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

BACKGROUND: Treatment failure in pneumococcal meningitis due to antibiotic resistance is an increasing clinical challenge and alternatives to antibiotics warrant investigation. Phage-derived endolysins efficiently kill gram-positive bacteria including multi-drug resistant strains, making them attractive therapeutic candidates. The current study assessed the therapeutic potential of the novel endolysin PlyAZ3aT in an infant rat model of ceftriaxone-resistant pneumococcal meningitis.
METHODS: Efficacy of PlyAZ3aT was assessed in a randomized, blinded and controlled experimental study in infant Wistar rats. Meningitis was induced by intracisternal infection with 5 x 107 CFU/ml of a ceftriaxone-resistant clinical strain of S. pneumoniae, serotype 19A. Seventeen hours post infection (hpi), animals were randomized into 3 treatment groups and received either (i) placebo (phosphate buffered saline [PBS], n = 8), (ii) 50 mg/kg vancomycin (n = 10) or (iii) 400 mg/kg PlyAZ3aT (n = 8) via intraperitoneal injection. Treatments were repeated after 12 h. Survival at 42 hpi was the primary outcome; bacterial loads in cerebrospinal fluid (CSF) and blood were secondary outcomes. Additionally, pharmacokinetics of PlyAZ3aT in serum and CSF was assessed.
RESULTS: PlyAZ3aT did not improve survival compared to PBS, while survival for vancomycin treated animals was 70% which is a significant improvement when compared to PBS or PlyAZ3aT (p<0.05 each). PlyAZ3aT was not able to control the infection, reflected by the inability to reduce bacterial loads in the CSF, whereas Vancomycin sterilized the CSF and within 25 h. Pharmacokinetic studies indicated that PlyAZ3aT did not cross the blood brain barrier (BBB). In support, PlyAZ3aT showed a peak concentration of 785 μg/ml in serum 2 h after intraperitoneal injection but could not be detected in CSF.
CONCLUSION: In experimental pneumococcal meningitis, PlyAZ3aT failed to cure the infection due to an inability to reach the CSF. Optimization of the galenic formulation e.g. using liposomes might enable crossing of the BBB and improve treatment efficacy.

Introduction

The emergence and the spread of antimicrobial resistance in the last decades are major threats to human health. Alternative treatment strategies targeting antibiotic-resistant bacteria are urgently needed. One such approach is the use of endolysins (or lysins). Lysins are bacteriophage-derived enzymes with peptidoglycan hydrolase activity and are thereby able to degrade the bacterial cell wall. Recent in vitro and in vivo results suggest that endolysins might be promising antimicrobial agents. First, they rapidly lyse bacterial cells including those that are multi-drug resistant [1-6]. Second, they act very specifically, killing only strains from closely related species and do not affect off-target commensal bacteria, which makes them less likely to disturb the physiologic microflora [1, 4, 7]. Third, the frequency of resistance emergence seems to be low [8, 9]. Finally, they appear safe; in 2 recent clinical trials, no detrimental effects were observed after topical [10] or intravenous [11] application in humans.

Streptococcus pneumoniae is an opportunistic human pathogen that can cause diseases like sinusitis, otitis media, pneumonia, septicemia, and meningitis. Meningitis due to pneumococci is associated with important morbidity and mortality. Despite the availability of appropriate antibiotic treatment, pneumococcal meningitis (PM) is associated with a high case-fatality rate of up to 37% in high-income countries [12, 13]. By the end of the 20th century, a concerning increase in antimicrobial resistance was recognized for S. pneumoniae [14]. This was mitigated by the implementation of the pneumococcal conjugate vaccines (PCV), which led to a concomitant decrease in the rate of invasive pneumococcal diseases (IPD) [15, 16] and overall antimicrobial resistance [17-19]. Yet, as high as 30% of infections caused by S. pneumoniae remain resistant to at least one antibiotic [20], and cephalosporin-resistance in PM represents an emerging threat [21]. Additionally, serotype redistribution caused by vaccine selection pressure has been observed [22-24], leading to the risk of the re-emergence and spread of non-vaccine serotypes that are resistant to antibiotics.

The efficacy of different pneumococcal endolysins (e.g. Cpl-1 and Pal) has been previously assessed in experimental in vivo models for pneumonia [25], endocarditis [26], bacteremia [4, 6], and otitis media [27]. In an animal model of PM, Cpl-1 reduced bacterial loads within the central nervous system (CNS) after a single intracisternal or intraperitoneal injection [28]. Overall, in principle endolysins seem attractive therapeutic candidates to treat pneumococcal infections.

So far, however, the translation of promising preclinical applications of endolysin into clinical trials has been strikingly slow. We adapted a well-established infant rat model of PM and evaluated the efficacy of a novel endolysin called PlyAZ3aT for the treatment of a ceftriaxone-resistant S. pneumoniae serotype 19A strain in a randomized, blinded, and controlled animal study. As PM affects in particular children under the age of 5 years, infant rats are especially well suited to mimic pediatric bacterial meningitis, since they reflect the developmental stage of this specific population.

Material and methods

Infecting bacterial strain

A ceftriaxone-resistant (in accordance with CLSI guidelines, MIC ≥ 2 μg/ml as determined by E-test, [Biomérieux, Nürtingen, Germany]) clinical isolate of S. pneumoniae, serotype 19A (identified as SPn28) was used. Bacteria were routinely grown overnight at 37°C in brain heart infusion broth (BHI, BD, USA), adjusted to an optical density (OD570) of 0.1, and then further diluted tenfold in fresh and pre-warmed BHI medium. The subculture was grown for 2 h to reach logarithmic growth phase. Colony forming units (CFU) were determined on Columbia sheep blood agar (CSBA) plates.

Expression and purification of PlyAZ3aT

Endolysin was produced as previously described, with minor modification [29, 30]. Briefly, the gene coding for PlyAZ3aT was amplified from the genome of Streptococcus tigurinus strain AZ3aT [31] by PCR using specific primers (5’- ATGAAGAAAAACGACTTATTCATCGACG-3’ and 5’-ATTTAGTGGTAACAATTAGTCCATCAGGTAATACATC-3’), cloned into plasmid pIN-IIIA and transformed into E. coli DH5α. Expression was induced by addition of 2% (w/v) lactose. PlyAZ3aT binds strongly to diethylaminoethyl (DEAE). Therefore, crude extracts of PlyAZ3aT were purified by fast protein liquid chromatography (FPLC) with HiTrap DEAE FF columns (GE Healthcare Bio-Science AB, Uppsala, Sweden) followed by endotoxin removal to < 1 endotoxin units (EU)/mg (ToxinEraser™ Endotoxin Removal Kit, GenScript, Leiden, Netherlands) and dialysis against phosphate-buffer saline (PBS, pH 7.4) (Slide-A-Lyzer™ Dialysis Flasks, ThermoFisher, Reinach, Switzerland). Residual endotoxin concentration was determined by ToxinSensor™ Chromogenic LAL Endotoxin Assay Kit (GenScript, Leiden, Netherlands) and endolysin concentrations using Pierce™ BCA Protein Assay Kits (ThermoFisher, Reinach, Switzerland).

Characterization of PlyAZ3aT

To assess similarities of PlyAZ3aT (GenBank: EMG32405.1) to the well-characterized pneumococcal endolysin Cpl-1 (GenBank: NP_044837.1) protein alignment was performed using BLASTp [32]. Killing efficacy of PlyAZ3aT was assessed on SPn28 and compared to vancomycin. SPn28 was grown to exponential phase and treated with either PlyAZ3aT (1 μg/ml, 100 μg/ml or 200 μg/ml), vancomycin (1 μg/ml) or PBS. Bacteria were incubated statically at 37°C with 5% CO2 and CFU were quantified before treatment, and 1, 2, 4 and 6 h after treatment. Prior to quantification on agar plates, 10 μl of PlyAZ3aT-treated samples was added to 90 μl of NaCl 0.85% containing 10% w/v choline (Sigma Aldrich, Buchs, Switzerland) in order to block further activity of the enzyme. Six biological replicates were performed for each condition. The effect of temperature and pH on enzymatic activity of PlyAZ3aT was evaluated as previously described by Son et al. [33].

Assessment of toxicity and inflammation in primary astroglial cell culture

The use of animal for cell culture experiments was approved by the animal care and experimentation committee of the canton of Bern, Switzerland (license no. BE 6/20). Primary astroglial cells were isolated and processed from 2-day old infant Wistar rats (central animal facility of the Medical Faculty of the University of Bern, Switzerland) as previously described [34, 35] with minor adaptations. After 11 days of cultivation, cells were seeded in 24-well plates (~250’000 cells/well) precoated with poly-L-ornithine (0.01 mg/ml in PBS, Buchs, Sigma-Aldrich). Three days later, astroglial cells were exposed for 24 h to (i) sterile PBS (pH 7.4), (ii) 10 μg/ml lipopolysaccharide (LPS) from E. coli 026:B6 (Sigma Aldrich, Switzerland), (iii) 100 μg/ml PlyAZ3aT or (iv) 2% Triton X-100 (Merck, Darmstadt, Germany). Production of NO2− was assessed in the supernatant of cell cultures as previously described [34, 35]. Viability of cells was assessed using an XTT assay, according to manufacturer’s instruction (Cell Proliferation Kit II, Sigma Aldrich, Buchs, Switzerland). The experiment was performed twice and in biological replicates of 18 for every condition.

Changes in inducible nitric oxide synthase (iNOS) expression were also assessed by immunofluorescence. Cells were fixed with 4% paraformaldehyde (PFA, Merck, Switzerland) diluted in PBS for 10 min at room temperature, followed by three washing cycles with sterile PBS. Permeabilization was done with 0.1% Triton X -100 (TX-100, Merck, Zug, Switzerland) for 5 min, followed by a 1 h blocking with blocking buffer (0.01% TX-100 + 2% bovine serum albumin, dissolved in PBS). Primary antibodies consisted of anti-GFAP mouse antibodies for astrocytes (dilution 1:1000; ThermoFisher, Reinach, Switzerland) and anti-NCOA3 rabbit antibodies for iNOS (dilution 1:500; Amsbio, Abingdon, UK). Secondary antibodies were goat anti-mouse Cy3 for astrocytes (dilution 1:500; ABCAM, Switzerland) and donkey anti-mouse Cy3 for iNOS (dilution 1:500; ThermoFisher, Switzerland), dissolved in blocking buffer. Secondary antibodies were added after overnight incubation of the primary antibodies at 4°C and three washing cycles with PBS and incubated for additional 2 h at room temperature. 4′,6′-diamidino-2-phenylindole (DAPI, ThermoFisher, Reinach, Switzerland) was used to visualize cell nuclei. Images were generated using fluorescence microscopy (OLYMPUS CKX53, Olympus LS, Germany).

Infant rat model of pneumococcal meningitis

All animal experiments were approved by the animal care and experimentation committee of the canton of Bern, Switzerland (license no. BE 5/20). Animals had access to water and food ad libitum and were kept in rooms with controlled temperature (22 ± 2°C) and natural light.

A well-established infant rat model for PM was used as previously described [34-38] and adapted to use the serotype 19A clinical isolate SPn28. SPn28 was passaged 3 times in infant rats before further investigations were performed.

Mixed-sex Wistar rats (outbred, 11-day old) and their dams were purchased from Charles River Laboratories (Sulzfeld, Germany). Bacteria in logarithmic growth phase were used as inoculum and were washed twice by centrifugation (3100xg, 10 min at 4°C) and resuspended in ice cold sterile saline (NaCl 0.85%), followed by dilution to reach the desired OD570 of 0.15.

Bacterial inoculation (10 μl containing 3.9 ± 0.25 x 107 CFU/ml SPn28) was performed by injection into the cisterna magna. This amount of inoculum was chosen according to the results of LD90 experiments (S1 Fig). CSF sampling was performed by puncture of the cisterna magna using a 30-gauge needle and blood sampling by puncturing the facial vein with a 20-gauge needle and collection of 1 drop (approximately 50 μl). Clinical signs of meningitis arose at 17 hours post infection (hpi), and this correlated with quantitative analysis of bacterial titers in the CSF. This timepoint was chosen for the first treatment dose. Clinical scoring and weighing were performed at 17, 24 and 42 hpi as described previously [36]. Briefly, clinical score was determined as follows: 5 = healthy with normal behavior; 4 = turns upright within 5 s, weight loss and/or appearance of fur; 3 = turns upright within 30 s; 2 = inability to turn upright; 1 = marked diminished motor activity, coma. Animals were sacrificed upon reaching a score of ≤ 2 to meet ethical requirements. At 42 hpi all remaining animals were sacrificed by intraperitoneal (i.p.) injection of pentobarbital (Esconarkon®, 150 mg/kg, Streuli Pharma AG, Switzerland) followed by perfusion with 4% PFA. Brains were collected for histomorphometric analysis of cortical damage and hippocampal apoptosis, which was performed as previously described [35, 39, 40].

Pharmacokinetic study

In order to assess pharmacokinetic properties of PlyAZ3aT, infected animals were treated with a single i.p. injection of either 100 mg/kg or 400 mg/kg b.w. PlyAZ3aT at 17 hpi. Animals were separated in 2 groups (n = 16 per group) with different sample withdrawal times, in order to avoid excessive sampling frequency from the same animals. CSF and blood samples were taken at 15 min, 4 h and 12 h for the first group and at 2 h, 6 h and 12 h for the second group. If animals reached a clinical score ≤ 2, samples were taken after sacrifice and included into the analysis. Blood was collected in serum Microvettes® 200 Z (Sarstedt, Nümbrecht, Germany).

Treatment study

The procedure for the randomized, blinded and controlled study of PM was as follows. CSF sampling was performed at 17 hpi to confirm meningitis and all animals were randomly allocated into 3 groups using GraphPad (https://www.graphpad.com/quickcalcs/randomize1.cfm). Treatment groups were (i) placebo (sterile PBS), (ii) PlyAZ3aT 400 mg/kg b.w. and (iii) Vancomycin 50 mg/kg b.w. (Vancocin, in saline, Teva, Petach Tikwa, Israel). Treatments were applied twice daily (12-hour time interval) through an i.p. injection. All persons involved in animal treatment were blinded regarding treatment groups until the end of the experiment. The primary endpoint was survival at 42 hpi. Secondary endpoints were (i) CFU in CSF and blood at 6 and 25 h post treatment and (ii) extent of cortical necrosis and hippocampal apoptosis in histopathological analysis.

The volume of CSF recovered from each animal during sampling was restricted and ranged from 5 to 15 μl. This resulted into a limit of detection for CFU quantification of 1 x 103 CFU/ml.

Animals displaying a clinical score of ≤ 2 prior to the time of randomization were sacrificed immediately and excluded from the analysis. Similarly, animals without established meningitis at randomization, defined by bacterial titers in CSF <1 x 104 CFU/ml at 17 hpi, were excluded from the analysis.

Quantification of PlyAZ3aT in CSF and blood samples

The amount of PlyAZ3aT in CSF and serum samples was assessed by quantitative near infrared Western Blot (NIR-WB). Proteins in CSF or serum were separated using 10% SDS-polyacrylamide gels. Western blotting and immunostaining were performed with WesternBrightTM MCF and MCF-IR kit (Advansta, San Jose, USA) according to manufacturer’s instructions. PlyAZ3aT was detected as a 41 kD band, by using a primary antibody (custom-produced PlyAZ3aT-specific polyclonal rabbit antibody, David’s Biotechnology, Regensburg, Germany). Fluorescence was measured using a Fusion FX6 EDGE System (Vilber Lourmat, Collégien, France) with an exposure time of 2 min. Quantification of PlyAZ3aT was performed by comparison to a standard curve of PlyAZ3aT ranging from 2–0.02 μg using FIJI (version 1.8). The limit of detection was 10 μg/ml.

Analysis of cytokines in cell culture supernatant

Inflammatory cytokines known to be upregulated in PM (IL-1β, IL-6 and TNF-α) were measured in cell supernatants and CSF samples using a magnetic multiplex assay (Rat Magnetic Luminex® Assay, R&D Systems, Bio-Techne) on a Bio-Plex 200 station (Bio-Rad Laboratory) as reported previously [35, 41].

Plate lysis assay for endolysin resistance testing

Plate lysis assay was performed as described previously [42-44] to assess resistance and activity against various pneumococcal serotypes. Bacteria recovered from animals was grown to exponential phase and plated on a CSBA to produce uniform bacterial lawns. PlyAZ3aT (0.5 μg) was spotted onto the plate and incubated overnight. Bacteria were classified as susceptible to PlyAZ3aT if a clear lysis zone was observed.

Statistical analysis

Statistical analyses were performed using GraphPad Prism® (San Diego, USA). Differences in survival were calculated using log-rank tests. Differences between means of non-normally distributed groups (cytokines, cortical necrosis, hippocampal apoptosis) were assessed using Kruskal-Wallis test with Dunn’s multiple comparison. Differences in CFU reduction in CSF and blood of animals were calculated using Fisher’s exact test. A p value < 0.05 was considered statistically significant.

Results

PlyAZ3aT lyses ceftriaxone-resistant S. pneumoniae in vitro

PlyAZ3aT is a bacteriophage-derived endolysin found within the genome of S. tigurinus strain AZ3aT (Accession number GCA_000344275.1). Alignment of the amino acid sequence of PlyAZ3aT and Cpl-1 (up to now the most extensively characterized pneumococcal endolysin) displayed 80% similarity for the putative catalytic domain and 47% similarity for the putative cell-wall binding domain (Fig 1A), suggesting PlyAZ3aT might be active against S. pneumoniae. Indeed, PlyAZ3aT exhibit a broad-spectrum lytic activity against S. pneumoniae, lysing 18 distinct serotypes including both PCV 13 (3, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F) and non-PCV 13 serotypes (11A, 15A, 17F, 20, 22F, 23B and 35B), as tested by plate lysis assay. No resistance to the endolysin activity could be detected among the tested isolates (n = 18). PlyAZ3aT lysed S. pneumoniae efficiently (>50% of maximal efficacy) at temperatures ranging from 25–42°C (Fig 1B) and for pH ranging from 5–7. Optimal activity occurred at pH 6, while pH ≥ 9 completely inactivated the enzyme (Fig 1B). In time-kill curve assays, PlyAZ3aT showed a dose-dependent killing of the ceftriaxone-resistant strain SPn28. The bacteria were not eradicated even after 6 h of exposure to the lysin at the highest concentration of 200 μg/ml (Fig 1C). In contrast, vancomycin displayed a bactericidal effect and reduced counts below the detection limit within the same time period (Fig 1C).

Fig 1

(A) Alignment of the amino acid sequences of PlyAZ3aT and Cpl-1. Each domain is highlighted in a different color. The catalytic domain in blue, the cell-wall binding domain in orange and the linker in green. PlyAZ3aT displays six choline binding repeats marked in red. (B) Lytic activity of PlyAZ3aT after exposure for 10 min to different temperatures and pH. Relative lytic activity was assessed by measuring the reduction in OD570 and comparing it to the condition with the maximal reduction (data point marked in red and defined as 100%). (C) Time-kill curves for S. pneumoniae strain SPn28 exposed to PlyAZ3aT or vancomycin. The dotted line represents the limit of detection. CFU, colony forming units. All experiments were performed in triplicate. Data are presented as mean ± standard deviation.

PlyAZ3aT failed to control ceftriaxone-resistant pneumococcal meningitis

In primary astroglial cell cultures, no direct toxicity of PlyAZ3aT was observed (S2A and S2B Fig). Exposure led to a minimal inflammatory response when compared to the inflammatory control LPS as inferred by NO2- and cytokine levels (S2C and S2D Fig), suggesting the protein was safe for animal administration. Overall, 38 infant Wistar rats were included in the treatment study, which was performed in 2 independent experiments. At the time of randomization (17 hpi), 8 animals had to be excluded due to a clinical score ≤ 2. The 30 remaining animals were randomly allocated to receive either (i) placebo (n = 8), (ii) PlyAZ3aT (n = 11) or (iii) vancomycin (n = 11) (S3 Fig). Post hoc additional 4 animals not having established meningitis (<1 x 104 CFU/ml in the CSF at 17 hpi) had to be excluded (n = 3 for PlyAZ3aT, n = 1 for vancomycin).

PlyAZ3aT was not superior to placebo in improving survival of animals with PM (p > 0.05, Fig 2A). In contrast, the standard-of-care antibiotic vancomycin significantly improved survival with a rate of 70% survival (p = 0.02 versus PBS and PlyAZ3aT, Fig 2A). PlyAZ3aT did not significantly reduce bacterial loads in the CSF and blood over the course of infection when compared to placebo (p > 0.05, Table 1). On the opposite, vancomycin-treated animals displayed a consistent reduction of bacterial loads in both CSF and blood within the first 6 h after start of therapy (CSF p = 0.0182 and blood p = 0.021 compared to placebo, Table 1). In surviving animals under vancomycin treatment, the infection was cleared within 25 h of treatment (S4 Fig). The kinetics of bacteria in blood of PlyAZ3aT and placebo treated animals are displayed in S4 Fig. Finally, the extent of cortical necrosis and the number of apoptotic cells of the subgranular zone of the dentate gyrus were similar among all groups (p > 0.05 for all comparisons) (S5A and S5B Fig).

Fig 2

Treatment of ceftriaxone-resistant pneumococcal meningitis using PlyAZ3aT.

(A) Kaplan-Meier survival curve of Wistar rats treated with either PBS (n = 8), 400 mg/kg PlyAZ3aT (n = 8) or 50 mg/kg Vancomycin (n = 10). Treatment was applied intraperitoneally twice daily. Significance was determined by log-rank test. (B) Anti-PlyAZ3aT near-infrared western blots of serum or CSF samples recovered from one animal treated with a single intraperitoneal injection at 15 min and 4 h after injection. (C) Pharmacokinetic assessment of PlyAZ3aT in serum of Wistar pups (n = 16 per group) after one single intraperitoneal injection. Limit of detection 10 μg/ml. Data are presented as mean ± standard deviation.

Table 1

Comparison of the decrease of CFU in CSF and blood between treatment groups within the first 6 h after treatment.

(A) Comparison of CFU reduction in CSF and (B) in blood. Statistical significance was determined using Fisher’s exact test. Animals displaying bacterial loads below the limit of detection throughout the experiment or missing follow-up sampling were excluded from the analysis.

A) Numbers of animals displaying decrease of CFU in CSF
CSF	Decrease	No Decrease	Comparison to PBS
PBS	1	3
PlyAZ3a T	2	4	p > 0.05
Vancomycin	8	0	p = 0.0182
B) Numbers of animals displaying decrease of CFU in blood
CSF	Decrease	No Decrease	Comparison to PBS
PBS	2	5
PlyAZ3a T	6	1	p > 0.05
Vancomycin	6	0	p = 0.021

Reduced bioavailability of PlyAZ3aT in CSF

We further investigated whether the lack of efficacy of PlyAZ3aT in the experimental meningitis model was associated either with the emergence of endolysin resistance or with an altered pharmacokinetic profile. First, bacteria recovered from PlyAZ3aT treated animals (60 colonies in total) were tested for resistance and all remained susceptible to lysis. Second, PlyAZ3aT was quantified from the serum and CSF of infected animals receiving a single dose of either 100 mg/kg or 400 mg/kg b.w. PlyAZ3aT. PlyAZ3aT was detected in the serum (Fig 2B), peaked after 2 h of intraperitoneal administration (268 μg/ml for 100 mg/kg group and 785 μg/ml for 400 mg/kg group), and was detected up to 12 h in the 400 mg/kg BW group, albeit at low levels (Fig 2C). In contrast, PlyAZ3aT was not detected in the CSF of any of the animals tested, at any time point (Fig 2B, limit of detection 10 μg/ml).

Discussion

Despite the introduction of the pneumococcal conjugate vaccine 13 (PCV13), S. pneumoniae serotype 19A remains a major causative of invasive pneumococcal disease and meningitis [22, 45]. In addition, serotype 19A is among the major serotypes associated with penicillin or cephalosporin resistance [22]. Having a wider range of treatment alternatives to combat ceftriaxone-resistant pneumococci is of necessity. Herein, we investigated whether a novel phage lysine recently isolated from S. tigurinus strain AZ3aT [31] would be effective for the treatment of S. pneumoniae serotype 19A either in vitro or in vivo, in an experimental bacterial meningitis model in infant rats. In vitro, PlyAZ3aT showed a broad host-range and was able to lyse all tested S. pneumoniae serotypes independently from their antibiotic resistance profile. PlyAZ3aT induced a quick and dose-dependent reduction of bacterial loads of the ceftriaxone-resistant serotype 19A. Furthermore, the endolysin was safe and did not induce any evident direct toxic effect in astroglial cell cultures. Only minor inflammatory reactions were observed, mostly related to residual endotoxin activity.

In vivo, PlyAZ3aT failed to improve survival, to reduce bacterial numbers in the CSF or to prevent systemic spreading of the infection in blood compared to placebo. On the other hand, vancomycin significantly increased survival to 70%, which is in accordance with survival rates observed in previous studies when using standard of care treatment in this animal model [34, 35, 38]. PlyAZ3aT showed an important pharmacokinetic limitation, as endolysin could not be detected in CSF of endolysin treated animals most likely as a result of its inability to cross the BBB or the low stability of the protein in this environment. A possible solution to bypass this limitation is the use of direct intracisternal application, which is not devoid of problems either, as shown in a previous investigation assessing the pneumococcal endolysin Cpl-1 in the similar experimental setting [28]. There, Cpl-1 exhibited a short half-life time of ~16 min after direct intracisternal application, leading to bacterial regrowth within 4 h and prompting the necessity of repeated intracisternal injections, which on their part where harmful [28]. An intracisternal-sustained release might help to optimize the pharmacokinetic profile. In bacterial meningitis, such an opportunity might be found when an external ventricular drain (EVD) is available, allowing for a continuous delivery of endolysin directly to the CNS thereby circumventing the BBB. Alternatively, innovative galenic formulations (e.g. using liposomes) might improve the ability of the endolysin to cross the BBB allowing for an intravenous administration.

Taken together, our results highlighted the pitfalls and drawbacks of the application of PlyAZ3aT, and possibly endolysins in general, in bacterial meningitis. Despite a well described break down of the BBB in bacterial meningitis the BBB seems to remain largely impermeable to such phage proteins. As such, endolysins might be more suitable for the treatment of other bacterial infections as it was shown for Cpl-1, such as bacteremia [4, 6], pneumonia [25] or endocarditis [26] where the endolysin can efficiently and readily reach the infection site.

Adaptation of a ceftriaxone-resistant pneumococcal strain (serotype 19A) to the infant rat model of meningitis.

Kaplan-Meier survival curves for LD90 finding of the new strain (A) before and (B) after 3 passages in infant rats. 10 μl of the corresponding inoculum were injected.

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PlyAZ3aT induces a minimal inflammatory reaction with no direct toxicity in confluent primary astroglial cell cultures.

Cells were exposed for 24 h to either PBS (negative control), LPS (positive control), PlyAZ3aT or TritonX (membrane disrupting agent). (A) Immunohistological staining in astroglial cell cultures, iNOS is stained in green (anti-iNOS), astroglia is stained in red (anti-GFAP) and cell nuclei in blue (4′,6-Diamidin-2-phenylindole, DAPI). (B) Viability of cells was confirmed by using an XTT-assay (the mean of PBS treated cells is defined as 100%). (C) Production of NO2- measured as an index for nitric oxide (NO) release. (D) Levels of IL-1β, TNF-α and IL-6. Data are presented as mean ± standard deviation. Statistical differences between groups were assessed using Kruskal-Wallis test with Dunn’s multiple comparisons; *p<0.05, **** p<0.0001.

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Flowchart of the randomized, blinded and controlled experimental study.

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Tracked course of bacterial loads in CSF (black) and blood (purple) in each single animal.

Tracking of bacterial loads displayed for the first six hours or until the end of the experiment (25 hours post treatment). Dots connected with lines represent repeated sampling in one single animal. CFU, colony forming unit.

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Histopathological assessment of cerebral complications after acute pneumococcal meningitis.

(A) Comparison in percentage of necrotizing cortex between groups and (B) number of apoptotic cells in the hippocampus. Animals reaching the end of the 42 hours trial are represented by closed circles and succumbed prematurely to the infection by crosses. Statistical significance was assessed using Kruskal-Wallis test with Dunn’s multiple comparisons, ns, not significant.

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Raw images of the western blots displayed in Fig 2B.

The red rectangle corresponds to the section displayed in Fig 2B. Lanes not included in the final figure are marked with an “X”.

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Overview of the raw data of CFU detected in CSF.

Bacterial loads are displayed as Log10 (CFU). Hpt, hours post treatment. N/A, not available due to failed sampling. Limit of detection is 3 Log10.

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Overview of the raw data of CFU detected in blood.

Bacterial loads are displayed as Log10 (CFU). Hpt, hours post treatment. N/A, not available due to failed sampling. Limit of detection is 3 Log10.

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Spreadsheet containing raw data to Figs 1 and 2.

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Spreadsheet containing raw data to supplemental figures.

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16 Feb 2022

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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 #2: No

Reviewer #3: No

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28 Mar 2022

RE: Response to review: Efficacy assessment of a novel endolysin PlyAZ3aT for the treatment of ceftriaxone-resistant pneumococcal meningitis in an infant rat model’.

Dear Dr. del Campo

We thank the editor and reviewers for their helpful suggestions. Below, we have included a point-by-point response to each comment.

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.

Answer: We appreciate the positive response of reviewer #1.

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.

Answer: Thank you for this pertinent comment. Even if the endolysin is not able to totally eradicate the infection in vitro we hypothesized, that, in vivo, reducing the bacterial loads in the initial phase of the disease would let enough time to the host to mount a sufficiently robust immune answer able to eradicate the remaining living bacteria. However, we do agree to the conclusion that this endolysin (in its current formulation) does not seem suitable in this infection setting and that the results demonstrated in this manuscript are in essence negative results.

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.

Answer: We indeed suggest another model for delivery across the blood brain barrier, using liposome or direct intracisternal injection. However, in the discussion, we also suggested that, as such, endolysins might be more suitable for the treatment of other bacterial infections such as bacteremia, pneumonia or endocarditis. The actual results we provide do not warrant enough ethical justification for the use of additional animals to test this endolysin in the same setting. Still, in our opinion, our negative results in the in vivo model of meningitis are of importance to the scientific community and this is why we submitted the current manuscript. To our opinion, it is important that negative results obtained from the use of animals are also published.

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.

Answer: The statement is true; however, we don’t perceive a question in it. To our opinion, the reason for the observed residual inflammatory reaction is the most plausible.

Finally, it should be explained why the detection limit of bacterial counts is 3 log10 (CFU/ml).

Answer: This is due to the small volume of CSF which can be recovered from infant wistar rat, which ranges from 5 to 15 �  l per sampling, limiting the amount of sample volume which can be used to quantify the bacterial loads. We routinely dilute 5ul CSF into 495ul PBS and plate out 100ul of the first dilution, this leads to the limit of detection used in this project.

Measure: We added the following text to better clarify this fact (lines 200 - 202): The volume of CSF recovered from each animal during sampling was restricted and ranged from 5 to 15 µl. This resulted into a limit of detection for CFU quantification of 1 x 103 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".

Answer: We acknowledge the impression that the sentence seems a headline.

Measure: We changed the sentence to (line 28). Efficacy of PlyAZ3aT was assessed in a 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.

Answer: We thank the reviewer for this useful comment.

Measure: The sentence has been changed accordingly (line 242). PlyAZ3aT is a bacteriophage-derived endolysin found within the genome of S. tigurinus strain AZ3aT.

Discussion

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

Answer: We thank the reviewer for this remark.

Measure: An additional sentence and the references have been added (lines 355 and 356). As such, endolysins might be more suitable for the treatment of other bacterial infections as it was shown for Cpl-1, such as bacteremia (4,27), pneumonia (25) or endocarditis (26) where the endolysin can efficiently and readily reach the infection site.

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.

Answer: We share the revisers concern and are thankful for the suggestion plot data from Table S1 in a graph.

Measure: Results of Table S1 and S2 are plotted in a graph shown in Figure S4. It is clearly stated that animals were excluded from this analysis and all the raw data is presented in Table S1 and S2.

The abbreviation N/A should be explained.

Answer: We concur

Measure: Additional details have been added (lines 569 and 573). Hpt, hours post treatment. N/A, not available due to failed sampling. Limit of detection is 3 Log10.

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).

Answer: we thank the reviewer for pointing out this issue in Fig.1A.

Measure: The figure was adapted accordingly, and additional information was added in the figure caption. Alignment of the amino acid sequences of PlyAZ3aT and Cpl-1. Each domain is highlighted in a different color. The catalytic domain in blue, the cell-wall binding domain in orange and the linker in green. PlyAZ3aT displays six choline binding repeats marked in red.

Reviewer #2:

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.

Answer: We appreciate the positive response of reviewer #2.

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.

Answer: We agree that this might be an interesting comparison. Unfortunately, we did not have access to Cpl-1 thus this comparison was not done. However, results from the study performed by Grandgirard et al. are hard to compare with this study due to several differences and thus it remains unclear whether there is a real difference between Cpl-1 and PlyAZ3aT. First, a different bacterial strain was used (ceftriaxone-susceptible serotype 3 vs. ceftriaxone-resistant serotype 19A) and PlyAZ3aT was not tested on pneumococcus serotype 3. Second, a different quantification method for the endolysin was used (NIR-WB vs. WB and spot densitometry) displaying different limits of detection.

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.

Answer: In vitro studies with CSF or "CSF-like" media are difficult to perform in static conditions, since the pH of CSF increases significantly after sampling from patients or animals and that these changes in pH after sampling of CSF may affect survival, growth and antibiotic killing of bacterial strains in vitro (DOI: 10.1159/000444263). To tackle this issue, we have performed in vitro tests using a wide range of pHs and temperatures, and found that the activity was preserved in those conditions that can be estimated as similar in the CSF. We agree that, in analogy to Cpl-1, the half-life of PlyAZ3 is probably short. In order to prevent issues with the short half-life time we chose to apply higher amounts of the endolysin by intraperitoneal injections. By choosing this approach we were able to achieve a sustained release lasting for more than six hours as displayed in Figure 2C.

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

Answer: We thank the reviewer for this suggestion.

Measure: The sentence has been changed accordingly (line 339). PlyAZ3aT showed an important pharmacokinetic limitation, as endolysin could not be detected in CSF of endolysin treated animals most likely as a result of its 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?

Answer: We agree, it might well be that specific conditions of the CSF have an impact. However, as can be seen in Figure S4 bacteria do actively grow in CSF and thus it is not likely that they are in a metabolically dormant state.

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.

Answer: We agree with the comments. Nevertheless, we think it is of utter importance to publish negative results because it is a crucial step towards limiting publication bias. Especially, considering that animals were used for these experiments, it is ethically important that the scientific community have access the results, even if negative by essence. We had a clear hypothesis and tested it adequately. Our results inform the scientific community that the use of endolysins as therapeutic agents can have some pitfalls which have to be considered, one of them being limited penetration into the CSF.

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.

Answer: We agree with the reviewer’s comment

Measure: In line 241 we stated that the endolysin was found within the genome of S. tigurinus AZ3aT. Thus, it is most likely that this endolysin derives from a temperate phage. Additional details have been added (line 242). PlyAZ3aT is a bacteriophage-derived endolysin found within the genome of S. tigurinus strain AZ3aT (Accession number GCA_000344275.1).

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”?

Answer: Similar to Cpl-1, also PlyAZ3aT displays six choline binding repeats with the consensus sequence TGW-b-(K,Q)DNGSWYYLN-x-SG-z-M-x1-2.

Measure: This information was also included in Figure 1A.

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.

Answer: Our data does not allow such a conclusion as we were not able to test Cpl-1 on the given strain (ceftriaxone-resistant serotype 19A) and in the same setting.

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.

Answer: We agree to this and our search for better treatment options in bacterial meningitis will continue.

In light of these improvements, we hope that the manuscript is now ready for publication in PLOS ONE.

Sincerely yours,

Stephen L. Leib

Submitted filename: Response to Reviewers_V2_PONE-D-22-01149.docx

Click here for additional data file.

30 Mar 2022

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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PLOS ONE

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Reviewers' comments:

4 Apr 2022

PONE-D-22-01149R1

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

Dear Dr. Leib:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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