Aeromonas species are ideal to
investigate due to the varying interactions with hosts: symbiosis,
opportunistic pathogenicity and high pathogenicity. Reith et al (2008) looked
into the genetic differences between the only strain exhibiting pathogenicity
in healthy salmonids in low quantities (Aeromonas salmonicida subsp. Salmonicida),
9 other strains of A. salmonicida, and A. hydrophila.
Genomic sequencing, annotation, and frameshift
analysis were carried out on a previously isolated sample of Aeromonas
salmonicida subsp. salmonicida A449, from Brown trout in the
Eure river, in 1975. Sequencing was carried out of the entire genome, hyro-sheared
and end-repaired sequences (Shot gun library), and the partially digested
genome (BAC library). Contigs (genes with overlapping sequences) were replicated
(fosmid library) and joined so they represented genome of the bacteria. This
was done using PCR-based methods and direct sequencing. Genomic PCR products
were sequenced for validity reasons. PCR was attempted at least 3 times for
those regions that initially failed, with lower annealing temperatures. The chloramphenicol resistant plasmid,
pAsa4, was transformed into E. coli from
A449, where it was cloned, and used to check the locations of the sequences
during the joining process.
This process was repeated for 10 Aeromonas
species, or subspecies of A. salmonicida,
including A. hydrophila (see paper for specific strains), using isolates
from the American Type Culture Collection (ATCC). These were grown in tryptc soy
broth prior to DNA extraction and replication.
For A. salmonicida subsp salmonicida, the genes were
annotated using the software Perl, and by hand, from which framshifts were identified.
The software ‘Mummer’ was used to compare the chromosomes of A. salmonicida and
A. hydrophila.
A. salmonicida subsp salmonicida contains similar genes for toxins, secretion
systems and mechanisms for iron uptake and utilization to other A. salmonicida subspecies, and additional
genes that are present in other bacteria. None of the
Aeromonas strains investigated show the current capability for motility,
although partial genes were found in A. salmonicida
subsp salmonicida. The presence of more than 25 genes for multidrug resistance, including streptomycin and tetracycline, and also resistance to mercury, were observed in A. salmonicida subsp salmonicida. There were also coding regions for 9
operons, 2 copies of one, multiple copies of genes coding for tRNAs (110 in
total), which act as suppressors. The genes are known in other bacteria classified under another genus, including Vibrio and Pseudomonus. An
additional difference is the increase in insertion sequences (88 total) and pseudogenes (170 total).
Insertion sequences have been observed within genes presumably taken up from
other bacteria, leading to pseudogenes that would originally have lead to
expression of virulent factors. There are 7 pathways for quorum sensing named,
although no comparison to the other species.
This study identifies key traits that increase pathogenicity as: resistance,
adhesive abilities, toxin secretion, efficient iron utilization and uptake,
adaptive abilities and quorum sensing abilities. These have been demonstrated
as possible in A. salmonicida subsp salmonicida, judging by the genome. The
presence of genes coding for mercury and microbial resistance lead to lower
mortality rates than the strains without. The mercury resistance is particularly
important for fish pathogens considering the high mercury content of salmonids.
Although the lack of motility is not important for virulence, it could play
a role in the range of organisms that can be infected. If the genes for
motility were expressed, this could potentially become omnipresent in the way
that the Vibrio species are. At
present this strain is not capable, however the high numbers of genes for
conjugative transfer and transposons suggest this has potential to become a
major pathogen.
Genes originally present in the ancestor of A. salmonicida subsp
salmonicida, as well as those accumulated from other bacterial species, have
been found yet are dysfunctional due to insertion sequences. This coincides with
the theory of gene loss leading to host specificity (their insertions with the
original genes) and potential yet not currently expressible traits that
increase virulence. The host specificity could be a reason why it does not
infect a variety of organisms.
This study also demonstrates the need to re-evaluate the view of A. salmonicida sub salmonicida as ‘typical’ for A.
salmonicida subspecies, considering the now realized differences between
the subspecies.
The samples were quite old, so are likely to have evolved since then. It
would be useful to compare the genetics of the sample A. salmonicida subsp salmonicida
used here and a present day bacteria. This is particularly important since the
changes observed have been noted as ‘recent’. Another experiment leading from this could look
into the extent to which a gene is important for virulence by creating mutants
lacking certain genes present in A. salmonicida
subsp salmonicida but not in other
subspecies of A. salmonicida and
observing the infectious effects on fish or other organisms. This would have to
be highly controlled in order to prevent accidentally releasing a more
pathogenic strain.
Overall, this paper is informative on traits related to virulence, even if the
genomic data presented for the strains is now outdated.
The genome of Aeromonas salmonicida subsp. salmonicida A449: insights into the evolution of a fish pathogen
Reith, M. E; Singh, R. K; Curtis, B; Boyd, J. M; Bouevitch, A; Kimball, J; Munholland, J; Murphy, C; Sarty, D; Williams, J; Nash, J. H. E; Johnson, S. C; and Brown, L. L;
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2556355/#ui-ncbiinpagenav-2
hey meg, nothing really to say just a quick ask, was it just mercury resistant genes found or was it a case of numerous genes found but the highest abundance of these resistant genes code for mercury resistance? if that makes sense, because surely although mercury is high in salmonids if they have these specific genes they must have hidden or unexpressed genes for more common chemicals encountered.
ReplyDeletecheersies,
Ollie
Hi Ollie,
ReplyDeleteThank you for your question. In short: there are more than 25 genes that code for resistance to multiple antibiotics, as well as genes present that are responsible for specific resistance, such as the resistance to mercury.
There was also an integron from E. coli, which is a whole ‘cassette’ of genes, enabling resistance to multiple antibiotics and some compounds. These are major contributors in terms of the transfer of antimicrobial resistance in bacteria due the lateral gene transfer. If you wish to know more about integrons I recommend reading a paper on the Vibrio genus such as that by Rowe-Magnus et al (2003), as they are considered a model system. (Boucher et al, 2006)
Your point about the mercury content is very good, if I understand correctly. It is possible that all the species that can inhabit the fish must be able to withstand mercury to some degree, however this is dependent on the distribution of mercury in the fish tissue. It is possible for the bacteria in colonise regions on the fish where mercury levels are low, that exhibit enhanced immune responses or a mechanism that represses virulence. If the region that is infected by A. salmonicida subsp salmonicida is high in mercury, this would make sense as to why this subspecies can infect the fish when other subspecies cannot.
If that was not was you were considering please feel free to re-phrase the comment and I will explain as much as I can!
Megan
Boucher, Y; Nesbø, C. L; Joss, M. J; Robinson, A; Mabbutt, B. C; Gillings, M. R; Doolittle, W. F; and Stokes, H. W (2006) Recovery and evolutionary analysis of complete integron gene cassette arrays from Vibrio, BMC Evolutionary Biology 2006, 6:3
Rowe-Magnus, D. A; Guerout A. M; Biskri, L; Bouige, P; and Mazel D. (2003) Comparative analysis of superintegrons: engineering extensive genetic diversity in the Vibrionaceae., Genome Res.13(3):428-42.
Thats perfect, thankyou very much. What a useful species, being so resistant to numerous things, dont see that too much.
ReplyDeleteOllie.
Hi Meg, great blog post!
ReplyDeleteIm facinated with your suggestion of comparing old isolated samples with wild field samples. Just wondering, how old were the samples and are there limited numbers of these isolated bacteria? or many samples? I ask because it would be interesting to see how the pan-genome has evolved, especially if the samples were isolated before we started pumping antibiotics into aquaculture systems.
Thanks, Myles
Hi Myles, thank you for your interesting comment.
DeleteThe samples of Aeromonas salmonicida subsp. salmonicida A449 were isolated in 1975, which is obviously a very long time ago! This was done during another experiment (Michel, 1979) that I cannot find a full version of the paper of, so regrettably cannot answer you last question. The paper also did not state how many samples were used.
The point you made about the increased application of antibiotics is valid as a factor that would effect the direction of evolution, particularly causing convergence to populations that are resistant to certain antibiotics as the species that are not resistant are 'killed off'. However, in terms of marine microbes, I am not sure how frequently they come into contact with antibiotics. The use of (some) antibiotics has been banned in practices like aquaculture due to awareness of the environmental impact becoming increasingly noted. The frequency of application depends on the legislations around the specific practices. Of course, there was a large period in which the antibiotics were commonly used in close contact with marine organisms, over which time period antibiotic resistance may have increased. The difficulty comes when attempting to test the antibiotic-resistance of the bacteria, considering a large proportion of marine bacteria are not culturable when using the traditional culture based methods.
Megan
Michel C. (1979) Furunculosis of salmonids: vaccination attempts in rainbow trout (Salmo gairdneri) by formalin-killed germs. Ann Rech Vet. 10:33–40.