Impact of prolonged fructose and fructooligosaccharides stress on the adaptive growth, antimicrobial susceptibility, and genomic mutation of Klebsiella pneumoniae

Impact of prolonged fructose and fructooligosaccharides stress on the adaptive growth, antimicrobial susceptibility, and genomic mutation of Klebsiella pneumoniae | 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 Research Article Impact of prolonged fructose and fructooligosaccharides stress on the adaptive growth, antimicrobial susceptibility, and genomic mutation of Klebsiella pneumoniae Yunhui HE, Bo XIONG, Fanlu ZOU, Kewei FAN, Jintuan LIN, Yong XIANG, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9145216/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Klebsiella pneumoniae ( K.pneumoniae ) can make use of various carbohydrates (glucose, fructose, sucrose et al ) as nutrient sources for sustaining its cononization and adaptive growth in human body. Fructose and fructooligosaccharides (FOS) are commonly used as dietary supplements and can be metabolized by gut probiotics, thereby promoting intestinal health and nutrient absorption, whereas the impact of prolonged exposure with fructose or FOS on the adaptive growth and antimicrobial susceptibility of K.pneumoniae remains unclear. Here, fructose- or FOS-induced K.pneumoniae strains were selected from the parental isolates K2044, EKP108 and EKP19. Our data indicated that two FOS-induced strains (K2044-0.5FOS-90G and EKP108-0.5FOS-90G) exhibited the best planktonic growth under different concentrations of fructose or FOS, suggesting the reshaping possibility of the growth adaption of K.pneumonia under fructose or FOS pressure. Interestingly, compared with the media with varying concentrations of fructose of FOS, the planktonic growth of fructose- or FOS-induced K.pneumoniae strains was significantly decreased with the culture media supplemented with low concentration of glucose (1%-2%), whereas high concentrations of glucose (8%,16%) could reactivate their adaptive growth. Moreover, K2044 knockout strain (K2044-Δ envZ mutant) showed the enhanced adaptive growth compared to K2044 WT under different concentrations of fructose, FOS or glucose, whereas a significantly prolonged lag phase was observed for K2044-Δ envZ with sucrose exposure, compared to the control. Notably, EKP108-0.5FOS-90G showed decreased resistance to levofloxacin (LEV), whereas the K2044-Δ envZ mutant displayed increased resistance to gentamicin (GEN) and tigecycline (TGC). Both EKP108-0.5FOS-90G and K2044-Δ envZ demonstrated increased outer membrane permeability and distinct alterations in membrane phospholipid composition. The genetic mutation between the parental strain EKP108 and its FOS-induced derivative (EKP108-0.5FOS-90G) were determined and we identified mutations in genes encoding members of the carbohydrate kinase family, which is primarily associated with carbohydrate metabolism and transport. Collectively, the growth adaptation, antimicrobial susceptibility changes, and membrane remodeling of K. pneumoniae following fructose or FOS exposure might be isolate-specific. Furthermore, isolates exposed to high carbohydrate concentrations do not necessarily exhibit enhanced growth adaptability. Moreover, knockout of the envZ gene may also promote the adaptive growth of K. pneumoniae , and involve membrane remodeling and impact the antimicrobial susceptibility. Klebsiella pneumoniae Adaptive growth Fructose Fructooligosaccharides Biofilm envZ Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 INTRODUCTION Klebsiella pneumoniae ( K.pneumoniae ) belongs to the family Enterobacteriaceae , which mainly colonizes the nasopharynx and gastrointestinal tract of humans and primates, and is prone to developing resistance to multiple antibiotics [ 1 ] . K.pneumoniae is classified into classical K. pneumoniae (cKP) and hypervirulent K. pneumoniae (hvKP) on the basis of virulence and mucoviscosity phenotype [ 2 ] . HvKp strains exhibit enhanced mucosal adherence, higher virulence compared to cKP, and are associated with more severe infections [ 3 , 4 ] . An important virulence factor contributing to K. pneumoniae pathogenicity is its ability to form a biofilm, which enhances tolerance to environmental stressors and antimicrobial agents [ 5 ] . K. pneumoniae is capable of utilizing a broad range of sugars as carbon sources. Fructose, a monosaccharide and isomer of glucose, is commonly used in the food industry as a sweetener and nutritional additive [ 6 , 7 ] ( Fig. 1 , A ) . Fructooligosaccharides (FOS), contain a variable number of β-D-fructofuranosyl units with one glucosyl unit, is a naturally occurring carbohydrate found in various plants ( Fig. 1 , B ) , which are indigestible by humans but are metabolized by gut probiotics, thereby promoting intestinal health [ 8 ] . There are three main transport pathways for fructose utilization in bacteria: Pathway A : transport via the phosphoenolpyruvate-dependent phosphotransferase system (PTS), specifically through the transmembrane enzymes EII^Fru (encoded by fruA and fruB ), leading to intracellular phosphorylation to fructose-1,6-bisphosphate; Pathway B : the mannose transport system (MTS), which is encoded by the manXYZ gene; Pathway C : this pathway does not involve the carbohydrate PTS transport system, but requires relatively high concentrations of fructose to function [ 9 , 10 ] . FOS uptake is mediated by several transport systems, including the ABC transporter system, found mainly in Lactobacillus ; the PTS transporter system, found mainly in Bacillus and Lactococcus ; and the permease system, found mainly in Bifidobacterium [ 11 , 12 ] . Biofilm formation is a key mechanism contributing to the drug resistance of K. pneumoniae . Biofilms are structured communities of bacteria, often composed of one or more species, embedded in a self-produced extracellular matrix consisting of proteins, polysaccharides, and nucleic acids derived from both the bacteria and the host, and the bacteria in the biofilm are protected from host immune defenses and antimicrobial agents [ 5 ] . The outer membrane of K.pneumoniae is composed of phospholipids, lipopolysaccharides, and proteinsthat collectively act as a barrier to antibiotic entry. In drug-resistant strains, the reduced expression or absence of outer membrane porins can significantly alter membrane permeability, thereby limiting antibiotic uptake and diminishing therapeutic efficacy [ 13 ] . The two-component regulatory system (TCS) is the most important mechanism for regulating physiological behavior of bacteria, and plays an important role in bacterial pathogenicity, antibiotic resistance, virulence factor regulation, and environmental adaptation [ 14 , 15 ] . The envZ gene encodes a histidine protein kinase (HK) in the two-component system EnvZ/OmpR, which plays a key role in sensing environmental signals and regulating bacterial adaptive responses [ 16 ] . The EnvZ protein kinase is located in the bacterial cell membrane, and when the bacterial surroundings are altered, EnvZ activates the expression of specific genes by phosphorylating the OmpR protein, thus enabling the bacteria to adapt to these unfavorable conditions [ 17 , 18 ] . In addition, mutations in envZ have been shown to influence both bacterial virulence and antibiotic resistance [ 18 – 20 ] . Previous research has primarily focused on the types of carbohydrates available to K.pneumoniae , with little attention paid to how different concentrations of a single carbohydrate—especially under sustained exposure—affect bacterial adaptation. This study aims to explore the adaptive growth, antimicrobial susceptibility, biofilm formation, and membrane phospholipid composition of K.pneumoniae under fructose and FOS pressure. Additionally, we investigated genomic mutations induced by long-term FOS exposure. To further examine the regulatory role of envZ , we analyzed the adaptive growth of K. pneumoniae strains lacking envZ under fructose and FOS stress, as well as the phenotypic changes associated with envZ deletion. These findings may provide new insights for understanding bacterial adaptation and improving clinical strategies against K. pneumoniae -associated infections. MATERIALS AND METHODS Bacterial strains and growth conditions In this study, two clinical isolates from inpatients of Shenzhen Nanshan People’s Hospital, China, and one strain of NTUH-K2044 (Hereinafter referred to as K2044) were collected. K.pneumoniae was grown in Luria-Bertani medium at 37 °C with shaking unless otherwise stated. For antimicrobial susceptibility testing, strains were grown in cation-adjusted Mueller-Hinton broth (CAMHB) at 37 °C with shaking. K.pneumoniae K2044, EKP19 and EKP108 were cultured in vitro under control conditions (without sugar), as well as in medium supplemented with fructose or FOS at concentrations of 0.5% and 8%, resepectively and serially passaged for 90 generations to obtain 3 subpassaged strains (K2044-W90, EKP19-W90, EKP108-W90) and 12 induced strains (K2044-0.5frut-90G, K2044-8frut-90G, K2044-0.5FOS-90G and K2044-8FOS-90G, same for EKP19 and EKP108). Similar to K2044, two clinical K.pneumoniae isolates (EKP19 and EKP108) were also used to establish the induced isolates with De Man, Rogosa and Sharp control media (MRS, purchased from Topbiol, Shangdong, China, consists of peptone (10.0g/L), beef extract (8.0g/L), yeast extract (4.0g/L), tween 80 (1.0g/L), sodium acetate·H 2 O (5.0g/L), K 2 HPO 4 ·7H 2 O (2.0g/L), ammonium citrate tribasic (2.0g/L), MgSO 4 ·7H 2 O (0.2g/L) and MnSO 4 ·4H 2 O (0.05g/L) for non-supplemented carbon conditions), and the media with concentrations of 0.5% and 8% fructose or FOS. All strains used in this work are listed in Table S1 . Antimicrobials and reagents Ceftazidime (MB1334, 94%-105%), gentamicin (MB1331, ≥590IU/mg), levofloxacin (MB1576, ＞98.5%), meropenem (MB1129, ＞98%), and tigecycline (MB1246, ＞99%) were purchased from MeilunBio, Dalian, China. Glucose and sucrose were purchased from Biosharp, Beijing, China. D-fructose and N-Phenyl-1-naphthylamine (NPN) were purchased from aladdin, Shanghai, China. Fructooligosaccharides was purchased from macklin, Shanghai, China. DL5000 DNA marker, bacterial DNA extraction kits, and 2X taq plus master mix II were purchased from Vanzyme, Nanjing, China. Growth curves Overnight cultures of K. pneumoniae was diluted 1:200 in fresh MRS (without sugar), combined with 1%, 2%, 4%, 8%, 16%, 32% fructose or FOS, and inoculated in Honeycomb plates (100 wells, 200 μL/well); MRS without sugar was used as untreated control. OD600 was determined by a Bioscreen C system (Lab Systems Helsinki Finland). The experiment was recorded for 24h. Each assay was performed in triplicate at least three times. Antimicrobial susceptibility test ing Minimal inhibit concentrations (MICs) were determined by the broth macrodilution method in CAMHB according to Clinical and Laboratory Standards Institute guidelines (CLSI-M100-S34). The range of concentrations tested for antimicrobials was 0.25–256 μg/mL. No antimicrobial was added as control and E.coli ACTCC 25922 was the quality control strain. Antimicrobial susceptibility results were confirmed based on CLSI-M100-S34 (Table S2) . All experiments were performed in triplicate. Biofilm biomass determination Overnight cultures of K. pneumoniae was diluted 1:100 in Luria-Bertani broth, combined with 2% glucose, and inoculated in 96-well polystyrene microtiter plates (200 μL/well). After incubation at 37℃ for 24h, the bacterial solution was aspirated, washed with PBS buffer PH 7.4 for 3 times, dried and fixed with 95% methanol for 15min. Methanol was removed and then stained with 1% crystal violet for 10min. Crystal violet was dissolved in 95% ethanol and OD570 was determined. This experiment was performed in triplicate at least three times. Determination of bacterial outer membrane permeability K.pneumoniae grown to logarithmic phase was resuspended in PBS buffer and diluted to 0.5 McFarland turbidity. N-phenyl-1-naphthaline (NPN) fluorescent dye was added to the bacterial suspension, making a final concentration of NPN of 10 μM, and incubated for 30 min. 420 nm fluorescence was recorded over time (excitation wavelength of 350 nm) using high-content analysis until no further change in fluorescence was observed. Construction of knockout strains Firstly, using high-fidelity DNA polymerase and the relevant primer sequences listed in Table S3 , amplify the upstream and downstream homologous arms of the envZ gene from the K.pneumoniae (NUTH-K2044) genome. Amplify the gentamicin (GEN) resistance gene from the pJQ200SK plasmid. The upstream and downstream homologous arms of the envZ gene were connected to the GEN resistance gene via fusion PCR, yielding the fusion fragment Δ envZ ::GEN (upstream homologous arm-GEN resistance gene-downstream homologous arm). This was then cloned into the pCVD442 plasmid to obtain the knockout vector pCVD442-Δ envZ ::GEN. Transform pCVD442-Δ envZ ::GEN into E.coli β2155 to obtain the donor strain β2155/pCVD442-Δ envZ ::GEN. Conjugation experiment was performed between the β2155/pCVD442-Δ envZ ::GEN and K2044. A K2044 monoclonal strain exhibiting GEN resistance was screened on GEN plates and designated K2044/pCVD442-Δ envZ ::GEN. Select several K2044/pCVD442-Δ envZ ::GEN monoclonal colonies and individually inoculate them onto LB plates containing 10% sucrose for cultivation. Screen these clones using PCR technology to obtain K2044-Δ envZ . Membrane phospholipid extraction and quantification K2044-Δ envZ and EKP108-0.5FOS-90G were selected for membrane phospholipid analysis, with K2044 WT and EKP108 WT serving as respective controls. Bacterial cultures were grown in MRS broth with shaking until the logarithmic growth phase was reached. Cells were harvested by centrifugation at 4,500 rpm for 15 minutes, the supernatant was discarded, and the bacterial pellets were flash-frozen at -80℃ for 10 minutes. For lipid extraction, 9.5 mL of a mixture consisting of chloroform: methanol: 0.3% NaCl (v/v/v = 1:2:0.8) was added to each sample. The mixture was incubated in a water bath at 80℃ for 15 minutes and then shaken at 220 rpm for 1 hour. After centrifugation (15 minutes), the supernatant was transferred to a new centrifuge tube. 5 mL each of chloroform and 0.3% NaCl were added to the supernatant and mixed gently. Following another centrifugation step (15 minutes), the upper aqueous phase was discarded. A 4.5 mL aliquot of the lower organic phase was collected and evaporated to dryness under a stream of nitrogen gas.The dried lipid extract was redissolved in 3 mL of methanol and filtered through a 0.22 μm membrane filter. The prepared samples were subjected to membrane phospholipid quantification using liquid chromatography–tandem mass spectrometry (LC-MS/MS). Whole genome sequencing Genomic DNA was extracted from EKP108-0.5FOS-90G and EKP108 WT with a bacterial DNA extraction kits, respectively. Whole genomes were sequenced in an Illumina HiSeq2500 sequencer. Genomic alignments were performed with MUMmer4 tools. Single nucleotide polymorphisms (SNPs), insertions, deletions, and structural variation annotations were identified based on inter-sample alignment results with BLAST, TRF and Repeatmask. Graphing and statistical analysis Statistics, data analysis, and graphing were performed using GraphPad Prism 8.0 software (La Jolla, CA). Data were processed using multiple comparisons of one-way ANOVA and two-way ANOVA. RESULTS The adaptive growth of K. pneumoniae with fructose pressure. Here, MRS (without sugar)-induced K. pneumoniae (K2044-W90, EK19-W90, EK108-W90), fructose-induced K. pneumoniae (K2044-0.5frut-90G, K2044-8frut-90G, EK19-0.5frut-90G, EK19-8frut-90G, EK108-0.5frut-90G, EK108-8frut-90G) and FOS-induced K. pneumoniae (K2044- 0.5FOS-90G, K2044-8FOS-90G, EK19-0.5FOS-90G, EK19-8FOS-90G, EK108-0.5FOS-90G, EK108-8FOS-90G) were selected and identified firstly. K2044-∆ envZ was constructed and identified with envZ gene knockout. The planktonic growth of K2044 and its respective derivatives (K2044-W90, K2044-0.5frut-90G, K2044-8frut-90G, K2044-0.5FOS-90G, K2044-8FOS-90G) was evaluated in media supplemented with various concentrations (1%, 2%, 4%, 8%, 16%, and 32%) of fructose, respectively. Overall, most effectively planktonic growth of fructose- or FOS-induced strains were found in media with 4% or 8% of fructose, whereas 16% of fructose significantly downregulated the K.pneumoniae growth at the logarithmic period compared with that with concentrations≤8%. In media with 32% of fructose, K2044 and its respective derived strains (K2044-W90, K2044-0.5frut-90G, K2044-8frut-90G, K2044-8FOS-90G) lost their ability of planktonic growth (Figure 2, G; Figure S2, A,B,H) , whereas K2044-0.5FOS-90G showed a slight proliferative ability after incubating for over 18 hours (Figure S2, G). We also compared the planktonic growth of K2044 and its derivatives under each concentration of fructose. K2044-W90 and K2044-0.5FOS-90G showed the better adaptive growth than other derivatives at each concentration, whereas K2044-8frut-90G and K2044 WT demonstrated the lowest planktonic growth (Figure 2) , indicating that continuous induction with 0.5% FOS might rapidly promote the adaptive growth of K2044 under fructose exposure, in contrast, high concentration (8%) of fructose exposure exerted a minimal effect on reshaping the adaptive growth of K2044. In order to avoid the isolate-spectific characteristic of the K2044 adaptation under fructose or FOS pressure, two additional K. pneumoniae clinical isolates, EKP19 and EKP108, along with their respective derivatives, were selected to investigate the effects of fructose and FOS exposure on the planktonic growth. Similar to K2044, the planktonic growth of EKP19 and its respective derived strains (EKP19-W90, EKP19-0.5frut-90G, EKP19-8frut-90G, EKP19-0.5FOS-90G, EKP19-8FOS-90G) was evaluated in media supplemented with various concentrations (1%, 2%, 4%, 8%, 16%, and 32%) of fructose, respectively. We found that the planktonic growth of EKP19 and EKP108, and their respective derived strains was also completely inhibited at 32% concentrations of fructose (Figure S2, C-F, I-L) . The similar planktonic growth ability was found between the EKP19 and its derivatives in media supplemented with various concentrations, suggesting that the high or low concentration of fructose or FOS exposure seemed not to affect the adaptive growth of EK19 derivatives (Figure 3) . Notably, comparison of planktonic growth among EKP108 and its derivatives (EKP108-W90, EKP108-0.5Frut-90G, EKP108-8Frut-90G, EKP108-0.5FOS-90G, and EKP108-8FOS-90G) revealed the markedly enhanced adaptive growth of EKP108-0.5FOS-90G under fructose pressure. however, the adaptive growth of other derivatives (EKP108-W90, EKP108-0.5Frut-90G, EKP108-8Frut-90G, EKP108-0.5FOS-90G) displayed similar tendency without impovement (Figure 4) , suggesting that sustained 0.5% FOS exposure reshaped the adaptive growth capacity of EKP108 under fructose pressure. The envZ gene belongs to the EnvZ/OmpR two-component system and participates in sensing environmental signals and regulating bacterial adaptive responses in K.pneumoniae [16] . Compared with wild-type NTUH-K2044 (K2044 WT), K2044-Δ envZ exhibited significantly enhanced adaptive growth under fructose exposure across all tested concentrations, indicating that deletion of the envZ gene might facilitate K.pnumoniae to make use of fructose for planktonic growth. (Figure 2, H-I) . The adaptive growth of K. pneumoniae with FOS pressure. The planktonic growth of K2044 and its respective derivatives was also determined in media supplemented with various concentrations (1%, 2%, 4%, 8%, 16%, and 32%) of FOS, respectively. We found that 4% or 8% of FOS most effectively promoted the planktonic growth of fructose or FOS-induced strains. However, unlike under fructose stress, the adaptive growth of K2044 derivatives was not completely inhibited at 32% concentrations of FOS (Figure S2, M-P) . Similar to under fructose stress, compared with K2044-WT, all K2044-derived strains showed the better planktonic growth under FOS exposure(1%, 2%, 4%, 8%, 16%). Additionally, K2044-W90 and K2044-0.5FOS-90G exhibited the best adaptive growth among all derived strains under 4%, 8%, 16% of FOS (Figure 5, A-C, G) , respectively, whereas the planktonic growth of K2044-W90 outcompeted K2044-0.5FOS-90G under 1% of 2% of FOS (Figure 5, D-E, H) . Similar to the observation under fructose stress, the nearly identical tendency of the planktonic growth ability of EKP19 WT and its derived strains was found under various concentrations of FOS (Figure S3) . For EKP108, EKP108-0.5FOS-90G exhibited better adaptive growth than other EKP108-derived strains across all tested concentrations of FOS. In contrast, EKP108-8frut-90G showed the lowest planktonic growth under FOS exposure with the concentrations of 1%, 2%, 4%, 8%. (Figure S4) . Moreover, at the same concentration of FOS, K2044-∆ envZ exhibited superior adaptive growth compared to the wild-type strain (Figure 5) , indicating that the deletion of the envZ gene might promote the utilization of FOS by K.pneumoniae. In summary, knockout of the envZ gene and continuous induction with 0.5% FOS may promote the adaptive growth of K. pneumoniae. The adaptive growth of FOS- or fructose-induced K. pneumoniae under glucose or sucrose pressure. The adaptive growth of K2044, EKP19, EKP108 and their derivatives under glucose (Glc) or sucrose (Scr) exposure was further explored. K2044-0.5frut-90G and K2044-0.5FOS-90G exhibited better growth under glucose (Glc) or sucrose, and K2044 W90 demonstrated superior adaptive growth in glucose other than sucrose (Figure 6, G; Figures S6, A-F, S) . And fructose or FOS-induced strains showed better adaptive growth capacity under high concentrations of glucose (4%, 8% and 16%) (Figure 6, A-C) . Interestingly, compared to K2044-WT and K2044 W90, the slight planktonic growth of fructose or FOS-induced K2044 (K2044-0.5frut-90G, K2044-0.5FOS-90G, K2044-8frut-90G and K2044-8FOS-90G) was found under 1% of Glc, and conversely, with 2% of Glc exposure, K2044-8frut-90G showed enhanced planktonic growth ability and other K2044 derivatives remained inhibited growth (Figure 6, D-E) . For EKP19, the growth tendency of EKP19 WT and EKP19-W90 were nearly identical under different concentrations of glucose (Figure, S5) . Fructose or FOS-induced strains showed better adaptive growth capacity under 8% and 16% of glucose (Figure S5, A-B) . Moreover, there was also limited planktonic growth of fructose or FOS-induced EKP19 (EKP19-0.5frut-90G, EKP19-0.5FOS-90G, EKP19-8frut-90G and EKP19-8FOS-90G) under 1% or 2% of Glc, while the corresponding WT and W90 strains remained unaffected (Figure S5, D-E) . Conversely, at 4% or 8% of Glc exposure, EKP19-0.5frut-90G exhibited favourable planktonic growth among the other induced strains (Figure 6, H; Figure S5, B-C) . Additionally, we found that EKP108-0.5FOS-90G still showed excellent adaptive growth under glucose exposure compared to its other induced derivatives (Figure 6, I; Figures S5, G-L) . Particularly under 8% glucose stress, among the other induced EKP108 strains, only EKP108-0.5FOS-90G maintained optimal growth (Figure S5, H) . At 1%–4% of glucose, the planktonic growth capacity of fructose or FOS-induced strains of EKP108 was also limited compared to the EKP108 WT and EKP108 W90 (Figure S5, I-K) . We also observed the adaptive growth capacity of K2044, EKP19 and EKP108, and their derivatives under different concentrations of sucrose. In K2044, K2044-0.5frut-90G and K2044-0.5FOS-90G remained the best adaptation of planktonic growth under varying concentrations of sucrose (Figure S6, A-E, S) . There was no significant difference in planktonic growth of EKP19 fructose and FOS-induced derivatives under different concentrations of sucrose, showing the similar tendency of the adaptive growth with that under fructose exposure (Figure S6, G-L) , and these induced strains exhibited superior planktonic growth compared to EKP19 WT and EKP19 W90 under various sucrose concentrations (Figure S6, T) . Notably, EKP108-0.5FOS-90G still exhibited excellent adaptive growth under sucrose exposure (Figure S6, M-R and U). Furthermore, the planktonic growth of K2044-Δ envZ was also markedly inhibited at glucose concentrations of 1–2% (Figure 6, D-E) . Interestingly, a significantly prolonged lag phase was observed for K2044-Δ envZ in MRS medium supplemented with sucrose, compared to the control (MRS without sugar) (Figure S6, A-F) . The antimicrobial susceptibility test ing of K. pneumoniae. The antimicrobial susceptibility of K2044, EKP108, and their respective derivative strains was evaluated against ceftazidime (CAZ), gentamicin (GEN), levofloxacin (LEV), meropenem (MEM), and tigecycline (TGC). EKP108-0.5FOS-90G showed a reduced MIC value for LEV (a 16-fold decrease). In contrast, compared to K2044 WT, K2044-Δ envZ demonstrated a 128-fold and 4-fold increase in MICs for GEN and TGC, respectively, indicating acquired resistance to both antibiotics (Table 1) . Table 1 The MICs of K. pneumoniae (μg / mL) Strains CAZ GEN LEV MEM TGC K2044-WT 0.25 0.25 0.5 0.25 2 K2044-W90 0.5 0.5 0.5 0.25 2 K2044-0.5frut-90G 0.5 0.5 0.25 0.25 4 K2044-8frut-90G 0.25 0.25 0.125 0.25 2 K2044-0.5FOS-90G 0.25 0.5 0.125 0.25 2 K2044-8FOS-90G 0.25 0.5 0.125 0.25 2 K2044-∆ envZ 0.25 32* 0.125 0.25 8* EKP108-WT 16 0.5 16 0.5 32 EKP108-W90 16 0.5 16 0.5 32 EKP108-0.5frut-90G 16 0.5 16 0.5 32 EKP108-8frut-90G 16 0.5 16 0.5 32 EKP108-0.5FOS-90G 16 0.5 1* 0.5 16 EKP108-8FOS-90G 16 0.5 16 0.5 32 ATCC 25922 4 2 ＜0.0625 0.125 2 *: The change of MIC value≥4-fold, compared to the Wild type. The antimicrobial susceptibility breakpoints are shown in Table S2. ATCC 25922 serves as the reference strain. The biofilm formation, outer membrane permeability and membrane phospholipids of K. pneumoniae. Biofilm formation and membrane permeability analysis revealed distinct phenotypic differences among the tested strains. K2044 and its derivatives, including both induced and envZ knockout strains, exhibited robust biofilm-forming capacity. In contrast, EKP19-derived strains demonstrated reduced biofilm formation relative to EKP19 WT, while no significant differences were observed among the EKP108 series strains (Figure 7, A). Given that outer membrane permeability is a critical factor limiting the influx and efflux of compounds in gram-negative bacteria , we assessed the outer membrane permeability of K2044-Δ envZ and EKP108-0.5FOS-90G using the NPN uptake assay to investigate the reasons for the growth changes and drug sensitivity changes produced by both strains. Results showed that both K2044-Δ envZ and EKP108-0.5FOS-90G had significantly increased membrane permeability compared to their respective wild-type strains (Figure 7, D) . Interestingly, polymyxin B (PB) treatment increased outer membrane permeability in K2044 WT, EKP108 WT, and EKP108-0.5FOS-90G. However, the membrane permeability of K2044-Δ envZ was not significantly increased upon exposure to PB (Figure 7, D) . To determine whether the membrane permeability changes were associated with alterations in membrane phospholipid composition, LC-MS/MS was used to quantify key lipid species including phosphatidylglycerol (PG), phosphatidylcholine (PC), and lysophosphatidylethanolamine (LPE). K2044-Δ envZ showed reduced levels of PG-16:0-22:6 and LPE (Figure 7, B) . In EKP108-0.5FOS-90G, PG-16:0-22:6 , PC, and LPE levels were significantly increased compared to EKP108 WT (Figure 7, C) . Genomic mutation of EKP108-0.5FOS-90G . There were significant changes in adaptive growth, antimicrobial susceptibility, outer membrane permeability, and membrane phospholipids in EKP108-0.5FOS-90G, to further investigate the mechanisms underlying the impact of FOS on K. pneumoniae , whole genome sequencing was performed on EKP108-0.5FOS-90G and compared to its parental strain, EKP108. A total of 13 genes were mutated, of which 2 genes were synonymous and 11 genes were non-synonymous, and the most mutation sites were found on the gene encoding the conjugated transfer relaxase/helicase enzyme TraI, with 18 mutation sites (Table 2) . Table 2 Whole gene sequencing comparison between EKP108-0.5FOS-90G and EKP108 WT. Ref_gene_ID Base change Amino acids Ref_gene_product Ref_gene_function GM004699 GA EK SidA/IucD/PvdA family monooxygenase - GM005147 CT TC TC GA TG TG AG AG CT AG TC GC TC TC AG GA TG GA MI MV VV PS QP NT SS SP GG VA SS GG LL AA RR LL AA LF Conjugative transfer relaxase/helicase TraI Horizontal gene transfer,prokaryotic genetics GM000718 GT HQ Type VI secretion protein, VC_A0111 family Antibiotic resistance and virulence factor transmission GM005287 TC WR DUF262 domain-containing protein - GM001195 GA WX PucR family transcriptional regulator ligand-binding domain-containing protein Purine catabolism GM001228 GT GV Siderophore enterobactin receptor FepA The outer membrane receptor encoding the siderophore enterobacterin receptor, colicin GM001774 AT DV NADH-quinone oxidoreductase subunit NuoI - GM001861 CT MI CidB/LrgB family autolysis modulator - GM002855 CG TR F imbrial protein - GM002999 CA GC Carbohydrate kinase family protein Metabolism and transport of carbohydrates GM003927 GT AS Thiamine pyrophosphate enzyme, central domain - DISCUSSION K. pneumoniae commonly colonizes the mucosal surfaces of the gastrointestinal tract in animals and healthy humans, and microbial carbohydrate metabolism plays a crucial role in the process of gastrointestinal colonization [ 21 ] . Long-term carbohydrates pressure in the gut may induce adaptive evolution in K. pneumoniae , thereby affecting the adaptive growth phenotype, virulence, drug susceptibility, and metabolic stability [ 22 , 23 ] . In this study, 0.5% FOS-induced strains of K. pneumoniae (K2044-0.5FOS-90G and EKP108-0.5FOS-90G) showed enhanced growth capacity in a series of concentrations of fructose, FOS and other carbohydrates (glucose and sucrose), whereas K2044-8frut-90G demonstrated the lowest planktonic growth at each concentration, suggesting that long-term induction with fructose or FOS at appropriate concentrations may enhance the adaptive growth capacity of K. pneumoniae , however, excessively high concentrations do not promote the growth adaptation of strains for utilizing carbon sources. Moreover, there was no significant difference in the adaptive growth of EKP19 derived strains under different concentrations of fructose or FOS. In our previous studies, we found that xylose-induced EKP19 series strains exhibited essentially consistent planktonic growth under varying xylose concentrations [ 24 ] . Similarly, glucose- or sucrose-induced EKP108 series strains showed no significant differences in adaptive growth under different glucose or sucrose concentrations [ 23 ] , demonstrating the sustained stability of some strains and the isolate-specific characteristics of K.pneumonia growth adaptation under carbohydrates exposure. Furthermore, we were surprised to find that the planktonic growth of fructose or FOS-induced K.pneumoniae was slightly inhibited under 1–2% concentrations of glucose. Many evidences have supported that several common functional proteins play key roles in the metabolism of both fructose and glucose [ 9 , 25 ] , such as GLUT2 and hexokinase (HK). This may be explained by the fact that, under prolonged fructose pressure, K. pneumoniae strains downregulate the expression of functional proteins involved in efficient glucose transport and metabolism, including components of the glucose-specific phosphotransferase system (PTS) and high-affinity glucose transporters. Such changes may arise through regulatory mechanisms, such as carbon catabolite repression (CCR), or through genetic mutations, ultimately promoting survival and more efficient fructose utilization. Moreover, under higher glucose concentration conditions (8%-16%), the planktonic growth capacity of these fructose- or FOS-induced strains was restored, which may be attributed to the higher concentration of carbon sources providing energy to the bacteria, thereby facilitating the reactivation of glucose metabolic pathways. However, further validation will be required through RT-qPCR to elucidate the differences in the expression levels of glucose and fructose transporters, as well as key metabolic enzyme-encoding genes in K.pneumonia (e.g., glk, pfkA, fruK ). The isolation of K.pneumoniae in China ranked second only to E.coli , and the rate of resistance to antimicrobial drugs is increasing in recent years, which suggests the severity of K.pneumoniae infections worldwide [ 26 ] . This study found that the MIC of LEV against EKP108-0.5FOS-90G was markedly decreased. LEV exerts its antibacterial effect by inhibiting bacterial DNA replication and disrupting cell membrane permeability [ 27 , 28 ] . And the outer membrane permeability of gram-negative bacteria is primarily determined by the composition of membrane phospholipids, the type and expression levels of porins, and adaptive responses of bacteria to environmental stimuli [ 29 , 30 ] , alterations in phospholipid profiles can profoundly affect bacterial physiology and phenotype [ 31 , 32 ] . In this study, EKP108-0.5FOS-90G showed increased membrane permeability and elevated phospholipid content following FOS induction, which may be contributing factors to its reduced MIC value against LEV. In contrast, the MICs of GEN and TGC in K2044-∆ envZ showed a 128-fold and 4-fold increase, respectively, suggesting that knockout of the envZ gene may affect the expression of the membrane protein OmpR by disrupting the EnvZ/OmpR two-component signaling system, thereby causing the strain to become resistant to aminoglycoside and tetracycline antibiotics, which is consistent with the findings of the previous report [ 18 ] , and the underlying resistance mechanisms require further experimental validation. Whole-genome sequencing and SNP analysis revealed that the gene encoding the binding transfer relaxase/helix-unwinding enzyme TraI, a DNA endonuclease primarily involved in horizontal gene transfer and prokaryotic genetics between bacteria [ 33 , 34 ] , harbored the highest number of mutations in EKP108-0.5FOS-90G. In addition, mutations were identified in genes encoding members of the carbohydrate kinase family, which are mainly associated with carbohydrate metabolism and transport. These genetic alterations may underlie the enhanced growth capacity of EKP108-0.5FOS-90G under sugar stress conditions. CONCLUSION In summary, this study demonstrates that K.pneumoniae can undergo adaptive growth under FOS and fructose stress, with optimal growth typically observed at 4–8% concentrations. Notably, K2044-0.5FOS-90G and EKP108-0.5FOS-90G exhibited the best planktonic growth under different concentrations of fructose or FOS. However, the growth of the induced strains was inhibited under 1%-2% glucose. Moreover, K2044-Δ envZ mutant showed the enhanced adaptive growth compared to K2044 WT under different concentrations of fructose and FOS, whereas a significantly prolonged lag phase was observed for K2044-Δ envZ with sucrose exposure, compared to the control. Additionally, EKP108-0.5FOS-90G further showed reduced resistance to LEV, while K2044-∆ envZ exhibited increased resistance to GEN and TGC. Both EKP108-0.5FOS-90G and K2044-∆ envZ also displayed elevated outer membrane permeability and altered membrane phospholipid profiles, with EKP108-0.5FOS-90G showing increased levels of PG, PC, and LPE. Whole-genome sequencing of EKP108-0.5FOS-90G identified 13 gene mutations, including extensive changes in traI , a gene involved in conjugation. These findings indicate that prolonged exposure to low concentrations of FOS (0.5%) or deletion of envZ gene induces multifaceted adaptive responses in K. pneumoniae , involving growth advantage, membrane remodeling, and shifts in antimicrobial susceptibility, which provide important insights into how dietary carbohydrates and bacterial signaling systems may influence colonization dynamics and clinical treatment outcomes. Declarations Supplementary Information The online version contains supplementary material available at. Author contributions Y.H. and B.X. performed the strains induction experiment, MIC assay, growth curves analysis, biofilm biomass determination and determination of bacterial outer membrane permeability, and drafted the manuscript. F.Z. constructed the knockout strain. K.F performed the membrane phospholipid extraction and quantification. J.L and Y.X are responsible for data organization and whole-genome sequencing analysis. B.C., T.H. and Z.Y conceived and designed the project and revised the manuscript. All authors have read and approved the manuscript.All authors participated in data analysis. Funding This work was supported by the following grants: National Natural Science Foundation of China (82572621); Sanming Project of Medicine in Shenzhen (SZSM202303037; SZSM202103014); Shenzhen Medical Key Discipline Construction Fund; Science, Technology and Innovation Commission of Shenzhen Municipality of basic research funds (JCYJ20240813114518024, KJZD20240903103500002) and the Shenzhen Nanshan District Scientific Research Program of the People’s Republic of China (NS2025012, NSZD2024036, NSZD2024023, NSZD2024032, NSZD2025001, NSZD2025005,YN2025011,YN2025018). Data availability statement The raw whole-genome sequencing data was posted in the Sequence Read Archive (SRA) database under accession number SRR29409842 and SRR29409843 (http://www.ncbi.nlm.nih.gov/sra). Ethics approval All methods were carried out in accordance with relevant guidelines and regulations and were approved by the Ethics Committee of Shenzhen Nanshan People’s Hospital and the 1964 Helsinki declaration and its later amendments, or comparable ethical standards. The biosafety approval number is 0125F300106. All experimental procedures involving human subjects were approved by the institutional ethical committee of Shenzhen Nanshan People’s Hospital. Bacterial strains were obtained from stored samples of hospitalized patients, collected as part of the routine clinical management of patients, according to the national guidelines in China. Therefore, informed consent was not sought, and informed consent waiver was approved by the institutional ethical committee of Shenzhen Nanshan People’s Hospital. Consent for publication Not applicable. Competing interests The authors declare no competing interests. References WYRES K L, LAM M M C, HOLT K E. 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Fructose metabolism and metabolic disease [J]. J Clin Invest, 2018, 128(2): 545-55. WU K G, LI T H, PENG H J. Lactobacillus salivarius plus fructo-oligosaccharide is superior to fructo-oligosaccharide alone for treating children with moderate to severe atopic dermatitis: a double-blind, randomized, clinical trial of efficacy and safety [J]. Br J Dermatol, 2012, 166(1): 129-36. LUO Y, ZHANG T, WU H. The transport and mediation mechanisms of the common sugars in Escherichia coli [J]. Biotechnol Adv, 2014, 32(5): 905-19. JECKELMANN J M, ERNI B. Transporters of glucose and other carbohydrates in bacteria [J]. Pflugers Arch, 2020, 472(9): 1129-53. CHEN C Z F, REN J. Advances in the metabolic mechanism of Lactobacillus utilizing oligofructose. [J]. Food and Fermentation Industries, 2013: 10:156-60. SAULNIER D M, MOLENAAR D, DE VOS W M, et al. Identification of prebiotic fructooligosaccharide metabolism in Lactobacillus plantarum WCFS1 through microarrays [J]. 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Emerging Transcriptional and Genomic Mechanisms Mediating Carbapenem and Polymyxin Resistance in Enterobacteriaceae: a Systematic Review of Current Reports [J]. mSystems, 2020, 5(6). XIAO G, ZHENG X, LI J, et al. Contribution of the EnvZ/OmpR two-component system to growth, virulence and stress tolerance of colistin-resistant Aeromonas hydrophila [J]. Front Microbiol, 2022, 13: 1032969. KO D, CHOI S H. Mechanistic understanding of antibiotic resistance mediated by EnvZ/OmpR two-component system in Salmonella enterica serovar Enteritidis [J]. J Antimicrob Chemother, 2022, 77(9): 2419-28. KHAN I, BAI Y, ZHA L, et al. Mechanism of the Gut Microbiota Colonization Resistance and Enteric Pathogen Infection [J]. Front Cell Infect Microbiol, 2021, 11: 716299. KIM K, HOU C Y, CHOE D, et al. Adaptive laboratory evolution of Escherichia coli W enhances gamma-aminobutyric acid production using glycerol as the carbon source [J]. Metab Eng, 2022, 69: 59-72. HE Y, LIU F, LI C, et al. The adaptive growth and mechanisms of Klebsiella pneumoniae under sucrose and glucose exposure [J]. Microbiol Spectr, 2025, 13(12): e0160325. YI R, ZHENG J, XU Z, et al. Klebsiella pneumoniae under xylose pressure: the growth adaptation, antimicrobial susceptibility, global proteomics analysis and role of XylA and XylB proteins [J]. BMC Microbiol, 2025, 25(1): 257. ZHAO M, LONE J, REGHUPATY S, et al. Progress in Understanding the Regulation of Glucose and Fructose Metabolism [J]. Annu Rev Nutr, 2025. DING L G Y, WU S, ET AL. CHINET 2024 bacterial resistance surveillance results [Z]. CHINET. 2024 ROY R, TIWARI M, DONELLI G, et al. Strategies for combating bacterial biofilms: A focus on anti-biofilm agents and their mechanisms of action [J]. Virulence, 2018, 9(1): 522-54. LU Q, YANG Q. Study on the Mechanism of Levofloxacin Combined with Imipenem Against Pseudomonas aeruginosa [J]. Appl Biochem Biotechnol, 2024, 196(2): 690-700. ZGURSKAYA H I, RYBENKOV V V. Permeability barriers of Gram-negative pathogens [J]. Ann N Y Acad Sci, 2020, 1459(1): 5-18. LEUS I V, ADAMIAK J, CHANDAR B, et al. Functional Diversity of Gram-Negative Permeability Barriers Reflected in Antibacterial Activities and Intracellular Accumulation of Antibiotics [J]. Antimicrob Agents Chemother, 2023, 67(2): e0137722. STRAHL H, ERRINGTON J. Bacterial Membranes: Structure, Domains, and Function [J]. Annu Rev Microbiol, 2017, 71: 519-38. DENICH T J, BEAUDETTE L A, LEE H, et al. Effect of selected environmental and physico-chemical factors on bacterial cytoplasmic membranes [J]. J Microbiol Methods, 2003, 52(2): 149-82. LARKIN C, HAFT R J F, HARLEY M J, et al. Roles of active site residues and the HUH motif of the F plasmid TraI relaxase [J]. J Biol Chem, 2007, 282(46): 33707-13. GUZMáN-HERRADOR D L, LLOSA M. The secret life of conjugative relaxases [J]. Plasmid, 2019, 104: 102415. Additional Declarations No competing interests reported. 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Chemical structural formula for other carbonhydrates (glucose, sucrose) are shown in \u003cstrong\u003eFigure S1\u003c/strong\u003e.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-9145216/v1/1cea8715a72379b78b8e421c.png"},{"id":106093084,"identity":"b46fd893-366c-4717-9193-c67b29d7a7eb","added_by":"auto","created_at":"2026-04-03 11:33:49","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":346960,"visible":true,"origin":"","legend":"\u003cp\u003eGrowth curves of K2044 and its derivatives (K2044-WT, K2044-W90, K2044-0.5frut-90G, K2044-8frut-90G, K2044-0.5FOS-90G, K2044-8FOS-90G, K2044-∆\u003cem\u003eenvZ\u003c/em\u003e) at the same initial fructose concentration. MRS medium supplemented with 16% fructose\u003cstrong\u003e (A)\u003c/strong\u003e, 8% fructose \u003cstrong\u003e(B)\u003c/strong\u003e, 4% fructose\u003cstrong\u003e (C)\u003c/strong\u003e, 2% fructose\u003cstrong\u003e (D)\u003c/strong\u003e, 1% fructose\u003cstrong\u003e (E)\u003c/strong\u003e. MRS without sugar as control\u003cstrong\u003e (F). \u003c/strong\u003eGrowth curves of NTUH-K2044 WT under different initial fructose concentrations \u003cstrong\u003e(G)\u003c/strong\u003e. Comparison of OD600 values of K2044 series strains at logarithmic period (12\u003csup\u003eth\u003c/sup\u003e hour), n=3, *\u003cem\u003eP\u003c/em\u003e＜0.05, **\u003cem\u003eP\u003c/em\u003e＜0.01, ***\u003cem\u003eP\u003c/em\u003e＜0.001, ****\u003cem\u003eP\u003c/em\u003e＜0.0001 \u003cstrong\u003e(H-I)\u003c/strong\u003e.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-9145216/v1/fb8c39864ea7c6df2d20f94e.png"},{"id":106093163,"identity":"e9bf1111-ebc3-4a13-ae3c-c033778fc7d6","added_by":"auto","created_at":"2026-04-03 11:35:23","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":227965,"visible":true,"origin":"","legend":"\u003cp\u003eGrowth curves of EKP19 and its derivatives (EKP19-WT, EKP19-W90, EKP19-0.5frut-90G, EKP19-8frut-90G, EKP19-0.5FOS-90G, EKP19-8FOS-90G) at the each concentration of fructose. MRS medium supplemented with 16% fructose\u003cstrong\u003e (A)\u003c/strong\u003e, 8% fructose \u003cstrong\u003e(B)\u003c/strong\u003e, 4% fructose\u003cstrong\u003e (C)\u003c/strong\u003e, 2% fructose\u003cstrong\u003e (D)\u003c/strong\u003e, 1% fructose\u003cstrong\u003e (E)\u003c/strong\u003e. MRS without sugar as control\u003cstrong\u003e (F). \u003c/strong\u003eComparison of OD600 values of EKP19 series strains at logarithmic period (12\u003csup\u003eth\u003c/sup\u003e hour), n=3, ns: no significance\u003cstrong\u003e (G)\u003c/strong\u003e.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-9145216/v1/f90f9875b0548250f8533f64.png"},{"id":106092989,"identity":"fd24cfb3-4029-4dd6-9dcc-238867e6b440","added_by":"auto","created_at":"2026-04-03 11:32:01","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":235035,"visible":true,"origin":"","legend":"\u003cp\u003eGrowth curves of EKP108 and its derivatives (EKP108-WT, EKP108-W90, EKP108-0.5frut-90G, EKP108-8frut-90G,EKP108-0.5FOS-90G, EKP108-8FOS-90G) at the each concentration of fructose. MRS medium supplemented with 16% fructose\u003cstrong\u003e (A)\u003c/strong\u003e, 8% fructose \u003cstrong\u003e(B)\u003c/strong\u003e, 4% fructose\u003cstrong\u003e (C)\u003c/strong\u003e, 2% fructose\u003cstrong\u003e (D)\u003c/strong\u003e, 1% fructose\u003cstrong\u003e (E)\u003c/strong\u003e. MRS without sugar as control\u003cstrong\u003e (F). \u003c/strong\u003eComparison of OD600 values of EKP108 series strains at logarithmic period (12\u003csup\u003eth\u003c/sup\u003e hour), n=3, ****\u003cem\u003eP\u003c/em\u003e＜0.0001\u003cstrong\u003e (G)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-9145216/v1/ba1a220691a8f1943e7bc5cf.png"},{"id":105931775,"identity":"59e67645-6029-4849-b8a6-311dfab4a10e","added_by":"auto","created_at":"2026-04-01 14:22:37","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":311378,"visible":true,"origin":"","legend":"\u003cp\u003eGrowth curves of K2044 and its derivatives (K2044-WT, K2044-W90, K2044-0.5frut-90G, K2044-8frut-90G, K2044-0.5FOS-90G, K2044-8FOS-90G, K2044-∆\u003cem\u003eenvZ\u003c/em\u003e) at the each concentration of FOS. MRS medium supplemented with 16% FOS\u003cstrong\u003e (A)\u003c/strong\u003e, 8% FOS \u003cstrong\u003e(B)\u003c/strong\u003e, 4% FOS \u003cstrong\u003e(C)\u003c/strong\u003e, 2% FOS \u003cstrong\u003e(D)\u003c/strong\u003e, 1% FOS \u003cstrong\u003e(E)\u003c/strong\u003e. MRS without sugar as control\u003cstrong\u003e (F). \u003c/strong\u003eComparison of OD600 values of K2044 series strains at logarithmic period (12\u003csup\u003eth\u003c/sup\u003e hour), n=3, *\u003cem\u003eP\u003c/em\u003e＜0.05, **\u003cem\u003eP\u003c/em\u003e＜0.01, ***\u003cem\u003eP\u003c/em\u003e＜0.001, ****\u003cem\u003eP\u003c/em\u003e＜0.0001 \u003cstrong\u003e(G-H)\u003c/strong\u003e.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-9145216/v1/002fe85669188c5a16280b10.png"},{"id":105931777,"identity":"175b6ca3-7825-4d76-9bfa-c3a0869b7020","added_by":"auto","created_at":"2026-04-01 14:22:37","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":349582,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGrowth curves of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eK. pneumoniae \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eand their derivatives at the same initial glucose concentration.\u003c/strong\u003e Growth curves of K2044 and its derivatives (K2044-WT, K2044-W90, K2044-0.5frut-90G, K2044-8frut-90G, K2044-0.5FOS-90G, K2044-8FOS-90G, K2044-∆\u003cem\u003eenvZ\u003c/em\u003e) at the same initial glucose concentration \u003cstrong\u003e(A-F)\u003c/strong\u003e.\u003cstrong\u003e \u003c/strong\u003eMRS without sugar as control. Comparison of OD600 values of K2044\u003cstrong\u003e (G)\u003c/strong\u003e, EKP19\u003cstrong\u003e (H)\u003c/strong\u003e, EKP108\u003cstrong\u003e (I)\u003c/strong\u003e series strains at logarithmic period (12\u003csup\u003eth\u003c/sup\u003e hour), n=3, *\u003cem\u003eP\u003c/em\u003e＜0.05, **\u003cem\u003eP\u003c/em\u003e＜0.01, ***\u003cem\u003eP\u003c/em\u003e＜0.001, ****\u003cem\u003eP\u003c/em\u003e＜0.0001.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-9145216/v1/d60ed55ffc5ccdd0f812e49f.png"},{"id":105931778,"identity":"a7b2fda4-00bb-485d-8a1a-44e070e3adf1","added_by":"auto","created_at":"2026-04-01 14:22:37","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":369856,"visible":true,"origin":"","legend":"\u003cp\u003eBiofilm formation in K2044, EKP19 and EKP108, “Blank” means without bacteria. \u003cstrong\u003e(A)\u003c/strong\u003e. Comparison of the phospholipid composition of K2044 membranes \u003cstrong\u003e(B)\u003c/strong\u003e. Comparison of the phospholipid composition of EKP108 membranes \u003cstrong\u003e(C). \u003c/strong\u003eChanges in outer membrane permeability in \u003cem\u003eK.pneumoniae\u003c/em\u003e, “Control” is the outer membrane permeability before dosing \u003cstrong\u003e(D)\u003c/strong\u003e.\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-9145216/v1/f2aba5ba2837e34c2af72da6.png"},{"id":108977296,"identity":"b9f63598-c6b2-489d-860c-edb98cd55d63","added_by":"auto","created_at":"2026-05-11 11:31:16","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1918735,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9145216/v1/2cac9655-0a78-4bc5-a532-84168a9036d7.pdf"},{"id":105931774,"identity":"e10be5bf-8f55-4f12-bdde-d4b949585320","added_by":"auto","created_at":"2026-04-01 14:22:37","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":2949515,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-9145216/v1/656985f087ba14d3865743ac.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Impact of prolonged fructose and fructooligosaccharides stress on the adaptive growth, antimicrobial susceptibility, and genomic mutation of Klebsiella pneumoniae","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003e \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e (\u003cem\u003eK.pneumoniae\u003c/em\u003e) belongs to the family \u003cem\u003eEnterobacteriaceae\u003c/em\u003e, which mainly colonizes the nasopharynx and gastrointestinal tract of humans and primates, and is prone to developing resistance to multiple antibiotics\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. \u003cem\u003eK.pneumoniae\u003c/em\u003e is classified into classical \u003cem\u003eK. pneumoniae\u003c/em\u003e (cKP) and hypervirulent \u003cem\u003eK. pneumoniae\u003c/em\u003e (hvKP) on the basis of virulence and mucoviscosity phenotype\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. HvKp strains exhibit enhanced mucosal adherence, higher virulence compared to cKP, and are associated with more severe infections\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. An important virulence factor contributing to \u003cem\u003eK. pneumoniae\u003c/em\u003e pathogenicity is its ability to form a biofilm, which enhances tolerance to environmental stressors and antimicrobial agents\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cem\u003eK. pneumoniae\u003c/em\u003e is capable of utilizing a broad range of sugars as carbon sources. Fructose, a monosaccharide and isomer of glucose, is commonly used in the food industry as a sweetener and nutritional additive\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, A\u003cb\u003e)\u003c/b\u003e. Fructooligosaccharides (FOS), contain a variable number of β-D-fructofuranosyl units with one glucosyl unit, is a naturally occurring carbohydrate found in various plants \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, B\u003cb\u003e)\u003c/b\u003e, which are indigestible by humans but are metabolized by gut probiotics, thereby promoting intestinal health\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. There are three main transport pathways for fructose utilization in bacteria: \u003cb\u003ePathway A\u003c/b\u003e: transport via the phosphoenolpyruvate-dependent phosphotransferase system (PTS), specifically through the transmembrane enzymes EII^Fru (encoded by \u003cem\u003efruA\u003c/em\u003e and \u003cem\u003efruB\u003c/em\u003e), leading to intracellular phosphorylation to fructose-1,6-bisphosphate; \u003cb\u003ePathway B\u003c/b\u003e: the mannose transport system (MTS), which is encoded by the \u003cem\u003emanXYZ\u003c/em\u003e gene; \u003cb\u003ePathway C\u003c/b\u003e: this pathway does not involve the carbohydrate PTS transport system, but requires relatively high concentrations of fructose to function\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. FOS uptake is mediated by several transport systems, including the ABC transporter system, found mainly in \u003cem\u003eLactobacillus\u003c/em\u003e; the PTS transporter system, found mainly in \u003cem\u003eBacillus\u003c/em\u003e and \u003cem\u003eLactococcus\u003c/em\u003e; and the permease system, found mainly in \u003cem\u003eBifidobacterium\u003c/em\u003e\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eBiofilm formation is a key mechanism contributing to the drug resistance of \u003cem\u003eK. pneumoniae\u003c/em\u003e. Biofilms are structured communities of bacteria, often composed of one or more species, embedded in a self-produced extracellular matrix consisting of proteins, polysaccharides, and nucleic acids derived from both the bacteria and the host, and the bacteria in the biofilm are protected from host immune defenses and antimicrobial agents\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. The outer membrane of \u003cem\u003eK.pneumoniae\u003c/em\u003e is composed of phospholipids, lipopolysaccharides, and proteinsthat collectively act as a barrier to antibiotic entry. In drug-resistant strains, the reduced expression or absence of outer membrane porins can significantly alter membrane permeability, thereby limiting antibiotic uptake and diminishing therapeutic efficacy\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe two-component regulatory system (TCS) is the most important mechanism for regulating physiological behavior of bacteria, and plays an important role in bacterial pathogenicity, antibiotic resistance, virulence factor regulation, and environmental adaptation\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. The \u003cem\u003eenvZ\u003c/em\u003e gene encodes a histidine protein kinase (HK) in the two-component system EnvZ/OmpR, which plays a key role in sensing environmental signals and regulating bacterial adaptive responses\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. The EnvZ protein kinase is located in the bacterial cell membrane, and when the bacterial surroundings are altered, EnvZ activates the expression of specific genes by phosphorylating the OmpR protein, thus enabling the bacteria to adapt to these unfavorable conditions\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. In addition, mutations in \u003cem\u003eenvZ\u003c/em\u003e have been shown to influence both bacterial virulence and antibiotic resistance\u003csup\u003e[\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003ePrevious research has primarily focused on the types of carbohydrates available to \u003cem\u003eK.pneumoniae\u003c/em\u003e, with little attention paid to how different concentrations of a single carbohydrate\u0026mdash;especially under sustained exposure\u0026mdash;affect bacterial adaptation. This study aims to explore the adaptive growth, antimicrobial susceptibility, biofilm formation, and membrane phospholipid composition of \u003cem\u003eK.pneumoniae\u003c/em\u003e under fructose and FOS pressure. Additionally, we investigated genomic mutations induced by long-term FOS exposure. To further examine the regulatory role of \u003cem\u003eenvZ\u003c/em\u003e, we analyzed the adaptive growth of \u003cem\u003eK. pneumoniae\u003c/em\u003e strains lacking \u003cem\u003eenvZ\u003c/em\u003e under fructose and FOS stress, as well as the phenotypic changes associated with \u003cem\u003eenvZ\u003c/em\u003e deletion. These findings may provide new insights for understanding bacterial adaptation and improving clinical strategies against \u003cem\u003eK. pneumoniae\u003c/em\u003e-associated infections.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cp\u003e\u003cstrong\u003eBacterial strains and growth conditions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn this study, two clinical isolates from inpatients of Shenzhen Nanshan People\u0026rsquo;s Hospital, China, and one strain of NTUH-K2044 (Hereinafter referred to as K2044) were collected. \u003cem\u003eK.pneumoniae \u003c/em\u003ewas grown in Luria-Bertani medium at 37 \u0026deg;C with shaking unless otherwise stated. For antimicrobial susceptibility testing, strains were grown in cation-adjusted Mueller-Hinton broth (CAMHB) at 37 \u0026deg;C with shaking.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eK.pneumoniae\u003c/em\u003e\u003cem\u003e \u003c/em\u003e\u003cspan id=\"_Toc9352\"\u003eK2044, EKP19 and EKP108 were cultured in vitro under control conditions (without sugar), as well as in medium supplemented with fructose or FOS at concentrations of 0.5% and 8%, resepectively and serially passaged for 90 generations to obtain 3 subpassaged strains (K2044-W90, EKP19-W90, EKP108-W90) and 12 induced strains (K2044-0.5frut-90G, K2044-8frut-90G, K2044-0.5FOS-90G and K2044-8FOS-90G, same for EKP19 and EKP108). Similar to K2044, two clinical \u003cem\u003eK.pneumoniae\u003c/em\u003e isolates (EKP19 and EKP108) were also used to establish the induced isolates with De Man, Rogosa and Sharp control media (MRS, purchased from Topbiol, Shangdong, China, consists of peptone (10.0g/L), beef extract (8.0g/L), yeast extract (4.0g/L), tween 80 (1.0g/L), sodium acetate\u0026middot;H\u003csub\u003e2\u003c/sub\u003eO (5.0g/L), K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e\u0026middot;7H\u003csub\u003e2\u003c/sub\u003eO (2.0g/L), ammonium citrate tribasic (2.0g/L), MgSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;7H\u003csub\u003e2\u003c/sub\u003eO (0.2g/L) and MnSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;4H\u003csub\u003e2\u003c/sub\u003eO (0.05g/L) \u003c/span\u003efor non-supplemented carbon conditions), and the media with concentrations of 0.5% and 8% fructose or FOS. All strains used in this work are listed in \u003cstrong\u003eTable S1\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAntimicrobials and reagents\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCeftazidime (MB1334, 94%-105%), gentamicin (MB1331, \u0026ge;590IU/mg), levofloxacin (MB1576, ＞98.5%), meropenem (MB1129, ＞98%), and tigecycline (MB1246, ＞99%) were purchased from MeilunBio, Dalian, China.\u003c/p\u003e\n\u003cp\u003eGlucose and sucrose were purchased from Biosharp, Beijing, China. D-fructose and N-Phenyl-1-naphthylamine (NPN) were purchased from aladdin, Shanghai, China. Fructooligosaccharides was purchased from macklin, Shanghai, China. DL5000 DNA marker, bacterial DNA extraction kits, and 2X taq plus master mix II were purchased from Vanzyme, Nanjing, China.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eGrowth curves\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOvernight cultures of \u003cem\u003eK. pneumoniae\u003c/em\u003e was diluted 1:200 in fresh MRS (without sugar), combined with 1%, 2%, 4%, 8%, 16%, 32% fructose or FOS, and inoculated in Honeycomb plates (100 wells, 200 \u0026mu;L/well); MRS without sugar was used as untreated control. OD600 was determined by a Bioscreen C system (Lab Systems Helsinki Finland). The experiment was recorded for 24h. Each assay was performed in triplicate at least three times.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eAntimicrobial susceptibility test\u003c/strong\u003e\u003cstrong\u003eing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMinimal inhibit concentrations (MICs) were determined by the broth macrodilution method in CAMHB according to Clinical and Laboratory Standards Institute guidelines (CLSI-M100-S34). The range of concentrations tested for antimicrobials was 0.25\u0026ndash;256 \u0026mu;g/mL. No antimicrobial was added as control and \u003cem\u003eE.coli\u003c/em\u003e ACTCC 25922 was the quality control strain. Antimicrobial susceptibility results were confirmed based on CLSI-M100-S34\u003cstrong\u003e (Table S2)\u003c/strong\u003e. All experiments were performed in triplicate.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBiofilm biomass determination\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOvernight cultures of \u003cem\u003eK. pneumoniae\u003c/em\u003e was diluted 1:100 in Luria-Bertani broth, combined with 2% glucose, and inoculated in 96-well polystyrene microtiter plates (200 \u0026mu;L/well). After incubation at 37℃ for 24h, the bacterial solution was aspirated, washed with PBS buffer PH 7.4 for 3 times, dried and fixed with 95% methanol for 15min. Methanol was removed and then stained with 1% crystal violet for 10min. Crystal violet was dissolved in 95% ethanol and OD570 was determined. This experiment was performed in triplicate at least three times.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eDetermination of bacterial outer membrane permeability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eK.pneumoniae\u003c/em\u003e grown to logarithmic phase was resuspended in PBS buffer and diluted to 0.5 McFarland turbidity. N-phenyl-1-naphthaline (NPN) fluorescent dye was added to the bacterial suspension, making a final concentration of NPN of 10 \u0026mu;M, and incubated for 30 min. 420 nm fluorescence was recorded over time (excitation wavelength of 350 nm) using high-content analysis until no further change in fluorescence was observed.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eConstruction of knockout strains\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFirstly, using high-fidelity DNA polymerase and the relevant primer sequences listed in \u003cstrong\u003eTable S3\u003c/strong\u003e, amplify the upstream and downstream homologous arms of the \u003cem\u003eenvZ\u003c/em\u003e gene from the \u003cem\u003eK.pneumoniae\u003c/em\u003e (NUTH-K2044) genome. Amplify the gentamicin (GEN) resistance gene from the pJQ200SK plasmid. The upstream and downstream homologous arms of the \u003cem\u003eenvZ\u003c/em\u003e gene were connected to the GEN resistance gene via fusion PCR, yielding the fusion fragment \u0026Delta;\u003cem\u003eenvZ\u003c/em\u003e::GEN (upstream homologous arm-GEN resistance gene-downstream homologous arm). This was then cloned into the pCVD442 plasmid to obtain the knockout vector pCVD442-\u0026Delta;\u003cem\u003eenvZ\u003c/em\u003e::GEN. Transform pCVD442-\u0026Delta;\u003cem\u003eenvZ\u003c/em\u003e::GEN into \u003cem\u003eE.coli \u003c/em\u003e\u0026beta;2155 to obtain the donor strain \u0026beta;2155/pCVD442-\u0026Delta;\u003cem\u003eenvZ\u003c/em\u003e::GEN. Conjugation experiment was performed between the \u0026beta;2155/pCVD442-\u0026Delta;\u003cem\u003eenvZ\u003c/em\u003e::GEN and K2044. A K2044 monoclonal strain exhibiting GEN resistance was screened on GEN plates and designated K2044/pCVD442-\u0026Delta;\u003cem\u003eenvZ\u003c/em\u003e::GEN. Select several K2044/pCVD442-\u0026Delta;\u003cem\u003eenvZ\u003c/em\u003e::GEN monoclonal colonies and individually inoculate them onto LB plates containing 10% sucrose for cultivation. Screen these clones using PCR technology to obtain K2044-\u0026Delta;\u003cem\u003eenvZ\u003c/em\u003e.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eMembrane phospholipid extraction and quantification\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eK2044-\u0026Delta;\u003cem\u003eenvZ\u003c/em\u003e and EKP108-0.5FOS-90G were selected for membrane phospholipid analysis, with K2044 WT and EKP108 WT serving as respective controls. Bacterial cultures were grown in MRS broth with shaking until the logarithmic growth phase was reached. Cells were harvested by centrifugation at 4,500 rpm for 15 minutes, the supernatant was discarded, and the bacterial pellets were flash-frozen at -80℃ for 10 minutes. For lipid extraction, 9.5 mL of a mixture consisting of chloroform: methanol: 0.3% NaCl (v/v/v = 1:2:0.8) was added to each sample. The mixture was incubated in a water bath at 80℃ for 15 minutes and then shaken at 220 rpm for 1 hour. After centrifugation (15 minutes), the supernatant was transferred to a new centrifuge tube. 5 mL each of chloroform and 0.3% NaCl were added to the supernatant and mixed gently. Following another centrifugation step (15 minutes), the upper aqueous phase was discarded. A 4.5 mL aliquot of the lower organic phase was collected and evaporated to dryness under a stream of nitrogen gas.The dried lipid extract was redissolved in 3 mL of methanol and filtered through a 0.22 \u0026mu;m membrane filter. The prepared samples were subjected to membrane phospholipid quantification using liquid chromatography\u0026ndash;tandem mass spectrometry (LC-MS/MS). \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWhole genome sequencing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGenomic DNA was extracted from EKP108-0.5FOS-90G and EKP108 WT with a bacterial DNA extraction kits, respectively. Whole genomes were sequenced in an Illumina HiSeq2500 sequencer. Genomic alignments were performed with MUMmer4 tools. Single nucleotide polymorphisms (SNPs), insertions, deletions, and structural variation annotations were identified based on inter-sample alignment results with BLAST, TRF and Repeatmask.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGraphing and statistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStatistics, data analysis, and graphing were performed using GraphPad Prism 8.0 software (La Jolla, CA). Data were processed using multiple comparisons of one-way ANOVA and two-way ANOVA.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003col\u003e\n \u003cli\u003e\u003cstrong\u003eThe adaptive growth of \u003cem\u003eK. pneumoniae\u003c/em\u003e with fructose pressure.\u003c/strong\u003e\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003eHere, MRS (without sugar)-induced \u003cem\u003eK. pneumoniae\u003c/em\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e(K2044-W90, EK19-W90, EK108-W90), fructose-induced \u003cem\u003eK. pneumoniae\u003c/em\u003e (K2044-0.5frut-90G, K2044-8frut-90G, EK19-0.5frut-90G, EK19-8frut-90G, EK108-0.5frut-90G, EK108-8frut-90G) and FOS-induced \u003cem\u003eK. pneumoniae\u003c/em\u003e (K2044- 0.5FOS-90G, K2044-8FOS-90G, EK19-0.5FOS-90G, EK19-8FOS-90G, EK108-0.5FOS-90G, EK108-8FOS-90G) were selected and identified firstly. K2044-∆\u003cem\u003eenvZ\u003c/em\u003e was constructed and identified with \u003cem\u003eenvZ\u0026nbsp;\u003c/em\u003egene knockout.\u003c/p\u003e\n\u003cp\u003eThe planktonic growth of K2044 and its respective derivatives (K2044-W90, K2044-0.5frut-90G, K2044-8frut-90G, K2044-0.5FOS-90G, K2044-8FOS-90G) was evaluated in media supplemented with various concentrations (1%, 2%, 4%, 8%, 16%, and 32%) of fructose, respectively. Overall, most effectively planktonic growth of fructose- or FOS-induced strains were found in media with 4% or 8% of fructose, whereas 16% of fructose significantly downregulated the \u003cem\u003eK.pneumoniae\u0026nbsp;\u003c/em\u003egrowth at the logarithmic period compared with that with concentrations\u0026le;8%. In media with 32% of fructose, K2044 and its respective derived strains (K2044-W90, K2044-0.5frut-90G, K2044-8frut-90G, K2044-8FOS-90G) \u0026nbsp;lost their ability of planktonic growth \u003cstrong\u003e(Figure 2, G; Figure S2, A,B,H)\u003c/strong\u003e, whereas K2044-0.5FOS-90G showed a slight proliferative ability after incubating for over 18 hours \u003cstrong\u003e(Figure S2, G).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe also compared the planktonic growth of K2044 and its derivatives under each concentration of fructose. K2044-W90 and K2044-0.5FOS-90G showed the better adaptive growth than other derivatives at each concentration, whereas K2044-8frut-90G and K2044 WT demonstrated the lowest planktonic growth \u003cstrong\u003e(Figure 2)\u003c/strong\u003e, indicating that continuous induction with 0.5% FOS might rapidly promote the adaptive growth of K2044 under fructose exposure, in contrast, high concentration (8%) of fructose exposure exerted a minimal effect on reshaping the adaptive growth of K2044.\u003c/p\u003e\n\u003cp\u003eIn order to avoid the isolate-spectific characteristic of the K2044 adaptation under fructose or FOS pressure, two additional \u003cem\u003eK. pneumoniae\u003c/em\u003e clinical isolates, EKP19 and EKP108, along with their respective derivatives, were selected to investigate the effects of fructose and FOS exposure on the planktonic growth. Similar to K2044, the planktonic growth of EKP19 and its respective derived strains (EKP19-W90, EKP19-0.5frut-90G, EKP19-8frut-90G, EKP19-0.5FOS-90G, EKP19-8FOS-90G) was evaluated in media supplemented with various concentrations (1%, 2%, 4%, 8%, 16%, and 32%) of fructose, respectively. We found that the planktonic growth of EKP19 and EKP108, and their respective derived strains was also completely inhibited at 32% concentrations of fructose \u003cstrong\u003e(Figure S2, C-F, I-L)\u003c/strong\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe similar planktonic growth ability was found between the EKP19 and its derivatives in media supplemented with various concentrations, suggesting that the high or low concentration of fructose or FOS exposure seemed not to affect the adaptive growth of EK19 derivatives \u003cstrong\u003e(Figure 3)\u003c/strong\u003e. Notably, comparison of planktonic growth among EKP108 and its derivatives (EKP108-W90, EKP108-0.5Frut-90G, EKP108-8Frut-90G, EKP108-0.5FOS-90G, and EKP108-8FOS-90G) revealed the markedly enhanced adaptive growth of EKP108-0.5FOS-90G under fructose pressure. however, the adaptive growth of other derivatives (EKP108-W90, EKP108-0.5Frut-90G, EKP108-8Frut-90G, EKP108-0.5FOS-90G) displayed similar tendency without impovement\u003cstrong\u003e\u0026nbsp;(Figure 4)\u003c/strong\u003e, suggesting that sustained 0.5% FOS exposure reshaped the adaptive growth capacity of EKP108 under fructose pressure.\u003c/p\u003e\n\u003cp\u003eThe \u003cem\u003eenvZ\u003c/em\u003e gene belongs to the EnvZ/OmpR two-component system and participates in sensing environmental signals and regulating bacterial adaptive responses in \u003cem\u003eK.pneumoniae\u003c/em\u003e\u003csup\u003e[16]\u003c/sup\u003e. Compared with wild-type NTUH-K2044 (K2044 WT), K2044-\u0026Delta;\u003cem\u003eenvZ\u003c/em\u003e exhibited significantly enhanced adaptive growth under fructose exposure across all tested concentrations, indicating that deletion of the \u003cem\u003eenvZ\u003c/em\u003e gene might facilitate \u003cem\u003eK.pnumoniae\u003c/em\u003e to make use of fructose for planktonic growth. \u003cstrong\u003e(Figure 2, H-I)\u003c/strong\u003e.\u003c/p\u003e\n\u003col start=\"2\"\u003e\n \u003cli\u003e\u003cstrong\u003eThe adaptive growth of \u003cem\u003eK. pneumoniae\u003c/em\u003e with FOS pressure.\u003c/strong\u003e\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003eThe planktonic growth of K2044 and its respective derivatives was also determined in media supplemented with various concentrations (1%, 2%, 4%, 8%, 16%, and 32%) of FOS, respectively. We found that 4% or 8% of FOS most effectively promoted the planktonic growth of fructose or FOS-induced strains. However, unlike under fructose stress, the adaptive growth of K2044 derivatives was not completely inhibited at 32% concentrations of FOS \u003cstrong\u003e(Figure S2, M-P)\u003c/strong\u003e. Similar to under fructose stress, compared with K2044-WT, all K2044-derived strains showed the better planktonic growth under FOS exposure(1%, 2%, 4%, 8%, 16%). Additionally, K2044-W90 and K2044-0.5FOS-90G exhibited the best adaptive growth among all derived strains under 4%, 8%, 16% of FOS \u003cstrong\u003e(Figure 5, A-C, G)\u003c/strong\u003e, respectively, whereas the planktonic growth of K2044-W90 outcompeted K2044-0.5FOS-90G under 1% of 2% of FOS \u003cstrong\u003e(Figure 5, D-E, H)\u003c/strong\u003e. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSimilar to the observation under fructose stress, the nearly identical tendency of the planktonic growth ability of EKP19 WT and its derived strains was found under various concentrations of FOS\u003cstrong\u003e\u0026nbsp;(Figure S3)\u003c/strong\u003e. For EKP108, EKP108-0.5FOS-90G exhibited better adaptive growth than other EKP108-derived strains across all tested concentrations of FOS.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eIn contrast, EKP108-8frut-90G showed the lowest planktonic growth under FOS exposure with the concentrations of 1%, 2%, 4%, 8%. \u003cstrong\u003e(Figure S4)\u003c/strong\u003e. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMoreover, at the same concentration of FOS, K2044-∆\u003cem\u003eenvZ\u003c/em\u003e exhibited superior adaptive growth compared to the wild-type strain \u003cstrong\u003e(Figure 5)\u003c/strong\u003e,\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eindicating that the deletion of the\u003cem\u003e\u0026nbsp;envZ\u0026nbsp;\u003c/em\u003egene might promote the utilization of FOS by \u003cem\u003eK.pneumoniae.\u0026nbsp;\u003c/em\u003eIn summary, knockout of the \u003cem\u003eenvZ\u003c/em\u003e gene and continuous induction with 0.5% FOS may promote the adaptive growth of \u003cem\u003eK. pneumoniae.\u003c/em\u003e\u003c/p\u003e\n\u003col start=\"3\"\u003e\n \u003cli\u003e\u003cstrong\u003eThe adaptive growth of FOS- or fructose-induced \u003cem\u003eK. pneumoniae\u003c/em\u003e under glucose or sucrose pressure.\u003c/strong\u003e\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003eThe adaptive growth of K2044, EKP19, EKP108 and their derivatives under glucose (Glc) or sucrose (Scr) exposure was further explored. K2044-0.5frut-90G and K2044-0.5FOS-90G exhibited better growth under glucose (Glc) or sucrose, and K2044 W90 demonstrated superior adaptive growth in glucose other than sucrose \u003cstrong\u003e(Figure 6, G; Figures S6, A-F, S)\u003c/strong\u003e. And fructose or FOS-induced strains showed better adaptive growth capacity under high concentrations of glucose (4%, 8% and 16%) \u003cstrong\u003e(Figure 6, A-C)\u003c/strong\u003e. Interestingly, compared to K2044-WT and K2044 W90, the slight planktonic growth of fructose or FOS-induced K2044 (K2044-0.5frut-90G, K2044-0.5FOS-90G, K2044-8frut-90G and K2044-8FOS-90G) was found under 1% of Glc, and conversely, with 2% of Glc exposure, K2044-8frut-90G showed enhanced planktonic growth ability and other K2044 derivatives remained inhibited growth \u003cstrong\u003e(Figure 6, D-E)\u003c/strong\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor EKP19, the growth tendency of EKP19 WT and EKP19-W90 were nearly identical under different concentrations of glucose \u003cstrong\u003e(Figure, S5)\u003c/strong\u003e. Fructose or FOS-induced strains showed better adaptive growth capacity under 8% and 16% of glucose \u003cstrong\u003e(Figure S5, A-B)\u003c/strong\u003e. Moreover, there was also limited planktonic growth of fructose or FOS-induced EKP19 (EKP19-0.5frut-90G, EKP19-0.5FOS-90G, EKP19-8frut-90G and EKP19-8FOS-90G) under 1% or 2% of Glc, while the corresponding WT and W90 strains remained unaffected \u003cstrong\u003e(Figure S5, D-E)\u003c/strong\u003e. Conversely, at 4% or 8% of Glc exposure, EKP19-0.5frut-90G exhibited favourable planktonic growth among the other induced strains \u003cstrong\u003e(Figure 6, H; Figure S5, B-C)\u003c/strong\u003e. Additionally, we found that EKP108-0.5FOS-90G still showed excellent adaptive growth under glucose exposure compared to its other induced derivatives \u003cstrong\u003e(Figure 6, I; Figures S5, G-L)\u003c/strong\u003e. Particularly under 8% glucose stress, among the other induced EKP108 strains, only EKP108-0.5FOS-90G maintained optimal growth \u003cstrong\u003e(Figure S5, H)\u003c/strong\u003e. At 1%\u0026ndash;4% of glucose, the planktonic growth capacity of fructose or FOS-induced strains of EKP108 was also limited compared to the EKP108 WT and EKP108 W90 \u003cstrong\u003e(Figure S5, I-K)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eWe also observed the adaptive growth capacity of K2044, EKP19 and EKP108, and their derivatives under different concentrations of sucrose. In K2044, K2044-0.5frut-90G and K2044-0.5FOS-90G remained the best adaptation of planktonic growth under varying concentrations of sucrose\u003cstrong\u003e\u0026nbsp;(Figure S6, A-E, S)\u003c/strong\u003e. There was no significant difference in planktonic growth of EKP19 fructose and FOS-induced derivatives under different concentrations of sucrose, showing the similar tendency of the adaptive growth with that under fructose exposure \u003cstrong\u003e(Figure S6, G-L)\u003c/strong\u003e, and these induced strains exhibited superior planktonic growth compared to EKP19 WT and EKP19 W90 under various sucrose concentrations \u003cstrong\u003e(Figure S6, T)\u003c/strong\u003e. Notably, EKP108-0.5FOS-90G still exhibited excellent adaptive growth under sucrose exposure \u003cstrong\u003e(Figure S6, M-R and U).\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFurthermore, the planktonic growth of K2044-\u0026Delta;\u003cem\u003eenvZ\u003c/em\u003e was also markedly inhibited at glucose concentrations of 1\u0026ndash;2% \u003cstrong\u003e(Figure 6, D-E)\u003c/strong\u003e. Interestingly, a significantly prolonged lag phase was observed for K2044-\u0026Delta;\u003cem\u003eenvZ\u003c/em\u003e in MRS medium supplemented with sucrose, compared to the control (MRS without sugar)\u003cstrong\u003e\u0026nbsp;(Figure S6, A-F)\u003c/strong\u003e. \u0026nbsp;\u003c/p\u003e\n\u003col start=\"4\"\u003e\n \u003cli\u003e\u003cstrong\u003eThe\u003c/strong\u003e \u003cstrong\u003eantimicrobial susceptibility test\u003c/strong\u003e\u003cstrong\u003eing of \u003cem\u003eK. pneumoniae.\u003c/em\u003e\u003c/strong\u003e\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003eThe antimicrobial susceptibility of K2044, EKP108, and their respective derivative strains was evaluated against ceftazidime (CAZ), gentamicin (GEN), levofloxacin (LEV), meropenem (MEM), and tigecycline (TGC). EKP108-0.5FOS-90G showed a reduced MIC value for LEV (a 16-fold decrease). In contrast, compared to K2044 WT, K2044-\u0026Delta;\u003cem\u003eenvZ\u003c/em\u003e demonstrated a 128-fold and 4-fold increase in MICs for GEN and TGC, respectively, indicating acquired resistance to both antibiotics \u003cstrong\u003e(Table 1)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1 \u0026nbsp; The MICs of \u003cem\u003eK. pneumoniae\u003c/em\u003e (\u0026mu;g / mL)\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"579\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 24.1796%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eStrains\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14.5078%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCAZ\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.0259%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGEN\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.0622%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eLEV\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.0259%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMEM\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.1986%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTGC\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 24.1796%;\"\u003e\n \u003cp\u003eK2044-WT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14.5078%;\"\u003e\n \u003cp\u003e0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.0259%;\"\u003e\n \u003cp\u003e0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.0622%;\"\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.0259%;\"\u003e\n \u003cp\u003e0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.1986%;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 24.1796%;\"\u003e\n \u003cp\u003eK2044-W90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14.5078%;\"\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.0259%;\"\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.0622%;\"\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.0259%;\"\u003e\n \u003cp\u003e0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.1986%;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 24.1796%;\"\u003e\n \u003cp\u003eK2044-0.5frut-90G\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14.5078%;\"\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.0259%;\"\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.0622%;\"\u003e\n \u003cp\u003e0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.0259%;\"\u003e\n \u003cp\u003e0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.1986%;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 24.1796%;\"\u003e\n \u003cp\u003eK2044-8frut-90G\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14.5078%;\"\u003e\n \u003cp\u003e0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.0259%;\"\u003e\n \u003cp\u003e0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.0622%;\"\u003e\n \u003cp\u003e0.125\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.0259%;\"\u003e\n \u003cp\u003e0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.1986%;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 24.1796%;\"\u003e\n \u003cp\u003eK2044-0.5FOS-90G\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14.5078%;\"\u003e\n \u003cp\u003e0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.0259%;\"\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.0622%;\"\u003e\n \u003cp\u003e0.125\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.0259%;\"\u003e\n \u003cp\u003e0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.1986%;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 24.1796%;\"\u003e\n \u003cp\u003eK2044-8FOS-90G\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14.5078%;\"\u003e\n \u003cp\u003e0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.0259%;\"\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.0622%;\"\u003e\n \u003cp\u003e0.125\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.0259%;\"\u003e\n \u003cp\u003e0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.1986%;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 24.1796%;\"\u003e\n \u003cp\u003eK2044-∆\u003cem\u003eenvZ\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14.5078%;\"\u003e\n \u003cp\u003e0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.0259%;\"\u003e\n \u003cp\u003e32*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.0622%;\"\u003e\n \u003cp\u003e0.125\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.0259%;\"\u003e\n \u003cp\u003e0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.1986%;\"\u003e\n \u003cp\u003e8*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 24.1796%;\"\u003e\n \u003cp\u003eEKP108-WT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14.5078%;\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.0259%;\"\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.0622%;\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.0259%;\"\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.1986%;\"\u003e\n \u003cp\u003e32\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 24.1796%;\"\u003e\n \u003cp\u003eEKP108-W90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14.5078%;\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.0259%;\"\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.0622%;\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.0259%;\"\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.1986%;\"\u003e\n \u003cp\u003e32\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 24.1796%;\"\u003e\n \u003cp\u003eEKP108-0.5frut-90G\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14.5078%;\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.0259%;\"\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.0622%;\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.0259%;\"\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.1986%;\"\u003e\n \u003cp\u003e32\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 24.1796%;\"\u003e\n \u003cp\u003eEKP108-8frut-90G\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14.5078%;\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.0259%;\"\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.0622%;\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.0259%;\"\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.1986%;\"\u003e\n \u003cp\u003e32\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 24.1796%;\"\u003e\n \u003cp\u003eEKP108-0.5FOS-90G\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14.5078%;\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.0259%;\"\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.0622%;\"\u003e\n \u003cp\u003e1*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.0259%;\"\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.1986%;\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 24.1796%;\"\u003e\n \u003cp\u003eEKP108-8FOS-90G\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14.5078%;\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.0259%;\"\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.0622%;\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.0259%;\"\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.1986%;\"\u003e\n \u003cp\u003e32\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 24.1796%;\"\u003e\n \u003cp\u003eATCC 25922\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14.5078%;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.0259%;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.0622%;\"\u003e\n \u003cp\u003e＜0.0625\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.0259%;\"\u003e\n \u003cp\u003e0.125\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.1986%;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e*: The change of MIC value\u0026ge;4-fold, compared to the Wild type. The antimicrobial susceptibility breakpoints are shown in Table S2. ATCC 25922 serves as the reference strain.\u003c/p\u003e\n\u003col start=\"5\"\u003e\n \u003cli\u003e\u003cstrong\u003eThe biofilm formation, outer membrane permeability and membrane phospholipids of\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eK. pneumoniae.\u003c/em\u003e\u003c/strong\u003e\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003eBiofilm formation and membrane permeability analysis revealed distinct phenotypic differences among the tested strains. K2044 and its derivatives, including both induced and \u003cem\u003eenvZ\u003c/em\u003e knockout strains, exhibited robust biofilm-forming capacity. In contrast, EKP19-derived strains demonstrated reduced biofilm formation relative to EKP19 WT, while no significant differences were observed among the EKP108 series strains \u003cstrong\u003e(Figure 7, A).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGiven that outer membrane permeability is a critical factor limiting the influx and efflux of compounds in gram-negative bacteria\u003c/strong\u003e, we assessed the outer membrane permeability of K2044-\u0026Delta;\u003cem\u003eenvZ\u0026nbsp;\u003c/em\u003eand EKP108-0.5FOS-90G using the NPN uptake assay to investigate the reasons for the growth changes and drug sensitivity changes produced by both strains. Results showed that both K2044-\u0026Delta;\u003cem\u003eenvZ\u003c/em\u003e and EKP108-0.5FOS-90G had significantly increased membrane permeability compared to their respective wild-type strains \u003cstrong\u003e(Figure 7, D)\u003c/strong\u003e. Interestingly, polymyxin B (PB) treatment increased outer membrane permeability in K2044 WT, EKP108 WT, and EKP108-0.5FOS-90G. However, the membrane permeability of K2044-\u0026Delta;\u003cem\u003eenvZ\u0026nbsp;\u003c/em\u003ewas not significantly increased upon exposure to PB \u003cstrong\u003e(Figure 7, D)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eTo determine whether the membrane permeability changes were associated with alterations in membrane phospholipid composition, LC-MS/MS was used to quantify key lipid species including phosphatidylglycerol (PG), phosphatidylcholine (PC), and lysophosphatidylethanolamine (LPE). \u003cstrong\u003eK2044-\u0026Delta;\u003cem\u003eenvZ\u003c/em\u003e showed reduced levels of PG-16:0-22:6 and LPE\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e(Figure 7, B)\u003c/strong\u003e. In EKP108-0.5FOS-90G, \u003cstrong\u003ePG-16:0-22:6\u003c/strong\u003e\u003cstrong\u003e, PC, and LPE levels were significantly increased\u003c/strong\u003e compared to EKP108 WT \u003cstrong\u003e(Figure 7, C)\u003c/strong\u003e.\u003c/p\u003e\n\u003col start=\"6\"\u003e\n \u003cli\u003e\u003cstrong\u003eGenomic mutation of EKP108-0.5FOS-90G\u003cem\u003e.\u003c/em\u003e\u003c/strong\u003e\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003eThere were significant changes in adaptive growth, antimicrobial susceptibility, outer membrane permeability, and membrane phospholipids in EKP108-0.5FOS-90G, to further investigate the mechanisms underlying the impact of FOS on \u003cem\u003eK. pneumoniae\u003c/em\u003e, whole genome sequencing was performed on EKP108-0.5FOS-90G and compared to its parental strain, EKP108. A total of 13 genes were mutated, of which 2 genes were synonymous and 11 genes were non-synonymous, and the most mutation sites were found on the gene encoding the conjugated transfer relaxase/helicase enzyme TraI, with 18 mutation sites \u003cstrong\u003e(Table 2)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eWhole gene sequencing comparison between EKP108-0.5FOS-90G and EKP108 WT.\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"98%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 15px;\"\u003e\n \u003cp\u003eRef_gene_ID\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003eBase change\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003eAmino acids\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 35px;\"\u003e\n \u003cp\u003eRef_gene_product\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22px;\"\u003e\n \u003cp\u003eRef_gene_function\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 15px;\"\u003e\n \u003cp\u003eGM004699\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003eG\u0026lt;-\u0026gt;A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003eE\u0026lt;-\u0026gt;K\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 35px;\"\u003e\n \u003cp\u003eSidA/IucD/PvdA family monooxygenase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 15px;\"\u003e\n \u003cp\u003eGM005147\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003eC\u0026lt;-\u0026gt;T\u003c/p\u003e\n \u003cp\u003eT\u0026lt;-\u0026gt;C\u003c/p\u003e\n \u003cp\u003eT\u0026lt;-\u0026gt;C\u003c/p\u003e\n \u003cp\u003eG\u0026lt;-\u0026gt;A\u003c/p\u003e\n \u003cp\u003eT\u0026lt;-\u0026gt;G\u003c/p\u003e\n \u003cp\u003eT\u0026lt;-\u0026gt;G\u003c/p\u003e\n \u003cp\u003eA\u0026lt;-\u0026gt;G\u003c/p\u003e\n \u003cp\u003eA\u0026lt;-\u0026gt;G\u003c/p\u003e\n \u003cp\u003eC\u0026lt;-\u0026gt;T\u003c/p\u003e\n \u003cp\u003eA\u0026lt;-\u0026gt;G\u003c/p\u003e\n \u003cp\u003eT\u0026lt;-\u0026gt;C\u003c/p\u003e\n \u003cp\u003eG\u0026lt;-\u0026gt;C\u003c/p\u003e\n \u003cp\u003eT\u0026lt;-\u0026gt;C\u003c/p\u003e\n \u003cp\u003eT\u0026lt;-\u0026gt;C\u003c/p\u003e\n \u003cp\u003eA\u0026lt;-\u0026gt;G\u003c/p\u003e\n \u003cp\u003eG\u0026lt;-\u0026gt;A\u003c/p\u003e\n \u003cp\u003eT\u0026lt;-\u0026gt;G\u003c/p\u003e\n \u003cp\u003eG\u0026lt;-\u0026gt;A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp\u003eM\u0026lt;-\u0026gt;I\u003c/p\u003e\n \u003cp\u003eM\u0026lt;-\u0026gt;V\u003c/p\u003e\n \u003cp\u003eV\u0026lt;-\u0026gt;V\u003c/p\u003e\n \u003cp\u003eP\u0026lt;-\u0026gt;S\u003c/p\u003e\n \u003cp\u003eQ\u0026lt;-\u0026gt;P\u003c/p\u003e\n \u003cp\u003eN\u0026lt;-\u0026gt;T\u003c/p\u003e\n \u003cp\u003eS\u0026lt;-\u0026gt;S\u003c/p\u003e\n \u003cp\u003eS\u0026lt;-\u0026gt;P\u003c/p\u003e\n \u003cp\u003eG\u0026lt;-\u0026gt;G\u003c/p\u003e\n \u003cp\u003eV\u0026lt;-\u0026gt;A\u003c/p\u003e\n \u003cp\u003eS\u0026lt;-\u0026gt;S\u003c/p\u003e\n \u003cp\u003eG\u0026lt;-\u0026gt;G\u003c/p\u003e\n \u003cp\u003eL\u0026lt;-\u0026gt;L\u003c/p\u003e\n \u003cp\u003eA\u0026lt;-\u0026gt;A\u003c/p\u003e\n \u003cp\u003eR\u0026lt;-\u0026gt;R\u003c/p\u003e\n \u003cp\u003eL\u0026lt;-\u0026gt;L\u003c/p\u003e\n \u003cp\u003eA\u0026lt;-\u0026gt;A\u003c/p\u003e\n \u003cp\u003eL\u0026lt;-\u0026gt;F\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 35px;\"\u003e\n \u003cp\u003eConjugative transfer relaxase/helicase TraI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22px;\"\u003e\n \u003cp\u003eHorizontal gene transfer,prokaryotic genetics\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 15px;\"\u003e\n \u003cp\u003eGM000718\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003eG\u0026lt;-\u0026gt;T\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003eH\u0026lt;-\u0026gt;Q\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 35px;\"\u003e\n \u003cp\u003eType VI secretion protein, VC_A0111 family\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22px;\"\u003e\n \u003cp\u003eAntibiotic resistance and virulence factor transmission\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 15px;\"\u003e\n \u003cp\u003eGM005287\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003eT\u0026lt;-\u0026gt;C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003eW\u0026lt;-\u0026gt;R\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 35px;\"\u003e\n \u003cp\u003eDUF262 domain-containing protein\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 15px;\"\u003e\n \u003cp\u003eGM001195\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003eG\u0026lt;-\u0026gt;A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003eW\u0026lt;-\u0026gt;X\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 35px;\"\u003e\n \u003cp\u003ePucR family transcriptional regulator ligand-binding domain-containing protein\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22px;\"\u003e\n \u003cp\u003ePurine catabolism\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 15px;\"\u003e\n \u003cp\u003eGM001228\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003eG\u0026lt;-\u0026gt;T\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003eG\u0026lt;-\u0026gt;V\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 35px;\"\u003e\n \u003cp\u003eSiderophore enterobactin receptor FepA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22px;\"\u003e\n \u003cp\u003eThe outer membrane receptor encoding the siderophore enterobacterin receptor, colicin\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 15px;\"\u003e\n \u003cp\u003eGM001774\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003eA\u0026lt;-\u0026gt;T\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003eD\u0026lt;-\u0026gt;V\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 35px;\"\u003e\n \u003cp\u003eNADH-quinone oxidoreductase subunit NuoI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 15px;\"\u003e\n \u003cp\u003eGM001861\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003eC\u0026lt;-\u0026gt;T\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003eM\u0026lt;-\u0026gt;I\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 35px;\"\u003e\n \u003cp\u003eCidB/LrgB family autolysis modulator\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 15px;\"\u003e\n \u003cp\u003eGM002855\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003eC\u0026lt;-\u0026gt;G\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003eT\u0026lt;-\u0026gt;R\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 35px;\"\u003e\n \u003cp\u003eF imbrial protein\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 15px;\"\u003e\n \u003cp\u003eGM002999\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003eC\u0026lt;-\u0026gt;A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003eG\u0026lt;-\u0026gt;C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 35px;\"\u003e\n \u003cp\u003eCarbohydrate kinase family protein\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22px;\"\u003e\n \u003cp\u003eMetabolism and transport of carbohydrates\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 15px;\"\u003e\n \u003cp\u003eGM003927\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003eG\u0026lt;-\u0026gt;T\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13px;\"\u003e\n \u003cp\u003eA\u0026lt;-\u0026gt;S\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 35px;\"\u003e\n \u003cp\u003eThiamine pyrophosphate enzyme, central domain\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003e \u003cem\u003eK. pneumoniae\u003c/em\u003e commonly colonizes the mucosal surfaces of the gastrointestinal tract in animals and healthy humans, and microbial carbohydrate metabolism plays a crucial role in the process of gastrointestinal colonization\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e. Long-term carbohydrates pressure in the gut may induce adaptive evolution in \u003cem\u003eK. pneumoniae\u003c/em\u003e, thereby affecting the adaptive growth phenotype, virulence, drug susceptibility, and metabolic stability\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. In this study, 0.5% FOS-induced strains of \u003cem\u003eK. pneumoniae\u003c/em\u003e (K2044-0.5FOS-90G and EKP108-0.5FOS-90G) showed enhanced growth capacity in a series of concentrations of fructose, FOS and other carbohydrates (glucose and sucrose), whereas K2044-8frut-90G demonstrated the lowest planktonic growth at each concentration, suggesting that long-term induction with fructose or FOS at appropriate concentrations may enhance the adaptive growth capacity of \u003cem\u003eK. pneumoniae\u003c/em\u003e, however, excessively high concentrations do not promote the growth adaptation of strains for utilizing carbon sources. Moreover, there was no significant difference in the adaptive growth of EKP19 derived strains under different concentrations of fructose or FOS. In our previous studies, we found that xylose-induced EKP19 series strains exhibited essentially consistent planktonic growth under varying xylose concentrations\u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. Similarly, glucose- or sucrose-induced EKP108 series strains showed no significant differences in adaptive growth under different glucose or sucrose concentrations\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e, demonstrating the sustained stability of some strains and the isolate-specific characteristics of \u003cem\u003eK.pneumonia\u003c/em\u003e growth adaptation under carbohydrates exposure.\u003c/p\u003e \u003cp\u003eFurthermore, we were surprised to find that the planktonic growth of fructose or FOS-induced \u003cem\u003eK.pneumoniae\u003c/em\u003e was slightly inhibited under 1\u0026ndash;2% concentrations of glucose. Many evidences have supported that several common functional proteins play key roles in the metabolism of both fructose and glucose\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e, such as GLUT2 and hexokinase (HK). This may be explained by the fact that, under prolonged fructose pressure, \u003cem\u003eK. pneumoniae\u003c/em\u003e strains downregulate the expression of functional proteins involved in efficient glucose transport and metabolism, including components of the glucose-specific phosphotransferase system (PTS) and high-affinity glucose transporters. Such changes may arise through regulatory mechanisms, such as carbon catabolite repression (CCR), or through genetic mutations, ultimately promoting survival and more efficient fructose utilization. Moreover, under higher glucose concentration conditions (8%-16%), the planktonic growth capacity of these fructose- or FOS-induced strains was restored, which may be attributed to the higher concentration of carbon sources providing energy to the bacteria, thereby facilitating the reactivation of glucose metabolic pathways. However, further validation will be required through RT-qPCR to elucidate the differences in the expression levels of glucose and fructose transporters, as well as key metabolic enzyme-encoding genes in \u003cem\u003eK.pneumonia\u003c/em\u003e (e.g., \u003cem\u003eglk, pfkA, fruK\u003c/em\u003e).\u003c/p\u003e \u003cp\u003eThe isolation of \u003cem\u003eK.pneumoniae\u003c/em\u003e in China ranked second only to \u003cem\u003eE.coli\u003c/em\u003e, and the rate of resistance to antimicrobial drugs is increasing in recent years, which suggests the severity of \u003cem\u003eK.pneumoniae\u003c/em\u003e infections worldwide\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. This study found that the MIC of LEV against EKP108-0.5FOS-90G was markedly decreased. LEV exerts its antibacterial effect by inhibiting bacterial DNA replication and disrupting cell membrane permeability\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e. And the outer membrane permeability of gram-negative bacteria is primarily determined by the composition of membrane phospholipids, the type and expression levels of porins, and adaptive responses of bacteria to environmental stimuli\u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e, alterations in phospholipid profiles can profoundly affect bacterial physiology and phenotype\u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e. In this study, EKP108-0.5FOS-90G showed increased membrane permeability and elevated phospholipid content following FOS induction, which may be contributing factors to its reduced MIC value against LEV. In contrast, the MICs of GEN and TGC in K2044-∆\u003cem\u003eenvZ\u003c/em\u003e showed a 128-fold and 4-fold increase, respectively, suggesting that knockout of the \u003cem\u003eenvZ\u003c/em\u003e gene may affect the expression of the membrane protein OmpR by disrupting the EnvZ/OmpR two-component signaling system, thereby causing the strain to become resistant to aminoglycoside and tetracycline antibiotics, which is consistent with the findings of the previous report\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e, and the underlying resistance mechanisms require further experimental validation.\u003c/p\u003e \u003cp\u003eWhole-genome sequencing and SNP analysis revealed that the gene encoding the binding transfer relaxase/helix-unwinding enzyme TraI, a DNA endonuclease primarily involved in horizontal gene transfer and prokaryotic genetics between bacteria\u003csup\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e, harbored the highest number of mutations in EKP108-0.5FOS-90G. In addition, mutations were identified in genes encoding members of the carbohydrate kinase family, which are mainly associated with carbohydrate metabolism and transport. These genetic alterations may underlie the enhanced growth capacity of EKP108-0.5FOS-90G under sugar stress conditions.\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eIn summary, this study demonstrates that \u003cem\u003eK.pneumoniae\u003c/em\u003e can undergo adaptive growth under FOS and fructose stress, with optimal growth typically observed at 4\u0026ndash;8% concentrations. Notably, K2044-0.5FOS-90G and EKP108-0.5FOS-90G exhibited the best planktonic growth under different concentrations of fructose or FOS. However, the growth of the induced strains was inhibited under 1%-2% glucose. Moreover, K2044-Δ\u003cem\u003eenvZ\u003c/em\u003e mutant showed the enhanced adaptive growth compared to K2044 WT under different concentrations of fructose and FOS, whereas a significantly prolonged lag phase was observed for K2044-Δ\u003cem\u003eenvZ\u003c/em\u003e with sucrose exposure, compared to the control. Additionally, EKP108-0.5FOS-90G further showed reduced resistance to LEV, while K2044-∆\u003cem\u003eenvZ\u003c/em\u003e exhibited increased resistance to GEN and TGC. Both EKP108-0.5FOS-90G and K2044-∆\u003cem\u003eenvZ\u003c/em\u003e also displayed elevated outer membrane permeability and altered membrane phospholipid profiles, with EKP108-0.5FOS-90G showing increased levels of PG, PC, and LPE. Whole-genome sequencing of EKP108-0.5FOS-90G identified 13 gene mutations, including extensive changes in \u003cem\u003etraI\u003c/em\u003e, a gene involved in conjugation. These findings indicate that prolonged exposure to low concentrations of FOS (0.5%) or deletion of \u003cem\u003eenvZ\u003c/em\u003e gene induces multifaceted adaptive responses in \u003cem\u003eK. pneumoniae\u003c/em\u003e, involving growth advantage, membrane remodeling, and shifts in antimicrobial susceptibility, which provide important insights into how dietary carbohydrates and bacterial signaling systems may influence colonization dynamics and clinical treatment outcomes.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eSupplementary Information\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe online version contains supplementary material available at.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eY.H. and B.X. performed the strains induction experiment, MIC assay, growth curves analysis, biofilm biomass determination and determination of bacterial outer membrane permeability, and drafted the manuscript. F.Z. constructed the knockout strain. K.F performed the membrane phospholipid extraction and quantification. J.L and Y.X are responsible for data organization and whole-genome sequencing analysis. B.C., T.H. and Z.Y conceived and designed the project and revised the manuscript. All authors have read and approved the manuscript.All authors participated in data analysis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the following grants: National Natural Science Foundation of China (82572621); Sanming Project of Medicine in Shenzhen (SZSM202303037; SZSM202103014); Shenzhen Medical Key Discipline Construction Fund; Science, Technology and Innovation Commission of Shenzhen Municipality of basic research funds (JCYJ20240813114518024, KJZD20240903103500002) and the Shenzhen Nanshan District Scientific Research Program of the People\u0026rsquo;s Republic of China (NS2025012, NSZD2024036, NSZD2024023, NSZD2024032,\u0026nbsp;NSZD2025001,\u0026nbsp;NSZD2025005,YN2025011,YN2025018).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe raw whole-genome sequencing data was posted in the Sequence Read Archive (SRA) database under accession number SRR29409842 and SRR29409843 (http://www.ncbi.nlm.nih.gov/sra).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll methods were carried out in accordance with relevant guidelines and regulations and were approved by the Ethics Committee of Shenzhen Nanshan People\u0026rsquo;s Hospital and the 1964 Helsinki declaration and its later amendments, or comparable ethical standards. The biosafety approval number is 0125F300106. All experimental procedures involving human subjects were approved by the institutional ethical committee of Shenzhen Nanshan People\u0026rsquo;s Hospital. Bacterial strains were obtained from stored samples of hospitalized patients, collected as part of the routine clinical management of patients, according to the national guidelines in China. Therefore, informed consent was not sought, and informed consent waiver was approved by the institutional ethical committee of Shenzhen Nanshan People\u0026rsquo;s Hospital.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eWYRES K L, LAM M M C, HOLT K E. Population genomics of Klebsiella pneumoniae [J]. Nat Rev Microbiol, 2020, 18(6): 344-59.\u003c/li\u003e\n\u003cli\u003eWALKER K A, MILLER V L. The intersection of capsule gene expression, hypermucoviscosity and hypervirulence in Klebsiella pneumoniae [J]. Curr Opin Microbiol, 2020, 54: 95-102.\u003c/li\u003e\n\u003cli\u003eGONZALEZ-FERRER S, PE\u0026ntilde;ALOZA H F, BUDNICK J A, et al. Finding Order in the Chaos: Outstanding Questions in Klebsiella pneumoniae Pathogenesis [J]. Infect Immun, 2021, 89(4).\u003c/li\u003e\n\u003cli\u003eCHOBY J E, HOWARD-ANDERSON J, WEISS D S. Hypervirulent Klebsiella pneumoniae - clinical and molecular perspectives [J]. J Intern Med, 2020, 287(3): 283-300.\u003c/li\u003e\n\u003cli\u003eGUERRA M E S, DESTRO G, VIEIRA B, et al. Klebsiella pneumoniae Biofilms and Their Role in Disease Pathogenesis [J]. Front Cell Infect Microbiol, 2022, 12: 877995.\u003c/li\u003e\n\u003cli\u003eWALKER A R, PHAM D N, NOEPARVAR P, et al. Fructose activates a stress response shared by methylglyoxal and hydrogen peroxide in Streptococcus mutans [J]. mBio, 2025, 16(5): e0048525.\u003c/li\u003e\n\u003cli\u003eHANNOU S A, HASLAM D E, MCKEOWN N M, et al. Fructose metabolism and metabolic disease [J]. 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Annu Rev Nutr, 2025.\u003c/li\u003e\n\u003cli\u003eDING L G Y, WU S, ET AL. CHINET 2024 bacterial resistance surveillance results [Z]. CHINET. 2024\u003c/li\u003e\n\u003cli\u003eROY R, TIWARI M, DONELLI G, et al. Strategies for combating bacterial biofilms: A focus on anti-biofilm agents and their mechanisms of action [J]. Virulence, 2018, 9(1): 522-54.\u003c/li\u003e\n\u003cli\u003eLU Q, YANG Q. Study on the Mechanism of Levofloxacin Combined with Imipenem Against Pseudomonas aeruginosa [J]. Appl Biochem Biotechnol, 2024, 196(2): 690-700.\u003c/li\u003e\n\u003cli\u003eZGURSKAYA H I, RYBENKOV V V. Permeability barriers of Gram-negative pathogens [J]. Ann N Y Acad Sci, 2020, 1459(1): 5-18.\u003c/li\u003e\n\u003cli\u003eLEUS I V, ADAMIAK J, CHANDAR B, et al. Functional Diversity of Gram-Negative Permeability Barriers Reflected in Antibacterial Activities and Intracellular Accumulation of Antibiotics [J]. Antimicrob Agents Chemother, 2023, 67(2): e0137722.\u003c/li\u003e\n\u003cli\u003eSTRAHL H, ERRINGTON J. Bacterial Membranes: Structure, Domains, and Function [J]. Annu Rev Microbiol, 2017, 71: 519-38.\u003c/li\u003e\n\u003cli\u003eDENICH T J, BEAUDETTE L A, LEE H, et al. Effect of selected environmental and physico-chemical factors on bacterial cytoplasmic membranes [J]. J Microbiol Methods, 2003, 52(2): 149-82.\u003c/li\u003e\n\u003cli\u003eLARKIN C, HAFT R J F, HARLEY M J, et al. Roles of active site residues and the HUH motif of the F plasmid TraI relaxase [J]. J Biol Chem, 2007, 282(46): 33707-13.\u003c/li\u003e\n\u003cli\u003eGUZM\u0026aacute;N-HERRADOR D L, LLOSA M. The secret life of conjugative relaxases [J]. Plasmid, 2019, 104: 102415.\u003cstrong\u003e\u003c/strong\u003e\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Klebsiella pneumoniae, Adaptive growth, Fructose, Fructooligosaccharides, Biofilm, envZ","lastPublishedDoi":"10.21203/rs.3.rs-9145216/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9145216/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e (\u003cem\u003eK.pneumoniae\u003c/em\u003e) can make use of various carbohydrates (glucose, fructose, sucrose \u003cem\u003eet al\u003c/em\u003e) as nutrient sources for sustaining its cononization and adaptive growth in human body. Fructose and fructooligosaccharides (FOS) are commonly used as dietary supplements and can be metabolized by gut probiotics, thereby promoting intestinal health and nutrient absorption, whereas the impact of prolonged exposure with fructose or FOS on the adaptive growth and antimicrobial susceptibility of \u003cem\u003eK.pneumoniae\u003c/em\u003e remains unclear. Here, fructose- or FOS-induced \u003cem\u003eK.pneumoniae\u003c/em\u003e strains were selected from the parental isolates K2044, EKP108 and EKP19. Our data indicated that two FOS-induced strains (K2044-0.5FOS-90G and EKP108-0.5FOS-90G) exhibited the best planktonic growth under different concentrations of fructose or FOS, suggesting the reshaping possibility of the growth adaption of \u003cem\u003eK.pneumonia\u003c/em\u003e under fructose or FOS pressure. Interestingly, compared with the media with varying concentrations of fructose of FOS, the planktonic growth of fructose- or FOS-induced \u003cem\u003eK.pneumoniae\u003c/em\u003e strains was significantly decreased with the culture media supplemented with low concentration of glucose (1%-2%), whereas high concentrations of glucose (8%,16%) could reactivate their adaptive growth. Moreover, K2044 knockout strain (K2044-Δ\u003cem\u003eenvZ\u003c/em\u003e mutant) showed the enhanced adaptive growth compared to K2044 WT under different concentrations of fructose, FOS or glucose, whereas a significantly prolonged lag phase was observed for K2044-Δ\u003cem\u003eenvZ\u003c/em\u003e with sucrose exposure, compared to the control. Notably, EKP108-0.5FOS-90G showed decreased resistance to levofloxacin (LEV), whereas the K2044-Δ\u003cem\u003eenvZ\u003c/em\u003e mutant displayed increased resistance to gentamicin (GEN) and tigecycline (TGC). Both EKP108-0.5FOS-90G and K2044-Δ\u003cem\u003eenvZ\u003c/em\u003e demonstrated increased outer membrane permeability and distinct alterations in membrane phospholipid composition. The genetic mutation between the parental strain EKP108 and its FOS-induced derivative (EKP108-0.5FOS-90G) were determined and we identified mutations in genes encoding members of the carbohydrate kinase family, which is primarily associated with carbohydrate metabolism and transport. Collectively, the growth adaptation, antimicrobial susceptibility changes, and membrane remodeling of \u003cem\u003eK. pneumoniae\u003c/em\u003e following fructose or FOS exposure might be isolate-specific. Furthermore, isolates exposed to high carbohydrate concentrations do not necessarily exhibit enhanced growth adaptability. Moreover, knockout of the \u003cem\u003eenvZ\u003c/em\u003e gene may also promote the adaptive growth of \u003cem\u003eK. pneumoniae\u003c/em\u003e, and involve membrane remodeling and impact the antimicrobial susceptibility.\u003c/p\u003e","manuscriptTitle":"Impact of prolonged fructose and fructooligosaccharides stress on the adaptive growth, antimicrobial susceptibility, and genomic mutation of Klebsiella pneumoniae","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-01 14:22:32","doi":"10.21203/rs.3.rs-9145216/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"90959481-cc4f-449e-8bae-d59a3c7cf943","owner":[],"postedDate":"April 1st, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Rejected","date":"2026-05-11T06:34:01+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-08T16:55:46+00:00","index":85,"fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-07T08:28:04+00:00","index":84,"fulltext":""},{"type":"reviewerAgreed","content":"71099268730396883193893672574203947606","date":"2026-05-04T16:22:27+00:00","index":83,"fulltext":""},{"type":"reviewerAgreed","content":"128058237267178102938923061093213112011","date":"2026-05-03T09:46:26+00:00","index":81,"fulltext":""},{"type":"reviewerAgreed","content":"318089282324476497312343583355298901012","date":"2026-04-30T16:33:21+00:00","index":78,"fulltext":""},{"type":"reviewerAgreed","content":"157585140590506090790816030700252259381","date":"2026-04-30T13:35:38+00:00","index":75,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-05-11T06:45:03+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-01 14:22:32","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9145216","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9145216","identity":"rs-9145216","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}