Carbapenem-resistant Acinetobacter baumannii (CRAB): metabolic adaptation and transcriptional response to human urine (HU)

Carbapenem-resistant Acinetobacter baumannii (CRAB): metabolic adaptation and transcriptional response to human urine (HU) | 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 Carbapenem-resistant Acinetobacter baumannii (CRAB): metabolic adaptation and transcriptional response to human urine (HU) Jenny Escalante, Mase Hamza, Brent Nishimura, Meghan Melecio, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4415275/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 19 Aug, 2024 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract Carbapenem-resistant Acinetobacter baumannii (CRAB) is a major human pathogen and a research priority for developing new antimicrobial agents. CRAB is a causative agent of a variety of infections in different body sites. One of the manifestations is catheter-associated urinary tract infection, which exposes the bacteria to the host's urine, creating a particular environment. Exposure of two CRAB clinical isolates, AB5075 and AMA40, to human urine (HU) resulted in the differential expression levels of 264 and 455 genes, respectively, of which 112 were common to both strains. Genes within this group play roles in metabolic pathways such as phenylacetic acid (PAA) catabolism, the Hut system, the tricarboxylic acid (TCA) cycle, and other processes like quorum sensing and biofilm formation. These results indicate that the presence of HU induces numerous adaptive changes in gene expression of the infecting bacteria. These modifications presumably help bacteria establish and thrive in the hostile conditions in the urinary tract. These analyses advance our understanding of CRAB's metabolic adaptations to human fluids, as well as expanding knowledge on bacterial responses to distinct human fluids containing different concentrations of human serum albumin (HSA). Biological sciences/Microbiology/Microbial genetics/Bacterial genetics Biological sciences/Microbiology Acinetobacter baumannii human urine cefiderocol human serum albumin carbapenem-resistance Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Acinetobacter baumannii is a Gram negative non-fermentative coccobacillus which has emerged as an important human pathogen mainly due to its capacity to persist in hospital settings as well as to resist multiple antimicrobials. Infections with A. baumannii are challenging to treat as per an increase in the incidence of multi-drug (MDR) and extensively-drug (XDR) resistant strains 1 . The World Health Organization has placed carbapenem-resistant strains of A. baumannii as critical priority for the research and development of new antimicrobials 2 . A. baumannii can cause pneumonia, as well as infections in the bloodstream, skin, soft-tissue, and urinary tract. Acinetobacter spp. infections in the clinical setting can be associated to the use of medical devices such as ventilation tubes and central venous and urinary catheters, as well as to surgery, invasive procedures and prolonged treatment with broad spectrum antimicrobials 3 . Studies carried out in intensive care units indicated A. baumannii as the main cause of catheter-associated urinary infection (CAUTI) in that setting 4,5 . Up to 20 per cent of all A. baumannii isolates are obtained from urinary sources 6 and recently it was proposed that secondary urinary tract infection (UTI) after re-catheterization could be caused by an intracellular reservoir of A. baumannii in bladder epithelial cells in a murine model 7 . A. baumannii has a versatile metabolism that allows it to acquire nutrients, survive and ultimately replicate in a low nutrient environment like the one present in the host during an infection 1,3 . Also, the interaction with the host's environment can trigger A. baumannii metabolic responses that activate antimicrobial resistance and immunomodulatory effects 1,8 . An example of the latter is the catabolism of the alpha amino acid histidine, that in A. baumannii is done through the Hut system. The Hut system has an important role in A. baumannii infections, as it is implicated in multiple metabolic pathways including zinc homeostasis, biofilm formation, and histamine synthesis 1,9 . Then, studying the physiological responses of A. baumannii caused by exposure to environmental conditions that mimic those of the host, is key to understanding its metabolism during infection and, ultimately, for the development of new methods to control those infections 1 . The catabolism of organic acids has been regarded as essential for the virulence and immunomodulation of A. baumannii 1 . Thus, phenylacetic acid (PAA) metabolism plays a key role in A. baumannii infection. The PAA catabolic pathway, that is encoded in the paa operon, is an important route in the catabolism of aromatic compounds that will lately converge in the Krebs cycle 10,11 . A. baumannii mutations in the ppaE gene resulted in lesser virulence in a murine septicemia model 10 . Conversely, GacS is a global virulence regulator of A. baumannii , and deletion of its gene leads to high repression of the paa operon and accumulation of phenylacetate (PA) 8 . In a zebra fish infection model, inhibition of the A. baumannii paa operon led to migration of polymorphonuclear neutrophiles to the infection site, with the ultimate consequence of a reduction in bacterial burden and attenuated disease 8 . Another set of experiments demonstrated that subinhibitory concentrations of antimicrobials upregulated the paa operon; as well as interfering with PAA metabolism increased susceptibility to antibiotics and hydroxide peroxide treatment 12 . Also, the blockage of PAA catabolism resulted in attenuated virulence in a murine catheter-associated urinary tract infection (CAUTI) model 12 . Previous results indicated that exposure to human pleural fluid (HPF), a fluid with high content of human serum albumin (HSA), can alter the expression of A. baumannii genes related with survival and persistence 13 and elicit metabolic changes that enhance cytotoxicity and immune evasion 14 and DNA uptake 15 . Moreover, HPF triggers the differential expression of genes related to processes such as antimicrobial resistance, biofilm formation, motility, osmotic stress, and DNA-damage control, thus acting as an adaptative response to environmental stressors 16 . Still, when A. baumannii is exposed to fluids with low HSA content like cerebrospinal fluid (CSF), global changes in gene expression are triggered including the increase of metabolism and virulence expression factors 17 . In this study, we evaluate the transcriptomic response followed by phenotypic analysis to human urine (HU), a fluid low in HSA content, of two carbapenem-resistant A. baumannii (CRAB) strains, AB5075 18 and the AMA40 19 , belonging to different genetic lineages and harboring different cabapenemases 18,19 . Results Complete metabolic pathways are modulated by human urine (HU) RNA-seq analyses were performed using two carbapenem-resistant A. baumannii (CRAB) strains, AB5075 and AMA40, belonging to different genetic lineages and possessing different carbapenemases 18,19 , in the presence or absence of human urine (HU). RNA-Seq analysis revealed 264 and 455 differentially expressed genes (DEGs) in AB5075 and AMA40, respectively, upon exposure to HU. In presence of HU, AB5075 had 148 genes upregulated, and 116 genes downregulated. In the case of AMA40 strain, 262 genes were upregulated and 193 were downregulated (Supplementary Fig. S1 ). In terms of gene ontology, the genes corresponded with a variety of functions, such as catalytic activity and metabolic process (Fig. 1 A, B). In addition, it was found that of these DEG, 112 were shared by both strains upon HU exposure. Notably, the majority of up- and downregulated genes fell into metabolic pathways. When comparing the data obtained with HU, we observed different results in the expression of genes compared to those obtained with fluids high in HSA or pure HSA 13,14,16,20 . A key pathway is the tricarboxylic acid (TCA) cycle, which is strongly linked to pathogen virulence and has significant influence on energy production, biosynthesis, and adaptation to the host environment. TCA cycle intermediates function as signalling molecules, orchestrating the regulation of virulence genes, and exerting a pivotal impact on host-pathogen interactions, thereby unveiling potential targets for therapeutic interventions 21,22,23, 24 . In both AB5075 and AMA40, a total of 7 out of 18 TCA genes were found to be upregulated. Additionally, 3 out of 18 and 4 out of 18 downregulated TCA genes were identified in AB5075 and AMA40, respectively. Furthermore, in both strains, the glyoxylate pathway exhibited upregulation, with 2 out of 2 and 1 out of 2 genes being upregulated in AB5075 and AMA40, respectively. Notably, the expression of the aceB gene was not detected in both strains (Fig. 1 C). In addition, genes upregulated in the presence of HU for both A. baumannii strains AB5075 and AMA40 correspond to other metabolic pathways such as those for benzoate, acetoin, iron uptake, PAA and Phe catabolic pathways, and the catabolism of central aromatic intermediates (Figs. 2 – 4 and Supplementary Figs. S1-S4). Regarding genes that are downregulated in association with metabolic pathways in the presence of HU for both strains, these include a decrease in DGE in the high-affinity Potassium transporter Kdp operon (Fig. 4 C), as well as in the cysteine (Fig. 4 E) and arginine succinyl transferase pathways (Supplementary Fig. S2), along with the taurine and alkanesulfonate transport system pathways (Supplementary Fig. S2). A distinctive difference is observed in the arsenic metabolism where in AMA40 gene expression is clearly downregulated while in AB5075 most genes remain upregulated or slightly downregulated (Supplementary Fig. S2). Finally, the lipid metabolism was upregulated in both strains under HU condition (Supplementary Fig. S3). These results may indicate that under this condition, which resembles a minimal medium, lipid catabolism is increased. In the following section we describe some of the most relevant pathways affected by the presences of HU in both CRAB studied in this work. The PAA degradation pathway of CRAB strains is induced in HU Considering the relevance of the PAA catabolic pathway in virulence and immune evasion, we decided to assess the expression level of genes involved in the PAA and Phe catabolic pathways in the carbapenem-resistant A. baumannii strains AB5075 and AMA40. For these, both CRAB strains were cultured in CAMHB with or without supplementation with HU, and the transcriptome analysis showed that the genes encoding enzymes of the PAA and Phe catabolic pathway are induced under HU in both strains AB5075 (Fig. 2 A) and AMA40 (Fig. 2 C). Further qRT-PCR experiments confirmed that HU significantly induced the expression of genes of those pathways, such as paaA, paaB, paaE, paaG, paaK, paaZ and feaB in the strain AB5075 (Fig. 2 B). Similar qRT-PCR results were observed in the strain AMA40 (Fig. 2 D), confirming that PAA degradation pathway was upregulated by the presence of HU in both CRAB strain evaluated. It has been shown that if the PAA metabolic pathway is inhibited, the accumulation of metabolic by-products acts as a direct attractant of neutrophils, one of the main immune cells involved in the response to bacterial infections 8 . Therefore, neutrophil chemotaxis assays were performed to evaluate whether HU affects this phenomenon. To this end, both CRAB strains were grown in the presence of PAA, and in the presence or absence of HU. The results obtained were consistent with what was observed at the transcriptional level. Under HU treatment, both strains increased PAA catabolism and attracted significantly fewer neutrophils than the control condition, 2.32-fold and 1.46-fold decrease for AB5075 and AMA40 strains, respectively (Fig. 2 E). Histidine catabolism gene expression in AB5075 and AMA40 strains is increased under HU condition The catabolism of the alpha amino acid histidine is done through the Hut system in A. baumannii . As mentioned in the introduction, the Hut system is implicated in multiple metabolic pathways that include biofilm formation, zinc homeostasis, and histamine synthesis. The RNA-seq results showed an up-regulation in the hut operon for AB5075 strain, while for AMA40 strain, the hutCDGHITU genes were down-regulated (although not significantly) (Fig. 3 A). Also, for both strains, qRT-PCR assays were carried after being cultured in HU respect to CAMHB. Results indicated that the expression of genes linked to histidine catabolism was enhanced in the presence of HU, for AB5075 and AMA40 (Fig. 3 B). Other metabolic genes of interest in A. baumannii are modulated by HU As described above in the transcriptomic results, all genes of the benzoate pathway were upregulated when both CRAB strains growth in HU (Fig. 4 A). To corroborate this, the expression level of benA gene was evaluated by qRT-PCR, resulting in a significant enhanced expression in the presence of HU in both AB5075 and AMA40 (Fig. 4 B). Also, RNA-seq results indicated that the expression of high-affinity K + transporter (Kdp) genes was down-regulated in HU conditions (Fig. 4 C). In this case, the expression level for kdpA gene, was evaluated for RT-qPCR and was obtained as a result a 0.24-fold and 0.22-fold decrease for AB5075 and AMA40, respectively (Fig. 4 D). Finally, the cysteine pathway composed of cysDNPTW genes was completely repressed under HU conditions in both CRAB strains, according to RNA-seq results (Fig. 4 E). To confirm this repression, the evaluation of cysT gene was assessed by RT-qPCR, resulting significantly reduced by 5-fold, in both strains, when grown in the presence of HU (Fig. 4 F). Exposure to human urine affects biofilm formation in CRAB The lifestyle changes from planktonic to biofilm, and conversely, are key for environmental adaptation and survival of bacteria. Previous studies in animal model indicated that biofilm formation may play a role by increasing virulence in infections by A. baumannii 25 . RNA-seq analyses performed in the two carbapenem-resistant A. baumannii strains AB5075 and AMA40, showed differential gene expression regarding the genes involved in biofilm formation. Analyses of differentially expressed genes showed that for both strains AB5075 and AMA40 in the presence of HU, genes from the csu locus were downregulated. However, in the presence of HU, the genes from the pga locus were mostly upregulated in AMA40 while a different pattern can be seen for AB5075 where some genes are slightly downregulated, other slightly downregulated and some show no significant changes (Fig. S4). Biofilm experiments were performed and quantified using crystal violet. As results, a small significant difference was found for the AMA40 strain, while differences were not found in AB5075 between CAMHB and CAMHB + 50% HU conditions (Fig. S4). In addition, CFU recovered from biofilm did not show significant differences (Fig. S4). Finally, the transcriptomic analyses revealed that Quorum Quenching (QQ) related genes were upregulated in the presence of HU in both CRAB strains. While most of Quorum Sensing (QS) associated genes were significantly upregulated or slightly downregulated in AB5075 in presence of HU, they were mostly downregulated in AMA40 (Fig. S4). Discussion CRAB strains can cause CAUTI that are difficult to treat. The ability of A. baumannii to survive in a low-nutrient environment, such as the one in the host during an infection, could be explained by the bacterium versatile metabolism. In this work, we have described the effects of exposure to human urine (HU) of two genetically different CRAB strains, AB5075 and AMA40. As a result of HU exposure, we established changes to the differential expression of 264 and 455 genes in strains AB5075 and AMA40, respectively. Further, both strains shared 112 DEGs, suggesting common transcriptional responses to HU. This common transcriptional response of the studied CRABs to HU exposure includes DEGs in various metabolic pathways. Among these, genes coding for TCA cycle and glyoxylate pathway showed upregulation in both strains, implying potential adaptations in energy production and biosynthesis. In addition, the TCA cycle is linked to the virulence of pathogens through various mechanisms including metabolic adaptation to diverse nutrients allowing biosynthesis and growth, regulation of virulence genes, redox balance, iron acquisition, tissue colonization and immune evasion, and host immune response modulation 21,22 . Another response to HU in both strains tested, is the downregulation of the transcription of genes present in the kdp operon, which encodes the high-affinity Potassium transporter Kdp. In E. coli , the Kdp transporter is encoded by the kdpABC operon, and its expression is regulated by the products of kdpD and kdpE . Previous observations indicate that the expression of Kdp is affected by the concentration of K + in the medium, resulting in a repression of the operon when the K + concentration in the medium is high 26 . A further study confirmed that in A. baumannii the transcription of the five components of the Kdp system is linked, and that kdpE is notably upregulated under K + limiting conditions, as well as an important factor for pathogenicity in a murine pneumonia model 27 . Also, the PAA degradation pathway was induced under HU conditions in both strains, as evidenced by upregulation of genes in the PAA and Phe catabolic pathways. Accordingly, in neutrophil assays, the exposure to HU attracted significantly fewer neutrophils in both CRAB analyzed. These results are consistent with previous observations indicating that PAA metabolism in A. baumannii affects infection outcomes by directly influencing neutrophil chemotaxis 8 . Hence, we can conclude that HU is an environmental signal that affects A. baumannii metabolism, providing favorable conditions for this pathogen to evade the host immune system. Finally, lipid metabolism was upregulated in both strains under HU conditions, suggesting increased lipid catabolism in a minimal medium-like environment. It was established that up to 20% of A. baumannii clinical isolates are obtained from urinary sources 6 , and CRAB are considered a critical priority for the research and development of new antimicrobials 2 . The findings in this study contribute to a better understanding of metabolic changes undergone by CRAB when exposed to HU. The human fluids that A. baumannii may encounter while infecting its host, possess different compositions regarding proteins, metabolites and other solutes. In this sense, a characteristic that distinguishes HU from other fluids is its low content of human serum albumin (HSA). It was previously reported that A. baumannii responds to components of human fluids by modifying its transcriptional and phenotypic profiles 16,17,20 . HSA and human pleural fluid (HPF) can modulate the expression of genes associated with iron uptake systems, biofilm formation, antibiotic resistance, DNA acquisition and metabolism. Previous results indicated that most genes of the paa locus were downregulated when exposed to HPF 14 , while as showed in this study paa locus is upregulated in presence of HU in both CRAB strains, thus suggesting a differential role of HSA in the regulation of the PAA metabolic pathway. In a similar direction, on A. baumannii strain A118 cultured with HSA led to a downregulation of many genes of the paa locus with the repressor paaX being upregulated 13 . HSA appears to act as a host-derived signal, inducing adaptive mechanisms that enhance virulence-associated gene expression 49 . Studies on other bacteria like Bordetella pertussis and Pseudomonas aeruginosa have shown that purified HSA produces outcomes similar to those of serum, and that removing albumin through membrane filtration decreases the serum-mediated effects. This suggests that HSA is the main component responsible for these phenotypes 28,49 . Additionally, previous research has demonstrated that the effectiveness of cefiderocol, a novel chlorocatechol-substituted siderophore antibiotic used to treat cUTI, is reduced when exposed to human fluids containing HSA, such as Human Serum (HS) and HPF 30,31 . Ferric siderophore transporters facilitate the uptake of cefiderocol into bacterial cells, and the presence of HSA or HSA-containing fluids is associated with a decrease in the expression of genes linked to high-affinity siderophore-mediated iron uptake systems 30 . In contrast, studies have shown that exposure to HU, a fluid with little to no HSA or free-iron content, did not significantly alter the minimum inhibitory concentration (MIC) values for CRABs under the conditions tested 32 . Additionally, genes involved in iron uptake were upregulated. These results support the hypothesis that an unknown mechanism triggers a regulatory response in A. baumannii when exposed to human fluids, enabling it to thrive in different environments. Thus, the HSA content in the fluid may play a crucial role in eliciting a differential adaptive response. In conclusion, this report shows that exposure to HU induces widespread changes in the transcriptome of CRAB strains, impacting various metabolic pathways, as well as genes coding for antibiotic resistance, biofilm formation, and quorum sensing functions. These findings contribute to a better understanding of the adaptive responses of CRAB strains to urinary environments, providing insights that may guide future therapeutic interventions and infection control methods. Material and Methods Bacterial strains . Two CRAB strains were used in the present study: the multidrug and hypervirulent AB5075 (OXA-23) strain and AMA40 (NDM-1) (REF). RNA extraction. A. baumannii AB5075 and AMA40 were cultured in CAMHB and CAMHB supplement with 50% human urine (HU) from healthy individuals obtained from a certified vendor (Innovative Research Inc., MI, USA) and incubated with agitation for 18 h at 37°C. Then, overnight cultures were then diluted 1:10 in fresh Cation Adjusted Mueller-Hinton Broth (CAMHB), supplemented with HU, and incubated at 37°C with agitation during 7 h. RNA extraction was carried out with Direct-zol RNA Kit (Zymo Research, Irvine, CA, USA), in triplicate. The RNA samples obtained were subjected to DNase treatment (Thermo Fisher Scientific, Waltham, MA, USA) following manufacturer’s instruction, afterwards a PCR amplification of the 16S rDNA gene was performed to confirm there was no DNA contamination. Then, from three independent replicates per sample, ribosomal RNA-depletion was done using the Ribo-Zero kit (Illumina) followed by the construction of the cDNA library with the TruSeq Stranded Total RNA Library Prep kit (Illumina). The RNA sequencing was outsourced to Novogene (Novogene Corporation, Sacramento, CA, USA). RNA-seq analysis . The quality control of the Illumina reads, trimming of low-quality bases and removal of Illumina adapters was performed as described previously ( 36 ). Reads were aligned to the genome of A. baumannii AB5075 or AMA40, using Burrows-Wheeler Alignment (BWA) software (v0.7.17) BWA and visualized using the Integrative Genomics Viewer (IGV). Read counts per gene were calculated using FeatureCounts ( 37 ). Differential expression analysis was performed using DEseq2 and the Differentially Expressed Genes (DEGs) were defined as those displaying an FDR adjusted P value of 1. In addition, to represent metabolic pathways in our RNA sequencing data, the Omics Dashboard Tool provided by both BioCyc 33 and MetaCyc 34 was employed. Enrichment or depletion of metabolic pathways was assessed utilizing the Fisher's exact test hypothesis, with significance determined at P values below 0.05. Enrichment or depletion scores (represented as -log 10 P values) for each pathway within the dashboard were subsequently acquired through download and analyzed. The RNA-seq data generated in the current study are available in the NCBI repository with the GEO accession No GSE201259. qRT-PCR assays . The cDNA was prepared using the iScript Reverse Transcription Supermix for qRT-PCR (BioRad, Hercules, CA, USA) and the quantitative PCR was performed using iQ™SYBR Green Supermix (BioRad, Hercules, CA, USA), in both cases following the recommendation of the manufacturer. Different primers to confirm RNA-seq results and also study the expression of genes associated with virulence and antimicrobial resistance were used (Table S1 ). Experiments were performed in technical and biological triplicates. The results were analyzed using the qBASE method 35 with recA and rpoB genes as normalizers 36,37 . Data are presented as NRQ (normalized relative quantities). Differences were determined by two-way ANOVA followed by Tukey’s multiple comparison test ( P < 0.05) using GraphPad Prism (GraphPad software version 10.0.0, San Diego, CA, USA). Neutrophil Chemotaxis Assay . For this assay, a previously published protocol with modifications was used 14 . AB5075 or AMA40 cultured overnight in CAMHB supplement with 50% human urine with or without 0.02% phenylacetic acid (PAA) were tested (Millipore) ( 2 ). Controls for this assay constituted of CAMHB and CAMHB + 0.02% PA with performance in parallel. First, 100 µl an overnight culture of A. baumannii AB5075 or AMA40 in the tested conditions were combined with 100 µl of chemotaxis buffer (CF). Then, each mix was transferred to a twenty-four-well Olympus polycarbonate tissue culture plate with 8-µm pore size membranes (Genesee Scientific). Prior incubation 100 µl of neutrophils (10 x 10 6 cells/ml) from iQ Biosciences was added to the semipermeable well inserts. Then, plates were incubated for 1 hour in 5% CO 2 at 37°C. Neutrophils were then counted from each sample tested. Migration chemotaxis index was calculated by the number of neutrophils in the test well by number of neutrophils in control. All conditions were performed in triplicates. The chemotaxis buffer (CF) has the following components: 25 ml of Roswell Park Memorial Institute Medium (Thermo Fisher), 10% fetal calf serum (FCS) (Thermo Fisher), 500 µl of 100 U/ml penicillin-streptomycin (Sigma Aldrich), and 22 ml of Hanks’ balanced salt solution (HBSS) (Thermo Fisher). Biofilm assay. First, A. baumannii AB5075 and AMA40 strains were cultured in fresh CAMHB medium, or CAMHB supplemented with 50% HU in static conditions at 37 ºC for 18 h. Then, tubes were emptied, washed three times with 1X phosphate-buffered saline (PBS) and stained with 1% crystal violet (CV) for 15 min. Excess CV was removed by washing three more times with 1X PBS. The biofilm assays were performed in triplicate (absorbance determination at 580 and 660 nm as well CFU/ml), with at least three technical replicates per biological replicate (REF). Statistical analysis. Experiments performed at least in triplicates were statistically analyzed by one- or two-way ANOVA followed by Tukey’s multiple comparison tests using GraphPad Prism (GraphPad software, San Diego, CA, USA). A P value < 0.05 was considered significant. All procedures performed in this study were in accordance with the CSUF Institutional Biosafety Committee Approval plan (DBH117-01) and follow the NIH, CDC, OSHA and other environmental and occupational regulations. Declarations Data availability The datasets generated and analyzed during the current study are available in the Gene Expression Omnibus (GEO) repository (GEO accession No GSE201259). Author Contributions: G.M.T, L.A., R.S., M.E.T, R.A.B, and M.S.R. conceived the study and designed the experiments. J.E., M.H., B.N., M.M., C.P., T.S., M.R.T, R.S., G.M.T., and M.S.R performed the experiments and genomics and bioinformatics analyses. M.R.T., T.S., R.S., C.D.S, L.A., and M.S.R. analyzed the data and interpreted the results. M.E.T., G.M.T., and M.S.R. contributed reagents/materials/analysis tools. C.D.S., G.M.T, L.A., R.S., M.E.T, R.A.B, and M.S.R. wrote and revised the manuscript. All authors read and approved the final manuscript. Acknowledgements: The authors' work was supported by NIH SC3GM125556 to M.S.R., R01AI100560, R01AI063517, R01AI072219 to R.A.B., and 2R15 AI047115 to M.E.T. This study was supported in part by funds and facilities provided by the Cleveland Department of Veterans Affairs, Award Number 1I01BX001974 to R.A.B from the Biomedical Laboratory Research & Development Service of the VA Office of Research and Development and the Geriatric Research Education and Clinical Center VISN 10 to R.A.B. J.E. was supported by grant MHRT 2T37MD001368 from the National Institute on Minority Health and Health Disparities, National Institute of Health. The content is solely the authors' responsibility and does not necessarily represent the official views of the National Institutes of Health or the Department of Veterans. M.R.T., T.S., and R.S are members of the CONICET Research carreer. Conflicts of Interest: The authors declare no conflict of interest. References Ren, X. & Palmer, L. D. Acinetobacter Metabolism in Infection and Antimicrobial Resistance. Infect Immun 91, e00433-22 (2023). Tacconelli, E. et al. Discovery, research, and development of new antibiotics: the WHO priority list of antibiotic-resistant bacteria and tuberculosis. The Lancet Infectious Diseases 18, 318–327 (2018). Wong, D. et al. Clinical and Pathophysiological Overview of Acinetobacter Infections: a Century of Challenges. Clin Microbiol Rev 30, 409–447 (2017). Ding, R., Li, X., Zhang, X., Zhang, Z. & Ma, X. The Epidemiology of Symptomatic Catheter-associated Urinary Tract Infections in the Intensive Care Unit: A 4-year Single Center Retrospective Study. 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Acinetobacter baumannii virulence is enhanced in Galleria mellonella following biofilm adaptation. Journal of Medical Microbiology 61, 470–477 (2012). Asha, H. & Gowrishankar, J. Regulation of kdp operon expression in Escherichia coli : evidence against turgor as signal for transcriptional control. J Bacteriol 175, 4528–4537 (1993). Samir, R. et al. Adaptation to Potassium-Limitation Is Essential for Acinetobacter baumannii Pneumonia Pathogenesis. J Infect Dis. 214, 2006–2013 (2016). Gonyar, L. A., Gray, M. C., Christianson, G. J., Mehrad, B. & Hewlett, E. L. Albumin, in the Presence of Calcium, Elicits a Massive Increase in Extracellular Bordetella Adenylate Cyclase Toxin. Infect Immun 85, e00198-17 (2017). Kruczek, C. et al. Serum albumin alters the expression of iron-controlled genes in Pseudomonas aeruginosa . Microbiology 158, 353–367 (2012). Escalante, J. et al. The Iron Content of Human Serum Albumin Modulates the Susceptibility of Acinetobacter baumannii to Cefiderocol. Biomedicines 11, 639 (2023). Le, C. et al. Human Serum Proteins and Susceptibility of Acinetobacter baumannii to Cefiderocol: Role of Iron Transport. Biomedicines 10, 600 (2022). Nishimura, B. et al. Acinetobacter baumannii response to cefiderocol challenge in human urine. Sci Rep 12, 8763 (2022). Keseler, I. M. et al. The EcoCyc database: reflecting new knowledge about Escherichia coli K-12. Nucleic Acids Res 45, D543–D550 (2017). Caspi, R. et al. The MetaCyc database of metabolic pathways and enzymes and the BioCyc collection of pathway/genome databases. Nucleic Acids Research 40, D742–D753 (2012). Hellemans, J., Mortier, G., De Paepe, A., Speleman, F. & Vandesompele, J. [No title found]. Genome Biol 8, R19 (2007). Mezcord, V. et al. Induced Heteroresistance in Carbapenem-Resistant Acinetobacter baumannii (CRAB) via Exposure to Human Pleural Fluid (HPF) and Its Impact on Cefiderocol Susceptibility. IJMS 24, 11752 (2023). Escalante, J. et al. Human serum albumin (HSA) regulates the expression of histone-like nucleoid structure protein (H-NS) in Acinetobacter baumannii . Sci Rep 12, 14644 (2022). Additional Declarations No competing interests reported. Supplementary Files SupplementaryMaterialSciReports2024Urine.pdf Supplementary Figures Supplementary Fig. S1. A) Venn diagram showing the comparative analysis of the transcriptional response of A. baumannii AB5075 and AMA40 cells cultured in CAMHB or CAMHB containing 50% HU. Heatmap outlining the differential expression of genes associated with the acetoin catabolic pathway (B) and the catabolic aromatic intermediate pathway (C). The asterisks represent the DEGs (adjusted P 1). Supplementary Fig. S2. Heatmap outlining the differential expression of genes associated with A) arginine succinyltransferase pathway, B) taurine and alkanesulfonate transport systems pathway and C) arsenic metabolism in the presence of HU in CRAB strains AB5075 and AMA40. The asterisks represent the DEGs (adjusted P 1). Supplementary Fig. S3. Heatmap outlining the differential expression of genes associated with lipid metabolism by the AB5075 and AMA40 stains in response to the presence of 50% of HU. The asterisks represent the DEGs (adjusted P 1). Supplementary Fig. S4. A) Heatmap outlining the differential expression of genes associated with biofilm in the presence of HU in CRAB strains AB5075 and AMA40. The asterisks represent significant DEGs (adjusted P 1). (B and C) Biofilm assays performed with and without HU in AB5075 and AMA40 strains represented by B) OD580/OD600 and C) CFU recovered. Experiments were performed in triplicate, with at least three technical replicates per biological replicate. Statistical analysis (ANOVA two-way) was performed using GraphPad Prism (GraphPad software, San Diego, CA, USA), and a P < 0.05 was considered significant. D) Heatmap outlining the differential expression of genes associated with quorum sensing and quorum quenching in the presence of HU in CRAB strains AB5075 and AMA40. The asterisks represent the DEGs (adjusted P 1). Cite Share Download PDF Status: Published Journal Publication published 19 Aug, 2024 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 09 Jul, 2024 Reviews received at journal 08 Jul, 2024 Reviews received at journal 01 Jun, 2024 Reviewers agreed at journal 23 May, 2024 Reviewers agreed at journal 23 May, 2024 Reviewers invited by journal 23 May, 2024 Editor assigned by journal 23 May, 2024 Editor invited by journal 19 May, 2024 Submission checks completed at journal 15 May, 2024 First submitted to journal 13 May, 2024 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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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-4415275","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":306508284,"identity":"85fef89e-9979-4bf2-9071-d5f890c34f3f","order_by":0,"name":"Jenny Escalante","email":"","orcid":"","institution":"California State University Fullerton","correspondingAuthor":false,"prefix":"","firstName":"Jenny","middleName":"","lastName":"Escalante","suffix":""},{"id":306508285,"identity":"afbabf78-4309-426d-83bd-bec02f3790f1","order_by":1,"name":"Mase Hamza","email":"","orcid":"","institution":"California State University Fullerton","correspondingAuthor":false,"prefix":"","firstName":"Mase","middleName":"","lastName":"Hamza","suffix":""},{"id":306508288,"identity":"77bc333f-1f7b-4671-847d-4afa691c76b2","order_by":2,"name":"Brent Nishimura","email":"","orcid":"","institution":"California State University Fullerton","correspondingAuthor":false,"prefix":"","firstName":"Brent","middleName":"","lastName":"Nishimura","suffix":""},{"id":306508289,"identity":"4490152f-df31-4996-8f8d-b83041088487","order_by":3,"name":"Meghan Melecio","email":"","orcid":"","institution":"California State University Fullerton","correspondingAuthor":false,"prefix":"","firstName":"Meghan","middleName":"","lastName":"Melecio","suffix":""},{"id":306508290,"identity":"7119f192-31a1-4998-af7e-3056d5425065","order_by":4,"name":"Carol Davies-Sala","email":"","orcid":"","institution":"California State University Fullerton","correspondingAuthor":false,"prefix":"","firstName":"Carol","middleName":"","lastName":"Davies-Sala","suffix":""},{"id":306508295,"identity":"a3d7bb9d-3362-4930-bb2a-c2a99a1f0131","order_by":5,"name":"Marisel R. Tuttobene","email":"","orcid":"","institution":"Universidad Nacional de Rosario","correspondingAuthor":false,"prefix":"","firstName":"Marisel","middleName":"R.","lastName":"Tuttobene","suffix":""},{"id":306508296,"identity":"a54a49b7-d103-48ec-b341-d13023f97295","order_by":6,"name":"Tomás Subils","email":"","orcid":"","institution":"Universidad Nacional de Rosario","correspondingAuthor":false,"prefix":"","firstName":"Tomás","middleName":"","lastName":"Subils","suffix":""},{"id":306508299,"identity":"1b0b7785-542a-4a34-bb76-dc2f4ddc6a5e","order_by":7,"name":"German M. Traglia","email":"","orcid":"","institution":"Universidad de la República","correspondingAuthor":false,"prefix":"","firstName":"German","middleName":"M.","lastName":"Traglia","suffix":""},{"id":306508300,"identity":"3f9967bc-c583-454f-a29b-c1d56323a7c0","order_by":8,"name":"Chloe Pham","email":"","orcid":"","institution":"California State University Fullerton","correspondingAuthor":false,"prefix":"","firstName":"Chloe","middleName":"","lastName":"Pham","suffix":""},{"id":306508301,"identity":"276a09cc-b0c4-4eab-a525-8856c1548e65","order_by":9,"name":"Rodrigo Sieira","email":"","orcid":"","institution":"Fundación Instituto Leloir-IIBBA CONICET","correspondingAuthor":false,"prefix":"","firstName":"Rodrigo","middleName":"","lastName":"Sieira","suffix":""},{"id":306508302,"identity":"d4b7e5fe-93de-45fc-b0e1-a9b97b4f51fe","order_by":10,"name":"Luis Actis","email":"","orcid":"","institution":"Miami University","correspondingAuthor":false,"prefix":"","firstName":"Luis","middleName":"","lastName":"Actis","suffix":""},{"id":306508303,"identity":"f4bbd38c-53dc-4c81-95be-9558b5153a83","order_by":11,"name":"Robert A. Bonomo","email":"","orcid":"","institution":"Case Western Reserve UniversitySchool of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Robert","middleName":"A.","lastName":"Bonomo","suffix":""},{"id":306508304,"identity":"27f12260-29a6-4e91-9791-ecb65c0f63a9","order_by":12,"name":"Marcelo E. Tolmasky","email":"","orcid":"","institution":"California State University Fullerton","correspondingAuthor":false,"prefix":"","firstName":"Marcelo","middleName":"E.","lastName":"Tolmasky","suffix":""},{"id":306508305,"identity":"18279f24-c050-49e0-851f-769d180fe72e","order_by":13,"name":"María Soledad Ramirez","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5ElEQVRIiWNgGAWjYNACAyBmb3zA8KAAJsJGjBaewwYMCUAGD3FaQEAimUgtuv1nH366UXBPTn7mYzaJBAObfHv23gMMH8oO49RidiPdWDrHoNiYcXYySEuaZQ/PuQTGGefwaWFjAGpJSGyWzj8G1HLYgEcix4CZtw2PlvPHmH8DtdS3SR4G2fIfouUvPi0H0thAtiTwSDCDtByAaGHEp+VGGps1UIvhDJ5kZosEg2QDnjNnDA72nEvH67DbOX8S5OXbDzPe+FBhZ8De3mP44EeZNU4t2MEBEtWPglEwCkbBKEADAGBvS8H+U+0UAAAAAElFTkSuQmCC","orcid":"","institution":"California State University Fullerton","correspondingAuthor":true,"prefix":"","firstName":"María","middleName":"Soledad","lastName":"Ramirez","suffix":""}],"badges":[],"createdAt":"2024-05-13 21:08:26","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4415275/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4415275/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-024-70216-w","type":"published","date":"2024-08-19T15:57:40+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":57453498,"identity":"9f0a36b2-6619-4245-b48c-76f9153e0758","added_by":"auto","created_at":"2024-05-30 22:01:14","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1346442,"visible":true,"origin":"","legend":"\u003cp\u003eEnriched Gene Ontology terms of the most representative differentially expressed genes. Gene ontology enrichment analysis of the DEGs of strains AB5075 (A) or AMA40 (B) incubated in the presence or in the absence of human urine. Data were obtaining using the Fisher’s test (FDR \u0026lt; 0.05) with the Blast2GO software. C) \u003cem\u003eA. baumannii\u003c/em\u003e TCA-glyoxylate pathways in response to HU. Reactions and intermediates of the TCA and glyoxylate cycle are represented and based on BioCyc and MetaCyc. Green and red represents up and down-regulation, respectively. *, Indicates statically significant differential expressed genes (DEGs).\u003c/p\u003e","description":"","filename":"Fig.1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4415275/v1/18c451587fde8c804d91f549.jpg"},{"id":57453499,"identity":"5683d355-37ad-4725-b0ef-6accec08eb4b","added_by":"auto","created_at":"2024-05-30 22:01:14","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":665625,"visible":true,"origin":"","legend":"\u003cp\u003ePAA degradation pathway is induced in CRAB by exposure to HU and affects neutrophile chemotaxis. Differential gene expression of genes in PAA and Phe pathways of \u003cem\u003eA. baumannii\u003c/em\u003e AB5075 when exposed to HU in transcriptomic analyses (A) and by qRT-PCR (B). Differential gene expression of genes in PAA and Phe pathways of \u003cem\u003eA. baumannii\u003c/em\u003e AMA40 when exposed to HU in transcriptomic analyses (C) and by qRT-PCR (D). Neutrophile chemotaxis assays (E) indicate a reduced neutrophile recruitment when both CRAB strains are exposed to HU. Statistical significance (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) was determined by two-way ANOVA followed by Tukey’s multiple comparison test. Significance was indicated as follows: ***, \u003cem\u003eP \u0026lt;\u003c/em\u003e 0.001; ** \u003cem\u003eP \u003c/em\u003e\u0026lt;0.01, and * \u003cem\u003eP \u003c/em\u003e\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"Fig.2paa.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4415275/v1/0b6223d1684b9c4756390c47.jpg"},{"id":57453502,"identity":"57648e56-c92e-4d67-adee-89c0b7d65956","added_by":"auto","created_at":"2024-05-30 22:01:14","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":657481,"visible":true,"origin":"","legend":"\u003cp\u003eDifferential expression of the genes involved on histidine catabolism in CRAB strains exposed to HU. (A) Heatmap outlining the differential expression of genes associated with histidine catabolism for AB5075 and AMA40 strains. (B) qRT-PCR results of the Hut system genes in AB5075 (A) and AMA40 (B). Statistical significance (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) was determined by two-way ANOVA followed by Tukey’s multiple comparison test. Significance was indicated as follows: *** indicates \u003cem\u003ep\u0026lt;\u003c/em\u003e 0.001; ** \u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, and * \u003cem\u003ep\u003c/em\u003e\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"Fig.3hutgenes.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4415275/v1/31d34e05e2914c2c14304b8d.jpg"},{"id":57453686,"identity":"b0096c48-d4f3-41b7-b7d9-bc0c569b2708","added_by":"auto","created_at":"2024-05-30 22:09:14","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":692197,"visible":true,"origin":"","legend":"\u003cp\u003eImpact of HU on the expression of CRAB genes coding for K+ transport and benzoate and cysteine pathway functions. Benzoate pathway genes are upregulated in both strains, AB5075 and AMA 40, in transcriptomics studies (A) and corroborated by qRT-PCR (B). Expression of genes involved in the high-affinity potassium transporter are downregulated in both strains of \u003cem\u003eA. baumannii\u003c/em\u003e studied (C and D). Cysteine metabolic pathway genes are also downregulated in the presence of HU for both strains AB5075 and AMA40 shown by transcriptomic analyses (E) and by qPCR (F). Statistical significance (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) was determined by two-way ANOVA followed by Tukey’s multiple comparison test. Significance was indicated as follows: *** indicates \u003cem\u003eP \u0026lt;\u003c/em\u003e 0.001; ** \u003cem\u003eP \u003c/em\u003e\u0026lt;0.01, and * \u003cem\u003eP \u003c/em\u003e\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"Fig.4othergenes.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4415275/v1/044ca40a212ae414069a5bf7.jpg"},{"id":63300680,"identity":"9e807727-1934-497e-8088-86a274510303","added_by":"auto","created_at":"2024-08-26 16:16:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3938962,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4415275/v1/c821ba18-ea6a-44c5-940c-70dcbcf4bf6e.pdf"},{"id":57453687,"identity":"3d1b2cdd-fecd-4b1d-ad5a-08bc80351811","added_by":"auto","created_at":"2024-05-30 22:09:14","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":487692,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figures\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Fig. S1.\u003c/strong\u003e A) Venn diagram showing the comparative analysis of the transcriptional response of \u003cem\u003eA. baumannii\u003c/em\u003e AB5075 and AMA40 cells cultured in CAMHB or CAMHB containing 50% HU. Heatmap outlining the differential expression of genes associated with the acetoin catabolic pathway (B) and the catabolic aromatic intermediate pathway (C). The asterisks represent the DEGs (adjusted \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 with log\u003csub\u003e2\u003c/sub\u003efold change \u0026gt; 1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Fig. S2\u003c/strong\u003e. Heatmap outlining the differential expression of genes associated with A) arginine succinyltransferase pathway, B) taurine and alkanesulfonate transport systems pathway and C) arsenic metabolism in the presence of HU in CRAB strains AB5075 and AMA40. The asterisks represent the DEGs (adjusted \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 with log\u003csub\u003e2\u003c/sub\u003efold change \u0026gt; 1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Fig. S3\u003c/strong\u003e. Heatmap outlining the differential expression of genes associated with lipid metabolism by the AB5075 and AMA40 stains in response to the presence of 50% of HU. The asterisks represent the DEGs (adjusted \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 with log\u003csub\u003e2\u003c/sub\u003efold change \u0026gt; 1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Fig. S4\u003c/strong\u003e. A) Heatmap outlining the differential expression of genes associated with biofilm in the presence of HU in CRAB strains AB5075 and AMA40. The asterisks represent significant DEGs (adjusted \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 with log2fold change \u0026gt; 1). (B and C) Biofilm assays performed with and without HU in AB5075 and AMA40 strains represented by B) OD580/OD600 and C) CFU recovered. Experiments were performed in triplicate, with at least three technical replicates per biological replicate. Statistical analysis (ANOVA two-way) was performed using GraphPad Prism (GraphPad software, San Diego, CA, USA), and a \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 was considered significant. D) Heatmap outlining the differential expression of genes associated with quorum sensing and quorum quenching in the presence of HU in CRAB strains AB5075 and AMA40. The asterisks represent the DEGs (adjusted \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 with log\u003csub\u003e2\u003c/sub\u003efold change \u0026gt; 1).\u003c/p\u003e","description":"","filename":"SupplementaryMaterialSciReports2024Urine.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4415275/v1/693668551791942b50445f83.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Carbapenem-resistant Acinetobacter baumannii (CRAB): metabolic adaptation and transcriptional response to human urine (HU)","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e is a Gram negative non-fermentative coccobacillus which has emerged as an important human pathogen mainly due to its capacity to persist in hospital settings as well as to resist multiple antimicrobials. Infections with \u003cem\u003eA. baumannii\u003c/em\u003e are challenging to treat as per an increase in the incidence of multi-drug (MDR) and extensively-drug (XDR) resistant strains\u003csup\u003e1\u003c/sup\u003e. The World Health Organization has placed carbapenem-resistant strains of \u003cem\u003eA. baumannii\u003c/em\u003e as critical priority for the research and development of new antimicrobials\u003csup\u003e2\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cem\u003eA. baumannii\u003c/em\u003e can cause pneumonia, as well as infections in the bloodstream, skin, soft-tissue, and urinary tract. \u003cem\u003eAcinetobacter\u003c/em\u003e spp. infections in the clinical setting can be associated to the use of medical devices such as ventilation tubes and central venous and urinary catheters, as well as to surgery, invasive procedures and prolonged treatment with broad spectrum antimicrobials\u003csup\u003e3\u003c/sup\u003e. Studies carried out in intensive care units indicated \u003cem\u003eA. baumannii\u003c/em\u003e as the main cause of catheter-associated urinary infection (CAUTI) in that setting\u003csup\u003e4,5\u003c/sup\u003e. Up to 20 per cent of all \u003cem\u003eA. baumannii\u003c/em\u003e isolates are obtained from urinary sources\u003csup\u003e6\u003c/sup\u003e and recently it was proposed that secondary urinary tract infection (UTI) after re-catheterization could be caused by an intracellular reservoir of \u003cem\u003eA. baumannii\u003c/em\u003e in bladder epithelial cells in a murine model\u003csup\u003e7\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cem\u003eA. baumannii\u003c/em\u003e has a versatile metabolism that allows it to acquire nutrients, survive and ultimately replicate in a low nutrient environment like the one present in the host during an infection\u003csup\u003e1,3\u003c/sup\u003e. Also, the interaction with the host's environment can trigger \u003cem\u003eA. baumannii\u003c/em\u003e metabolic responses that activate antimicrobial resistance and immunomodulatory effects\u003csup\u003e1,8\u003c/sup\u003e. An example of the latter is the catabolism of the alpha amino acid histidine, that in \u003cem\u003eA. baumannii\u003c/em\u003e is done through the Hut system. The Hut system has an important role in \u003cem\u003eA. baumannii\u003c/em\u003e infections, as it is implicated in multiple metabolic pathways including zinc homeostasis, biofilm formation, and histamine synthesis\u003csup\u003e1,9\u003c/sup\u003e. Then, studying the physiological responses of \u003cem\u003eA. baumannii\u003c/em\u003e caused by exposure to environmental conditions that mimic those of the host, is key to understanding its metabolism during infection and, ultimately, for the development of new methods to control those infections\u003csup\u003e1\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe catabolism of organic acids has been regarded as essential for the virulence and immunomodulation of \u003cem\u003eA. baumannii\u003c/em\u003e\u003csup\u003e1\u003c/sup\u003e. Thus, phenylacetic acid (PAA) metabolism plays a key role in \u003cem\u003eA. baumannii\u003c/em\u003e infection. The PAA catabolic pathway, that is encoded in the \u003cem\u003epaa\u003c/em\u003e operon, is an important route in the catabolism of aromatic compounds that will lately converge in the Krebs cycle\u003csup\u003e10,11\u003c/sup\u003e. \u003cem\u003eA. baumannii\u003c/em\u003e mutations in the \u003cem\u003eppaE\u003c/em\u003e gene resulted in lesser virulence in a murine septicemia model\u003csup\u003e10\u003c/sup\u003e. Conversely, GacS is a global virulence regulator of \u003cem\u003eA. baumannii\u003c/em\u003e, and deletion of its gene leads to high repression of the \u003cem\u003epaa\u003c/em\u003e operon and accumulation of phenylacetate (PA)\u003csup\u003e8\u003c/sup\u003e. In a zebra fish infection model, inhibition of the \u003cem\u003eA. baumannii paa\u003c/em\u003e operon led to migration of polymorphonuclear neutrophiles to the infection site, with the ultimate consequence of a reduction in bacterial burden and attenuated disease\u003csup\u003e8\u003c/sup\u003e. Another set of experiments demonstrated that subinhibitory concentrations of antimicrobials upregulated the \u003cem\u003epaa\u003c/em\u003e operon; as well as interfering with PAA metabolism increased susceptibility to antibiotics and hydroxide peroxide treatment\u003csup\u003e12\u003c/sup\u003e. Also, the blockage of PAA catabolism resulted in attenuated virulence in a murine catheter-associated urinary tract infection (CAUTI) model\u003csup\u003e12\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003ePrevious results indicated that exposure to human pleural fluid (HPF), a fluid with high content of human serum albumin (HSA), can alter the expression of \u003cem\u003eA. baumannii\u003c/em\u003e genes related with survival and persistence\u003csup\u003e13\u003c/sup\u003e and elicit metabolic changes that enhance cytotoxicity and immune evasion\u003csup\u003e14\u003c/sup\u003e and DNA uptake\u003csup\u003e15\u003c/sup\u003e. Moreover, HPF triggers the differential expression of genes related to processes such as antimicrobial resistance, biofilm formation, motility, osmotic stress, and DNA-damage control, thus acting as an adaptative response to environmental stressors\u003csup\u003e16\u003c/sup\u003e. Still, when \u003cem\u003eA. baumannii\u003c/em\u003e is exposed to fluids with low HSA content like cerebrospinal fluid (CSF), global changes in gene expression are triggered including the increase of metabolism and virulence expression factors\u003csup\u003e17\u003c/sup\u003e. In this study, we evaluate the transcriptomic response followed by phenotypic analysis to human urine (HU), a fluid low in HSA content, of two carbapenem-resistant \u003cem\u003eA. baumannii\u003c/em\u003e (CRAB) strains, AB5075\u003csup\u003e18\u003c/sup\u003e and the AMA40\u003csup\u003e19\u003c/sup\u003e, belonging to different genetic lineages and harboring different cabapenemases\u003csup\u003e18,19\u003c/sup\u003e.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eComplete metabolic pathways are modulated by human urine (HU)\u003c/h2\u003e \u003cp\u003eRNA-seq analyses were performed using two carbapenem-resistant \u003cem\u003eA. baumannii\u003c/em\u003e (CRAB) strains, AB5075 and AMA40, belonging to different genetic lineages and possessing different carbapenemases\u003csup\u003e18,19\u003c/sup\u003e, in the presence or absence of human urine (HU). RNA-Seq analysis revealed 264 and 455 differentially expressed genes (DEGs) in AB5075 and AMA40, respectively, upon exposure to HU. In presence of HU, AB5075 had 148 genes upregulated, and 116 genes downregulated. In the case of AMA40 strain, 262 genes were upregulated and 193 were downregulated (Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). In terms of gene ontology, the genes corresponded with a variety of functions, such as catalytic activity and metabolic process (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, B). In addition, it was found that of these DEG, 112 were shared by both strains upon HU exposure. Notably, the majority of up- and downregulated genes fell into metabolic pathways. When comparing the data obtained with HU, we observed different results in the expression of genes compared to those obtained with fluids high in HSA or pure HSA\u003csup\u003e13,14,16,20\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eA key pathway is the tricarboxylic acid (TCA) cycle, which is strongly linked to pathogen virulence and has significant influence on energy production, biosynthesis, and adaptation to the host environment. TCA cycle intermediates function as signalling molecules, orchestrating the regulation of virulence genes, and exerting a pivotal impact on host-pathogen interactions, thereby unveiling potential targets for therapeutic interventions\u003csup\u003e21,22,23, 24\u003c/sup\u003e. In both AB5075 and AMA40, a total of 7 out of 18 TCA genes were found to be upregulated. Additionally, 3 out of 18 and 4 out of 18 downregulated TCA genes were identified in AB5075 and AMA40, respectively. Furthermore, in both strains, the glyoxylate pathway exhibited upregulation, with 2 out of 2 and 1 out of 2 genes being upregulated in AB5075 and AMA40, respectively. Notably, the expression of the \u003cem\u003eaceB\u003c/em\u003e gene was not detected in both strains (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). In addition, genes upregulated in the presence of HU for both \u003cem\u003eA. baumannii\u003c/em\u003e strains AB5075 and AMA40 correspond to other metabolic pathways such as those for benzoate, acetoin, iron uptake, PAA and Phe catabolic pathways, and the catabolism of central aromatic intermediates (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003e and Supplementary Figs. S1-S4). Regarding genes that are downregulated in association with metabolic pathways in the presence of HU for both strains, these include a decrease in DGE in the high-affinity Potassium transporter Kdp operon (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eC), as well as in the cysteine (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eE) and arginine succinyl transferase pathways (Supplementary Fig. S2), along with the taurine and alkanesulfonate transport system pathways (Supplementary Fig. S2). A distinctive difference is observed in the arsenic metabolism where in AMA40 gene expression is clearly downregulated while in AB5075 most genes remain upregulated or slightly downregulated (Supplementary Fig. S2).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFinally, the lipid metabolism was upregulated in both strains under HU condition (Supplementary Fig. S3). These results may indicate that under this condition, which resembles a minimal medium, lipid catabolism is increased. In the following section we describe some of the most relevant pathways affected by the presences of HU in both CRAB studied in this work.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eThe PAA degradation pathway of CRAB strains is induced in HU\u003c/h2\u003e \u003cp\u003eConsidering the relevance of the PAA catabolic pathway in virulence and immune evasion, we decided to assess the expression level of genes involved in the PAA and Phe catabolic pathways in the carbapenem-resistant \u003cem\u003eA. baumannii\u003c/em\u003e strains AB5075 and AMA40. For these, both CRAB strains were cultured in CAMHB with or without supplementation with HU, and the transcriptome analysis showed that the genes encoding enzymes of the PAA and Phe catabolic pathway are induced under HU in both strains AB5075 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA) and AMA40 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Further qRT-PCR experiments confirmed that HU significantly induced the expression of genes of those pathways, such as \u003cem\u003epaaA, paaB, paaE, paaG, paaK, paaZ\u003c/em\u003e and \u003cem\u003efeaB\u003c/em\u003e in the strain AB5075 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Similar qRT-PCR results were observed in the strain AMA40 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD), confirming that PAA degradation pathway was upregulated by the presence of HU in both CRAB strain evaluated.\u003c/p\u003e \u003cp\u003eIt has been shown that if the PAA metabolic pathway is inhibited, the accumulation of metabolic by-products acts as a direct attractant of neutrophils, one of the main immune cells involved in the response to bacterial infections\u003csup\u003e8\u003c/sup\u003e. Therefore, neutrophil chemotaxis assays were performed to evaluate whether HU affects this phenomenon. To this end, both CRAB strains were grown in the presence of PAA, and in the presence or absence of HU. The results obtained were consistent with what was observed at the transcriptional level. Under HU treatment, both strains increased PAA catabolism and attracted significantly fewer neutrophils than the control condition, 2.32-fold and 1.46-fold decrease for AB5075 and AMA40 strains, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eHistidine catabolism gene expression in AB5075 and AMA40 strains is increased under HU condition\u003c/h2\u003e \u003cp\u003eThe catabolism of the alpha amino acid histidine is done through the Hut system in \u003cem\u003eA. baumannii\u003c/em\u003e. As mentioned in the introduction, the Hut system is implicated in multiple metabolic pathways that include biofilm formation, zinc homeostasis, and histamine synthesis. The RNA-seq results showed an up-regulation in the \u003cem\u003ehut\u003c/em\u003e operon for AB5075 strain, while for AMA40 strain, the \u003cem\u003ehutCDGHITU\u003c/em\u003e genes were down-regulated (although not significantly) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Also, for both strains, qRT-PCR assays were carried after being cultured in HU respect to CAMHB. Results indicated that the expression of genes linked to histidine catabolism was enhanced in the presence of HU, for AB5075 and AMA40 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eOther metabolic genes of interest in A. baumannii are modulated by HU\u003c/h2\u003e \u003cp\u003eAs described above in the transcriptomic results, all genes of the benzoate pathway were upregulated when both CRAB strains growth in HU (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). To corroborate this, the expression level of \u003cem\u003ebenA\u003c/em\u003e gene was evaluated by qRT-PCR, resulting in a significant enhanced expression in the presence of HU in both AB5075 and AMA40 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Also, RNA-seq results indicated that the expression of high-affinity K\u003csup\u003e+\u003c/sup\u003e transporter (Kdp) genes was down-regulated in HU conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). In this case, the expression level for \u003cem\u003ekdpA\u003c/em\u003e gene, was evaluated for RT-qPCR and was obtained as a result a 0.24-fold and 0.22-fold decrease for AB5075 and AMA40, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Finally, the cysteine pathway composed of \u003cem\u003ecysDNPTW\u003c/em\u003e genes was completely repressed under HU conditions in both CRAB strains, according to RNA-seq results (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). To confirm this repression, the evaluation of \u003cem\u003ecysT\u003c/em\u003e gene was assessed by RT-qPCR, resulting significantly reduced by 5-fold, in both strains, when grown in the presence of HU (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eF).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eExposure to human urine affects biofilm formation in CRAB\u003c/h2\u003e \u003cp\u003eThe lifestyle changes from planktonic to biofilm, and conversely, are key for environmental adaptation and survival of bacteria. Previous studies in animal model indicated that biofilm formation may play a role by increasing virulence in infections by \u003cem\u003eA. baumannii\u003c/em\u003e\u003csup\u003e25\u003c/sup\u003e. RNA-seq analyses performed in the two carbapenem-resistant \u003cem\u003eA. baumannii\u003c/em\u003e strains AB5075 and AMA40, showed differential gene expression regarding the genes involved in biofilm formation. Analyses of differentially expressed genes showed that for both strains AB5075 and AMA40 in the presence of HU, genes from the \u003cem\u003ecsu locus\u003c/em\u003e were downregulated. However, in the presence of HU, the genes from the \u003cem\u003epga\u003c/em\u003e locus were mostly upregulated in AMA40 while a different pattern can be seen for AB5075 where some genes are slightly downregulated, other slightly downregulated and some show no significant changes (Fig. S4). Biofilm experiments were performed and quantified using crystal violet. As results, a small significant difference was found for the AMA40 strain, while differences were not found in AB5075 between CAMHB and CAMHB\u0026thinsp;+\u0026thinsp;50% HU conditions (Fig. S4). In addition, CFU recovered from biofilm did not show significant differences (Fig. S4). Finally, the transcriptomic analyses revealed that Quorum Quenching (QQ) related genes were upregulated in the presence of HU in both CRAB strains. While most of Quorum Sensing (QS) associated genes were significantly upregulated or slightly downregulated in AB5075 in presence of HU, they were mostly downregulated in AMA40 (Fig. S4).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eCRAB strains can cause CAUTI that are difficult to treat. The ability of \u003cem\u003eA. baumannii\u003c/em\u003e to survive in a low-nutrient environment, such as the one in the host during an infection, could be explained by the bacterium versatile metabolism. In this work, we have described the effects of exposure to human urine (HU) of two genetically different CRAB strains, AB5075 and AMA40. As a result of HU exposure, we established changes to the differential expression of 264 and 455 genes in strains AB5075 and AMA40, respectively. Further, both strains shared 112 DEGs, suggesting common transcriptional responses to HU.\u003c/p\u003e \u003cp\u003eThis common transcriptional response of the studied CRABs to HU exposure includes DEGs in various metabolic pathways. Among these, genes coding for TCA cycle and glyoxylate pathway showed upregulation in both strains, implying potential adaptations in energy production and biosynthesis. In addition, the TCA cycle is linked to the virulence of pathogens through various mechanisms including metabolic adaptation to diverse nutrients allowing biosynthesis and growth, regulation of virulence genes, redox balance, iron acquisition, tissue colonization and immune evasion, and host immune response modulation\u003csup\u003e21,22\u003c/sup\u003e. Another response to HU in both strains tested, is the downregulation of the transcription of genes present in the \u003cem\u003ekdp\u003c/em\u003e operon, which encodes the high-affinity Potassium transporter Kdp. In \u003cem\u003eE. coli\u003c/em\u003e, the Kdp transporter is encoded by the \u003cem\u003ekdpABC\u003c/em\u003e operon, and its expression is regulated by the products of \u003cem\u003ekdpD\u003c/em\u003e and \u003cem\u003ekdpE\u003c/em\u003e. Previous observations indicate that the expression of Kdp is affected by the concentration of K\u003csup\u003e+\u003c/sup\u003e in the medium, resulting in a repression of the operon when the K\u003csup\u003e+\u003c/sup\u003e concentration in the medium is high\u003csup\u003e26\u003c/sup\u003e. A further study confirmed that in \u003cem\u003eA. baumannii\u003c/em\u003e the transcription of the five components of the Kdp system is linked, and that \u003cem\u003ekdpE\u003c/em\u003e is notably upregulated under K\u003csup\u003e+\u003c/sup\u003e limiting conditions, as well as an important factor for pathogenicity in a murine pneumonia model\u003csup\u003e27\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAlso, the PAA degradation pathway was induced under HU conditions in both strains, as evidenced by upregulation of genes in the PAA and Phe catabolic pathways. Accordingly, in neutrophil assays, the exposure to HU attracted significantly fewer neutrophils in both CRAB analyzed. These results are consistent with previous observations indicating that PAA metabolism in \u003cem\u003eA. baumannii\u003c/em\u003e affects infection outcomes by directly influencing neutrophil chemotaxis\u003csup\u003e8\u003c/sup\u003e. Hence, we can conclude that HU is an environmental signal that affects \u003cem\u003eA. baumannii\u003c/em\u003e metabolism, providing favorable conditions for this pathogen to evade the host immune system. Finally, lipid metabolism was upregulated in both strains under HU conditions, suggesting increased lipid catabolism in a minimal medium-like environment.\u003c/p\u003e \u003cp\u003eIt was established that up to 20% of \u003cem\u003eA. baumannii\u003c/em\u003e clinical isolates are obtained from urinary sources\u003csup\u003e6\u003c/sup\u003e, and CRAB are considered a critical priority for the research and development of new antimicrobials\u003csup\u003e2\u003c/sup\u003e. The findings in this study contribute to a better understanding of metabolic changes undergone by CRAB when exposed to HU. The human fluids that \u003cem\u003eA. baumannii\u003c/em\u003e may encounter while infecting its host, possess different compositions regarding proteins, metabolites and other solutes. In this sense, a characteristic that distinguishes HU from other fluids is its low content of human serum albumin (HSA).\u003c/p\u003e \u003cp\u003eIt was previously reported that \u003cem\u003eA. baumannii\u003c/em\u003e responds to components of human fluids by modifying its transcriptional and phenotypic profiles\u003csup\u003e16,17,20\u003c/sup\u003e. HSA and human pleural fluid (HPF) can modulate the expression of genes associated with iron uptake systems, biofilm formation, antibiotic resistance, DNA acquisition and metabolism. Previous results indicated that most genes of the \u003cem\u003epaa\u003c/em\u003e locus were downregulated when exposed to HPF\u003csup\u003e14\u003c/sup\u003e, while as showed in this study \u003cem\u003epaa\u003c/em\u003e locus is upregulated in presence of HU in both CRAB strains, thus suggesting a differential role of HSA in the regulation of the PAA metabolic pathway. In a similar direction, on \u003cem\u003eA. baumannii\u003c/em\u003e strain A118 cultured with HSA led to a downregulation of many genes of the \u003cem\u003epaa\u003c/em\u003e locus with the repressor \u003cem\u003epaaX\u003c/em\u003e being upregulated\u003csup\u003e13\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eHSA appears to act as a host-derived signal, inducing adaptive mechanisms that enhance virulence-associated gene expression\u003csup\u003e49\u003c/sup\u003e. Studies on other bacteria like \u003cem\u003eBordetella pertussis\u003c/em\u003e and \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e have shown that purified HSA produces outcomes similar to those of serum, and that removing albumin through membrane filtration decreases the serum-mediated effects. This suggests that HSA is the main component responsible for these phenotypes\u003csup\u003e28,49\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAdditionally, previous research has demonstrated that the effectiveness of cefiderocol, a novel chlorocatechol-substituted siderophore antibiotic used to treat cUTI, is reduced when exposed to human fluids containing HSA, such as Human Serum (HS) and HPF\u003csup\u003e30,31\u003c/sup\u003e. Ferric siderophore transporters facilitate the uptake of cefiderocol into bacterial cells, and the presence of HSA or HSA-containing fluids is associated with a decrease in the expression of genes linked to high-affinity siderophore-mediated iron uptake systems\u003csup\u003e30\u003c/sup\u003e. In contrast, studies have shown that exposure to HU, a fluid with little to no HSA or free-iron content, did not significantly alter the minimum inhibitory concentration (MIC) values for CRABs under the conditions tested\u003csup\u003e32\u003c/sup\u003e. Additionally, genes involved in iron uptake were upregulated. These results support the hypothesis that an unknown mechanism triggers a regulatory response in \u003cem\u003eA. baumannii\u003c/em\u003e when exposed to human fluids, enabling it to thrive in different environments. Thus, the HSA content in the fluid may play a crucial role in eliciting a differential adaptive response.\u003c/p\u003e \u003cp\u003eIn conclusion, this report shows that exposure to HU induces widespread changes in the transcriptome of CRAB strains, impacting various metabolic pathways, as well as genes coding for antibiotic resistance, biofilm formation, and quorum sensing functions. These findings contribute to a better understanding of the adaptive responses of CRAB strains to urinary environments, providing insights that may guide future therapeutic interventions and infection control methods.\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cp\u003e \u003cb\u003eBacterial strains\u003c/b\u003e. Two CRAB strains were used in the present study: the multidrug and hypervirulent AB5075 (OXA-23) strain and AMA40 (NDM-1) (REF).\u003c/p\u003e \u003cp\u003e \u003cb\u003eRNA extraction.\u003c/b\u003e \u003cem\u003eA. baumannii\u003c/em\u003e AB5075 and AMA40 were cultured in CAMHB and CAMHB supplement with 50% human urine (HU) from healthy individuals obtained from a certified vendor (Innovative Research Inc., MI, USA) and incubated with agitation for 18 h at 37\u0026deg;C. Then, overnight cultures were then diluted 1:10 in fresh Cation Adjusted Mueller-Hinton Broth (CAMHB), supplemented with HU, and incubated at 37\u0026deg;C with agitation during 7 h. RNA extraction was carried out with Direct-zol RNA Kit (Zymo Research, Irvine, CA, USA), in triplicate. The RNA samples obtained were subjected to DNase treatment (Thermo Fisher Scientific, Waltham, MA, USA) following manufacturer\u0026rsquo;s instruction, afterwards a PCR amplification of the 16S rDNA gene was performed to confirm there was no DNA contamination. Then, from three independent replicates per sample, ribosomal RNA-depletion was done using the Ribo-Zero kit (Illumina) followed by the construction of the cDNA library with the TruSeq Stranded Total RNA Library Prep kit (Illumina). The RNA sequencing was outsourced to Novogene (Novogene Corporation, Sacramento, CA, USA).\u003c/p\u003e \u003cp\u003e \u003cb\u003eRNA-seq analysis\u003c/b\u003e. The quality control of the Illumina reads, trimming of low-quality bases and removal of Illumina adapters was performed as described previously (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). Reads were aligned to the genome of \u003cem\u003eA. baumannii\u003c/em\u003e AB5075 or AMA40, using Burrows-Wheeler Alignment (BWA) software (v0.7.17) BWA and visualized using the Integrative Genomics Viewer (IGV). Read counts per gene were calculated using FeatureCounts (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). Differential expression analysis was performed using DEseq2 and the Differentially Expressed Genes (DEGs) were defined as those displaying an FDR adjusted \u003cem\u003eP\u003c/em\u003e value of \u0026lt;\u0026thinsp;0.05 and log2 fold change\u0026thinsp;\u0026gt;\u0026thinsp;1.\u003c/p\u003e \u003cp\u003eIn addition, to represent metabolic pathways in our RNA sequencing data, the Omics Dashboard Tool provided by both BioCyc\u003csup\u003e33\u003c/sup\u003e and MetaCyc\u003csup\u003e34\u003c/sup\u003e was employed. Enrichment or depletion of metabolic pathways was assessed utilizing the Fisher's exact test hypothesis, with significance determined at \u003cem\u003eP\u003c/em\u003e values below 0.05. Enrichment or depletion scores (represented as -log\u003csub\u003e10\u003c/sub\u003e \u003cem\u003eP\u003c/em\u003e values) for each pathway within the dashboard were subsequently acquired through download and analyzed. The RNA-seq data generated in the current study are available in the NCBI repository with the GEO accession No GSE201259.\u003c/p\u003e \u003cp\u003e \u003cb\u003eqRT-PCR assays\u003c/b\u003e. The cDNA was prepared using the iScript Reverse Transcription Supermix for qRT-PCR (BioRad, Hercules, CA, USA) and the quantitative PCR was performed using iQ\u0026trade;SYBR Green Supermix (BioRad, Hercules, CA, USA), in both cases following the recommendation of the manufacturer. Different primers to confirm RNA-seq results and also study the expression of genes associated with virulence and antimicrobial resistance were used (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Experiments were performed in technical and biological triplicates. The results were analyzed using the qBASE method\u003csup\u003e35\u003c/sup\u003e with \u003cem\u003erecA\u003c/em\u003e and \u003cem\u003erpoB\u003c/em\u003e genes as normalizers\u003csup\u003e36,37\u003c/sup\u003e. Data are presented as NRQ (normalized relative quantities). Differences were determined by two-way ANOVA followed by Tukey\u0026rsquo;s multiple comparison test (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) using GraphPad Prism (GraphPad software version 10.0.0, San Diego, CA, USA).\u003c/p\u003e \u003cp\u003e \u003cb\u003eNeutrophil Chemotaxis Assay\u003c/b\u003e. For this assay, a previously published protocol with modifications was used\u003csup\u003e14\u003c/sup\u003e. AB5075 or AMA40 cultured overnight in CAMHB supplement with 50% human urine with or without 0.02% phenylacetic acid (PAA) were tested (Millipore) (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Controls for this assay constituted of CAMHB and CAMHB\u0026thinsp;+\u0026thinsp;0.02% PA with performance in parallel. First, 100 \u0026micro;l an overnight culture of \u003cem\u003eA. baumannii\u003c/em\u003e AB5075 or AMA40 in the tested conditions were combined with 100 \u0026micro;l of chemotaxis buffer (CF). Then, each mix was transferred to a twenty-four-well Olympus polycarbonate tissue culture plate with 8-\u0026micro;m pore size membranes (Genesee Scientific). Prior incubation 100 \u0026micro;l of neutrophils (10 x 10\u003csup\u003e6\u003c/sup\u003e cells/ml) from iQ Biosciences was added to the semipermeable well inserts. Then, plates were incubated for 1 hour in 5% CO\u003csub\u003e2\u003c/sub\u003e at 37\u0026deg;C. Neutrophils were then counted from each sample tested. Migration chemotaxis index was calculated by the number of neutrophils in the test well by number of neutrophils in control. All conditions were performed in triplicates. The chemotaxis buffer (CF) has the following components: 25 ml of Roswell Park Memorial Institute Medium (Thermo Fisher), 10% fetal calf serum (FCS) (Thermo Fisher), 500 \u0026micro;l of 100 U/ml penicillin-streptomycin (Sigma Aldrich), and 22 ml of Hanks\u0026rsquo; balanced salt solution (HBSS) (Thermo Fisher).\u003c/p\u003e \u003cp\u003e \u003cb\u003eBiofilm assay.\u003c/b\u003e First, \u003cem\u003eA. baumannii\u003c/em\u003e AB5075 and AMA40 strains were cultured in fresh CAMHB medium, or CAMHB supplemented with 50% HU in static conditions at 37 \u0026ordm;C for 18 h. Then, tubes were emptied, washed three times with 1X phosphate-buffered saline (PBS) and stained with 1% crystal violet (CV) for 15 min. Excess CV was removed by washing three more times with 1X PBS. The biofilm assays were performed in triplicate (absorbance determination at 580 and 660 nm as well CFU/ml), with at least three technical replicates per biological replicate (REF).\u003c/p\u003e \u003cp\u003e \u003cb\u003eStatistical analysis.\u003c/b\u003e Experiments performed at least in triplicates were statistically analyzed by one- or two-way ANOVA followed by Tukey\u0026rsquo;s multiple comparison tests using GraphPad Prism (GraphPad software, San Diego, CA, USA). A \u003cem\u003eP value\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered significant.\u003c/p\u003e \u003cp\u003eAll procedures performed in this study were in accordance with the CSUF Institutional Biosafety Committee Approval plan (DBH117-01) and follow the NIH, CDC, OSHA and other environmental and occupational regulations.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated and analyzed during the current study are available in the Gene Expression Omnibus (GEO) repository (GEO accession No GSE201259).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u003c/strong\u003e G.M.T, L.A., R.S., M.E.T, R.A.B, and M.S.R. conceived the study and designed the experiments. J.E., M.H., B.N., M.M., C.P., T.S., M.R.T, R.S., G.M.T., and M.S.R performed the experiments and genomics and bioinformatics analyses. M.R.T., T.S., R.S., C.D.S, L.A., and M.S.R. analyzed the data and interpreted the results. M.E.T., G.M.T., and M.S.R. contributed reagents/materials/analysis tools. C.D.S., G.M.T, L.A., R.S., M.E.T, R.A.B, and M.S.R. wrote and revised the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements:\u003c/strong\u003e The authors' work was supported by NIH SC3GM125556 to M.S.R., R01AI100560, R01AI063517, R01AI072219 to R.A.B., and 2R15 AI047115 to M.E.T. This study was supported in part by funds and facilities provided by the Cleveland Department of Veterans Affairs, Award Number 1I01BX001974 to R.A.B from the Biomedical Laboratory Research \u0026amp; Development Service of the VA Office of Research and Development and the Geriatric Research Education and Clinical Center VISN 10 to R.A.B. \u0026nbsp;J.E. was supported by grant MHRT 2T37MD001368 from the National Institute on Minority Health and Health Disparities, National Institute of Health. The content is solely the authors' responsibility and does not necessarily represent the official views of the National Institutes of Health or the Department of Veterans. M.R.T., T.S., and R.S are members of the CONICET Research carreer.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest:\u003c/strong\u003e The authors declare no conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eRen, X. \u0026amp; Palmer, L. 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Genome Biol 8, R19 (2007).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMezcord, V. \u003cem\u003eet al.\u003c/em\u003e Induced Heteroresistance in Carbapenem-Resistant \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e (CRAB) via Exposure to Human Pleural Fluid (HPF) and Its Impact on Cefiderocol Susceptibility. IJMS 24, 11752 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEscalante, J. \u003cem\u003eet al.\u003c/em\u003e Human serum albumin (HSA) regulates the expression of histone-like nucleoid structure protein (H-NS) in \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e. Sci Rep 12, 14644 (2022).\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":"Acinetobacter baumannii, human urine, cefiderocol, human serum albumin, carbapenem-resistance","lastPublishedDoi":"10.21203/rs.3.rs-4415275/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4415275/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCarbapenem-resistant \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e (CRAB) is a major human pathogen and a research priority for developing new antimicrobial agents. CRAB is a causative agent of a variety of infections in different body sites. One of the manifestations is catheter-associated urinary tract infection, which exposes the bacteria to the host's urine, creating a particular environment. Exposure of two CRAB clinical isolates, AB5075 and AMA40, to human urine (HU) resulted in the differential expression levels of 264 and 455 genes, respectively, of which 112 were common to both strains. Genes within this group play roles in metabolic pathways such as phenylacetic acid (PAA) catabolism, the Hut system, the tricarboxylic acid (TCA) cycle, and other processes like quorum sensing and biofilm formation. These results indicate that the presence of HU induces numerous adaptive changes in gene expression of the infecting bacteria. These modifications presumably help bacteria establish and thrive in the hostile conditions in the urinary tract. These analyses advance our understanding of CRAB's metabolic adaptations to human fluids, as well as expanding knowledge on bacterial responses to distinct human fluids containing different concentrations of human serum albumin (HSA).\u003c/p\u003e","manuscriptTitle":"Carbapenem-resistant Acinetobacter baumannii (CRAB): metabolic adaptation and transcriptional response to human urine (HU)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-30 22:01:09","doi":"10.21203/rs.3.rs-4415275/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-07-09T06:02:19+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-08T20:09:27+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-06-01T23:38:33+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"30113748125142097676062155279164502192","date":"2024-05-23T12:21:18+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"301965603516349118279965884417385475900","date":"2024-05-23T12:08:56+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-05-23T07:49:51+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-05-23T07:49:01+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-05-19T10:04:50+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-05-15T10:30:15+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-05-13T20:58:13+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":"52c3087e-9a1d-40a6-89f7-7a4a4a9f1140","owner":[],"postedDate":"May 30th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":32378582,"name":"Biological sciences/Microbiology/Microbial genetics/Bacterial genetics"},{"id":32378583,"name":"Biological sciences/Microbiology"}],"tags":[],"updatedAt":"2024-08-26T16:09:13+00:00","versionOfRecord":{"articleIdentity":"rs-4415275","link":"https://doi.org/10.1038/s41598-024-70216-w","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2024-08-19 15:57:40","publishedOnDateReadable":"August 19th, 2024"},"versionCreatedAt":"2024-05-30 22:01:09","video":"","vorDoi":"10.1038/s41598-024-70216-w","vorDoiUrl":"https://doi.org/10.1038/s41598-024-70216-w","workflowStages":[]},"version":"v1","identity":"rs-4415275","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4415275","identity":"rs-4415275","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}