycosis and sporotrichosis. Moreover the geographic distribution of these fungi, which are known to be climate sensitive and/or limited to endemic tropical and subtropical areas, is highlighted. In turn, dermatophytes cause superficial fungal infections of skin, hairs and nails, which are the most prevalent mycoses worldwide with a high economic burden. Therefore, the possibility of causing zoonoses and reverse zoonoses by dermatophytes is highly important. In conclusion, the article illustrates the current issues of the epidemiology and distribution of fungal diseases, emphasizing the lack of public programmes for prevention and control of these types of infection.

Are dogs and cats a reservoir of resistant and virulent Enterococcus faecalis strains and a potential threat to public health?

AIMS: The aim of the study was to assess resistance and virulence of Enterococcus faecalis isolated from the gastrointestinal tract of dogs and cats, analyse their genotypic variability and estimate the correlation between the occurrence of antimicrobial resistance, virulence determinants and genotypic profiles. METHODS AND RESULTS: The susceptibility of E. faecalis to penicillin, ampicillin, vancomycin, erythromycin, tetracycline, ciprofloxacin, gentamicin, streptomycin and kanamycin was determined by the broth microdilution method. The isolates were tested for the presence of selected genes encoding resistance to macrolides, tetracyclines, aminoglycosides and glycopeptides as well as genes encoding virulence factors. Genotyping was performed using the ADSRRS-fingerprinting method. The highest percentage of resistant strains was observed in relation to erythromycin (96%), ciprofloxacin (93%) and tetracycline (82%). High percentage of strains resistant to high-level aminoglycosides was noted (kanamycin-33%, gentamicin-29%, streptomycin-24%), as well as multidrug-resistant (78%). The genotypic analysis of E. faecalis showed high heterogeneity of genotypic profiles (37) correlating with some resistance profiles. The most common virulence genes amongst E. faecalis were efaAfs (93%), cpd, ccf and cob (86%). SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this study confirm that companion animals should be considered as a reservoir of E. faecalis carrying resistance and virulence determinants.

A global view on fungal infections in humans and animals: opportunistic infections and microsporidioses.

After cardiovascular diseases, infectious diseases are the second most common cause of death worldwide. Although these infections are caused mainly by viruses or bacteria, a systematically growing prevalence of human and animal opportunistic fungal infections is noticeable worldwide. More attention is being paid to this problem, especially due to the growing frequency of recalcitrant and recurrent mycoses. The latter are classically divided into superficial, which are the most common type, subcutaneous, and systemic. This work discusses opportunistic fungal pathogens without proven horizontal transmission between different animal species including humans and microsporidia as spore-forming unicellular parasites related to fungi; however, with a yet undetermined taxonomic position. The review also mentions aetiological agents, risk factors, epidemiology, geographical distribution, and finally symptoms characteristic for individual disease entities. This paper provides insight into fungal infections from a global perspective and simultaneously draws attention to emerging pathogens, whose prevalence is continuously increasing. Finally, this work also takes into consideration the correct nomenclature of fungal disease entities and the importance of secondary metabolites in the pathogenesis of fungal infections.

Assessment of the subtilisin gene profile in Trichophyton verrucosum isolated from human and animal dermatophytoses in two-stage multiplex PCR.

AIMS: Keratin is a fibrous and recalcitrant structural protein and the third most abundant polymer in nature after cellulose and chitin. Subtilisin-like proteases (SUB) are a group of serine endoproteases, coded by seven genes (SUB1-7), which decompose keratin structures and have been isolated from dermatophytes. Herein, we identified the SUB genes in 30 clinical isolates of Trichophyton verrucosum obtained from human and animal dermatophytosis as well as asymptomatic animal carriers. METHODS AND RESULTS: We designed and proposed a two-stage multiplex PCR technique to detect all seven genes encoding serine proteases in dermatophytes. The analysis revealed the presence SUB1 and SUB2 amplicons in all strains regardless of the host. In the group of isolates obtained from humans, all seven subtilisin genes were shown in 40% of the strains. In T. verrucosum from asymptomatic animals, none of the isolates showed the presence of all seven subtilisin genes, and only 30% had six genes. In turn, 10% of the isolates from symptomatic animals demonstrated all seven subtilisins amplicons. CONCLUSIONS: In conclusion, the severity of infection and ability of T. verrucosum to cause dermatophytosis in humans may not be related to specific genes but their accumulation and synergistic effects of their products. SIGNIFICANCE AND IMPACT OF THE STUDY: Dermatophytes are pathogenic filamentous fungi with capacity to attack keratinized structures such as skin, hair and nails, causing cutaneous superficial infections. Indeed, a biological characteristic of dermatophytes is their ability to invade keratin-rich tissues by producing enzymes. Various degrees of inflammatory responses can be induced exactly by the enzymes. Subtilisin-like proteases are endoproteases, which decompose keratin structures. Our study identifies SUB genes in clinical isolates of T. verrucosum obtained from human and animal dermatophytosis as well as asymptomatic animal carriers.

Detection and identification of dermatophytes based on currently available methods - a comparative study.

AIMS: Accurate identification of dermatophytes is essential for implementing appropriate antifungal treatment and epidemiological analysis. However, the limitations of conventional diagnostics are a frequently discussed topic, and new diagnostic techniques are constantly expanding. In this study, we assess the suitability of conventional diagnostic techniques in comparison to the real-time PCR assay and MALDI-TOF MS in detection and identification of dermatophytes. METHODS AND RESULTS: Strains included in this study were obtained from human and animals with symptomatic, and asymptomatic infection. A direct examination revealed that 31·7 and 60·9% of samples from symptomatic patients, and 25·7 and 60% from asymptomatic animals were positive, as shown by light and fluorescence microscopy respectively. In turn, dermatophytes were isolated from 90·2 and 71·4% of these samples. The pan-dermatophyte primers in real-time PCR assay facilitated detection in 85·3 and 82·9% of the symptomatic and asymptomatic dermatophytoses respectively. Additionally, species-specific PCR assays were positive in 70·7 and 37·1% of these samples. The MALDI-TOF MS analysis yielded positive results consistent with conventional techniques in 97·2 and 72% of symptomatic and asymptomatic infections respectively. CONCLUSIONS: Our study revealed that there is no universal diagnostic method that would be ideal in each of the cases considered. Nonetheless, conventional techniques are still the most effective and reliable tools for mycological diagnostics. SIGNIFICANCE AND IMPACT OF THE STUDY: Dermatologists and veterinarians have difficulties in making a diagnosis of dermatophytoses based only on observed symptoms of fungal infections, as they mimic symptoms of other dermatoses. In this context, a comparative analysis of the results of diagnostics performed using conventional methods and new technologies are crucial for implementing these pioneer methods into routine laboratory practice.

Major challenges and perspectives in the diagnostics and treatment of dermatophyte infections.

Dermatophytes are the aetiological factors of a majority of superficial fungal infections. What distinguishes them from other pathogenic filamentous fungi is their unique ability to degrade keratin. The remarkable ability of this group of fungi to survive in different ecosystems results from their morphological and ecological diversity as well as high adaptability to changing environmental conditions. Paradoxically, despite the progress in medicine, the prevalence of dermatophyte infections is increasing from year to year. At the beginning of the third millennium, practical diagnostic and therapeutic options are still very limited. This review focuses on understanding the major problems in this aspect of dermatophyte infections and indicates future strategies and perspectives for novel approaches to identification and drugs for elimination of dermatophytes. Particular importance is placed on development of a strategy for a diagnostic pathway and implementation of rapid and reliable diagnostics methods designed by international teams. Furthermore, among compounds that currently arouse great interest, representatives of terpenoids, alkaloids, saponins, flavonoids and essential oils deserve attention. Many of these compounds are undergoing clinical trials as potential antifungal agents, and future research should focus on attempts at determination of the applicability of tested substances. Finally, the advantages and disadvantages in implementation of new diagnostic paths and medicinal substances for routine use are indicated.

Changes in growth hormone secretion and leptin receptor mRNA expression under the influence of leptin and adrenocorticotropin in pituitary cells of early weaned ewe lambs.

Early weaning of ewe lambs strongly stimulates the hypothalamic-pituitary-adrenal axis and is associated with suppressed growth rate despite the increased food intake. At the same time, plasma leptin concentration increases only slightly or undetectably. To better understand this atypical interdependence among somatic stress, leptin, and lamb growth rate, we analyzed impact of leptin and/or adrenocorticotropic hormone (ACTH) on growth hormone (GH) secretion as well as the effect of ACTH on mRNA expression of two splice variants of leptin receptor (LEPRa, LEPRb) in pituitary cells isolated from early weaned ewe lambs. The GH secretion under the influence of leptin and/or ACTH depended on the timing of exposure and hormone concentration. After 6 - 30 h, GH secretion increased under 10-11 - 10-8 M leptin (P ≤ 0.05). However, after 24 - 30 h, GH secretion significantly increased only in cells exposed to both leptin and ACTH compared to culture with leptin only. Simultaneously, there was a significant (P ≤ 0.05) decrease in leptin receptor mRNA expression under the influence of ACTH at 10-8 - 10-6 M after 12 - 30 and 24 - 30 h for LEPRa and LEPRb, respectively. ACTH-related downregulation of LEPR mRNA was associated with a significant (P ≤ 0.05) reduction in leptin-stimulated GH secretion, also after 24 - 30 hours. Thus, the timing of ACTH exposure, followed by decreased leptin receptor mRNA, converged with the timing of decreased GH secretion under the influence of leptin with ACTH. The ACTH-induced downregulation of LEPR mRNA therefore may underlie the decrease in GH. These results show a direct role for leptin, ACTH, and leptin receptor expression in modulation of pituitary GH secretion in early weaned ewe lambs. During the early weaning-induced stress response, the ACTH-mediated decrease in sensitivity of pituitary cells to leptin may abolish a stimulatory effect of leptin on GH secretion and explain in part, the reduction in lamb growth rate.

Adropin, nesfatin-1 and angiotensin II receptor expression in the abdominal aorta in ovariectomized rats after nesfatin-1 treatment.

The aim of the research was to assess the effect of nesfatin-1 on the structure, flexibility parameters, and expression of adropin, nesfatin-1, and angiotensin II receptor type 1 (AT1R) in the abdominal aorta in ovariectomized rats. Fragments of aortas were collected after euthanasia of female sham-operated (CONT) and ovariectomized Wistar rats (EXP), which were administered intraperitoneal injection of physiological saline (CONT, n = 7; EXP-O, n = 7) or nesfatin-1 (EXP-N, n = 7) in an amount of 2 µg/kg b.w. once a day for 8 weeks. The samples of aortas were collected for measurement of elasticity as well as histomorphometric, immunohistochemical, FTIR, and Raman spectroscopy analysis. The ovariectomy caused a significant increase in the thickness of the total wall and its particular layers in the aorta, in comparison to the CONT and EXP-N groups. However, the ovariectomy led to a decrease in the amount of elastin, collagen (mature, immature collagen, collagen maturity ratio 1660 - 1690 cm-1), and amides, with a simultaneous increase in lipids, especially in the tunica intima-media of the abdominal aorta compared to the other groups. The use of nesfatin-1 significantly increased the amount of collagen, elastin and amides with a simultaneous decrease in the amount of lipids and the expression of AT1R, adropin and nesfatin-1 in the abdominal aorta of ovariectomized rats. In conclusion, our study showed that the ovariectomy surgery induced changes in the abdominal aorta wall characteristic for aging females. Application of nesfatin-1 may prevent the negative consequences in the vessel wall structure in females in conditions of estrogen deficiency and prevent atherosclerotic changes in the cardiovascular system.

Phenotypic characterization of enzymatic activity of clinical dermatophyte isolates from animals with and without skin lesions and humans.

AIMS: The pathogenesis of dermatophytoses is associated with the secretion of enzymes degrading the infected tissue components. Although many studies on enzymatic activity of dermatophytes have been conducted over the years, there have been no concrete proposals on the construction of the profile of enzymes characteristic of individual species, genus or ecological types of dermatophytes. The aim of this study was to assess the capability of clinical dermatophyte isolates from both symptomatic and asymptomatic animals and humans to produce different enzymes. METHODS AND RESULTS: Clinical isolates of 234 dermatophyte strains collected during routine examination of animal health were used in this study. The enzymatic production of keratinase, elastase, phospholipase, lipase, protease, DNase and gelatinase as well as the haemolytic activity were evaluated using specific test media. The overall degree of enzymatic activity of the analysed clinical isolates of the dermatophytes was 67%. All tested clinical isolates of different species of dermatophytes showed keratinase activity and 96% additionally exhibited phospholipase activity. The weakest activity among the tested enzymes was demonstrated for elastase and gelatinase. 83% of the isolates of the dermatophytes showed haemolytic activity. CONCLUSION: Our data indicate that clinical isolates of dermatophytes from different species produce enzymes with different levels of activities. SIGNIFICANT AND IMPACT OF THE STUDY: Profile of enzymes characteristic of individual species, genus or ecological types of dermatophytes is possibly dependent upon factors related to the host. The relationship between each enzyme and the occurrence of skin lesions in animals and humans or asymptomatic animal carriers varies on whether the infection is caused by Trichophyton mentagrophytes, Trichophyton verrucosum or Microsporum canis. Interestingly, only keratinase seems to be correlated with the appearance of dermatophyte infections, irrespective of the pathogen species, and elastase is a characteristic enzyme for dermatophyte strains infecting humans. Haemolysis seems to be dependent on host factors and is more common in the case of human dermatophyte isolates.

Evaluation of growth conditions and DNA extraction techniques used in the molecular analysis of dermatophytes.

AIMS: Recent molecular methods for diagnosis of superficial mycoses have determined the need for a rapid and easy method of extracting DNA. The aim of study was to determine growth conditions and techniques of DNA extraction for Microsporum canis, Trichophyton mentagrophytes and T. verrucosum. METHODS AND RESULTS: Samples were prepared of each of the DNA extraction methods (phenol-chloroform, CTAB and four different kits) for all of the incubation periods (4, 7 and 10 days) of the cultures on the solid and in the liquid medium. The highest DNA concentrations were obtained using the phenol-chloroform method. The concentration of DNA extracted with the CTAB method accounted for 62·21%, for kits it corresponded from 35·53 to 15·41%. The analysis of the DNA weight yield revealed the highest isolation efficiency of the phenol-chloroform method, 1 mg of mycelium yielded 223·8 µg DNA. Lower DNA yield (by 39·32%) was obtained with the CTAB method; in the case of kits by 68·46-85·32%. In most of the techniques, the DNA yield on the solid medium was higher. CONCLUSION: In summary, the highest DNA yield was noted in the 7-day cultures and extraction with the phenol-chloroform method. Importantly, the type of culture was not relevant for the diagnostic result. SIGNIFICANCE AND IMPACT OF THE STUDY: Most mycoses are caused by fungi that reside in nature. The severity of the infection depends on the pathogenic attributes, socioeconomic factors and local environmental conditions. Recent diagnosis increasingly relies on not only the clinical features. Molecular identifications have determined the need for a rapid and easy method of extracting DNA. Usually two factors have to be considered: maximize the DNA yield and ensure that the extracted DNA is susceptible to enzymatic reactions. These data suggest that phenol-chloroform methods and a 7-day culture period may be useful for validation and constitute the first step of molecular diagnosis of dermatophytes.

Antimicrobial activity of some plant extracts against bacterial pathogens isolated from faeces of red deer (Cervus elaphus).

Antibacterial activity is the most widely studied aspect of plant extracts. Antibiotics extensively produced and consumed in large quantities, have proved to be problematic due to various types of adverse effects. The development of bacterial resistance to currently available antibiotics has necessitated the search for new antibacterial agents. One of the alternative strategies for fighting antibiotic- resistant bacteria is the use of natural antimicrobial substances such as plant extracts. We tested the antimicrobial activity of nine extracts from different plants against pathogenic bacteria isolated from the faeces of red deer (Cervus elaphus). Selected bacteria commonly contaminated the natural environment and constitute a source of infection in other animals and humans. Extracts obtained from the following plants were tested: Hypericum perforatum L., Chamomilla recutita L., Achillea millefolium L., Salvia officinalis L., Thymus vulgaris L., Pinus sylvestris L., Mentha x piperita L., Valeriana officinalis L. and Foeniculum vulgare Mill. The highest degree of antibacterial properties was observed for Mentha x piperita L., narrower spectrum of activity possessed Hypericum perforatum L. Extracts of Achillea millefolium L. had the lowest spectrum of antibacterial activity. Our study confirms that many plant extracts shows in vitro antibacterial activity.

Determination of resistance and virulence genes in Enterococcus faecalis and E. faecium strains isolated from poultry and their genotypic characterization by ADSRRS-fingerprinting.

The aim of this study was to determine the antimicrobial resistance of E. faecalis and E. faecium strains isolated from poultry and to carry out genotypic characterization thereof with the ADSRRS-fingerprinting method (amplification of DNA fragments surrounding rare restriction sites) and analysis of the genetic relatedness between the isolates with different resistance and virulence determinants. Samples were collected from 70 4-week-old chickens and tested for Enterococcus. Minimum inhibitory concentrations of 11 antimicrobials were determined using the broth microdilution method. Detection of antibiotic resistance and virulence genes was performed using PCR, and molecular analysis was carried out using the ADSRRS-fingerprinting method. The highest percentage of strains was resistant to tetracycline (60.5%) and erythromycin (54.4%), and a large number exhibited high-level resistance to both kanamycin (42.1%) and streptomycin (34.2%). Among 8 genes encoding AME, the tested strains showed mainly the presence of [aph(3΄)-IIIa], [ant(6)-Ia], [aac(6΄)-Ie-aph(2΄΄)-Ia], and [ant(9)-Ia] genes. Phenotypic resistance to erythromycin was encoded in 98.4% strains by the ermB gene. Genotypic resistance to tetracycline in E. faecium was associated with the presence of tetM and tetL (respectively, in 95.5 and 57.7% of the isolates); in contrast, E. faecalis strains were characterized mainly by the presence of tetO (83.3%). The virulence profile was homogenous for all E. faecium strains and included only efaAfm and ccf genes. All E. faecalis strains exhibited efaAfs, gelE, and genes encoding sex pheromones. The strains tested exhibited 34 genotypic profiles. Comparative analysis of phenotypic and genotypic resistance and virulence profiles and confrontation thereof with the genotypes of the strains tested showed that strains assigned to a particular genotype have an identical phenotypic resistance profile and a panel of resistance and virulence genes. The results of this study confirm that poultry can be a reservoir of resistant E. faecium and E. faecalis strains with multiple combinations of resistance and virulence genes, whose specific panel determines not only phenotypic characteristics but also has a strong correlation with the genotypic profiles of the strains.

Experimental studies of microbial populations and incidence of zoonotic pathogens in the faeces of red deer (Cervus elaphus).

UNLABELLED: Wild animals can serve as hosts, amplifiers or reservoirs for various zoonotic diseases. Most species of deer in highly fragmented agricultural landscapes, search out maximum cover from intrusive human activity. Hence, the likelihood of zoonosis transmission is likely to increase the more humans and wildlife interact. In our study, we conducted a comparative analysis of bacteria isolated from the faeces of red deer (Cervus elaphus) living in their natural environment in south-western Poland and brought in from Hungary and Slovakia under a species reintroduction programme. The faecal bacterial flora from 120 specimens of deer were examined, with particular attention to potentially pathogenic agents. We isolated 458 micro-organisms, of which 13 (2·84%) were identified as EHEC (Enterohaemorrhagic Escherichia coli) strains, and of these one strain, produced the Shiga toxin. No strain was identified as having ESBL (Extended-Spectrum Beta-Lactamase) resistance. Other bacteria that are important in terms of the health of humans and animals included Yersinia enterocolitica (4, 0·67%) and Staphylococcus aureus (4, 0·67%), but without methicillin resistance, and Listeria monocytogenes (8, 1·75%). Of all the micro-organisms 138 (30·13%) were bacteria of the genus Enterococcus, including 12 (2·62%) of the species Enterococcus faecium. The results of the study indicate that red deer may play an important role in the environmental maintenance of zoonotic pathogens. SIGNIFICANCE AND IMPACT OF THE STUDY: A particularly important factor in the epidemiology of bacterial infections is the introduction of pathogens posing a risk to other animals and humans into the soil, plants and especially water, as contaminants together with faeces. Our study presents screening of potentially pathogenic bacteria in different populations of deer that were displaced under reintroduction programmes. Based on our own research and the literature data, it seems that wild ruminants play an important role in the maintenance of zoonotic pathogens and information about zoonoses from red deer will become increasingly important as deer populations continue to grow, especially in Europe.

Susceptibility testing of Aspergillus niger strains isolated from poultry to antifungal drugs--a comparative study of the disk diffusion, broth microdilution (M 38-A) and Etest methods.

The aim of this study was to determine the sensitivity of Aspergillus niger strains isolated from birds to available antifungal drugs using different in vitro assays--classical disk diffusion, Etest and broth microdilution NCCLS/CLSI M 38-A. The study material consisted of about 2.000 swabs and samples from different species of birds. A. niger (n=10) was accounted for 6.81% of the total pool of strains isolated. Determinations were made for 13 antifungal drugs using the disk diffusion method. The A. niger exhibited high susceptibility to enilconazole, terbinafine, voriconazole, tioconazole and ketoconazole, low susceptibility to clotrimazole, miconazole and nystatin, and resistance to amphotericin B, itraconazole, pimaricin, fluconazole and 5-fluorocytosine. Minimum inhibitory concentration (MIC) was determined for 9 antifungal drugs using the micromethod of duplicate serial dilutions in a liquid medium. A. niger strains were most susceptible to enilconazole and voriconazole. MIC ranged from 0.0625 to 0.5 microg/ml for enilconazole, with MIC90-0.5 microg/ml and MIC50-0.125 microg/ml. The corresponding values for voriconazole were 0.25-1 microg/ml, 1 microg/ml and 0.5 microg/ml. MIC for amphotericin B and terbinafine ranged from 0.5 to 4 microg/ml, while the values for the remaining drugs were highly varied. MIC was measured by the gradient diffusion method using Etest for 5 antifungal drugs: amphotericin B, fluconazole, itraconazole, ketoconazole and voriconazole. By far the highest susceptibility was obtained in the case of voriconazole, with MIC ranging from 0.0625 to 1 microg/ml. MIC for amphotericin B ranged from 0.25 to 4 microg/ml, for itraconazole and ketoconazole ranging from 0.5 to 16 microg/ml. Methods available for this purpose are not always applicable in field conditions. The present results indicate that the Etest technique, due to its high percentage of agreement with the M 38-A microdilution method, should find application in medical and veterinary practice.