New Results

Phage Steering in the Presence of a Competing Bacterial Pathogen

Sean M. Czerwinski, James Gurney
doi: https://doi.org/10.1101/2024.09.28.615603

Abstract

The rise of antibiotic-resistant bacteria has necessitated the development of alternative therapeutic strategies such as bacteriophage therapy, where viruses specifically infect bacteria. However, rapid bacterial resistance to phage treatment remains a critical challenge, often leading to failure. Phage steering, which leverages the co-evolutionary dynamics between phage and bacteria, offers a novel solution by driving bacteria to evolve away from virulence factors or resistance mechanisms. In this study, we examined whether phage steering using bacteriophage Luz19 could function in the presence of a competing pathogen, Staphylococcus aureus (USA300), while targeting Pseudomonas aeruginosa (PAO1). Through in vitro co-evolution experiments with and without the competitor, we observed that Luz19 consistently steered P. aeruginosa away from the Type IV Pilus (T4P), a key virulence factor, without interference from S. aureus. Genomic analyses revealed mutations in T4P-associated genes, including pilR and pilZ, which conferred phage resistance. Our findings suggest that Phage Steering remains effective even in polymicrobial environments, providing a promising avenue for enhancing bacteriophage therapy efficacy in complex infections.

Introduction

The increasing prevalence of antibiotic-resistant bacteria necessitates the exploration of alternative therapeutic approaches. Bacterio(phage) therapy, which utilizes viruses that infect and kill bacteria to combat drug-resistant infections, offers a promising avenue. However, the rapid emergence of phage-resistance in vitro raises alarms over its long-term efficacy [1, 2]. The antagonistic co-evolutionary dynamics of bacteria-phage interactions generates diversity in both the bacterial and phage populations, with reciprocal evolution of defense vs attack alleles occurring in an almost turn-based competition known as Arms Race Dynamics (ARD) [3-5] wherein bacterial resistance to phage emerges, followed by the evolution of phage infectivity [6-11]. Ultimately, in the lab, ARD culminates in the emergence of a resistant genotype that the phages cannot overcome [8], in most cases coming in the form of de novo mutations resulting in the loss or structural and functional attenuation of the specific surface receptors in which the bacteriophage binds [8]. The shortcoming of bacteriophages as a miracle treatment is to be expected, as Lenski and Levin proposed, there is an asymmetry in the evolutionary potential between the bacterium and its phage [8, 9]. This asymmetry is due, in part, to a single spontaneous de novo mutation resulting in significant receptor structure alteration and therefore acquisition of resistance for the bacterium, whereas the bacteriophage would need multiple successive de novo mutations in order to overcome resistance [12, 13]. Phage Steering seeks to exploit the directional selection of parasite-host coevolution. In the event that resistance evolves against the phage, the de novo mutations that result from ARD, should lead to the loss or functional attenuation of the binding receptor [9, 14, 15]. Phage steering utilizes bacteriophages with known receptors, or factors, that are implicated in antimicrobial resistance or in virulence [14].

The intricate dynamics of bacteria-phage interactions, particularly the rapid emergence of phage resistance, pose significant challenges to the long-term efficacy of bacteriophage therapy. This is especially critical when considering pathogens like Pseudomonas aeruginosa (PA), a notorious Gram-negative bacterium known for its intrinsic multi-drug resistance and its role in chronic, often life-threatening infections. Furthermore, PA infections often occur in a poly-microbial context, most notably alongside Staphylococcus aureus (SA), significantly complicating treatment strategies. These two pathogens are the most prevalent in cystic fibrosis infections, with studies indicating enhanced virulence when they coexist [16, 17]. The dynamic and often synergistic interactions between PA and SA in co-infection models [16, 18-21], emphasize the complexity of PA-associated polymicrobial infections. The community interactions found within a polymicrobial infection has the potential to greatly impact phage therapies and steering. As previously mentioned, PA is an opportunistic pathogen, and did not evolve its vast armament of virulence factors and antimicrobial resistance mechanisms in order to colonize a human host, but rather to outcompete other micro-organisms in the environment. Thus, the question remains whether or not phage steering will prove efficacious in the presence of competing co-infecting organisms that exert their own selective pressure via competition or otherwise interaction.

In this study, we focus on the interplay between Pseudomonas aeruginosa - PAO1 (PA) and Staphylococcus aureus - USA300 (SA) and the phage Luz19. We employ the phage Luz19 to steer PAO1 away from the expression or function of its receptor, the Type IV pilus (T4P) (Fig. 1) [22, 23]. By a series of co-evolution lines with and without the phage and SA present we determined if Luz19 is able to perform Phage Steering on PA during bacterial competition. Our phenotypic assays of T4P activity and whole genome sequencing of evolved lines show that Phage Steering still works under competition with SA in LB, it remains to be seen if in media that replicate infection conditions or in vivo allow for the action of Phage Steering.

Fig. 1.
Fig. 1. Phage-steering-mediated selection of Pa populations away from Type IV pilus expression.

An initial bacterial population dominated by Pa with Type IV pilus (T4P) expression (red) and circulating low T4P-expressing genotypes (green). The T4P-binding bacteriophage, Luz19, acts as a strong selective pressure, selecting for reduced expression of its binding receptor. After phage steering, surviving bacteria are now compromised with reduced expression of the target virulence factor (green population).

Materials and Methods

Bacterial culture and phage preparation

Pseudomonas aeruginosa (Nottingham PAO1) was grown at 37°C with agitation (shaking at 200 rpm) in Lysogeny Broth (LB) media. For phage stock quantification, Luz19 phage was diluted in 1X Phosphate Buffer Saline (PBS) and spot plated onto PAO1 soft lawns on Lysogeny Broth Agar (LBA) and incubated at 37°C overnight. Plaque Forming Units (PFUs) were counted, and PFU/mL was calculated. Phage stock was diluted in 1X PBS. The final concentration of phage stock prior to initial inoculation was approximately 3.0e9 PFU/mL. Phage inoculation dosage was calculated for a multiplicity of infection (MOI) of 3, from estimated Colony Forming Unit (CFU) per mL by multiplying the Optical Density at a wavelength of 600 nm OD600 by 8.0e8 (Supplemental data).

In vitro evolution experiment

We investigated bacterial evolutionary response to an anti-virulence bacteriophage, Luz19, over the course of 10 serial transfers (10 days) in the context of presence or absence of a common competitor, Staphylococcus aureus (SA). A total of three co-evolution conditions were used, PA with SA, PA with Luz19 phage, and PA with both phage and SA. A control for serial transfer culturing was included with only PA serially transferred for the total 10 transfers. All co-evolution cultures were conducted in 1 ml LB in 24 well plates (Corning)®) and incubated at 37°C, shaking, for 24 hours. Bacteria colony forming unit (CFU) estimates for initial and subsequent (for SA) inoculations were made using OD600 measurements. For accurate CFU estimation from OD600, PAO1 and USA300 suspended cultures were spectrophotometrically measured and then serially diluted and CFU plate counted. An OD600=1.0 was found to contain approximately 8e^8 and 1e8 CFU/mL for PAO1 and USA300 respectively. 50 µL of PAO1 (1.64e7 CFU) was inoculated into wells containing 900 µL of LB media. In conditions with phage, bacteria were exposed to the 16 µL of the initial ancestral Luz19 phage at an MOI of 3:1 (4.91e7 PFU); from Luz19 stock at 3.0e^9 PFU/mL at time point 0 (T0). Stock phage concentrations were previously determined via PFU.

In conditions with competing SA, 50 µL of USA300 (2.04 e6 CFU) was inoculated into the wells containing 900uL of LB media and 50 µL of newly inoculated PAO1. In +SA conditions, 50 µL of SA, 5.0e^6 CFUs, was re-introduced at each transfer to maintain the selective pressure generated by consistent competition.

In pilot experiments, we found that SA went extinct after no more than 3 transfers when co-cultured with PAO1 at comparable relative concentrations (data not shown). After 24 hours, 100 µL was transferred into 900 µL of LB media. 300 µL was stored in 25% final concentration glycerol at – 80°C.

In the case that a replicate culture needed to be restarted, the frozen stock was used to resume the replicate from the most recently stored time point. 1 µl of Frozen culture was inoculated into 1 mL of LB and incubated with shaking for 4 hours. 100µL was then transferred into 900 µL of LB and normal culturing conditions resumed. Upon culmination of co-evolution culturing, all Transfer 10 replicates were frozen in glycerol (25% final) and stored at -80°C.

Preparation for Twitching Motility and Growth Rate Assays

Frozen replicates were cultured in LB at 37°C shaking overnight. The overnight culture was then transferred into a 1.5 mL microfuge tube, vortexed to break up aggregates, and then centrifuged at 17,000 RCF for 2 minutes to pellet the cells then washed 2 times in 1X PBS and re-pelleted. Cells were resuspended in 1 mL of LB and further diluted in LB to an OD600 of 0.05. In the case of USA300-positive co-cultures, cultures were streaked onto Pseudomonas Isolation Agar (PIA) and incubated at 37°C overnight to remove SA. Single colonies of isolated PA were incubated overnight at 37°C in LB.

qPCR of Phages

qPCR of phage stock was performed using the QuantStudio 3 rt PCR system and analyzed with the ThermoFisher applied biosystems app. See SI for list of primers used.

Twitching Motility Assays

We performed a twitching motility assay adapted from Alm & Mattick’s 1996 protocol, with the following modifications [24]. A p20 pipette tip was introduced to the cultures then pushed through a LB agar plate until the pipette tip contacted plastic at the base of the plate and then promptly removed. Plates were incubated at 37°C for 20 h. Agar was removed to reveal the imprint of PA growth. Imprints were stained with Crystal Violet for 15 min. Excess stain was gently washed off using sterile diH2O. Plates were then air dried. Once dry, the stained imprints were measured across at the widest point. All phenotypic assays were done in triplicate for each co-evolution replicate for a total of 15 assay replicates-per-condition.

Data transformations (log and Box-Cox using the car package for R 4.2.2 [25]) were attempted, but normality was not achieved across all treatments according to a Shapiro-Wilk test. Due to heteroskedasticity we used the Kruskal-Wallis test. When significant differences were found, post-hoc analyses were performed using the Games-Howell test for Welch’s ANOVA.

Growth Rate Analysis

Analysis of growth rate assays was performed using the Growthcurver package for R [26]. Growthcurver fits the data points collected for each individual replicates growth curve to a standard form of the logistics equation used in ecology and evolution [26]. Growth rate curves had the area under the logistic growth curves differentiated for each co-evolution condition subject to four different growth rate treatments, with standard error bars. AUC(l) is calculated as the area under the logistic curve obtained by integrating the logistic equation using the growthcurver package [26].

The growth rates of (PA) from different co-evolution conditions under various treatments was assessed via area under the curve AUC(l). Statistical analyses were performed to determine significant differences in growth between conditions and treatments. Data transformations (log and Box-Cox using ‘car’ and ‘MASS’ packages for R 4.2.2 respectively[25]) were attempted but did not achieve normality across all treatments. The Kruskal-Wallis test was used when significant differences were found, post-hoc analyses were performed using Games-Howell test for Welch’s ANOVA and Dunn’s test with Bonferroni correction for Kruskal-Wallis. There was no difference in the control evolution line (SI Fig. 1) We also performed the same analysis on growth rate (r) and carrying capacity (k) (SI Fig. 2 & 3).

DNA Extraction & Purification

Single colonies were cultured in LB shaking overnight at 37°C. DNA extraction, isolation, and purification was performed using the Promega Wizard ® Genomic DNA Purification Kit following the manufacturer’s instructions.

Sequencing

Whole genome sequencing of replicates was performed by SeqCoast Genomics (Portsmouth, NH, USA). SeqCoast prepared samples using the Illumina DNA Prep tagmentation kit and unique dual indexes. Sequencing was performed on the Illumina NextSeq2000 platform using a 300-cycle flow cell kit to produce a 2×150bp paired reads. 1-2$ PhiX control was spiked into the run to support optimal base calling. Read demultiplexing, read trimming, and run analytics were performed using DRAGEN v3.10.12, an on-board analysis software on the NextSeq2000.

Genomic Analysis

Genomic analyses were completed using breseq 0.38.3 [27] computational pipeline to identify mutations relative to the reference PAO1 genome Pseudomonas aeruginosa PAO1 [28]. Sequencing of our ancestral nPAO1 genome and subsequent breseq analysis was used to identify single nucleotide polymorphisms (SNPs) that are not present in the reference genome [28]. We compared the ancestral nPAO1 to the evolved PA to remove SNPs that were a result of adaptation to the media.

Results

Twitching Motility

Selection by Luz19 phage attenuates twitching motility function in the presence of competing SA (Fig. 2). Twitching motility was attenuated in phage-positive conditions in the presence and absence of competing SA (p < 0.05 for both conditions), relative to the ancestral WT. There was not significant difference between the two positive-phage conditions nor between the WT ancestor and the negative/negative culture control. PA that was co-cultured with SA did not have a significant difference in twitching motility relative to the WT control (p > 0.05).

Fig. 2.
Fig. 2. Twitching motility compared across evolution conditions

PA twitching motility decreased significantly (p < 0.05) in conditions exposed to Luz19 phage relative to the ancestor WT, with no significant difference in twitching motility measured between the positive-phage groups with/without Sa (p > 0.05) or between non-phage conditions and the ancestral control p > 0.05).

Growth rate assays and area under the curve

Our statistical analysis asked two questions. First – did strains evolved under phage selection develop resistance. We found no significant difference in the area under the curve of lines evolved with the phage compared when tested with and without the phage present. When compared to the ancestor there was a significant reduction in growth in the ancestor when phages were present (Fig. 3). Second, did the evolution in the media or in the presence of SA alter phage resistance. Our analysis revealed significant differences in the area under the growth curve among the co-evolution conditions (χ2=54.54, df = 4, p = 4.06e−11) (Fig 4). Subsequent Dunn’s post-hoc tests with Bonferroni correction were performed to identify specific condition differences within the assay treatments. When exposed to the ancestral phage, our data shows that only lines evolved in the presence of the phage demonstrated increased resistance, as measured by area under the curve. This increased resistance was observed in both the +Luz19 and +SA+Luz19 treatments compared to the ancestral PAO1 strain (p = 2.17 e-04 and p = 3.47e-05, respectively). Importantly, the presence of S. aureus during evolution did not significantly alter phage resistance. There was no significant difference between the +Luz19 and +SA+Luz19 treatments (p > 0.05), indicating that co-evolution with S. aureus did not alter resistance against the phage. Neither S. aureus co-culture nor the media alone increased resistance to the phage (p > 0.05). These results consistently demonstrate the enhanced phage resistance in evolved lines, regardless of the presence of SA during evolution.

Fig 3.
Fig 3. Growth with and without phage as measured by area under the curve, comparing isolates evolved in the presence of the phage to the ancestor.

Bacteria evolved in the presence of the phage developed resistance to the phage. Area under the curve (AUC_I) was not significantly different with the addition of the ancestor phage when the bacteria had evolved under phage selection.

Fig. 4.
Fig. 4. Growth rate assays and area under the curve (AUC(l)).

Growth rate assay mean curves for each co-evolution treatments subject to two different growth rate treatments +Luz19 or +Luz19&SA. Area under individual logistic growth curves (AUC(l)), calculated by growthcurver plotted as box plots A.) Luz19 treatment, growth curve of the five conditions with Luz19 added to initial inoculation point of cultures at a MOI of 3 (2.67*10^5 PFU). B.) Luz19 treatment, AUC(l). (Kruskal-Wallis, χ2 (4) = 54.54, p = 4.055*10^-11) +Luz19 and +SA+Luz19 showed significantly greater AUC(l) in comparison to all other conditions that had not been co-evolved with the phage (p < 0.0005). There was no significant difference between +Luz19 and +Sa+Luz19 (p > 0.05), nor between the non-phage-resistant conditions (p > 0.05) C.) S. aureus & Luz19 treatment, growth curves of the five conditions with the aforementioned PA:SA ratio and CFUs and Luz19 MOI and PFU. D) S. aureus & Luz19 treatment, AUC(l). (Kruskal-Wallis, χ2 (4) = 56.992, p = 1.242*10^-11) Similarly to the Luz19 treatment (A,B), +Luz19 and +SA+Luz19 showed significantly greater AUC(l) in comparison to all other conditions that had not been co-evolved with the phage (p < 0.005). There was no significant difference between +Luz19 and +Sa+Luz19 (p > 0.05), nor between the non-phage-resistant conditions (p > 0.05).

Genomics

Sequencing revealed a clear signature of evolution in the presence of the phage (Fig. 5). Three SNP variations between two genes involved in Type 4 Pilus biogenesis, pilR and pilZ, were defined in the conditions subject to coevolution with the phage. In the two conditions with phage, 8 out of 10 replicates (5/5 +Phage, 3/5 +Phage/+SA) had SNPs in pilR, the response regulator element of the PilSR two component system, responsible for activation of pilA transcription [29].

Fig. 5.
Fig. 5.

Chromosomal single nucleotide polymorphisms (SNPs) defined for all coevolution condition replicates and the ancestral wildtype PAO1. SNPs were determined using Breseq. pilR. SNPs of two variations in 5 out of 5 (5/5) PAO1 + Luz19 co-evo replicates and 3/5 PAO1 + Sa + Luz19 co-evo replicates. In all but one of these replicate sequences, the SNP in pilR is C192Y (TGC→TAC). Replicate b of the +Luz19 condition had a different SNP to pilR, P189Q (CCG→CAG). Of the replicates in the two conditions that were subject to co-evolution with Luz19, two lacked a SNP in pilR, both in the +Sa+Luz19 condition. Replicate b instead had a Δ197 bp deletion in pilZ. Replicate a of +Sa+Luz19 was the only replicate co-evolved alongside Luz19 that sequencing did not reveal a pil gene mutation, instead, a PQS system mutation was identified in pqsR.

Of the remaining two replicates, one (replicate A) had a 197 bp deletion in pilZ. The Δ197bp pilZ replicate showed significantly attenuated twitching motility (p < 0.05) with no significant motility difference between it and the pilR mutants (Fig. 5).

Replicate B of the +Phage+SA condition lacked detectable SNPs in genes directly related to Type 4 pilus biogenesis, structure, or function. However, it did have a unique SNP in mvfR, also known as pqsR, which is a global virulence regulator implicated in quorum sensing [30]. It is currently unclear what specific impact the mvfR mutation has on T4P expression or function, though the replicate did show roughly the same attenuation of twitching motility (Fig. 2) and followed resistant growth curves (Fig. 5)

Discussion

Phage Steering is the use of phage to reduce or remove pathogenic behaviors from a bacterial population. Fundamentally, it works by phage making use of a bacterial product, typically an outer membrane expressed structure, in some way to complete a full infection cycle. Bacteria are therefore under a selective pressure to modify the structure – engendering resistance to the phage. This modification also typically leads to a reduction in effectiveness of the structure in its evolved function. Should the structure not provide an actual fitness benefit in the environment in which the phage is applied there is no selective pressure to restore the phenotype. In infections like those seen in the Cystic Fibrosis lung, many virulence factors of PA do not provide a fitness benefit when considered in isolation, only contributing to nonadaptive virulence, increasing morbidity of people with CF. This is likely due to PA not having evolved in the CF lung, as the CF lung itself is, in evolutionary terms, incredibly young. Before 1930s the majority of people with CF died before lung became chronically infected with PA [31]. However, virulence factors such as the Type 4 Pili likely did evolve to help bacteria deal with inter species competition. Therefore, a central requirement of Phage Steering, the lack of fitness consequence during phage application might not hold true if PA is competition with other bacteria at the time of phage application.

We aimed to test the question of how phage steering works during multispecies competition, we did so in a simple experimental setup, effectively providing Phage Steering with the most favorable conditions to work. A simple co-evolutionary experiment with either the phage, Luz19, or SA, or in combination or in the absence of either. The major result of our work is that regardless of the presence of SA, the phage still steered PA away from the expression of type 4 pili, there was also no clear example of mutations outside the those that control type 4 pili in either case when SA was present. We measured the phenotypic changes in T4P with twitching motility, we confirmed phage resistance emerged as effectively in both phage treatments, indicating again the presence of SA did not alter this outcome. Finally, we genomically characterized individual isolates after the evolution experiment and found the mutations selected by Luz19 were found in pili structural and regulatory genes.

Examining these specific mutations in more detail, the gene product of pilA, is the major subunit, pilin (PilA) that composes the Type 4 pilus shaft [32, 33]. PilS is located within the inner membrane of PA and is responsible for the detection of excess pilin/PilA subunits localized to the inner membrane [29]. In the absence of excess PilA, PilS autophosphorylates, and subsequently transfers the phosphate group to the regulator protein, PilR [29]. PilR then binds to a specific sequence proximal to the promoter of pilA; and with the assistance of the alternative sigma factor, σ54, pilA is transcribed [29] (Fig. 6). Two unique SNPs in pilR were found in the phage-positive replicates, both point substitutions that result in amino acid changes. Of the eight pilR SNPs, seven were C192Y(TGC→TAC) with the sole outlier, P189Q (CCG→CAG). There was no significant difference between the attenuation of twitching motility between the two pilR mutation variants. The gene product of pilZ is another regulatory element of type 4 pilus biogenesis [34]. PilZ interacts with PilB, an ATPase localized to the cytoplasmic face of the inner membrane that provides the energy necessary to transfer the pilin subunits from the inner membrane to the base of the growing pilus polymer [34]. The exact mechanism by which PilZ manipulates PilB ATPase activity is not yet clear, however the necessity of PilZ binding to PilB for T4P biogenesis has been demonstrated [34] (Fig. 7).

Fig 6.
Fig 6. Nominal structure and regulation of the Type IV pilus (T4P) serves as the binding site for Luz19 phage.

The assembled T4P shaft is composed of PilA (red), the major pilin subunit, and its regulatory mechanisms. The PilSR two-component system controls pilA transcription through a phosphorylation signal, with PilS (pink) phosphorylating PilR (purple) when low PilA concentration localized to the inner-membrane is detected, which then activates pilA expression. PilZ (orange) is shown interacting with the ATPase, PilB (green), which provides energy for shaft assembly. Luz19 adsorption is dependent of the T4P shaft. This figure represents the wild-type scenario before mutations that confer phage resistance.

Fig 7.
Fig 7. Disrupted Type IV pilus shaft assembly confers phage resistance.

This figure demonstrates two key mechanisms preventing pilus formation: (1) Inactive PilR (purple) fails to activate pilA transcription, leading to absence of PilA pilin subunits. (2) Failure of PilZ (orange) binding renders PilB (green) inactive, halting energy production necessary for shaft assembly. The absence of the Type IV pilus shaft is shown, with Luz19 unable to adsorb to the bacterium. Mutations in pilR and pilZ genes resulted in phage resistance, likely, by preventing pilus formation.

Slightly unexpected is the appearance of a Quorum sensing mutation in the mvfR regulator, which is also known as pqsR, a regulator in the Pseudomonas Quinolone Signal (PQS) quorum sensing system. The phage pf, also a T4P binding phage has been shown to generate mutations within the PQS system [35], therefore there is likely some interaction between the PQS system and T4P. Guo et al showed that while adding exogenous PQS did not alter the twitching motility, pqsR mutants had attenuated twitching [36]. It is somewhat surprising this mutation was seen in the SA positive line. PQS is a pivotal quorum sensing molecule in PA that regulates biofilm formation along with a plethora of virulence factors [37]. The role that PQS plays in the killing of SA is complex with many PQS regulated factors contributing to PA’s competitive advantage. In brief, PQS has antimicrobial properties against SA, via the action of 2-heptyl-4-hydroxyquinoline (HHQ) and 2-heptyl-4-hydroxyquinoline N-oxide (HQNO) [38-43]. In PQS mutants (ΔpqsA and ΔpqsH), a reduced ability to kill SA has been shown relative to WT PA strains [30, 39]. While PQS plays a critical role in PA’s ability to kill SA, it is part of a complex network of QS molecules and virulence factors influenced by the PQS quorum sensing system.

Notably, the antimicrobial activity of PQS and its derivatives is modulated by environmental factors, particularly iron availability [41]. Consequently, competition studies and further investigations using media that more accurately mimic in vivo nutrient-limited environments, such as SCFM2, may illuminate potential evolutionary trade-offs or reveal vulnerabilities in Luz19-resistant PA harboring pqsR mutations.

These phenotypic and genomic data point us towards the conclusion that Phage Steering was able to work in the presence of SA when targeting the T4P. In future work we will expand this to include other bacterial pathogens and commensals, as well as working in media which better mimics infection sites, such as SCFM2. We know that the essential genome of bacteria, as measured by TN-seq, is dependent on environment, both the biotic (inter-species competition) and the abiotic [44]. It is possible that infection environments which include competition from pathogens and commensals, a host immune system, and nutritional limitations might impose to a fitness cost on lost structures which phage can target.

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Posted September 29, 2024.
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Phage Steering in the Presence of a Competing Bacterial Pathogen
Sean M. Czerwinski, James Gurney
bioRxiv 2024.09.28.615603; doi: https://doi.org/10.1101/2024.09.28.615603
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