Bacterial virulence plays a crucial role in MRSA sepsis

Gordon Y C Cheung; Justin S Bae; Ryan Liu; Rachelle L Hunt; Yue Zheng; Michael Otto

doi:10.1371/journal.ppat.1009369

. 2021 Feb 25;17(2):e1009369. doi: 10.1371/journal.ppat.1009369

Bacterial virulence plays a crucial role in MRSA sepsis

Gordon Y C Cheung ¹, Justin S Bae ¹, Ryan Liu ¹, Rachelle L Hunt ^1,^¤, Yue Zheng ¹, Michael Otto ^1,^*

Editor: Michael R Wessels²

PMCID: PMC7942999 PMID: 33630954

Abstract

Bacterial sepsis is a major global cause of death. However, the pathophysiology of sepsis has remained poorly understood. In industrialized nations, Staphylococcus aureus represents the pathogen most commonly associated with mortality due to sepsis. Because of the alarming spread of antibiotic resistance, anti-virulence strategies are often proposed to treat staphylococcal sepsis. However, we do not yet completely understand if and how bacterial virulence contributes to sepsis, which is vital for a thorough assessment of such strategies. We here examined the role of virulence and quorum-sensing regulation in mouse and rabbit models of sepsis caused by methicillin-resistant S. aureus (MRSA). We determined that leukopenia was a predictor of disease outcome during an early critical stage of sepsis. Furthermore, in device-associated infection as the most frequent type of staphylococcal blood infection, quorum-sensing deficiency resulted in significantly higher mortality. Our findings give important guidance regarding anti-virulence drug development strategies for the treatment of staphylococcal sepsis. Moreover, they considerably add to our understanding of how bacterial sepsis develops by revealing a critical early stage of infection during which the battle between bacteria and leukocytes determines sepsis outcome. While sepsis has traditionally been attributed mainly to host factors, our study highlights a key role of the invading pathogen and its virulence mechanisms.

Author summary

Bacterial infections often develop sepsis as a complication. Sepsis is a severe blood infection and one of the main reasons for death, especially in hospitals of the developed world. Sepsis is believed to be due to an overshooting immune reaction to structures that most bacteria share. However, this model fails to explain why some bacteria cause more frequent and severe sepsis than others. In our study, which we performed with the main sepsis-causing bacteria, Staphylococcus aureus, we show that the outcome of sepsis depends on the battle between bacteria and white blood cells that happens early during infection. Many of the weapons that bacteria use for that battle are controlled by a quorum-sensing regulator, which implies that so-called anti-virulence strategies directed at that regulator may work to treat sepsis. However, we also show that targeting quorum-sensing is counterproductive when sepsis originates from biofilms on implanted catheters, which it often does. Our study shows that bacterial weaponry plays a key role in sepsis and gives important advice on how to use this finding for alternative drug development.

Introduction

Sepsis results from bacteremia, the invasion of bacteria into the bloodstream, and represents one of the most frequent causes of death. Globally, an estimated ~ 50 million cases of sepsis are reported with ~ 11 million fatalities [1]. In the United States, sepsis is the predominant cause of death in hospitalized patients and costs the healthcare system ~ $ 24 million annually [2,3].

Sepsis is a life-threatening organ dysfunction syndrome that traditionally is believed to result from an overwhelming and dysregulated immune response to bacterial intruders [4–6]. One of the hallmarks of the inflammatory septic response is the production of specific pro-inflammatory cytokines, such as IL-6 or TNF-α, while anti-inflammatory cytokines, such as IL-4, are also produced to keep the inflammatory reaction under control [7]. However, the pathophysiology of sepsis is still not completely understood and there is considerable controversy regarding the role of immune suppression during sepsis [8–13] and whether cytokines are appropriate sepsis biomarkers [14–17].

Phylogenetically widely conserved bacterial surface structures, such as lipopolysaccharide (LPS) in Gram-negative and lipoteichoic acid and lipopeptides in Gram-positive bacteria, are commonly regarded as the primary triggers of the host inflammatory response to bacterial infection [18,19]. However, this model cannot explain why some bacteria cause more severe and frequent cases of sepsis than others; and more recent reports have suggested a role of toxin-mediated bacterial virulence in the progression and outcome of sepsis [20–22].

Staphylococcus aureus is the leading cause of blood infections in industrialized nations and the pathogen causing the highest mortality [23,24]. Antibiotic resistance as present in methicillin-resistant S. aureus (MRSA) leads to worsened clinical outcome of staphylococcal bacteremia [25]. Importantly, most MRSA blood infections originate from infections on indwelling medical devices, such as in the case of intravascular catheter-related S. aureus bacteremia [26,27], and characteristically involve biofilm formation on the infected devices [28].

The quorum-sensing system Agr (accessory gene regulator) controls most virulence determinants in S. aureus, including toxins that lyse leukocytes and other immune evasion factors [29]. For that reason and in the light of frequent antibiotic resistance in S. aureus, quorum-quenching approaches that target Agr functionality are often proposed as potential alternatives to antibiotic-based treatment of S. aureus infection [30]. However, clinical and experimental data regarding the impact of Agr quorum-sensing on sepsis are somewhat contradictory. Most [31–34], yet not all [35], studies using isogenic S. aureus agr mutants in experimental bacteremia indicate a strong impact of Agr on mortality. Then again, there is increased incidence and association with disease severity of naturally occurring functionally Agr-deficient isolates from clinical cases of bacteremia [36–38]. What further complicates the matter is the fact that Agr also impacts biofilm formation via strict control of the phenol-soluble modulin (PSM) surfactant peptides [39,40], which leads to increased biofilm extension and resistance to immune clearance of Agr-deficient strains during infection of indwelling medical devices [41–43].

Thus, as is the case for most bacteria causing bloodstream infections, the role of virulence and quorum-sensing in S. aureus sepsis remains incompletely understood [44]. This is to a large part due to the lack of detailed investigation of the events accompanying bacterial sepsis, such as interaction with leukocytes and cytokine production. Such investigation–particularly during early stages of sepsis—is hardly possible in the clinic and requires animal models. Furthermore, despite recently obtained insight into the role of quorum-sensing in S. aureus biofilm-associated infection [29,41,42,45], biofilm-associated infection models have not yet been used to investigate the role of quorum-sensing in sepsis as its main clinically important complication. As a consequence, the often-claimed potential of quorum-sensing blockers for systemic S. aureus and MRSA infection lacks thorough experimental confirmation.

Here, to understand the role of virulence and quorum-sensing in S. aureus sepsis, including sepsis of device-associated origin, we performed animal models of non-catheter-associated and catheter-associated sepsis using the MRSA strain USA300 (LAC), the most frequent cause of S. aureus infections in the Unites States [46]. In contrast to most previously published S. aureus infection studies, which were performed in mice, we focused on a rabbit model for the following reasons. First, only use of a larger animal allows repeated drawing of sufficient blood to monitor the development of sepsis-related parameters. Second, mice–in contrast to rabbits—are not sensitive to several S. aureus leukocidins [47], making rabbits a better choice for the analysis of the general impact of Agr, Agr-regulated virulence factors, and overall S. aureus virulence during systemic infection.

Our results demonstrate a critical role of bacterial virulence, and particularly of S. aureus-neutrophil interaction, for the outcome of sepsis and highlight fundamental differences between device- and non-device-related origins of sepsis regarding the role of the impact of quorum-sensing. Overall, they discourage the use of quorum-quenching but underline the potential of approaches directly targeting leukocyte-bacteria interaction for the treatment of S. aureus sepsis.

Results

The quorum-sensing virulence regulator Agr strongly impacts mortality in a mouse sepsis model

Mouse models of acute, systemic S. aureus infection that produce a sepsis scenario and pronounced mortality within days post infection have frequently been used before. This includes studies that compared agr mutants and corresponding isogenic parental strains [31–34]. To study the role of virulence and quorum-sensing in S. aureus sepsis, we here also first performed a mouse study comparing mortality due to infection with wild-type LAC versus isogenic Δagr bacteria. Additionally, we tested the impact of cyclophosphamide (CY), which depletes animals of leukocytes [48]. This analysis specifically addresses the impact of S. aureus virulence, because (i) phagocytosis by leukocytes, and among them particularly neutrophils, is known to represent the primary factor controlling S. aureus infection [49] and (ii) a wide array of S. aureus cytolytic toxins that target leukocytes, such as the bicomponent leukocidins, α-toxin and PSMs, are Agr-controlled and represent major virulence determinants of S. aureus, as demonstrated by the significant effects they have on the outcome of S. aureus infection [47,50–52].

Mice inoculated with equal amounts of the S. aureus wild-type strain LAC succumbed to infection much more rapidly than those infected with the isogenic Δagr mutant (p<0.0001) (Fig 1A). In CY-treated mice, in contrast to non-treated mice, there was no significant difference in mortality between wild-type and Δagr-infected animals (Fig 1B), confirming the idea of a major impact of bacteria-induced leukocyte lysis on mortality in this model. These results indicate that bacterial virulence and resulting leukocyte numbers have a determining impact on the outcome of S. aureus sepsis.

Fig 1 — (A) Scheme of experimental setup and survival experiment. Survival of female C57BL/6NCrl mice following intravenous challenge with ~2–3 × 10⁸ CFU of S. *aureus* LAC or its isogenic *agr* deletion strain (n = 10/group) was recorded. Animals were monitored for survival up to 52 hours. Statistical analysis is by log-rank (Mantel-Cox) test. (B) Survival experiment under cyclophosphamide (CY) treatment. Scheme of experimental setup and survival data. Survival of female C57BL/6NCrl mice following intravenous challenge with 10⁶ CFU of S. *aureus* LAC or its isogenic Δ*agr* deletion strain (n = 10/group) was recorded. Statistical analysis is by log-rank (Mantel-Cox) test. Mouse picture is from openclipart.org.

The impact of Agr on sepsis-related mortality is strongly dependent on whether the infection is device-associated

As already mentioned, the mouse is a suboptimal model organism to study the impact of virulence on sepsis caused by S. aureus. We therefore performed all following experimentation in rabbits to confirm and further elaborate on the findings we had achieved in mice. In these experiments, we included a catheter-associated model to assess the specific impact of device-associated infection as the most frequent type of staphylococcal blood infection.

In a non-catheter-related infection setup, essentially reproducing the above mouse model in rabbits (Fig 2A), we observed a slightly increased mortality rate in animals infected with the wild-type as compared to those infected with the Δagr strain (Fig 2B). This difference was only statistically significant in the Gehan-Breslow-Wilcoxon analysis, which attributes more weight to early time points (p = 0.038), but not in the usual log-rank test (p = 0.15). In contrast, in a setup with an inserted silastic central venous catheter (CVC), the Δagr strain produced significantly higher mortality using both statistical analysis methods (p = 0.0011, log-rank; p = 0.0013, Gehan-Breslow-Wilcoxon) (Fig 2C). CFU over the time course of infection in the blood and organs reflected the mortality results, inasmuch as they were (i) increased in moribund animals, at least in the blood and some organs, (ii) similar between wild-type and Δagr-infected mice in the non-catheter-associated model, and (iii) overall higher in Δagr-infected rabbits in the catheter-associated model (Figs 3A, 3B, 4A and 4B). There were no apparent differences in weight loss but we observed decreased rectal temperature in moribund animals (S1 Fig). Notably, most (10/12) rabbits infected with the Δagr strain developed biofilms on the device and of these rabbits, almost all (9/10) showed high blood and organ CFU and succumbed to the infection, whereas only one (1/12) rabbit infected with the wild-type strain died (Fig 4A and 4C). (The latter finding contrasts the results in the non-catheter-associated model considering that the same inoculum was used, but is probably to be explained by the catheters sequestering the bacteria and thus initially removing them from the bloodstream.) Only one rabbit infected with the wild-type strain developed a biofilm. There were highly significant correlations between negative Agr status and death (Fisher’s exact test, p = 0.0028) and between biofilm formation and death (Fisher’s exact test, p = 0.0005). Together, these results show that (i) in rabbits the observed impact of Agr on the outcome of sepsis is much weaker than in mice and (ii) the effect is reversed in device-associated infection, where Agr deficiency causes higher mortality in apparent association with biofilm formation on the device.

Fig 3 — (A) Bacterial CFU in the blood over time. (B) Bacterial CFU at the time of death or the end of the experiment in the indicated organs. Empty symbols represent animals that died during the observation period.

Fig 4 — (A) Bacterial CFU in the blood over time. (B) Bacterial CFU at the time of death or the end of the experiment in the indicated organs. (C) Bacterial CFU in the lumina of implanted catheters. Data are from the second batch of rabbits only (n = 6/group). The dotted line depicts the threshold above which CFU values were considered to reflect biofilm formation in both batches of rabbits. (**A-C**) Rabbits that developed catheter biofilms are marked by open symbols and light-colored borders.

Early neutropenia is associated with a fatal outcome of sepsis

Leukocyte numbers decreased in the early hours of infection in the non-catheter-related infection model, while they increased again in survivors (Figs 5A, 5B and S2). This was most pronounced in neutrophils (Fig 5B), but less obvious in the catheter-related model, probably due to attachment of bacteria to the device and slow release from the formed biofilm (Fig 5C and 5D). We then analyzed whether early leukopenia and specifically neutropenia is correlated with outcome of infection and Agr status. Numbers of total leukocytes and all leukocyte types except monocytes were significantly lower in animals that ultimately died during early infection in the catheter-related model, and neutrophil numbers were significantly lower in both models (Figs 5E–5H and S2). The most important conclusion from this analysis is that early neutropenia is correlated with fatality in both device- and non-device associated sepsis. Notably, confirming the opposite impact of Agr status on infection outcome in catheter- versus non-catheter-related infection, total leukocyte and neutrophil numbers early during infection were significantly lower in the wild-type- as compared to Δagr-infected animals in the non-catheter-related model, while they were significantly higher in the catheter-related model (Fig 5I–5L).

Fig 5 — (**A-D**) WBC and neutrophil numbers over time in the non-catheter-associated (**A,B**) and catheter-associated (**C,D**) model. (**C,D**) Rabbits that developed catheter biofilms are marked by open symbols and light-colored borders. (**E-H**) Analysis of death versus survival outcome based on the averages of leukocyte numbers of every animal in the time window 6–24 h (grey shading) in panels A-D. (**I-L**) Analysis of impact of Agr status on the averages of WBC and neutrophil numbers in the time window 6–24 h (grey shading) in panels A through D. (**E-L**) Statistical analysis is by unpaired two-tailed t-tests. Error bars show the mean ± SD. NS, not significant (p≥0.05). See **S2 Fig** for monocyte and lymphocyte analyses.

A “cytokine storm” is often regarded a hallmark of sepsis [7]. We therefore analyzed the concentration over time of specific cytokines often reported to change in concentration during sepsis. IL-6 concentrations were very strongly increased over the course of infection in both infection models and were generally higher in moribund animals than in those that recovered (Fig 6A and 6G). Concentrations of TNF-α followed a similar pattern but with much lower overall changes, and this was only observed in the non-catheter-associated model (Fig 6B and 6H). In contrast, concentrations of the anti-inflammatory cytokine IL-4 were overall reduced compared to baseline (Fig 6C and 6I). These findings reflect previous reports on cytokine concentrations during sepsis [7]. However, cytokine concentrations during the analyzed initial time window were not correlated with infection outcome, indicating that they are not an early predictor of infection outcome (Fig 6D, 6E, 6F, 6J, 6K and 6L). Consistently with the lack of correlation between cytokine concentration and death, there was also no significant correlation with Agr status (S3 Fig).

Fig 6 — (A-C) Fold-changes of the indicated cytokines over time in the non-catheter-associated model. (D-F) Analysis of death versus survival outcome based on average cytokine fold-changes in the time window 6–24 h (grey shading) in panels A-C. (G-I) Fold-changes of the indicated cytokines over time in the catheter-associated model. (J-L) Analysis of death versus survival outcome based on average cytokine fold-changes in the time window 6–24 h (grey shading) in panels G-I. (D-F, J-L) Statistical analysis is by unpaired two-tailed t-tests. Error bars show the geometric mean and geometric SD. NS, not significant (p≥0.05). See **S3 Fig** for the corresponding analysis of Agr status for both rabbit models.

Discussion

Bacterial virulence has not traditionally been regarded as a primary factor determining the outcome of sepsis. This notion is based on conventional models that attribute sepsis to an overwhelming immune reaction to widely conserved bacterial surface structures [4–6] or more recent reports that emphasize the role of a later stage of immune suppression [8–13]. These models, which explain differences in sepsis outcome predominantly by host factors, can hardly explain the clinically observed increased morbidity and mortality that is associated with specific pathogens such as S. aureus [53]. In this study we hypothesized that bacterial virulence significantly affects sepsis development and outcome. To evaluate that hypothesis, we analyzed the role of the global virulence regulator Agr and the association of leukopenia with mortality in acute blood infection caused by MRSA as a leading cause of sepsis. Agr and leukocyte killing are considered major parameters of S. aureus and MRSA virulence: Agr is a major regulator of virtually all S. aureus toxins, among them many leukocidins [29,52], and while only directly demonstrated in a pneumonia model [54], leukopenia is the assumed in-vivo consequence of their activity. We chose these parameters as they allow for a better general evaluation of our hypothesis than the analysis of specific toxins, given the plethora of S. aureus toxins and their functional redundancy [55]. Our findings on mortality being associated with the virulence regulator Agr in addition to the absence of any effect under CY treatment indicate a significant impact of bacterial virulence on the outcome of sepsis that occurs via leukocyte killing. This notion is also supported by the kinetics of the observed leukopenia, because overall leukocyte and neutrophil numbers first increased, but then rapidly decreased, strongly suggesting that low leukocyte concentrations in moribund animals were a result of lysis rather than leukocyte migration into organs, which happens much earlier, at ~ 5 min post infection [56]. This migration-dependent leukopenia is short-timed and leukocyte numbers return to normal at 60 min, which is long before the effects we measured [56].

Our study revealed differences in mouse versus rabbit test animals regarding the relative impact of Agr during non-catheter-associated infection, inasmuch as the relative impact of Agr was stronger in mice. We believe this to be due to the fact that mice are immune to many leukocidins owing to the lack of responsive receptors [47]. While rabbits are therefore overall more sensitive to infection by S. aureus, the relative impact of Agr–which increases with the relative Agr dependence of toxin expression–is greater in mice, as in that species it is mostly dependent on PSMs, which are under much stricter Agr control than the bicomponent leukocidins [40,52]. As rabbits more closely resemble humans in their responsiveness to leukocidins, this finding indicates that the impact of Agr on systemic infection may be overestimated in the mouse infection model. Furthermore, we are aware of the fact that these toxins not only have cytolytic, but also pro-inflammatory capacities, often detected at concentrations much lower than those that are cytolytic [57,58]. Moreover, some toxins, such as PSMs and LukAB/GH, are assumed to contribute to leukocyte lysis after phagocytosis rather than by toxin release into the bloodstream [59,60]. Which specific leucocidin activities contribute to pathogenesis, including during different experimental setups that may differ for example in the initial bacterial inoculum, is not understood.

While our study was focused on assessing the role of bacterial virulence, it gave important information also regarding immune regulation in sepsis. Notably, in our animal studies we analyzed very early stages of sepsis, which is not commonly possible in the clinic. We found that early leukopenia (particularly neutropenia), but not early increase of pro-inflammatory cytokines, was strongly correlated with mortality. Our results indicate that immune suppression early during infection plays a critical role for the development of sepsis. This highlights the importance of different stages of sepsis regarding inflammation and immune suppression. Of note, the early immune suppression we observed, which is mediated by bacterial virulence, is different in character and timing from the previously described late immune suppression that is considered a host effect [8–13].

While many animal models of device-associated infection have been performed, particularly using S. aureus as pathogen [28,41,42,61–64], we here also performed what to the best of our knowledge represents the first experimental assessment of device-associated sepsis. As mentioned above, this is of particular importance given that most cases of staphylococcal sepsis originate from the infection of indwelling medical devices, which due to the constant seeding from the bacterial biofilm on the infected device represents a completely different scenario [26,27]. Results of the device-associated infection model generally reflected those of the non-device-associated model as for bacteria-leukocyte interaction, but were opposite regarding the role of Agr. Due to complete absence of the PSMs, which are important for biofilm structuring and turnover [39,42,65], Agr-deficient S. aureus are known to form extended biofilms in vitro [43] and on indwelling medical devices in vivo [41,42]. However, it has not been previously investigated how this situation impacts the development of sepsis as the most severe complication of device-associated infection. Our results demonstrated increased mortality, accompanied by increased bacterial presence in blood and organs, in rabbits infected with Agr-deficient as compared to those infected with wild-type MRSA, effects that were more pronounced in animals in which biofilms had formed on the device. Only one animal infected with wild-type bacteria developed a biofilm on the device, but did not show comparably severe disease parameters, in accordance with the notion that in-vivo biofilms formed by wild-type S. aureus are less extended and less resistant to removal by phagocytes than those formed by Agr-deficient strains [41]. These results show that Agr dysfunction and concomitant biofilm formation on the indwelling device causes increased development of sepsis, most likely caused by constant seeding of bacteria from the device. Notably, they help to explain the clinical observation of a high percentage of functionally Agr-deficient isolates found in cases of S. aureus blood infection [36].

Our results have important implications for the suggested application of quorum-sensing blockers for the treatment of S. aureus systemic infection. Such drugs have often been proposed and reported to have efficacy in local and occasionally systemic S. aureus infections [66–68]. An inherent problem with the interpretation of results achieved with quorum-sensing blockers is the frequently narrow window between genuine quorum-sensing blocking efficacy and bactericidal effects. The comparison of wild-type with isogenic Δagr S. aureus thus gives a better idea than the use of quorum-sensing blocking drugs about whether Agr is a promising target. The frequent involvement of devices in S. aureus bacteremia [26,27], the fact that clinical treatment even of sepsis without a device origin often requires placement of devices such as catheters, and the increased pathogenesis associated with Δagr S. aureus in device-associated systemic infection that we found argue against using quorum-sensing blockers for systemic S. aureus infections. Because strict control of PSMs by Agr is the underlying cause for these phenomena [40,42,65], specifically targeting PSMs also does not appear to be a promising drug development strategy for blood infections. Our results on the importance of leukocyte-bacteria interaction for the outcome of sepsis rather suggest that protection of leukocytes from bacterial attacks, such as by direct interference with non-PSM bacterial leukocidins [69–71], bears promise for the treatment of device- or non-device-associated S. aureus blood infection.

In conclusion, in this study we provide evidence for a major contribution of bacterial virulence to sepsis and show that early neutropenia is a predictor for sepsis outcome. These results call for an adjustment of the traditional notion that the severity of bacterial sepsis is predominantly attributable to host factors. In addition, our results give important mechanistic information regarding device-associated staphylococcal sepsis to guide the development of alternative anti-virulence strategies.

Materials and methods

Ethics statement

Mouse and rabbit experiments were performed under NIAID animal study protocols LB1E and LB2E, respectively. Both protocols were reviewed and approved by the Animal Care and Use Committee (NIAID, NIH) and are in accordance with the Animal Welfare Act of the United States (7 U.S.C. 2131 et. seq.).

Bacterial strains and growth conditions

S. aureus strains used in this study were LAC (CA-MRSA, pulsed-field type USA300) [72] and its isogenic agr mutant [57]. Bacteria were prepared for infections as described previously [20].

Mouse model of non-catheter-associated blood infection

The mouse sepsis model was performed using female, 8–10 week-old, C57BL/6NCrl mice (Charles River Laboratories) as described previously [73] using tail vein injection with the following modifications. Mice received ~ 2–3 × 10⁸ CFUs of live S. aureus. The health status of challenged mice was recorded every 2 hours for the first 12 hours, every 4 hours for the following 12 hours, and every 8 hours thereafter until the study ended (52 hours). Mice that prematurely reached the end point (defined as having a head tilt, microphthalmia, lack of response to prodding, immobility, or >20% weight loss from the pre-infection baseline) during the study and those that survived to the end of the study were anesthetized with isoflurane. For depletion of leukocytes in naïve mice, cyclophosphamide monohydrate (CY, Sigma), diluted in sterile PBS, was injected intraperitoneally at 250 mg/kg each day for 3 days prior to intravenous infection with 1 × 10⁶ CFUs of bacteria on day 4. This lower dose of bacteria was chosen because of generally increased sensitivity to infection of CY-treated animals. CBC analyses of blood from CY-treated mice confirmed significantly reduced numbers of WBCs (~ 20/ml) compared to untreated mice (~8000/ml). The health status of challenged mice was recorded as described above until the study ended.

Rabbit models of systemic catheter- and non-catheter-associated blood infection

Female, 2–3 kg, New Zealand White rabbits (Charles River Laboratories) were housed singly and allowed to acclimatize in an American Association for the Accreditation of Laboratory Animal Care (AAALAC)–approved NIH facility for a minimum of 7 days prior to introduction of catheters and/or infection with S. aureus.

Vascular catheterization was performed using in-house made sterilized central venous catheters (CVCs), composed of silastic catheters (1.02 × 2.16 mm, Dow Corning) attached to 18-gauge intramedic Luer stub adapters (Becton Dickenson). CVCs were sterilized with ethylene oxide gas and then cut to length for each rabbit. Hair, over the right cervical, right shoulder and scapular region for each rabbit was removed. Under anesthesia, a 2- to 3-cm-long skin incision was made on the right anterolateral cervical region to expose the external jugular vein. A small incision was then made through the vein wall and a sterile-saline filled CVC was threaded into the vein and forwarded until the catheter cuff was contiguous with the vein. The proximal end of the CVC was exteriorized by a subcutaneous tunnel created by threading it through a sterile trocar (1 × 34 cm) underneath the skin from the external jugular incision site to an exit site through the intrascapular skin. The skin incision over the external jugular vein was then closed and sutured. The proximal part of the catheter was attached to the metal end of the Luer stub and a needleless injection cap, which was secured to the skin exit with 2–0 silk sutures and tied together to prevent displacement [74]. Two-way flow was performed to confirm integrity of the catheter with up to 1 ml of sterile pharmaceutical-grade heparin in physiological saline solution (100 units/ml). Catheterized rabbits were placed on post-operative observation for 7 days post-surgery prior to intravenous infection with S. aureus.

For pre-infection baselines, weights and rectal temperatures were taken prior to infection. Additionally. blood samples were drawn from the ear artery or via the catheter of non-catheterized and catheterized rabbits, respectively. Blood samples were drawn from the ear artery and weights and rectal temperatures were taken prior to infection for pre-infection baselines. Rabbits (with and without catheters) were then infected via the marginal ear vein with 1 ml of bacteria at a concentration of 1 × 10⁸ CFUs/ml in sterile PBS. Approximately 0.5-ml volumes of blood were sampled 2, 4, 6, 8, 12, and 24 hours post infection from the marginal ear vein of non-catheterized rabbits, and 2, 4, 6, 8, 12, 16, 20 and 24 hours post infection via the catheter of catheterized rabbits, for quantitative blood culture, cytokine measurements and CBC analyses. The health status, weights and rectal temperatures were recorded every two hours post infection for the first 8 hours, every 4 hours for the following 16 hours, and then every 8 hours thereafter until the end of the study (96 hours). Rabbits prematurely reaching end-point (defined as having neurological signs, abdominal breathing, cyanosis, a lack of response to prodding, immobility, or >15% weight loss from the pre-infection baseline for two consecutive days) during the study and those that survived to the end of the study, were anesthetized with ketamine and xylazine and terminally exsanguinated. Rabbits were then euthanized with Beuthanasia D (Schering-Plough Animal Health Corp) and the livers, kidneys, spleens, lungs and any catheters were surgically removed. Enumeration of CFUs from organ homogenates was performed as previously described [75]. The catheter lumina were flushed several times with a total of 0.5 ml of sterile PBS each, homogenized, and serial dilutions of the contents were plated onto TSA plates for enumeration of CFU. Biofilm formation was defined as a result of > 7500 CFU/ml. No biofilm formation was defined as a result of < 200 CFU/ml (detection limit). There were no catheters with CFU between these two values.

CFU enumeration, cytokine measurements and complete blood count analyses from blood

Blood samples were distributed into tubes containing serum clotting gel, heparin, and ethylenediaminetetraacetic acid (EDTA) (Sarstedt) and then inverted immediately. The total volume of blood draws did not exceed those determined by Comparative Medicine Branch Standard Operating Protocol guidelines. The concentrations of IL-4, IL-6, and TNF-α in rabbit sera (stored at -20°C immediately after collection) were determined using Quantikine ELISA kits (R&D Systems) according to the manufacturer’s instructions. CFUs were enumerated from blood samples collected in heparin-containing tubes as described previously (20). CBCs were assessed in blood samples collected in EDTA-containing tubes within 3 hours of collection using a ProCyte Dx Hematology Analyzer (IDEXX technologies). Automatic gating, defined by the software algorithms, was used to identify white blood cell differentials on all uninfected samples. For blood samples collected from infected animals, manual gating was applied to identify white blood cell differentials. Identical gates were applied across samples collected at each time point.

Statistics

Statistical analysis was performed using Graph Pad Prism for MAC version 8.3.0. Statistical analysis is by Log-rank (Mantel-Cox) and/or Gehan-Breslow-Wilcoxon tests for survival curves. For the comparison of two groups, unpaired two-tailed t-tests were used. All error bars depict the mean and standard deviation for non-logarithmic, or the geometric mean and geometric standard deviation for logarithmic scales.

Supporting information

S1 Fig. Physiological parameters in the rabbit sepsis models.

Weight change and rectal temperature over time in the non-catheter-associated and the catheter-associated sepsis models. The transient dip at ~ 48 h in weight for some animals in the catheter-associated model was due to those rabbits not eating. After critical care (forced feeding) was activated, they recovered.

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S2 Fig. Rabbit sepsis models–leukocyte numbers (monocytes, lymphocytes).

(A-D) Monocyte and lymphocyte numbers over time in the non-catheter-associated (A,B) and catheter-associated (C,D) model. (C,D) Rabbits that developed catheter biofilms are marked by open symbols and light-colored borders. (E-H) Analysis of death versus survival outcome based on average monocyte and lymphocyte numbers for every animal in the time window 6–24 h (grey shading) in panels A-D. (I-L) Analysis of impact of Agr status on average monocyte and lymphocyte numbers for every animal in the time window 6–24 h (grey shading) in panels A through D. (C-L) Statistical analysis is by unpaired two-tailed t-tests. Error bars show the mean ± SD. NS, not significant (p≥0.05).

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S3 Fig. Impact of Agr status on cytokine levels early during infection.

Fold-changes in average cytokine levels for every animal in the rabbit infection models in the early 6–24 h infection window were analyzed dependent on infection group (wild-type versus Δagr-infected animals). Statistical analysis is by unpaired two-tailed t-tests. Error bars show the geometric mean and geometric SD. NS, not significant (p≥0.05).

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Acknowledgments

We would like to thank Dr. Binh A. Diep (UCSF, CA) for providing advice on the rabbit experiments, Dr. Marvin L. Thomas, Dr. Holly Habbershon, Mr. Keith Johnson, and Mr. John DeLeonardis at the Division of Veterinary Resources (DVR), NIH, for the surgical placement of catheters in rabbits and their invaluable technical expertise, and the technical staff at the DVR and Comparative Medicine Branch (CMB), NIH, for excellent technical support.

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

This study was supported by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases (NIAID), U.S. National Institutes of Health (NIH), project number ZIA AI000904 (to M.O.). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS Pathog. doi: 10.1371/journal.ppat.1009369.r001

Decision Letter 0

Michael H Malim, Michael R Wessels

5 Jun 2020

Dear Dr. Otto,

Thank you very much for submitting your manuscript "Bacterial virulence plays a crucial role in MRSA sepsis" for consideration at PLOS Pathogens. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. In light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' comments.

As you will see from the reviews, all referees thought the work has potentially important implications. However, all three were concerned about the relatively sparse data to support the major conclusions of the paper. In particular, R1 and R2 would like to see more thorough investigation of the catheter biofilms. All reviewers noted that the correlation between leukopenia and mortality was not strongly supported by the data, and R1 points out that the mechanism of leukopenia is not investigated. R3 raises important questions about the robustness of the statistical analysis and the hazards of drawing conclusions on the basis of apparently a single experiment with a small number of animals. Confirming the results of the rabbit experiment in another group of animals would greatly strengthen the conclusions.

We cannot make any decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript is also likely to be sent to reviewers for further evaluation.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Please prepare and submit your revised manuscript within 60 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. Please note that revised manuscripts received after the 60-day due date may require evaluation and peer review similar to newly submitted manuscripts.

Thank you again for your submission. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Michael R. Wessels

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

***********************

Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: Cheung and colleagues studied staphylococcal pathogenesis in models of catheter and non-catheter related sepsis both in mice and rabbit. To follow virulence functions regulated by agr, particularly toxins, they compared infection induced by WT and agr mutants. Overall, rabbits nicely modeled human sepsis and showed several clinical and lab features that approximated what is seen in human sepsis. For example, like in human infection, low WBC was shown to be a prognosticator of poor outcome, and agr negative S. aureus is associated with Staphylococcal bacteremia. The authors proposed that lysis of WBC by toxins accounted for the low WBC and drives poor outcome, although that needed better support. Finally, because negative agr is associated with worse outcome in catheter infection, the authors caution on the use of quorum sensing blockers for treatment of S. aureus sepsis. Overall, the study has potentially important clinical implications, but mechanistic aspects could be improved.

Reviewer #2: This paper examines the contribution of virulence factors during S. aureus sepsis.

The authors examine the possibility that sepsis is not only the result of excessive inflammation. The authors surmise that bacterial factors also contribute to sepsis. They hypothesize that secreted leukocidins may actively kill leukocytes early during infection exacerbating sepsis. Because of the redundancy of secreted leukocidins, the authors settle into comparing the infectivity of the wild type LAC strain and the isogenic mutant in the global regulator Agr which controls the expression of many leukocidin genes amongst many other genes. In addition, the authors use both a mouse and rabbit models of IV infection with and without catheter induced bacteremia. This is done because mouse leukocytes are in general resistant to S. aureus leukocidins unlike rabbit leukocytes.

The study addresses an important question in the field using a rational design. But, the interpretation of the results is constrained by the small number of data points.

Reviewer #3: Authors present findings of animal models of infection with WT MRSA and an isogenic agr-deletion strain to demonstrate the role of agr-mediated quorum-sensing in the pathogenesis and subsequent outcome of sepsis. Major conclusions are that bacterial leukocidins up-regulated via agr-mediated mechanisms during early sepsis contribute to leuko- and neutropenia that drives fatal outcomes. In addition, a model of IV infection in rabbits with a pre-inserted central venous catheter (CVC) surprisingly shows that agr-deficiency is associated with higher mortality that is associated with biofilm formation. Taken together, the implications of such findings could be highly significant, as they would suggest that treatment modalities targeting S. aureus virulence factors could effectively improve staphylococcal sepsis outcomes, but that targeting quorum-sensing may be the wrong approach in device- or biofilm-associated infection. The experiments were well-executed by researchers with deep experience in the methods and the field, although small numbers of animals, particularly in the catheter-associated infection model, hamper generation of statistically-supported conclusions in several experiments. Such high impact conclusions should be substantiated by more appropriate statistical analyses in some experiments, and this may not be feasible with the numbers of animals used in the experiments represented.

Authors describe two major experiments from which they ultimately draw their conclusions. First, using a mouse model of MRSA sepsis from IV infection with a WT (1 of 10 survived) or an isogenic agr-deletion LAC strain (all survived), they demonstrate the critical role of agr-mediated quorum-sensing in mortality. From blood draws early after infection, they show that survival was associated with higher leukocyte and leukocyte subset numbers. They repeat this experiment with cyclophosphamide administered prior to infection to show that leukocyte-depletion renders infection even with the agr-deletion strain fatal. Taken together, they conclude that leukopenia is associated with fatal outcome in this model, and that agr-regulated expression of leukocidins drives this critical deficit.

In their second experiment, they use a similar infection model with the same MRSA strains in rabbits with and without prior placement of a CVC. In the non-catheter-related infection, mortality was similar but delayed following infection with the agr-deletion strain (3 of 9 survived versus 2 of 9 survivors from WT infection). Surprisingly, in rabbits with a CVC, 6 of 6 animals survived IV infection with the WT strain while only 2 of 6 animals survived IV infection with the agr-deletion strain, and the 4 rabbits who succumbed had biofilm detected in their catheter by CFUs counted from postmortem catheter flushes (while the 2 survivors in this group had no biofilm). Survivors in both the non-catheter- and catheter-related models had higher neutrophil counts than non-survivors. Cytokine evaluation early after infection revealed elevated IL-6 amongst all animals with no difference among survivors and non-survivors but minimal change and no difference in TNFa levels. Taken together, authors conclude that (1) the impact of agr-mediated pathophysiology in non-catheter-related sepsis is weaker in rabbits than in mice and (2) the agr-mediated effect is reversed in catheter-associated sepsis in rabbits and this reversal is due to biofilm formation and (3) early leuko- and neutropenia is associated with mortality in non-catheter and catheter-related infection while levels of canonical inflammatory-associated cytokines were not.

**********

Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #1: 1) Reduced WBC as a prognosticator of poor outcome (a major point of this paper) is ascribed to toxin mediated cytolytic activity, but no supporting data is provided. The authors referred to a SA pneumonia paper where cytolysis is demonstrated. This needs to be better supported in the bloodstream infection model.

2) agr mutant caused greater mortality than WT S. aureus in the biofilm model. It makes sense that this could be secondary to slow and prolonged release from the catheter. Did the authors culture the catheter directly to demonstrate improved binding to the catheter, rather than indirectly through blood cultures, to correlate with the higher concentration in the blood?

Ideally blocking of biofilm related function within agr would lead to similar outcome as delta agr. At least discuss.

Reviewer #2: 1.Fig. 1A and 2B: How do the authors account for the difference in mortality associated with the agr mutant between mice and rabbits? The results seem counter-intuitive given the notion that the mouse should be insensitive to leukocidins controlled by Agr (as pointed by the authors).

2.l. 171-173 (Fig. 4): the authors state that 4 of 6 rabbits infected with the agr strain developed biofilms on the device and all these animals succumbed to infection. It would have been nice to report CFUs in biofilms or at least images of LAC and agr biofilms. Presumably some of the LAC infected animals also developed biofilm (Fig. 4A). It is hard to see the blue lines in this graph. Does this mean that biofilm in LAC infected animals do not release bacteria in the bloodstream?

3.Fig. 5: The authors conclude that neutropenia correlates with fatality in both device- and non-device associated sepsis (lines 186-187). However, when looking at Fig. 5D and Fig. 5H, it is hard to make such a conclusion. In panel 5D, animals survived with an average neutrophil count of 2000 per microliter. But in panel 5H, it seems that for the same average, all the animals died; rather 5000 neutrophils per microliter were required for survival.

4.The authors compare blood cell counts between surviving and dead animals throughout the manuscript. Do the authors observed any sort of cell count correlation when comparing the 2 strains used for infection?

Reviewer #3: 1.The data authors cite to justify the conclusion that leukopenia/neutropenia early after infection is associated with morbidity/death are not convincing as shown. If interpreting correctly from their methods description, they included repeated measures from the same 5-6 animals across several 2-hour intervals, but grouped all of these results stratified by survivors and non-survivors for comparison. That seems akin to having several technical replicates (repeated measures from the same animal) represent biological replicates for this critical comparison. This analysis should be more accurately represented, perhaps by measuring the fold-change in cell counts during the first 24 hours in each animal, or using an individual and consistent timepoint for each animal (ie- last CBC prior to moribund or last available for all animals) and comparing these between the groups.

2.In general, many of the conclusions inferred from the data in regards to recovered CFUs, WBC and neutrophil trends, and cytokine findings are based on trends without applied statistics (some examples are specifically cited as Minor Issues). This likely reflects lack of power to appropriately apply statistical comparisons between groups due to small numbers, but it generally weakens the strength of several of the major conclusions drawn by the authors.

3.The finding that rabbits with a CVC infected with the WT MRSA strain all survived the infection is very surprising and contrasts directly with the findings from the non-catheter-associated infection, yet there is no description from the authors to explain this finding. It is counter to the conclusion that agr-mediated leukocidin expression causes leuko- and neutropenia that leads to mortality and is not well explained by the possibility that bacteria seed the catheter early and are thus sequestered there since the leukocidins would be expected to be secreted. Likewise, explanation of the decreased impact of agr-deficiency on outcome in the non-catheter-associated infection in rabbits compared to mice is lacking.

**********

Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: 3) While cytolysis is a phenomenon that is readily observed in animals particularly when high inoculums of SA is injected, it is unclear how relevant that is with lower inoculum infections such as bloodstream infection (in humans). Toxins have important pro-inflammatory and cytolytic activity at different concentrations. This could be further discussed.

4) Line 204 – “Sepsis has not traditionally been thought to be impacted significantly by bacterial

205 virulence” – Not clear if this is a general consensus. Please provide a reference

5) Figure 3 and 4 - CFUs collected at time of death are not particularly useful. Probably better in supplement.

Reviewer #2: 1.Fig. 1B: Data was not collected at time 0 to ascertain the range of WBCs (neutrophils, monocytes, lymphocytes). The authors assume that all animals add the same number of neutrophils at time 0 but is this truly the case?

2.Fig. 2: Were bacterial inocula prepared similarly for infections shown in panels B and C?

3.The method regarding mouse infection (l. 302-303) should describe the preparation of S. aureus bacteria. Reference 20 describes a paper for S. epidermidis. It is okay to be specific here and confirm that authors performed tail vein injection.

4.l. 163: replace Dagr with “delta”agr.

5.Some of the sentences read awkwardly throughout the text especially in the Abstract and Author Summary.

Reviewer #3: 1.In Figure S1, p-values are described in the figure legend and referenced in the text but are not shown in the images.

2.In Figure 3B, could moribund animals be denoted with empty circles as in Fig S1? It would help clarify the statement that “CFU over the time course of infection in the blood and organs reflected the mortality results, inasmuch as they were (i) increased in moribund animals, at least in the blood and some organs” in lines 165-167.

3.The statement that “Leukocyte and neutrophil numbers decreased in the early hours of infection in the non-catheter-related infection model, while they increased again in survivors (Fig. 5A,B,E,F). This was not observed in the catheter-related model” in lines 179-181 is not clearly supported by the data shown.

4.The statement that “Concentrations of TNF-� followed a similar pattern but with much lower overall changes (Fig. 6B,H).” in lines 192-193 is not corroborated by the data shown, especially in the non-catheter-associated infection.

5.The statement that “However, we observed generally increased concentrations of pro- and decreased concentrations of anti-inflammatory cytokines over the course of the infection. Our results indicate that immune suppression early during infection plays a critical role for the development of sepsis, while later inflammatory phenomena appear associated with a moribund stage” in lines 231-235 are not corroborated by the data shown. In particular, the distinction of later inflammatory phenomena being associated with moribund stage are not supported by either the kinetics as shown or the lack of difference detected in survivors versus non-survivors.

**********

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PLoS Pathog. 2021 Feb 25;17(2):e1009369. doi: 10.1371/journal.ppat.1009369.r002

Author response to Decision Letter 0

24 Nov 2020

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PLoS Pathog. doi: 10.1371/journal.ppat.1009369.r003

Decision Letter 1

Michael R Wessels

1 Feb 2021

Dear Michael,

Thank you very much for submitting your manuscript "Bacterial virulence plays a crucial role in MRSA sepsis" for consideration at PLOS Pathogens. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by independent reviewers. The reviewers appreciated the attention to an important topic. Based on the reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the review recommendations.

The reviewers felt that the revised manuscript is substantially improved with addition of additional animal data and revisions to the text. However, I must agree with Reviewer 3 that a pervasive problem remains with the statistical analysis of the animal challenge experiments. Unless the reviewer and I misunderstand the methodology, multiple data points from an individual animal are treated as independent values, an approach which inflates the statistical power of comparisons between groups. I urge you to consult with a statistician to reanalyze these data using an appropriate method for repeated measures or another approach to define a single representative value for a particular time window for each animal. I understand that such an analysis may not yield statistical significance for every comparison, but I feel it is essential to use a rigorous statistical methodology.

Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to all review comments, and a description of the changes you have made in the manuscript.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Thank you again for your submission to our journal. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

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Mike

Michael Wessels

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

***********************

Reviewer Comments (if any, and for reference):

Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: The manuscript presents conceptual important findings.

Although the role of toxins has not been fully corroborated, the authors have satisfactorily addressed my concerns.

Reviewer #3: The authors’ revision addresses many of the questions and concerns raised in review of the original manuscript. Namely, the increased number of animals in the critical catheter-related infection model helps to strengthen many of the conclusions drawn about the differential role of agr-mediated effect on virulence in this model. The additional statistical analyses facilitated by these numbers (such as the correlations between agr status or biofilm formation and mortality) also bolster the strength of these conclusions. And the enhanced description of possible reasons for the differences in the role of agr in mice versus rabbits and in catheter- versus non-catheter-related infections substantiate these findings and provide a clearer understanding of what knowledge gaps remain to more fully understand the mechanisms of pathogenicity mediated by agr-regulated toxins.

**********

Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Reviewer #1: (No Response)

Reviewer #3: 1. In reference to the following comment from the original review: “The data authors cite to justify the conclusion that leukopenia/neutropenia early after infection is associated with morbidity/death are not convincing as shown. If interpreting correctly from their methods description, they included repeated measures from the same 5-6 animals across several 2-hour intervals, but grouped all of these results stratified by survivors and non-survivors for comparison. That seems akin to having several technical replicates (repeated measures from the same animal) represent biological replicates for this critical comparison. This analysis should be more accurately represented, perhaps by measuring the fold-change in cell counts during the first 24 hours in each animal, or using an individual and consistent timepoint for each animal (ie- last CBC prior to moribund or last available for all animals) and comparing these between the groups.” Authors’ cite the many challenges in comparing a similar timepoint or a standardized fold-change given difficulty in acquiring a complete set of data due to blood draw limits and technical feasibility in the rabbit experiments. While these challenges can certainly be appreciated, the chosen representation combining multiple timepoints from the same animal in the comparator groups (survived versus died or WT versus delta-agr) may be misleading. Since these data are cited to support one of the major findings from this work, that leukopenia early after infection predicts mortality from S. aureus sepsis, this analysis method should be reconsidered or should perhaps be cited as one possible limitation of the findings as presented.

**********

Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: (No Response)

Reviewer #3: 1. The light blue line and open triangle used to denote LAC (biofilm) is too light. This symbol seems to only account for one animal, but it is hard to see in any of the panels.

2. It seems in Figure 3, the intent is to denote animals that survived with closed symbols and those that did not with open, but this should be clarified either on the graphs or in the legend for Figure 3.

**********

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Reviewer #1: No

Reviewer #3: No

Figure Files:

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Reproducibility:

PLoS Pathog. 2021 Feb 25;17(2):e1009369. doi: 10.1371/journal.ppat.1009369.r004

Author response to Decision Letter 1

9 Feb 2021

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PLoS Pathog. doi: 10.1371/journal.ppat.1009369.r005

Decision Letter 2

Michael R Wessels

10 Feb 2021

Dear Dr. Otto,

We are pleased to inform you that your manuscript 'Bacterial virulence plays a crucial role in MRSA sepsis' has been provisionally accepted for publication in PLOS Pathogens.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.

IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.

Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us now if you or your institution is planning to press release the article. All press must be co-ordinated with PLOS.

Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Pathogens.

Best regards,

Michael R. Wessels

Section Editor

PLOS Pathogens

Michael Wessels

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

***********************************************************

Reviewer Comments (if any, and for reference):

PLoS Pathog. doi: 10.1371/journal.ppat.1009369.r006

Acceptance letter

Michael R Wessels

18 Feb 2021

Dear Dr. Otto,

We are delighted to inform you that your manuscript, "Bacterial virulence plays a crucial role in MRSA sepsis," has been formally accepted for publication in PLOS Pathogens.

We have now passed your article onto the PLOS Production Department who will complete the rest of the pre-publication process. All authors will receive a confirmation email upon publication.

The corresponding author will soon be receiving a typeset proof for review, to ensure errors have not been introduced during production. Please review the PDF proof of your manuscript carefully, as this is the last chance to correct any scientific or type-setting errors. Please note that major changes, or those which affect the scientific understanding of the work, will likely cause delays to the publication date of your manuscript. Note: Proofs for Front Matter articles (Pearls, Reviews, Opinions, etc...) are generated on a different schedule and may not be made available as quickly.

Soon after your final files are uploaded, the early version of your manuscript, if you opted to have an early version of your article, will be published online. The date of the early version will be your article's publication date. The final article will be published to the same URL, and all versions of the paper will be accessible to readers.

Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Pathogens.

Best regards,

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig. Physiological parameters in the rabbit sepsis models.

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S2 Fig. Rabbit sepsis models–leukocyte numbers (monocytes, lymphocytes).

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S3 Fig. Impact of Agr status on cytokine levels early during infection.

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Data Availability Statement

All relevant data are within the manuscript and its Supporting Information files.

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PERMALINK

Bacterial virulence plays a crucial role in MRSA sepsis

Gordon Y C Cheung

Justin S Bae

Ryan Liu

Rachelle L Hunt

Yue Zheng

Michael Otto

Roles

Abstract

Author summary

Introduction

Results

The quorum-sensing virulence regulator Agr strongly impacts mortality in a mouse sepsis model

Fig 1. Mouse sepsis model.

The impact of Agr on sepsis-related mortality is strongly dependent on whether the infection is device-associated

Fig 2. Rabbit sepsis models–experimental setup and survival.

Fig 3. Rabbit non-catheter-associated sepsis model–CFU in blood and organs.

Fig 4. Rabbit catheter-associated sepsis model–CFU in blood, organs, and on catheters.

Early neutropenia is associated with a fatal outcome of sepsis

Fig 5. Rabbit sepsis models–leukocyte numbers.

Fig 6. Rabbit sepsis model–cytokine levels.

Discussion

Materials and methods

Ethics statement

Bacterial strains and growth conditions

Mouse model of non-catheter-associated blood infection

Rabbit models of systemic catheter- and non-catheter-associated blood infection

CFU enumeration, cytokine measurements and complete blood count analyses from blood

Statistics

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Michael H Malim

Michael R Wessels

Roles

Author response to Decision Letter 0

Decision Letter 1

Michael R Wessels

Roles

Author response to Decision Letter 1

Decision Letter 2

Michael R Wessels

Roles

Acceptance letter

Michael R Wessels

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

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