Streptococcus suis is a major porcine pathogen and an emerging zoonotic agent. Nevertheless, the lack of a global collection has hindered understanding of its epidemiological characteristics. Here, we provide a genomic overview of S. suis, encompassing 195 newly sequenced isolates and 7359 publicly available genomes. Applying cgMLST scheme and UMAP embedding approach, a total of twenty high-density clusters were identified. The clustering exhibited discordance with the serotype and MLST schemes due to recombination, indicating the limitations of classical identification methods. We also demonstrate that S. suis has an intricate global distribution and significant geographical dispersion. Serotype 2, 1/2, and 9 are top three predominant serotypes globally and displayed distinct genomic profiles. Genome reduction occurred in global clusters rather than local epidemic clusters in SS2 and SS1/2, and the loss of 89 K pathogenicity island (PAI) is a contributing factor to the SS2 reduction process. Various crucial transmission hubs and patterns have been identified for SS2 in China and SS1/2 in Europe and Japan. Concurrently, SS9 manifested remarkably distinct characteristics. Two SS9 clusters have been identified in China and Europe, respectively, and no genetic relationship was observed between them, indicating local expansion in each region of SS9. Moreover, the distribution patterns of resistance and virulence genes were closely correlated with different clusters. These results enhance our understanding of the antimicrobial resistance, pathogenicity, and transmission characteristics in a large international collection of S. suis genomes and demonstrate the genomic framework of epidemic clones at local and global scales.

Hypervirulent Klebsiella pneumoniae (hvKp) infection often induces sepsis characterised by remarkable phenotypic and functional diversity among neutrophils. However, the roles of distinct lung neutrophil compartments involved in hvKp infection remain poorly understood. Here, utilising single-cell RNA sequencing, we identified four neutrophil subsets in the lungs of hvKp-infected mice: N1, N2, N3a, and N3b, which were organised along a maturation trajectory. Functionally, early-stage subsets exhibited proinflammatory and phagocytic activities with high levels of activation, whereas terminal subsets displayed enhanced pathogen sensing and immunoregulatory properties. Notably, the N3b population showed pronounced immunosuppressive features and was predicted to inhibit lymphocyte responses through Lgals9 and Ceacam1 ligands, potentially associated with hvKp-induced lymphopenia. Projection onto NeuMap further validated the functional states of identified subsets, linking N1 to proliferative programmes, N2 to granules and hypoxia-associated metabolic activity, N3a to antigen-presentation signatures and N3b to immunosuppressive hubs. Morphologically, N3a/b were predicted to be enriched for segmented neutrophils, consistent with a more mature phenotype compared with N1 and N2. Notably, segmented neutrophils were markedly expanded in infected lungs, suggesting their association with hvKp pathogenesis. Together, our findings delineate the lung neutrophil landscape in hvKp-induced sepsis, providing insights for understanding hvKp pathogenesis and developing targeted therapeutic interventions at a neutrophil subset level.

As antimicrobial resistance emerges as a critical global health threat, food-grade bacteriocin, a kind of antimicrobial peptide (AMPs), offers promising new therapies but is hampered by poor stability and water solubility. To address this, we engineered a carrier-free self-assembly strategy: a novel bacteriocin from lactic acid bacteria in fermented food was modified to increase its hydrophobicity, enabling spontaneous formation of nano-antimicrobial bacteriocins (NAMBs) in TSB, LB, and MH media. These NAMBs exhibit a broader antimicrobial spectrum and enhanced potency against both Gram-positive and Gram-negative pathogens, including Listeria monocytogenes, Acinetobacter baumannii, and Vibrio parahaemolyticus, as evidenced by markedly reduced minimum inhibitory concentrations in vitro and superior therapeutic efficacy in infected mice in vivo. Mechanistic investigations reveal targeted disruption of cell envelope metabolism: in L. monocytogenes, NAMBs fortify the peptidoglycan layer while depleting wall teichoic acids and lipoteichoic acids, impairing carbohydrate metabolism and membrane transport; in A. baumannii, they downregulate fatty acid synthesis, disorder phospholipid composition, and weaken lipopolysaccharide integrity, culminating in membrane destabilization and cell death. These dual actions-disordering metabolic processes and remodeling bacterial cell walls or membranes-highlight the versatility of NAMBs. Our carrier-free self-assembly approach thus overcomes AMP stability and solubility limitations and paves the way for next-generation antimicrobial therapies.

Foodborne infections pose an increasing public health challenge worldwide. The problem has been aggravated by the dissemination of antimicrobial resistance genes among zoonotic pathogens, which results in a sharp increase in antibiotic resistance rate recorded among the major foodborne pathogens. To obtain an overview of the extent to which food products purchased in the markets in Hong Kong were contaminated by foodborne pathogens, we collected 95 raw meat samples from wet markets and isolated 236 bacterial strains of various species, with Escherichia coli being the most dominant species (131 strains). Contamination of food products by multiple foodborne pathogens was commonly observed. These include both Gram-positive and Gram-negative bacteria that exhibit various levels of resistance, with some possessing multiple clinically important antibiotic resistance genes. Seventeen bacterial strains of various species isolated from three food samples were comprehensively analysed by the Oxford Nanopore R10.4 technology. Novel conjugative plasmids carrying antimicrobial resistance gene-bearing mobile genetic elements were commonly detectable in the test strains. Some of the plasmids were shown to have originated from other environmental sources or other bacterial species, indicating that raw foods in the local market may serve as a reservoir of resistance-encoding genetic elements from which such elements are disseminated to various microbial pathogens. These findings suggest a need to perform periodic but comprehensive surveillance of multidrug-resistant bacterial pathogens and the major antimicrobial resistance genes in common food products, so as to disrupt the transmission routes of such organisms and the resistance-encoding genetic elements that they harbour.

The escalating global prevalence of inflammatory bowel disease (IBD) parallels widespread dietary exposure to microplastics (MPs), yet causal mechanisms linking polydisperse MPs to colitis remain elusive. Here, we show that polydisperse polystyrene microspheres (PS-MS) exacerbate dextran sulfate sodium (DSS)-induced colitis in mice by disrupting a microbiota-butyrate-PPARγ signaling axis. Mechanistically, PS-MS treatment alone does not directly induce colon inflammation in healthy mice; however, it suppresses intestinal Muc2 protein expression and impairs the mucus barrier by reducing the abundance of Lachnospiraceae_NK4A136_group and butyrate levels, thereby inhibiting PPARγ signaling and aggravating colitis. An antibiotic cocktail (ABX)-mediated microbiota ablation abolishes PS-MS-induced colitis aggravation, whereas fecal microbiota transplantation (FMT) from PS-MS-exposed donors transmits susceptibility to antibiotic-treated mice, confirming microbiota-dependent pathogenesis. Exogenous sodium butyrate supplementation restores mucosal homeostasis via PPARγ activation, as evidenced by the abolition of protection following administration of the PPARγ antagonist GW9662, and by the comparable efficacy of the PPARγ agonist 5-ASA. Our findings establish the microbiota-butyrate-PPARγ axis as a critical target for counteracting the adverse effects of environmental MPs and propose butyrate-boosting therapies as a translatable strategy against IBD.

Antibiotic resistance represents a pressing global health challenge, with polymyxins serving as one of the last lines of defense against multidrug-resistant Gram-negative pathogens. Among them, polymyxin B (PMB) has gained renewed attention; however, its clinical use is limited by systemic toxicity. This study developed porous maize starch microparticles with tunable structure and surface chemistry to modulate PMB loading and release. Porous normal maize starch (NMS) and high-amylose maize starch (HAMS) were prepared by enzymatic treatment and modified by 2,2,6,6-Tetramethylpiperidine-1-oxyl (TEMPO)-mediated oxidation to introduce anionic carboxyl groups for PMB binding. Structural characterization revealed distinct pore morphology and lamellar organization in NMS- and HAMS-derived microparticles, while oxidation increased surface charge without disrupting granular integrity. TEMPO oxidation substantially enhanced PMB loading, suggesting that electrostatic interactions between PMB and oxidized starch contributed to adsorption. In an enzyme-assisted in vitro release model, oxidized porous starches exhibited biphasic profiles, with an initial rapid release followed by a plateau, indicating heterogeneous PMB binding. Oxidized porous NMS (NMS-P-T) and oxidized porous HAMS (HAMS-P-T) showed more sustained PMB release than non-oxidized particles. PMB-loaded HAMS-P-T maintained prolonged antibacterial activity against Escherichia coli in vitro relative to NMS-based matrices. These findings demonstrate that structural modification and surface-charge influence PMB loading and release behavior in starch microparticles, enabling the design of starch-based antimicrobial peptide delivery systems.

Introduction Non-typhoidal Salmonella (NTS) is a significant bacterial pathogen causing foodborne diseases, with the potential for severe bloodstream infections (BSI) leading to complications such as sepsis and high mortality rates, particularly in vulnerable populations. The rise of BSI associated NTS strains, notably ST11 S. Enteritidis, has raised public health concerns. Objectives This study aimed to investigate the epidemiological and genetic characteristics of Salmonella BSI strains in China, focusing on the adaptation of ST11 S. Enteritidis from animal and food sources to clinical settings, and to analyze correlations between clinical data and genetic profiles. Methods A total of 120 non-duplicated NTS strains isolated from bacteremia patients across nine hospitals in China between 2017 and 2022 were investigated using antimicrobial susceptibility testing, whole-genome sequencing and bioinformatics analysis. Results The average annual prevalence of bloodstream infections (BSI) due to Salmonella remained stable at 0.77%, based on 31,177 positive blood cultures, with 96.8% of infections caused by NTS strains, primarily Salmonella enterica serovar Enteritidis (41.9%) and Typhimurium variants (16.1%). WGS results revealed significant genetic features, including mutations and multiple copies of the 5S rRNA gene, which were identified as key drivers for host adaptation and systemic dissemination, particularly in immunocompetent adults. Notably, 40% of foodborne strains carried these genetic traits, exhibiting a higher genomic potential for invasiveness compared to animal-derived strains within this specific dataset. Additionally, we demonstrated the resistance levels of BSI Salmonella to first-line antibiotics and identified a novel plasmid that mediates the co-transmission of cefotaxime resistance and virulence factors, complicating treatment options. These factors significantly contribute to the transition of diarrheagenic strains to types with an enhanced potential for bloodstream invasion. Conclusion These findings underscore the ongoing public health concern associated with NTS infections and highlight the need for continuous surveillance and effective intervention strategies to manage these emerging threats.

Antimicrobial resistance (AMR) poses a major global health threat, yet the effectiveness of national action plans (NAPs) remains uncertain. Here we developed a multidimensional One Health governance index through a structured Delphi expert consultation to evaluate AMR governance across 193 countries (2017-2022), integrating 269 policy documents, expert-weighted indicators and multinational survey and surveillance datasets. Difference-in-differences and joinpoint regression analyses were used to link governance to antimicrobial use, AMR prevalence and AMR-related mortality. Global governance scores improved from 30.7 to 44.5/100, although implementation and monitoring lagged behind policy design, particularly in animal and environmental sectors. A significant increase in AMR prevalence scores was observed only 5 years after NAP adoption (two-stage difference-in-differences, β = 2.43, 95% confidence interval (CI) 1.02-3.85, P < 0.05), underscoring the delayed impact of policy. Multisector engagement (early-adopting countries, β = 0.05, 95% CI 0.02-0.08, P < 0.01) and antimicrobial use surveillance system (early-adopting countries, β = 0.05, 95% CI 0.03-0.07, P < 0.01) showed the strongest associations with improvement in AMR outcomes. As the 2026 Global Action Plan update approaches, sustained financing and integrated One Health surveillance, with stronger environmental and agricultural engagement, are essential for translating NAPs into sustained reductions in resistance.

ABSTRACT The polymicrobial infections caused by Vibrio species, especially multidrug‐resistant isolates, are posing increasing threats to the health of coastal residents. Herein, a novel dodecapeptide, denoted as D‐zp37, was designed and synthesized, aiming to combat these notorious pathogens. Experimental results proved that the simple chirality conversion of the parent peptide, zp37, could significantly boost the antibacterial activity and proteolytic stability. To a specific cephalosporin‐resistant Vibrio alginolyticus, the minimal inhibitory concentration value of D‐zp37 was as low as 0.5 µm. Beyond the strong bacteriolytic effect against planktonic Vibrio alginolyticus, Vibrio parahaemolyticus and Vibrio vulnificus strains, D‐zp37 was found to restrain the biofilm establishment of Vibrio mixtures via inhibiting the efflux of intracellular polysaccharides. Mechanistic studies hinted that D‐zp37 impeded the electron transport chain, impaired the membrane stress response by downregulating the phage shock protein family, and blocked amino acid biosynthesis in Vibrio cells. Furthermore, three different infection models (shrimp, Galleria mellonella larvae and immunosuppressed mice) collaboratively confirmed the anti‐Vibrio efficacy of D‐zp37 in vivo. In addition, the intragastric administration of D‐zp37 was found to be beneficial in restoring the imbalanced gut microbiota induced by the polymicrobial Vibrio invasion. In summary, this work investigated the antibacterial mechanisms and in vivo efficacy of a novel peptide lead compound, proposing a new therapeutic against Vibrio infections.

Rapid and accurate detection of antibiotics is crucial for ensuring food safety. Given the different inherited chemical properties of various antibiotics, it remains challenging to design a universal system that can be expanded easily to a new target. To address this challenge, we propose a modularized strategy for antibiotic identification by leveraging the trans-cleavage activity of CRISPR-associated protein 14a (CRISPR/Cas14a), integrating a specific probe-based recognition module and a universal CRISPR/Cas amplification module for signal generation. Thus, the same single guide RNA (sgRNA) can be employed for different antibiotic targets, making the expansion of the method much more straightforward. The CRISPR/Cas signal-generation module is conservatively loyal to the presence of the trigger DNA, which is blocked in the sensing probe at the very beginning and released only in response to specific target-aptamer binding. As a proof of principle, we successfully detected tetracycline (TC) and enrofloxacin (ENR) using the same CRISPR/Cas amplification module. The effect of crowded conditions on the detection system was also explored, and BSA was found to enhance the stability of the system. The inter-batch coefficient of variation was reduced from 13.8% to 2.9%. Furthermore, the modularized system demonstrated excellent performance for TC and ENR detection, achieving limits of detection of both at 50 fg/mL, respectively, in spiked milk samples. This work introduces a strategy for small molecule detection that combines modularized sensing and CRISPR/Cas signal production, offering new perspectives for the design of simple, cost-effective, and universal detection systems.

Objective To investigate the clinical diagnostic value of the endothelial glycocalyx injury biomarker syndecan-1(SDC-1)for necrotizing enterocolitis(NEC)in preterm infants.Methods A multicenter,prospective study was conducted from February to July 2025 at the First Affiliated Hospital of Army Medical University,Sichuan Maternal and Child Health Hospital,and Liaocheng People's Hospital.Preterm infants with Bell stage Ⅱ-Ⅲ NEC were enrolled as the NEC group(n=38),and contemporaneous non-NEC preterm infants were selected in a 1∶1 ratio as the non-NEC group(n=38).Perinatal data and measurements of complete blood counts,SDC-1,and high-sensitivity C-reactive protein(hs-CRP)were collected.Multivariable logistic regression was used to evaluate risk factors for NEC.Receiver operating characteristic(ROC)curves were used to assess diagnostic performance of SDC-1.Results Neutrophil count,SDC-1,and hs-CRP levels were significantly higher in the NEC group than in the non-NEC group(P<0.05),while platelet count was significantly lower(P<0.05).Elevated SDC-1(OR=1.081,95%CI:1.028-1.137;P<0.05)and hs-CRP(OR=1.267,95%CI:1.051-1.527;P<0.05)were independent risk factors for NEC.ROC analysis showed that SDC-1(cutoff 125 ng/mL)and hs-CRP(cutoff 6.56 mg/L)yielded areas under the curve(AUCs)of 0.882 and 0.863,respectively.Their combination achieved an AUC of 0.938 with a sensitivity of 76.3%and a specificity of 97.4%.Conclusions SDC-1 is a potential biochemical biomarker for diagnosing NEC in preterm infants,but its clinical utility requires further validation in larger-sample studies.

Objectives To investigate the prevalence, genomic relatedness, and resistance characteristics of carbapenemase-gene-positive Morganella spp. (CRM) across human, animal, fly, and aquatic sources. Methods A total of 163 Morganella isolates were collected from humans (n=124), animals (n=5), flies (n=21), aquatic environment (n=13) across 13 provinces or municipalities during 2018-2024. A subset of 71 representative isolates was subjected to antimicrobial susceptibility testing (AST), whole-genome sequencing and conjugation experiments. Results Among 163 isolates, 18 were carbapenemase-gene-positive: 15 carried blaNDM-1 alone, two carried blaKPC-2 alone, and one carried both genes. They were recovered from humans, flies, and hospital sewage. Five isolates carried blaPER-4; four carbapenemase-negative carriers were resistant to both ceftazidime/avibactam and aztreonam/avibactam. The aac(3)-IV gene was associated with high apramycin MICs and was most frequent in animal- and fly-derived isolates. Phylogenetic analysis showed diverse lineages, with limited low-SNP links between human and urban-river isolates. blaNDM-1 was transferred successfully from 11 of 16 donor isolates. Conclusion CRM occur across multiple One Health niches. The findings highlight environmental and non-human reservoirs as potential contributors to their dissemination and identify blaPER-4 and aac(3)-IV as resistance-associated genes requiring further study.

ObjectiveTo analyze the antimicrobial resistance and molecular characteristics of Vibrio parahaemolyticus isolated from aquatic products in Taiyuan’s supermarkets.MethodsOne hundred and two aquatic samples were collected from major supermarkets in Taiyuan City for isolates identification by National Food Safety Standard - Food Microbiological Examination - Vibrio parahaemolyticus （ GB 4789.7-2013）， and then the purified isolates were carried out for whole genome sequencing. The sequencing data were applied to perform multilocus sequence typing （MLST）， O- and K-antigen genotyping， screening of virulence and antibiotic resistance genes， as well as functional annotation and comparison of plasmids for the isolates. A phylogenetic tree was constructed for analysis including 21 clinical and environmental isolates.ResultsThe total detection rate of Vibrio spp. in the samples was 26.47%， with V. parahaemolyticus at 13.73%. The highest V. parahaemolyticus detection rate was observed in shrimp （23.53%）， followed by mollusks （10.71%）. No V. parahaemolyticus was detected in fish samples. Antimicrobial susceptibility testing results showed that all isolates were resistant against sulfamethoxazole-trimethoprim， and the resistance rates to ampicillin and tetracycline were 52.94% and 47.06%， respectively. All isolates were susceptible to amikacin， meropenem， gentamicin， polymyxin B， tigecycline， and azithromycin.MLST analysis showed that the 17 isolates were classified into 10 sequence types （STs）， with ST722 the dominant type. The strains exhibited high genetic diversity without obvious origin-specific clustering. Gene screening revealed that the β-lactam resistance gene blaCARB-21 was the most prevalent. The same blaCARB gene subtype was found in the same ST type. Furthermore， the tetracycline resistance gene tet（B） was located on a plasmid containing the IS10 insertion sequence， indicating a potential risk of horizontal gene transfer. As for virulence genes， all isolates carried the V. parahaemolyticus specific gene tlh， and genes related to T3SS1 and T2SS. However， none of the 17 isolates carried the core virulence genes tdh or trh， nor was the T3SS2 related genes detected.ConclusionThere is a certain degree of V. parahaemolyticus contamination in aquatic products from supermarkets in Taiyuan. The isolated V. parahaemolyticus strains exhibit genetic diversity and carry various resistance and virulence genes， with some resistance genes possessing the potential for horizontal gene transfer.

The microbial community plays a crucial role in plant health and sustainable agricultural development by influencing plant physiology and development. This study aimed to explore the biocontrol potential of Bacillus subtilis A9, an endophytic bacterium isolated from Morchella sextelata, against rot disease caused by Lecanicillium aphanocladii. Metagenomic and metabolomic analyses were conducted on Morchella sextelata samples sprayed with B. subtilis A9 and a control group sprayed with LB medium. Metagenomic sequencing revealed that B. subtilis A9 significantly altered the microbial community structure and functional composition of Morchella sextelata, enriching genes related to biofilm formation, arginine and proline metabolism, and sulfur metabolism. Metabolomic analysis indicated significant upregulation of stress-resistant metabolites such as L-proline, ketoleucine, and pelargonic acid. Integrated multi-omics analysis demonstrated a strong correlation between the microbial community structure and the metabolomic profile, suggesting that B. subtilis A9 may be related to the disease-resistance response in the Morchella sextelata microecosystem. These findings provide a theoretical basis for the biological control of Morchella sextelata rot disease and support the development of green biocontrol strategies.

Abstract The tigecycline resistance gene tet (X4) is prevalent in Enterobacteriaceae, particularly in Escherichia coli . To our knowledge, no study has reported the dissemination dynamics of tet (X4) in Vibrio spp. Herein, we isolated and characterized a first tet (X4)-positive non-O1/O139 Vibrio cholerae isolate from retail pork. Genomic sequencing identified a novel tet (X4) variant in the V. cholerae chromosome, harboring a G568A nucleotide substitution that resulted in an Ala190Thr (A190T) amino acid substitution in Tet(X4). While this Tet(X4)-A190T variant conferred lower phenotypic resistance to tetracyclines (including tigecycline) than the wild-type Tet(X4), its overall catalytic efficiency against these antibiotics was paradoxically enhanced despite a reduced substrate affinity. Genomic comparisons revealed that two copies of IS CR2 flanked the variant gene, and the structure was IS CR2 - hp - hp - abh - tet (X4) G568A -IS CR2 , which is highly homologous to the reported E. coli plasmids carrying tet (X4). In addition, it confirmed the presence of an IS CR2 -mediated circular intermediate, proving this module’s capacity for horizontal transfer of the tet (X4) G568A variant. Furthermore, the IS CR2 – tet (X4) genetic structure carrying the G568A substitution was integrated within a chimeric SXT/R391-like integrative and conjugative element (ICE), which is also serving as a vehicle for genetic dissemination. As per our knowledge, this is the first report on the emergence of SXT/R391-like ICE carrying tet (X4) in Vibrio strains. Our finding demonstrates that the clinically relevant tigecycline resistance gene tet (X4), previously confined mainly to Enterobacterales from humans and livestock, is now actively spreading into environmental Vibrio populations. This cross-species transfer highlights a previously underappreciated ecological and public health concern in aquatic ecosystems. Importance Tigecycline serves as a vital last-resort antibiotic against severe multidrug-resistant bacterial infections, but its clinical efficacy is currently threatened by the rapid global dissemination of resistance genes like tet (X4). While land-based agriculture is a well-recognized reservoir for these genes, the role of aquatic ecosystems and environmental pathogens, such as V. cholerae , in harboring tet (X) determinants remains largely unexplored. In this study, we characterize a non-O1/non-O139 V. cholerae isolate from retail pork that harbors a naturally occurring, chromosomally integrated tet (X4) G568A variant. This novel variant exhibits elevated catalytic efficiency against tetracycline antibiotics. The tet (X4) G568A allele is embedded in a highly conserved structural module (IS CR2 – tet (X4)– abh – hp – hp- IS CR2 ) flanked by two IS CR2 repeats, which is integrated into an SXT/R391-like ICE at the chromosomal prfC locus. These findings provide the first high-confidence genomic evidence of tet (X4) in V. cholerae , highlighting aquatic Vibrio species as critical environmental reservoirs for clinically significant antimicrobial resistance genes and emphasizing the urgent need for continuous genomic surveillance.

Pseudomonas aeruginosa is a significant opportunistic pathogen, particularly prevalent in intensive care units (ICUs). Through a comprehensive genomic and phenotypic analysis of 518 ICU and healthy isolates collected over a 13-year period (2010-2022), we found that ICU strains, despite reduced sequence type (ST) diversity, are dominated by persistent high-risk clones, notably ST463 and ST1076. Despite possessing a lower overall prophage content, ICU isolates show a broader and more diverse repertoire of anti-phage defense systems. This strengthened defense capacity correlates with ICU strains, demonstrating heightened resistance to phage challenge and contributing to lower historical phage exposure. Concurrently, ICU isolates harbor a significantly higher antimicrobial resistance (AMR) gene burden. Our detailed genomic analysis shows that this increased AMR is primarily driven by plasmid acquisition. Importantly, AMR genes associated with prophage elements are exclusively found in ICU isolates, highlighting their selective retention and functional contribution to resistance in this high-pressure environment. Furthermore, specific virulence genes, including exoU, pilA, and rhsP2, are more prevalent in ICU strains, indicating enhanced pathogenicity. Collectively, these findings underscore a qualitative distinction in ICU P. aeruginosa: their dominance and persistence stem from highly adapted clones, robust anti-phage defenses, rapid plasmid-mediated AMR acquisition, and clinically selected prophage-borne AMR.

The unresponsiveness of bacterial tolerant cells to antibiotics has been attributed to physiological dormancy triggered by environmental stresses. Interestingly, after a 24 - hour treatment, the tolerant cells became susceptible to ciprofloxacin and nitrofurantoin, which target DNA, as well as gentamicin, which targets protein synthesis, with distinct combinational effects. However, they remained resistant to ampicillin, which targets the cell wall. The mechanism study revealed that the activities of protein synthesis and DNA repair in tolerant bacteria formed during nutrient starvation remained at a high level and only gradually decreased during six days of starvation. Meanwhile, the reduction in energy production (ATP level), antioxidant defense (ROS level), and efflux functions led to an increased susceptibility of the bacterial tolerant subpopulation to antibiotics. These findings confirm that bacterial tolerant cells remain physiologically active, and inhibiting protein synthesis and DNA repair presents promising opportunities for combating bacterial tolerance with existing antibiotics.