Solithromycin, the fourth generation of ketolides, has been demonstrated potent antibacterial effect against commonly-isolated gram-positive strains. However, Staphylococcus aureus (S. aureus) strains with a higher solithromycin MIC have already been emerged, the mechanism of which is unknown. Antimicrobial susceptibility test was performed on 266 strains of S. aureus. The antibiotic resistance phenotype of erm-positive strain was determined by D-zone test. Spontaneous mutation frequency analysis was performed to compare the risk levels for solithromycin resistance among different strains. Efflux pumps and mutational analysis of ribosomal fragments as well as erm(B) gene domains were detected. Quantitative reverse transcription polymerase chain reaction was conducted to compare the transcriptional expression of the erm gene between the constitutive macrolide-lincosamide-streptogramin B (cMLSB)- and inducible MLSB (iMLSB)-phenotypes. In the erm-positive S. aureus strains, the minimum inhibitory concentration (MIC)50/90 of solithromycin (2/> 16 mg/L) was significantly higher than that in the erm-negative strains (0.125/0.25 mg/L). Of note, the MIC50 value of the strains with iMLSB (0.25 mg/L) was significantly lower than that of the strains with cMLSB (4 mg/L). A comparison among strains demonstrated that the median mutational frequency in isolates with cMLSB (> 1.2 × 10− 4) was approximately > 57-fold and > 3333-fold higher than that in iMLSB strains (2.1 × 10− 6) and in erythromycin-sensitive strains (3.6 × 10− 8), respectively. The differential antibiotic in vitro activity against strains between cMLSB and iMLSB could not be explained by efflux pump carriers or genetic mutations in the test genes. The expression of the erm genes in strains with cMLSB did not differ from that in strains with iMLSB. The reduced susceptibility to solithromycin by S. aureus was associated with the cMLSB resistance phenotype mediated by erm.

Over the last few decades, Staphylococcus aureus infection remain a major medical challenge and health concern worldwide. Biofilm formation and antibiotic resistance caused by S. aureus make it difficult to be eradicated from bacterial infections in clinics. In this study, our data demonstrated the antibacterial and excellent anti-biofilm activity of entrectinib against S. aureus. Entrectinib also exhibited the good safety, suggesting no toxicity with antibacterial concentration of entrectinib toward the erythrocytes and mammalian 239 T cells. Moreover, entrectinib significantly reduced the bacterial burden of septic tissue in a murine model of MRSA infection. Global proteomic analysis of S. aureus treated with entrectinib showed significant changes in the expression levels of ribosomal structure-related (rpmC, rpmD, rplX, and rpsT) and oxidative stress-related proteins (Thioredoxin system), suggesting the possible inhibition of bacterial protein biosynthesis with entrectinib exposure. The increased production of reactive oxygen species (ROS) was demonstrated in the entrectinib-treated S. aureus, supported the impact of entrectinib on the expression changes of ROS-correlated proteins involved in oxidative stress. Furthermore, entrectinib-induced resistant S. aureus clone was selected by in vitro induction under entrectinib exposure and 3 amino acid mutations in the entrectinib-induced resistant S. aureus strain, 2 of which were located in the gene encoding Type II NADH: quinoneoxidoreductase and one were found in GTP pyrophosphokinase family protein. Finally, the bactericidal action of entrectinib on S. aureus were confirmed by disrupting the bacterial cell membrane. Conclusively, entrectinib exhibit the antibacterial and anti-biofilm activity by destroying cell membrane against S. aureus.

Klebsiella pneumoniae can be cultured in medium with xylose as the sole carbon source. However, the effect of xylose exposure on its growth adaptation, virulence, antimicrobial susceptibility, and proteomic response remain unclear. Here, we show that low concentrations of xylose (≤ 2%) promote the planktonic growth of three K. pneumoniae isolates (K2044, EKP19, and EKP108) in a concentration-dependent manner, while 8% xylose consistently inhibits their planktonic growth. Notably, the xylose-induced isolate K2044−8Xyl−60G, when exposed to various xylose concentrations, exhibited the longest logarithmic growth phase and the highest optical density (OD) after logarithmic growth, compared to K2044. In contrast, the xylose-induced isolates EKP19 and EKP108 did not successfully reshape growth adaptation under persistent xylose pressure compared to K2044. Additionally, while the growth adaptation of K2044−8Xyl−60G under xylose pressure was confirmed, no amino acid mutations were detected in the functional proteins of this xylose-induced isolate, suggesting that persistent xylose pressure does not cause nonsense mutations in the bacterial genome. Xylose exposure reduced the gentamicin minimum inhibitory concentration (MIC) in all three K. pneumoniae isolates (K2044, EKP108, and EKP19) and their xylose-induced derivatives. In a Galleria mellonella infection model, significantly decreased virulence was observed in the xylose-induced isolates of K2044 and EKP19. Proteomic analysis of K2044−8Xyl−60G treated with 8% xylose revealed upregulation of proteins involved in glycolysis, the pentose phosphate pathway, and transmembrane transport. We also constructed K2044-ΔxylA (with deletion of the xylA gene) and K2044-ΔxylB (with deletion of the xylB gene). Our data showed that K2044-ΔxylA exhibited enhanced planktonic growth compared to K2044 when exposed to xylose concentrations of ≥ 4%, while K2044-ΔxylB displayed significantly reduced growth capacity regardless of xylose exposure. The virulence of K2044-ΔxylA was also significantly reduced, as demonstrated by the increased survival rates in G. mellonella infection models. Additionally, xylose exposure strongly enhanced membrane depolarization in both K2044-ΔxylA and K2044-ΔxylB compared to the wild-type K2044. Proteomic analysis indicated that the deletion of xylA primarily affected functional proteins related to ribosomes, xylose transmembrane transporters and capsular polysaccharides, while the deletion of xylB impacted the expression of xylose metabolism-related proteins. In conclusion, xylose exposure can reshape the growth adaptation, virulence, and antimicrobial susceptibility of K. pneumoniae in an isolate-specific manner, with xylA playing a more critical role than xylB under high xylose concentrations.

Klebsiella pneumoniae commonly colonizes the gastrointestinal (GI) mucosa of animals and healthy humans. Successful GI colonization by K. pneumoniae requires overcoming the colonization resistance (CR) exerted by the gut microbiota. Although previous studies have demonstrated the role of microbial carbohydrate metabolism in K. pneumoniae colonization, the specific effects of individual carbohydrates, such as glucose and sucrose, particularly across concentration gradients or under sustained induction on the adaptive growth of K. pneumoniae remain unknown. Herein, we demonstrate that 4% or 8% glucose and sucrose are favorable for promoting adaptive growth of sugar-induced strains. Additionally, the growth response to glucose exhibited strain-specific variability. Sustained sugar induction did not significantly alter the hypermucoviscosity (HMV) phenotype but did affect antibiotic resistance of K. pneumoniae . Knockout of the scrA and scrY genes impaired the adaptive growth under sucrose and glucose conditions, yet did not significantly influence antimicrobial susceptibility or the HMV phenotype. These findings provide insights into the metabolic regulation of K. pneumoniae colonization and offer potential guidance for clinical treatment strategies targeting K. pneumoniae -associated infections. IMPORTANCE: This work elucidates the impact of single-carbon source gradients and sustained sugar induction on the adaptive growth and drug resistance of Klebsiella pneumoniae and preliminarily reveals the roles of scrA and scrY in carbohydrate metabolism, suggesting a possible mechanism by which sucrose and glucose affect the adaptive growth of K. pneumoniae . These findings contribute to the theoretical understanding of CCR and provide insights that may inform clinical management of K. pneumoniae -related infections.

Volume 13, no. 12, e01603-25, 2025, https://doi.org/10.1128/spectrum.01603-25. The following footnote to the byline stating “Jiayan Wu, Kewei Fan, Zewen Wen, Duoyun Li, Zhijian Yu, and Tieying Hou contributed equally to this article” should be deleted.

Staphylococcus aureus infections, particularly those caused by multidrug-resistant strains and associated with biofilm formation, pose a major therapeutic challenge in clinical practice. The objective of this study was to evaluate the antibacterial and antibiofilm activity of mocravimod (KRP-203), an FDA-approved S1P receptor modulator, against clinical S. aureus isolates and to explore its underlying mechanism of action. The antibacterial activity of KRP-203 was assessed against methicillin-susceptible S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) using MIC determination, time-kill assays, and biofilm inhibition models. KRP-203 exhibited strong bactericidal activity against planktonic MSSA and MRSA, with MIC values ranging 6.25–50μM. Time-kill assays demonstrated rapid bacterial eradication at 8× MIC within 2 h, showing superior killing kinetics compared with vancomycin. At sub-inhibitory concentrations, KRP-203 inhibited biofilm formation by up to 70% and reduced viable bacterial counts in mature biofilms by >2.5 logs. To elucidate the antibacterial mechanism, whole-genome sequencing and quantitative proteomic analyses were performed. These analyses revealed mutations in membrane-associated genes, including glnQ and BCAT, and significant alterations in proteins related to membrane integrity and redox regulation. Consistently, functional assays confirmed that KRP-203 disrupts bacterial cell membrane, as evidenced by dose-dependent membrane depolarization, increased permeability, and direct binding to cardiolipin and phosphatidylglycerol. Molecular docking further predicted a favorable interaction between KRP-203 and GlnQ. In conclusion, KRP-203 demonstrated notable antibacterial and antibiofilm activity against S. aureus , likely through membrane integrity disruption. While these findings highlight its potential as a repurposed antibacterial agent, further studies are required to fully elucidate its molecular targets, optimize antibacterial efficacy, and evaluate its in vivo safety profile. IMPORTANCE: Antibiotic resistance and the formation of biofilms, which protect bacteria from medications and immunological responses, present the significant challenges for the clinical treatment of Staphylococcus aureus infections. This study reveals mocravimod hydrochloride (KRP-203), a clinically approved drug initially intended to treat leukemia, as a viable new candidate against S. aureus infection. KRP-203 quickly kills both drug-susceptible and resistant S. aureus , including difficult-to-treat biofilm-associated cells. Its membrane-disrupting activity quickly kills drug-resistant bacteria while also destroying biofilm formations, presenting a dual action rarely accomplished by conventional antibiotics. Critically, KRP-203’s established safety profile in human studies may hasten its repurposing as a new weapon against biofilm-associated infections, providing possible solutions for chronic and drug-resistant S. aureus infections where existing treatments commonly fail.