The larger costs to the bacteria 46. In

The emergence of phage-resistant mutants during phage infection has been
reported in many studies 11,38–42 but the mechanisms of bacteria resistance to phages
are not yet completely understood. A previous study of our group 42 showed that the agent of furunculosis can be efficiently
inactivated by the phage AS-A (reduction of 4 Log CFU mL-1 after 8 h
of treatment). However, some bacteria survived to the infection by the phage
due to the development of phage-resistance (2.24 x 10-4) 42. The frequency of resistance was, nevertheless,
limited as already reported in previous studies 13,43,44.

So, with these two studies we verified that although
1) a specific phage against the agent of the furunculosis can control
efficiently the bacterial growth, 2) some phage-resistant bacteria emerge after
treatment; 3) being the resistant colonies after 4 and 5 streak-plating steps clearly
distinct from earlier streaking steps, (steps 1, 2 and 3); 4) showing a
significant modification in the expression of intracellular proteins when
compared with the phage sensitive bacteria; 4) but these modifications affect
distinct proteins after the first and the fifth streak-plating steps; 5)
allowing “lysis from without” (positive spot test) after the forth streak-plating
step contrarily to that observed for bacteria from 1, 2 and 3 streak-plating

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It has been stated in the literature that resistance to phage can be overcome
by the phage itself because it evolves along with the host 45. Moreover, it has been also stated
that resistance to phages entails larger costs to the bacteria 46. In fact, as observed for other
phages, colonies of AS-A phage-resistant mutants were smaller than colonies
formed by the non-phage added control 14. These results suggest that the
remaining bacterial mutants (forming small size colonies) maintained their
viability in the presence of phages but their phenotypes were affected. These
decrease in the bacterial growth after phage exposure could be fitness cost,
which can contribute to their elimination from the environment faster than their
wild-type parents.

In this study, as already observed for other phages 14,15, it was also detected that phage-resistant bacteria
also mutate after successive streak-plating steps. Although the spot tests
showed negative results until the fourth streak-plating step, at the fourth and
fifth steps, the spot test was positive, as already observed in other studies 14,15. These results were confirmed by infrared spectroscopy
data of the whole cells. Infrared spectroscopy results shows that the spectra
obtained from the fourth and fifth streak-plating days colonies are similar to ones
from phage-sensitive control colonies, suggesting that these colonies are more similar
to control phage-sensitive bacteria than the colonies from 1, 2 and 3
streak-plating steps. It seems that the resistant bacteria somehow “recovered”,
being more similar to control bacterial populations, which are sensitive to the
phage infection. The infrared peaks that contributed to these results were
found to be majorly associated with proteins. Taking this into account, we
focused the further studies on protein analysis with 1D SDS PAGE gels.

Regarding the presumptively identified proteins with
differential expression on first streak-plating phage-resistant clones, a
decrease in band 8 is noticeable, being this band associated with a phage
transcriptional protein with regulation function in the transcription of phage
genes 47. This may be a response of the bacteria to the viral
infection, preventing the transcription of the viral genome. Similarly, the
expression of the protein corresponding to 9 band in first streak-plating
clones decreased when compared to the control. This protein, phage-shock B
protein, is involved in a regulation system that responds to aggressions,
habitually to phage secretins, promoting the defensive response of the bacteria
48. This protein has been previously detected in the
response of other bacteria, however, this response mechanism is not yet
completely understood 48,49. In our case this protein is less expressed in the
phage-resistant clones, which seems contradictory. Nevertheless, it was stated
that bacteria synthesise phage shock proteins after being infected with phage,
that, in the case of the resistant clones, could not happen 50. Contrarily, the protein associated to band 13, tatA, increased in A. salmonicida first streak-plating clones. This protein belongs to
tat system
(twin-arginine-translocation) which is responsible to the transport of various
substances at the membrane level, against the concentration gradient of the
cytoplasm to the extracellular space, namely proteins, being associated to the
bacterial pathogenicity in the secretion of virulence factors 51,52. This increase suggests that these first streak-plating
clones can be more virulent than control bacteria However, some
studies showed the phage-resistant clones are less pathogenic than phage
sensitive bacteria 19,53. This suggest
that the increase in the expression of this protein can be associated to other
mechanisms not related with pathogenicity.

Regarding the proteins with differential expression on
phage-resistant clones in the fifth streak-plating, that have a positive
spot-test, band 16 suggests the expression of a transposase that is decreased
in these clones when comparing to control phage-sensitive bacteria. These type
of enzymes have the function to facilitate the transference of the genetic
material between organisms 54. The bacteria can decrease the expression of this
protein as a defence mechanism in order to prevent the phage replication. Band
18, corresponds to a toxin-antitoxin protein, which is implied in the
maintenance of plasmids, stress regulation and adaptation, as well as in the
growth control and programed cellular death 55,56, also decrease in the clones of the fifth
streak-plating when compared to the control. As this protein decrease in this
study, it can suggest that in the fifth streak-plating clones the stress caused
by the phage decrease. In fact, for the clones of the fifth streak-plating the
spot test was positive, which suggest that the changes in the proteins turn the
host cells more sensitive to the phage suspension. However, the efficiency of
plating (EOP) results indicates that the fifth streak-plating clones do not replicate
the phage. Other authors 57 obtained similar results, designating this situation
of “lysis from without”. The spot test lysis when the phage is not replicated
by the host (EOP is zero) has been described as a plausible mechanism which
happens when an overload of phages simultaneously infects a bacterium leading
to lysis either from the action of phage lysins or from rapid depletion of the
cells resources 58. As in the spot test the same volume of phage
suspension was used and lysis was only observed for the clones of the fourth
and fifth streak-plating, the hypothesis of rapid depletion of the cells
resources does not seems plausible. However, the lysis can be due to the
presence of lysins, explaning the positive spot test for the clones of the
fourth and fifth streak-plating and the negative spot test for the clones of
the first, second and third streak-plating. The modifications in the proteins
along the successive streak-plating can allow the clones to recover the
sensitivity to the phage lysins. This is in agreement with the infrared
spectroscopy results which show that the fourth and fifth streak-plating clones
were similar to the phage-sensitive bacteria (control), but clearly different
from those of the clones of the first, second and third streak-plating.

Taking into account all these results, we noticed that
the different analysed clones present significant modifications in
intracellular proteins related to phage infection, either in the first and fifth streak-plating. However, there are more proteins that
are differentially expressed in clones of the first streak-plating
than in clones of the fifth the
fifth streak-plating, which is in accordance with infrared
spectroscopy results. The fact that the phage-sensitive control bacteria have
infrared spectra that are more similar to the fourth and fifth streak-plating clones may be because the cellular envelope, used by the
phages to infect the bacteria, became more similar in these cases. This may be
related to the fact that the spot test turns positive again for the fourth and fifth streak-plating clones, which can be due to
phenotypical similarities in the cell envelope. In order to confirm these
results, the identification of the proteins that show differential expression
between the clones should be performed and future studies are being done.


4. Materials and Methods


4.1. Bacteria
and phage

The bacteria A.
salmonicida CECT 894 was used in this study. Fresh plate bacterial cultures
were maintained in solid Tryptic Soy Agar medium (TSA; Liofilchem, Italy) at 4
°C. Before each assay, one isolated colony was aseptically transferred to 10 mL
of Tryptic Soy Broth medium (TSB; Liofilchem, Italy) and was grown overnight at
25 °C. An aliquot of this culture (100 ?L) was aseptically transferred to 10 mL
of fresh TSB medium (Liofilchem, Italy) and grown overnight at 25 °C to reach
an optical density (O.D. 600) of 0.8, corresponding to about 109
cells mL?1.

Phage AS-A was isolated from sewage water from a lift
station of the sewage network of Aveiro, Portugal (station EEIS9 of SIMRIA
Multi Sanitation System of Ria de Aveiro) using A. salmonicida as host, according to 42. The phage stocks were stored at 4 °C and 1%
chloroform (final volume) (Scharlau, Spain) was added. The phage suspension
titre was determined by the double-layer agar method using TSA (Liofilchem,
Italy) as culture medium 59. The plates were incubated at 25 °C for 12 h and the
number of lysis plaques was counted. The results were expressed as plaque
forming units per millilitre (PFU mL-1).


4.2. Isolation
of A. salmonicida phage-resistant

Only bacterial colonies that were resistant to the
phage were used (bacteria that developed inside phage plates). For this,
bacteria A. salmonicida and phage
AS-A were plated by the double layer agar method and the plates were incubated
for 24 h at 25 °C. After that, several colonies that grew inside the phage
plates, thus, resistant to phage infection, were visible. Three individualized
colonies (A, B and C) were picked and used in the subsequent assays.


4.3. Detection
of bacteria sensitivity to the phage after one cycle of phage contact

The phage resistant colonies obtained in the section
4.2. were used. The colonies were inoculated in TSB medium for 24 h at 25 °C.
After that, the culture was used to perform a spot test and was also plated in
TSA medium. This procedure was done 4 more times, making a total of 5 streak
plating steps. This procedure was made for the 3 selected colonies.


4.4. Efficiency
of platting (EOP)

The efficiency of plating was determined for bacteria
that shown positive spot tests (clear lysis area), i.e. for the bacteria from
fourth and fifth streak-plating steps, according to Pereira et al. 14 using the double-agar method 59. The EOP was calculated (average PFU on target
bacteria/ average PFU on host bacteria), three independent assays were


4.5. Phage

The determination of phage adsorption was performed
according to Pereira et al. 14. Briefly, ten microliters of phage suspension of
about 106 PFU/mL were added to 10 mL of A. salmonicida culture of about 109 CFU/mL
corresponding to an optical density (600 nm) of 0.8 60 and incubated at 25 °C. Aliquots of this culture were
collected after 0, 5, 10, 15, 20, 25, 30, 40, 50, 60 and 70 minutes of
incubation and chloroform was added to a final concentration of 1%. The mixture
was centrifuged at 12000g for 5 minutes, after that the supernatants were
filtered using 0.2 µL membranes (Millipore, Bedford, USA). The filtrates
containing unadsorbed phages were then diluted and titrated. The plates were
then incubated at 25 °C and observed after 8h for plaques formation. The values
were calculated as decrease of phage titre in supernatant (percentage) compared
with time zero. Three independent assays were performed.


4.6. Infrared

In order to access the spectral differences of
sensitive A. salmonicida colonies and
phage resistant mutant colonies, mid-infrared spectroscopy was used, as it was
previously described 14,25. They were used the A. salmonicida phage resistant colonies A, B and C (from section 43).

To analyse the whole cells, colonies A, B and C were analysed along the
5 days of streaking (section 4.3), as well as control sensitive colonies Ct1
and CT5 (after 1 and 5 streak plating steps). The colonies were collected with
a loop and placed in the crystal of a horizontal single reflection ATR
accessory. The colonies were gently air dried and the spectra were acquired.

Spectra were done in a MIR (Bruker ALPHA FTIR spectrometer, Germany)
with a resolution of 4 cm-1 and 32 scans, in the infrared region
(4000 to 600 cm-1). At least 5 replicate spectra were performed for
each colony. Mid-infrared spectra were obtained in OPUS format (OPUS 6.5,
Bruker, Germany) and transferred via JCAMP.DX format for use in a house
developed data analysis software (CATS build 97). The spectra were SNV
(standard normal deviate) corrected previous to multivariate analysis.
Principal component analysis (PCA) was done in order to find the major sources
of variability in the spectra and to detect groups.


4.7. Intracellular
proteins extraction and quantification

The proteins extracts were obtained from the growth
until the late exponential phase of the strains (OD 0.9 at 550 nm) in Luria
Bertani Broth (Merck, Germany). The cells were separated from the supernatant
by centrifugation at 8000xg for 10 min at 4 °C. The protein extractions were
made in three independent experiments per each strain and the protein
quantification was performed in triplicate.

The cell pellets were washed three times in 10mM
phosphate buffered saline pH 7.4. After that they were resuspended in 1 mL of
lysis buffer solution 7 M urea, 2 M thiourea, 4% cholamidopropyl
dimethylammonio-1-propanesulfonate (CHAPS), 30 mM Tris base, pH 8.5. Crude
cell-free extracts were obtained by sonication in ice to minimize protein
damage, during 2min, using a 30% duty cycle, 2 s pulses with intervening
periods of 3 s. The intracellular protein solution was incubated with 1 mg.mL-1
of Dnase I (GE Healthcare, Sweden) and 10mM of protease inhibitor mix (GE
Healthcare, Sweden) during 1 h at 15 °C . The final solution was
collected by centrifugation at 20000xg for 40min at 4 °C and then,
the protein concentration was measured using the 2-D Quant Kit  (GE Healthcare, Sweden), following the
manufacturer’s instructions. The procedure was performed in triplicate.


4.8. Protein
separation by 1-D electrophoresis

were separated by 12.5 % SDS-PAGE 61, in a
Mini-PROTEAN 3 Cell (Bio-Rad, USA),
for 50 min at 150 V. Proteins were visualized by colloidal Coomassie Brilliant
BlueG-250 (CBB) staining 62. Gel images
were acquired using the Gel DocTM XR+ (Bio-Rad, USA). The comparative analysis
of the acquired images was performed in Image Lab v3.0 software (Biorad, USA)
and based in the optical density measurement of each band. The result was
expressed in band percentage, resulting from the value of the optical density
of a given band in the total of the bands per lane x 100. The comparison of the
differential expression of the intracellular proteins of the different tested A. salmonicida clones in the different
analysis times was made through a two-way ANOVA, using GraphPad Prism software
v7 (USA). The differences were considered statistically significant when p < 0.05.   4.9. Presumptive identification of the proteins in differentially expressed bands The molecular weight of the bands that were differentially expressed between control and A. salmonicida clones on day 1 and between control and day 5, recurring to databases UniProtKB ( and NCBI ( allowed us to presumptively identify the proteins and their function, based on the deposited genome of Aeromonas salmonicida A449.   


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