Daily rhythms modulate Acinetobacter baumannii physiology impacting infection outcome and antibiotic-inactivating capacity

Daily rhythms modulate Acinetobacter baumannii physiology impacting infection outcome and antibiotic-inactivating capacity | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Daily rhythms modulate Acinetobacter baumannii physiology impacting infection outcome and antibiotic-inactivating capacity Rocío Anabel Giordano, Valentín Permingeat, Bárbara Perez Mora, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8649481/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 11 You are reading this latest preprint version Abstract We recently demonstrated that the human pathogen Acinetobacter baumannii exhibits light-regulated daily and circadian rhythms, suggesting that the physiological state of the bacterium varies along the day. Because this temporal dimension may influence host–pathogen interactions, we investigated here whether key bacterial processes relevant to pathogenesis fluctuate according to light–dark–induced rhythms and whether these fluctuations impact infection outcome. Using a murine skin-wound infection model, we show that both prior bacterial entrainment and the time of infection critically determine disease progression. Specifically, higher bacterial titers were recovered when light-dark entrained bacteria infected mice at the end of the dark phase (morning) with respect to infections caused by arrhythmic bacteria, while the opposite occurred in the evening. In addition, β-lactamase activity displayed significant daily variation in several multidrug-resistant strains, with higher activity at the end of the light phase compared to the dark phase, in light-dark entrained cultures. Consistent with these findings, macrocolony ring formation also followed a rhythmic pattern. Together, these results demonstrate that A. baumannii displays diurnal regulation of physiological and pathogenic traits that influence infection dynamics and antibiotic-inactivating activity, introducing a new temporal dimension to bacterial pathogenesis with important implications for understanding disease progression and treatment strategies. Health sciences/Diseases Biological sciences/Microbiology Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction It has been now extensively shown that light is a global modulator of the physiology of non-phototrophic bacteria, including critical human pathogens such as Acinetobacter baumannii 1 – 5 . In this microorganism, blue light modulates iron uptake, antibiotic tolerance susceptibility, desiccation resistance, competition, antioxidant defenses, metabolism, biofilm formation, and quorum sensing 1–4,6−21 . In A. baumannii , light responses at environmental temperatures are mainly governed by the BLUF photoreceptor BlsA, the only canonical photoreceptor encoded in its genome 1 . BlsA is a global regulator that switches conformation upon illumination, enabling its interaction with different transcriptional regulators such as Fur, the iron metabolism repressor, and AcoN, the acetoin catabolism repressor, in a light-dependent manner, leading to differential physiological responses upon illumination 7 , 16 . At 37ºC, BlsA-independent light-regulation of virulence in a human keratinocyte epithelial infection model, motility, biofilm formation and quorum sensing have been reported 13 , 22 . The two component system BfmRS has also been shown to transduce a light signal both at 23 and 37ºC 6 . Recently, we identified daily rhythms displaying a robust response to light, and endogenous circadian rhythms in blsA promoter activity at 23ºC in A. baumannii , with BlsA being required for synchronization to light-dark cycles 11 . Daily rhythms reflect environmentally driven temporal variation in bacterial physiology, whereas endogenous rhythms indicate the evolution of intrinsic mechanisms that allow anticipation of predictable daily environmental cycles. Only a few articles describe the existence of circadian rhythms in non-photosynthetic bacteria, but none have investigated their role in bacterial pathogenesis. Recently, it has been shown that non-photosynthetic soil microorganisms, such as Bacillus subtilis , exhibit a light-dependent circadian system that behaves in a complex fashion, similar to the circadian systems of multicellular eukaryotes 23 , 24 . Moreover, it was shown that a clinical isolate of the gut bacterium Klebsiella aerogenes exhibited melatonin and temperature-dependent rhythms in the expression of the motility gene motA 25 . Although the relevance of these circadian rhythms in pathogenesis has not been explored, these findings suggest that the circadian clock of K. aerogenes may entrain to host cues in vivo . Finally, sustained temporal oscillations in lipid droplets synchronized by temperature cycles 26 , as well as cyclic changes in light exposure and temperature on redox metabolism in biofilms have been reported in Pseudomonas aeruginosa 27 . Knowledge accumulated regarding A. baumannii’s lifestyle and pathophysiology present it as the paradigm of resist-persist pathogen, highlighting the importance of the organism's environmental tenacity, resistance to desiccation, and initial physiological state at the onset of infection, in its success as pathogen 28 . In fact, the pathogen’s ability to survive, persist and thrive in clinical settings is directly linked to acquisition and selection of multi-drug resistance leading to hospital outbreaks 29 – 31 . The direct transmission of the microorganism to the immunocompromised patient tissues through medical devices such as endotracheal tubes, catheters, etc., which circumvent natural host barriers, facilitate environmental transmission and infection establishment and constitute one of the most widespread mechanisms leading to disease in the clinical setting. Lifestyle strategies supporting A. baumannii persistence in the environment include the formation of biofilms, a condition that is more favored at environmental temperatures of 25–30ºC than at 37ºC 29 , 30 , 32 – 34 and entering into the viable but non-culturable (VBNC) state 35 . Despite being elusive for decades, advances in genotypic typification have contributed to identification of A. baumannii environmental reservoirs, for example in soil contaminated with petroleum hydrocarbons, as well as in vegetables collected in supermarkets, greengrocers, and private gardens, inanimate surfaces that are often in contact with humans, like tables in parks, game console, mobile phones, and animals 28 , 36 . In fact, systems specifically adapted for its environmental lifestyle have been described, such as the light-sensing and response system BlsA. The bacterial initial physiological state at the moment of infection is a critical determinant of infection outcome and host fate 28 , 37 , 38 . In fact, this initial state directly influences bacterial virulence and antibiotic susceptibility, which in turn dictate the effectiveness of early antimicrobial therapy—one of the most important determinants of patient survivability 28 . In this work, we studied bacterial physiologic responses under light-dark (LD) photoperiod entrainment constituting daily rhythms in A. baumannii , and whether these rhythms -which could modulate the bacterial physiological state during the environmental and pre-infectious bacterial life-, can produce variations along the day in important processes such as infection progress and antibiotic-inactivating activity. Our results show that previous bacterial entrainment as well as the time-of-infection defined infection outcome in a mouse skin infection model. In fact, higher bacterial titers were recovered when LD entrained bacteria infected mice at the end of the dark phase (morning) with respect to infections caused by arrhythmic bacteria, previously maintained under constant illumination (LL), while the opposite occurred in the evening, reflecting prevalence of the pathogen and host’s rhythms contributions, respectively. Moreover, skin lesions also showed time-of infection effect, which do not depend solely on the mice's clock, but instead also rely on the pathogen's daily rhythm. Moreover, β-lactamase activity measured as nitrocefin hydrolysis fluctuates in some multidrug-resistant strains, showing higher values at the end of the light phase (evening) with respect to the dark phase (morning), in LD entrained cultures. Finally, ring formation in macrocolonies was also found to be rhythmic. These findings represent a paradigm shift in bacterial pathogenesis, strongly suggesting that infection treatment has a new temporal dimension. Results Pathogen’s entrainment and time-of-infection shape the outcome of A. baumannii –mouse interactions. Given our recent demonstration that A. baumanni i exhibits daily and circadian rhythms 11 , we asked whether these temporal programs influence infection outcomes, i.e., whether disease progression varies depending on the time of day at which infection is initiated, as these rhythms may determine the bacterium’s physiological state at the onset of infection. To this end, we designed a murine skin-wound infection model in which A. baumannii V15 cells were inoculated into lesions after being cultured under 12 h blue light followed by 12 h darkness photoperiod (12bL:12D; LD). Under this condition, we previously showed a robust response to the light zeitgeber, which configures light-driven daily and circadian rhythms 11 . In fact, A. baumannii V15 pLPV1Z-p blsA :: luc clone 1, which expresses a luciferase reporter under the control of the blsA promoter from the pLPV1Z plasmid, showed rhythmic oscillations when the bacteria was entrained under LD for 6 days (Fig. 1 A). Conversely, when the bacteria were cultured under continuous blue light illumination (LL), no rhythmic oscillations were observed in populations of V15 pLPV1Zp blsA :: luc clone 1 (Fig. 1 B), indicating the absence of endogenous circadian rhythms under these conditions. Thus, under LL A. baumannii V15 is arrhythmic, a condition further used as control for studying the contribution of the pathogen’s entrainment. This approach allowed us to assess whether the temporal context -both in terms of host and bacterial daily entrainment to environmental LD cycles- could influence infection, by measuring bacterial recovery from the lesion and wound healing. To this end, A. baumannii V15 cultures were incubated under each entrainment protocol for 6 days at 23°C, mimicking conditions at which the bacteria thrive in the environment. At 7 pm of day 5 (LD5) and 7 am of day 6 (LD6) (Fig. 2 A), 5 × 10⁹ CFU were collected from LD or LL entrained cultured, and subsequently used to infect mice maintained under a white-light LD photoperiod for 3 weeks. We then analyzed the impact of bacterial entrainment in infection outcome by quantifying bacterial titers in infected tissues at 4- and 7-days post-infection (dpi). Our data show that, at the 4 dpi, the number of bacteria recovered was higher when infection was performed at the end of the dark phase, in the morning, using A. baumannii previously entrained under LD compared to arrhythmic bacteria maintained under LL (Fig. 2 B, left panel). A similar trend was observed between both groups at 7 dpi (Fig. 2 B, right panel). Conversely, when infections were initiated in the evening significantly higher bacterial loads were observed when bacteria were previously cultured under LL with respect to LD entrainment at 7 dpi (Fig. 2 B, right panel). It should be noted that differences in the infection outcomes dependent on time of the day of infection and on bacterial previous entrainment were evident at different dpi, i.e., at the 4th dpi for infections performed in the morning, while at 7 dpi for infections performed in the evening (Fig. 2 B). Moreover, and as expected, by 7 dpi, the proportion of animals in which no bacterial load was observed (clearance) was higher than on 4 dpi, regardless of the group evaluated, e.g., in the LD group at 7 pm, 0/10 animals showed no bacterial load on day 4, compared with 8/11 on day 7 (Fig. 2 B). Taken together, these results indicate that both bacterial entrainment history and the time of day at which infection occurs critically influence infection outcome, beyond the contribution of the host’s circadian rhythm. We then analyzed the maximum wound diameter after 4 and 7 dpi, as an indicator of the cicatrization ability of wounds infected with LL and LD entrained bacteria. Our data show significant differences at different time-of-infection for each entrainment condition at 4dpi (LLe vs LLm and LDe vs LDm) (Fig. 3 A left panel), with higher wound cicatrization in the morning for both conditions (Fig. 3 A, left panel), likely reflecting the contribution of the host. Differences among different entrainments in the evening (LLe vs LDe) were also detected at 7 dpi, suggesting contribution of the pathogen’s rhythm (Fig. 3 A, right panel). Histological analysis of wound sections at 4 and 7 dpi was consistent with the macroscopic observations (Fig. 3 B-D). Wounds exhibiting greater macroscopic closure showed advanced stages of healing, characterized by well-developed granulation tissue, reduced inflammatory infiltrates, and limited tissue edema. In contrast, wounds with larger diameters displayed delayed healing features, including persistent inflammatory foci, superficial crust formation, hemorrhagic exudate, and interstitial edema, indicating an incomplete resolution of the inflammatory phase. Detailed histology score is shown in Table S1 . Time of day-dependent nitrocefin hydrolysis. A. baumannii is the paradigm of bacterial multidrug resistance, as circulating strains are invariably resistant to last-generation antibiotics, including the β-lactams carbapenems, which seriously complicate therapeutics. As we previously showed the existence of daily rhythms in this microorganism 11 , we decided to evaluate next whether antibiotic inactivating activity also oscillates along the day. For this purpose, A. baumannii cells were grown under an LD photoperiod for 5 days, and samples were collected at at 7 am and 7 pm on the 4th and 5th days of entrainment (LD4 and LD5), to quantify nitrocefin hydrolysis, a chromogenic cephalosporin, as an indirect measure of β-lactamase activity. It should be noted that this assay allows an instantaneous measurement at each timepoint in a growth-independent manner, as is based on spectrophotometric determinations of hydrolyzed vs. non-hydrolyzed nitrocefin, which turns from yellow to red upon hydrolysis. We used the A. baumannii multidrug resistant Ab825 39,40 , Ab1914 and Ab1915 strains, three clinical isolates recovered from Hospital Provincial del Centenario (HPC) (Table S2). Also included in the analyses are V15 and ATCC 19606, which are sensitive to multiple drugs, despite 19606 is resistant to ampicillin 41 . Our results show oscillations in nitrocefin inactivating activity levels along the day both in LD4 and LD5 for Ab825, Ab1914 and 19606 strains, which was higher at the end of the light phase (7pm) with respect to the end of the dark phase (7 am)(Fig. 4 A-C). However, we did not observe such daily oscillation in strains V15 and Ab1915 (Fig. 4 D-E), indicating a strain-specific behavior. These results indicate that oscillations are observed not only at the gene expression level but also in cellular processes as important in A. baumannii lifestyle as is antibiotic-inactivating activity. Rhythmic rings in macrocolonies. We have previously shown that light inhibits motility at 23ºC with the bacteria remaining at the inoculation point, while covering the whole plate in the dark, in a BlsA-dependent manner in A. baumannii 1 . Moreover, we have shown that biofilms are also modulated by light at 23ºC, being inhibited by light, also in a BlsA-dependent manner. As blsA expression oscillates in a pattern consistent with daily and circadian rhythms 11 we hypothesized that motility-biofilms observed in swimming plates would also be subjected to circadian regulation. To test this hypothesis, we inoculated different A. baumannii strains on top of swimming agar plates and further incubated them in LD photoperiod, or under LL, for 5 days. Mixtures of Congo red and Coomassie blue dyes are frequently used to detect extracellular matrix components in agar-grown macrocolonies of various bacterial species 42 . Here, we supplemented swimming agar medium with this dye mixture as an indicator of matrix production during potential swimming-to-sessile state transitions. Interestingly, we observed rhythmic population patterns in the macrocolonies characterized by red-brownish concentric rings stained by the dyes, suggesting accumulation of extracellular matrix material, which alternated with unstained white rings, in ATCC 19696, Ab1915 and Ab825 strains (Fig. 5 A). Furthermore, the number of rings formed under different illumination conditions corresponded to the total number of days under LD cycles (Fig. 5 A). This alternating ring pattern, attributable to successive dynamic periods of motility and sessility within the macrocolonies, including the local production and accumulation of non-diffusible, stainable extracellular matrix components, indicative of a biofilm-like state (Fig. 5 A). The repetitive transition between motile and biofilm-like states is consistent with oscillations governed by LD cycles, suggesting a robust and rapid reprogramming of gene expression in A. baumannii under rhythmic conditions. Conversely, this rhythmic ring pattern was lost when the bacteria were incubated under LL (Fig. 5 B), further supporting the correlation between rhythmic rings and cyclic conditions. It should be noted that some strains including V15 and Ab1914 did not show rhythmicity for this trait (Fig. 5 A), consistent with the known physiological heterogeneity among A. baumannii strains. Discussion In this work, we studied whether key bacterial processes relevant to pathogenesis fluctuate according to LD–induced rhythms and whether these fluctuations impact infection outcome. Interestingly, infections performed using bacterial cultures maintained under continuous illumination conditions resulting in arrhythmicity (LLe vs LLm), showed differences at the end of the light vs dark phases (evening vs morning), reflecting the host’s circadian rhythm. In fact, this is consistent with previous data indicating that mice infected with the Gram-negative S. Typhimurium were colonized to higher levels and developed a higher proinflammatory response during the early resting period for mice (ZT4), compared with other times of the day, pointing to a clock-regulated mechanism of activation of the host immune response against this enteric pathogen 43 . Given the robust light-driven rhythms we previously documented at 23°C in LD and the consistent time-of-day divergence in CFU and wound healing observed with respect with the arrhythmic LL condition, we infer that differences among different entrainments at the same time of infection would reflect the effect of the pathogen’s rhythm, as a result of differential physiological bacterial temporal state at inoculation. Our results show an effect of the pathogen’s rhythm in infection outcome determined as bacterial load recovered from lesion and wound healing, with opposing effects with respect to the host’s rhythm. In the absence of pathogen’s rhythm contribution, the host better solves infections initiated in the morning, at the end of the dark phase with respect to those initiated in the evening, at the end of the light phase. Thus, the pathogen’s rhythm switches infection outcome, and this is likely the result of a differential initial physiological state at the onset of infection. Since entrainment occurred at 23°C and infections were performed at 37°C, we did not directly test whether bacterial phase persists after transfer to the host environment, which will certainly be pursued in the near future. Moreover, we found that antibiotic-inactivating capacity varies predictably along the day suggesting fluctuating beta-lactamase activity. In fact, we observed higher nitrocefin hydrolysis activity at the end of the illuminated phase (evening) with respect to the end of the dark phase (morning), in LD entrained cultures. This is consistent with a mechanism evolving improved host response for infection control in the morning. This further provides a temporal window at which antibiotic treatment would be more efficient. This data could represent a paradigm shift in bacterial pathogenesis. We decided to use direct approaches such as nitrocefin hydrolysis measurement, which provides a rapid and growth-independent indirect estimation of β-lactamase activity at each time point. Growth-dependent approaches such as antibiograms or MIC determination in liquid media could mask the effect of differential rhythmicity along the day. This difference in total β-lactamase activity could derive either from fluctuations at the enzyme activity or transcriptional levels, the latter being the most probable. Thus, our data reveal a previously unrecognized temporal regulation of antibiotic-inactivating activity in A. baumannii . Also interesting is the fact that rhythmicity can vary among strains for a specific trait, likely as a result of complex and differential regulatory cascades. In fact, we observed variations in rhythmicity among strains both for antibiotic-inactivating activity as well as ring formation in macrocolonies, with some strains showing non-rhythmic constant β-lactamase inactivating activity along the day, e.g. V15 and Ab1915, multidrug sensitive and resistant strains, respectively. It should be noted that daily and circadian rhythms were previously observed for blsA promoter activity in V15 strain, while no rhythmicity in ring formation was observed in this strain. On the contrary, Ab1915 showed rhythmic ring formation on macrocolonies, while Ab1914 showed rhythmic β-lactamase inactivating activity but no rhythmic ring formation on macrocolonies. In turn, Ab825 showed both β-lactamase activity rhythmic pattern as well as ring formation. Now that we have shown that bacterial critical pathogens such as A. baumannii exhibit diurnal and circadian rhythms, which could shape infection dynamics and antibiotic-inactivating capacity, the current concepts of infection and disease acquire a new temporal dimension. This notion is conceptually innovative, as only a handful of articles have described the existence of rhythms in non-photosynthetic bacteria, but none has addressed their potential role in pathogenesis, establishing a new paradigm. Predictable fluctuations in pathogens’ vulnerability along the day would pose a novel concept in infectious disease management: alignment of treatment with pathogen’s circadian vulnerability, and ultimately design of new antimicrobials targeting microbial clock components to reduce the global burden of antimicrobial resistance (AMR). Daily rhythm’s structure physiology and behavior across the 24-h day; when these rhythms are generated by an endogenous circadian clock, they persist under constant condition. In addition, our results highlight the importance of considering A. baumanii infection as a temporally structured interaction between a rhythmic pathogen and a rhythmic host. Thus, the outcome of A. baumannii infection seems to be shaped not only by the host’s circadian immune responses 44 – 47 , but also by the bacterium’s prior entrainment state, which determines its physiological condition at the onset of infection 28 , 37 , 38 . Bacteria entrained under LD cycles and infecting at different times of day encounter distinct host´ immune landscapes, suggesting that bacterial rhythms may have evolved to optimize infection, modulating growth, survival, and virulence factor expression to synchronize to oscillatory host defenses rather than constant immune pressure. Importantly, the differential bacterial loads and wound resolution observed depending on both bacterial entrainment history (LL or LD) and infection timing (am versus pm) suggest that A. baumannii is not a passive target of host circadian rhythms but an active participant whose internal temporal programs can influence its ability to withstand circadian fluctuations in tissue repair capacity. Depending on its internal temporal state, it is also conceivable that the bacterium may also exploit rhythmic features of the host environment in a manner analogous to that described for viruses, which have been shown to take advantage of host circadian clock–regulated pathways to enhance their replication 48 . These findings support a model in which infection progression emerges from the alignment -or misalignment- between bacterial light-driven temporal programs and host circadian immunity. Notably, identifying time windows of increased bacterial vulnerability may offer opportunities to temporally target antimicrobial therapies 49 , potentially enhancing treatment efficacy while reducing selective pressure that drives the development of bacterial resistance. Methods Light settings. Bacterial samples were exposed to blue light emitted by nine-LED (light-emitting diode) arrays with an intensity of 6 to 10 µmol photons/m 2 /s and peak emission centered at 462 nm 1 . Light intensity was measured using a radiometer/photometer (Flame-T, OceanOptics). Temperature was set at 23ºC and fluctuations in the incubator were less than 0.5°C. Zeitgeber (i.e., “time giver” or entraining agent) time 0 or ZT0 (7:00 am) indicates the time at which lights were turned on. Photo and thermal conditions were controlled with an I-291PF incubator (INGELAB, Argentina) and temperature was monitored using DS1921H-F5 iButton Thermochrons (Maxim Integrated, USA). Bacterial culture and light entrainment conditions. Bacteria were initially cultured from a single isolated colony in LB liquid medium and grown overnight at 37°C with constant shaking. The overnight culture was then diluted to an optical density (OD 660 ) of 0.05 in fresh LB medium and inoculated into 24-well plates under static conditions. For light entrainment, cultures were exposed to 12-hour blue light:12-hour dark (12bL:12D, LD) cycles or to constant light (LL) conditions at 23°C for 5 days. For animal infections, bacterial cultures retrieved at 7 pm of the 5th and 7 am of the 6th days of entrainment were centrifuged at 5,000 rpm for 10 minutes, washed twice with phosphate-buffered saline (PBS), and approximately 10⁹ CFU were resuspended in 16 ul of the same buffer to be used for infections. Animals. C57BL/6 female mice (6–8 weeks old, 3–5 per group) were obtained from the animal facilities at the Facultad de Ciencias Veterinarias de la Universidad Nacional de La Plata (LAE-FCVUNLP) and kept in HEPA-ventilated cages at the facilities of the Centro de Investigación y Producción de Reactivos Biológicos, Facultad de Ciencias Médicas, Universidad Nacional de Rosario (CIPReB-FCM-UNR), both in Argentina. All protocols for animal studies were approved by the Institutional Animal Care and Use Committee (Resolution Nº 2141/2024), according to the institutional guidelines and carried out following the National Institutes of Health’s ‘Guide for the Care and Use of Animals. All experiments were conducted in a biosafety level II facility. Mice were housed in sealed cages with ad libitum access to food and water and placed in HEPA-ventilated racks designed to prevent microbial exchange between the environment and neighboring cages. Mice were maintained under a 12 white light–12hdark (12wL:12D) cycle, with lights being turned on at 7:00 am and off at 7:00 pm, controlled by an automated timer. Light conditions followed those previously described by Aiello et al. (2020) 50 and Casiraghi et al. (2012) 51 . The average light intensity at cage level was approximately 300 lux. Skin Wound Infection Model with Acinetobacter baumannii. Mice were anesthetized via intraperitoneal injection with a 50%:10% Ketamine–Xylazine solution (acquired from Holliday–Scott S.A., Béccar, Argentina and PharmaVet, Carole Park, Australia, respectively). The dorsal neck region was shaved (~ 1 cm × 1 cm in each side) using an electric shaver. The skin was surgically excised down to the muscle layer to generate two circular wounds of 0.6 cm in diameter in each side (Fig. 3 B). Each wound was then infected with 16 µL of an Acinetobacter baumannii V15 suspension containing ~ 10⁹ CFU total. Control animals received an identical wounding procedure followed by the application of 16 µL of sterile phosphate-buffered saline (PBS). Non-infected animals were used as controls for histological analyses for each experiment. Wound diameters were measured after 4- and 7-days post-infection (dpi) using a digital caliper, measuring in millimeters the X axis or the longest point spanning the length of the wound. Assessment of Bacterial Burden in Wound Tissue. At 4- and 7-days post-infection (dpi), tissue samples from the wound site were aseptically collected and homogenized in phosphate-buffered saline (PBS) using sterile disposable Piston Pellet Eppendorf. The resulting homogenates were subjected to serial dilutions in LB medium, and aliquots were plated on LB agar for bacterial enumeration. Plates were incubated at 37°C for 18–24 hours, and colony-forming units (CFUs) were quantified to assess bacterial load. Bacterial loads were normalized to tissue weight. As control, we verified that animals infected at (t = 0 dpi) presented similar bacterial loads among the different groups. Nitrocefin hydrolysis determination along the day in LD cultures. A. baumannii Ab825 cells were grown overnight in LB at 37 ºC in the dark, and then inoculated in fresh new LB media at a DO 600 = 0.05. The bacteria were then grown under LD photoperiod for 4 days at 23ºC. At 7 am and 7 pm of the 3rd and 4th days (LD3 and LD4), samples were retrieved and processed using a nitrocefin-based colorimetric method for β-lactamase activity detection following the manufacturer recommendations (Amplite Colorimetric Beta-Lactamase Activity, AAT Bioquest). The reactions were incubated at room temperature, with the plate protected from light, and after 60 minutes absorbances at 490, 380, and 600 nm were determined using a microplate reader (Bio Tek InstrumentsEPOCH2T). A 490/380 ratio corresponding to hydrolyzed vs intact nitrocefin was calculated using these values and normalized to the OD 600 corresponding to the time point for each sample. At least three independent experiments were carried out. Macrocolonies. Bacteria were inoculated directly from an overnight plate grown at 37°C using a sterile wooden stick in swimming agarose plates 1 supplemented with Congo red (40 mg/l) and Coomassie brilliant blue G-250 (20 mg/l). Plates were incubated at the indicated temperatures and entrainment schedules, and the development of colony morphology and coloration was analyzed. Statistical analysis. Statistical analyses were performed using parametric or non-parametric tests as appropriate, depending on data distribution and variance. Statistical analyses for the skin-wound infection model were performed using non-parametric ANOVA followed by the Mann–Whitney U test, as appropriate, and Fisher’s exact test for frequency comparisons. For the analysis of antibiotic susceptibility, the effect of measurement time on themean of ratio/OD was analyzed using a mixed effects model with replicates included as a random effect. Residuals of the fitted model confirmed that necessary assumptions were met. Post hoc pairwise comparisons between groups were conducted using Tukey’s multiple comparisons test. Statistical significance was set at p < 0.05. Declarations Competing interests. The authors declare no competing interests. Funding Declaration. This work was supported by grants from the Secretaría de Ciencia, Tecnología e Innovación (Provincia de Santa Fe) to MAM (PEIC I + D 2023-255). Author Contribution Conceptualization: R.G., V.P., B.P.M., D.A.G., A.R.P. and M.A.M. Formal analysis: V.P., B.E.P.M., M.L.M., N.A., M.F., D.A.G., A.R.P. and M.A.M. Funding acquisition: M.A.M. Investigation: R.G., V.P., B.P.M., N.A., T.B., A.R.P. and M.A.M. Methodology: R.G., V.P., B.P.M., M.L.M., N.A., T.B., M.F., S.D., D.A.G., A.R.P. and M.A.M. Project administration: M.A.M. Visualization: R.G., V.P., B.P.M., T.B., M.F., D.A.G., A.R.P. and M.A.M. (Writing—original draft: M.A.M and A.R.P. Writing—review & editing: R.G., V.P., B.P.M., D.S. and D.A.G. Acknowledgement This work was supported by grants from the Secretaría de Ciencia, Tecnología e Innovación (Provincia de Santa Fe) to MAM (PEIC I+D 2023-255). BPM, DS, DAG, ARP and MAM, are career investigators of CONICET, while RG, VP and NA are fellows from the same institution. We thank Cecilia Farré for technical support with animals at CiPReB. Data Availability Source data are provided in Supplementary Data 1 and 2. Any other data or material is available from the corresponding authors (or other sources, as applicable) on reasonable request. References Mussi, M. A. et al. The opportunistic human pathogen Acinetobacter baumannii senses and responds to light. J. 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PloS one . 9 , e96462. 10.1371/journal.pone.0096462 (2014). Additional Declarations No competing interests reported. Supplementary Files Supplementaryinformation.pdf Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 31 Mar, 2026 Reviews received at journal 30 Mar, 2026 Reviewers agreed at journal 12 Mar, 2026 Reviewers agreed at journal 27 Feb, 2026 Reviews received at journal 26 Feb, 2026 Reviewers agreed at journal 26 Feb, 2026 Reviewers invited by journal 23 Feb, 2026 Editor invited by journal 12 Feb, 2026 Editor assigned by journal 21 Jan, 2026 Submission checks completed at journal 21 Jan, 2026 First submitted to journal 20 Jan, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8649481","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":598351466,"identity":"4a4d7b39-5b73-4c57-af22-a68cdde305fd","order_by":0,"name":"Rocío Anabel Giordano","email":"","orcid":"","institution":"Centro de Estudios Fotosintéticos y Bioquímicos (CEFOBI, CONICET-UNR)","correspondingAuthor":false,"prefix":"","firstName":"Rocío","middleName":"Anabel","lastName":"Giordano","suffix":""},{"id":598351469,"identity":"dbc3f839-40f7-4e53-8d9c-a41bc10e616a","order_by":1,"name":"Valentín Permingeat","email":"","orcid":"","institution":"Centro de Estudios Fotosintéticos y Bioquímicos (CEFOBI, CONICET-UNR)","correspondingAuthor":false,"prefix":"","firstName":"Valentín","middleName":"","lastName":"Permingeat","suffix":""},{"id":598351476,"identity":"d6df9c72-f297-4988-a1d5-b88c76f7ff4e","order_by":2,"name":"Bárbara Perez Mora","email":"","orcid":"","institution":"Centro de Estudios Fotosintéticos y Bioquímicos (CEFOBI, CONICET-UNR)","correspondingAuthor":false,"prefix":"","firstName":"Bárbara","middleName":"Perez","lastName":"Mora","suffix":""},{"id":598351483,"identity":"c315b987-4d09-4b50-8ee0-6f30cc435f8f","order_by":3,"name":"Natalia Arana","email":"","orcid":"","institution":"Centro de Estudios Fotosintéticos y Bioquímicos (CEFOBI, CONICET-UNR)","correspondingAuthor":false,"prefix":"","firstName":"Natalia","middleName":"","lastName":"Arana","suffix":""},{"id":598351484,"identity":"6c53ef3c-e09a-454c-85ab-aa054634085f","order_by":4,"name":"Tomás Braccialarghe","email":"","orcid":"","institution":"Centro de Estudios Fotosintéticos y Bioquímicos (CEFOBI, CONICET-UNR)","correspondingAuthor":false,"prefix":"","firstName":"Tomás","middleName":"","lastName":"Braccialarghe","suffix":""},{"id":598351488,"identity":"bf566a80-4a7d-497a-b099-1ac917900519","order_by":5,"name":"Matías Fusini","email":"","orcid":"","institution":"Cátedra de Histología y Embriología Humana. Facultad de Cs. Médicas. Universidad Nacional de Rosario.","correspondingAuthor":false,"prefix":"","firstName":"Matías","middleName":"","lastName":"Fusini","suffix":""},{"id":598351489,"identity":"35f7c087-69dc-45cf-b220-f9f0f1e332f6","order_by":6,"name":"Diego Serra","email":"","orcid":"","institution":"Instituto de Biología Celular y Molecular de Rosario (IBR), CONICET, Rosario, Argentina.","correspondingAuthor":false,"prefix":"","firstName":"Diego","middleName":"","lastName":"Serra","suffix":""},{"id":598351490,"identity":"b7f8aa8d-8d2c-4f54-b112-053a39a07011","order_by":7,"name":"María Laura Migliori","email":"","orcid":"","institution":"Departamento de Ciencia y Tecnología, Universidad Nacional de Quilmes, Bernal, Argentina.","correspondingAuthor":false,"prefix":"","firstName":"María","middleName":"Laura","lastName":"Migliori","suffix":""},{"id":598351491,"identity":"ec64e1d0-8482-4a94-a725-2d2fdd61b79b","order_by":8,"name":"Diego Andrés Golombek","email":"","orcid":"","institution":"Laboratorio Interdisciplinario del Tiempo (LITERA), Universidad de San Andrés/CONICET.","correspondingAuthor":false,"prefix":"","firstName":"Diego","middleName":"Andrés","lastName":"Golombek","suffix":""},{"id":598351493,"identity":"625e6a71-8fc7-4d2a-9edc-651e441d98f0","order_by":9,"name":"Ana Rosa Perez","email":"","orcid":"","institution":"Instituto de Inmunología Clínica y Experimental de Rosario (IDICER-CONICET)","correspondingAuthor":false,"prefix":"","firstName":"Ana","middleName":"Rosa","lastName":"Perez","suffix":""},{"id":598351494,"identity":"52e1f4f8-9bdb-455e-bb86-8c0c5c1cf898","order_by":10,"name":"María Alejandra Mussi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABDElEQVRIie2SMUvDQBTH3y3J8jDrTfETCAmFTKGf5R6Fy1JcXBwK3hSX6NxB6rfo6slBu1TnjpGCk0O6FangacUsOa1bkfsNBw/ej/97jwPweA4VcQ4QKgT9UQT7KQsA1K3C1K8KK3fKd/2jcjIuVs3TpB9jeHWvYZSfHoVmXsNrTip8qLuUbDnMOE0HPcRHoWEmzwKUpNi1fbBIHEoANNV0x4eJeVOGSo6pYpXpAcjOwbKlHYxu9EVlFc0+lWi9U6JnhyISTkoLbBW0629MDNyRsnjJuJgN0uprFypRpmNSMg64I2VerNabUf/YXsw09mJ0e2nqptnmGEXdKQ6o3PcbtGz/2O/xeDz/mXdW8mGm290EkQAAAABJRU5ErkJggg==","orcid":"","institution":"Centro de Estudios Fotosintéticos y Bioquímicos (CEFOBI, CONICET-UNR)","correspondingAuthor":true,"prefix":"","firstName":"María","middleName":"Alejandra","lastName":"Mussi","suffix":""}],"badges":[],"createdAt":"2026-01-20 13:13:58","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8649481/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8649481/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":103782918,"identity":"30191691-a49a-4450-b475-ec0cd241a386","added_by":"auto","created_at":"2026-03-02 21:25:37","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":86034,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eblsA\u003c/em\u003e promoter–driven luminescence under different bacterial illumination entrainments used in this work. Luminescence reporter activity of \u003cem\u003eA. baumannii\u003c/em\u003e V15 pLPV1Z-p\u003cem\u003eblsA\u003c/em\u003e::\u003cem\u003eluc\u003c/em\u003e clone 1 populations under LD (\u003cstrong\u003eA\u003c/strong\u003e) or LL (\u003cstrong\u003eB\u003c/strong\u003e) for 6 days. The graphs show baseline detrended, normalized luminescence including all traces and average. A representative experiment from two biological replicates is shown. The parameters were determined using CircaLuc (freely available at https://ispiousas.shinyapps.io/circaluc/)\u003csup\u003e52\u003c/sup\u003e and BioDare2\u003csup\u003e53\u003c/sup\u003e, respectively. Bacteria were grown at a constant temperature of 23 °C. 1 sample = 1 well of a 96-well plate. n rhythmic = number of samples exhibiting circadian rhythmicity under free-running conditions \u003csup\u003e11\u003c/sup\u003e.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8649481/v1/49bd0118ba2a031bbdf6d7c2.png"},{"id":103782921,"identity":"fb827f9f-d4cd-4a3b-bbfd-1176d16e5239","added_by":"auto","created_at":"2026-03-02 21:25:37","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":227057,"visible":true,"origin":"","legend":"\u003cp\u003ePrevious bacterial entrainment and time-of-infection determine infection outcome. \u003cstrong\u003eA.\u003c/strong\u003e Bacteria were entrained under LD or LL illumination conditions. At 7 pm LD5 (ZT12) and 7 am LD6 (ZT0), or the corresponding timepoints under LL, aliquots equivalent to 5 × 10⁹ bacteria were retrieved and used to infect skin-wounded mice (see Methods for details), which were then further incubated under normal 12wL:12D photoperiod. At the 4th and 7th dpi, skin slices were recovered from infected wound tissues, and the bacteria were enumerated. Bacterial loads were normalized to tissue weight at the indicated times points. For each day evaluated, three independent experimental rounds were conducted (n = 3–4 animals per group per round). Results shown include these three rounds, yielding a total of N = 10 animals per group at day 4 and N = 11 animals at day 7. Dotted lines represent the limit of detection, which corresponds to 20 CFU in these experiments. Determinations below the limit of detection line received the arbitrary value of 1, for plotting purposes. Data are shown as mean ± SD. Statistical analyses were performed using the Mann–Whitney test, **\u003cem\u003ep\u003c/em\u003e\u0026lt; 0,0001, *\u003cem\u003ep\u003c/em\u003e=0,0014. dpi: days post-infection.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8649481/v1/bef01bb6f08c7c1244064d6f.jpeg"},{"id":103782919,"identity":"66984e0f-a314-4f9e-9887-b8ebc977758f","added_by":"auto","created_at":"2026-03-02 21:25:37","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":670982,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA. \u003c/strong\u003eMaximum wound diameter measured in infected mice at 4 and 7 dpi. The data are the means and standard deviations of three pooled independent experiments (n=10-11/group/day). Dotted lines indicate average initial diameter size. Statistical analyses between morning and evening and between LL and LD groups were performed using Student’s \u003cem\u003et\u003c/em\u003e-test\u003cstrong\u003e. B- \u003c/strong\u003eMice infected at 0 dpi, as reference for cicatrization in skin-wound infection model. \u003cstrong\u003eC-D. \u003c/strong\u003e\u003cu\u003eTop:\u003c/u\u003eRepresentative macroscopic images of mouse skin wounds undergoing the healing process in the different experimental groups at 4 and 7 dpi, respectively. \u003cu\u003eBottom:\u003c/u\u003eRepresentative light micrographs of wound sites in an advanced stage of healing, showing underlying granulation tissue (black arrowheads) and residual or scarce inflammatory foci (black arrows). In some infected groups, superficial crust formation (red arrows) and persistence of hemorrhagic exudate and interstitial edema (red arrowheads) are still observed. LM, 4X, H\u0026amp;E.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8649481/v1/7bc059e928ca0a7f64e314f4.jpeg"},{"id":103782937,"identity":"d072bd06-23cf-4f18-a290-bcd8b343c1ce","added_by":"auto","created_at":"2026-03-02 21:25:38","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":174939,"visible":true,"origin":"","legend":"\u003cp\u003eNitrocefin hydrolysis activity fluctuates along the day. The bacteria were grown under LD for 5 days at 23ºC. At 7 am and 7 pm of the 4rd and 5th days (LD4 and LD5), samples were retrieved and processed using a nitrocefin-based colorimetric method for β-lactamase activity detection, based on spectrophotometric measurements of hydrolyzed (490 nm) vs. non-hydrolyzed nitrocefin (380 nm), which turns from yellow to red upon hydrolysis. Colored lines in each time point show mean values± SD from at least three independent experiments. Statistical analysis was performed using a mixed-effects model with replicate as a random effect, followed by Tukey’s multiple comparisons test for post hoc pairwise comparisons. Asterisks indicate significant differences *(\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05); **(\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01); ***(\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8649481/v1/69786dc22757b6973fb755f1.jpeg"},{"id":103782920,"identity":"13d0a852-bedf-40cc-b78b-4d7cf3066428","added_by":"auto","created_at":"2026-03-02 21:25:37","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":690343,"visible":true,"origin":"","legend":"\u003cp\u003eRhythmic ring pattern in macrocolonies of some \u003cem\u003eA. baumannii \u003c/em\u003estrains under bLD entrainment for 5 days at 23ºC. \u003cstrong\u003eA.\u003c/strong\u003e \u003cem\u003eA. baumannii\u003c/em\u003e wild type macrocolonies showing the appearance of rings consistent with the number of days incubated under LD (upper row). Inverted transformation performed with Fiji (ImageJ) (middel row). The differential grey values were quantified from the center to the end of the macrocolony, showing rhythmic patterns (lower row). \u003cstrong\u003eB\u003c/strong\u003e. Same as in A, except that constant light (LL) was used as entrainment.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8649481/v1/c233d721df515cde86862775.jpeg"},{"id":103782941,"identity":"33042568-7151-4691-8116-fc545846cc29","added_by":"auto","created_at":"2026-03-02 21:25:49","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2676273,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8649481/v1/f7836e14-39c2-4a45-99b3-d494b18659a7.pdf"},{"id":103782922,"identity":"42a1d565-0331-49ea-9f32-9a21f7de40b5","added_by":"auto","created_at":"2026-03-02 21:25:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":689808,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryinformation.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8649481/v1/bd1bfabb5c15018fcaabf4f4.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Daily rhythms modulate Acinetobacter baumannii physiology impacting infection outcome and antibiotic-inactivating capacity","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIt has been now extensively shown that light is a global modulator of the physiology of non-phototrophic bacteria, including critical human pathogens such as \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e \u003csup\u003e\u003cspan additionalcitationids=\"CR2 CR3 CR4\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. In this microorganism, blue light modulates iron uptake, antibiotic tolerance susceptibility, desiccation resistance, competition, antioxidant defenses, metabolism, biofilm formation, and quorum sensing \u003csup\u003e1\u0026ndash;4,6\u0026minus;21\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn \u003cem\u003eA. baumannii\u003c/em\u003e, light responses at environmental temperatures are mainly governed by the BLUF photoreceptor BlsA, the only canonical photoreceptor encoded in its genome \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. BlsA is a global regulator that switches conformation upon illumination, enabling its interaction with different transcriptional regulators such as Fur, the iron metabolism repressor, and AcoN, the acetoin catabolism repressor, in a light-dependent manner, leading to differential physiological responses upon illumination \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. At 37\u0026ordm;C, BlsA-independent light-regulation of virulence in a human keratinocyte epithelial infection model, motility, biofilm formation and quorum sensing have been reported \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. The two component system BfmRS has also been shown to transduce a light signal both at 23 and 37\u0026ordm;C \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Recently, we identified daily rhythms displaying a robust response to light, and endogenous circadian rhythms in \u003cem\u003eblsA\u003c/em\u003e promoter activity at 23\u0026ordm;C in \u003cem\u003eA. baumannii\u003c/em\u003e, with BlsA being required for synchronization to light-dark cycles \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Daily rhythms reflect environmentally driven temporal variation in bacterial physiology, whereas endogenous rhythms indicate the evolution of intrinsic mechanisms that allow anticipation of predictable daily environmental cycles.\u003c/p\u003e \u003cp\u003eOnly a few articles describe the existence of circadian rhythms in non-photosynthetic bacteria, but none have investigated their role in bacterial pathogenesis. Recently, it has been shown that non-photosynthetic soil microorganisms, such as \u003cem\u003eBacillus subtilis\u003c/em\u003e, exhibit a light-dependent circadian system that behaves in a complex fashion, similar to the circadian systems of multicellular eukaryotes \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Moreover, it was shown that a clinical isolate of the gut bacterium \u003cem\u003eKlebsiella aerogenes\u003c/em\u003e exhibited melatonin and temperature-dependent rhythms in the expression of the motility gene \u003cem\u003emotA\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Although the relevance of these circadian rhythms in pathogenesis has not been explored, these findings suggest that the circadian clock of \u003cem\u003eK. aerogenes\u003c/em\u003e may entrain to host cues \u003cem\u003ein vivo\u003c/em\u003e. Finally, sustained temporal oscillations in lipid droplets synchronized by temperature cycles \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e, as well as cyclic changes in light exposure and temperature on redox metabolism in biofilms have been reported in \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eKnowledge accumulated regarding \u003cem\u003eA. baumannii\u0026rsquo;s\u003c/em\u003e lifestyle and pathophysiology present it as the paradigm of resist-persist pathogen, highlighting the importance of the organism's environmental tenacity, resistance to desiccation, and initial physiological state at the onset of infection, in its success as pathogen \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. In fact, the pathogen\u0026rsquo;s ability to survive, persist and thrive in clinical settings is directly linked to acquisition and selection of multi-drug resistance leading to hospital outbreaks \u003csup\u003e\u003cspan additionalcitationids=\"CR30\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. The direct transmission of the microorganism to the immunocompromised patient tissues through medical devices such as endotracheal tubes, catheters, etc., which circumvent natural host barriers, facilitate environmental transmission and infection establishment and constitute one of the most widespread mechanisms leading to disease in the clinical setting. Lifestyle strategies supporting \u003cem\u003eA. baumannii\u003c/em\u003e persistence in the environment include the formation of biofilms, a condition that is more favored at environmental temperatures of 25\u0026ndash;30\u0026ordm;C than at 37\u0026ordm;C \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan additionalcitationids=\"CR33\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e and entering into the viable but non-culturable (VBNC) state \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Despite being elusive for decades, advances in genotypic typification have contributed to identification of \u003cem\u003eA. baumannii\u003c/em\u003e environmental reservoirs, for example in soil contaminated with petroleum hydrocarbons, as well as in vegetables collected in supermarkets, greengrocers, and private gardens, inanimate surfaces that are often in contact with humans, like tables in parks, game console, mobile phones, and animals \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. In fact, systems specifically adapted for its environmental lifestyle have been described, such as the light-sensing and response system BlsA.\u003c/p\u003e \u003cp\u003eThe bacterial initial physiological state at the moment of infection is a critical determinant of infection outcome and host fate \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e,\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. In fact, this initial state directly influences bacterial virulence and antibiotic susceptibility, which in turn dictate the effectiveness of early antimicrobial therapy\u0026mdash;one of the most important determinants of patient survivability \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn this work, we studied bacterial physiologic responses under light-dark (LD) photoperiod entrainment constituting daily rhythms in \u003cem\u003eA. baumannii\u003c/em\u003e, and whether these rhythms -which could modulate the bacterial physiological state during the environmental and pre-infectious bacterial life-, can produce variations along the day in important processes such as infection progress and antibiotic-inactivating activity. Our results show that previous bacterial entrainment as well as the time-of-infection defined infection outcome in a mouse skin infection model. In fact, higher bacterial titers were recovered when LD entrained bacteria infected mice at the end of the dark phase (morning) with respect to infections caused by arrhythmic bacteria, previously maintained under constant illumination (LL), while the opposite occurred in the evening, reflecting prevalence of the pathogen and host\u0026rsquo;s rhythms contributions, respectively. Moreover, skin lesions also showed time-of infection effect, which do not depend solely on the mice's clock, but instead also rely on the pathogen's daily rhythm. Moreover, β-lactamase activity measured as nitrocefin hydrolysis fluctuates in some multidrug-resistant strains, showing higher values at the end of the light phase (evening) with respect to the dark phase (morning), in LD entrained cultures. Finally, ring formation in macrocolonies was also found to be rhythmic. These findings represent a paradigm shift in bacterial pathogenesis, strongly suggesting that infection treatment has a new temporal dimension.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003ePathogen\u0026rsquo;s entrainment and time-of-infection shape the outcome of\u003c/b\u003e\u003cb\u003eA. baumannii\u003c/b\u003e\u003cb\u003e\u0026ndash;mouse interactions.\u003c/b\u003e Given our recent demonstration that \u003cem\u003eA. baumanni\u003c/em\u003ei exhibits daily and circadian rhythms \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e, we asked whether these temporal programs influence infection outcomes, i.e., whether disease progression varies depending on the time of day at which infection is initiated, as these rhythms may determine the bacterium\u0026rsquo;s physiological state at the onset of infection. To this end, we designed a murine skin-wound infection model in which \u003cem\u003eA. baumannii\u003c/em\u003e V15 cells were inoculated into lesions after being cultured under 12 h blue light followed by 12 h darkness photoperiod (12bL:12D; LD). Under this condition, we previously showed a robust response to the light zeitgeber, which configures light-driven daily and circadian rhythms \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. In fact, \u003cem\u003eA. baumannii\u003c/em\u003e V15 pLPV1Z-p\u003cem\u003eblsA\u003c/em\u003e::\u003cem\u003eluc\u003c/em\u003e clone 1, which expresses a luciferase reporter under the control of the \u003cem\u003eblsA\u003c/em\u003e promoter from the pLPV1Z plasmid, showed rhythmic oscillations when the bacteria was entrained under LD for 6 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Conversely, when the bacteria were cultured under continuous blue light illumination (LL), no rhythmic oscillations were observed in populations of V15 pLPV1Zp\u003cem\u003eblsA\u003c/em\u003e::\u003cem\u003eluc\u003c/em\u003e clone 1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), indicating the absence of endogenous circadian rhythms under these conditions. Thus, under LL \u003cem\u003eA. baumannii\u003c/em\u003e V15 is arrhythmic, a condition further used as control for studying the contribution of the pathogen\u0026rsquo;s entrainment.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThis approach allowed us to assess whether the temporal context -both in terms of host and bacterial daily entrainment to environmental LD cycles- could influence infection, by measuring bacterial recovery from the lesion and wound healing.\u003c/p\u003e \u003cp\u003eTo this end, \u003cem\u003eA. baumannii\u003c/em\u003e V15 cultures were incubated under each entrainment protocol for 6 days at 23\u0026deg;C, mimicking conditions at which the bacteria thrive in the environment. At 7 pm of day 5 (LD5) and 7 am of day 6 (LD6) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), 5 \u0026times; 10⁹ CFU were collected from LD or LL entrained cultured, and subsequently used to infect mice maintained under a white-light LD photoperiod for 3 weeks. We then analyzed the impact of bacterial entrainment in infection outcome by quantifying bacterial titers in infected tissues at 4- and 7-days post-infection (dpi).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOur data show that, at the 4 dpi, the number of bacteria recovered was higher when infection was performed at the end of the dark phase, in the morning, using \u003cem\u003eA. baumannii\u003c/em\u003e previously entrained under LD compared to arrhythmic bacteria maintained under LL (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, left panel). A similar trend was observed between both groups at 7 dpi (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, right panel). Conversely, when infections were initiated in the evening significantly higher bacterial loads were observed when bacteria were previously cultured under LL with respect to LD entrainment at 7 dpi (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, right panel). It should be noted that differences in the infection outcomes dependent on time of the day of infection and on bacterial previous entrainment were evident at different dpi, i.e., at the 4th dpi for infections performed in the morning, while at 7 dpi for infections performed in the evening (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Moreover, and as expected, by 7 dpi, the proportion of animals in which no bacterial load was observed (clearance) was higher than on 4 dpi, regardless of the group evaluated, e.g., in the LD group at 7 pm, 0/10 animals showed no bacterial load on day 4, compared with 8/11 on day 7 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eTaken together, these results indicate that both bacterial entrainment history and the time of day at which infection occurs critically influence infection outcome, beyond the contribution of the host\u0026rsquo;s circadian rhythm.\u003c/p\u003e \u003cp\u003eWe then analyzed the maximum wound diameter after 4 and 7 dpi, as an indicator of the cicatrization ability of wounds infected with LL and LD entrained bacteria. Our data show significant differences at different time-of-infection for each entrainment condition at 4dpi (LLe vs LLm and LDe vs LDm) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA left panel), with higher wound cicatrization in the morning for both conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, left panel), likely reflecting the contribution of the host. Differences among different entrainments in the evening (LLe vs LDe) were also detected at 7 dpi, suggesting contribution of the pathogen\u0026rsquo;s rhythm (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, right panel). Histological analysis of wound sections at 4 and 7 dpi was consistent with the macroscopic observations (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB-D). Wounds exhibiting greater macroscopic closure showed advanced stages of healing, characterized by well-developed granulation tissue, reduced inflammatory infiltrates, and limited tissue edema. In contrast, wounds with larger diameters displayed delayed healing features, including persistent inflammatory foci, superficial crust formation, hemorrhagic exudate, and interstitial edema, indicating an incomplete resolution of the inflammatory phase. Detailed histology score is shown in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eTime of day-dependent nitrocefin hydrolysis.\u003c/b\u003e \u003cem\u003eA. baumannii\u003c/em\u003e is the paradigm of bacterial multidrug resistance, as circulating strains are invariably resistant to last-generation antibiotics, including the β-lactams carbapenems, which seriously complicate therapeutics. As we previously showed the existence of daily rhythms in this microorganism \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e, we decided to evaluate next whether antibiotic inactivating activity also oscillates along the day. For this purpose, \u003cem\u003eA. baumannii\u003c/em\u003e cells were grown under an LD photoperiod for 5 days, and samples were collected at at 7 am and 7 pm on the 4th and 5th days of entrainment (LD4 and LD5), to quantify nitrocefin hydrolysis, a chromogenic cephalosporin, as an indirect measure of β-lactamase activity. It should be noted that this assay allows an instantaneous measurement at each timepoint in a growth-independent manner, as is based on spectrophotometric determinations of hydrolyzed vs. non-hydrolyzed nitrocefin, which turns from yellow to red upon hydrolysis. We used the \u003cem\u003eA. baumannii\u003c/em\u003e multidrug resistant Ab825 \u003csup\u003e39,40\u003c/sup\u003e, Ab1914 and Ab1915 strains, three clinical isolates recovered from Hospital Provincial del Centenario (HPC) (Table S2). Also included in the analyses are V15 and ATCC 19606, which are sensitive to multiple drugs, despite 19606 is resistant to ampicillin \u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Our results show oscillations in nitrocefin inactivating activity levels along the day both in LD4 and LD5 for Ab825, Ab1914 and 19606 strains, which was higher at the end of the light phase (7pm) with respect to the end of the dark phase (7 am)(Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-C). However, we did not observe such daily oscillation in strains V15 and Ab1915 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD-E), indicating a strain-specific behavior. These results indicate that oscillations are observed not only at the gene expression level but also in cellular processes as important in \u003cem\u003eA. baumannii\u003c/em\u003e lifestyle as is antibiotic-inactivating activity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eRhythmic rings in macrocolonies.\u003c/b\u003e We have previously shown that light inhibits motility at 23\u0026ordm;C with the bacteria remaining at the inoculation point, while covering the whole plate in the dark, in a BlsA-dependent manner in \u003cem\u003eA. baumannii\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Moreover, we have shown that biofilms are also modulated by light at 23\u0026ordm;C, being inhibited by light, also in a BlsA-dependent manner. As \u003cem\u003eblsA\u003c/em\u003e expression oscillates in a pattern consistent with daily and circadian rhythms \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e we hypothesized that motility-biofilms observed in swimming plates would also be subjected to circadian regulation. To test this hypothesis, we inoculated different \u003cem\u003eA. baumannii\u003c/em\u003e strains on top of swimming agar plates and further incubated them in LD photoperiod, or under LL, for 5 days.\u003c/p\u003e \u003cp\u003eMixtures of Congo red and Coomassie blue dyes are frequently used to detect extracellular matrix components in agar-grown macrocolonies of various bacterial species \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Here, we supplemented swimming agar medium with this dye mixture as an indicator of matrix production during potential swimming-to-sessile state transitions. Interestingly, we observed rhythmic population patterns in the macrocolonies characterized by red-brownish concentric rings stained by the dyes, suggesting accumulation of extracellular matrix material, which alternated with unstained white rings, in ATCC 19696, Ab1915 and Ab825 strains (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Furthermore, the number of rings formed under different illumination conditions corresponded to the total number of days under LD cycles (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). This alternating ring pattern, attributable to successive dynamic periods of motility and sessility within the macrocolonies, including the local production and accumulation of non-diffusible, stainable extracellular matrix components, indicative of a biofilm-like state (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). The repetitive transition between motile and biofilm-like states is consistent with oscillations governed by LD cycles, suggesting a robust and rapid reprogramming of gene expression in \u003cem\u003eA. baumannii\u003c/em\u003e under rhythmic conditions. Conversely, this rhythmic ring pattern was lost when the bacteria were incubated under LL (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB), further supporting the correlation between rhythmic rings and cyclic conditions. It should be noted that some strains including V15 and Ab1914 did not show rhythmicity for this trait (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA), consistent with the known physiological heterogeneity among \u003cem\u003eA. baumannii\u003c/em\u003e strains.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this work, we studied whether key bacterial processes relevant to pathogenesis fluctuate according to LD\u0026ndash;induced rhythms and whether these fluctuations impact infection outcome.\u003c/p\u003e \u003cp\u003eInterestingly, infections performed using bacterial cultures maintained under continuous illumination conditions resulting in arrhythmicity (LLe vs LLm), showed differences at the end of the light vs dark phases (evening vs morning), reflecting the host\u0026rsquo;s circadian rhythm. In fact, this is consistent with previous data indicating that mice infected with the Gram-negative \u003cem\u003eS. Typhimurium\u003c/em\u003e were colonized to higher levels and developed a higher proinflammatory response during the early resting period for mice (ZT4), compared with other times of the day, pointing to a clock-regulated mechanism of activation of the host immune response against this enteric pathogen \u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. Given the robust light-driven rhythms we previously documented at 23\u0026deg;C in LD and the consistent time-of-day divergence in CFU and wound healing observed with respect with the arrhythmic LL condition, we infer that differences among different entrainments at the same time of infection would reflect the effect of the pathogen\u0026rsquo;s rhythm, as a result of differential physiological bacterial temporal state at inoculation.\u003c/p\u003e \u003cp\u003eOur results show an effect of the pathogen\u0026rsquo;s rhythm in infection outcome determined as bacterial load recovered from lesion and wound healing, with opposing effects with respect to the host\u0026rsquo;s rhythm. In the absence of pathogen\u0026rsquo;s rhythm contribution, the host better solves infections initiated in the morning, at the end of the dark phase with respect to those initiated in the evening, at the end of the light phase. Thus, the pathogen\u0026rsquo;s rhythm switches infection outcome, and this is likely the result of a differential initial physiological state at the onset of infection. Since entrainment occurred at 23\u0026deg;C and infections were performed at 37\u0026deg;C, we did not directly test whether bacterial phase persists after transfer to the host environment, which will certainly be pursued in the near future.\u003c/p\u003e \u003cp\u003eMoreover, we found that antibiotic-inactivating capacity varies predictably along the day suggesting fluctuating beta-lactamase activity. In fact, we observed higher nitrocefin hydrolysis activity at the end of the illuminated phase (evening) with respect to the end of the dark phase (morning), in LD entrained cultures. This is consistent with a mechanism evolving improved host response for infection control in the morning. This further provides a temporal window at which antibiotic treatment would be more efficient. This data could represent a paradigm shift in bacterial pathogenesis.\u003c/p\u003e \u003cp\u003eWe decided to use direct approaches such as nitrocefin hydrolysis measurement, which provides a rapid and growth-independent indirect estimation of β-lactamase activity at each time point. Growth-dependent approaches such as antibiograms or MIC determination in liquid media could mask the effect of differential rhythmicity along the day. This difference in total β-lactamase activity could derive either from fluctuations at the enzyme activity or transcriptional levels, the latter being the most probable. Thus, our data reveal a previously unrecognized temporal regulation of antibiotic-inactivating activity in \u003cem\u003eA. baumannii\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eAlso interesting is the fact that rhythmicity can vary among strains for a specific trait, likely as a result of complex and differential regulatory cascades. In fact, we observed variations in rhythmicity among strains both for antibiotic-inactivating activity as well as ring formation in macrocolonies, with some strains showing non-rhythmic constant β-lactamase inactivating activity along the day, e.g. V15 and Ab1915, multidrug sensitive and resistant strains, respectively. It should be noted that daily and circadian rhythms were previously observed for \u003cem\u003eblsA\u003c/em\u003e promoter activity in V15 strain, while no rhythmicity in ring formation was observed in this strain. On the contrary, Ab1915 showed rhythmic ring formation on macrocolonies, while Ab1914 showed rhythmic β-lactamase inactivating activity but no rhythmic ring formation on macrocolonies. In turn, Ab825 showed both β-lactamase activity rhythmic pattern as well as ring formation.\u003c/p\u003e \u003cp\u003eNow that we have shown that bacterial critical pathogens such as \u003cem\u003eA. baumannii\u003c/em\u003e exhibit diurnal and circadian rhythms, which could shape infection dynamics and antibiotic-inactivating capacity, the current concepts of infection and disease acquire a new temporal dimension. This notion is conceptually innovative, as only a handful of articles have described the existence of rhythms in non-photosynthetic bacteria, but none has addressed their potential role in pathogenesis, establishing a new paradigm. Predictable fluctuations in pathogens\u0026rsquo; vulnerability along the day would pose a novel concept in infectious disease management: alignment of treatment with pathogen\u0026rsquo;s circadian vulnerability, and ultimately design of new antimicrobials targeting microbial clock components to reduce the global burden of antimicrobial resistance (AMR). Daily rhythm\u0026rsquo;s structure physiology and behavior across the 24-h day; when these rhythms are generated by an endogenous circadian clock, they persist under constant condition.\u003c/p\u003e \u003cp\u003eIn addition, our results highlight the importance of considering \u003cem\u003eA. baumanii\u003c/em\u003e infection as a temporally structured interaction between a rhythmic pathogen and a rhythmic host. Thus, the outcome of \u003cem\u003eA. baumannii\u003c/em\u003e infection seems to be shaped not only by the host\u0026rsquo;s circadian immune responses \u003csup\u003e\u003cspan additionalcitationids=\"CR45 CR46\" citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e, but also by the bacterium\u0026rsquo;s prior entrainment state, which determines its physiological condition at the onset of infection \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e,\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Bacteria entrained under LD cycles and infecting at different times of day encounter distinct host\u0026acute; immune landscapes, suggesting that bacterial rhythms may have evolved to optimize infection, modulating growth, survival, and virulence factor expression to synchronize to oscillatory host defenses rather than constant immune pressure. Importantly, the differential bacterial loads and wound resolution observed depending on both bacterial entrainment history (LL or LD) and infection timing (am versus pm) suggest that \u003cem\u003eA. baumannii\u003c/em\u003e is not a passive target of host circadian rhythms but an active participant whose internal temporal programs can influence its ability to withstand circadian fluctuations in tissue repair capacity. Depending on its internal temporal state, it is also conceivable that the bacterium may also exploit rhythmic features of the host environment in a manner analogous to that described for viruses, which have been shown to take advantage of host circadian clock\u0026ndash;regulated pathways to enhance their replication \u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. These findings support a model in which infection progression emerges from the alignment -or misalignment- between bacterial light-driven temporal programs and host circadian immunity. Notably, identifying time windows of increased bacterial vulnerability may offer opportunities to temporally target antimicrobial therapies \u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e, potentially enhancing treatment efficacy while reducing selective pressure that drives the development of bacterial resistance.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e \u003cb\u003eLight settings.\u003c/b\u003e Bacterial samples were exposed to blue light emitted by nine-LED (light-emitting diode) arrays with an intensity of 6 to 10 \u0026micro;mol photons/m\u003csup\u003e2\u003c/sup\u003e/s and peak emission centered at 462 nm \u003csup\u003e1\u003c/sup\u003e. Light intensity was measured using a radiometer/photometer (Flame-T, OceanOptics). Temperature was set at 23\u0026ordm;C and fluctuations in the incubator were less than 0.5\u0026deg;C.\u003c/p\u003e \u003cp\u003eZeitgeber (i.e., \u0026ldquo;time giver\u0026rdquo; or entraining agent) time 0 or ZT0 (7:00 am) indicates the time at which lights were turned on. Photo and thermal conditions were controlled with an I-291PF incubator (INGELAB, Argentina) and temperature was monitored using DS1921H-F5 iButton Thermochrons (Maxim Integrated, USA).\u003c/p\u003e \u003cp\u003e \u003cb\u003eBacterial culture and light entrainment conditions.\u003c/b\u003e Bacteria were initially cultured from a single isolated colony in LB liquid medium and grown overnight at 37\u0026deg;C with constant shaking. The overnight culture was then diluted to an optical density (OD\u003csub\u003e660\u003c/sub\u003e) of 0.05 in fresh LB medium and inoculated into 24-well plates under static conditions. For light entrainment, cultures were exposed to 12-hour blue light:12-hour dark (12bL:12D, LD) cycles or to constant light (LL) conditions at 23\u0026deg;C for 5 days. For animal infections, bacterial cultures retrieved at 7 pm of the 5th and 7 am of the 6th days of entrainment were centrifuged at 5,000 rpm for 10 minutes, washed twice with phosphate-buffered saline (PBS), and approximately 10⁹ CFU were resuspended in 16 ul of the same buffer to be used for infections.\u003c/p\u003e \u003cp\u003e\u003cb\u003eAnimals.\u003c/b\u003e C57BL/6 female mice (6\u0026ndash;8 weeks old, 3\u0026ndash;5 per group) were obtained from the animal facilities at the Facultad de Ciencias Veterinarias de la Universidad Nacional de La Plata (LAE-FCVUNLP) and kept in HEPA-ventilated cages at the facilities of the Centro de Investigaci\u0026oacute;n y Producci\u0026oacute;n de Reactivos Biol\u0026oacute;gicos, Facultad de Ciencias M\u0026eacute;dicas, Universidad Nacional de Rosario (CIPReB-FCM-UNR), both in Argentina. All protocols for animal studies were approved by the Institutional Animal Care and Use Committee (Resolution N\u0026ordm; 2141/2024), according to the institutional guidelines and carried out following the National Institutes of Health\u0026rsquo;s \u0026lsquo;Guide for the Care and Use of Animals. All experiments were conducted in a biosafety level II facility. Mice were housed in sealed cages with \u003cem\u003ead libitum\u003c/em\u003e access to food and water and placed in HEPA-ventilated racks designed to prevent microbial exchange between the environment and neighboring cages. Mice were maintained under a 12 white light\u0026ndash;12hdark (12wL:12D) cycle, with lights being turned on at 7:00 am and off at 7:00 pm, controlled by an automated timer. Light conditions followed those previously described by Aiello \u003cem\u003eet al.\u003c/em\u003e (2020)\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e and Casiraghi \u003cem\u003eet al.\u003c/em\u003e (2012)\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. The average light intensity at cage level was approximately 300 lux.\u003c/p\u003e \u003cp\u003e \u003cb\u003eSkin Wound Infection Model with\u003c/b\u003e \u003cb\u003eAcinetobacter baumannii.\u003c/b\u003e Mice were anesthetized via intraperitoneal injection with a 50%:10% Ketamine\u0026ndash;Xylazine solution (acquired from Holliday\u0026ndash;Scott S.A., B\u0026eacute;ccar, Argentina and PharmaVet, Carole Park, Australia, respectively). The dorsal neck region was shaved (~\u0026thinsp;1 cm \u0026times; 1 cm in each side) using an electric shaver. The skin was surgically excised down to the muscle layer to generate two circular wounds of 0.6 cm in diameter in each side (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Each wound was then infected with 16 \u0026micro;L of an \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e V15 suspension containing\u0026thinsp;~\u0026thinsp;10⁹ CFU total. Control animals received an identical wounding procedure followed by the application of 16 \u0026micro;L of sterile phosphate-buffered saline (PBS). Non-infected animals were used as controls for histological analyses for each experiment. Wound diameters were measured after 4- and 7-days post-infection (dpi) using a digital caliper, measuring in millimeters the X axis or the longest point spanning the length of the wound.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAssessment of Bacterial Burden in Wound Tissue.\u003c/b\u003e At 4- and 7-days post-infection (dpi), tissue samples from the wound site were aseptically collected and homogenized in phosphate-buffered saline (PBS) using sterile disposable Piston Pellet Eppendorf. The resulting homogenates were subjected to serial dilutions in LB medium, and aliquots were plated on LB agar for bacterial enumeration. Plates were incubated at 37\u0026deg;C for 18\u0026ndash;24 hours, and colony-forming units (CFUs) were quantified to assess bacterial load. Bacterial loads were normalized to tissue weight. As control, we verified that animals infected at (t\u0026thinsp;=\u0026thinsp;0 dpi) presented similar bacterial loads among the different groups.\u003c/p\u003e \u003cp\u003e \u003cb\u003eNitrocefin hydrolysis determination along the day in LD cultures.\u003c/b\u003e \u003cem\u003eA. baumannii\u003c/em\u003e Ab825 cells were grown overnight in LB at 37 \u0026ordm;C in the dark, and then inoculated in fresh new LB media at a DO\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.05. The bacteria were then grown under LD photoperiod for 4 days at 23\u0026ordm;C. At 7 am and 7 pm of the 3rd and 4th days (LD3 and LD4), samples were retrieved and processed using a nitrocefin-based colorimetric method for β-lactamase activity detection following the manufacturer recommendations (Amplite Colorimetric Beta-Lactamase Activity, AAT Bioquest). The reactions were incubated at room temperature, with the plate protected from light, and after 60 minutes absorbances at 490, 380, and 600 nm were determined using a microplate reader (Bio Tek InstrumentsEPOCH2T). A 490/380 ratio corresponding to hydrolyzed vs intact nitrocefin was calculated using these values and normalized to the OD\u003csub\u003e600\u003c/sub\u003e corresponding to the time point for each sample. At least three independent experiments were carried out.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMacrocolonies.\u003c/b\u003e Bacteria were inoculated directly from an overnight plate grown at 37\u0026deg;C using a sterile wooden stick in swimming agarose plates \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003esupplemented with Congo red (40 mg/l) and Coomassie brilliant blue G-250 (20 mg/l). Plates were incubated at the indicated temperatures and entrainment schedules, and the development of colony morphology and coloration was analyzed.\u003c/p\u003e \u003cp\u003e \u003cb\u003eStatistical analysis.\u003c/b\u003e Statistical analyses were performed using parametric or non-parametric tests as appropriate, depending on data distribution and variance. Statistical analyses for the skin-wound infection model were performed using non-parametric ANOVA followed by the Mann\u0026ndash;Whitney U test, as appropriate, and Fisher\u0026rsquo;s exact test for frequency comparisons. For the analysis of antibiotic susceptibility, the effect of measurement time on themean of ratio/OD was analyzed using a mixed effects model with replicates included as a random effect. Residuals of the fitted model confirmed that necessary assumptions were met. Post hoc pairwise comparisons between groups were conducted using Tukey\u0026rsquo;s multiple comparisons test. Statistical significance was set at \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eCompeting interests.\u003c/h2\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003ch2\u003eFunding \u003cstrong\u003eDeclaration.\u003c/strong\u003e\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eThis work was supported by grants from the Secretar\u0026iacute;a de Ciencia, Tecnolog\u0026iacute;a e Innovaci\u0026oacute;n (Provincia de Santa Fe) to MAM (PEIC I\u0026thinsp;+\u0026thinsp;D 2023-255).\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eConceptualization: R.G., V.P., B.P.M., D.A.G., A.R.P. and M.A.M. Formal analysis: V.P., B.E.P.M., M.L.M., N.A., M.F., D.A.G., A.R.P. and M.A.M. Funding acquisition: M.A.M. Investigation: R.G., V.P., B.P.M., N.A., T.B., A.R.P. and M.A.M. Methodology: R.G., V.P., B.P.M., M.L.M., N.A., T.B., M.F., S.D., D.A.G., A.R.P. and M.A.M. Project administration: M.A.M. Visualization: R.G., V.P., B.P.M., T.B., M.F., D.A.G., A.R.P. and M.A.M. (Writing\u0026mdash;original draft: M.A.M and A.R.P. Writing\u0026mdash;review \u0026amp;amp; editing: R.G., V.P., B.P.M., D.S. and D.A.G.\u003c/p\u003e\n\u003ch2\u003eAcknowledgement\u003c/h2\u003e\n\u003cp\u003eThis work was supported by grants from the Secretar\u0026iacute;a de Ciencia, Tecnolog\u0026iacute;a e Innovaci\u0026oacute;n (Provincia de Santa Fe) to MAM (PEIC I+D 2023-255). BPM, DS, DAG, ARP and MAM, are career investigators of CONICET, while RG, VP and NA are fellows from the same institution. We thank Cecilia Farr\u0026eacute; for technical support with animals at CiPReB.\u003c/p\u003e\n\u003ch2\u003eData Availability\u003c/h2\u003e\n\u003cp\u003eSource data are provided in Supplementary Data 1 and 2. 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Strengths and limitations of period estimation methods for circadian data. \u003cem\u003ePloS one\u003c/em\u003e. \u003cb\u003e9\u003c/b\u003e, e96462. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1371/journal.pone.0096462\u003c/span\u003e\u003cspan address=\"10.1371/journal.pone.0096462\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2014).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-8649481/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8649481/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eWe recently demonstrated that the human pathogen \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e exhibits light-regulated daily and circadian rhythms, suggesting that the physiological state of the bacterium varies along the day. Because this temporal dimension may influence host\u0026ndash;pathogen interactions, we investigated here whether key bacterial processes relevant to pathogenesis fluctuate according to light\u0026ndash;dark\u0026ndash;induced rhythms and whether these fluctuations impact infection outcome. Using a murine skin-wound infection model, we show that both prior bacterial entrainment and the time of infection critically determine disease progression. Specifically, higher bacterial titers were recovered when light-dark entrained bacteria infected mice at the end of the dark phase (morning) with respect to infections caused by arrhythmic bacteria, while the opposite occurred in the evening. In addition, β-lactamase activity displayed significant daily variation in several multidrug-resistant strains, with higher activity at the end of the light phase compared to the dark phase, in light-dark entrained cultures. Consistent with these findings, macrocolony ring formation also followed a rhythmic pattern. Together, these results demonstrate that \u003cem\u003eA. baumannii\u003c/em\u003e displays diurnal regulation of physiological and pathogenic traits that influence infection dynamics and antibiotic-inactivating activity, introducing a new temporal dimension to bacterial pathogenesis with important implications for understanding disease progression and treatment strategies.\u003c/p\u003e","manuscriptTitle":"Daily rhythms modulate Acinetobacter baumannii physiology impacting infection outcome and antibiotic-inactivating capacity","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-02 21:25:32","doi":"10.21203/rs.3.rs-8649481/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-03-31T15:05:26+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-30T17:00:41+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"32453478790475348691354915295037918550","date":"2026-03-13T01:39:18+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"220584207767044225614244073484872478887","date":"2026-02-27T20:27:12+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-26T20:14:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"105487809874562609092131417373971032899","date":"2026-02-26T19:25:08+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-24T00:46:54+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-02-12T06:07:14+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-22T01:15:20+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-22T01:14:53+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2026-01-20T12:19:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"307b46b6-aeb1-4d78-bead-d5182074f48d","owner":[],"postedDate":"March 2nd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":63678360,"name":"Health sciences/Diseases"},{"id":63678361,"name":"Biological sciences/Microbiology"}],"tags":[],"updatedAt":"2026-05-13T09:57:45+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-02 21:25:32","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8649481","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8649481","identity":"rs-8649481","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}