Growing Staphylococcus aureus in Synthetic Cystic Fibrosis Medium Promotes Colonization in a Murine Pneumonia Model

Staphylococcus aureus, mouse model, cystic fibrosis 19 Running Title: S. aureus in SCFM2 increases mouse lung infection 20 Word count: abstract (234), Importance (147), text (5520) 21 22 .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted October 14, 2025. ; https://doi.org/10.1101/2025.10.14.682363doi: bioRxiv preprint

23 Staphylococcus aureus is a leading cause of bacterial infections worldwide and can lead to 24 diseases such as osteomyelitis, skin, and lung infections in humans. Murine mouse models have 25 been essential to the study of S. aureus virulence but are not without their limitations. In murine 26 pneumonia models, colonization of the lungs by S. aureus are generally not well maintained. To 27 increase the level and duration of S. aureus respiratory colonization, various methods have been 28 employed including embedding the bacteria in agar beads and suppressing the mouse’s immune 29 system. These modifications have improved colonization, but they do not accurately represent 30 clinical infections of diseases such as cystic fibrosis (CF). We hypothesize that culturing S. 31 aureus in a different media such as Synthetic CF Medium 2 (SCFM2) would increase 32 colonization compared to growing the bacteria on standard rich media agar plates. We observed 33 that culturing 6 different S. aureus strains in SCFM2 led to either a neutral or increased level of 34 lung colonization compared to agar plates. For one strain, WU1, culturing in SCFM2 improved 35 colonization in the oropharynx compared to agar plates and led to a sustained long-term infection 36 in the lungs. Finally, when cultured in SCFM2 compared to agar plates, infection with WU1 led 37 to increased inflammation in both the left and right lung lobe. Overall, we have shown that 38 culturing S. aureus in different conditions prior to infection impacts colonization and host 39 response. 40 IMPORTANCE 41 Staphylococcus aureus is a bacterial pathogen that can infect multiple anatomical sites in 42 humans. To study S. aureus virulence, murine mouse models have been an essential tool. 43 However, it has been difficult to establish and maintain these infections. To improve S. aureus 44 pneumonia murine models, changes to the bacterial dose and mice have been utilized but limits 45 .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted October 14, 2025. ; https://doi.org/10.1101/2025.10.14.682363doi: bioRxiv preprint its accuracy to represent clinical infections. In this study, we compared how culturing S. aureus 46 in two different conditions prior to infection impacts colonization. We found that the effects of 47 growing S. aureus in these different media on acute infections is strain specific. Following up 48 with one of these strains, we showed that Synthetic Cystic Fibrosis Medium 2 (SCFM2) 49 increases S. aureus colonization in the oropharynx and leads to a sustain long-term infection in 50 the lungs. Additionally, we showed how culturing S. aureus in these conditions impacts the host 51 response. 52

53 Staphylococcus aureus is a Gram-positive pathogenic bacterium and a leading cause of bacterial 54 infections worldwide (1). This bacterium can colonize a variety of mammals including humans 55 (2). In humans, S. aureus can persist in multiple anatomical sites and can cause a wide variety of 56 infections such as osteomyelitis, endocarditis, skin, and lung infections (3). Additionally, 57 approximately 30% of the human population is asymptomatically colonized with S. aureus in 58 their nasal cavity (4). 59 Murine models, particularly mice, have been an essential tool to study S. aureus in the 60 context of infections (5). They offer many advantages such as small size, relative ease of 61 breeding, cost efficiency, a well-characterized immune system, as well as the ability to generate 62 transgenic strains. For these reasons, mice have been used to study infections induced by S. 63 aureus such as sepsis, osteomyelitis, endocarditis, and pneumonia (6–9). However, in murine 64 pneumonia infection models, there are difficulties with maintaining bacterial colonization which 65 leads to issues with assessing potential treatments (10). When testing S. aureus strains in mice in 66 the context of lung infections, most studies have had to administer supraphysiological (10 7-108 67 .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted October 14, 2025. ; https://doi.org/10.1101/2025.10.14.682363doi: bioRxiv preprint CFUs) doses intranasally to mice to establish infection (9–13); doses below this threshold 68 generally result in clearance rather than colonization. 69 To improve the sustainability of S. aureus during pneumonia infections in mice, 70 modifications to the preparation of the bacterial dose and the strains of mice used have been 71 explored. Administration of S. aureus embedded in agar beads intrathecally to mice have led to 72 prolonged infections that were sustained for at least 21 days (14, 15). Cyclophosphamide, an 73 immunosuppressive chemotherapy that leads to the depletion of neutrophils, has also been used 74 to render mice immunocompromised and more susceptible to S. aureus infections (16–18). 75 Additionally, changing the choice of bacteria used in studies have been evaluated. The use of 76 mouse-derived S. aureus strains has been shown to more persistently colonize the nasopharynx 77 compared to human-derived S. aureus strains (19, 20). These approaches have been greatly 78 beneficial to advancing S. aureus infection models; however, these modifications have important 79

which have restricted their adoption. In particular, while embedding S. aureus in agar 80 beads improves infection sustainability, it does not accurately represent a clinical infection for 81 diseases such as cystic fibrosis (CF). 82 CF is a multi-organ genetic disease that impacts more than 120,000 people worldwide 83 (21). This disease is caused by mutations in the CF transmembrane regulator, a sodium and 84 bicarbonate ion channel, which negatively impacts mucus clearing in the lungs and leads to the 85 buildup of sputum (22). Microbial pathogens can utilize sputum as a nutrient source to establish 86 and maintain chronic infections (23). As a result, CF patients are frequently infected with 87 respiratory pathogens throughout their life (24). These chronic bacterial respiratory infections 88 remain the leading cause of morbidity in these patients. S. aureus is the most frequently isolated 89 bacteria from respiratory samples of CF patients (8) and persistently colonizes these patients (25, 90 .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted October 14, 2025. ; https://doi.org/10.1101/2025.10.14.682363doi: bioRxiv preprint 26). Thus, models to study S. aureus in the context of lung infections are paramount to 91 understanding its impact in CF infections. 92 Here, we studied the growth of S. aureus in two different culture conditions prior to 93 infection and monitored its impact on murine colonization and host response. Traditionally, to 94 prepare S. aureus for infection, bacteria are cultured in rich laboratory media (such as tryptic soy 95 or lysogeny) on plates or in broth prior to infection. Rich media is relatively inexpensive, widely 96 used and studied, and offers consistency between laboratories. However, this growth condition 97 does not replicate the nutrient composition of a human environment. To address this issue, a 98 defined laboratory medium that mimics the nutrient composition of the sputum of CF patients 99 was developed called Synthetic CF Medium 2 (SCFM2) (27). A previous study by Ibberson and 100 Whiteley revealed that when comparing the gene expression of S. aureus cultured in rich media 101 to human CF sputum, the rich media transcriptomes clustered independently of human samples 102 (28). Further they observed that when cultured in SCFM2, the S. aureus transcriptomes were 103 more similar to S. aureus from CF sputum compared to rich laboratory media (28). Here, we 104 propose that the choice of environmental conditions of S. aureus growth prior to infection may 105 improve S. aureus colonization during murine pneumonia models and hypothesize that culturing 106 S. aureus in SCFM2 prior to infection will increase lung colonization. 107 To test this, we compared the effects of culturing 6 different S. aureus strains using rich 108 media (lysogeny broth; LB) agar plates and in SCFM2 on lung colonization in 109 immunocompetent mice. LB is a commonly used rich laboratory media composed of tryptone, 110 yeast extract, and sodium chloride. As mentioned, SCFM2 contains components meant to mimic 111 sputum from patients with CF; these include mucin, DNA, and amino acids. We chose strains 112 from both human and mouse infections as well as strains from different clonal complexes (CC). 113 .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted October 14, 2025. ; https://doi.org/10.1101/2025.10.14.682363doi: bioRxiv preprint S. aureus isolates are grouped into different CCs based on their similarity of specific 114 housekeeping genes. In the United States, CC8 and CC5 are the dominate strains isolated from 115 patients and we included strains to represent both (29). We observed that while the effects are 116 strain specific, SCFM2 either had a neutral impact or increased the strains’ ability to colonize the 117 murine lung. In one S. aureus strain known to persistently colonize the oropharynx in mice, 118 WU1, we found that culturing in SCFM2 improved colonization levels in the oropharynx. We 119 also discovered that culturing WU1 in SCFM2 leads to sustained colonization in the lungs for at 120 least 4 days. Furthermore, infections with this strain cultured in SCFM2 led to increased 121 inflammation and altered cytokine levels in the left and right lung lobes compared to growing 122 this strain on agar plates. Overall, we have shown the impact of culturing S. aureus in SCFM2 123 compared to agar plates on colonization and host response in a murine pneumonia model. 124

125 S. aureus strains used in this study exhibit both clinical and genomic diversity. 126 The goal of this study is to characterize how altering culture conditions impacts S. aureus 127 colonization during murine pneumonia. To achieve this, we sought to test a diverse set of strains 128 under two growth conditions. We used 6 S. aureus strains: 5 strains isolated from human 129 infections and 1 strain isolated from a mouse infection (Table 1). JE2 and Newman, both CC8 130 strains, are frequently used as model strains for methicillin-resistant (MRSA) and methicillin-131 sensitive (MSSA) strains, respectively (30, 31). JE2 is a derivative of a strain isolated from a 132 skin abscess, while Newman was isolated from a tubercular osteomyelitis infection. N315, an 133 MRSA pharyngeal isolate, was chosen as a representative CC5 strain (32). Sa_CFBR_43 134 (MRSA) and Sa_CFBR_46 (MSSA) was isolated from respiratory samples of patients with CF 135 (33). Sa_CFBR_43 belongs to CC8 while Sa_CFBR_46 belongs to CC45, a lineage of S. aureus 136 .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted October 14, 2025. ; https://doi.org/10.1101/2025.10.14.682363doi: bioRxiv preprint that has two classes of S. aureus quorum sensor regulators (34). WU1 (MSSA) was isolated from 137 an outbreak of preputial gland infections in a mouse breeding facility and belongs to CC88, a CC 138 that is primarily isolated from rodents in the Western World (20, 35). We determined the 139 diversity of these strains genomically through average nucleotide analysis comparisons (Table 140 S1) (Figure 1). Perhaps not surprisingly JE2, Newman, and Sa_CFBR_43, all being CC8, shared 141 greater than 99.9% similarity. Sa_CFBR_46 was the most genetically distant compared to the 142 other strains followed by WU1. 143 Culturing S. aureus strains in SCFM2 leads to similar growth kinetics compared to 144 culturing in LB. 145 LB and SCFM2 differ in nutrient composition which could have an impact on S. aureus growth 146 and physiology (27). To determine whether the difference in nutrient composition impacts 147 growth, all strains were cultured statically in either LB or SCFM2 in 6-well plates and sampled 148 for colony forming units (CFUs) every 2 hours for 8 hours as well as at 24 hours (Figure 2). 149 During the first 8 hours of growth, all strains exhibited similar growth kinetics in both media. At 150 the 24-hour time point, there was equal number of CFUs for all strains cultured in both growth 151 media, except for Newman. Newman had a higher CFUs when cultured in LB (10 8.5 CFUs) 152 compared to SCFM2 (108.2 CFUs) at 24-hours. These results indicate that the strains investigated 153 here exhibit similar growth kinetics in LB and SCFM2. 154 Culturing S. aureus in SCFM2 increases lung colonization in most examined strains. 155 We hypothesized that culturing S. aureus in SCFM2 would increase the level of colonization of 156 the mouse lung compared to LB. To test this hypothesis, we infected 8–10-week-old C57BL/6 or 157 Balb/c mice with ~1 x 10 8 CFUs of each S. aureus strain, prepared through our agar plate and 158 SCFM2 workflow (Figure 3). At 24-hours post-infection, the mice were sacrificed and S. aureus 159 .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted October 14, 2025. ; https://doi.org/10.1101/2025.10.14.682363doi: bioRxiv preprint colonization in both the lungs (Figure 4) and nasal cavity (Supplemental Figure 1) was 160 determined. 161 Our results revealed that culturing strains Sa_CFBR_43, Sa_CFBR_46, N315, and WU1 162 in SCFM2 significantly increased lung colonization in both C57BL/6 and Balb/c mice compared 163 to culturing on agar plates (Figure 4C, 4D, 4E, 4F). In contrast, there was no significant 164 difference in colonization for JE2 and Newman between the culture conditions in either mouse 165 strain (Figure 4A, 4B). When cultured in SCFM2, N315 had the highest average colonization in 166 C57BL/6 mice while WU1 had the highest average colonization in Balb/c mice. The effects of 167 SCFM2 on nasal cavity colonization were more variable (Supplemental Figure 1). While there 168 were no significant differences in JE2 colonization in either mouse strains, Newman had 169 significantly lower colonization in the nasal passage of Balb/c mice when cultured in SCFM2 170 compared to agar plates while no significant differences in C57BL/6 mice. Both CF isolates, 171 Sa_CFBR_43 and Sa_CFBR_46, had significantly lower colonization in the nasal cavity of 172 either mouse strain. Finally, N315 and WU1 had significantly higher colonization in the nasal 173 cavity independent of mouse strains. Overall, our results indicate that while the effects of 174 growing S. aureus in SCFM2 on lung and nasal cavity colonization is strain-specific, it can 175 improve colonization levels in the lungs. 176 Culturing WU1 in SCFM2 increases recovery from the throat of mice 2 days post infection 177 and from the lungs at 4 days post infection compared to LB. 178 Since we have observed that the effects of SCFM2 on S. aureus colonization were strain specific, 179 we used S. aureus strain WU1 as a model system for subsequent studies. WU1 had the highest 180 and most consistent level of colonization in Balb/c mice when cultured in SCFM2, therefore, we 181 chose to concentrate our subsequent analysis on WU1 and Balb/c mice. Previous studies have 182 .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted October 14, 2025. ; https://doi.org/10.1101/2025.10.14.682363doi: bioRxiv preprint only characterized WU1’s ability to persistently colonize mice oropharynx during respiratory 183 infections (20, 36, 37) and we aimed to determine if SCFM2 can improve the level and duration 184 of both the throat and lung colonization. We monitored colonization in both the throat and the 185 lungs of Balb/c mice over time after they intranasally infected with ~1 x 10 8 C F U s o f W U 1 186 grown using either culture condition (Figure 5). In one cohort of mice, throat colonization was 187 determined by swabbing the oropharynx of the same mice on 1, 2, 3-, 7-, 10-, and 14-days post 188 infection (Figure 5A). There was significantly higher oropharynx colonization by WU1 when 189 grown in SCFM2 compared to on agar plates at day 1 and 2 post infection. No significant 190 differences were observed on days 3, 7, 10, and 14 post infection. This suggests that the initial 191 improvement in WU1 oropharynx colonization provided by SCFM2 occurs within 2 days post-192 infection. Next, we monitored WU1 colonization in lungs over time using separate cohorts of 193 Balb/c mice (Figure 5B). Every day for 4 days, colonization levels in the lungs of the mice were 194 determined. We observed significantly higher and consistent lung colonization up to 4 days post 195 infection when WU1 was grown in SCFM2 compared to agar plates. Overall, our results indicate 196 that, in addition to increasing colonization during early infections, culturing WU1 in SCFM2 197 increases oropharynx colonization 2 days post infection and leads to sustained lung colonization 198 for 4 days post infection. 199 Infections with WU1 cultured in SCFM2 leads to increased inflammation and altered 200 cytokine profile compared to agar conditions. 201 We next sought to determine how culturing WU1 in SCFM2 vs. agar plates impacts the host 202 response through histology and cytokine analysis in our pneumonia model. Balb/c mice were 203 intranasally infected with ~1 x 10 8 CFUs of WU1 prepared through either culture methods; 1X 204 PBS, and SCFM2 alone served as “vehicle controls". For these studies, mice were sacrificed 24-205 .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted October 14, 2025. ; https://doi.org/10.1101/2025.10.14.682363doi: bioRxiv preprint hours post infection, and the bacterial load in each lung lobe was determined. We observed a 206 relatively even distribution of CFUs in the left or right lung lobes of mice infected with WU1 207 cultured in SCFM2 (Supplemental Figure 2B). In WU1 cultured from agar plates, we observed 208 slightly higher CFUs recovered from the left compared to the right lung lobes. Overall, we 209 observed higher levels of colonization in both lung lobes when infected with WU1 cultured in 210 SCFM2 compared to agar plates. 211 We wanted to determine how WU1 growth conditions prior to infection impacted the 212 response in the lung. To do this, we used two parallel sets of mice for histology and cytokine 213 analysis (Supplemental Figure 2A). In the first set, the left lung lobe was processed for histology 214 while the right lobe was processed for cytokine analysis. In the other set, the opposite lobe was 215 processed for histology and cytokine analysis. Thus, for all these comparisons, the left and right 216 lungs were from different mice. 217 We assessed the histopathological changes (Figure 6) and cytokine levels (Figure 7) in 218 each lung lobe following infection with WU1 cultured in SCFM2 vs. on agar plates. Overall, we 219 did not observe a major difference between the left and right lobes under any of the conditions 220 tested. First, we monitored the effects of the “vehicle controls” and found that administration of 221 1X PBS led to scores within the normal histological limits while SCFM2 led to the recruitment 222 of primarily lymphocytes (Figure 6A, 6B, 6E, 6F). These results of the vehicle control were used 223 as the baseline when assessing the histology of the infected lungs. While WU1 cultured from 224 either SCFM2 or agar plates led to inflammation composed of PMN cells, WU1 cultured in 225 SCFM2 led to a higher degree of inflammation as seen by the multifocal and coalescing areas of 226 PMN cells in both lung lobes (Figure 6C, 6D, 6G and 6H). Inflammation of lungs infected with 227 .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted October 14, 2025. ; https://doi.org/10.1101/2025.10.14.682363doi: bioRxiv preprint WU1 cultured from agar plates was scored as moderate to severe while lungs infected with WU1 228 cultured in SCFM2 was scored as severe. 229 Next, we measured the cytokine levels in the other lung lobe. Overall, in both lungs, we 230 generally observed higher cytokine levels in lungs infected with WU1 under either growth 231 conditions compared to the vehicle controls (Figure 7). The only exception was that there was 232 slightly more IL-5 detected in the right lung that was administered the PBS control compared to 233 WU1 cultured on agar plates, however all these levels were very low. In looking specifically at 234 the right lungs, we observed significantly higher levels of the cytokines IL-6, TNF- /i1, KC/GRO, 235 IL-1β , IL-2, and IL-5 in the mice infected with WU1 cultured in SCFM2 compared to agar plates 236 (Figure 7B). On the other hand, IL-12p70, IL-10, or IFN-/g2011 had lower or no significant difference 237 between conditions. For the left lungs, significantly higher levels of KC/GRO were detected in 238 the lungs infected with WU1 cultured in SCFM2 compared to agar plates. TNF- /i1 , IL-12p70, 239 and IFN- /g2011 were significantly lower between these conditions while there was no significant 240 differences between the levels of IL-6, IL-1 β , IL-10, IL-2, and IL-5. Overall, we observed a 241 cytokine profile that does correlate with the higher levels of inflammation and neutrophil 242 recruitment observed in the histology between the culture conditions. Our findings indicate that 243 culturing WU1 in SCFM2 leads to increased inflammation in both lung lobes that is reflected 244 both in histopathology and cytokine levels. 245

246 Laboratory mice have often been used as an in vivo model to study S. aureus infections 247 such as bacteremia, sepsis, pneumonia, and many others (5). These models have been essential in 248 advancing our understanding of S. aureus virulence and host response but have important 249 limitations. When studying S. aureus induced pneumonia in murine models, the level of 250 .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted October 14, 2025. ; https://doi.org/10.1101/2025.10.14.682363doi: bioRxiv preprint colonization is not well sustained (9, 13, 38, 39). Groups have explored the use of different 251 concentrations of inoculum to induce S. aureus pneumonia infection. Parker et al. administered 1 252 x 107 CFUs of S. aureus strain USA300 intranasally to C57BL/6J mice and observed a recovery 253 of 0.5 x 10 7 CFUs from lung homogenates after 24 hours (38). Torres et al. observed a similar 254 recovery after administering a lethal dose of 3.78 x 108 CFUs of S. aureus strain Newman via the 255 same route to C57BL/6J mice after 18 hours (13). Finally, Urso et al. recovered 5 x 10 5 CFUs of 256 Newman after 24 hours following administration of 1 x 10 8 CFUs intranasally to C57BL/6 mice 257 (39). Additional modifications include using mouse-adapted strains, altered inoculum 258 preparation methods, and depletion of the mouse’s innate immune response (9, 14, 16–19, 40). 259 While these methods have resulted in infections, the levels of colonization could be improved, 260 especially in immunocompetent mice. Here, we hypothesized that culturing S. aureus in a media 261 to mimic the lung environment (SCFM2), would increase colonization. 262 Our study reveals that improving S. aureus colonization during a murine respiratory 263 infection relies on the interactions between several factors: the choice of S. aureus strain, the 264 choice of mouse strain, and the growth conditions of the bacteria inoculum. Of the 6 S. aureus 265 strains tested, we observed the greatest improved of colonization in N315 for C57BL/6 mice and 266 WU1 for Balb/c mice. WU1’s sustained colonization could be attributed to it being isolated from 267 a mouse and may already be more adapted to evade this specific host response. In mice, culturing 268 JE2 and Newman, two commonly studied S. aureus strains, in SCFM2 did not improve 269 colonization in mice while all other strains (Sa_CFBR_43, Sa_CFBR_46, N315, and WU1) 270 showed increased levels of colonization. Except for WU1, all S. aureus strains tested were 271 isolated from human infection but only strains isolated from respiratory infections 272 (Sa_CFBR_43, Sa_CFBR_46, and N315) had increased colonization levels in SCFM2. 273 .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted October 14, 2025. ; https://doi.org/10.1101/2025.10.14.682363doi: bioRxiv preprint Interestingly, of the 3 CC8 strains tested, only Sa_CFBR_43 had a significantly higher lung 274 colonization in both mouse strains when cultured in SCFM2 compared to agar plates despite 275 being highly similar genomically to JE2 (99.99%) and Newman (99.85%). In future studies, 276 exploring the genetic differences between JE2 and Sa_CFBR_43 might reveal why SCFM2 277 increases Sa_CFBR_43 colonization in mice. Similar studies carried out by Sun et al. revealed 278 that the Staphylococcal protein A (spA) is required for persistent colonization in the nasal 279 passage of WU1 (20). JE2 and Sa_CFBR_43 share genomically identical sequences for spA but 280 whether differences in expression of this protein contributes to colonization was not investigated 281 in our study. While more strains must be tested, adaptations to the infection site where the strains 282 were isolated from might influence the colonization of S. aureus. When developing models for 283 infection and choosing a strain to test, we recommend testing strains isolated from different 284 infection sites as that might impact colonization. 285 When assessing how different culture conditions impacts colonization of the 6 S. aureus 286 strains, we utilized both C57BL/6 and Balb/c mice. These two strains are the most commonly 287 used inbred mouse strains in the field, are the genetic backbone of many transgenic strains, and 288 allow the study of different innate immune responses. C57BL/6 mice exhibit a stronger Th1 289 immune response while Balb/c exhibit a stronger Th2 immune response (41, 42). Previous 290 studies of WU1 have primarily focused on its ability to persistently colonize the nasopharynx in 291 C57BL/6 mice (20, 36, 37). Our study characterized the ability of WU1 to colonize the lungs of 292 Balb/c mice not only during an acute short-term infection but also during a longer-term infection. 293 Compared to infections in C57BL/6, we found that WU1 colonizes the lungs of Balb/c mice at a 294 higher and more consistent level. We propose that Balb/c mice may be the preferable choice to 295 study WU1 in the context of lung colonization in SCFM2. Additionally, our findings compare 296 .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted October 14, 2025. ; https://doi.org/10.1101/2025.10.14.682363doi: bioRxiv preprint the colonization distribution, histology, and cytokine recruitment between the left and right lobes 297 of mice infected with S. aureus. The distribution of S. aureus in one or another mouse lung lobe 298 following intranasal administration has not been previously studied according to our literature 299 search. We found that WU1 had similar levels of colonization in both the left and right lung lobe 300 as well as similar histopathology scores but a different proinflammatory cytokine profiles. When 301 cultured on agar plates, we observed that WU1 colonization in the left lung was slightly higher 302 than the right lung while the distribution of WU1 cultured in SCFM2 was more even between 303 lobes. Similar pathohistological scoring was between the right and left lungs when infected with 304 WU1 cultured in both conditions. Previous studies had shown that S. aureus induced pneumonia 305 in mice leads to inflammation and the recruitment of polymorphonuclear (PMN) cells and our 306

were consistent with those findings (9, 43, 44). While both culture conditions resulted in 307 inflammation, the degree of inflammation was higher when WU1 was cultured in SCFM2 308 compared to agar plates. The cytokine profile was generally consistent between the lobes under 309 both culture conditions but there were some variations. Levels of proinflammatory cytokines (IL-310 1β , IL-6, TNF-/i1 , IFN-/g2011, IL-2, and IL-5) were generally lower in the right lungs compared to the 311 left when infected with WU1 cultured on agar plates but higher when infected with WU1 312 cultured in SCFM2. In our infection model, there was not a significant difference in WU1 313 colonization observed but a different host response between the left and right lungs. This 314 suggests that harvesting individual lung lobes may be useful for measuring colonization to 315 decrease the number of mice used but the whole lung may be more appropriate for measuring the 316 host response. 317 Finally, our results compare the impact that culturing S. aureus in SCFM2 compared to 318 agar plates has on both bacterial colonization and host response during S. aureus induced murine 319 .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted October 14, 2025. ; https://doi.org/10.1101/2025.10.14.682363doi: bioRxiv preprint pneumonia. The agar plate preparation has been traditionally used in the lab to prepare bacteria 320 for murine pneumonia infections (45–47). The choice of growing S. aureus in SCFM2 in wells 321 was adapted from previous studies that used 4-well microchamber slides to study S. aureus 322 transcriptomics, physiology, and spatial biogeography (28, 48). A 6-well plate format was 323 chosen to maximize the surface area and increase the overall number of S. aureus . The 324 comparison between the agar plate, our traditional bacterial method, and the SCFM2 preparation 325 was adapted from a study from our group that compared the transcriptomics of Pseudomonas 326 aeruginosa during murine pneumonia infections (49). When comparing growth kinetics of each 327 strain in LB to SCFM2 in 6-well plates, no significant differences were observed between the 328 two growth mediums. This indicates that the nutritional environment of the media did not 329 significantly alter the growth kinetics of the bacteria and was not responsible for the differences 330 observed in colonization. Based on our results, SCFM2 either had a neutral or beneficial impact 331 on S. aureus’ ability to colonize the lungs during acute pneumonia when compared to our 332 standard laboratory protocol of culturing bacteria on agar plates. For WU1 in particular, we 333 observed that growth in SCFM2 results in higher colonization in both the throat and lungs of 334 Balb/c mice compared to agar plates. WU1’s ability to colonize the oropharynx has been 335 described and our results indicate growth in SCFM2 improves colonization levels for 2 days post 336 infection. WU1’s colonization in the lungs have not previous been explored and here we report 337 that culturing WU1 in SCFM2 leads to a sustained long-term infection up to 4 days post 338 infection. For all S. aureus strains tested here, while SCFM2 can improve lung colonization, the 339 overall CFUs recovered from lungs were still lower than the number of originally administered 340 bacteria. This suggest that while S. aureus is typically cleared by the host, growth in SCFM2 341 may improve the bacteria’s ability to persist beyond 24 hours in the lungs. 342 .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted October 14, 2025. ; https://doi.org/10.1101/2025.10.14.682363doi: bioRxiv preprint In conclusion, we have characterized the potential benefits culturing S. aureus in SCFM2 343 has on colonization during murine respiratory infections as well as the host response compared to 344 LB media. While this study utilized six S. aureus strains, ultimately focusing on one, S. aureus is 345 known to infect multiple human body site and how SCFM2 impacts isolates from different 346 infection sites remains to be determined. Our study administered S. aureus to the mice 347 intranasally but the effect of commonly used methods of administration, such as intratracheal, on 348 the infection will be explored in future studies. Additionally, we report that WU1 infections in 349 the lungs were well maintained up to 4 days post-infection and future studies will identify how 350 long the infection can be maintained. The use of humanized mice has been shown to be more 351 susceptible to S. aureus infections and allowed the study of human response to infection (50, 51). 352 The infection outcome of humanized mice, such as CF humanized mice, when infected with S. 353 aureus cultured in SCFM2 might further improve the clinical relevance of this infection model. 354 Finally, while we observed that SCFM2 can increase colonization in the lungs, the mechanisms 355 responsible are not understood. In vivo proteomics and transcriptomics might provide insight into 356 the underlying benefits of SCFM2. Overall, our findings highlight the interplay between bacterial 357 strain, mouse strain, and culture conditions on S. aureus ability to colonize during a murine 358 pneumonia model and lays the groundwork for future studies. 359

360 Bacterial Strains and Comparative Analysis 361 Six S. aureus strains were used in this study (Table 1). To determine the diversity of the S. 362 aureus strains chosen, the whole genomes of each strain were compared bioinformatically. The 363 sequence assemblies for JE2, Newman, N315, Sa_CFBR_43, and Sa_CFBR_46 was retrieved 364 from NCBI. The whole genome sequence of WU1 was kindly provided by Dr. Dominique 365 .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted October 14, 2025. ; https://doi.org/10.1101/2025.10.14.682363doi: bioRxiv preprint Missiakas (University of Chicago). All assemblies were compared to each other, and average 366 nucleotide identity analysis was determined through pyani (52). 367 Bacterial Growth Curves in LB and SCFM2 in 6-well Plates 368 Strains were streaked on Staphylococcus Isolation Agar (SIA) (lysogeny broth (LB) (BD) + 7.5% 369 NaCl) and grown overnight at 37 /i2 . A single colony was transferred to a 2 ml of LB and grown 370 shaking at 37 /i2 . The cultures were then transferred to a 1.5 ml Eppendorf tube, washed 3 times 371 with sterile 1X phosphate buffer saline (PBS), and optical density was measured at 600 nm 372 (OD600). The cultures were adjusted to an OD 600 of 0.01 in 6 ml of LB or SCFM2 (Synthbiome) 373 and were and grown statically in a 6-well plate (Cellstar) at 37 /i2 . Aliquots of each culture were 374 removed every 2 hours for 8 hours as well as 24 hours, serially diluted, and plated on Lysogeny 375 agar (LA) plates. The plates were placed in at 37 /i2 and the number of colony-forming units 376 (CFUs) was determined the following day. 377 S. aureus Growth on Agar Media or SCFM2 Workflow 378 To prepare the S. aureus inoculum under our agar plate procedure, strains were streaked on SIA 379 and grown overnight at 37 /i2 . On the following day a small swab of each S. aureus was 380 transferred into a sterile Eppendorf tube containing sterile 1X PBS. The strains were washed 3 381 times and OD 600 was measured. Cultures were adjusted to an OD 600 of 6.5 in 1 ml of 1X PBS, 382 corresponding to ~1 x 108 colony-forming units (CFU) per 25 µl dose. An aliquot was removed, 383 serially diluted, and plated on SIA plates to determine colony forming units per dose. 384 For S. aureus inoculum preparation in SCFM2, strains were streaked on SIA and grown 385 overnight at 37 /i2 . A single colony was transferred to a culture tube with 2 ml of LB and grown 386 shaking at 37 /i2 overnight. The cultures were then pelleted, washed 3 times with 1X PBS, and 387 the OD600 was measured. The strains were then adjusted to an OD600 of 0.01 in 6 ml of SCFM2 in 388 .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted October 14, 2025. ; https://doi.org/10.1101/2025.10.14.682363doi: bioRxiv preprint a 6-well plate and were grown statically at 37 /i2 for 24 hours. After incubation, the cultures were 389 transferred to a 15 ml falcon tube, pelleted, and concentrated 15X in fresh SCFM2. An aliquot 390 was removed, serially diluted, and plated on SIA plates to determine CFU per dose. 391 Mice 392 All animal experiments were performed according to the guidelines of the Emory University 393 Institutional Animal Care and Use Committee under the approved protocol PROTO201700441. 394 8- to 10-week-old female C57BL/6 and Balb/c mice were purchased from Jackson Laboratories 395 (Bar Harbor, ME) and were acclimated for at least a week at Emory University before 396 experiments. Prior to infection, all mice were anesthetized with an intraperitoneal injection of a 397 0.2 ml mixture of ketamine (6.7 mg/ml) and xylazine (1.3 mg/ml). All mice were euthanized by 398 CO2 asphyxiation. 399 Lung Infection 400 Anesthetized C57BL/6 and Balb/c mice were intranasally administered 25 µl (12.5 µl per nostril) 401 of each strain cultured either on agar plates or in SCFM2 preparation corresponding to about 1 x 402 108 CFUs. Mice were humanely euthanized at specified time points and whole lungs and nasal 403 wash were aseptically collected. For the nasal wash, 1 ml of sterile 1X PBS was flushed through 404 the nasal passage using an 18-G catheter placed at the nasopharyngeal opening. The lungs were 405 weighed and homogenized in 1 ml of sterile 1X PBS in a bullet blender storm 5 (Next Advance). 406 Both the nasal wash and lungs were serially diluted and plated on SIA. Plates were then 407 incubated at 37 /i2 overnight and CFUs were determined. 408 Oropharyngeal Colonization 409 Anesthetized Balb/c mice were intranasally administered 25 µl (12.5 µl per nostril) of WU1 410 prepared either through the agar plate or SCFM2 workflow. To track WU1 colonization in the 411 .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted October 14, 2025. ; https://doi.org/10.1101/2025.10.14.682363doi: bioRxiv preprint throat over time, mice were anesthetized with 3% isoflurane through an XGI-8 Gas Anesthesia 412 System (Caliper Life Sciences) and oropharyngeal swabs were collected from each mouse using 413 calcium alginate swabs (Puritan) on days 1, 2, 3, 7, 10, and 14. The swab was cut from the stick 414 with sterile scissors, transferred to a 1.5 ml Eppendorf tube with 500 µl PBS, resuspended, 415 serially diluted, and spread on SIA plates. The plates were then incubated at 37 /i2 overnight and 416 CFUs were determined. 417 Lung Preparation for Histology and Proinflammatory Cytokine Analysis 418 To prepare the left or right lung lobes for histology and cytokine analysis, anesthetized Balb/c 419 mice were intranasally administered 25 µl (12.5 µl per nostril) of S. aureus strain WU1 prepared 420 either through the agar plate or SCFM2 workflow. 1X PBS and SCFM2 were also administered 421 as a “vehicle control”. After 24 hours, mice were euthanized, and the chest was opened. A sterile 422 clamp was used to separate the lung lobes, one lobe was inflated with 4% paraformaldehyde, and 423 removed for histology. The other lobe was processed for cytokine analysis. To determine the 424 bacterial loads in each lung, a parallel group of mice (n=2) were used. Each lobe was separated, 425 weighed, and homogenized in 1 ml of sterile 1X PBS. The lung lobes were plated on SIA, 426 incubated overnight at 37 /i2 , and CFUs were counted. 427 Histopathology 428 Lung specimen from mice were insufflated with, and fixed in, 4% paraformaldehyde (PFA), 429 processed, and blocked in paraffin for histological analysis. All samples were sectioned at 5 µm 430 and stained with hematoxylin-eosin (H&E) for routine histopathology. Samples were evaluated 431 by a board-certified veterinary pathologist in a blinded manner. Sections were examined under 432 light microscopy using an Olympus BX51 microscope and photographs were taken using an 433 Olympus DP73 camera. 434 .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted October 14, 2025. ; https://doi.org/10.1101/2025.10.14.682363doi: bioRxiv preprint Cytokine Analysis 435 Lung lobes were weighed, homogenized in 1 ml of sterile 1X PBS supplemented with ProBlock 436 Protease Inhibitor Cocktail (Goldbio), the supernatant was collected through centrifugation, and 437 stored at -80 /i2 . Cytokine levels were measured through the MesoScale Discovery (MSD) 438 PlatformV-PLEX proinflammatory Panel 1 Mouse Kit. The cytokines measured through this kit 439 were IFN-/i1 , IL-1β , IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p70, KC/GRO, and TNF-/i1 . The cytokine 440 levels were quantified and analyzed at the Emory Multiplex Immunoassay Core with MESO 441 QuickPlex SQ 120 following the manufacturer’s instructions. Results were normalized to lung 442 weight. Results for IL-4 were not included as the results from all groups were below the limit of 443 detection. 444 Statistical Analysis 445 Statistical analyses were performed for all experiments using GraphPad Prism 9. 446

447 We thank Dr. Dominique Missiakas for providing the S. aureus WU1 strain as well as its 448 associated genomic sequences. Additionally, the N315 strain was kindly provided by Dr. 449 Timothy Read. We thank Dr. Christine Bojanowski for the fruitful discussions. We also thank 450 the Emory Histology and Molecular Pathology Lab as well as the Emory Multiplexed 451 Immunoassay Core for their help with performing the histopathology and cytokine analysis, 452 respectively. Finally, we thank the Goldberg Lab for their helpful discussion and feedback on the 453 manuscript. 454 This work was supported by Cystic Fibrosis Foundation Grants WHITEL20A0 and 455 WHITEL24XX0. The pathology research reported in this publication was supported by the 456 Emory National Primate Research Center of the Office of Research Infrastructure Programs/OD 457 .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted October 14, 2025. ; https://doi.org/10.1101/2025.10.14.682363doi: bioRxiv preprint P51 OD011132. The content is solely the responsibility of the authors and does not necessarily 458 represent the official views of the National Institutes of Health. 459 460 .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted October 14, 2025. ; https://doi.org/10.1101/2025.10.14.682363doi: bioRxiv preprint

461 1. GBD 2019 Antimicrobial Resistance Collaborators. 2022. Global mortality associated 462 with 33 bacterial pathogens in 2019: a systematic analysis for the Global Burden of 463 Disease Study 2019. Lancet 400:2221–2248. 464 465 2. Haag AF, Fitzgerald JR, Penadés JR. 2019. Staphylococcus aureus in animals. Microbiol 466 Spectr 7. 467 468 3. David MZ, Daum RS. 2017. Treatment of Staphylococcus aureus infections, p. 325–383. 469 In . 470 471 4. Sakr A, Brégeon F, Mège J-L, Rolain J-M, Blin O. 2018. Staphylococcus aureus nasal 472 colonization: an update on mechanisms, epidemiology, risk factors, and subsequent 473 infections. Front Microbiol 9. 474 475 5. Kim HK, Missiakas D, Schneewind O. 2014. Mouse models for infectious diseases caused 476 by Staphylococcus aureus. J Immunol Methods 410:88–99. 477 478 6. Horst SA, Hoerr V, Beineke A, Kreis C, Tuchscherr L, Kalinka J, Lehne S, Schleicher I, 479 Köhler G, Fuchs T, Raschke MJ, Rohde M, Peters G, Faber C, Löffler B, Medina E. 2012. 480 A novel mouse model of Staphylococcus aureus chronic osteomyelitis that closely mimics 481 the human infection: an integrated view of disease pathogenesis. Am J Pathol 181:1206–482 14. 483 .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted October 14, 2025. ; https://doi.org/10.1101/2025.10.14.682363doi: bioRxiv preprint 484 7. Gibson GW, Kreuser SC, Riley JM, Rosebury-Smith WS, Courtney CL, Juneau PL, 485 Hollembaek JM, Zhu T, Huband MD, Brammer DW, Brieland JK, Sulavik MC. 2007. 486 Development of a mouse model of induced Staphylococcus aureus infective endocarditis. 487 Comp Med 57:563–9. 488 489 8. Sheykhsaran E, Abbasi A, Memar MY, Ghotaslou R, Baghi HB, Mazraeh FN, Laghousi 490 D, Sadeghi J. 2024. The role of Staphylococcus aureus in cystic fibrosis pathogenesis and 491 clinico-microbiological interactions. Diagn Microbiol Infect Dis 109:116294. 492 493 9. Bubeck Wardenburg J, Patel RJ, Schneewind O. 2007. Surface proteins and exotoxins are 494 required for the pathogenesis of Staphylococcus aureus pneumonia. Infect Immun 495 75:1040–4. 496 497 10. Tomlinson KL, Riquelme SA, Baskota SU, Drikic M, Monk IR, Stinear TP, Lewis IA, 498 Prince AS. 2023. Staphylococcus aureus stimulates neutrophil itaconate production that 499 suppresses the oxidative burst. Cell Rep 42:112064. 500 501 11. Kim G-L, Akoolo L, Parker D. 2020. The ClpXP protease contributes to Staphylococcus 502 aureus pneumonia. J Infect Dis 222:1400–1404. 503 504 .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted October 14, 2025. ; https://doi.org/10.1101/2025.10.14.682363doi: bioRxiv preprint 12. Gauguet S, D’Ortona S, Ahnger-Pier K, Duan B, Surana NK, Lu R, Cywes-Bentley C, 505 Gadjeva M, Shan Q, Priebe GP, Pier GB. 2015. Intestinal microbiota of mice influences 506 resistance to Staphylococcus aureus pneumonia. Infect Immun 83:4003–4014. 507 508 13. Torres VJ, Attia AS, Mason WJ, Hood MI, Corbin BD, Beasley FC, Anderson KL, Stauff 509 DL, McDonald WH, Zimmerman LJ, Friedman DB, Heinrichs DE, Dunman PM, Skaar 510 EP. 2010. Staphylococcus aureus fur regulates the expression of virulence factors that 511 contribute to the pathogenesis of pneumonia. Infect Immun 78:1618–28. 512 513 14. Cigana C, Bianconi I, Baldan R, De Simone M, Riva C, Sipione B, Rossi G, Cirillo DM, 514 Bragonzi A. 2018. Staphylococcus aureus impacts Pseudomonas aeruginosa chronic 515 respiratory disease in murine models. J Infect Dis 217:933–942. 516 517 15. Kriegeskorte A, Lorè NI, Bragonzi A, Riva C, Kelkenberg M, Becker K, Proctor RA, 518 Peters G, Kahl BC. 2015. Thymidine-dependent Staphylococcus aureus small-colony 519 variants are induced by trimethoprim-sulfamethoxazole (SXT) and have increased fitness 520 during SXT challenge. Antimicrob Agents Chemother 59:7265–72. 521 522 16. Zuluaga AF, Salazar BE, Rodriguez CA, Zapata AX, Agudelo M, Vesga O. 2006. 523 Neutropenia induced in outbred mice by a simplified low-dose cyclophosphamide 524 regimen: characterization and applicability to diverse experimental models of infectious 525 diseases. BMC Infect Dis 6:55. 526 527 .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted October 14, 2025. ; https://doi.org/10.1101/2025.10.14.682363doi: bioRxiv preprint 17. Hua L, Cohen TS, Shi Y, Datta V, Hilliard JJ, Tkaczyk C, Suzich J, Stover CK, Sellman 528 BR. 2015. MEDI4893* promotes survival and extends the antibiotic treatment window in 529 a Staphylococcus aureus immunocompromised pneumonia model. Antimicrob Agents 530 Chemother 59:4526–32. 531 532 18. Ibberson CB, Jones CL, Singh S, Wise MC, Hart ME, Zurawski D V, Horswill AR. 2014. 533 Staphylococcus aureus hyaluronidase is a CodY-regulated virulence factor. Infect Immun 534 82:4253–64. 535 536 19. Holtfreter S, Radcliff FJ, Grumann D, Read H, Johnson S, Monecke S, Ritchie S, Clow F, 537 Goerke C, Bröker BM, Fraser JD, Wiles S. 2013. Characterization of a mouse-adapted 538 Staphylococcus aureus strain. PLoS One 8:e71142. 539 540 20. Sun Y, Emolo C, Holtfreter S, Wiles S, Kreiswirth B, Missiakas D, Schneewind O. 2018. 541 Staphylococcal protein A contributes to persistent colonization of mice with 542 Staphylococcus aureus. J Bacteriol 200. 543 544 21. Guo J, Garratt A, Hill A. 2022. Worldwide rates of diagnosis and effective treatment for 545 cystic fibrosis. Journal of Cystic Fibrosis 21:456–462. 546 547 22. Brennan M-L, Schrijver I. 2016. Cystic fibrosis. The Journal of Molecular Diagnostics 548 18:3–14. 549 550 .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted October 14, 2025. ; https://doi.org/10.1101/2025.10.14.682363doi: bioRxiv preprint 23. LiPuma JJ. 2010. The changing microbial epidemiology in cystic fibrosis. Clin Microbiol 551 Rev 23:299–323. 552 553 24. Gilligan PH. 2014. Infections in patients with cystic fibrosis: diagnostic microbiology 554 update. Clin Lab Med 34:197–217. 555 556 25. Lu K-Y, Wagner NJ, Velez AZ, Ceppe A, Conlon BP, Muhlebach MS. 2023. Antibiotic 557 tolerance and treatment outcomes in cystic fibrosis methicillin-resistant Staphylococcus 558 aureus infections. Microbiol Spectr 11:e0406122. 559 560 26. Dasenbrook EC. 2010. Association between respiratory tract methicillin-resistant 561 Staphylococcus aureus and survival in cystic fibrosis. JAMA 303:2386. 562 563 27. Turner KH, Wessel AK, Palmer GC, Murray JL, Whiteley M. 2015. Essential genome of 564 Pseudomonas aeruginosa in cystic fibrosis sputum. Proc Natl Acad Sci U S A 112:4110–565 5. 566 567 28. Ibberson CB, Whiteley M. 2019. The Staphylococcus aureus transcriptome during cystic 568 fibrosis lung infection. mBio 10. 569 570 29. Planet PJ, Narechania A, Chen L, Mathema B, Boundy S, Archer G, Kreiswirth B. 2017. 571 Architecture of a species: phylogenomics of Staphylococcus aureus. Trends Microbiol 572 25:153–166. 573 .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted October 14, 2025. ; https://doi.org/10.1101/2025.10.14.682363doi: bioRxiv preprint 574 30. Kennedy AD, Otto M, Braughton KR, Whitney AR, Chen L, Mathema B, Mediavilla JR, 575 Byrne KA, Parkins LD, Tenover FC, Kreiswirth BN, Musser JM, DeLeo FR. 2008. 576 Epidemic community-associated methicillin-resistant Staphylococcus aureus: Recent 577 clonal expansion and diversification. Proceedings of the National Academy of Sciences 578 105:1327–1332. 579 580 31. Lorenz LL, Duthie ES. 1952. Staphylococcal Coagulase: Mode of Action and 581 Antigenicity. Microbiology (N Y) 6:95–107. 582 583 32. Okonogi K, Noji Y, Kondo M, Imada A, Yokota T. 1989. Emergence of methicillin-584 resistant clones from cephamycin-resistant Staphylococcus aureus. Journal of 585 Antimicrobial Chemotherapy 24:637–645. 586 587 33. Bernardy EE, Raghuram V, Goldberg JB. 2022. Staphylococcus aureus and Pseudomonas 588 aeruginosa isolates from the same cystic fibrosis respiratory sample coexist in coculture. 589 Microbiol Spectr 10:e0097622. 590 591 34. Raghuram V, Alexander AM, Loo HQ, Petit RA, Goldberg JB, Read TD. 2022. Species-592 wide phylogenomics of the Staphylococcus aureus agr operon revealed convergent 593 evolution of frameshift mutations. Microbiol Spectr 10. 594 595 .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted October 14, 2025. ; https://doi.org/10.1101/2025.10.14.682363doi: bioRxiv preprint 35. Yebra G, Mrochen D, Fischer S, Pfaff F, Ulrich RG, Pritchett-Corning K, Holtfreter S, 596 Fitzgerald JR. 2024. Bacteriophage-driven emergence and expansion of Staphylococcus 597 aureus in rodent populations. PLoS Pathog 20:e1012378. 598 599 36. Bobrovskyy M, Chen X, Missiakas D. 2023. The type 7b secretion system of S. aureus 600 and its role in colonization and systemic infection. Infect Immun 91:e0001523. 601 602 37. Shi M, Chen X, Sun Y, Kim HK, Schneewind O, Missiakas D. 2021. A protein A based 603 Staphylococcus aureus vaccine with improved safety. Vaccine 39:3907–3915. 604 605 38. Parker D, Ryan CL, Alonzo F, Torres VJ, Planet PJ, Prince AS. 2015. CD4+ T cells 606 promote the pathogenesis of Staphylococcus aureus pneumonia. J Infect Dis 211:835–45. 607 608 39. Urso A, Monk IR, Cheng Y-T, Predella C, Wong Fok Lung T, Theiller EM, Boylan J, 609 Perelman S, Baskota SU, Moustafa AM, Lohia G, Lewis IA, Howden BP, Stinear TP, 610 Dorrello N V., Torres V, Prince AS. 2024. Staphylococcus aureus adapts to exploit 611 collagen-derived proline during chronic infection. Nat Microbiol 9:2506–2521. 612 613 40. Trübe P, Hertlein T, Mrochen DM, Schulz D, Jorde I, Krause B, Zeun J, Fischer S, Wolf 614 SA, Walther B, Semmler T, Bröker BM, Ulrich RG, Ohlsen K, Holtfreter S. 2019. 615 Bringing together what belongs together: Optimizing murine infection models by using 616 mouse-adapted Staphylococcus aureus strains. International Journal of Medical 617 Microbiology 309:26–38. 618 .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted October 14, 2025. ; https://doi.org/10.1101/2025.10.14.682363doi: bioRxiv preprint 619 41. Fornefett J, Krause J, Klose K, Fingas F, Hassert R, Benga L, Grunwald T, Müller U, 620 Schrödl W, Baums CG. 2018. Comparative analysis of humoral immune responses and 621 pathologies of BALB/c and C57BL/6 wildtype mice experimentally infected with a highly 622 virulent Rodentibacter pneumotropicus (Pasteurella pneumotropica) strain. BMC 623 Microbiol 18:45. 624 625 42. Mills CD, Kincaid K, Alt JM, Heilman MJ, Hill AM. 2000. M-1/M-2 macrophages and 626 the Th1/Th2 paradigm. The Journal of Immunology 164:6166–6173. 627 628 43. Dietert K, Gutbier B, Wienhold SM, Reppe K, Jiang X, Yao L, Chaput C, Naujoks J, 629 Brack M, Kupke A, Peteranderl C, Becker S, von Lachner C, Baal N, Slevogt H, Hocke 630 AC, Witzenrath M, Opitz B, Herold S, Hackstein H, Sander LE, Suttorp N, Gruber AD. 631 2017. Spectrum of pathogen- and model-specific histopathologies in mouse models of 632 acute pneumonia. PLoS One 12:e0188251. 633 634 44. Ventura CL, Higdon R, Hohmann L, Martin D, Kolker E, Liggitt HD, Skerrett SJ, Rubens 635 CE. 2008. Staphylococcus aureus elicits marked alterations in the airway proteome during 636 early pneumonia. Infect Immun 76:5862–5872. 637 638 45. Mukherjee S, Moustafa D, Smith CD, Goldberg JB, Bassler BL. 2017. The RhlR quorum-639 sensing receptor controls Pseudomonas aeruginosa pathogenesis and biofilm development 640 .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted October 14, 2025. ; https://doi.org/10.1101/2025.10.14.682363doi: bioRxiv preprint independently of its canonical homoserine lactone autoinducer. PLoS Pathog 641 13:e1006504. 642 643 46. Moustafa DA, Wu AW, Zamora D, Daly SM, Sturge CR, Pybus C, Geller BL, Goldberg 644 JB, Greenberg DE. 2021. Peptide-conjugated phosphorodiamidate morpholino oligomers 645 retain activity against multidrug-resistant Pseudomonas aeruginosa in vitro and in vivo. 646 mBio 12. 647 648 47. Kalelkar PP, Moustafa DA, Riddick M, Goldberg JB, McCarty NA, García AJ. 2022. 649 Bacteriophage/i2 loaded poly(lactic/i2 co /i2 glycolic acid) microparticles mitigate 650 Staphylococcus aureus infection and cocultures of Staphylococcus aureus and 651 Pseudomonas aeruginosa. Adv Healthc Mater 11. 652 653 48. Barraza JP, Whiteley M. 2021. A Pseudomonas aeruginosa antimicrobial affects the 654 biogeography but not fitness of Staphylococcus aureus during coculture. mBio 12. 655 656 49. Duncan RP, Moustafa DA, Lewin GR, Diggle FL, Bomberger JM, Whiteley M, Goldberg 657 JB. 2024. Improvement of a mouse infection model to capture Pseudomonas aeruginosa 658 chronic physiology in cystic fibrosis. Proceedings of the National Academy of Sciences 659 121. 660 661 50. Parker D. 2017. Humanized mouse models of Staphylococcus aureus infection. Front 662 Immunol 8:512. 663 .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted October 14, 2025. ; https://doi.org/10.1101/2025.10.14.682363doi: bioRxiv preprint 664 51. Wang H, Parker D. 2025. Improved humanized mouse model of Staphylococcus aureus 665 infection. Mucosal Immunol https://doi.org/10.1016/j.mucimm.2025.05.001. 666 667 52. Pritchard L, Glover RH, Humphris S, Elphinstone JG, Toth IK. 2016. Genomics and 668 taxonomy in diagnostics for food security: soft-rotting enterobacterial plant pathogens. 669 Analytical Methods 8:12–24. 670 671 672 .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted October 14, 2025. ; https://doi.org/10.1101/2025.10.14.682363doi: bioRxiv preprint Table 1. Staphylococcus aureus strains utilized in this study. 673 Strain MRSA/ MSSA Clonal Complex Origin Host Reference JE2 MRSA 8 Skin abscess Human 31 Newman MSSA 8 Tubercular osteomyelitis Human 32 Sa_CFBR_43 MRSA 8 Respiratory sample Human (Cystic fibrosis) 34 Sa_CFBR_46 MSSA 45 Respiratory sample Human (Cystic Fibrosis) 34 N315 MRSA 5 Pharyngeal Human 33 WU1 MSSA 88 Preputial gland abscess Mouse 21 674 .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted October 14, 2025. ; https://doi.org/10.1101/2025.10.14.682363doi: bioRxiv preprint Figure 1. Strains used in this study exhibit genetic diversity. The genomic sequences of each S. aureus were compared to each other, and average nucleotide identity was determined utilizing pyani. The numeric results were used to generate the heatmap. .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted October 14, 2025. ; https://doi.org/10.1101/2025.10.14.682363doi: bioRxiv preprint Figure 2. Culturing S. aureus in SCFM2 led to similar growth kinetics compared to growth in LB for most strains. Each S. aureus strain was grown statically in either LB or SCFM2 in a 6 -well plate at a starting OD600 of 0.01. Aliquots were removed, serially diluted, and plated on LA plates every 2 hours for 8 hours as well as 24 hours. Each point represents an average of biological triplicates. Statistical analysis was performed on the 24 -hour time point through unpaired student t-test with Welch correction. **p < 0.01, ns=not significant. .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted October 14, 2025. ; https://doi.org/10.1101/2025.10.14.682363doi: bioRxiv preprint Figure 3. Schematic of the S. aureus preparation workflows used in this study. S. aureus strains were cultured on either an agar plate (left) or in SCFM2 (right). For the agar plate preparation, S. aureus was grown on Staphylococcus Isolation Agar (SIA) overnight at 37℃ , swabs of colonies were transferred to 1X PBS, washed 3 times in 1X PBS, and adjusted to an OD600 of 6.5. For the SCFM2 preparation, a single colony from S. aureus grown on SIA was grown in LB overnight with shaking at 37℃. The culture was washed 3 times in 1X PBS, back diluted in SCFM2 at a starting OD600 of 0.01 in a 6-well plate, grown statically for 24 hours, and concentrated 15X in fresh SCFM2. 12.5 µl of each condition was subsequently administered intranasally into each nostril (25 µl total) of anesthetized C57BL/6 or Balb/c mice, corresponding to ~1 x 108 CFUs. .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted October 14, 2025. ; https://doi.org/10.1101/2025.10.14.682363doi: bioRxiv preprint Figure 4. Culturing S. aureus in SCFM2 generally increases lung colonization in mice during acute pneumonia independent of mouse strain. S. aureus strains were cultured on an agar plate or in SCFM2 as described in Figure 3 and administered to either 8–10-week-old female C57BL/6 and Balb/c mice. Mice were sacrificed a t 24 hours post-infection, the lungs were aseptically removed and weighed, homogenized in 1X PBS, and plated on SIA. Each data point represents a mouse. The mean and standard deviation are represented by the error bars. Data were analyzed using two-way ANOVA with Šídák correction. ***p < 0.001, ****p < 0.0001, ns=not significant. .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted October 14, 2025. ; https://doi.org/10.1101/2025.10.14.682363doi: bioRxiv preprint Figure 5. Compared to agar plate preparation, c ulturing WU1 in SCFM2 improves colonization for the first 2 days post -infection in the throat and for 4 days post -infection in the lungs. S. aureus strain WU1 was cultured on an agar plate or in SCFM2 as described in Figure 3 and were administered to 8–10-week-old female Balb/c mice. A) The throats of the same mice were swabbed on 1, 2, 3-, 7-, 10-, and 14-days post infection, the swabs were resuspended in 1X PBS, serially diluted, and plated on SIA. Each point represents the average and standard deviation of 12 to 15 mice. B) A separate cohort of mice were sacrificed after 1-, 2-, 3-, and 4-days post-infection, the lungs were aseptically removed and weighed, homogenized in 1X PBS, and plated on SIA. Each data point represents a mouse. Statistical analysis for the throat swabs was performed on the 1- and 2-day time point through unpaired student t-test with Welsch correction. Multi-day lung colonization was analyzed using two -way ANOVA with Fisher’s LSD post -hoc test. *p < 0.05, ***p < 0.001, ns=not significant. .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted October 14, 2025. ; https://doi.org/10.1101/2025.10.14.682363doi: bioRxiv preprint Figure 6. Infections with WU1 cultured in SCFM2 leads to increased inflammation and neutrophil recruitment in both lung lobes compared to agar plate preparation. S. aureus strain WU1, cultured either on an agar plate or in SCFM2 as described in the workflow in Figure 3 , was administered to 8 –10-week-old female Balb/c mice. Mice were euthanized 24 hours post infection. For each culture workflow (Supplemental Figure S2) the left lung (A-D) or right lung (E-H) lobes were from each mouse (n=6) were inflated and fixed using 4% .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted October 14, 2025. ; https://doi.org/10.1101/2025.10.14.682363doi: bioRxiv preprint paraformaldehyde. The lungs were then processed, strained with H&E, and blindly assessed by a veterinary pathologist. .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted October 14, 2025. ; https://doi.org/10.1101/2025.10.14.682363doi: bioRxiv preprint Figure 7. Cytokine profile from lungs infected with WU1 cultured on agar plates and SCFM2. .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted October 14, 2025. ; https://doi.org/10.1101/2025.10.14.682363doi: bioRxiv preprint S. aureus strain WU1 cultured either on an agar plate or in SCFM2 as described in Figure 3, was administered to 8–10-week-old female Balb/c mice. Mice were euthanized 24 hours post infection. For each culture workflow (Supplemental Figure S2), the left lung or right lung lobes were excised from each mouse (n=6). The lungs were then homogenized in 1X PBS + 1X protease inhibitor, the cell free lysates were isolated, and cytokines were measured with the V -plex proinflammatory cytokine panel 1 (MSD). Quantification of each cyt okine was normalized to weight of the lungs and visualized in a heatmap (A). Proinflammatory cytokine profiles for lungs infected with WU1 cultured in both conditions were compared (B). The mean and standard deviation are represented by the error bars. Statistical analysis was performed using two -way ANOVA with Fisher’s LSD post-hoc test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns=not significant. .CC-BY-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted October 14, 2025. ; https://doi.org/10.1101/2025.10.14.682363doi: bioRxiv preprint