Arginine-Enhanced Antimicrobial Activity of Nanozymes against Gram-Negative Bacteria.

The continuous reduction of clinically available antibiotics has made it imperative to exploit more effective antimicrobial therapies, especially for difficult-to-treat Gram-negative pathogens. Herein, it is shown that the combination of an antimicrobial nanozyme with the clinically compatible basic amino acid L-arginine affords a potent treatment for infections with Gram-negative pathogens. In particular, the antimicrobial activity of the antimicrobial nanozyme is dramatically increased by ≈1000-fold after L-arginine stimulation. Specifically, the combination therapy enhances bacterial outer and inner membrane permeability and promotes intracellular reactive oxygen species (ROS) generation. Moreover, the metabolomic and transcriptomic results reveal that combination treatment leads to the increased ROS-mediated damage by inhibiting the tricarboxylic acid cycle and oxidative phosphorylation, thereby inducing an imbalance of the antioxidant and oxidant systems. Importantly, L-arginine dramatically significantly accelerates the healing of infected wounds in mouse models of multidrug-resistant peritonitis-sepsis and skin wound infection. Overall, this work demonstrates a novel synergistic antibacterial strategy by combining the antimicrobial nanozymes with L-arginine, which substantively facilitates the nanozyme-mediated killing of pathogens by promoting ROS production.

Comparative Genomic Analysis of Hypervirulence Carbapenem-Resistant Klebsiella pneumoniae from Inpatients with Infection and Gut Colonization, China.

Background: The emergence and spread of hypervirulent carbapenem-resistant Klebsiella pneumoniae (hv-CRKP) is a potential epidemiological threat that needs to be monitored. However, the transmission and pathogenic characteristics of hv-CRKP in China remain unclear. We investigated the epidemiological characteristics of gut colonized hv-CRKP in a hospital in Guangdong Province, China. Methods: A total of 46 gut colonized hv-CRKP isolates were collected from Sun Yat-Sen Memorial Hospital (Guangzhou, China) from August 31st to December 31st, 2021. Minimum inhibitory concentrations (MICs) were obtained for 15 antibiotics for 46 hv-CRKP isolates. BALB/C mice infection model and mucoviscosity assay was used to evaluate the virulence of the isolates. The characteristics of genome, phylogenetic relationship and the structure of the plasmid of 46 gut colonized hv-CRKP isolates were compared with pathogenic isolates from GeneBank based on whole-genome data. Results: The hv-CRKP isolation rate of all gut colonized carbapenem-resistant Klebsiella pneumoniae was 17% (46/270), and the intestinal colonization rate of hv-CRKP was irrelevant to the sex, age, department of hospitalization, and history of antibiotic use of the host. The gut colonized hv-CRKP showed pandrug resistance and hypervirulence. The gut colonized hv-CRKP and pathogenic hv-CRKP prevalent in China were mainly ST11 hv-CRKP and had two major epidemic clades. The similarities in genomic characteristics between gut colonized hv-CRKP and pathogenic hv-CRKP were consistent. The gut colonized hv-CRKP carried an incomplete structure pK2044 virulence plasmid from hypervirulent K. pneumoniae NTUH-K2044 by analyzing the virulence plasmid structure. Conclusion: Our results suggest that the gut colonized ST11 hv-CRKP may serve as a reservoir for the clinical pathogenic ST11 HV-CRKP. It is necessary to further strengthen the monitoring of gut colonized hv-CRKP and research the potential mechanism of infection caused by gut colonized hv-CRKP.

Predominance of Escherichia-Shigella in Gut Microbiome and Its Potential Correlation with Elevated Level of Plasma Tumor Necrosis Factor Alpha in Patients with Tuberculous Meningitis.

Tuberculous meningitis (TBM), the most lethal and disabling form of tuberculosis (TB), may be related to gut microbiota composition, warranting further study. Here we systematically compared gut microbiota compositions and blood cytokine profiles of TBM patients, pulmonary TB patients, and healthy controls. Notably, the significant gut microbiota dysbiosis observed in TBM patients was associated with markedly high proportions of Escherichia-Shigella species as well as increased blood levels of tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6). Next, we obtained a fecal bacterial isolate from a TBM patient and administered it via oral gavage to mice in order to develop a murine gut microbiota dysbiosis model for use in exploring mechanisms underlying the observed relationship between gut microbial dysbiosis and TBM. Thereafter, cells of commensal Escherichia coli (E. coli) were isolated and administered to model mice by gavage and then mice were inoculated with Mycobacterium tuberculosis (M. tuberculosis). Subsequently, these mice exhibited increased blood TNF-α levels accompanied by downregulated expression of tight junction protein claudin-5, increased brain tissue bacterial burden, and elevated central nervous system inflammation relative to corresponding indicators in controls administered PBS by gavage. Thus, our results demonstrated that a signature dysbiotic gut microbiome profile containing a high proportion of E. coli was potentially associated with an increased circulating TNF-α level in TBM patients. Collectively, these results suggest that modulation of dysbiotic gut microbiota holds promise as a new strategy for preventing or alleviating TBM. IMPORTANCE As the most severe form of tuberculosis, the pathogenesis of tuberculous meningitis (TBM) is still unclear. Gut microbiota dysbiosis plays an important role in a variety of central nervous system diseases. However, the relationship between gut microbiota and TBM has not been identified. In our study, significant dysbiosis in gut microbiota composition with a high proportion of E. coli and increased levels of TNF-α in plasma was noted in TBM patients. A commensal E. coli was isolated and shown to increase the plasma level of TNF-α and downregulate brain tight junction protein claudin-5 in the murine model. Gavage administration of E. coli aggravated the bacterial burden and increased the inflammatory responses in the central nervous system after M. tuberculosis infection. Dysbiosis of gut microbiota may be a promising therapeutic target and biomarker for TBM prevention or treatment.

A Peptidomic Approach to Identify Novel Antigen Biomarkers for the Diagnosis of Tuberculosis.

Background: Here, we conducted a peptidomic study in murine model to identify novel antigen biomarkers for the diagnosis of tuberculosis (TB) with improved performance. Methods: Four recombinant proteins, including Mycobacterium tuberculosis protein 32 (MPT32), Mycobacterium tuberculosis protein 64 (MPT64), culture filtrate protein 10 (CFP10), and phosphate ABC transporter substrate-binding lipoprotein (PstS1) were expressed and intravenously injected into BALB/c mice. The serum were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The concentrations of candidate peptides in serum of suspected TB patients were determined using competitive enzyme-linked immunosorbent assay. Results: A total of 65 peptides from 4 MTB precursor recombinant proteins were identified in mouse serum by LC-MS/MS, of which 5 peptides were selected as candidates for serological analysis. The concentrations of peptides MPT64-2, CFP10-2 and PstS1-2 in TB patients were significantly higher than those in non-TB patients. MPT64-2 exhibited the most promising sensitivity (81.4%), followed by PstS1-2 and CFP10-2. In addition, PstS1-2 had the highest specificity (93.3%), followed by CFP10-2 and MPT64-2. According to the area under the curve (AUC), MPT64-2 (AUC = 0.863), PstS1-2 (AUC = 0.812) and CFP10-2 (AUC = 0.809) exhibited better diagnostic validity. Conclusion: We develop an effective approach to identify new antigen biomarkers via LC-MS/MS-based peptidomics. Multiple peptides exhibit promising efficacy in diagnosis of active TB patients.

Bone Penetration of Cycloserine in Osteoarticular Tuberculosis Patients of China.

The cycloserine concentrations in plasma and bone that were collected during operations on 28 osteoarticular tuberculosis (TB) patients treated daily with a 500-mg cycloserine-containing regimen were determined. The median concentrations in plasma and bone were 16.29 µg/mL (interquartile range [IQR], 6.47 µg/mL) and 24.33 µg/g (IQR, 14.68 µg/g), respectively. The median bone/plasma penetration ratio was 0.76 (range, 0.33 to 1.98). Cycloserine could effectively penetrate bone and acquire concentrations comparable to those in plasma, which favors its usage in osteoarticular TB treatment.

In vitro Antimicrobial Activity Comparison of Linezolid, Tedizolid, Sutezolid and Delpazolid Against Slowly Growing Mycobacteria Isolated in Beijing, China.

BACKGROUND: The antimicrobial activities of some new oxazolidinones against slowly growing mycobacteria (SGM) have never been well evaluated. METHODS: We evaluate the in vitro susceptibility of 20 reference strains and 157 clinical isolates, pertaining different SGM species, against four oxazolidinones, ie, delpazolid, sutezolid, tedizolid and linezolid. In addition, the association of linezolid resistance and mutations in 23srRNA, rplC, rplD were also tested. RESULTS: Sutezolid presented the strongest antimicrobial activity against the clinical isolates of M. intracellulare than the other oxazolidinones, with MIC50 at 2 µg/mL and MIC90 at 4 µg/mL. MICs of sutezolid were usually 4- to 8-fold lower than these of linezolid against M. intracellulare and M. avium. The tested isolates of M. kansasii were susceptible to all of the four oxazolidinones. According to the multiple sequence alignment, novel 23srRNA mutations (A2267C and A2266G) in M. intracellulare and rplD mutations (Thr147Ala) in M. avium were identified in this study which have plausible involvement in rendering resistance against linezolid. CONCLUSION: This study showed that sutezolid harbors the strongest inhibitory activity against M. intracellulare, M. avium and M. kansasii in vitro, which provided important insights on the potential clinical application of oxazolidinones for treating SGM infections.

Comparison of the in vitro activity of linezolid, tedizolid, sutezolid, and delpazolid against rapidly growing mycobacteria isolated in Beijing, China.

BACKGROUND: The natural resistance of rapidly growing mycobacteria (RGM) to multiple antibiotics renders the treatment of the infections caused less successful. The objective of this study was to evaluate the in vitro susceptibilities of four oxazolidinones against different RGM species. METHODS: The microplate alamarBlue assay was performed to identify the minimum inhibitory concentrations (MICs) of four oxazolidinones - delpazolid, sutezolid, tedizolid, and linezolid - for 32 reference strains and 115 clinical strains of different RGM species. The MIC breakpoint concentration was defined as 16 µg/ml for linezolid. Next, the gene fragments associated with oxazolidinone resistance were amplified and sequenced, and mutations were defined in contrast with the sequences of the reference strains. RESULTS: Tedizolid showed the strongest inhibitory activity against the Mycobacterium abscessus isolates. Delpazolid exhibited better antimicrobial activity against the Mycobacterium fortuitum isolates when compared to linezolid, with 4-fold lower MIC values. The protein alignment and structure-based analysis showed that there might be no correlation between oxazolidinone resistance and mutations in the rplC, rplD, and 23S rRNA genes in the tested RGM. CONCLUSIONS: Tedizolid had the strongest inhibitory activity against M. abscessus in vitro, while delpazolid presented the best inhibitory activity against M. fortuitum. This provides important insights into the potential clinical application of oxazolidinones to treat RGM infections.

Bone penetration of linezolid in osteoarticular tuberculosis patients of China.

BACKGROUND: Linezolid presents strong antimicrobial activity against multidrug-resistant (MDR) pulmonary tuberculosis (TB), but its application in osteoarticular tuberculosis treatment remains understudied. Our objective was to analyze the bone penetration efficiency of linezolid in osteoarticular TB patients. METHODS: Osteoarticular TB patients, treated with 600 mg q 24 h linezolid-containing regimens and undergoing surgery, were prospectively and consecutively enrolled. One dose linezolid was administered before surgery. Blood and bone samples were collected simultaneously during operation, and their linezolid concentrations were then detected using high-performance liquid chromatography-tandem mass spectrometry. Pus samples were subjected to mycobacterial culture and GeneXpert MTB/RIF assay. The minimum inhibition concentrations (MICs) and drug susceptibility testing were performed with the recovered isolates. RESULTS: A total of 36 eligible osteoarticular TB patients were enrolled, including five MDR/rifampicin-resistant cases. All the 12 recovered isolates had MICs ≤0.5 µg/mL for linezolid. Mean concentrations in plasma, collected 100-510 min after the preoperative dosing, were 10.43 ± 4.83 µg/mL (range 3.29-22.26 µg/mL), and median concentrations in bone were 3.93 µg/mL (range 0.61-16.34 µg/mL). The median bone/plasma penetration ratio was 0.42 (range 0.14-0.95 µg/mL). Linezolid concentration in bone had a linear correlation with the drug concentration in plasma (r = 0.7873, p < 0.0001), while plasma concentration could explain 61.98% of the variation of concentration in bone (R2 = 0.6198). Notably, stratification analysis by sampling time demonstrated that samples collected 200-510 min after dosing had very good linear relationships between their bone and plasma concentrations (r = 0.9323). CONCLUSIONS: Linezolid penetrates from blood to bone efficiently, and the penetration further stabilizes ∼3 h after dosing.

Population Pharmacokinetic Analysis of Isoniazid among Pulmonary Tuberculosis Patients from China.

The blood concentration of isoniazid (INH) is evidently affected by polymorphisms in N-acetyltransferase 2 (NAT2), an enzyme that is primarily responsible for the trimodal (i.e., fast, intermediate, and slow) INH elimination. The pharmacokinetic (PK) variability, driven largely by NAT2 activity, creates a challenge for the deployment of a uniform INH dosage in tuberculosis (TB) patients. Although acetylator-specific INH dosing has long been suggested, well-recognized dosages according to acetylator status remain elusive. In this study, 175 blood samples were collected from 89 pulmonary TB patients within 0.5 to 6 h after morning INH administration. According to their NAT2 genotypes, 32 (36.0%), 38 (42.7%), and 19 (21.3%) were fast, intermediate, and slow acetylators, respectively. The plasma INH concentration was detected by liquid chromatography-tandem mass spectrometry. Population pharmacokinetic (PPK) analysis was conducted using NONMEM and R software. A two-compartment model with first-order absorption and elimination well described the PK parameters of isoniazid. Body weight and acetylator status significantly affected the INH clearance rate. The dosage simulation targeting three indicators, including the well-recognized efficacy-safety indicator maximum concentration in serum (Cmax; 3 to 6 µg/ml), the reported area under the concentration-time curve from 0 h to infinity (AUC0-∞; ≥10.52 µg·h/ml), and the 2-h INH serum concentrations (≥2.19 µg/ml), was associated with the strongest early bactericidal activity. The optimal dosages targeting the different indicators varied from 700 to 900 mg/day, 500 to 600 mg/day, and 300 mg/day for the rapid, intermediate, and slow acetylators, respectively. Furthermore, a PPK model for isoniazid among Chinese tuberculosis patients was established for the first time and suggested doses of approximately 800 mg/day, 500 mg/day, and 300 mg/day for fast, intermediate, and slow acetylators, respectively, after a trade-off between efficacy and the occurrence of side effects.

In Vitro Susceptibility Testing of GSK656 against Mycobacterium Species.

In this study, we aimed to assess the in vitro susceptibility to GSK656 among multiple mycobacterial species and to investigate the correlation between leucyl-tRNA synthetase (LeuRS) sequence variations and in vitro susceptibility to GSK656 among mycobacterial species. A total of 187 mycobacterial isolates, comprising 105 Mycobacterium tuberculosis isolates and 82 nontuberculous mycobacteria (NTM) isolates, were randomly selected for the determination of in vitro susceptibility. For M. tuberculosis, 102 of 105 isolates had MICs of ≤0.5 mg/liter, demonstrating a MIC50 of 0.063 mg/liter and a MIC90 of 0.25 mg/liter. An epidemiological cutoff value of 0.5 mg/liter was proposed for identification of GSK656-resistant M. tuberculosis strains. For NTM, the MIC50 and MIC90 values were >8.0 mg/liter for both Mycobacterium intracellulare and Mycobacterium avium In contrast, all Mycobacterium abscessus isolates had MICs of ≤0.25 mg/liter, yielding a MIC90 of 0.063 mg/liter. LeuRS from M. abscessus showed greater sequence similarity to M. tuberculosis LeuRS than to LeuRSs from M. avium and M. intracellulare Sequence alignment revealed 28 residues differing between LeuRSs from M. avium and M. intracellulare and LeuRSs from M. tuberculosis and M. abscessus; among them, 15 residues were in the drug binding domain. Structure modeling revealed that several different residues were close to the tRNA-LeuRS interface or the entrance of the drug-tRNA binding pocket. In conclusion, our data demonstrate significant species diversity in in vitro susceptibility to GSK656 among various mycobacterial species. GSK656 has potent efficacy against M. tuberculosis and M. abscessus, whereas inherent resistance was noted for M. intracellulare and M. avium.

Development and Clinical Validation of Multiple Cross Displacement Amplification Combined With Nanoparticles-Based Biosensor for Detection of Mycobacterium tuberculosis: Preliminary Results.

Tuberculosis is still a major threat to global public health. Here, a novel diagnosis assay, termed as multiple cross displacement amplification combined with nanoparticle-based lateral flow biosensor (MCDA-LFB), was developed to simultaneously detect IS6110 and IS1081 of Mycobacterium tuberculosis (MTB) in DNA extracted from reference strain H37Rv and clinical samples. The amplification can be finished within 30 min at a fixed temperature (67°C), thus the whole procedure, including rapid template preparation (15 min), isothermal reaction (30 min) and result reporting (2 min), can be completed within 50 min. The limit of detection of multiplex MCDA assay was 10 fg per reaction. By using the multiplex MCDA protocol, cross-reaction with non-mycobacteria and non-tuberculous mycobacteria (NTM) strains was not observed. Among clinically diagnosed TB patients, the sensitivity of liquid culture, Xpert MTB/RIF and multiplex MCDA assay was 42.0% (50/119), 49.6% (59/119), and 88.2% (105/119), respectively. Among culture positive samples, the sensitivity of Xpert MTB/RIF and multiplex MCDA assay was 86.0% (43/50) and 98.0% (49/50), respectively. Among culture negative samples, the sensitivity of Xpert MTB/RIF and multiplex MCDA assay was 23.2% (16/69) and 81.2% (56/69), respectively. The specificity was 100% (60/60) for Xpert MTB/RIF and 98.3% (59/60) for multiplex MCDA. Therefore, the multiplex MCDA assay for MTB detection is rapid, sensitive and easy to use and may be a promising test for early diagnosis of TB.

Penetration of linezolid into bone tissue 24 h after administration in patients with multidrug-resistant spinal tuberculosis.

BACKGROUND: Linezolid has shown strong antimicrobial activity against multidrug-resistant (MDR)/rifampin-resistant strains of Mycobacterium tuberculosis. Linezolid achieves clinical efficacy mainly through area under the concentration time curve/minimum inhibitory concentration ratio in the infected lesion site. Previous studies mainly focused on the relationship between linezolid concentrations in the blood and infected bone tissue when the blood drug concentration reached the peak 2 h after administration. However, we do not know whether linezolid can maintain the same bone/plasma ratio in infected bone tissue when the blood concentration reaches the trough level. Therefore, this study aimed to evaluate the penetrability of linezolid into bone tissue 24 h after administration in patients with MDR spinal tuberculosis (TB). METHODS: Nine MDR spinal TB patients, who received a treatment regimen including linezolid and underwent surgery, were enrolled prospectively from April 2017 to March 2019. Blood and diseased bone tissue specimens were collected simultaneously during operations 24 h after taking 600 mg of linezolid orally. Linezolid concentrations in plasma and diseased bone tissue specimens were determined by high-performance liquid chromatography-tandem mass spectrometry. RESULTS: Following a 600 mg oral administration of linezolid 24 h before surgery, median concentrations of linezolid in plasma and diseased bone tissue for the 9 patients were 1.98 mg/L (range 0.30-3.44 mg/L) and 0.60 mg/L (range 0.18-2.13 mg/L), respectively, at resection time. The median diseased bone/plasma linezolid concentration ratio was 0.48 (range 0.30-0.67). Pearson's correlation analysis showed that linezolid concentrations in the plasma were positively related to those in diseased bone tissue (r = 0.949, p < 0.001). CONCLUSIONS: After 24 h of medication, linezolid still had good penetrability into diseased bone tissue in patients with MDR spinal TB.

Para-aminosalicylic acid increases the susceptibility to isoniazid in clinical isolates of Mycobacterium tuberculosis.

Background: The purpose of this work was to assess the activity of para-aminosalicylic acid (PAS) in combination with isoniazid (INH) against clinical isolates of Mycobacterium tuberculosis (MTB). Materials and methods: A total of 72 MTB isolates with differential in vitro drug susceptibilities were included in this study, comprising 24 pan-susceptible, 24 MDR-TB, and 24 extensively drug-resistant (XDR) isolates. A microplate alamarBlue assay was performed to identify the minimal inhibitory concentrations (MICs) of MTB isolates. Results: The MIC50 of INH was 4 mg/L, and that of PAS was 0.063 mg/L against MTB isolates when single drug used. The combined use of INH and PAS resulted in 16-fold and 8-fold decrease in MIC50 for INH and PAS, respectively. The INH-PAS revealed synergistic activity in 94.4% of the isolates. In addition, there was no significant difference in the FIC index of the INH-PAS combination among individual isolates harboring different susceptibility pattern (P>0.05). Conclusion: The synergy between INH and PAS is demonstrated using non-multidrug-resistant (non-MDR) and MDR-TB strains, which will provide clinicians with useful hints to reuse this combination for treatment of TB patients in clinical practice.

Comparison of in vitro activity of the nitroimidazoles delamanid and pretomanid against multidrug-resistant and extensively drug-resistant tuberculosis.

Delamanid exhibited greater in vitro potency than pretomanid against multidrug-resistant (MDR-) and extensively drug-resistant tuberculosis (XDR-TB) isolates. The pretomanid minimum inhibitory concentration (MIC) values of four MDR-TB isolates were found to be resistant to delamanid ranging from 0.031 to 0.063 mg/L. A novel nonsynonymous mutation within the fbiA gene (Glu249Lys) may be contributing to high-level resistance to delamanid and pretomanid in Mycobacterium tuberculosis.

In Vitro Activity of PBTZ169 against Multiple Mycobacterium Species.

In this study, we demonstrate that PBTZ169 exhibits significant differences in in vitro activity against multiple Mycobacterium species. The amino acid polymorphism at codon 387 of decaprenylphosphoryl-beta-d-ribose oxidase (DprE1) can be used as a surrogate marker for in vitro susceptibility to PBTZ169 in mycobacteria. In addition, the amino acid substitution at codon 154 in DprE1 may be associated with acquired resistance to PBTZ169 in the Mycobacterium fortuitum mutants.

Comparison of In Vitro Activity and MIC Distributions between the Novel Oxazolidinone Delpazolid and Linezolid against Multidrug-Resistant and Extensively Drug-Resistant Mycobacterium tuberculosis in China.

Oxazolidinones are efficacious in treating mycobacterial infections, including tuberculosis (TB) caused by drug-resistant Mycobacterium tuberculosis In this study, we compared the in vitro activities and MIC distributions of delpazolid, a novel oxazolidinone, and linezolid against multidrug-resistant TB (MDR-TB) and extensively drug-resistant TB (XDR-TB) in China. Additionally, genetic mutations in 23S rRNA, rplC, and rplD genes were analyzed to reveal potential mechanisms underlying the observed oxazolidinone resistance. A total of 240 M. tuberculosis isolates were included in this study, including 120 MDR-TB isolates and 120 XDR-TB isolates. Overall, linezolid and delpazolid MIC90 values for M. tuberculosis isolates were 0.25 mg/liter and 0.5 mg/liter, respectively. Based on visual inspection, we tentatively set epidemiological cutoff (ECOFF) values for MIC determinations for linezolid and delpazolid at 1.0 mg/liter and 2.0 mg/liter, respectively. Although no significant difference in resistance rates was observed between linezolid and delpazolid among XDR-TB isolates (P > 0.05), statistical analysis revealed a significantly greater proportion of linezolid-resistant isolates than delpazolid-resistant isolates within the MDR-TB group (P = 0.036). Seven (53.85%) of 13 linezolid-resistant isolates were found to harbor mutations within the three target genes. Additionally, 1 isolate exhibited an amino acid substitution (Arg126His) within the protein encoded by rplD that contributed to high-level resistance to linezolid (MIC of >16 mg/liter), compared to a delpazolid MIC of 0.25. In conclusion, in vitro susceptibility testing revealed that delpazolid antibacterial activity was comparable to that of linezolid. A novel mutation within rplD that endowed M. tuberculosis with linezolid, but not delpazolid, resistance was identified.