Genomic epidemiology of antifungal resistance in human and avian isolates of Candida albicans: a pilot study from the One Health perspective.

Stress-induced genomic changes in Candida albicans contribute to the adaptation of this species to various environmental conditions. Variations of the genome composition of animal-origin C. albicans strains are largely unexplored and drug resistance or other selective pressures driving the evolution of these yeasts remained an intriguing question. Comparative genome analysis was carried out to uncover chromosomal aneuploidies and regions with loss of heterozygosity (LOH), two mechanisms that manage genome plasticity. We detected aneuploidy only in human isolates. Bird-derived isolates showed LOH in genes commonly associated with antifungal drug resistance similar to human isolates. Our study suggests that environmental fungicide usage might exert selective pressure on C. albicans infecting animals, thus contributing to the spread of potentially resistant strains between different hosts.

Metagenomic Identification of Novel Eukaryotic Viruses with Small DNA Genomes in Pheasants.

A panel of intestinal samples collected from common pheasants (Phasianus colchicus) between 2008 and 2017 was used for metagenomic investigation using an unbiased enrichment protocol and different bioinformatic pipelines. The number of sequence reads in the metagenomic analysis ranged from 1,419,265 to 17,507,704 with a viral sequence read rate ranging from 0.01% to 59%. When considering the sequence reads of eukaryotic viruses, RNA and DNA viruses were identified in the samples, including but not limited to coronaviruses, reoviruses, parvoviruses, and CRESS DNA viruses (i.e., circular Rep-encoding single-stranded DNA viruses). Partial or nearly complete genome sequences were reconstructed of at least three different parvoviruses (dependoparvovirus, aveparvovirus and chaphamaparvovirus), as well as gyroviruses and diverse CRESS DNA viruses. Generating information of virus diversity will serve as a basis for developing specific diagnostic tools and for structured epidemiological investigations, useful to assess the impact of these novel viruses on animal health.

Development of molecular assays for the analysis of genetic relationships of Mycoplasma iowae.

Mycoplasma iowae is a worldwide spread and economically important avian pathogen that mostly infects turkeys. Currently, multi-locus sequence typing (MLST) serves as the gold standard method for strain identification in M. iowae. However, additional robust genotyping methods are required to effectively monitor M. iowae infections and conduct epidemiological investigations. The first aim of this study was to develop genotyping assays with high resolution, that specifically target M. iowae, namely a multiple-locus variable number of tandem-repeats analysis (MLVA) and a core genome multi-locus sequence typing (cgMLST) schema. The second aim was the determination of relationships among a diverse selection of M. iowae strains and clinical isolates with a previous and the newly developed assays. The MLVA was designed based on the analyses of tandem-repeat (TR) regions in the six serotype reference strains (I, J, K, N, Q and R). The cgMLST schema was developed based on the coding sequences (CDSs) common in 95% of the examined 99 isolates. The samples were submitted for a previously published MLST assay for comparison with the developed methods. Out of 94 TR regions identified, 17 alleles were selected for further evaluation by PCR. Finally, seven alleles were chosen to establish the MLVA assay. Additionally, whole genome sequence analyses identified a total of 676 CDSs shared by 95% of the isolates, all of which were included into the developed cgMLST schema. The MLVA discriminated 19 distinct genotypes (GT), while with the cgMLST assay 79 sequence types (ST) could be determined with Simpson's diversity indices of 0.810 (MLVA) and 0.989 (cgMLST). The applied assays consistently identified the same main clusters among the diverse selection of isolates, thereby demonstrating their suitability for various genetic analyses and their ability to yield congruent results.

Molecular epidemiology of Candida albicans infections revealed dominant genotypes in waterfowls diagnosed with esophageal mycosis.

Fungal infections of animals could yield significant economic losses, especially in the poultry industry, due to their adverse effects on growth, feed intake, digestion, and reproduction. Previous investigations showed that Candida albicans plays the main etiological role in the esophageal mycosis of birds. In this study, we used multilocus sequence typing (MLST) to determine the population structure and molecular epidemiology of C. albicans isolated from geese and ducks in Hungary. Interestingly, only three known genotypes were identified among investigated flocks, namely, diploid sequence type (DST) 840, DST 656, and DST 605, suggesting the intra-species transmission of these genotypes. Additionally, two novel allele combinations (new DSTs) were found that have not been previously submitted to the MLST database. Phylogenetic analysis of isolates revealed a close relationship between DST 656 and DST 605 as well as between the two newly identified genotypes (designated DST 3670 and DST 3671). Although isolates from birds belonged to minor clades in contrast with most human isolates, no species-specificity was observed. Poultry-derived isolates were group founders or closely related to group founders of clonal complexes, suggesting that C. albicans is exposed to lesser selective pressure in animal hosts. The increasing number of genetic information in the C. albicans MLST database could help to reveal the epidemiological characteristics and evolutionary pathways that are essential for disease prevention strategies.

Marked Genotype Diversity among Reoviruses Isolated from Chicken in Selected East-Central European Countries.

The concern that the vaccines currently used against Avian orthoreovirus (ARV) infections are less efficient in the field justifies the need for the close monitoring of circulating ARV strains. In this study, we collected necropsy samples from various chicken breeds and tested for ARV by virus isolation, RT-PCR assay and sequence analysis. ARVs were isolated from birds showing runting-stunting syndrome, uneven growth, lameness or increased mortality, with relative detection rates of 38%, 35%, 6% and 25%, respectively. Partial σC gene sequences were determined for nearly 90% of ARV isolates. The isolates could be classified into one of the major genetic clusters. Interestingly, cluster 2 and cluster 5 were isolated from vaccinated broiler breeders, while clusters 1 to 4 were isolated from unvaccinated broilers. The isolates shared less than 75% amino acid identities with the vaccine strains (range, 44.3-74.6%). This study reaffirms the global distribution of the major genetic clusters of ARVs in chicken. The diversity of ARV strains isolated from unvaccinated broilers was greater than those detected from vaccinated animals, however, the relative importance of passive and active immunity on the selection of novel strains in different chicken breeds needs to be better understood.

Genome stability assessment of PRRS vaccine strain with new ARTIC-style sequencing protocol.

A tiling amplicon sequencing protocol was developed to analyse the genome sequence stability of the modified live PRRSV vaccine strain, Porcilis MLV. The backbone of the ARTIC-style protocol was formed by 34 individual primer pairs, which were divided into two primer pools. Primer pairs were designed to amplify 532 to 588 bp fragments of the corresponding genomic region. The amplicons are suitable for sequencing on Illumina DNA sequencers with available 600-cycle sequencing kits. The concentration of primer pairs in the pools was optimized to obtain a balanced sequencing depth along the genome. Deep sequencing data of three vaccine batches were also analysed. All three vaccine batches were very similar to each other, although they also showed single nucleotide variations (SNVs) affecting less than 1 % of the genome. In the three vaccine strains, 113 to 122 SNV sites were identified; at these sites, the minority variants represented a frequency range of 1 to 48.7 percent. Additionally, the strains within the batches contained well-known length polymorphisms; the genomes of these minority deletion mutants were 135 to 222 bp shorter than the variant with the complete genome. Our results show the usefulness of ARTIC-style protocols in the evaluation of the genomic stability of PRRS MLV strains.

Animal Models Used in Monkeypox Research.

Monkeypox is an emerging zoonotic disease with a growing prevalence outside of its endemic area, posing a significant threat to public health. Despite the epidemiological and field investigations of monkeypox, little is known about its maintenance in natural reservoirs, biological implications or disease management. African rodents are considered possible reservoirs, although many mammalian species have been naturally infected with the monkeypox virus (MPXV). The involvement of domestic livestock and pets in spillover events cannot be ruled out, which may facilitate secondary virus transmission to humans. Investigation of MPXV infection in putative reservoir species and non-human primates experimentally uncovered novel findings relevant to the course of pathogenesis, virulence factors and transmission of MPXV that provided valuable information for designing appropriate prevention measures and effective vaccines.

Development and evaluation of temperature-sensitive Mycoplasma anserisalpingitidis clones as vaccine candidates.

Mycoplasma anserisalpingitidis is economically the most important pathogenic Mycoplasma species of waterfowl in Europe and Asia. The lack of commercially available vaccines against M. anserisalpingitidis had prompted this study with the aim to produce temperature-sensitive (ts+) clones as candidates for an attenuated live vaccine. The production of ts+ clones was performed by N-methyl-N'-nitro-N-nitrosoguanidine (NTG)-induced mutagenesis of Hungarian M. anserisalpingitidis field isolates. The clones were administered via eye-drop and intracloacally to 33-day-old geese. Colonization ability was examined by PCR and isolation from the trachea and cloaca, while the serological response of the birds was tested by ELISA. Pathological and histopathological examinations were performed in the eighth week after inoculation. Whole-genome sequence (WGS) analysis of the selected clone and its parent strain was also performed. NTG-treatment provided three ts+ mutants (MA177/1/11, MA177/1/12, MA271). MA271 was detected at the highest rate from cloacal (86.25%) and tracheal (30%) samples, while MA177/1/12 and MA271 elicited remarkable serological responses with 90% of the birds showing seroconversion. Re-isolates of MA271 remained ts+ throughout the experiment. Based on these properties, clone MA271 was found to be the most promising vaccine candidate. WGS analysis revealed 59 mutations in the genome of MA271 when compared to its parent strain, affecting both polypeptides involved in different cellular processes and proteins previously linked to bacterial fitness and virulence. Although further studies are needed to prove that MA271 is in all aspects a suitable vaccine strain, it is expected that this ts+ clone will contribute to the control of M. anserisalpingitidis infection.RESEARCH HIGHLIGHTS Three M. anserisalpingitidis ts+ vaccine candidates were produced by NTG-mutagenesis.Clone MA271 was able to colonize geese and induce a serological response.MA271 re-isolates remained ts+ during the 8-week-long experiment.WGS analysis revealed 59 mutations in the genome of MA271.

Molecular Phylogenetic Analysis of Candida krusei.

Revealing the phylogenetic relationships of Candida krusei strains (sexual form Pichia kudriavzevii) is a prerequisite for understanding the evolution of its virulence-associated mechanisms and ecological lifestyles. Molecular phylogenetic analyses based on entire internal transcribed spacer region (ITS) and multilocus sequence typing (MLST) data were carried out with sequences available in public databases and Hungarian isolates from animals obtained for the study. The ITS haplotype network yielded a high frequency haplotype at the centre of the network (H1; n = 204) indicating that various selective pressure might resulted in population expansion from H1. MLST analysis identified three new genotypes among animal-derived isolates, therefore overall 203 sequence types were investigated to determine the population structure of C. krusei. The most commonly encountered sequence types were ST 17 and ST 67. Phylogenetic analyses showed diverse genetic construction of C. krusei population. Evidence of potential recombination events were also observed that might play some role in high intraspecies genetic variability among strains, however, the limited data of C. krusei genotypes from different countries prevented us to identify accurate evolutionary routes of commensal and pathogenic strains or species-specific lineages. Further expansion of C. krusei MLST database may promote the better understanding of the mixed evolutionary history of this species.

COVID-19-Associated Fungal Infections: An Urgent Need for Alternative Therapeutic Approach?

Secondary fungal infections may complicate the clinical course of patients affected by viral respiratory diseases, especially those admitted to intensive care unit. Hospitalized COVID-19 patients are at increased risk of fungal co-infections exacerbating the prognosis of disease due to misdiagnosis that often result in treatment failure and high mortality rate. COVID-19-associated fungal infections caused by predominantly Aspergillus and Candida species, and fungi of the order Mucorales have been reported from several countries to become significant challenge for healthcare system. Early diagnosis and adequate antifungal therapy is essential to improve clinical outcomes, however, drug resistance shows a rising trend highlighting the need for alternative therapeutic agents. The purpose of this review is to summarize the current knowledge on COVID-19-associated mycoses, treatment strategies and the most recent advancements in antifungal drug development focusing on peptides with antifungal activity.

Phylogeny of Hungarian EBLV-1 strains using whole-genome sequence data.

European bat lyssavirus 1 (EBLV-1) is a widespread lyssavirus across Europe, whose epizootic cycle is linked to a few bat species. Occasionally, EBLV-1 infection may occur in domestic animals and humans. EBLV-1 can be classified into two subtypes, where subtype EBLV-1a shows a wide geographic distribution between France and Russia whereas subtype EBLV-1b is distributed between Spain and Poland. In this study, we determined the genome sequence of two recent EBLV-1a strains detected in Hungary and analysed their adaptive evolution and phylodynamics. The data set that included 100 EBLV-1 genome sequences identified positive selection at selected sites in genes coding for viral proteins (N, codon 18; P, 141 and 155; G, 244 and 488; L, 168, 980, 1597 and 1754). A major genetic clade containing EBLV-1a isolates from Hungary, Slovakia, Denmark and Poland was estimated to have diverged during the 19th century whereas the divergence of the most recent ancestor of Hungarian and Slovakian isolates dates back to 1950 (time span, 1930 to 1970). Phylogeographic analysis of the EBLV-1a genomic sequences demonstrated strong evidence of viral dispersal from Poland to Hungary. This new information indicates that additional migratory flyways may help the virus spread, a finding that supplements the general theory on a west-to-east dispersal of EBLV-1a strains. Long-distance migrant bats may mediate the dispersal of EBLV-1 strains across Europe; however, structured surveillance and extended genome sequencing would be needed to better understand the epizootiology of EBLV-1 infections in Europe.

Unexpected Diversity of Yeast Species in Esophageal Mycosis of Waterfowls.

This study was performed to evaluate the diversity and prevalence of yeasts associated with esophageal mycosis in domestic ducks and geese. Fungi were isolated from esophageal lesions of dead animals sent for microbiologic laboratory diagnosis. Species identification using a culture-dependent method was carried out by sequencing of the internal transcribed spacer (ITS)1-5.8S rRNA-ITS2 region. The most frequently isolated yeast was Candida albicans (43.1%) followed by Saccharomyces cerevisiae (17.6%), Candida kefyr (11.7%), Kazachstania bovina (11.7%), Candida lambica (3.9%), and single isolates (1.9%) representing Candida inconspicua, Candida rugosa, Candida pelliculosa, Candida krusei, Magnusiomyces capitatus, and Trichosporon asahii. Our results indicate that a number of potentially pathogenic yeast species can be isolated from esophageal mycosis of waterfowls, but additional studies are needed to make conclusions regarding their possible etiologic role in disease.

Phylogenetic Analysis of Lednice Orthobunyavirus.

Lednice virus (LEDV) has been detected in Culex modestus mosquitoes in several European countries within the last six decades. In this study, phylogenetic analyses of the complete genome segments confirm that LEDV belongs to the Turlock orthobunyavirus (Orthobunyavirus, Peribunyaviridae) species and is closely related to Umbre, Turlock, and Kedah viruses.

Novel Orthobunyavirus Causing Severe Kidney Disease in Broiler Chickens, Malaysia, 2014-2017.

During 2014-2017, we isolated a novel orthobunyavirus from broiler chickens with severe kidney lesions in the state of Kedah, Malaysia; we named the virus Kedah fatal kidney syndrome virus (KFKSV). Affected chickens became listless and diarrheic before dying suddenly. Necropsies detected pale and swollen kidneys with signs of gout, enlarged and fragile livers, and pale hearts. Experimental infection of broiler chickens with KFKSV reproduced the disease and pathologic conditions observed in the field, fulfilling the Koch's postulates. Gene sequencing indicated high nucleotide identities between KFKSV isolates (99%) and moderate nucleotide identities with the orthobunyavirus Umbre virus in the large (78%), medium (77%), and small (86%) genomic segments. KFKSV may be pathogenic for other host species, including humans.

The in vitro and in vivo efficacy of fluconazole in combination with farnesol against Candida albicans isolates using a murine vulvovaginitis model.

Farnesol is a quorum-sensing molecule that inhibits biofilm formation in Candida albicans. Previous in vitro data suggest that, in combination with certain antifungals, farnesol may have an adjuvant anti-biofilm agent. However, the in vivo efficacy of farnesol is very questionable. Therefore, the in vitro and in vivo activity of fluconazole combined with farnesol was evaluated against C. albicans biofilms using fractional inhibitory concentration index (FICI) determination, time-kill experiments and a murine vulvovaginitis model. The median biofilm MICs of fluconazole-sensitive C. albicans isolates ranged between 4 -> 512 mg/L and 150-300 µM for fluconazole and farnesol, respectively. These values were 512 -> 512 mg/L and > 300 µM for fluconazole-resistant clinical isolates. Farnesol decreased the median MICs of fluconazole by 2-64-fold for biofilms. Based on FICI, synergistic interaction was observed only in the case of the sessile SC5314 reference strain (FICIs: 0.16-0.27). In time-kill studies, only the 512 mg/L fluconazole and 512 mg/L fluconazole + 75 µM farnesol reduced biofilm mass significantly at each time point in the case of all isolates. The combination reduced the metabolic activity of biofilms for all isolates in a concentration- and time-dependent manner. Our findings revealed that farnesol alone was not protective in a murine vulvovaginitis model. Farnesol was not beneficial in combination with fluconazole for fluconazole-susceptible isolates, but partially increased fluconazole activity against one fluconazole-resistant isolate, but not the other one.

Killing Rates of Caspofungin in 50 Percent Serum Correlate with Caspofungin Efficacy Against Candida albicans in a Neutropenic Murine Model.

Previous studies suggested that caspofungin dose escalation against Candida species is more beneficial than currently used lower daily doses. Thus, we determined in vitro and in vivo activity of caspofungin against six wild-type C. albicans clinical isolates, the ATCC 10231 strain and an echinocandin resistant strain. MIC ranges of clinical isolates in RPMI-1640 with and without 50% serum were 0.125-0.25 and 0.015-0.06 mg/L, respectively. Two and three isolates showed paradoxical growth in MIC and time-kill tests, respectively, in RPMI-1640 but not in 50% serum. Caspofungin killing rate (k) in RPMI-1640 at 1 mg/L was higher than at 16 and 32 mg/L for all isolates (p<0.001). Killing rates for five of six isolates were concentration independent between 1-32 mg/L in 50% serum (p>0.05 for all comparisons), but for one isolate k value at 32 mg/L was significantly lower than at 1-16 mg/L. Although k values at 1-32 mg/L showed a great variability in 50% serum (the lowest and highest k value ranges were 0.085-0.109 and 0.882-0.985 1/h, respectively), daily 3, 5 and 15 mg/kg caspofungin was effective in a neutropenic murine model against all isolates, without significant differences between the effective doses. This study confirms that paradoxical growth does not affect the in vivo efficacy of caspofungin. We demonstrated that dose escalation did not increase the efficacy of caspofungin against C. albicans either in vitro or in vivo. These results are in concordance with the clinical experience that efficacy of echinocandins does not increase at larger doses.

Effect of caspofungin and micafungin in combination with farnesol against Candida parapsilosis biofilms.

The in vitro activities of caspofungin and micafungin were determined with and without farnesol against Candida parapsilosis biofilms. Drug interactions were examined using the XTT colorimetric assay-based broth microdilution chequerboard method. Drug-drug interactions were assessed utilising the FICI, Bliss independence models and time-kill experiments. Median sessile MICs of five C. parapsilosis clinical isolates ranged between 32-256 mg/L, 16-512 mg/L and >300 µM for caspofungin, micafungin and farnesol, respectively. Median MICs for caspofungin and micafungin in combination with farnesol showed 8-64- and 4-64-fold decreases, respectively. Paradoxical growth noticed with both echinocandins was eliminated by farnesol. Based on FICIs for sessile clinical isolates, synergism was observed for caspofungin (range of median FICIs, 0.155-0.5) and micafungin (range of median FICIs, 0.093-0.5). Concordantly, MacSynergy analysis and global fitting of non-linear regression based on a Bliss independence models also showed synergism for caspofungin and micafungin. In line with FICI findings and the Bliss independence model, synergistic interactions were confirmed by time-kill experiments. The metabolic activity of fungal cells was significantly inhibited by caspofungin+farnesol at all three tested combinations (4 mg/L+75 µM, 8 mg/L+75 µM and 16 mg/L+75 µM) between 3 and 24 h compared with the control (P<0.05-0.001). Significant inhibition was observed for micafungin+farnesol between 3 and 12h (P<0.001) but not at 24 h. Despite the favourable effect of farnesol in combination with echinocandins, further in vivo studies are needed to confirm its therapeutic advantage in catheter-associated infections caused by C. parapsilosis.

Dose escalation studies with caspofungin against Candida glabrata.

Echinocandins are recommended as first-line agents against invasive fungal infections caused by Candida glabrata, which still carry a high mortality rate. Dose escalation of echinocandins has been suggested to improve the clinical outcome against C. glabrata. To address this possibility, we performed in vitro and in vivo experiments with caspofungin against four WT C. glabrata clinical isolates, a drug-susceptible ATCC 90030 reference strain and two echinocandin-resistant strains with known FKS mutations. MIC values for the clinical isolates in RPMI 1640 were ≤ 0.03 mg l(-1 ) but increased to 0.125-0.25 mg l(-1 )in RPMI 1640+50% serum. In RPMI 1640+50% serum, the replication of C. glabrata was weaker than in RPMI 1640.Caspofungin in RPMI 1640 at 1 and 4 mg l(-1) showed a fungicidal effect within 7 h against three of the four clinical isolates but was only fungistatic at 16 and 32 mg l(-1) (paradoxically decreased killing activity). In RPMI 1640+50% serum, caspofungin at ≥ 1 mg l(-1) was rapidly fungicidal (within 3.31 h) against three of the four isolates. In a profoundly neutropenic murine model, all caspofungin doses (1, 2, 3, 5 and 20 mg kg(-1) daily) decreased the fungal tissue burdens significantly (P < 0.05-0.001) without statistical differences between doses, but the mean fungal tissue burdens never fell below 105 cells (g tissue)(-1). The echinocandin-resistant strains were highly virulent in animal models and all doses were ineffective. These results confirm the clinical experience that caspofungin dose escalation does not improve efficacy.

Efficacy of single large doses of caspofungin in a neutropenic murine model against the "psilosis" group.

We compared the in vivo efficacy of single large dose of caspofungin to that of daily smaller caspofungin doses (with same cumulative doses) against C. albicans (echinocandin susceptible and resistant isolates) and the â€Å“psilosisâ€? group in a neutropenic murine model. Seven treatment groups were formed for C. orthopsilosis, C. metapsilosis and C. albicans (no treatment, 1, 2 and 3 mg/kg caspofungin daily for five days; single 5, 10 and 15 mg/kg caspofungin doses). For C. parapsilosis there were five treatment groups (no treatment, 3 and 4 mg/kg caspofungin daily for five days; single 15 and 20 mg/kg caspofungin). Tissue burdens of C. orthopsilosis and C. parapsilosis were significantly decreased by daily 3 mg/kg and 10 or 15 mg/kg single caspofungin doses (P<0.05-0.01) and daily 4 mg/kg and by single 15 and 20 mg/kg caspofungin doses (P<0.05-0.01), respectively. Against C. metapsilosis all treatment arms except the daily 1 mg/kg were effective (P<0.05-<0.001). Against C. albicans all treatment doses were effective. Neither daily 16 mg/kg nor single 80 mg/kg were effective against the resistant C. albicans strain. Higher doses and less frequent administration of caspofungin were comparable or sometimes superior to the lower, daily-dose regimen against the â€Å“psilosisâ€? group supporting further studies with this therapeutic strategy.