PONE-D-22-01149Efficacy assessment of a novel endolysin PlyAZ3aT for the treatment of ceftriaxone-resistant pneumococcal meningitis in an infant rat modelPLOS ONE

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

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Reviewer #2: Yes

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Reviewer #2: Yes

Reviewer #3: No

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5. Review Comments to the Author

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Reviewer #1: In this work, the efficacy of the recombinant endolysin PlyAZ3aT identified in the genome of Streptococcus tigurinus AZ3aT was evaluated in an infant rat model of pneumococcal meningitis. In general, this is a well-organized manuscript whose scope is worthy of investigation for this journal. However, the results showed that the endolysin PlyAZ3aT did not improve the survival of infant rats when compared with animals treated with vancomycin. Although the use of endolysins has been reported as a promising solution against antibiotic resistant pathogenic bacteria, the results obtained in this work suggest that the endolysin PlyAZ3aT is not a real alternative to treat meningitis provoked by Streptococcus pneumoniae serotype 19A. In fact, the results showed that the use of the antibiotic vancomycin is a better alternative to treat the infection produced by this ceftriaxon-resistant pneumococcal strain in rats.

The main concern of this study is that PlyAZ3aT did not exert a bactericidal effect in vitro as the growth of S. pneumoniae was not inhibited after 6 h of exposure using the highest concentration in antibactericidal plate assays (200 ug/ml). Consequently, this fact hinders the possible antimicrobial effect of this endolysin in rats inoculated with S. pneumoniae. The second issue is the failure of this endolysin to cross the blood brain barrier as it could not be detected in cerebrospinal fluid. In the discussion section, it was proposed the use of liposomes for endolysin delivery across the blood brain barrier in order to improve its efficacy and enhance its action in meningitis treatment. Therefore, the manner in which this work is presented is not enough to have an own identity as a primary research article. In my opinion, a new assay using liposomes or other mechanisms as endolysin delivery vehicles should be tested in an infant rat model of pneumococcal meningitis to elucidate whether PlyAZ3aT is active in vivo against S. pneumoniae. Otherwise, this manuscript should be considered as a letter to editor to explain the ineffectiveness of this endolysin and avoid developing uselessness assays with this endolysin against S. pneumoniae. The third problem is related to the inflammatory response observed in supernatants from primary astroglial cell culture treated with a low dose of PlyAZ3aT (100 ug/ml). In this sense, levels of cytokines (IL-1β, IL-6 and TNF-α) were statistically significant when compared with those obtained in control cultures treated with PBS and it was attributed to residual endotoxin activity. Finally, it should be explained why the detection limit of bacterial counts is 3 log10 (CFU/ml).

There are other specific comments:

Abstract

- Line 30. The following sentence seems a headline: "Randomized, blinded and controlled experimental study in infant Wistar rats".

Results

- Line 240. It should be specified that PlyAZ3aT is a bacteriophage-derived endolysin found in the genome of Streptococcus tigurinus.

Discussion

- The discussion should include more studies regarding the application of endolysin in animal models.

Tables

- The comparison of the decrease of CFU in CSF and blood shown in Table 1 is not representative. However, data from Table S1 should be plotted in a graph. The abbreviation N/A should be explained.

Figures

- Figure 1. The alignment of amino acid sequences of the catalytic and cell-wall binding domains of endolysins PlyAZ3aT and Cp-1 should be included in Fig.1A instead of the schematic structure of PlyAZ3aT in order to support the information explained in lines 240-244 (Results).

Reviewer #2: The manuscript submitted by Valente and colleagues describes the use of the streptococcal endolysin PlyAZ3a to treat pneumococcal meningitis in an animal model. Disease was induced in animals by inoculation of Streptococcus pneumoniae and 17 h later animals were treated with endolysin. Survival of treated animals was not higher than those treated with PBS (negative control). Additionally, it was shown that endolysin was not found in the cerebrospinal fluid, which would explain the failure in the animals’ recovery.

General comments: the manuscript is well written and organized. The subject of the work is of interest due the huge problem derived from multiresistant bacteria. All experiments have been done with proper controls. Although not positive results were obtained from this work the conclusions are very valuable.

Specific comments:

Lane 115. The antimicrobial activity of endolysin was compared with vancomicyn. Did you compare with endolysin Cpl-1? How different is the activity of PlyAZ3a and Cpl-1? It might explain the different activity in vivo.

Line 279. PlyAZ3 did not reduced the bacteria in CFS. Did you test the activity of endolysin in vitro in a solution similar to CFS? A low activity or stability of the protein in CFS would explain this result. Actually Cpl-1 has a short half-life time.

Line 334. The inability to cross the BBB or the low stability of the protein in this environment.

Specific conditions in CFS also might explain the low activity of the protein but in terms of bacteria metabolic state. Are bacteria growing actively in CFS?

Reviewer #3: The manuscript "Efficacy assessment of a novel endolysin…" by Valente et al, deals with the evaluation of the novel endolysin PlyAZ3aT as a therapeutic agent for treating pneumococcal meningitis in an animal model of infection. The objective is undoubtedly important because of the high incidence of mortality caused by this type of infection, especially in children, and by the increase of antibiotic resistance in certain pathogenic bacterial strains. A key factor adding to the difficulty of the challenge posed is the fact that the niche where the pneumococci causing the infection develop is the cerebrospinal fluid (CSF), which is protected by the blood-brain barrier (BBB).

Faced with this real challenge, the authors have tested the efficacy of the new endolysin in some in vitro experiments, and then in a rat model. But the conclusions of these assays could not be clearer. In their own words:

"PlyAZ3aT was not superior to placebo in improving survival of animals with pneumococcal meningitis“ (line 276).

"PlyAZ3aT did not significantly reduce bacterial loads in the CSF and blood over the course of infection when compared to placebo” (line 278).

“In contrast, PlyAZ3aT was not detected in the CSF in any of the animals tested, at any time point” (line 312).

These negative results do not admit any other interpretation and authors can only speculate on the reasons for these data and discuss possible alternatives to try to make this enzyme effective with another experimental protocol.

Because of these strong negative results, this manuscript does not provide any promising data to meet the proposed objectives. It is evident that it could only be evaluated positively if the authors succeed in developing an alternative method of enzyme delivery that reaches the CSF, and demonstrates that the chosen endolysin has sufficiently potent bactericidal effect to achieve a bacterial lethality that would make the treatment successful.

Although the substance of the evaluation is reflected above, the manuscript contains other serious flaws that deserve some comments:

· Authors claim that the endolysin PlyAZ3aT “was found within the genome of a particular strain of Streptococcus tigurinus”, but it is no comment whether the origin was the proper bacterium or a temperate phage integrated in the chromome.

· According to the alignment between the amino acid sequences of PlyAZ3aT and Cpl-1 (Fig. 1A), both enzymes displayed 47% amino acid similarity for the putative cell wall binding domain (CBD). But the CBD of Cpl-1 is built on six choline binding repeats, which have been demonstrated essential to be fully active for the whole enzyme, as well as in other examples of pneumococcal murein hydrolases, both from bacterial and phage origin. In the case of PlyAZ3aT a single “Cholin_bind_3” (from residue 225 to 296) is depicted in such Fig. 1A. Does it mean that there are no other choline binding repeats in this domain? What are the relevant characteristics of this domain beyond the single “Cholin_bind_3”?

· From the in vitro results presented in this study, it appears evident that PlyAZ3aT is much less active than Cpl-1, in terms of bactericidal activity. The reasons come, most likely, from the comments explained above. Thus, the eventual use of PlyAZ3aT as therapeutic agent against pneumococci does not represent any improvement compared with other endolysins already published.

In summary, the success of the ambitious goal set by the authors would only be achieved with an enzyme (endolysin) very active against pneumococcus and with a formulation capable to cross the BBB and reach the CSF.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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Efficacy assessment of a novel endolysin PlyAZ3aT for the treatment of ceftriaxone-resistant pneumococcal meningitis in an infant rat model

PONE-D-22-01149R1

Dear Dr. Leib,

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Kind regards,

Rosa del Campo

Academic Editor

PLOS ONE

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