Cryptococcus gattii is an environmental yeast capable of causing severe infections in immunocompetent hosts. Its virulence mechanisms remain less characterized than those of C. neoformans. This study aimed to evaluate the expression of major virulence factors and the immunomodulatory potential of eight C. gattii strains representing five genotypes and multiple sequence types (STs). Virulence traits—including capsule thickness, melanin and laccase production, phospholipase, hemolysin, urease, and DNase activities—were assessed using standardized in vitro assays. In vivo pathogenicity was evaluated in Galleria mellonella, and macrophage responses were analyzed via nitric oxide (NO) production and cytokine gene expression in J774.A1 cells. Capsule and melanin production varied significantly across strains, with VGI/ST106 and VGII/ST7 showing the highest levels, respectively. VGIV/ST336, VGI/ST106, and VGVI/ST556 induced greater larval mortality than the reference C. neoformans strain (VNI/ST5), while VGII strains exhibited ST-dependent virulence. Most strains inhibited NO production in macrophages, except VGI/ST106, which enhanced it. Cytokine expression remained low in the absence of inflammatory priming. These findings reveal substantial phenotypic variability in virulence and immune modulation among C. gattii strains. Further investigation into strain-specific host interactions may helpthe development of targeted antifungal strategies and improve clinical management of cryptococcosis.

Hybrid AD strains of the human pathogenic Cryptococcus neoformans species complex have been reported from many parts of the world. However, their origin, diversity, and evolution are incompletely understood. In this study, we analyzed 102 AD hybrid strains representing 21 countries on five continents. For each strain, we obtained its mating type and its allelic sequences at each of the seven loci that have been used for genotyping haploid serotypes A and D strains of the species complex by the Cryptococcus research community. Our results showed that most AD hybrids exhibited loss of heterozygosity at one or more of the seven analyzed loci. Phylogenetic and population genetic analyses of the allelic sequences revealed multiple origins of the hybrids within each continent, dating back to one million years ago in Africa and up to the present in other continents. We found evidence for clonal reproduction and long-distance dispersal of these hybrids in nature. Comparisons with the global haploid serotypes A and D strains identified new alleles and new haploid multi-locus genotypes in AD hybrids, consistent with the presence of yet-to-be discovered genetic diversity in haploid populations of this species complex in nature. Together, our results indicate that AD hybrids can be effectively genotyped using the same multi-locus sequencing type approach as that established for serotypes A and D strains. Our comparisons of the AD hybrids among each other as well as with the global haploid serotypes A and D strains revealed novel genetic diversity as well as evidence for multiple origins and dynamic evolution of these hybrids in nature.

This study was conducted in Côte d’Ivoire to determine the respective prevalence of Candida species and to study their susceptibility profile to antifungal agents to identify the emergence of resistance.This retrospective study was conducted from 2017 to 2019 at the Institute Pasteur of Côte d’Ivoire on patients with fungal disease. The yeasts isolated and identified in Côte d’Ivoire were subcultured on a chromogenic medium and identified with MALDI-TOF-MS. Sensitivity tests were performed using the CLSI method and evaluated by the M27. In total, 227 Candida spp. were isolated from 1941 patients. Most of the samples were vaginal swabs (120/228). Using the conventional method, C. albicans was prevalent (52.6%). There were mixtures of 2 to 4 species in 36.8% of the samples. Thus MALDI-TOF-MS identified in 315 isolates 13 distinct species, C. tropicalis was prevalent at 35.2%, and the species in both C. albicans and C. parapsilosis complexes were differentiated. This study highlights the hurdle of correct yeast identification in developing countries. The transition from conventional to modern MALDI-TOF based identification of these life-threatening opportunistic pathogens is mandatory and should be emphasised as a public health priority.

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.

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.

Abstract S8.5 Genotyping of Cryptococcus neoformans and C. gattii, September 23, 2022, 3:00 PM - 4:30 PM Objectives In a previous study a set of new molecular-type specific primers were designed to apply the standard ISHAM consensus multi-locus sequence typing (MLST) scheme to Cryptococcus neoformans AD hybrids. In the present study, we report the preliminary results of the investigation by MLST of a large number of AD hybrids with the aim to identify the circulating genotypes, their phylogenesis, and population genetics. Methods A total of 50 AD-hybrid isolates from different parts of the world and from different sources were genotyped by MLST. Minimum spanning trees using GoeBurst algorithm were generated by comparing hybrid genotypes and by comparing separately either allele-A and allele-D portions of the hybrid genotypes to the haplotypes recorded in the MLST global database. Results Analysis identified 32 hybrid genotypes grouped in three distinct main clusters (CC12, CC21, and CC30) including 12 isolates each. Both CC12 and CC21 clusters included isolates from different countries and continents but the former grouped only isolates with mating type aADalpha whereas the latter those with mating type alphaADa. Cluster CC30 included only isolates from Ivory Coasts. Heterozygous allelic combinations in each of the seven MLST loci presented two or three combinations more frequent than the other ones. In some isolates, one or more alleles were not amplified after multiple attempts, and therefore, they were considered as lacking. A total of 22 MLST profiles were identified by analyzing separately the allele-A combinations of the hybrids. Comparison with all MLST profiles of VNI, VNII, and VNB included in the MLST global database showed that the allele-A portion of the hybrid genotypes was grouped in few VNI or VNB clusters. In none of the investigated hybrids, the allele-A portion originated from VNII genotypes. Similarly, when the MLST profile of allele-D portion of hybrids was compared to all VNIV genotypes present in the global MLST database, few clusters were identified but, in this case, mostly originated from genotypes not yet found among VNIV haplotypes. Conclusions These preliminary results suggest that the AD hybrids here investigated originated from the mating of A haploids very common in both clinical and environmental isolates and D haploids that are not circulating at present or are very rare. Therefore, it is likely that hybrids originated in the environment where VNIV genotypic diversity is higher and suitable AD combinations can occur. Sequencing of further AD hybrids is in progress to confirm these results.

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.

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 mu g/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.

INTRODUCTION:Cryptococcus gattii species complex is endemic to tropical and subtropical regions and is described as a causative agent of cryptococcosis in immunocompetent individuals. CASE PRESENTATION:We describe the first case of cryptococcosis in a HIV-negative patient from Ivory Coast infected by Cryptococcus gattii sensu stricto VGI. Isolates were recovered from cerebrospinal fluid (CSF) prior to systemic antifungal treatment up to 42 days after detection of the presence of yeasts in the CSF. Eighteen isolates were recovered, genetic diversity and antifungal susceptibility analyses were performed. All the isolates belonged to the Cryptococcus gattii sensu stricto (B;VGI) and were identified as a new sequence type (ST) 553 by Multilocus Sequence Typing (MLST) analyses. Susceptibility testing showed that all the strains had a wild-type phenotype for fluconazole, amphotericin B and flucytosine. Treatment with fluconazole (1200mg/day) was initiated with success. CONCLUSION:This is the first case report of the presence of C. gattii sensu stricto VGI in a HIV-negative ivorian patient and the second report of the presence of species from the C. gattii complex species in this country.

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 (SHAM 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.

OBJECTIVES:Fluconazole (FCZ), either alone or in combination, is often administered for treatment of cryptococcal meningitis, especially in sub-Saharan Africa. Its extensive use has led to the emergence of FCZ-resistant strains. The mechanisms underlying FCZ resistance are poorly documented for yeasts belonging to the Cryptococcus gattii species complex. The literature suggests that resistance could be due to mutations in and/or overexpression of the ERG11 gene (encoding the 14-α-demethylase) and efflux pumps such as MDR and AFR (two subclasses of ABC transporters). Here we highlight the presence of genotype VGII strains (Cryptococcus deuterogattii) from the Ivory Coast with a rare sequence type (ST173) associated with high FCZ minimum inhibitory concentrations (MICs) compared with strains originating from the Pacific Northwest (USA).METHODS:Mechanisms of FCZ resistance were investigated in 28 Ivorian clinical C. deuterogattii isolates recovered from three patients during their antifungal treatment and follow-up.RESULTS:The results demonstrated that: (i) these strains exhibited no mutations in the ERG11 gene; (ii) some strains had increased ERG11 and MDR1 mRNA expression, whilst AFR1 and AFR2 were not overexpressed in strains with high FCZ MICs compared with the expression levels for strains with low FCZ MICs; and (iii) exposure to FCZ in strains with high MICs induced AFR1 mRNA overexpression.CONCLUSION:This study demonstrated that the FCZ resistance mechanism commonly described in Cryptococcus neoformans was not responsible for resistance to FCZ in rare subtype strains.

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.

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.

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.

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.

Aeromonas species are environmental organisms that are responsible for numerous infections in humans and animals. Their antimicrobial susceptibility is usually evaluated using Enterobacteriaceae breakpoints. Although disk diffusion and minimum inhibitory concentration (MIC)-based methods are important for infectious disease management and epidemiological surveys of resistance, comparisons between these two methods have not been extensively studied for Aeromonas isolates. We propose the first extensive comparison of agar dilution and disk diffusion susceptibility testing methods, performed for 20 antimicrobial agents, including unevaluated or incompletely evaluated antibiotics (ticarcillin with or without clavulanic acid, ertapenem, tigecycline), on 146 Aeromonas isolates affiliated with six Aeromonas species via molecular means. We evaluated the level of agreement between Enterobacteriaceae breakpoints-based methods. Reliable agreement (>95%) was observed for piperacillin, cefotaxime, cefepime, nalidixic acid, ofloxacin, ciprofloxacin, gentamicin, amikacin, tetracycline and cotrimoxazole, whereas marked inconsistencies between the methods were noted for carbapenems, amoxicillin–clavulanic acid, ticarcillin, ticarcillin–clavulanic acid, tobramycin and tigecycline. The results indicate that beta-lactam and aminoglycoside susceptibility testing should be limited to piperacillin, cephems, gentamicin and amikacin. Co-amoxiclav should be avoided given the lack of agreement between the two methods. Adjusting the zone diameter breakpoints for tigecycline and cefoxitin could also improve the agreement to >95% and reduce the error rates to acceptable levels.

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.

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.