Four uncommon clinical fungi, Lodderomyces elongisporus, Kodamaea ohmeri, Cyberlindnera fabianii and Wickerhamomyces anomalus, isolated in superficial samples from Côte d'Ivoire.

AIMS: The rare yeast species Lodderomyces elongisporus, Kodamaea ohmeri, Cyberlindnera fabianii, and Wickerhamomyces anomalus are increasingly implicated in severe mycoses in immunocompromised patients. This study aimed to assess the prevalence of uncommon yeast species in Côte d'Ivoire. METHODS: The yeast isolates from superficial samples, mainly vaginal swabs, were collected at the Pasteur Institute of Abidjan in a study on the molecular epidemiology of clinical yeast species. Identification relied on MALDI-TOF MS and ITS sequence analysis. Antifungal susceptibility testing was performed using the CLSI method. RESULTS: Of the 315 strains analysed from 227 outpatients, 14 belonged to 4 uncommon species: Lodderomyces elongisporus, Kodamaea ohmeri, Cyberlindnera fabianii, and Wickerhamomyces anomalus. None exhibited elevated fluconazole, amphotericin B, caspofungin, ketoconazole, or flucytosin MIC. CONCLUSIONS: The presence of these rare yeasts represents a risk in immunocompromised people. Their adequate and timely identification is a priority. Overall, enhancing the mycoses diagnostic capacities in Côte d'Ivoire, and more generally in African clinical laboratories with limited resources is a critical aim.

Fluconazole Resistance and Virulence in In Vitro Induced-Fluconazole Resistant Strains and in Clinical Fluconazole Resistant Strain of Cryptococcus deuterogattii.

Neuromeningeal cryptococcosis is a life-threatening infection of the central nervous system, caused by encapsulated yeast belonging to the Cryptococcus neoformans and Cryptococcus gattii species complexes. Recent data showed that virulence and antifungal resistance are variable for yeasts belonging to the C. gattii species complex. There is an increase in resistance to fluconazole for yeasts of the C. gattii species complex and the virulence is variable according to the genotype. In the present study, (i) we explored and compared the mechanisms of resistance to fluconazole between C. deuterogattii clinically resistant strains and induced fluconazole-resistant strains by exposure to fluconazole in vitro, and (ii) we studied their virulence in the Galleria mellonella study model. We demonstrated that the fluconazole resistance mechanisms involved were different between clinically resistant strains and induced resistant strains. We also demonstrated that fluconazole-induced resistant strains are less virulent when compared to the original susceptible strains. On the contrary, the clinically resistant strain tested maintains its virulence compared to fluconazole-susceptible strains of the same sequence type.

Exposure of Cryptococcus neoformans to Seven Commonly Used Agricultural Azole Fungicides Induces Resistance to Fluconazole as Well as Cross-Resistance to Voriconazole, Posaconazole, Itraconazole and Isavuconazole.

BACKGROUND: Cross-resistance to medical azoles by exposure to azole pesticides is well documented for Aspergillus family fungi but is poorly evaluated for other environmental pathogen fungi, particularly for yeasts belonging to the Cryptococcus neoformans/Cryptococcus gattii species complexes. METHODS: One thousand C. neoformans yeast were exposed to various concentrations of seven different commonly used azole pesticides. Clones surviving exposure were picked randomly, and their minimal inhibitory concentrations (MICs) of fluconazole, voriconazole, posaconazole, itraconazole and isavuconazole were assessed. RESULTS: Depending on the pesticide used for exposure, up to 13.3% of selected Cryptococcus colonies showed a phenotype of resistance to fluconazole, and among them, several showed cross-resistance to another or several other medical azoles. Molecular mechanisms involved in the resistance setups seem to be dependent on ERG11 and AFR1 gene overexpression. CONCLUSION: Exposure to any of the seven azole pesticides tested is capable of increasing the MIC of fluconazole in C. neoformans, including up to the level of the fluconazole-resistant phenotype, as well as generating cross-resistance to other medical azoles in some cases.

Search for Cryptococcus neoformans/gattii Complexes and Related Genera (Filobasidium, Holtermanniella, Naganishia, Papiliotrema, Solicoccozyma, Vishniacozyma) spp. Biotope: Two Years Surveillance of Wild Avian Fauna in Southern France.

Fungi belonging to the Cryptococcus genus and related genera (Filobasidium, Holtermanniella, Naganishia, Papiliotrema, Solicoccozyma, Vishniacozyma) are encapsulated yeasts found in either the environment or animal sources. However, the precise biotopes of most species remain poorly defined. To assess whether wild birds from southern France can carry or spread the most pathogenic species (i.e., species belonging to the C. neoformans and C. gattii complexes), as well as lesser-studied species (non-neoformans/gattii Cryptococcus and former Cryptococcus spp.), 669 birds belonging to 89 species received for care over a two-year period at the Centre de Protection de la Faune Sauvage of Villeveyrac (Bird Protection League nongovernmental organization (NGO) care center) were sampled. Samples were cultured, and Cryptococcus and former Cryptococcus yeasts were identified by PCR sequencing. The purpose was to evaluate whether there was any health risk to local populations or care personnel in aviaries and gather new data on the ecological niches of lesser-known species. One hundred and seven birds (16%) were found to be positive for at least one Cryptococcus or former Cryptococcus species. No yeasts belonging to the highly pathogenic C. neoformans or C. gattii complexes were isolated. However, diversity was notable, with 20 different Cryptococcus or former Cryptococcus species identified. Furthermore, most bird-yeast species associations found in this study have never been described before.

Multilocus sequence typing of strains from the Cryptococcus gattii species complex from different continents.

BACKGROUND: Cryptococcus neoformans and Cryptococcus gattii species complexes are pathogens causing cryptococcal meningitis, a fungal infection that leads to death unless treated. Worldwide, it is estimated to kill over 180,000 individuals annually. OBJECTIVES: We aim to investigate the molecular diversity of C. gattii isolates from strains isolated from 1995 to the present day from different continents. METHOD: In this study, we analysed the molecular diversity by MLST and antifungal susceptibility by using the broth microdilution method according to the CLSI M27-A4 protocol of a total of 26 strains from Cryptococcus gattii species complex from both clinical and environmental sources. RESULTS: Genotyping showed that most of the strains (17/26; 65.4%) belonged to serotype B and were distributed between three genotypes: VGI (13/17; 76.5%), VGII (3/17; 17.6%) and VGVI (1/17; 5.9%). The serotype C strains (9/26; 34.6%) were distributed between the VGIII (1/9; 11.1%) and VGIV (8/9; 88.9%) genotypes. The 26 strains belonged to 17 different MLST subtypes, and we highlight four new MLST genotypes (ST553, 554, 555 and 556). The two environmental strains were identified as serotype B and genotype VGI, but were of ST 51 and 154. All isolates have wild-type MIC of fluconazole and flucytosine. Regarding amphotericin B, five VGI strains showed MICs to AMB equal to 1 µg/ml, and according to the ECV for these genotypes, they were considered non-wild-type strains. CONCLUSIONS: The current study reveals the genetic diversity and new sequence types among strains from the C. gattii complex species.

Reducing indoor virus transmission using air purifiers.

Air purifiers are limited to small polluting airborne particles and poor air circulation (fan) for bringing airborne particles inside the device. Thus, the optimal utility of domestic air purifiers (DAPs) for eliminating airborne viruses is still ambiguous. This paper addresses the above limitations using computational fluid dynamics modeling and simulations to investigate the optimal local design of a DAP in an indoor space. We also investigate the integrated fan system and the local transport of airborne viruses. Three different scenarios of using standard DAP equipment ( 144 m 3 / h ) are explored in an indoor space comprising a furnished living room 6 × 6 × 2.5 m 3 . We show that the local positioning of a purifier indoors and the fan system embedded inside it can significantly alter the indoor airborne virus transmission risk. Finally, we propose a new indoor air circulation system that better ensures indoor airborne viruses' local orientation more efficiently than a fan embedded in a standard DAP.

New multilocus sequence typing primers to enable genotyping of AD hybrids within the Cryptococcus neoformans species complex.

Although AD hybrids within the Cryptococcus neoformans species complex represent about 20% of the isolates identified in Europe, phylogenetic and population genetic studies are lacking due to the inability to use the standardized typing method. The aim of the present study was to design new molecular type specific primers in order to apply the standard ISHAM consensus multilocus sequence typing (MLST) scheme to AD hybrids. The new primers are able to specifically amplify VNI and VNIV alleles of the seven MLST loci in both haploid and diploid or aneuploid hybrid strains. This study forms the basis for future molecular epidemiology studies of AD hybrids. LAY ABSTRACT: We designed and tested new specific primers to amplify the two alleles of each of the seven MLST loci in C. neoformans species complex hybrids. The sequences obtained from hybrids can be compared with those present in the Cryptococcus global MLST database for future molecular epidemiology studies.

Cryptococcus genetic diversity and mixed infections in Ivorian HIV patients: A follow up study.

Genetic diversity analyses were performed by sero-genotyping and multi-locus sequence typing on 252 cryptococcal isolates from 13 HIV-positive Ivorian patients followed-up for cryptococcal meningitis. Antifungal susceptibility analyses were performed according to the CLSI M27A3 method. The majority (67.8%) of the isolates belonged to the Cryptococcus neoformans (serotype A) species complex, with 93% being VNI and 7% being VNII. Cryptococcus deuterogattii VGII (serotype B) represented 16.7% of the strains, while C. neoformans/C. deneoformans VNIII (serotype AD) hybrids accounted for 15.1% of the strains. One strain (0.4%) was not identifiable. Nine different sequence types (STs 5, 6, 23, 40, 93, 207, 311, and a new ST; 555) were identified in the C. neoformans population, while the C. deuterogattii population comprised the single ST 173. The distribution of the strains showed that, while the majority of patients (9/13) harboured a single sequence type, 4 patients showed mixed infections. These patients experienced up to 4 shifts in strain content either at the species and/or ST level during their follow-up. This evolution of diversity over time led to the co-existence of up to 3 different Cryptococcus species and 4 different ST within the same individual during the course of infection. Susceptibility testing showed that all strains were susceptible to amphotericin B while 3.6% of them had a none-wild type phenotype to 5-flucytosine. Concerning fluconazole, 2.9% of C.neoformans serotype A strains and 2.4% of C. deuterogattii had also respectively a none-wild type phenotype to this molecule. All C. neoformans x C. deneoformans serotype AD hybrids had however a wild type phenotype to fluconazole. The present study showed that mixed infections exist and could be of particular importance for care outcomes. Indeed, (i) the different Cryptococcus species are known to exhibit different virulence and different susceptibility patterns to antifungal drugs and (ii) the strains genetic diversity within the samples may influence the susceptibility to antifungal treatment.

Comparative typing analyses of clinical and environmental strains of the Cryptococcus neoformans/Cryptococcus gattii species complex from Ivory Coast.

PURPOSE: The aim of this study was to assess the biotope of the Cryptococcus neoformans/Cryptococcus gattii species complex from Ivory Coast, and clarify the possible epidemiological relationship between environmental and clinical strains. METHODOLOGY: Samples from Eucalyptus camaldulensis (n=136), Mangifera indica (n=13) and pigeon droppings (n=518) were collected from different sites close to the living environment of Ivorian HIV patients with cryptococcosis (n=10, 50 clinical strains). Clinical and environmental strains were characterized by molecular serotyping and genotyping [RFLP analysis of the URA5 gene, (GACA)4, (GTG)5 and M13 PCR fingerprinting] and compared.Results/Key findings. Environmental strains were recovered only from the pigeon droppings. In vitro susceptibility profiles showed that all strains were susceptible to fluconazole, flucytosine and amphotericin B. All environmental strains consisted of C. neoformans (A, AFLP1/VNI), whereas clinical strains included C. neoformans (A, AFLP1/VNI), C. neoformans x Cryptococcus deneoformans hybrids (AD, AFLP3/VNIII) and Cryptococcus deuterogattii (B, AFLP6/VGII). Two patients were co-infected with both C. neoformans and C. neoformans x C. deneoformans hybrids. We noticed a low genetic diversity among the environmental samples compared to the high diversity of the clinical samples. Some clinical strains were genetically more similar to environmental strains than to other clinical strains, including those from the same patient. CONCLUSION: These results provide new information on the ecology and epidemiology of the C. neoformans/C. gattii species complex in Ivory Coast.

Distinct Aeromonas Populations in Water Column and Associated with Copepods from Estuarine Environment (Seine, France).

Aeromonas spp. are ubiquitous bacteria primarily recovered from aquatic ecosystems. They are found in fresh water as well as estuarine and marine waters, and in association with numerous autochthonous aquatic organisms in these environments. However, aeromonads are also etiologic agents of fish diseases and are now recognized as emerging pathogens in humans. The estuary is therefore a key environment, harboring autochthonous aeromonads, and aeromonads originating from humans and animals, mainly released by treated WWTP effluent or watershed run-off via tributaries. The present study compares the abundance and the diversity of Aeromonas populations. Over 2 years of monitoring (eight campaigns from February 2013 to November 2015), the occurrence of Aeromonas was investigated within the water column (water and fluid mud) and in association with copepods. Moreover, the diversity of Aeromonas populations was ascertained by analyzing gyrB and radA sequences, and the antibiotic-resistance phenotypes were determined using the disk diffusion method. This study shows, for the first time, the presence of Aeromonas spp. in water (1.1 × 102 to 1.2 ± 0.3 × 103 CFU.100 mL-1), fluid mud (2.6 ± 2.6 × 102 to 9.8 ± 0.9 × 103 CFU.g-1) and in association with living copepods (1.9 ± 0.7 × 102 to >1.1 × 104 CFU.g-1) in the Seine estuary. Moreover, the diversity study, conducted on 36 strains isolated from the water column and 47 strains isolated from copepods, indicates distinct populations within these two compartments. Strains distributed in five clusters corresponding to A. bestiarum (n = 6; 5.45%), A. encheleia (n = 1; 0.91%), A. media (n = 22; 20.0%), A. rivipollensis (n = 34; 30.91%) and A. salmonicida (n = 47; 42.73%). A. salmonicida is the most abundant species associated with Eurytemora affinis (n = 35; 74.47%). In contrast, A. salmonicida accounts for only 30.56% (n = 11) of isolates in the water column. This study shows the coexistence of distinct populations of Aeromonas in the oligohaline area of an anthropized estuary. Moreover, A. media, a putative human pathogen, present in the water column and abundant in the WWTP samples, was not detected in association with living copepods.

Delineation of Taxonomic Species within Complex of Species: Aeromonas media and Related Species as a Test Case.

Aeromonas media is an opportunistic pathogen for human and animals mainly found in aquatic habitats and which has been noted for significant genomic and phenotypic heterogeneities. We aimed to better understand the population structure and diversity of strains currently affiliated to A. media and the related species A. rivipollensis. Forty-one strains were included in a population study integrating, multilocus genetics, phylogenetics, comparative genomics, as well as phenotypics, lifestyle, and evolutionary features. Sixteen gene-based multilocus phylogeny delineated three clades. Clades corresponded to different genomic groups or genomospecies defined by phylogenomic metrics ANI (average nucleotide identity) and isDDH (in silico DNA-DNA hybridization) on 14 whole genome sequences. DL-lactate utilization, cefoxitin susceptibility, nucleotide signatures, ribosomal multi-operon diversity, and differences in relative effect of recombination and mutation (i.e., in evolution mode) distinguished the two species Aeromonas media and Aeromonas rivipollensis. The description of these two species was emended accordingly. The genome metrics and comparative genomics suggested that a third clade is a distinct genomospecies. Beside the species delineation, genetic and genomic data analysis provided a more comprehensive knowledge of the cladogenesis determinants at the root and inside A. media species complex among aeromonads. Particular lifestyles and phenotypes as well as major differences in evolution modes may represent putative factors associated with lineage emergence and speciation within the A. media complex. Finally, the integrative and populational approach presented in this study is considered broadly in order to conciliate the delineation of taxonomic species and the population structure in bacterial genera organized in species complexes.

Microstructural and Mechanical Implications of Microscaled Assembly in Droplet-based Multi-Material Additive Manufacturing.

To reveal the potential and limits of multi-material three-dimensional (3D) printed parts in droplet-based additive manufacturing, a study combining tensile experiments and 3D imaging technique is proposed. A polymeric composite structure made of acrylonitrile butadiene styrene and thermoplastic polyurethane is manufactured using a two extrusion head printer. The quality of the interface between the two thermoplastics is quantified by adjusting the number of intertwining droplets at the interface. Tensile experiments assisted with digital image correlation are performed with two-interface orientation to discriminate shearing and traction at the interface. The 3D imaging results, which are based on X-ray micro-tomography, show the distinct features of droplet-based additive manufacturing in terms of porosity content and connectivity. Interface properties are found to control, in an incomparable way, the mechanical response. It is found that the interface quality is determinant for enhancing the ultimate performance whereas the interface orientation is found to be the perfect leverage for varying the slope of the linear part.

Ribosomal multi-operon diversity: an original perspective on the genus Aeromonas.

16S rRNA gene (rrs) is considered of low taxonomic interest in the genus Aeromonas. Here, 195 Aeromonas strains belonging to populations structured by multilocus phylogeny were studied using an original approach that considered Ribosomal Multi-Operon Diversity. This approach associated pulsed-field gel electrophoresis (PFGE) to assess rrn operon number and distribution across the chromosome and PCR-temporal temperature gel electrophoresis (TTGE) to assess rrs V3 region heterogeneity. Aeromonads harbored 8 to 11 rrn operons, 10 operons being observed in more than 92% of the strains. Intraspecific variability was low or nul except for A. salmonicida and A. aquariorum suggesting that large chromosomic rearrangements might occur in these two species while being extremely rarely encountered in the evolution of other taxa. rrn operon number at 8 as well as PFGE patterns were shown valuable for taxonomic purpose allowing resolution of species complexes. PCR-TTGE revealed a high rate of strains (41.5%) displaying intragenomic rrs heterogeneity. Strains isolated from human samples more frequently displayed intragenomic heterogeneity than strains recovered from non-human and environmental specimens. Intraspecific variability ranged from 0 to 76.5% of the strains. The observation of species-specific TTGE bands, the recovery of identical V3 regions in different species and the variability of intragenomic heterogeneity (1-13 divergent nucleotides) supported the occurrence of mutations and horizontal transfer in aeromonad rrs evolution. Altogether, the presence of a high number of rrn operon, the high proportion of strains harboring divergent rrs V3 region and the previously demonstrated high level of genetic diversity argued in favor of highly adaptative capabilities of aeromonads. Outstanding features observed for A. caviae supported the ongoing process of adaptation to a specialized niche represented by the gut, previously hypothesized. 16S rRNA gene is an informative marker in the genus Aeromonas for both evolutionary and polyphasic taxonomic studies provided that multi-operon fingerprinting approaches are used.

Multilocus genetics to reconstruct aeromonad evolution.

BACKGROUND: Aeromonas spp. are versatile bacteria that exhibit a wide variety of lifestyles. In an attempt to improve the understanding of human aeromonosis, we investigated whether clinical isolates displayed specific characteristics in terms of genetic diversity, population structure and mode of evolution among Aeromonas spp. A collection of 195 Aeromonas isolates from human, animal and environmental sources was therefore genotyped using multilocus sequence analysis (MLSA) based on the dnaK, gltA, gyrB, radA, rpoB, tsf and zipA genes. RESULTS: The MLSA showed a high level of genetic diversity among the population, and multilocus-based phylogenetic analysis (MLPA) revealed 3 major clades: the A. veronii, A. hydrophila and A. caviae clades, among the eleven clades detected. Lower genetic diversity was observed within the A. caviae clade as well as among clinical isolates compared to environmental isolates. Clonal complexes, each of which included a limited number of strains, mainly corresponded to host-associated subsclusters of strains, i.e., a fish-associated subset within A. salmonicida and 11 human-associated subsets, 9 of which included only disease-associated strains. The population structure was shown to be clonal, with modes of evolution that involved mutations in general and recombination events locally. Recombination was detected in 5 genes in the MLSA scheme and concerned approximately 50% of the STs. Therefore, these recombination events could explain the observed phylogenetic incongruities and low robustness. However, the MLPA globally confirmed the current systematics of the genus Aeromonas. CONCLUSIONS: Evolution in the genus Aeromonas has resulted in exceptionally high genetic diversity. Emerging from this diversity, subsets of strains appeared to be host adapted and/or "disease specialized" while the A. caviae clade displayed an atypical tempo of evolution among aeromonads. Considering that A. salmonicida has been described as a genetically uniform pathogen that has adapted to fish through evolution from a variable ancestral population, we hypothesize that the population structure of aeromonads described herein suggested an ongoing process of adaptation to specialized niches associated with different degrees of advancement according to clades and clusters.

From environment to man: genome evolution and adaptation of human opportunistic bacterial pathogens.

Environment is recognized as a huge reservoir for bacterial species and a source of human pathogens. Some environmental bacteria have an extraordinary range of activities that include promotion of plant growth or disease, breakdown of pollutants, production of original biomolecules, but also multidrug resistance and human pathogenicity. The versatility of bacterial life-style involves adaptation to various niches. Adaptation to both open environment and human specific niches is a major challenge that involves intermediate organisms allowing pre-adaptation to humans. The aim of this review is to analyze genomic features of environmental bacteria in order to explain their adaptation to human beings. The genera Pseudomonas, Aeromonas and Ochrobactrum provide valuable examples of opportunistic behavior associated to particular genomic structure and evolution. Particularly, we performed original genomic comparisons among aeromonads and between the strictly intracellular pathogens Brucella spp. and the mild opportunistic pathogens Ochrobactrum spp. We conclude that the adaptation to human could coincide with a speciation in action revealed by modifications in both genomic and population structures. This adaptation-driven speciation could be a major mechanism for the emergence of true pathogens besides the acquisition of specialized virulence factors.

Accuracy of 6 commercial systems for identifying clinical Aeromonas isolates.

We compared the accuracy of 6 commercial systems for Aeromonas identification by testing 87 clinical isolates in routine conditions, using partial rpoB gene sequencing as the reference standard. The systems were API-20E, API-32GN, the ID-GN card with the Vitek2 system (bioMérieux, Marcy l'Etoile, France), the identification portion of the NFC47 panel (MicroScan Walk/Away system; Siemens Healthcare, Sacramento, CA), ID69 (Phoenix system; BD Diagnostic Systems, Sparks, MD), and GN2 microplates (Omnilog system; Biolog, Hayward, CA), for which 67 (77.1%), 80 (91.9%), 72 (82.7%), 70 (80.5%), 64 (73.5%), and 59 (67.8%) isolates, respectively, were correctly identified at the genus and species level. Confusion with Vibrio affected 6.9% and 16.1% of results obtained with NFC47 and API-20E, respectively. Overall, the accuracy of identification for aeromonads was hampered by outdated databases and taxonomy, weak algorithms, and impractical additional tests. Commercial identification systems should be redesigned to make Aeromonas identification algorithms more robust and to cover infrequent clinical species of this genus.

Negativicoccus succinicivorans gen. nov., sp. nov., isolated from human clinical samples, emended description of the family Veillonellaceae and description of Negativicutes classis nov., Selenomonadales ord. nov. and Acidaminococcaceae fam. nov. in the bacterial phylum Firmicutes.

Three strains of a hitherto unknown, Gram-negative, tiny, anaerobic coccus were collected from human clinical samples originating from skin and soft tissues. The three isolates displayed at least 99.9 % identity in their 16S rRNA gene sequences and more than 99.8 % identity in their dnaK gene sequences. The isolates were affiliated to the family Veillonellaceae, the coccobacillus Dialister micraerophilus being the most closely related species, but there was no more than 91.1 % identity in the 16S rRNA gene sequence between this species and the three isolates. Phylogeny based on the 16S rRNA gene confirmed that the three strains represent a novel and robust lineage within the current family Veillonellaceae. A similar genomic structure was demonstrated for the three isolates by PFGE-based analysis. Morphology and metabolic end products, as well as genotypic and phylogenetic data supported the proposal of the novel genus Negativicoccus gen. nov., with the novel species Negativicoccus succinicivorans sp. nov. [type strain ADV 07/08/06-B-1388(T) (=AIP 149.07(T)=CIP 109806(T)=DSM 21255(T)=CCUG 56017(T)) as type species]. Phylogenetic analyses based on the 16S rRNA gene sequences of members of the phylum Firmicutes and other phyla indicated that the family Veillonellaceae forms a robust lineage clearly separated from those of the classes 'Bacilli', 'Clostridia', Thermolithobacteria and 'Erysipelotrichi' in the phylum Firmicutes. Therefore, we propose that this family is a class-level taxon in the phylum Firmicutes, for which the name Negativicutes classis nov. is proposed, based on the Gram-negative type of cell wall of its members, with the type order Selenomonadales ord. nov. In this order, a novel family, Acidaminococcaceae fam. nov., is proposed and description of the family Veillonellaceae is emended.