Aquaculture This can favour the arise of resistant bacteria,

Aquaculture produces around
30% of the seafood for human consumption, being an increasingly important food
fish source worldwide 1. Generally, fish aquaculture is subjected to a
greater stress than wild conspecifics, which affects their natural immune
system and often favours bacterial infection, namely during early life stages.
This happens because of the high organic content and low concentration of
dissolved oxygen often recorded in culture water, as well as the proximity of
cultured individuals (a result of production amplification through the use of
high stocking densities). Thus, opportunistic infections can easily emerge,
causing significant economic losses to producers.

Food and Agriculture Organization
(FAO) and most aquaculture organizations recommend the decrease, or even the
avoidance, of antibiotics in aquaculture, though they are still often used by
the industry worldwide 1. This can favour the arise of resistant bacteria,
antibiotherapy inefficacy and dispersal of antibiotic resistance in the
environment, indirectly affecting bacterial species that are not associated
with disease (non-target), allowing resistant strains to enter the human food
chain 2,3.

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Although
vaccination is considered the best approach for the prevention of fish
infections, it is practically impossible to employ during fish early life
stages, due to their small size and low capacity to develop immunity 4,5. Consequently, the development and application of
innovative treatment technologies are demanded by the fish farming industry in
order to increase the efficacy of aquaculture production, by lowering
production costs and fish mortality, with reduced environmental impacts.

Aeromonas salmonicida, the
causative agent of furunculosis, is one of the most relevant fish pathogens in
aquaculture. This disease causes high mortality and morbidity in a broad variety of fish,
with significant economic losses in worldwide aquaculture 6. The chronic form usually occurs in older
fish, and the manifestation of skin ulcers in weakened fish make them
unsuitable for human consumption 7. The acute form is more common in juveniles,
usually leading to septicaemia, being fatal in two to three days 8,9.

Phage therapy is an alternative
approach to diminish fish infections, being based on the use of bacteriophages
(viruses that infect bacteria) to inactivate pathogenic bacteria. Compared to
conventional methods, it presents several advantages: (a) phages are target
specific; (b) serious or irreversible side effects of phage addition are not
known; (c) phages can be chosen to have no environmental impact and to infect
specific bacteria; (d) phages are resistant to various environmental conditions;
(e) limited regrowth of resistant cells (f) phage therapy is a flexible, fast
and inexpensive technology 10,11. Consequently, phage therapy appears to be
a promising and environmentally friendly methodology to reduce the risk of
development of antibiotic resistant bacteria. However, there are some studies
reporting the development of phage-resistance by some bacteria 12–15. This resistance may
be due to the modification or loss of the bacterial cell surface receptors,
blocking of the receptors by the bacterial extracellular matrix, production of
modified restriction endonucleases that degrade the phage DNA, inhibition of
phage DNA penetration or intracellular development 16. The most frequent cause of
bacterial phage resistance appear to be genetic mutations affecting phage
receptors, restriction
modification or abortive infection associated with the presence of clustered
regularly interspaced short palindromic repeats (CRISPRs) in the bacterial
genome 16,17. However, currently,
there is an increasing recognition that the emergence of phage-resistant
mutants can be due to phenotypic resistance. 11,18,19.  Phenotypic resistance may be: i) induced – the
products of phage-lysed bacteria result in a change in uninfected bacterial
gene expression, thus reducing adsorption; ii) intrinsic – reduced adsorption
is due to a physiological or gene expression state that happens prior to the
phage introduction; and iii) dynamic – degradation or blocking of bacterial
receptors by phage proteins released during cell lysis 18. Another phenotypic resistance
that was not yet associated to the development of phage-resistant bacteria may
be a phenomenon designed by pseudo-lysogeny (i.e., false lysogeny). In this
condition, bacterial cells can coexist in an unstable association with
infective phage and some are resistant to phage infection like in case of
lysogeny 20,21. As very little is known
about the effects of the phage infection in the bacterial cells, it is
important to understand the inactivation mechanisms and which modifications are
induced by bacteriophages in the host cell in order to obtain knowledge and
probably a solution to the problem of phage-resistant bacteria.

Infrared spectroscopy (IR) has proven to be a valuable
method for detection and distinction of microbial cells and has also been
successfully used to detect modification in proteins and lipids extracted from
bacteria after exposure to a stress 22 and to study DNA 23.  Another advantage is the
possibility of studying the whole cells, without the need to extract cellular
components 24,25. This methodology was already used to discriminate phage-resistant from
phage-susceptible bacteria 14. The infrared absorbance spectrum represents a “fingerprint”
that is characteristic of a chemical or biological substance. The main reasons
for the wide acceptance of this method are the speed with which samples can be
characterized with almost no handling, the flexibility of the equipment, the
minimum sample amount required and the low cost of the analysis 26. The analytical information of the spectra can be interpreted using a
multivariate analysis that relates the spectra obtained with the properties of
the object of study, thus facilitating data interpretation 27.

The main objective of this study was to understand the
cellular modifications that occur on host targets after phage therapy using as
a model the causative agent of the furunculosis, A. salmonicida, and its specific  phage AS-A

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