Pulsed dosing and extended daily dosing of oral vancomycin do not facilitate clearance of Clostridioides difficile colonization in mice

ABSTRACT

Vancomycin taper and pulse regimens are commonly used to treat recurrent Clostridioides difficile infections, but the mechanism by which these regimens might reduce recurrences is unclear. Here, we used a mouse model to test the hypothesis that pulse dosing of vancomycin after a 10-day treatment course enhances clearance of C. difficile from the intestinal tract. Mice with C. difficile colonization received 10 days of once-daily oral vancomycin followed by 20 days of treatment with saline (controls), daily vancomycin, or pulse dosing of vancomycin every 2 or 3 days. Stool samples were collected to measure the concentration of C. difficile during and after treatment, vancomycin concentrations, and growth of vegetative C. difficile during every 3 days dosing. Pulse dosing of vancomycin was not effective in maintaining suppression of C. difficile (P > 0.05 in comparison to saline controls); growth of vegetative C. difficile occurred between pulse doses when vancomycin decreased to undetectable levels. Daily dosing of vancomycin suppressed C. difficile during treatment, but recurrent colonization occurred after treatment in more than 75% of mice, and by post-treatment day 14, there was no significant difference among the control, pulse dosing, and daily dosing groups (P > 0.05). These findings demonstrate that pulse dosing of vancomycin every 2 or 3 days does not facilitate the clearance of C. difficile spores in mice. Studies are needed to examine the impact of vancomycin taper and pulsed regimens in patients.

INTRODUCTION

Oral vancomycin administered as a tapered and/or pulsed regimen is commonly used for the treatment of recurrent Clostridioides difficile infection (CDI) (1, 2). These regimens typically include a 10- to 14-day course of oral vancomycin at a dose of 125 mg four times per day followed by a tapering dose over 2 weeks followed by “pulsed” dosing with 125 mg once every 2 or 3 days for 2 to 8 weeks (1). Standard vancomycin regimens achieve persistent high concentrations in stool (1,000 to 2,000 µg/g stool) and cause marked disruption of the intestinal microbiota (3 – 5). It has been proposed that tapered and pulsed regimens might reduce the risk for recurrence because the pulsed dosing results in lower vancomycin concentrations and antibiotic-free periods that may allow restoration of the indigenous microbiota (2). However, in a mouse model, a vancomycin taper and pulse regimen resulted in an alteration of the microbiota providing colonization resistance to exogenously introduced C. difficile and vancomycin-resistant enterococci that persisted during and for at least 18 days after treatment (6).

Even in the absence of microbiota recovery, vancomycin taper and pulse regimens could potentially reduce the risk for recurrence by facilitating clearance of spores. There is some evidence that persistent detection of C. difficile spores in stool at the end of treatment is associated with an increased frequency of CDI relapse (2). Taper and pulse regimens might be beneficial in providing increased time for clearance of spores. It has also been proposed that pulse dosing might facilitate clearance of spores by allowing germination during drug-free intervals with subsequent killing of germinated spores upon re-dosing (2). Here, we used a mouse model to test the hypothesis that pulse dosing of vancomycin after a 10-day course of daily treatment enhances clearance of C. difficile from the intestinal tract. For comparison, we evaluated the impact of a 10-day course of vancomycin with no subsequent dosing and a 10-day course followed by extended daily dosing for the same duration with the same total drug exposure.

RESULTS

Concentrations of vancomycin in stool

The mean concentration of vancomycin in stool on day 10 of the initial treatment was 1,923 µg/g (range, 439 to 3,704 µg/g). Figure 1 shows concentrations of vancomycin in the stool of mice after dosing during pulsing in the pulse every 2-day (10X) and pulse every 3-day (10X) groups. Vancomycin concentrations [mean ± standard error (SE)] peaked 8 hours after dosing at 168 ± 32 µg/g in the pulse every 2-day group and 253 ± 58 µg/g in the pulse every 3-day group. At 24 hours post-dosing, no vancomycin was detectable in the stool of the pulse every 2-day group, and a mean of 0.4 µg/g was detected in the pulse every 3-day group. No vancomycin was detectable in stool specimens collected 48 hours after dosing for either group.

Fig 1.

Concentrations of vancomycin in stool of mice treated with a 10-day course of oral vancomycin followed by pulsed dosing every 2 or 3 days for 20 days (N = 6 mice per group). Stool samples were collected at 4, 8, 24, and 48 hours after a pulse dose between days 10 and 20 of the pulse dosing period. Mean concentrations of vancomycin (N = 5 mice) are shown at each time point. The bars show the vancomycin dose in milligram per day. Error bars show standard error.

Impact of vancomycin pulse dosing and extended daily dosing on clearance of C. difficile

Figure 2 provides an overview of the treatment groups and timeline for dosing of vancomycin. After completion of the initial 10 days of vancomycin treatment, mice were divided into the treatment groups including three levels of cumulative extended daily dosing and three levels of pulsing (none, every 2 days, and every 3 days) intended to compare extended daily dosing versus every 2- or 3-day dosing while controlling for total vancomycin exposure. In Fig. 2, X indicates a dose of 1.125 mg or 37.5 mg/kg (i.e., one-fourth of the dose used for initial treatment and equivalent to 125 mg once daily in humans). Thus, a 10X dose indicates that mice received a cumulative total dose of 10 times X or 1,250 mg during the extended daily/pulse dosing period. The rationale for the daily cumulative doses of 10X and 7X was to provide total doses equivalent to the total doses when vancomycin was dosed every 2 days (10X) or every 3 days (7X); the 20X daily dosing group was included to test whether a higher daily dosage of vancomycin would be more effective than lower daily doses for clearance of colonization.

Fig 2.

Timeline showing treatment groups and vancomycin dosing regimens. X indicates a dose of 1.125 mg or 37.5 mg/kg (i.e., one-fourth of the dose used for initial treatment and equivalent to 125 mg once daily in humans).

Figure 3 shows the effects of the 10X cumulative vancomycin treatment regimens on initial colonization and subsequent clearance of C. difficile versus controls including the average (+SE) concentration of C. difficile (A) and the percentage of mice with detectable colonization (B). Results for the 20X and 7X daily dosing treatment regimens were similar to the 10X daily dosing regimen, and results for the 7X pulse every 2-day and 7X pulse every 3-day regimens were similar to the 10X pulse regimens (Supplementary material). All mice became colonized with high concentrations of C. difficile after ingestion of spores in conjunction with subcutaneous clindamycin for 2 days. During the 10-day course of oral vancomycin treatment, C. difficile concentrations decreased to undetectable levels in all mice except one mouse in the pulse every 3-day (7X) group which had 2.4log₁₀ CFU. During the 10-day oral vancomycin treatment, there were no significant differences between the control, extended daily dosing, and pulse dosing groups (P > 0.05).

Fig 3.

Effect of vancomycin treatment on concentration of Clostridioides difficile (A) in stool of mice and the percentage of mice with positive cultures for C. difficile (B). After the establishment of colonization, all mice received 10 days of once-daily oral vancomycin and then were divided into treatment groups including control (saline), pulse dosing every 2 or 3 days, and extended daily dosing. X indicates a dose of 1.125 mg which was equivalent on a mg/kg basis to a 125 mg dose in humans. Concentrations of C. difficile were measured in stool specimens collected on days 5, 10, 15, and 20 of the extended daily/pulse dosing and on days 2, 7, and 14 after the last extended daily/pulse dose. Error bars show standard error. Vanco, vancomycin.

During the extended daily and pulse dosing period, recurrent colonization occurred in 11 of 13 (85%) control mice and in >62% of mice in the groups receiving pulse dosing of vancomycin, and there was no significant difference in the concentrations of C. difficile for control versus pulse dosing mice (P > 0.05). In contrast, nearly all mice in the three groups receiving extended daily dosing of vancomycin maintained undetectable C. difficile in stool throughout the 20-day extended daily/pulse period (P < 0.001 for extended daily versus control and pulse dosing groups).

During the post-treatment period after completion of the extended daily or pulse dosing, the concentration of C. difficile was significantly higher in the pulse dosing groups versus the control and extended daily dosing groups on post-treatment days 2 and 7 (P < 0.01). However, by post-treatment day 14, there was no significant difference in the concentration of C. difficile in the control, pulse dosing, and extended daily dosing groups (P > 0.05). For the four pulse dosing groups, colonization was detected on post-treatment day 14 in >75% of mice. For the control group, 8 of 13 (62%) mice remained colonized on post-treatment day 14 (34 days after the last vancomycin dose), but the level of colonization was substantially reduced (mean ± SE, 2 ± 0.6log₁₀CFU/g stool).

Evaluation of spore germination and outgrowth during the period of pulse dosing

Of the 7 mice in the pulse every 3-day (10X) group evaluated, vegetative C. difficile (1.3 to 3log₁₀ CFU/g stool) was detected in the stool of 3 (43%), 4 (57%), and 5 (71%) mice at 24, 48, and 72 hours after vancomycin dosing, respectively.

Effect of the antibiotics on indigenous facultative Gram-negative bacilli by culture

In addition to increasing susceptibility to colonization by exogenous microorganisms, antibiotic-induced alteration of colonization resistance results in overgrowth of indigenous components of the intestinal microbiota that are resistant to the administered antibiotics (3 – 6). Because vancomycin does not have significant activity against indigenous facultative Gram-negative bacilli, we measured concentrations of this component of the microbiota as a measure of whether vancomycin treatment altered colonization resistance.

Figure 4 shows the effects of the 10X cumulative vancomycin treatment regimens on indigenous facultative Gram-negative bacilli. Results for the 20X and 7X daily dosing treatment regimens were similar to the 10X daily dosing regimen, and results for the 7X pulse every 2-day and 7X pulse every 3-day regimens were similar to the 10X pulse regimens (Supplementary material). Vancomycin treatment resulted in overgrowth of Gram-negative bacilli during the initial 10-day vancomycin course with no difference in concentration for the different groups (P > 0.05). In the control group, levels of Gram-negative bacilli in stool decreased from 8 to 5.1log₁₀ CFU/g stool by 15 days after discontinuation of vancomycin and was maintained at ~5log₁₀ CFU/g stool through the end of the study. For the vancomycin extended daily and pulse dosing groups, Gram-negative bacilli were maintained at significantly higher concentrations than the control group at all time points after the initial 10-day treatment course (P < 0.001). All Gram-negative bacilli recovered from the highest plated dilution were lactose-fermenting organisms and all isolates subjected to identification were Escherichia coli.

Fig 4.

Effect of antibiotic treatment on the concentrations of facultative Gram-negative bacilli in stool by culture. Mice received oral vancomycin for 10 days followed by vancomycin extended daily dosing or pulse dosing every 2 or 3 days or daily sterile water (control) for 20 days. X indicates a dose of 1.125 mg which was equivalent on a mg/kg basis to 125 mg dose in humans. Error bars show standard error. CFU, colony-forming unit.

Effect of antibiotic treatment on the indigenous microbiota by deep-sequencing analysis

Figure 5 shows the impact of treatment on the total bacterial diversity in the stool of mice in the saline control, extended daily dose (20X cumulative dose), extended daily dose (10X cumulative dose), pulse every other day (10X cumulative dose), and pulse every 3 days (10X cumulative dose) groups. Alpha (Shannon diversity index) analysis of 16S rRNA gene amplicon-sequencing data (Fig. 5A) revealed a marked reduction in diversity for all groups on day 10 after completion of the initial vancomycin treatment. The daily dosing and pulse dosing vancomycin groups had significantly reduced diversity in comparison to the control group (P < 0.05) on day 22 (day 12 of the extended daily/pulse regimens) but not on day 44 (14 days after the last dose of the extended daily/pulse regimens).

Fig 5.

Comparison of stool microbiota of mice (N = 6 per group at each time point) by 16S deep-sequencing analysis before, during, and after treatment with oral vancomycin. (A) Shannon diversity index. (B) Beta diversity is visualized using a non-metric multidimensional scaling (NMDS) plot. X indicates a dose of 1.125 mg which was equivalent on a mg/kg basis to 125 mg dose in humans. Stool samples were collected at baseline, day 10 (end of the initial 10-day vancomycin treatment), day 22 (day 12 of the extended daily/pulse regimens), and day 44 (14 days after the last dose of the extended daily/pulse regimens).

Beta diversity for samples from day 22 and day 44 visualized using a non-metric multidimensional scaling plot (Fig. 5B) revealed differential community shift patterns in the daily and pulsed vancomycin groups in comparison to the saline control group. There were statistically significant differences between the control group and the daily dose 20X, daily dose 10X (.5X in comparison to the extended daily 20X dose), and the pulse every 3 days (10X) groups (P < 0.05).

DISCUSSION

Taper and pulse vancomycin regimens are commonly used for the treatment of recurrent CDI (1). However, limited information is available on potential mechanisms by which these regimens might offer a benefit over other treatment options. We previously reported that the intestinal microbiota that provide colonization resistance to C. difficile in mice did not recover during vancomycin taper and pulse treatment (6). Here, we found that pulse dosing of vancomycin every 2 or 3 days resulted in persistent disruption of the microbiota and did not facilitate clearance of C. difficile spores despite evidence of germination of spores during vancomycin-free intervals based on the presence of vegetative C. difficile. The concentration of C. difficile was significantly higher in the pulse dose groups on days 2 and 7 after completion of pulse treatment, and there was no difference between these groups on day 14 after pulse treatment. For the four pulse dosing groups, colonization was detected in >75% of mice on day 14 after pulse treatment.

One notable finding from our study was that daily dosing of vancomycin for the same duration as the pulse dosing regimens with the same total drug exposure resulted in effective suppression of C. difficile during treatment. However, by day 14 post-treatment, there was no significant difference in the C. difficile concentration for the extended daily dosing groups and the control and pulse dosing groups. These data suggest that daily dosing regimens might be more effective for suppression of C. difficile than every other day or every third-day regimens.

Our results expand on other recent evidence that vancomycin taper and pulse regimens may have suboptimal effectiveness in reducing the risk for recurrence of CDI after treatment. In a human gut model, vancomycin taper and pulse regimens caused disruption of the microbiota that persisted after discontinuation of treatment and in 1 of 2 regimens was associated with recurrent growth of vegetative C. difficile (7). In a propensity score-matched analysis, vancomycin taper regimens did not provide benefit over vancomycin regimens without taper in preventing additional CDI recurrence in patients with first or second recurrent episodes (8). Although relatively long taper and pulse regimens could potentially be beneficial to provide more time to eliminate residual spores from the intestinal tract, Sirbu et al. (9) reported similar cure rates in patients receiving vancomycin taper regimens of <10 weeks and >10 weeks in total duration.

Our study has some limitations. Findings in healthy mice may differ from findings in humans given differences in diet, microbiota, and intestinal transit time. There is a need for data in patients receiving oral vancomycin taper and pulse regimens, including evaluation of the levels of vancomycin in stool during pulse dosing. Only one strain of C. difficile was studied. However, we have previously shown that other C. difficile strains give similar results in our mouse model (4, 10). We studied relatively short 20-day pulsed dosing and extended daily dosing regimens and cannot exclude the possibility that longer courses of treatment might facilitate elimination of C. difficile. We did not compare the efficacy of vancomycin and fidaxomicin extended and pulsed dosing regimens. There is some evidence that fidaxomicin extended course and pulse regimens may be effective in the treatment of patients with multiple recurrences (11 – 13). We did not include a regimen with an initial taper dose followed by pulse dosing. In a recent systematic review and meta-analysis of 10 studies, Sehgal et al. (14) found that vancomycin taper and pulse regimens were superior to taper-alone or pulse-alone regimens in achieving resolution of CDI (83%, 68%, and 54%, respectively). Finally, the mouse model evaluates colonization but not infection. Our findings do not provide an explanation for the fact that vancomycin taper and pulse regimens are effective in achieving clinical resolution of CDI in many patients (9, 14). Many CDI patients do not develop recurrent symptoms despite persistent shedding of spores after treatment (15).

In summary, we found that pulse dosing of vancomycin every 2 or 3 days resulted in persistent disruption of the microbiota and did not facilitate clearance of C. difficile spores. Future studies are needed to examine the impact of vancomycin tapered and pulsed regimens in patients. There is a need to identify treatment strategies that allow the indigenous microbiota of patients with multiple recurrences to recover during therapy.

MATERIALS AND METHODS

C. difficile test strain

VA17 is an epidemic ribotype 027 C. difficile strain with vancomycin minimum-inhibitory concentration of 0.25 µg/mL. Spores were prepared according to the Environmental Protection Agency standard operating procedure for the production of C. difficile spores (16).

Bioassay for vancomycin concentrations

The concentration of vancomycin in stool was determined by an agar diffusion assay with Bacillus subtilis as the indicator strain (4, 17). The limit of detection was 2 µg/g of stool.

Quantification of C. difficile in stool

C. difficile concentrations in stool specimens were quantified as described previously (4 – 6). Stool specimens were emulsified in 10-fold (weight/volume) pre-reduced phosphate-buffered saline (PBS). Serially diluted aliquots were inoculated onto pre-reduced cycloserine-cefoxitin-brucella agar containing 0.1% taurocholic acid and 5 mg/mL lysozyme (CDBA) (17). The number of colony-forming units (CFU) per gram of stool was calculated.

Impact of vancomycin extended daily and pulse treatment regimens on clearance of C. difficile

The Animal Care Committee of the Cleveland VA Medical Center approved the experimental protocol. Female CF-1 outbred white mice (87 total) weighing ~30 g (Harlan Sprague-Dawley, Indianapolis, IN) were housed in individual cages. For all experiments, cages were changed daily during and after treatment to reduce re-exposure to contaminated bedding and cage material. All mice received subcutaneous clindamycin 2 mg in 0.1 mL PBS once daily for 2 days to disrupt the microbiota that provide colonization resistance to C. difficile (5, 6) followed by oral gavage of 10⁴ CFU of VA 17 C. difficile spores in 0.2 mL of sterile water. Stool pellets were collected 1 day later, and C. difficile was quantified to confirm the establishment of colonization. In this model, colonization with toxigenic C. difficile does not result in any overt adverse effects, including no diarrhea, weight loss, or altered behavior (6).

All mice received initial treatment with oral vancomycin once daily for 10 days. The dose of vancomycin for the initial treatment was 150 mg/kg/day or 4.5 mg/day. The dose of vancomycin was based on preliminary experiments demonstrating that stool concentrations of vancomycin were 1,000 to 2,000 µg/g of stool in mice treated once daily with this dose for 10 days, providing levels within the range measured in the stool of humans receiving oral vancomycin 125 mg four times daily (i.e., 500 to 2,000 µg/g stool) (3, 6). Vancomycin was administered once daily on Bacon Yummie food treats (Bio-Serve, Prospect, CT); 10 µL of solution containing the antibiotic dose was pipetted onto the food treat and allowed to air dry prior to placement in the cage bottom for consumption. Control mice received food treats inoculated with 10 µL of sterile saline. We have previously demonstrated that administration of antibiotics on food treats results in stool concentrations in mice that are equivalent to concentrations after oral gavage (authors’ unpublished data). Stool pellets were collected to quantify C. difficile after 1, 5, and 10 days of vancomycin treatment.

After completion of the initial 10 days of vancomycin treatment, mice were divided into eight treatment groups. Control mice received no additional vancomycin. Three groups received extended daily dosing of vancomycin for 20 days (experiment days 11 through 30) at doses of 1.13 mg (37.5 mg/kg) daily (i.e., equivalent to 125 mg once daily in humans), 0.56 mg daily, and 0.39 mg daily (cumulative doses 22.5 mg, 11.3 mg, and 7.9 mg, respectively). Two groups received pulse dosing of vancomycin every 2 days for 20 days (experiment days 11 through 30) at doses of 1.13 mg and 0.79 mg (cumulative doses 11.3 mg and 7.9 mg, respectively). Two groups received pulse dosing of vancomycin every 3 days for 20 days at doses of 1.61 mg and 1.13 mg (cumulative doses 11.3 mg and 7.9 mg, respectively). Concentrations of C. difficile were measured in stool specimens collected on days 5, 10, 15, and 20 of the extended daily/pulse dosing and on days 2, 7, and 14 after the last extended daily/pulse dose.

For six mice in the pulse every 2-day (10X) and pulse every 3-day (10X) groups, stool specimens were collected at 0, 4, 8, 24, and 48 hours after a dose between days 10 and 20 of the pulse regimens. Vancomycin concentrations were measured in these specimens as described previously (4, 6).

Evaluation of spore germination and outgrowth during the period of pulse dosing

For seven mice in the pulse every 3-day (10X) group with positive stool cultures, stool specimens were collected at 24, 48, and 72 hours after a vancomycin dose between days 10 and 20 of the taper/pulse regimen. Stool suspensions in PBS (1:4 weight:volume) were heated to 90°C for 10 minutes to kill vegetative C. difficile but not dormant spores or germinated spores that have not progressed to outgrowth. Suspensions with and without heat inactivation were plated on selective media to quantify C. difficile. Vegetative C. difficile was calculated as the total concentration without heat inactivation (spores plus vegetative C. difficile) minus the concentration with heat inactivation (spores). Detection of >1log₁₀CFU of vegetative C. difficile during the pulse dosing phase of treatment when vancomycin was not detectable was considered an indication that spores are germinating with outgrowth during the drug-free intervals.

Effect of the antibiotics on aerobic and facultative Gram-negative bacilli

Stool specimens collected for C. difficile quantification were also plated to quantify facultative and aerobic Gram-negative bacilli. Serially diluted specimens were plated onto MacConkey agar (Difco Laboratories, Detroit). Organisms recovered on MacConkey agar were characterized as lactose-fermenting or non-lactose-fermenting Gram-negative bacilli, and for a subset of plates, a colony growing at the highest dilution was subjected to identification using standard methods.

DNA extraction and 16S rRNA amplicon sequencing

To assess the impact of the treatments on the microbiota, stool samples (~100 mg total) were collected for sequencing analysis from four groups [control, daily dose (20X), daily dose (10X), pulse every 2 days (10X), and pulse every 3 days (10X)] (N = 6 mice per group) at baseline, day 10 (end of the initial 10-day vancomycin treatment), day 32 (day 12 of the extended daily/pulse regimens), and day 44 (14 days after the last dose of the extended daily/pulse regimens). DNA extraction and 16S rRNA amplicon sequencing were performed as described previously (18, 19). Briefly, DNA was isolated from stool samples using the QIAamp DNA Microbiome kit (Qiagen). The isolated microbial gDNA was checked for signs of degradation and quantified using the Bio-analyzer (Agilent) to ensure accurate sample input for the initial PCR step. A nested PCR method was used for amplification of the V4 region of the 16S rRNA gene and the addition of Illumina Nextera Unique Dual indexes. Afterward, each library underwent standard quality control procedures checking for sample concentration and sample quality. Each library was pooled together ensuring equal sample distribution among sequencing reads. Amplicon sequencing was performed on an Illumina MiSeq with a 2 × 150 read length.

Data analysis

For C. difficile, we compared concentrations in stool during three time periods: (i) initial colonization and vancomycin treatment days 1, 5, and 10; (ii) extended daily/pulse regimens days 5, 10, 15, and 20; and (iii) post-extended daily/pulse treatment days 2, 7, and 14. Within each period, we predicted C. difficile concentration with time, treatment with extended daily dosing, pulse dosing, or neither, and their interaction. We considered total dose over the extended daily/pulse period as a continuous variable ranging from 0 to 2,500 mg and adjusted for dose. Given repeated measurements within the mouse, we used a linear mixed-effects model with random intercepts for each mouse. When an interaction of time and treatment was detected (F-test P < 0.05), we performed Tukey-adjusted post hoc pairwise tests of treatments within each time to identify when and how the treatments differed.

For Gram-negative bacilli, we compared the extended daily and pulse groups with the control group during the initial 10-day vancomycin treatment period and during the subsequent periods. We estimated a linear mixed-effects model predicting Gram-negative bacilli CFU over time and treatment (control versus extended daily or pulse), fitting a random intercept for each mouse. Given a detected interaction of time and treatment, we identified the times at which the two treatment groups did and did not differ significantly. Data analysis was performed in R Version 4.2.2 using nlme and emmeans packages for model and contrast estimation.

For analysis of the sequencing data, individual fastq files without non-biological nucleotides were processed using the Divisive Amplicon Denoising Algorithm (DADA) pipeline (20). The output of the dada2 pipeline (feature table of amplicon sequence variants) was processed for alpha and beta diversity analysis using phyloseq (21), and microbiomeSeq (http://www.github.com/umerijaz/microbiomeSeq) packages in R. Alpha diversity estimates were measured within group categories using estimate richness function of the phyloseq package.

DATA AVAILABILITY

The sequence data for this study can be accessed through the open repository Zenodo at https://zenodo.org/records/10337605. The sequence data have also been deposited in the European Nucleotide Archive under accession number PRJEB67315, and it is anticipated that they will soon be available there as well.

SUPPLEMENTAL MATERIAL

The following material is available online at https://doi.org/10.1128/aac.00903-23.

Fig 3, Fig 4. Showing additional treatment groups. aac.00903-23-s0001.docx.

Supplementary material showing additional treatment groups for Figs 3 and 4.

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