As d-amino acids play important roles in the physiological metabolism of bacteria, combination of d-amino acids with antibiotics may provide synergistic antibacterial activity. The aim of the study was to evaluate in vitro and in vivo activity of d-serine alone and in combination with β-lactams against methicillin-resistant Staphylococcus aureus (MRSA) strains, and to explore the possible sensitization mechanisms. The activity of d-serine, β-lactams alone and in combinations was evaluated both in vitro by standard MICs, time-kill curves and checkerboard assays, and in vivo by murine systemic infection model as well as neutropenic thigh infection model. An in vitro synergistic effect was demonstrated with the combination of d-serine and β-lactams against MRSA standard and clinical strains. Importantly, the combinations enhanced the therapeutic efficacy in the animal models as compared to β-lactam alone groups. Initial mechanism study suggested possible revision of d-alanine-d-alanine residue to d-alanine-d-serine in peptidoglycan by adding of d-alanine in the medium, which may cause decreased affinity to PBPs during transpeptidation. In conclusion, d-serine had synergistic activity in combination with β-lactams against MRSA strains both in vitro and in vivo. Considering the relatively good safety of d-serine alone or in combination with β-lactams, d-serine is worth following up as new anti-MRSA infection strategies.

Background: Antibiotic-resistant H. pylori was increasingly found in infected individuals, which resulted in treatment failure and required alternative therapeutic strategies. Daphnetin, a coumarin-derivative compound, has multiple pharmacological activities. Methods: The mechanism of daphnetin on H. pylori was investigated focusing on its effect on cell morphologies, transcription of genes related to virulence, adhesion, and cytotoxicity to human gastric epithelial (GES-1) cell line. Results: Daphnetin showed good activities against multidrug resistant (MDR) H. pylori clinical isolates, with minimal inhibitory concentration (MIC) values ranging from 25 to 100 μg/mL. In addition, daphnetin exposure resulted in H. pylori morphological changes. Moreover, daphnetin caused increased translocation of phosphatidylserine (PS), DNA damage, and recA expression, and RecA protein production vs. control group. Of great importance, daphnetin significantly decreased H. pylori adhesion to GES-1 cell line vs. control group, which may be related to the reduced expression of colonization related genes (e.g., babA and ureI). Conclusions: These results suggested that daphnetin has good activity against MDR H. pylori. The mechanism(s) of daphnetin against H. pylori were related to change of membrane structure, increase of DNA damage and PS translocation, and decrease of H. pylori attachment to GES-1 cells.

Introduction: The clinical and molecular characteristics of hypervirulent Klebsiella pneumoniae (hvKp) in various provinces of China have been reported, however, there have been few reports in Hebei Province, North China. Methodology: The hvKp was identified by PCR amplification of hypervirulence-related genes, the hypermucoviscous phenotype was determined by the "string test", the drug susceptibility analysis was performed using the VITEK® 2 Compact Bacterial Identification and Monitoring System. Logistic regression was used to identify risk factors for hvKp infection. The molecular epidemiological characteristics of the strains were analyzed by pulsed-field gel electrophoresis (PFGE), and the capsular serotype of hvKp strain was detected by PCR. Results: Overall, 52.21% (59/113) of K. pneumoniae isolates were hvKp, and the ratios of patients with older ages or a higher PMN cell count among hvKp infection were higher than those among classical Klebsiella pneumoniae (cKp) infection. hvKp are more susceptible to antibacterial drugs than cKp, and one ESBLs-producing hvKp strain was detected. The main capsular serotype of hvKp were K2, K57 and K1. PFGE indicated that the 59 strains of hvKp could be classified into 51 PFGE band types, forming 6 PFGE clusters. Conclusions: In this study, the detection rate of hvKp was 52.21% (59/113) identified by virulence genes. People with older ages or a higher PMN cell count are more likely to gain hvKp infection. ESBLs-producing hvKp is emerging, indicating the importance of epidemiologic surveillance and clinical awareness of this pathogen in this region.

Carbapenem-resistant Pseudomonas aeruginosa (CRPA) is a global threat, but the mechanism of non-carbapenemase carbapenem resistance is still unclear. In the current study, we investigated the contributions of point mutations in mexR , oprD , and ftsI to carbapenem resistance in P. aeruginosa during in vivo evolution studies with consecutive clinical isolates. Real-time qPCR and Electrophoretic Mobility Shift Assay demonstrated that MexR (Gln55Pro) mutation increased MexAB efflux pump genes expression by altering MexR’s binding capacity, leading to a four- to eight-fold increase in meropenem MIC in the Pae d1 Green ∆ mexR and PAO1∆ mexR mutants. The OprD (Trp415*) truncation affected porin structure, and the constructed mutant Pae d1 Green oprD Trp415* increased meropenem MIC by 16-fold (from 0.25 to 4 µg/mL). The contribution of ftsI mutation to meropenem resistance was confirmed by clinical linkage analysis and was estimated to cause a two-fold increase in meropenem MIC by comparing the resistant clinical isolate with the Pae d1 Green oprD Trp415*∆ mexR double mutant. The study found that the oprD Trp415* allele alone accounts for the imipenem MIC in clinical isolates, while the ∆ mexR and ftsI Arg504Cys alleles do not contribute to imipenem resistance. In conclusion, we identified and explored the contributions of mexR , oprD, and ftsI mutations to high level non-carbapenemase carbapenem resistance in P. aeruginosa . These findings highlight the interplay of different mutations in causing non-carbapenemase carbapenem-resistance in P. aeruginosa . IMPORTANCE: The emergence of carbapenem-resistant Pseudomonas aeruginosa (CRPA) poses a significant global health threat, complicating treatment options for infections caused by this pathogen. Understanding the mechanisms behind non-carbapenemase carbapenem resistance is critical for developing effective therapeutic strategies. This study provides crucial insights into how specific point mutations in key genes- mexR , oprD , and ftsI -contribute to carbapenem resistance, particularly the MexR (Gln55Pro) mutation’s effect on efflux pump expression and the OprD (Trp415*) truncation’s impact on porin structure. The findings elucidate the complex interplay of these mutations, highlighting their roles in conferring high-level resistance, and underscore the imperative for continued research to inform therapeutic strategies against CRPA infections.

MvaT and MvaU are global transcriptional regulators belonging to the H-NS family, and pyocyanin is an important virulence factor produced by Pseudomonas aeruginosa. mvaT mvaU double knockout mutant of P. aeruginosa PAO1 demonstrated pyocyanin abolishment in the previous study. Here, we further explored the mechanism. Two main directions were studied: pyocyanin biosynthesis pathway and QS system. The effect on the expression of the pyocyanin biosynthesis genes was evaluated by promoter strength determination and Real-Time PCR assay, and significant changes leading to low pyocyanin production were found. The effect on the QS system was studied by signal molecule quantification using LC-MS/MS and related gene expression measurements using Real-Time PCR. In mvaT mvaU double knockout, the production of 3-oxo-C12-HSL obviously increased, while those of C4-HSL and PQS obviously decreased, and the changes can be recovered by mvaT or mvaU complementation. The expressions of transcriptional activator genes binding with QS system signal molecules were all decreased, resulting in decreased formation of signal-transcriptional activator complexes. And the decreased expression of rhlR and pqsE also led to the lower expression of phzA1 and phzA2. Further exploration found that QS system downregulation may be related to QsrO, a QS system repressor, which was highly upregulated with mvaT mvaU double knockout. Hence, the synthesis of pyocyanin was suffocated and the biofilm formation ability was decreased. These results were also confirmed by transcriptome analysis, which demonstrated similar gene expression changes of the aforementioned genes together with decreased expression of other virulence factor genes regulated by QS system.