Acetylcysteine increases sensitivity of ceftazidime-avibactam-resistant enterobacterales with different enzymatic resistance to ceftazidime-avibactam in vitro and in vivo.

BACKGROUND: Ceftazidime-avibactam (CZA) improves treatment outcomes for infections caused by carbapenem-resistant organisms, but has led to serious bacterial resistance. Acetylcysteine (NAC) is an approved medication that protects the respiratory tract through antioxidant and anti-inflammatory effects. RESULTS: This study found that NAC combined with CZA effectively inhibits the growth of CZA-resistant clinical Enterobacterales strains. The CZA/NAC combination inhibits biofilm formation in vitro and decreases bacterial burden in a mouse thigh infection model. The combination is biocompatible and primarily increases cell membrane permeability to cause bacterial death. CONCLUSIONS: These findings prove that the CZA/NAC combination has potential as a treatment for CZA-resistant Enterobacterales infections.

Clinical and Molecular Characteristics and Antibacterial Strategies of Klebsiella pneumoniae in Pyogenic Infection.

Treatment of Klebsiella pneumoniae causing pyogenic infections is challenging. The clinical and molecular characteristics of Klebsiella pneumoniae causing pyogenic infections are poorly understood, and antibacterial treatment strategies are limited. We analyzed the clinical and molecular characteristics of K. pneumoniae from patients with pyogenic infections and used time-kill assays to reveal the bactericidal kinetics of antimicrobial agents against hypervirulent K. pneumoniae (hvKp). A total of 54 K. pneumoniae isolates were included, comprising 33 hvKp and 21 classic K. pneumoniae (cKp) isolates, and the hvKp and cKp isolates were identified using five genes (iroB, iucA, rmpA, rmpA2, and peg-344) that have been applied as hvKp strain markers. The median age of all cases was 54 years (25th and 75th percentiles, 50.5 to 70), 62.96% of individuals had diabetes, and 22.22% of isolates were sourced from individuals without underlying disease. The ratios of white blood cells/procalcitonin and C-reactive protein/procalcitonin were potential clinical markers for the identification of suppurative infection caused by hvKp and cKp. The 54 K. pneumoniae isolates were classified into 8 sequence type 11 (ST11) and 46 non-ST11 strains. ST11 strains carrying multiple drug resistance genes have a multidrug resistance phenotype, while non-ST11 strains carrying only intrinsic resistance genes are generally susceptible to antibiotics. Bactericidal kinetics revealed that hvKp isolates were not easily killed by antimicrobials at susceptible breakpoint concentrations compared with cKp. Given the varied clinical and molecular features and the catastrophic pathogenicity of K. pneumoniae, it is critical to determine the characteristics of such isolates for optimal management and effective treatment of K. pneumoniae causing pyogenic infections. IMPORTANCE Klebsiella pneumoniae may cause pyogenic infections, which are potentially life-threatening and bring great challenges for clinical management. However, the clinical and molecular characteristics of K. pneumoniae are poorly understood, and effective antibacterial treatment strategies are limited. We analyzed the clinical and molecular features of 54 isolates from patients with various pyogenic infections. We found that most patients with pyogenic infections had underlying diseases, such as diabetes. The ratio of white blood cells to procalcitonin and the ratio of C-reactive protein to procalcitonin were potential clinical markers for differentiating hypervirulent K. pneumoniae strains from classical K. pneumoniae strains that cause pyogenic infections. K. pneumoniae isolates of ST11 were generally more resistant to antibiotics than non-ST11 isolates. Most importantly, hypervirulent K. pneumoniae strains were more tolerant to antibiotics than classic K. pneumoniae isolates.

Carbapenemase-loaded outer membrane vesicles protect Pseudomonas aeruginosa by degrading imipenem and promoting mutation of antimicrobial resistance gene.

AIMS: To investigate the effect of Klebsiella pneumoniae carbapenemase (KPC)-loaded outer membrane vesicles (OMVs) in protecting Pseudomonas aeruginosa against imipenem treatment and its mechanism. METHODS: The OMVs of carbapenem-resistant Klebsiella pneumonia (CRKP) were isolated and purified from the supernatant of bacterial culture by using ultracentrifugation and Optiprep density gradient ultracentrifugation. The transmission electron microscope, bicinchoninic acid, PCR and carbapenemase colloidal gold assays were applied to characterize the OMVs. Bacterial growth and larvae infection experiments were performed to explore the protective function of KPC-loaded OMVs for P. aeruginosa under imipenem treatment. Ultra-performance liquid chromatography, antimicrobial susceptibility testing, whole-genome sequencing and bioinformatics analysis were used to investigate the mechanism of P. aeruginosa resistance phenotype mediated by OMVs. RESULTS: CRKP secreted OMVs loaded with KPC, which protect P. aeruginosa from imipenem through hydrolysis of antibiotics in a dose- and time-dependent manner. Furthermore, carbapenem-resistant subpopulations were developed in P. aeruginosa by low concentrations of OMVs that were confirmed to inadequately hydrolyze imipenem. Interestingly, none of the carbapenem-resistant subpopulations obtained the exogenous antibiotic resistance genes, but all of them possessed OprD mutations, which was consistent with the mechanism of P. aeruginosa induced by sub-minimal inhibitory concentrations of imipenem. CONCLUSIONS: OMVs containing KPC provide a novel route for P. aeruginosa to acquire an antibiotic-resistant phenotype in vivo.

Clinical and Microbiological Characterization of Bloodstream Infections Caused by Mycoplasma hominis: An Overlooked Pathogen.

INTRODUCTION: Bloodstream infection (BSI) is associated with high mortality rates. Mycoplasma hominis, which rarely causes extragenital infections, has been shown to induce BSI and presents a clinical diagnostic and therapeutic challenge. METHODS: In this study, we investigated the clinical characteristics, antibiotic resistance, and multilocus sequence typing (MLST) of eight BSI cases caused by M. hominis in South China from January 2018 to October 2021. RESULTS: Underlying immunosuppression and genitourinary tract surgery are important risk factors for M. hominis BSI. The most prevalent clinical symptoms and signs were fever. Additional findings included elevated neutrophil count and C-reactive protein level. Furthermore, in this study, all the patients had erythrocytopenia. M. hominis harbored the highest rate of resistance to levofloxacin (75.0%), followed by sparfloxacin (50.0%), and gatifloxacin (37.5%). gyrA S153L was the most frequent mutation in levofloxacin-resistant strains, followed by parC S91I. parC K144R may be related to resistance to gatifloxacin and sparfloxacin. Eight strains showed sensitivity to all the other antibiotics analyzed (doxycycline, minocycline, josamycin, and clindamycin). MLST was performed in seven isolates, and seven new sequence types were described. We compared our isolates with all M. hominis strains from the PubMLST database, and one major clonal complex and eight singletons were identified. CONCLUSIONS: Our study clarified and expanded the clinical features and antibiotic resistance of M. hominis BSI. These findings are useful for the clinical diagnosis and control of M. hominis BSI.

Activation of unfolded protein response overcomes Ibrutinib resistance in diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is the most widespread type of non-Hodgkin lymphoma (NHL). As the most aggressive form of the DLBCL, the activated B-cell-like (ABC) subtype is often resistant to standard chemotherapies. Bruton's tyrosine kinase (BTK) inhibitor ibrutinib provides a potential therapeutic approach for the DLBCL but fails to improve the outcome in the phase III trial. In the current study, we investigated the molecular mechanisms underlying ibrutinib resistance and explored new combination therapy with ibrutinib. We generated an ibrutinib-resistant ABC-DLBCL cell line (OCI-ly10-IR) through continuous exposure to ibrutinib. Transcriptome analysis of the parental and ibrutinib-resistant cell lines revealed that the ibrutinib-resistant cells had significantly lower expression of the unfolded protein response (UPR) marker genes. Overexpression of one UPR branch-XBP1s greatly potentiated ibrutinib-induced apoptosis in both sensitive and resistant cells. The UPR inhibitor tauroursodeoxycholic acid (TUDCA) partially reduced the apoptotic rate induced by the ibrutinib in sensitive cells. The UPR activator 2-deoxy-D-glucose (2-DG) in combination with the ibrutinib triggered even greater cell growth inhibition, apoptosis, and stronger calcium (Ca2+) flux inhibition than either of the agents alone. A combination treatment of ibrutinib (15 mg·kg-1·d-1, po.) and 2-DG (500 mg/kg, po, b.i.d.) synergistically retarded tumor growth in NOD/SCID mice bearing OCI-ly10-IR xenograft. In addition, ibrutinib induced the UPR in the sensitive cell lines but not in the resistant cell lines of the DLBCL. There was also a combined synergistic effect in the primary resistant DLBCL cell lines. Overall, our results suggest that targeting the UPR could be a potential combination strategy to overcome ibrutinib resistance in the DLBCL.

Haemophilus seminalis sp. nov., isolated from human semen.

Two Haemophilus-like isolates with similar biochemical characteristics, designated strains SZY H1T and SZY H2, were isolated from human semen specimens. Cells were Gram-negative, non-motile, non-acid-fast, pleomorphic rods or coccobacilli. The major fatty acids (>10 %) were C16 : 0, C14 : 0, iso-C16 : 0 and/or C14 : 0 3-OH and C16 : 1 ω6c and/or C16 : 1 ω7c. The polar lipids were determined to be phosphatidylethanolamine, phosphatidylglycerol, an unidentified phospholipid, an unidentified aminophospholipid, two unidentified polar lipids and four unidentified aminolipids. The major polyamine was found to be cadaverine. The near-full-length (1462 nt) 16S rRNA gene sequences analysis showed the two isolates were nearly identical (>99.8 %), and closely matched Haemophilus haemolyticus ATCC 33390T with 98.9-99.1 % sequence similarities. Phylogenetic analysis based on 16S rRNA gene sequences and concatenation of 30 protein markers also revealed that the isolates clustered together with H. haemolyticus ATCC 33390T, and formed a distinct lineage well separated from the other members of the genus Haemophilus. Further, the average nucleotide identity values between the two isolates and their related species were below the established cut-off values for species delineation (95 %). Based on these findings, the two isolates are considered to represent a new species of the genus Haemophilus, for which name Haemophilus seminalis sp. nov. is proposed. The type strain is SZY H1T (=NBRC 113782T=CGMCC 1.17137T).

Design, synthesis, and biological evaluation of novel phenol ether derivatives as non-covalent proteasome inhibitors.

A series of novel phenol ether derivatives were designed, synthesized, and evaluated as non-covalent proteasome inhibitors. Most compounds exhibited moderate to excellent proteasome inhibitory activity. In particular, compound 18x proved to be the most potent compound (chymotrypsin-like: IC50 = 49 nM), exhibiting a 2-fold higher potency compared to the reported PI-1840. Besides, compound 18x exhibited excellent metabolic stability and selective anti-proliferative activity against solid cancer cell lines including HepG2 and HGC27, providing incentive for the further development as a potential anticancer agent against solid cancers.

Development of Macrocyclic Peptides Containing Epoxyketone with Oral Availability as Proteasome Inhibitors.

Macrocyclization has been frequently utilized for optimizing peptide or peptidomimetic-based compounds. In an attempt to obtain potent, metabolically stable, and orally available proteasome inhibitors, 30 oprozomib-derived macrocyclic peptides with structural diversity in their N-terminus and linker were successively designed and synthesized for structure-activity relationship (SAR) studies. As a consequence, the macrocyclic peptides with N-methyl-pyrazole (24p, 24x), imidazole (24t), and pyrazole (24v) as their respective N-termini exhibited favorable in vitro activity and metabolic stability, which translated into their potent in vivo proteasome inhibitory activity after oral administration. In particular, compound 24v, as the most distinguished one among this series, displayed excellent chymotrypsin-like (ChT-L, ß5) inhibitory potency (IC50 = 16 nM), low nanomolar antiproliferative activity against all three of the tested cell lines, and superior metabolic stability in mouse liver microsome (MLM), as well as favorable inhibition against ChT-L compared to that of oprozomib in BABL/c mice following po administration at a comparatively low dose, thereby representing a promising candidate for further development.

Treponema pallidum flagellin FlaA2 induces IL-6 secretion in THP-1 cells via the Toll-like receptor 2 signaling pathway.

Treponema pallidum subsp. pallidum membrane proteins are considered as potent inducers in the initiation and development of inflammation. In the present study, the mechanism that leads to the production of interleukin 6 (IL-6), one of the key proinflammatory cytokines, by human monocytic THP-1 cells when these cells are treated with T. pallidum flagellin FlaA2 was investigated. Stimulation with flagellin FlaA2 can induce IL-6 expression in human monocytes and augment the phosphorylation of ERK, p38, and NF-κB, but has no effect on the phosphorylation of JNK. Likewise, FlaA2-induced IL-6 production was found to be attenuated by inhibitors for ERK, p38, and NF-κB, but not by JNK inhibitor. Immunofluorescence analysis showed that flagellin FlaA2 could stimulate the translocation of IκBα from the cytosol to the nucleus, and this phenomenon could be inhibited by the specific inhibitor BAY11-7082. FlaA2-induced IL-6 expression was also proved to be abrogated by transfection with dominant negative (DN) plasmid of MyD88. We further demonstrated that transfection with DN-TLR2 was sufficient to attenuate IL-6 expression and the phosphorylation of ERK, p38, and IκBα. These results suggest that flagellin FlaA2 induces IL-6 production via signaling pathways involving TLR2, MyD88, ERK, p38, and NF-κB in monocytes, which could contribute to the pathogenesis of T. pallidum.