RESUMO
Renibacterium salmoninarum (Rs) is the etiological agent of bacterial kidney disease (BKD), which significantly affects farmed and wild salmonids worldwide. Although the whole genome of Rs (~3.1 million nucleotides) is highly conserved, genomic epidemiology analyses have identified four sub-lineages from Chilean isolates. A total of 94 Rs genomes from the BIGSdb aquaculture database were aligned and compared using bioinformatics tools, identifying 2199 independent single-nucleotide polymorphisms (SNPs) spread along the genome. A detailed analysis of the distribution of the SNPs showed five local zones of a length in the range of 10-15 kbp that should be used to unambiguously identify a specific sub-lineage. Based on the Rs type strain DSM 20767T , we designed multiplex PCR primers that produce specific amplification products which were further sequenced by the Sanger method to obtain the genotype of the sub-lineage. For the genetic typing, we evaluated 27 Rs isolates recovered from BKD outbreaks from different fish species and regions of Chile. Based on the findings reported here, we propose the PCR approach as a valuable tool for the rapid and reliable studying of the relationships between Rs isolates and the different sub-lineages without requiring the sequencing of the entire genome.
Assuntos
Doenças dos Peixes , Micrococcaceae , Animais , Salmão , Chile , Doenças dos Peixes/microbiologia , AquiculturaRESUMO
Renibacterium salmoninarum, a slow-growing facultative intracellular pathogen, is the causative agent of bacterial kidney disease, a chronic, progressive and granulomatous infection that threatens farmed and wild salmonids worldwide. Pathogenic R. salmoninarum colonizes tissues and invades the host through cell surface-associated and secreted proteins. While correlations between iron acquisition genes and virulence have been demonstrated in vitro, these mechanisms have not undergone proteomic characterization. The present study applied a proteomic approach to elucidate the differences between the virulent Chilean R. salmoninarum H-2 strain and the type strain ATCC 33209T . Analyses were conducted under normal (control) and iron-limited conditions (DIP) emulating the host environment. Interestingly, strain H-2 apparently responded better to the iron-limited condition-for example, only this strain presented a significantly enriched iron ion homeostasis pathway. Furthermore, key virulence factors related to an iron-limited environment were more abundant in strain H-2. Importantly, the lack of iron favoured the expression of the 57-kDa protein in strain H-2, the principal virulence factor for R. salmoninarum. Our findings can be employed in the design and development of treatments targeted to iron uptake mechanisms (e.g. siderophore synthesis or haem uptake), which represents a promising therapeutic approach for treating this persistent fastidious bacterium.
Assuntos
Doenças dos Peixes , Micrococcaceae , Animais , Ferro , Proteômica , RenibacteriumRESUMO
Renibacterium salmoninarum is the causative agent of bacterial kidney disease (BKD), which is a commercially important disease of farmed salmonids. Typing by conventional methods provides limited information on the evolution and spread of this pathogen, as there is a low level of standing variation within the R. salmoninarum population. Here, we apply whole-genome sequencing to 42 R. salmoninarum isolates from Chile, primarily from salmon farms, in order to understand the epidemiology of BKD in this country. The patterns of genomic variation are consistent with multiple introductions to Chile, followed by rapid dissemination over a 30 year period. The estimated dates of introduction broadly coincide with major events in the development of the Chilean aquaculture industry. We find evidence for significant barriers to transmission of BKD in the Chilean salmon production chain that may also be explained by previously undescribed signals of host tropism in R. salmoninarum. Understanding the genomic epidemiology of BKD can inform disease intervention and improve sustainability of the economically important salmon industry. This article contains data hosted by Microreact.
Assuntos
Aquicultura , Micrococcaceae/isolamento & purificação , Salmão/microbiologia , Animais , Chile , Micrococcaceae/classificação , Micrococcaceae/genética , Epidemiologia Molecular , Filogenia , Salmonidae , Sequenciamento Completo do GenomaRESUMO
Renibacterium salmoninarum is the causative agent of bacterial kidney disease, which significantly affects salmonid farming worldwide. Despite this impact, there is scarce data on its iron uptake ability, a factor of pathogenesis. This study investigated the iron acquisition mechanisms of R. salmoninarum and its capacity to uptake iron from different sources. Thirty-two Chilean isolates and the DSM20767T type strain grew in the presence of 2,2'-Dipyridyl at varying concentrations (250-330 µm), and all isolates positively reacted on chrome azurol S agar. Subsequently, inocula of four Chilean isolates and the type strain were prepared with or without 200 µm of 2,2'-Dipyridyl for uptake assays. Assay results revealed differences between the isolates in terms of iron acquisition. While a prior iron-limited environment was, for most isolates, not required to activate the uptake of iron (II) sulphate, ammonium iron (III) citrate or iron (III) chloride at higher concentrations (100 µm), it did facilitate growth at lower iron concentrations (10 µm and 1 µm). An exception was the H-2 isolate, which only grew with 100 µm of iron sulphide. In turn, 100 µm of haemin was toxic when isolates were grown in normal KDM-2. In silico R. salmoninarumATCC 33209T genome analysis detected various genes coding iron uptake-related proteins. This is the first study indicating two iron acquisition systems in R. salmoninarum: one involving siderophores and another involving haem group utilization. These data represent a first step towards fully elucidating this virulence factor in the pathogenic R. salmoninarum.