We collected respiratory specimens from 128 pediatric patients diagnosed with pneumonia in Beijing in late 2023. Mycoplasma pneumoniae was detected in 77.3% (99/128) patients, with 36.4% (4/11), 82.9% (34/41), 80.3% (61/76) in children aged less than 3 years, 3–6 years, over 7 years, respectively. Mycoplasma pneumoniae (M. pneumoniae) was characterized using P1 gene typing, MLVA typing and sequencing of domain V of the 23S rRNA gene. P1 gene type 1 (P1-1; 76.1%, 54/71) and MLVA type 4-5-7-2 (73.7%, 73/99) were predominant. MLVA identified a new genotype: 3–4–6-2. Macrolide resistance-associated mutations were detected in 100% of samples, with A2063G accounting for 99% and A2064G for 1%. The positive rate of M. pneumoniae was higher compared to previous reports, especially in children less than 3 years, suggesting a M. pneumoniae epidemic showing a younger age trend occurred in late 2023 in Beijing, China. Higher proportions of macrolide-resistant M. pneumoniae, P1-1 and 4-5-7-2 genotype M. pneumoniae indicated increased macrolide resistance rate and genotyping shift phenomenon, which might be attributable to this epidemic. Additionally, complete clinical information from 73 M. pneumoniae pneumonia inpatients were analyzed. The incidence of severe M. pneumoniae pneumonia was 56.2% (41/73). Mycoplasma pneumoniae pneumonia patients exhibited longer duration of fever, with a median value of 10.0 days (IQR, 8.0–13.0), and higher incidence of complications (74.0%, 54/73). However, in this cohort, we found that the severity of M. pneumoniae pneumonia, co-infection, or complications were not associated with M. pneumoniae P1 gene or MLVA types. Clinicians should be aware that patients infected with macrolide-resistant M. pneumoniae exhibited more severe clinical presentations.

Introduction: Mycoplasma pneumoniae (M. pneumoniae), a common pathogen of community-acquired pneumonia in school-age children and adolescents, can cause epidemics worldwide. In late 2023, the incidence of M. pneumoniae infection among children reached a high level.Methods: We investigated the in vitro antimicrobial susceptibility of 62 M. pneumoniae isolates obtained from children with pneumonia in Beijing between 2021 and 2023, and analyzed the correlation of antimicrobial susceptibility with molecular characteristics of isolates and clinical manifestations of patients.Results: The resistance rates of M. pneumoniae isolates against erythromycin and azithromycin were both 100% (62/62). The minimum inhibitory concentration (MIC) of acetylspiramycin (16-membered macrolides) was lower than that of erythromycin and azithromycin. The MIC of azithromycin in 2023 was notably higher compared to 2021 and 2022. No resistance to tetracycline and levofloxacin was observed. Genotypes P1 type 1 and P1 type 2 were identified in 74.2% and 25.8% of isolates, and M4-5-7-2 (61.3%) and M3-5-6-2 (22.6%) were predominant multi-locus variable-number tandem-repeat analysis (MLVA) types. The A2063G mutation was present in all isolates (100%). Among the patients, 45/59 cases (76.3%) had severe M. pneumoniae pneumonia, and 14/59 cases (23.7%) presented co-infection. The duration of fever was 12 days (1-30 days) and the fever duration after initiation of macrolide antibiotics treatment was 8 days (1-22 days).Discussion: Our study showed that macrolide-resistant M. pneumoniae (MRMP) with high in vitro antimicrobial resistance level may be the causative factor of the M. pneumoniae epidemic in late 2023 in Beijing, China. It is urgent to pay more attention to MRMP and the antibiotics choose.

Capsular polysaccharides are critical virulence factors of Klebsiella pneumoniae, enabling the bacterium to evade host immune recognition and exacerbate infection. Phage-derived depolymerases, which specifically degrade these capsular polysaccharides, are increasingly recognized as a highly promising strategy for the treatment of bacterial infections. In the present study, we isolated and characterized a lytic Klebsiella pneumoniae phage, named phiTH1, and sequenced its genome. The K30-type capsular polysaccharide was identified as the receptor for phiTH1 infection. A tail fiber protein with a pectate lyase domain, Dop5, was then recognized as a potential K30-type depolymerase. Therefore, the recombinant protein Dop5 was expressed in Escherichia coli and purified, and its in vitro capsular depolymerase activity was demonstrated. Further, by using a murine aspiration pneumonia model induced by K30-type Klebsiella pneumoniae TH1, we found that Dop5 protected 80% of mice from lethal challenge with Klebsiella pneumoniae. After Dop5 treatment, the pathological damage in multiple organs of mice was alleviated, the bacterial load was reduced, and serum levels of inflammatory cytokines and complement C3 decreased, along with a significant reduction in the pathological score of the lungs. Hence, this study revealed the potential of the depolymerase Dop5 for the treatment of Klebsiella pneumoniae infections.

Background: High-alcohol-producing Klebsiella pneumoniae (HiAlc Kpn ) can cause metabolic dysfunction-associated steatotic liver disease (MASLD) through sustained alcohol overflow in the microenvironment. As a probiotic, Bifidobacterium bifidum ( B. bifidum ) exhibits unique anti-inflammatory properties; however, whether and how it alleviate MASLD induced by HiAlc Kpn requires further investigation. Methods: MASLD mouse model was constructed by gavage administration with HiAlc Kpn W14 to assess the therapeutic effect of B. bifidum CIP-01 in vivo. Cell infection models, metabolomics sequencing, and in vitro antibacterial assays were integrated to systematically elucidate the mechanism by which B. bifidum CIP-01 mitigates HiAlc Kpn W14-induced cell damage. Results: B. bifidum CIP-01 was able to ameliorate MASLD induced by HiAlc Kpn through a multi-target mechanism. Compared to pair-fed mice, HiAlc Kpn W14 disrupted gut barrier and promoted inflammatory cytokines release. While, supplementation with B. bifidum CIP-01 reversed these effects by a) restoring intestinal integrity via upregulating tight junction proteins (ZO-1/Occludin) and mucin protein MUC-2, reducing reactive oxidative stress (ROS) and apoptosis in colonic cells, and b) rescuing hepatic cytochrome P450 2E1 (CYP2E1)-driven oxidative injury (ROS/Caspase-3) while promoting mitochondrial β -oxidation, as well as c) directly suppressing HiAlc Kpn proliferation and biofilm formation. Metabolomics and 16S rRNA of fecal samples analyses revealed B. bifidum CIP-01-mediated metabolic regulation: depletion of toxic branched-chain amino acids (BCAAs) intermediates and restoration of energy homeostasis and antioxidant defense alongside increased short-chain fatty acids (SCFAs)-associated pathways. Conclusion: Our findings highlight B. bifidum CIP-01 as a novel therapeutic candidate for HiAlc Kpn -induced MASLD, operating through a triad of pathogen suppression, gut–liver axis repair, and metabolic regulation.

This study aimed to use quantitative phosphoproteomics to explore phosphorylation characteristics in Klebsiella pneumoniae (Kpn)-induced liver abscess (KPLA) formation and liver injury. The Kpn strain LA-Kpn006 was phenotypically and genotypically characterized. A murine model of KPLA was established via intragastric inoculation. Phosphoproteomics and bioinformatic analyses were conducted to identify and quantify phosphosites and phosphoproteins. LA-Kpn006 displayed a hypermucoviscous phenotype, serotype K1, ST23 genotype, and harbored six major virulence genes. Inoculation induced liver colonization and typical histopathological abscess lesions. We quantified 3017 phosphoproteins covering 12,798 phosphosites (dominated by serine phosphorylation); 1723 proteins were upregulated and 425 downregulated. Bioinformatic analyses revealed remodeling in metabolism, stress response, signal transduction, and cytoskeleton organization. Upregulated proteins converged on fatty acid elongation, inositol phosphate metabolism, and the tricarboxylic acid cycle; downregulated proteins were enriched in PI3K–Akt, IL-17 signaling, and T-cell differentiation. Protein–protein interaction network analysis identified 10 key proteins (Src, Rac1, Actb, Hsp90aa1, Hsp90ab1, Egfr, Rps6, Pik3CA, Itgb1, and Fyn) that mediate inflammatory signaling, cytoskeleton remodeling, and immune infiltration. Dysregulated phosphorylation networks drive pathological metabolic adaptation, suppressed immune homeostasis, and cytoskeletal disorganization, collectively facilitating KPLA progression. The identified hub proteins and pathways represent high-value mechanistic targets and candidate therapeutic vulnerabilities for KPLA.

Background: Klebsiella pneumoniae ( K. pneumoniae ) strains exhibit diverse virulence phenotypes influenced by serotype (K-type) and sequence type (ST). However, the interplay between bacterial molecular/biological characteristics and specific pathogenicity remains poorly understood. Methods: We characterized 11 clinical K. pneumoniae isolates from liver abscess patients with varying serotypes (K1, K2, K5, K20, K57, K80), STs (including novel variants), and virulent factors. Phenotypic assays included the determination of growth curves, assessment of biofilm formation, and observation via electron microscopy. The Vitek 2 automated system was used to evaluate the minimum inhibitory concentration (MIC) of 11 clinical Klebsiella pneumoniae strains against common antibiotics. Mouse infection models were used to assess survival rates, analyze organ pathology, and detect hematological changes; these experimental data were complemented by clinical patient data. Results: Eleven clinically isolated Klebsiella pneumoniae strains displayed serotype/genotype-associated virulence and short bacilli morphology under TEM, with seven being hypermucoviscosity-positive. Growth and biofilm phenotypes varied: K2-ST65 (S2-029) had superior proliferation, K2-ST86 (S2-048) the weakest growth; K1-ST23 (S1-001) formed the strongest biofilm, while K57-ST218 (S57-077) the weakest. Phylogenetic analysis based on core SNPs derived from whole-genome sequencing of 11 K. pneumoniae strains revealed that S5-105 and S5-036 formed a distinct independent clade, significantly separated from the remaining strains. Notably, S5-105 and S5-036 exhibited significantly higher levels of differentiation compared to the other strains. Antibiotic susceptibility testing showed 9/11 of strains were fully sensitive, with overall low resistance. All strains were 100% susceptible to carbapenems, cephalosporins, aminoglycosides, and aztreonam. Only S5-105 was ciprofloxacin-resistant/levofloxacin-intermediate among quinolones; two strains had sulbactam-related non-susceptibility (S5-036: intermediate; S57-066: piperacillin-intermediate + sulbactam-resistant). Notably, two novel ST-type strains exhibited unique pathogenicity: K1-novel ST (S1-009) caused rapid systemic infection, 50% 72 h survival, severe liver abscess, and neutrophilic inflammation; K80-novel ST selectively induced pulmonary abscesses without hepatic involvement, an atypical tropism. K2 strain virulence correlated with ST: hypervirulent K2-ST65 led to 50% survival, while K2-ST86 resulted in 100% survival. Other serotypes showed distinct pathogenicity: K20 caused liver damage, K57 was nearly non-pathogenic, and slow-growing K5 induced mild tissue injury. Conclusion: Specific serotype-STs combination potentiates lethality under certain circumstances (e.g., K2-ST65), but its impact is complicated. Novel STs correlate with acute lethality (K1) or atypical tropism (K80 lung—specific virulence). These findings highlight strain—specific virulence mechanisms, which are critical for the risk stratification of K. pneumoniae infection.