I quote meticulously from this scientific work :
"Phage therapy presents several potential advantages for the treatment of
BU patients, namely phages present lytic activity against extracellular
bacteria which predominate in advanced lesions; phages may be used for
the treatment of ulcerative lesions where the necrotic infection site
would be accessible; and phages may be administered topically [28]."
"In the present study, following the screening of the lytic activity of
several mycobacteriophages, the therapeutic effect of the selected
mycobacteriophage D29 was evaluated against M. ulcerans in the
mouse footpad model of infection. The progression of
macroscopic/microscopic pathology and bacterial load, as well as the
cytokine profile, in both the footpad and the draining lymph node (DLN),
were evaluated after mycobacteriophage D29 administration."
"In order to select mycobacteriophages active against M. ulcerans strains, we first selected representative isolates of M. ulcerans from endemic BU areas, based on their genetic and phenotypic characteristics, including the type of mycolactone produced [3], [32], [33] and their virulence in mice [7], [34] (see Table 1). The strains were obtained from the collection of the Institute of Tropical Medicine (ITM), Antwerp, Belgium."
Table 1. Characteristics of M. ulcerans isolates used.
"This host-range determination was done by adapting a spot-test technique described elsewhere [35], [36]. Briefly, M. ulcerans was grown to an OD600
of 1.0 and clumps were dispersed by passing the bacterial suspension
several times through a 25-gauge needle. The suspension was plated on
Middlebrook 7H9 agar medium (Becton, Dickinson and Company). For each
mycobacteriophage, serial dilutions were prepared in phage buffer (MPB)
(10 mM Tris, pH 7.5, 1 mM MgSO4, 70 mM NaCl) and were plated onto the M. ulcerans lawn and the spots were allowed to dry completely. Plates were incubated at 32°C for approximately 6–8 weeks. "
Animals
"A total of 120 (per
experience) eight-week-old female BALB/c mice were obtained from
Charles River (Barcelona, Spain) and were housed under
specific-pathogen-free conditions with food and water ad libitum."
Footpad mouse model of M. ulcerans infection
"M. ulcerans 1615 is a mycolactone A/B producing strain isolated in Malaysia from an ulcerative case [7]. The isolate was grown on Middlebrook 7H9 agar medium at 32°C for approximately 6–8 weeks. For the preparation of inoculum, M. ulcerans
was recovered, diluted in phosphate-buffered saline (PBS) and vortexed
using glass beads. The number of acid-fast bacilli (AFB) in inocula were
determined as described previously using Ziehl-Neelsen (ZN) staining [37]. Mice were infected in the left hind footpad with 0.03 ml of M. ulcerans suspension containing 5.5 log10 AFB."
Treatment of M. ulcerans-infected mice with mycobacteriophage D29
" D29 particles were propagated in Mycobacterium smegmatis mc2155 (ATCC), as described elsewhere [36]. In brief, approximately 105 phage particles and 250 µl of M. smegmatis mc2155 (ATCC) (OD600
of 1.0) were plated on Middlebrook 7H9 overlays (0.6% agar) and
incubated at 37°C overnight. Phage particles were extracted with 3 ml of
MPB and harvested filtering through a 0.2 µm pore-size filter. Phages
were concentrated through polyethylene glycol (PEG) precipitation and
purified using a CsCl equilibrium density gradient centrifugation. Phage
titers (PFU/ml) were determined by serial dilution and plaque assays by
the soft overlay technique with some modifications [35]. Briefly, phage dilutions were spotted onto Middlebrook 7H9 overlays (0.6% agar) with M. smegmatis mc2155 (ATCC) and incubated at 37°C overnight.
The
treatment was initiated at day 33 post-infection, when the footpad of
mice were swollen to 3.0 mm, and was performed by subcutaneous injection
in the infected footpad with a single dose of mycobacteriophage D29
containing 8 log10 PFU. MPB was given to control (non-treated) mice."
Assessment of footpad swelling
"Footpad
swelling was monitored throughout the experiment, as an index of lesion
development, by using a caliper to measure the diameter of the frontal
area of the footpad. For ethical reasons, the non-treated mice were
sacrificed after the emergence of ulceration at day 68 post-infection,
and no further parameters were evaluated for this group."
Bacterial and phage growth
"M. ulcerans
growth and phage proliferation were evaluated in footpad tissues and in
the DLN. Briefly, footpad tissue specimens were minced, resuspended in
PBS (Sigma) and vortexed with glass beads to obtain homogenized
suspensions. DLN were homogenized, the cell numbers were counted and
then suspensions were lysed with saponin 0.1%. Serial dilutions of the
footpad and DLN homogenates were plated on Middlebrook 7H9 agar medium. M. ulcerans
numbers were counted after 6 to 8 weeks of incubation at 32°C and
expressed as colony forming units (CFU/ml). Homogenized samples were
also centrifuged for 10 min at 5000 rpm, supernatant was used for phage
determination by the soft overlay technique [35]
and expressed as plaque forming units (PFU/ml). Phage dissemination was
also investigated by detecting phages in the spleen and sera of mice."
Detection of cytokines
"The
levels of the cytokines tumor necrosis factor (TNF), interleukin
(IL)-6, gamma interferon (IFN-γ) and IL-10 in the supernatant of
homogenized suspensions from DLN and footpad tissues of control-infected
and mycobacteriophage D29 treated mice were quantified by using a
Quantikine Murine ELISA kit (eBioscience Inc), according to the
manufacturer's instructions."
Histological studies
"Mouse
footpads and DLN were harvested, fixed in 10% phosphate-buffered
formalin and embedded in paraffin. Light microscopy studies were
performed on tissue sections stained with hematoxylin and eosin (HE) or
Ziehl-Neelsen (ZN). Images were obtained with an Olympus BX61
microscope."
Statistical analysis
"Differences
between the means of experimental groups were analyzed with the
two-tailed Student t test. Differences with a P value of ≤0.05 were
considered significant."
Mycobacteriophage D29 shows a broad lytic activity against M. ulcerans isolates in vitro
"We first tested the lytic activity of different mycobacteriophages against several M. ulcerans isolates. The results for the plaque formation on the tested M. ulcerans strains are given in Table 2.
We observed that some phages were more strain-specific, such as the
phages Adjutor, Kostya and Brujita, and others presented a more narrow
lytic host range spectrum (L5, Chah and Phaedrus). A cluster of three
phages, namely D29, Bxz2 and Tweety, displayed the broadest lytic host
range spectrum and highest lytic activity against representative strains
of M. ulcerans. In line with a previous report [36], D29 phage showed the broadest lytic host range spectrum amongst the tested mycobacteriophages, affecting M. ulcerans isolates with genetic heterogeneity, variable phenotypic characteristics and from different geographic origins (Table 1). Based on these results, we selected mycobacteriophage D29 for in vivo therapeutic studies against infection with M. ulcerans 1615, a well characterized and stable strain that presents a mycolactone profile identical to that of African strains [32]."
Table 2. Sensitivity of phages to M. ulcerans isolates.
.......
.After there is the discussion of the results.
My contribution:
-This scientific work is good but I should have remember to the authors what is the objective of Phage therapy in Buruli: it is the lysis of M.ulcerans in the lesion.
-This scientific work shows for different phage a problem about the host
range spectrum.
There are for L5 and Bxz2 contrary results if I compare the table above with the table below from this scientific work :
I have the confirmation about the presence of the problem in host
range spectrum.
- Why there is not a list and a table with only lytic mycobacteriophages?
-lysogenic mycobacteriophages must be eliminated.
- In theory I should have used a mini cocktail with two lytic phage: ( D29 + ?) but in the table is there another lytic phage?
- I should have used 240 (per
experience) eight-week-old female BALB/c mice divided like this: 120 for M. ulcerans 1615 and 120 for M. ulcerans 94-1327 and I should have used the cocktail in both.
-When two different infections (two groups) by different M. ulcerans strains are controlled by the same cocktail in both groups it would have been the best result.
