Elizabethkingia species are emerging multidrug-resistant opportunistic pathogens associated with high mortality in critically ill patients. However, the genomic epidemiology and mechanisms of fluoroquinolone resistance in respiratory Elizabethkingia isolates remain incompletely understood. The objective of this study was to characterize the clinical, genomic, and resistance features of respiratory Elizabethkingia isolates from a tertiary pulmonary hospital and to explore the structural basis of fluoroquinolone non-susceptibility associated with DNA gyrase subunit A (GyrA) S83I substitution. A total of 18 non-duplicate Elizabethkingia isolates from 16 patients with severe pulmonary disease at a tertiary hospital (2024–2025) were included. Whole-genome sequencing was performed for species identification, pangenome analysis, and core-genome SNP-based phylogenetic reconstruction. Antimicrobial susceptibility testing was conducted using the VITEK® 2 system. Mutations in the quinolone resistance-determining region (QRDR) were analyzed, and the structural impact of the GyrA S83I substitution was evaluated by molecular docking. Among the 18 isolates, 15 were identified as Elizabethkingia anophelis and 3 as Elizabethkingia meningoseptica. Pangenome analysis demonstrated an open genome structure (γ = 0.156). Core-genome phylogeny revealed distinct clade-specific clustering, with seven highly related E. anophelis isolates in Clade B, suggesting potential nosocomial transmission. All isolates exhibited broad resistance to multiple antimicrobial classes, whereas minocycline retained full in vitro activity. Fluoroquinolone non-susceptibility was strongly associated with lineage and significantly correlated with the GyrA S83I substitution. Molecular docking analysis showed that this substitution reduced the binding affinity of ciprofloxacin and levofloxacin to GyrA (ΔΔG ≈ 3–6 kcal/mol). However, because GyrA S83I was largely lineage-restricted, its independent contribution could not be disentangled from clade background, and several non-susceptible isolates lacked S83I, indicating additional mechanisms, our integrated genomic and structural findings underscore the critical interplay between localized clonal expansion and target-site adaptation in driving fluoroquinolone resistance in clinical Elizabethkingia strains.

The escalating prevalence of antibiotic resistance in Staphylococcus aureus has underscored the urgent necessity for groundbreaking anti-infective therapies that target novel pathways. Metabolic intermediates, such as those in the TCA cycle, have been known to be linked to bacterial virulence. L-Malic acid (L-MA), a natural TCA cycle intermediate, is known for its antimicrobial and antioxidant properties; however, its effect on the virulence of Staphylococcus aureus has yet to be explored. It was found that L-malic acid not only inhibited bacterial growth but also markedly reduced the production of crucial virulence factors, including staphyloxanthin and α-hemolysin. The suppression of pigment synthesis was primarily due to the competitive antagonism of CrtO, a key oxidoreductase responsible for carotenoid biosynthesis in Staphylococcus aureus, coupled with enhanced flux through the TCA cycle that redirected acetyl-CoA pools away from the mevalonate pathway. Moreover, hemolytic activity was decreased by lowering the expression of α-hemolysin. In vivo studies using mouse infection models further demonstrated that L-malic acid effectively mitigated the pathogenicity of Staphylococcus aureus, significantly reducing the formation of skin and liver abscesses. These findings position L-malic acid as a promising agent targeting Staphylococcus aureus infections, highlighting the need for further research into its therapeutic potential.

Abstract Background Citrobacter braakii (C. braakii) is a gram-negative bacterium associated with hospital-acquired infections such as respiratory tract infections and bacteremia. There has been a gradual increase in the number of C. braakii infection cases in recent years. The antimicrobial resistance level of C. braakii has been steadily increasing, and the coexistence of multiple resistance genes further complicates the selection of appropriate clinical antibiotic therapies. Results we reported a multidrug-resistant C. braakii W221 co-harboring bla NDM−1, bla IMP−4, and bla OXA−181 with four key resistance encoding plasmids (pW221-1, pW221-2, pW221-4 and pW221-5). The results of antimicrobial susceptibility testing indicated that W221 exhibited high-level resistance to aminoglycosides, carbapenems and ceftazidime-avibactam. Conjugation assays indicated that plasmid pW221-1, bla NDM−1-carrying plasmid pW221-4 and bla OXA−181-carrying plasmid pW221-5 were transferrable to Escherichia coli (E. coli). In addition, bla NDM−1-carrying plasmid pW221-4 and bla OXA−181-carrying plasmid pW221-5 also could transfer to Klebsiella pneumoniae. Notably, mobilizable plasmid pW221-1 not only carried multiple resistance elements (such as sul1, qnrA1, etc.) but also possessed virulence factors (vipA/tssB). We also found that bla NDM−1, rmtC and sul1 resistance genes and virulence factor htpB co-occurred on the same mobilizable plasmid pW221-4. Detailed genetic analysis showed that multiple transposons (Tns) and insertion sequences (ISs) were found surrounding the vital resistant genes, which could stimulate mobilization of resistant determinants. bla IMP−4 was located on the class 1 integron In823. In addition, the fosA3-bla SHV−12-sul2-aph(3’’)-Ib-aph(6)-Id -qnrS1 antibiotic resistance island (ARI) in pW221-2 was surrounded by Tn3, IS26, IS5075, ISKpn19, and Tn5403. Moreover, bla NDM−1-carrying plasmid pW221-4 was typed as IncFII plasmid, which was known to have high-efficiency transmissibility. The bla OXA−181 gene was characterized by the following structure: IS26-ISEc63-IS3000-bla OXA−181-ISKpn19-ISMex22-qnrS1-ISAs17-IS26. Conclusions we isolated a C. braakii W221 co-existing bla NDM−1, bla IMP−4, and bla OXA−181, and this was first reported in the world. The presence of multiple transferrable and mobilizable plasmids carrying key resistance determinants suggested that this strain may have high potential for horizontal gene transfer and rapid dissemination. These findings suggesting that clinical settings should be vigilant against the further emergence, spread and prevalence of such novel multidrug-resistant strains.

Abstract The globally disseminated high-risk CRKP clone ST5422, though frequently harboring multiple carbapenemase genes, exhibits only sporadic occurrence in China. We report a Shanghai genetic clustering involving seven ST5422 CRKP isolates harboring blaKPC-2. Comprehensive characterization through antimicrobial susceptibility testing, whole-genome sequencing, phylogenetic reconstruction, and virulence/fitness assessments revealed all strains co-harbored blaCTX-M-15 with homogeneous resistance profiles: high-level resistance to carbapenems, ceftazidime/avibactam, and tigecycline, while maintaining susceptibility to colistin and amikacin. Phylogenetic analysis of all 13 ST5422 strains available in NCBI resolved three distinct clades. Subsequent characterization revealed that our clinical isolates exhibited significantly enhanced virulence and antimicrobial resistance relative to these reference strains. ST5422 isolates showed a higher virulence and certain adaptability compared with NUTH-K2044 and HS11286 (ST11). We conclude that localized dissemination of ST5422 CRKP carrying blaKPC-2 in China constitutes a significant public health concern requiring coordinated containment strategies. Importance K. pneumoniae is one of the most relevant opportunistic pathogens and causes various human infections. CRKP is not only known for its resistance to various antibiotics but also for the emergence of highly virulent strains. ST11 CRKP is the dominant clone, accounting for 60

BackgroundCitrobacter braakii (C. braakii) is a gram-negative bacterium associated with hospital-acquired infections such as respiratory tract infections and bacteremia. There has been a gradual increase in the number of C. braakii infection cases in recent years. The antimicrobial resistance level of C. braakii has been steadily increasing, and the coexistence of multiple resistance genes further complicates the selection of appropriate clinical antibiotic therapies.Resultswe reported a multidrug-resistant C. braakii W221 co-harboring blaNDM-1, blaIMP-4, and blaOXA-181 with four key resistance encoding plasmids (pW221-1, pW221-2, pW221-4 and pW221-5). The results of antimicrobial susceptibility testing indicated that W221 exhibited high-level resistance to aminoglycosides, carbapenems and ceftazidime-avibactam. Conjugation assays indicated that plasmid pW221-1, blaNDM-1-carrying plasmid pW221-4 and blaOXA-181-carrying plasmid pW221-5 were transferrable to Escherichia coli (E. coli). In addition, blaNDM-1-carrying plasmid pW221-4 and blaOXA-181-carrying plasmid pW221-5 also could transfer to Klebsiella pneumoniae. Notably, mobilizable plasmid pW221-1 not only carried multiple resistance elements (such as sul1, qnrA1, etc.) but also possessed virulence factors (vipA/tssB). We also found that blaNDM-1, rmtC and sul1 resistance genes and virulence factor htpB co-occurred on the same mobilizable plasmid pW221-4. Detailed genetic analysis showed that multiple transposons (Tns) and insertion sequences (ISs) were found surrounding the vital resistant genes, which could stimulate mobilization of resistant determinants. blaIMP-4 was located on the class 1 integron In823. In addition, the fosA3-blaSHV-12-sul2-aph(3'')-Ib-aph(6)-Id -qnrS1 antibiotic resistance island (ARI) in pW221-2 was surrounded by Tn3, IS26, IS5075, ISKpn19, and Tn5403. Moreover, blaNDM-1-carrying plasmid pW221-4 was typed as IncFII plasmid, which was known to have high-efficiency transmissibility. The blaOXA-181 gene was characterized by the following structure: IS26-ISEc63-IS3000-blaOXA-181-ISKpn19-ISMex22-qnrS1-ISAs17-IS26.Conclusionswe isolated a C. braakii W221 co-existing blaNDM-1, blaIMP-4, and blaOXA-181, and this was first reported in the world. The presence of multiple transferrable and mobilizable plasmids carrying key resistance determinants suggested that this strain may have high potential for horizontal gene transfer and rapid dissemination. These findings suggesting that clinical settings should be vigilant against the further emergence, spread and prevalence of such novel multidrug-resistant strains.

Given the global crisis of antimicrobial resistance, the dual anticancer and antibacterial properties of certain agents have attracted increasing attention as a potential alternative to conventional antimicrobial approaches. In this study, we characterized the bactericidal activity of an anticancer molecule (Pan-Ras-In-1) against Staphylococcus aureus, a pathogen associated with refractory infection. In vitro assays revealed that Pan-Ras-In-1 has a more pronounced bactericidal effect on S. aureus cells than the traditional antibiotics vancomycin and levofloxacin. Furthermore, Pan-Ras-In-1 displayed potential therapeutic efficacy in vivo, as assessed using the murine systemic infection and skin abscess models, suggesting its potential for subsequent clinical applications. Exposure of S. aureus to Pan-Ras-In-1 activates CidA-associated peptidoglycan hydrolase systems, which partially mediate bacterial programmed cell death. Peptide-centric local stability analyses revealed that Pan-Ras-In-1 binds to TarA protein, suppressing wall teichoic acid polymer production. The combined action of two mechanisms enables Pan-Ras-In-1 to compromise bacterial cell wall integrity.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) infections have emerged as a major global public health threat. Cefiderocol is a novel siderophore-conjugated cephalosporin with potent activity to treat severe infections caused by CRKP. In this study, we investigated the underlying mechanisms leading to cefiderocol resistance in an ST11-KL64 hypervirulent K. pneumoniae FK3023 isolated from a patient with infective fever and chronic kidney disease in China. Whole genome sequencing (WGS), amino acid sequences alignment, cirA gene complementation and lactamases genes cloning assays were performed to elucidate the molecular basis of cefiderocol resistance. Pathogenicity was evaluated by quantitative siderophore production, string test, capsule quantification, and a Galleria mellonella infection model. This strain carried a hybrid plasmid that contained both virulence and resistance determinants, including key virulence factors (iutA-iucABCD operon and rmpA gene) and β-lactamases genes (blaKPC−2, blaTEM−1B, blaCTX−M−3, blaCTX−M−65). Compared to the wild-type siderophore receptor CirA, K. pneumoniae FK3023 carried 119 nucleotide deletion (c.1830_1948del), leading to a frameshift, followed by a premature stop codon (Val611TyrfsTer32), resulting in a novel mutation in CirA. K. pneumoniae FK3023 complemented with the wild-type cirA showed a substantial decrease in cefiderocol MIC, further confirming the function of CirA. Each of the six identified β-lactamases genes in K. pneumoniae FK3023 was cloned into a pUC vector, and then expressed in Escherichia coli cells. The E. coli strains carrying blaSHV−12 showed an increase in cefiderocol MIC compared to the E. coli strains carrying empty vectors. In conclusion, we described a clinical ST11-KL64 hypervirulent K. pneumoniae isolate exhibiting cefiderocol resistance likely mediated by a novel CirA truncation and blaSHV−12. K. pneumoniae FK3023 also carried the hybrid plasmid and two additional resistance plasmids, highlighting the convergence of hypervirulence and multidrug resistance and underscoring the challenges for clinical management.

The emergence and spread of carbapenem-resistant Klebsiella pneumoniae (CRKP) ST37 necessitates urgent reassessment of its pathogenic potential. This study aims to characterize the genomic and phenotypic features of ST37 isolates collected from a tertiary hospital in eastern China, from December 2020 to August 2023. Fifteen CRKP isolates underwent antimicrobial susceptibility testing, whole-genome sequencing (WGS), and phylogenetic analysis for resistance/virulence gene profiling. Phenotypic virulence assays, including quantitative siderophore production, string test, mucoviscosity, capsule quantification, serum resistance, and Galleria mellonella in vivo infection model were designed to evaluate the level of pathogenicity of the isolates. All ST37 Klebsiella pneumoniae isolates exhibited resistance to carbapenems, with 60.0% (9/15) harboring blaKPC-2 alongside extensive ESBL genes. Critically, a significant subset (26.7%, 4/15; YF1062/YF1072/YF2197/YF2203; KL25/O5) demonstrated hypervirulent phenotypes, evidenced by elevated siderophore production, aerobactin gene carriage (iucABCD/iutA), and extreme lethality in Galleria mellonella, confirming convergent ST37 CRKP-hvKP emergence. This study confirms the emergence of hypervirulent strains within the carbapenem-resistant ST37 lineage, representing a significant convergent threat necessitating urgent surveillance and novel interventions.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) poses a significant threat to global public health. Identifying high-risk clones that facilitate the global dissemination of carbapenemases is essential for developing effective strategies to address this challenge. Here, we collected 29 ST307 CRKP isolates harbouring the bla KPC-2 and/or bla NDM-5 genes from three hospitals. bla NDM-5 was carried by the IncX3 plasmid, and bla KPC-2 was located on an IncFII plasmid. All of them were horizontally transmissible as verified by plasmid conjugation assays. Phylogenetic analysis revealed a clonal outbreak involving 27 of the isolates in this study and further demonstrated an emerging trend of bla KPC-2 - and bla NDM-5 -positive CRKP strains within the ST307 lineages in China since 2023. Resistance to ‘last-resort’ antibiotics mediated by genetic mutations is a major contributor to treatment failure in CRKP infections. In this study, we functionally confirmed that the plasmid-borne tet (A) I235F mutation confers resistance to both tigecycline and eravacycline. The results of the murine bloodstream infection model further demonstrated that the tet (A) I235F mutation increased the treatment cost of tigecycline. Furthermore, we found colistin resistance in ST307 CRKP primarily mediated by mutations in the mgrB and pmrB genes. Specific mutation patterns, including mgrB disruption by IS 5D , mgrB W20* and pmrB S203P substitutions, were identified in nine colistin-resistant ST307 CRKP isolates in this study. These findings highlight the need for enhanced surveillance and control measures to prevent the potential widespread outbreak of extensively drug-resistant ST307 isolates in China.

OBJECTIVES:This study aimed to investigate the anti-virulence effects of subinhibitory concentration of clarithromycin on Staphylococcus aureus and to explore the underlying molecular mechanism. METHODS:Haemolytic activity was measured using rabbit erythrocytes. α-haemolysin production was assessed by Western blot analysis. Transcriptional changes were analysed by RT-qPCR and promoter activity assays. To verify the target mechanism, a ΔsaeR mutant and a complemented strain were constructed. Molecular docking and DARTS assay were employed to evaluate the potential clarithromycin-SaeR interaction. Substitution of a single residue, Arg154, with alanine was performed to determine its functional role. In vivo efficacy of clarithromycin was assessed using the Galleria mellonella infection model. RESULTS:Clarithromycin at 1/8MIC reduced haemolytic activity in methicillin-resistant S. aureus strains MR200 and MR271 from 83.61% ± 0.86% to 9.05% ± 4.13% and from 85.12% ± 1.95% to 45.24% ± 1.06%, respectively, and significantly decreased α-haemolysin production. Clarithromycin selectively downregulated saeR, with a 1.87-fold reduction in MR200 and 1.54-fold reduction in MR271, while the agr pathway remained largely unaffected. Promoter activity assays confirmed suppression of the saeR-P1 promoter. Haemolytic activity and α-haemolysin production were significantly reduced in the ΔsaeR mutant, and clarithromycin caused no additional inhibition, and these effects were restored upon saeR complementation. Molecular docking and DARTS assay suggested a potential SaeR-clarithromycin binding. Arg154 substitution eliminated both SaeR regulatory activity and drug-mediated virulence suppression. In vivo, 1/8MIC clarithromycin improved larval survival to over 90%. CONCLUSIONS:This study demonstrated that clarithromycin exerted an anti-virulence effect against S. aureus by targeting the SaeR, highlighting a potential strategy for repurposing antibiotics to mitigate bacterial pathogenicity.

The prevalence of mupirocin resistance in MRSA severely limits therapeutic options for skin and soft tissue infections. This study aimed to evaluate the potential synergistic activity between mupirocin and protocatechuic acid ethyl ester (EDHB) through in vitro and in vivo investigations. Clinical S. aureus isolates were characterized for antibiotic resistance profiles and molecular features via antimicrobial susceptibility testing, MLST, and spa typing. For MRSA isolates, checkerboard and time-kill assays were performed to assess in vitro synergy. The potential interference of EDHB with bacterial membrane integrity and efflux pumps was investigated using propidium iodide and ethidium bromide, respectively. The disk diffusion method was applied to test the retained antimicrobial activity of mupirocin and EDHB in ointment formulations. A murine dermal wound model was established to evaluate in vivo efficacy by topical application of mupirocin and EDHB, alone or in combination, on infected wounds. EDHB alone exhibited limited activity but synergistically reduced mupirocin MICs by 4-8-fold in most strains. Checkerboard analysis revealed synergistic or partial synergistic interactions against MRSA. Time-kill curves further indicated that combining these two drugs can effectively inhibit the planktonic S. aureus. EDHB rapidly disrupts cytoplasmic membrane integrity via concentration-dependent propidium iodide influx, independent of norA/mepA efflux pump modulation. The enhanced antibacterial activity of mupirocin and EDHB was sustained in ointment formulations, resulting in superior therapeutic outcomes with combination therapy compared to monotherapy. EDHB acts as a membrane-disrupting adjuvant that synergizes with mupirocin against MuR-MRSA, offering a promising strategy to combat recalcitrant S. aureus infections through localized combination therapy.

The global emergence of multidrug-resistant (MDR) Klebsiella pneumoniae, particularly carbapenem-resistant K. pneumoniae (CRKP), presents a severe public health threat, limiting available treatment options. Tigecycline and eravacycline, have been considered a last-resort therapeutic against MDR Enterobacteriaceae. However, strains were resistant to these antibiotics increased recently. The tmexCD-toprJ, a plasmid-encoded resistance-nodulation-division (RND)-type efflux pump, has emerged as a critical factor conferring resistance to tigecycline and eravacycline. In this study, we reported the emergence of 11 CRKP isolates harboring tmexCD-toprJ, isolated from two lung transplant patients in a tertiary hospital in eastern China. Most of the isolates (82%) exhibited high-level resistance to tigecycline and eravacycline, along with other common antibiotics. Whole-genome sequencing (WGS) and phylogenetic analysis indicated these strains are not clonal, and resistance phenotypes were associated with the tmexCD-toprJ operon and other crucial resistance elements. We also found the tmexCD-toprJ operon was located on a conjugative plasmid, sharing high sequence similarity with the operon identified in Pseudomonas aeruginosa. Our results showed that the tmexCD-toprJ-harboring plasmid is efficiently transferable, which contributes to the dissemination of tigecycline and eravacycline resistance. At the same time, the plasmid can coexist with the blaKPC-2-carrying plasmid, which may cause multidrug resistance. The emergence of tmexCD-toprJ-positive CRKP in lung transplant patients highlights the potential for rapid nosocomial dissemination and reduced treatment efficacy of last-line antimicrobials. Our findings emphasize the need for enhanced genomic surveillance, infection control measures, and alternative therapeutic strategies to combat the spread of tmexCD-toprJ-mediated resistance in clinical settings.

Background:The global emergence of carbapenem-resistant Klebsiella pneumoniae (CRKP) has become a critical public health threat. However, the epidemiological significance of certain sequence types (STs) remains underappreciated. Among these, ST792-a lineage rarely documented in global surveillance studies-has recently emerged as a concerning threat in southern China. In this study, we characterized the epidemiological features and antimicrobial resistance mechanisms of CRKP ST792 isolates collected during a dissemination in a hospital in southern China. Methods:Seven separate clinical isolates were collected from hospitalized patients between January 2021 and March 2022. Bacterial isolates were identified, and antimicrobial susceptibility testing was conducted using the VITEK-2 compact automated system. Whole-genome sequencing (WGS) was performed on all seven isolates to confirm the presence of resistance genes. Additionally, a representative strain (G5) was selected for in-depth genomic characterization using long-read sequencing to analyze its genetic features and mobile genetic elements. Conjugation experiments were conducted to assess the transferability of the resistance plasmids. Results:All isolated strains were identified as ST792-type CRKP carrying blaKPC - 2 through whole-genome sequencing. The strains harbored additional resistance genes including blaSHV - 148, blaCTX - M-3, blaTEM - 1B, qnrS1, OqxA and OqxB. Genomic characterization of representative strain G5 revealed a circular chromosome and three resistance plasmids. The blaKPC - 2 gene was located on a 102,257 bp IncFIB(pQil) plasmid with a Tn3-TnpR-ISKpn27-ISKpn28-blaKPC - 2-ISKpn6 genetic structure. Conjugation experiments demonstrated successful transfer of two accessory plasmids (p[G5]-2 and p[G5]-3) to Escherichia coli EC600, confirming their mobility and potential role in resistance gene dissemination. Conclusion:This study characterizes the nosocomial dissemination of KPC-2-producing K. pneumoniae ST792 strains, elucidating their antimicrobial resistance patterns and plasmid-mediated transmission mechanisms to inform infection control strategies. The urgent need for enhanced surveillance and strict implementation of infection control measures is underscored to mitigate the spread of hospital-acquired multidrug-resistant pathogens.

ABSTRACT Staphylococcus aureus sequence type 965 (ST965) represents a significant member of clonal complex 5. However, there are still significant gaps in research regarding the resistance, virulence, and distribution characteristics of methicillin-resistant S. aureus (MRSA) ST965. Here, we conducted a comprehensive analysis of 27 ST965-MRSA genomes from three different countries, including 20 genomes sequenced in this study and 7 genomes retrieved from public databases. Our results indicate that ST965-MRSA mainly originates from China (92.6%, 25 out of 27) and was first reported in 2013. SCCmec IV typing (75%, 21 out of 27) and spa t062 typing (75%, 21 out of 27) constituted its predominant clonal characteristics. Among ST965-MRSA clinical isolates, bloodstream infection samples accounted for 35%. A novel multidrug-resistant plasmid (pYF965) was identified in ST965-MRSA, which contains erm genes integrated via Tn551 into pTZ2162 and simultaneously contains aac(6′)-Ie and blaI-blaR-blaZ. Through the acute pathogenicity model, ST965-MRSA exhibited higher hemolytic activity and mortality rates compared to N315 (ST5), yet their overall virulence remained significantly lower than the highly virulent strain USA300-LAC (ST8) (P < 0.0001). Notably, in terms of persistent infection, the bacterial colonization levels of ST965-MRSA in the heart, lungs, and spleen in vivo were significantly higher than those of N315 (ST5) and USA300-LAC (ST8) (P < 0.05). Particularly, its immune evasion ability and intracellular survival ability were superior to those of ST5 and ST8 (P < 0.05). This study elucidated the biological characteristics of ST965-MRSA for the first time, expanding our understanding of the molecular epidemiology of the ST965 clone.IMPORTANCEAs an emerging clonal lineage, ST965 has been predominantly discovered in Asian regions. However, research regarding the ST965-MRSA remains highly limited at present. Therefore, we conducted a detailed analysis of the genomic characteristics of ST965-MRSA and, combined with phenotypic experiments, further revealed their potential pathogenic risks. This study initially uncovers the potential global transmission and genetic and phenotypic features of ST965-MRSA and offers valuable insights for controlling and preventing persistent hospital infections.

Carbapenem-resistant Klebsiella pneumoniae (CRKP), particularly strains producing Klebsiella pneumoniae carbapenemase (KPC), pose a severe global health challenge due to limited therapeutic options and high mortality. While ST11 is the predominant KPC-producing lineage in China, the clinical and epidemiological significance of ST656 remains largely unexplored. We conducted a genomic and epidemiological investigation of twenty-five KPC-2-producing K. pneumoniae ST656 isolates collected from patients with severe pulmonary comorbidities in a tertiary hospital in Eastern China between July 2020 and October 2021. Antimicrobial susceptibility testing, whole-genome sequencing (WGS), phylogenetic analysis, plasmid profiling and conjugation assays were performed to determine resistance mechanisms, clonal relationships and plasmid transferability. Clinical information showed all 25 isolates originated from distinct patients in a tertiary hospital in Eastern China, predominantly older male post-lung transplantation cases with severe pulmonary disease, from intensive care units and thoracic surgery wards. All isolates displayed highly similar multidrug-resistant profiles, being resistant to most β-lactams, levofloxacin and trimethoprim-sulfamethoxazole, with limited susceptibility to amikacin and tigecycline. Genomic characterization of representative strain CRE132 showed that blaKPC−2 was located on a conjugative IncFII plasmid, and that this plasmid could transfer carbapenem resistance to Escherichia coli in conjugation assays. ST656 isolates in our study exhibited modest increase in virulence-associated traits, growth rates and certain adaptability compared with ST11 isolates. This study represents the first genomic and epidemiological investigation demonstrating nosocomial clonal dissemination of KPC-2–producing K. pneumoniae ST656 in China. These findings highlight the importance of continued genomic surveillance and improved understanding of this emerging lineage.

The objective of this research was to investigate the genomic epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) in a pediatric population in Shanghai, China. Whole-genome sequencing was conducted for 492 randomly selected MRSA isolates obtained from a pediatric hospital between 2013 and 2022. ST59 (37.4%), ST398 (22.4%), ST88 (5.7%), and ST22 (5.5%) were the predominant lineages among these children. While ST59 maintained a dominant annual proportion before 2017, the proportion of ST398 gradually increased from 2013 to 2016, with ST398 ultimately emerging as a prevalent clone with a proportion comparable to that of ST59 after 2017. Among the prevalent STs, the spa-SCCmec structure also experienced dynamic changes. Within ST59, the t437-IV subtype experienced a decline and has even been replaced by t172-IV in recent years. In ST398, the t011-V subtype appeared in 2014 and rapidly became the leading subtype. The antibiotic resistance profiles and virulence factors exhibited clone-related features. Compared with other prevalent lineages, ST59 presented high resistance to erythromycin and clindamycin, whereas ST398 presented relatively low resistance to common antimicrobial agents and fewer virulence determinants. Panton-Valentine leucocidin was more common in ST338 and ST1232, whereas toxic shock syndrome toxin was closely associated with ST1 and ST5. The MRSA cases could also be classified into community- and hospital-associated cases, with highly significant differences between the two in terms of demographic characteristics, clindamycin susceptibility, and virulence genes. In conclusion, this study revealed high genetic diversity and dynamic changes in the molecular epidemiology of pediatric MRSA isolates from Shanghai collected over a decade. IMPORTANCE:Methicillin-resistant Staphylococcus aureus (MRSA) has emerged as a significant global health concern. Previous research on MRSA epidemiology has predominantly focused on adult populations or targeted specific infection sites, while there was limited research on the long-term evolution of MRSA from the pediatric population. This study addresses this knowledge gap by conducting a comprehensive, 10-year surveillance of pediatric MRSA isolates using whole-genome sequencing. We characterized the molecular typing, as well as the phenotypic and genotypic antimicrobial resistance profiles, and virulence factors present in MRSA isolates obtained from children. Our results highlight the imperative for continuous, vigilant monitoring of MRSA within the pediatric demographic to track its evolving genetic landscape.

Staphylococcus aureus, an opportunistic pathogen of global health concern, presents a significant clinical challenge due to its escalating antibiotic resistance and biofilm-forming capacity. The biofilm matrix of S. aureus is enriched with carotenoids, primarily staphyloxanthin (STX), which function as virulence factors by scavenging reactive oxygen species and inhibiting antimicrobial peptides. In this study, we examined the impact of the methanol extract of S. aureus (MES) on biofilm formation. Our findings revealed that MES enhanced biofilm formation in S. aureus strains with inherently weak biofilm-forming ability by upregulating key adhesion genes (fibronectin-binding protein A/fnbB, serine-aspartate repeat-containing protein D, clumping factors A/B, elastin-binding protein, and fib) and downregulating autolysis-associated genes (lytR and lrgA). Furthermore, MES augmented the resistance of these strains to whole blood-mediated killing and improved their antioxidant capacity. To elucidate the role of STX, methanolic extracts were prepared from crtM and crtN mutants of the USA300 LAC strain and applied to biofilm-impaired strains. These experiments provided indirect evidence that STX in the methanolic extract is a critical mediator of biofilm promotion in vitro. Collectively, our results suggest a potential mechanistic link between STX in S. aureus methanolic extract and biofilm formation, offering novel insights for therapeutic strategies against S. aureus infections.IMPORTANCEOur findings demonstrate that the methanolic extract of S. aureus, predominantly comprising STX, augments biofilm formation and antioxidant capacity in vitro. These results not only offer novel insights into potential therapeutic strategies for S. aureus infections but also underscore the potential role of microbial secondary metabolites in interstrain interactions.

Objectives: Methicillin-resistant Staphylococcus aureus (MRSA) poses a significant threat to global public health, prompting the exploration of alternative strategies to mitigate its virulence. This study investigates the impact of subminimum inhibitory concentrations (sub-MICs) of rifampicin on MRSA virulence, aiming to provide insights for optimizing antibiotic treatment strategies. Methods: Enzyme-linked immunosorbent assay and western blot analysis were used to assess a- hemolysin expression. Transcriptomic sequencing and RT-qPCR analyzed gene expression changes in MRSA treated with sub-MICs of rifampicin. Mutant strains (DsaeR and DargGH) were constructed to validate the roles of the SaeRS system and arginine metabolism. Thermal shift assays evaluated the interaction between L-arginine and SaeR protein. In vivo murine models and Galleria mellonella infection models were used to assess the anti-virulence effects of rifampicin. Results: Our findings reveal that sub-MICs of rifampicin significantly reduce the expression of MRSA a- hemolysin. Transcriptomic sequencing and RT-qPCR analysis suggest a dual-pathway mechanism, wherein rifampicin suppresses virulence by indirectly inhibiting the SaeRS two-component system and disrupting arginine metabolism-related pathways. The construction of a saeR knockout mutant (DsaeR) and an arginine biosynthesis deficient mutant ( DargGH) further supports this mechanism. Notably, exogenous L- arginine supplementation reverses rifampicin's inhibitory effect on a-hemolysin expression, underscoring the pivotal role of L-arginine metabolism in MRSA virulence regulation. Thermal shift assays demonstrate a direct interaction between L-arginine and SaeR protein, elucidating the intricate interplay between metabolic pathways and virulence regulation. In vivo studies confirm that sub-MICs of rifampicin attenuate the severity of skin abscesses in a murine model, improve survival rates in bloodstream infection models, and mitigate inflammation in both skin and lung tissues. Conclusion: This study highlights the potential of rifampicin as an anti-virulence agent and pave the way for the development of innovative therapeutic strategies targeting MRSA infections. (c) 2025 Elsevier Ltd and International Society of Antimicrobial Chemotherapy. All rights are reserved, including those for text and data mining, AI training, and similar technologies.

Intestinal colonization is a critical precursor to invasive Klebsiella pneumoniae infection, yet the host-derived metabolic cues that license this transition remain unclear. Here, we identify arginine as a consistently enriched amino acid across inflammation-, diet-, and antibiotic-induced gut perturbations. Arginine markedly enhances capsular polysaccharide production and virulence in both hypervirulent ST23-KL1 and carbapenem-resistant ST11-KL64 lineages under conditions reflective of the gut microenvironment. Mechanistically, arginine must be transported by the ArtP ATP-binding transporter to activate the regulator ArgR, which directly binds capsular polysaccharide operon promoters and indirectly upregulates rmpA , forming a conserved ArtP–ArgR–capsule signaling axis. In mouse models, elevated intestinal arginine increases gut colonization, accelerates mucosal invasion, and promotes systemic dissemination, whereas arginase-mediated arginine depletion or loss of ArtP/ArgR abrogates these effects. Together, our findings reveal intestinal arginine as a key host-derived signal that drives K. pneumoniae pathogenic progression and identify arginine metabolism as a tractable therapeutic target.