Multilocus genetics to reconstruct aeromonad evolution

Frdric Roger 1 Hlne Marchandin 0 1 Estelle Jumas-Bilak 1 Angeli Kodjo the colBVH study group Brigitt L my 0 1 0 Laboratoire de Bacteriologie, Centre Hospitalier Regional Universitaire de Montpellier (Hopital Arnaud de Villeneuve) , 371, Avenue du Doyen Gaston Giraud, 34295 Montpellier Cedex 5 , France 1 Laboratoire de Bacteriologie-Virologie (UMR 5119 - Equipe Pathogenes et Environnements), Universite Montpellier 1 , 15, Avenue Charles Flahault, BP 14491, 34093 Montpellier Cedex 5 , France 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. - Background Aeromonads are ubiquitous free-living organisms found in aquatic environments with a strong ability to quickly colonize an exceptionally wide variety of habitats and hosts, ranging from hostile environments, such as polluted or chlorinated water, to leeches, insects, fish, mollusks, and mammals, including man [1]. They are opportunistic pathogens involved in various types of infections in a wide range of hosts. This versatility is supported by a large variety of genes involved in metabolic fitness and virulence; thus, Aeromonas hydrophila is referred to as a jack-of-alltrades [2]. Despite the adaptability of A. hydrophila, very few mobile genetic elements, which are usually associated with rapid adaptation, have been found in the complete genomic sequence of the pathogenic strain A. hydrophila ATCC 7966T [2]. Additionally, because some hosts may only be either colonized or infected, the concept that only specific subsets of Aeromonas strains within species might actually be pathogenic for humans was proposed [3,4]. In this setting, the question has arisen of whether isolates causing infectious diseases are exceptional and can be distinguished from other strains. Comparative analyses including environmental and clinical isolates showed that clinical strains are well differentiated from strains collected in the environment based on multilocus enzyme electrophoresis (MLEE) [5]. Other studies employing phenotypic, genotypic and virulence analyses have failed to distinguish isolates involved in infectious diseases from those that are not [3,6-8]. However, this situation is complex because the pathogenesis of Aeromonas infection is multifactorial and is associated with multiple sources of variability (e.g., a wide variety of virulence factor genes and the influence of environmental conditions); these studies are usually limited either by the sampling strategy applied (e.g., including a low number of isolates, species or types of infection), incomplete virulence factor analyses, or an absence of virulence gene expression analysis. Overall, in the case of generalist opportunistic pathogens, which do not meet all of the criteria Kochs postulate, the link between virulence-related genes and infection is not clearly established, and this opportunistic pathogenic behavior may instead be considered to represent an adaptation to human ecology [9-11]. There is evidence that genetic clusters can correspond to ecologically distinct populations and/or host-adapted populations, even when genes that are not related to virulence are considered [9,11-14]. In this context, in an attempt to improve the understanding of human aeromonosis, we investigated whether clinical isolates displayed specific characteristics among a large population of Aeromonas spp. from various origins. Because the 3 main Aeromonas species recovered from human clinical infectious diseases are A. caviae, A. hydrophila and A. veronii biovar sobria, we particularly focused on isolates belonging to these 3 taxa. The aim of this work was to determine the genetic characteristics, population structure and mode of evolution in a large population of aeromonads using a comparative approach that examined human, nonhuman animal and environmental strains. For this purpose, we developed a multilocus sequence analysis (MLSA) scheme specific for aeromonads, representing the third MLSA scheme to be described for this genus [15,16]. This strategy provided 4 new genes and produced new information on the mode of evolution, recombination rates and horizontal gene transfer in these species. This study, which was based on a large human clinical strain collection, provides interesting insight regarding the mode of evolution of aeromonads linked with human infection. Methods Bacterial strains A total of 195 strains of Aeromonas spp., including 62 type and reference strains, were analyzed. The distribution of the origin of these strains was as follows: 115 human clinical strains, 39 non-human animal strains and 41 environmental strains (Table 1). Of the 115 human clinical isolates, 67 and 7 isolates were sampled in 2006 during a prospective study on aeromonosis involving (...truncated)