OBJECTIVE CRITERIA (QUALITY)
Quality: Experiments (1–3 scale) SCORE = 2 (hard to determine based
on incomplete Materials & Methods section)
Figure by figure, do experiments, as performed, have the proper
controls?
Figure 2: We found that Figure 2B was missing a control 0 µg/mL dose.
Figure 2C had a spike in biofilm formation at 2 µg/mL and it is unclear
whether this is a true biological event or technical issue. It is
unclear whether the “n” in this experiment represents individual
biological replicates or one experiment with eight technical replicates.
The authors should clarify this point. Figure 2D: While it was nice to
see a negative control in this experiment it was a positive control;
NH125 is commercially available has been previously proven to kill MRSA
persister cells
(https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4976882/).
Figure 3: It was not clear how cell growth rates were normalized in the
Tn mutant library screen. In general, further methodological details on
these experiments is required.
Figure 4: We could not read this figure due to poor resolution although
we were curious as to why there was a differing number of mice in the
vancomycin control and vehicle control groups.
Figure 5: Labelling on Figure 5D did not explain why there were three
different heat maps for each group of bacteria tested. Further details
on this figure are required to aid reader comprehension.
Are specific analyses performed using methods that are consistent with
answering the specific question?
While difficult to assess this due to insufficient information in the
Materials & Methods, we believe that the analyses performed were
consistent with other studies in the field.
Is there the appropriate technical expertise in the collection and
analysis of data presented?
We identified multiple deficiencies in data presentation in this paper:
- Axis labelling between Figure 2B and 2C were inconsistent.
- Figure 3 lacked details in general and would benefit from added detail
in the figure legend.
- We were unable to read Figure 4 (low resolution).
- Labelling in Figure 5 did not describe what the three different
heatmaps represented.
- Figure 1 could be improved by presenting the MIC curves for each
compound alongside its structure.
Do analyses use the best-possible (most unambiguous) available methods
quantified via appropriate statistical comparisons?
We did not identify any deficiencies in statistical analyses, but the
authors should explain why a different number of mice were used in
Figure 4.
Are controls or experimental foundations consistent with established
findings in the field? A review that raises concerns regarding
inconsistency with widely reproduced observations should list at least
two examples in the literature of such results. Addressing this question
may occasionally require a supplemental figure that, for example,
re-graphs multi-axis data from the primary figure using established axes
or gating strategies to demonstrate how results in this paper line up
with established understandings. It should not be necessary to defend
exactly why these may be different from established truths, although
doing so may increase the impact of the study and discussion of
discrepancies is an important aspect of scholarship.
Experiments and controls are consistent with other studies in the
field that show that Cannabis sativa has antimicrobial
properties.
Quality: Completeness (1–3 scale) SCORE = 1.5
Does the collection of experiments and associated analysis of data
support the proposed title- and abstract-level conclusions? Typically,
the major (title- or abstract-level) conclusions are expected to be
supported by at least two experimental systems.
The experiments do not adequately support the conclusions stated in
the abstract. The title is quite ambiguous.
Are there experiments or analyses that have not been performed but if
“true” would disprove the conclusion (sometimes considered a fatal
flaw in the study)? In some cases, a reviewer may propose an alternative
conclusion and abstract that is clearly defensible with the experiments
as presented, and one solution to “completeness” here should always be
to temper an abstract or remove a conclusion and to discuss this
alternative in the discussion section.
- While there are no experiments that come to mind that would disprove
the conclusions of this paper, we believe that a skin infection model
experiment would add significant value as polymyxin B and CBG could be
used as a topical agent against both Gram-positive and Gram-negative
bacterial infections.
- We noticed that in supplemental Figure S4C a number of genes involved
in vitamin biosynthesis and metabolic processes were also enriched. We
would like to see this added to the discussion as it could suggest an
alternative mode of action. Despite this, we remain convinced that at
least one of CBG’s modes of action was disruption of cytoplasmic
membranes.
Quality: Reproducibility (1–3 scale) SCORE = 3
Figure by figure, were experiments repeated per a standard of 3 repeats
or 5 mice per cohort, etc.?
As mentioned above, it is not clear whether the sample size (n) for
experiments in Figure 2 represent a single experiment with multiple
replicates or multiple different experiments.
Is there sufficient raw data presented to assess rigor of the analysis?
Yes, we believe enough raw data is presented.
Are methods for experimentation and analysis adequately outlined to
permit reproducibility?
- The methods section is lacking in detail required to reproduce the
results. A few specific examples are pointed out below but in general
almost all experiments require further clarification re: the methods.
- Experimental methods for the MIC 90 experiment were not included in
the methods at all. This should be addressed.
- The analysis of the resistant mutant data was lacking in detail. More
detailed information would aid understanding of how the authors
concluded that there was not a single mutant that was sensitized to
sublethal concentrations of CBG.
- Additional information is required to understand how the authors
obtained persister cells.
Quality: Scholarship (1–4 scale but generally not the basis for
acceptance or rejection) SCORE = 2
Has the author cited and discussed the merits of the relevant data that
would argue against their conclusion?
- For the most part, the authors cite other work appropriately. The
novelty of the first screen for antibiotic potential is unclear. It
would be nice to clarify this issue of novelty in the text, as we
found a previous study that identified antibiotic potential of whole
plant extracts (https://doi.org/10.1016/j.joim.2018.07.005).
- We think that the paper would benefit from further discussion on the
how these metabolites may protect the Cannabis sativa plant.
Elaboration on whether bacterial infection is common in this plant and
what bacteria are known to infect this plant may help give us an idea
of the role of CBG in its natural context.
Has the author cited and/or discussed the important works that are
consistent with their conclusion and that a reader should be especially
familiar when considering the work?
Yes, although their discussion on CBG as a natural defense against
plant pathogens could be broadened.
Specific (helpful) comments on grammar, diction, paper structure, or
data presentation (e.g., change a graph style or color scheme) go in
this section, but scores in this area should not to be significant bases
for decisions.
- As mentioned previously, multiple low-resolution figures need to be
recreated as they are currently difficult to interpret.
- The prose could be improved.
- We found a few typos that should be addressed including:
- “..” in line 219
- And “fig.2a in line 146 instead of “fig. 2a”