Negatively charged nanodiscs for the reduction of toxicity and enhanced efficacy of polymyxin B against Acinetobacter baumannii sepsis.

The treatment of sepsis caused by multidrug-resistant (MDR) Gram-negative bacterial infections remains challenging. With these pathogens exhibiting resistance to carbapenems and new generation cephalosporins, the traditional antibiotic polymyxin B (PMB) has reemerged as a critical treatment option. However, its severe neurotoxicity and nephrotoxicity greatly limit the clinical application. Therefore, we designed negatively charged high-density lipoprotein (HDL) mimicking nanodiscs as a PMB delivery system, which can simultaneously reduce toxicity and enhance drug efficacy. The negative charge prevented the PMB release in physiological conditions and binding to cell membranes, significantly reducing toxicity in mammalian cells and mice. Notably, nanodisc-PMB exhibits superior efficacy than free PMB in sepsis induced by carbapenem-resistant Acinetobacter baumannii (CRAB) strains. Nanodisc-PMB shows promise as a treatment for carbapenem-resistant Gram-negative bacterial sepsis, especially caused by Acinetobacter baumannii, and the nanodiscs could be repurposed for other toxic antibiotics as an innovative delivery system. STATEMENT OF SIGNIFICANCE: Multidrug-resistant Gram-negative bacteria, notably carbapenem-resistant Acinetobacter baumannii, currently pose a substantial challenge due to the scarcity of effective treatments, rendering Polymyxins a last-resort antibiotic option. However, their therapeutic application is significantly limited by severe neurotoxic and nephrotoxic side effects. Prevailing polymyxin delivery systems focus on either reducing toxicity or enhancing bioavailability yet fail to simultaneously achieve both. In this scenario, we have developed a distinctive HDL-mimicking nanodisc for polymyxin B, which not only significantly reduces toxicity but also improves efficacy against Gram-negative bacteria, especially in sepsis caused by CRAB. This research offers an innovative drug delivery system for polymyxin B. Such advancement could notably improve the therapeutic landscape and make a significant contribution to the arsenal against these notorious pathogens.

Deletion of the Mycobacterium tuberculosis cyp138 gene leads to changes in membrane-related lipid composition and antibiotic susceptibility.

Introduction: Mycobacterium tuberculosis (Mtb), the main cause of tuberculosis (TB), has brought a great burden to the world's public health. With the widespread use of Mtb drug-resistant strains, the pressure on anti-TB treatment is increasing. Anti-TB drugs with novel structures and targets are urgently needed. Previous studies have revealed a series of CYPs with important roles in the survival and metabolism of Mtb. However, there is little research on the structure and function of CYP138. Methods: In our study, to discover the function and targetability of CYP138, a cyp138-knockout strain was built, and the function of CYP138 was speculated by the comparison between cyp138-knockout and wild-type strains through growth curves, growth status under different carbon sources, infection curves, SEM, MIC tests, quantitative proteomics, and lipidomics. Results and discussion: The knockout of cyp138 was proven to affect the Mtb's macrophage infection, antibiotics susceptibility, and the levels of fatty acid metabolism, membrane-related proteins, and lipids such as triacylglycerol. We proposed that CYP138 plays an important role in the synthesis and decomposition of lipids related to the cell membrane structure as a new potential anti-tuberculosis drug target.

Polymyxin resistance caused by large-scale genomic inversion due to IS26 intramolecular translocation in Klebsiella pneumoniae.

Polymyxin B and polymyxin E (colistin) are presently considered the last line of defense against human infections caused by multidrug-resistant Gram-negative organisms such as carbapenemase-producer Enterobacterales, Acinetobacter baumannii, and Klebsiella pneumoniae. Yet resistance to this last-line drugs is a major public health threat and is rapidly increasing. Polymyxin S2 (S2) is a polymyxin B analogue previously synthesized in our institute with obviously high antibacterial activity and lower toxicity than polymyxin B and colistin. To predict the possible resistant mechanism of S2 for wide clinical application, we experimentally induced bacterial resistant mutants and studied the preliminary resistance mechanisms. Mut-S, a resistant mutant of K. pneumoniae ATCC BAA-2146 (Kpn2146) induced by S2, was analyzed by whole genome sequencing, transcriptomics, mass spectrometry and complementation experiment. Surprisingly, large-scale genomic inversion (LSGI) of approximately 1.1 Mbp in the chromosome caused by IS26 mediated intramolecular transposition was found in Mut-S, which led to mgrB truncation, lipid A modification and hence S2 resistance. The resistance can be complemented by plasmid carrying intact mgrB. The same mechanism was also found in polymyxin B and colistin induced drug-resistant mutants of Kpn2146 (Mut-B and Mut-E, respectively). This is the first report of polymyxin resistance caused by IS26 intramolecular transposition mediated mgrB truncation in chromosome in K. pneumoniae. The findings broaden our scope of knowledge for polymyxin resistance and enriched our understanding of how bacteria can manage to survive in the presence of antibiotics.

Protective effect of the novel cyclic peptide ASK0912 on mice with sepsis induced by Acinetobacter baumannii.

BACKGROUND: Sepsis has become a global health concern owing to its increasing incidence and high mortality rate. In the present study, we investigated a novel drug candidate ASK0912 on its protective effects in mice with Acinetobacter baumannii 20-1-induced sepsis, and studied the related mechanisms. MATERIAL AND METHODS: To analyze the protective effect of ASK0912 on septic mice, survival rates, body temperature, organ and blood bacterial loads, white blood cell and platelet counts, organ damage, and cytokine levels were determined. RESULTS: ASK0912 remarkably increased the survival rate of mice with sepsis induced by A. baumannii 20-1 at a low dose of 0.6 mg/kg. Rectal temperature measurements showed that ASK0912 treatment prevented the body temperature decrease of septic mice to some extent. Treatment with ASK0912 can notably reduce the organ and blood bacterial loads and alleviate platelet count reduction due to sepsis. ASK0912 attenuated organ damage, including reduced levels of total bile acids, urea, and creatinine, aggregation of inflammatory cells, and mitigation of structural changes in septic mice, as demonstrated by biochemical analysis and hematoxylin & eosin staining. Additionally, multiplex assay showed that abnormally increased cytokine levels (IL-1ß, IL-3, IL-5, IL-6, IL-10, IL-13, MCP-1, RANTES, KC, MIP-1α, MIP-1ß, and G-CSF) in septic mice decreased after ASK0912 treatment. CONCLUSIONS: ASK0912 can not only improve the survival rate, hypothermia, lower the bacterial loads in the organs and blood, but also alleviate the pathophysiological manifestations such as intravascular coagulation abnormalities, organ damages, and immune system disorder of sepsis mice induced by A. baumannii 20-1.

In vivo pharmacodynamic study of contezolid acefosamil, a prodrug of contezolid for oral and intravenous administration.

OBJECTIVES: Contezolid acefosamil is a novel O-acyl phosphoramidate prodrug of contezolid. In the current study, we aimed to systemically evaluate the efficacy of contezolid acefosamil against infections caused by multiple Gram-positive pathogens, and compare the efficacy of the prodrug by oral and intravenous administrations. METHODS: The in vivo pharmacodynamic efficacy of contezolid acefosamil was evaluated in mouse models of systemic (with five S. aureus, three S. pneumoniae and two S. pyogenes bacterial isolates) and thigh (with two S. aureus isolates) infections using linezolid as the reference agent. RESULTS: In both models, contezolid acefosamil administrated either orally or intravenously, demonstrated high antibacterial efficacy similar to linezolid, and the antibacterial efficacy of oral and intravenous contezolid acefosamil were comparable. CONCLUSIONS: The high aqueous solubility and great efficacy of contezolid acefosamil support its clinical development as an injectable and oral antibiotic suitable for serious Gram-positive infections.

Identification and Quantification of S-Sulfenylation Proteome of Mycobacterium tuberculosis under Oxidative Stress.

The ability to maintain redox homeostasis is critical for Mycobacterium tuberculosis (Mtb) to survive the redox stress of the host. There are many antioxidant systems in Mtb to ensure its normal replication and survival in the host, and cysteine thiols are one of them. S-sulfenylation is one of the reversible modifications of cysteine thiols to resist oxidative stress. In the study, we investigated the total cysteine thiols modification and S-sulfenylation modification of Mtb proteome under the oxidative stress provided by hydrogen peroxide. To determine and quantify the S-sulfenylation modified proteins, high specific IodoTMT6plex reagents and high resolution mass spectrometry were used to label and quantify the peptides and proteins modified. There are significant differences for the total cysteine modification levels of 279 proteins and S-sulfenylation modification levels of 297 proteins under hydrogen peroxide stress. Functional enrichment analysis indicated that these cysteine-modified proteins were involved in the oxidation-reduction process, fatty acid biosynthetic process, stress response, protein repair, cell wall, etc. In conclusion, our study provides a view of cysteine modifications of the Mtb proteome under oxidative stress, revealing a series of proteins that may play a role in maintaining redox homeostasis. IMPORTANCE With the continuous spread of drug-resistant tuberculosis, there is an urgent need for new antituberculosis drugs with new mechanisms. The ability of Mtb to resist oxidative stress is extremely important for maintaining redox homeostasis and survival in the host. The reversible modifications of cysteine residues have a dual role of protection from irreversible damage to protein functions and regulation, which plays an important role in the redox homeostasis system. Thus, to discover cysteine modification changes in the proteome level under oxidative stress is quintessential to elucidate its antioxidant mechanism. Our results provided a list of proteins involved in the antioxidant process that potentially could be considered targets for drug discovery and vaccine development. Furthermore, it is the first study to determine and quantify the S-sulfenylation-modified proteins in Mtb, which provided better insight into the Mtb response to the host oxidative defense and enable a deeper understanding of Mtb survival strategies.

Antibacterial Activity of an FtsZ Inhibitor Celastrol and Its Synergistic Effect with Vancomycin against Enterococci In Vitro and In Vivo.

Enterococci can cause various infectious diseases, including urinary tract infection, wound infection, and life-threatening endocarditis and meningitis. The emergence and transmission of vancomycin-resistant enterococci (VRE) have presented a challenge to clinical treatment. There is an urgent need to develop new strategies to fight against this pathogen. This study investigated the antibacterial and anti-biofilm activity of celastrol (CEL), a natural product originating from Tripterygium wilfordii Hook F, against enterococci, and its adjuvant capacity of restoring the susceptibility of VRE to vancomycin in vitro and in vivo. CEL inhibited all enterococcus strains tested, with MICs ranging from 0.5 to 4 µg/mL. More than 50% of biofilm was eliminated by CEL at 16 µg/mL after 24 h of exposure. The combination of CEL and vancomycin showed a synergistic effect against all 23 strains tested in checkerboard assays. The combination of sub-MIC levels of CEL and vancomycin showed a synergistic effect in a time-kill assay and exhibited significant protective efficacy in Galleria mellonella larval infection model compared with either drug used alone. The underlying mechanisms of CEL were explored by conducting biomolecular binding interactions and an enzyme inhibition assay of CEL on bacterial cell-division protein FtsZ. CEL presented strong binding and suppression ability to FtsZ, with Kd and IC50 values of 2.454 µM and 1.04 ± 0.17 µg/mL, respectively. CEL exhibits a significant antibacterial and synergic activity against VRE in vitro and in vivo and has the potential to be a new antibacterial agent or adjuvant to vancomycin as a therapeutic option in combating VRE. IMPORTANCE The emergence and transmission of VRE pose a significant medical and public health challenge. CEL, well-known for a wide range of biological activities, has not previously been investigated for its synergistic effect with vancomycin against VRE. In the present study, CEL exhibited antibacterial activity against enterococci, including VRE strains, and restored the activity of vancomycin against VRE in vitro and in vivo. Hence, CEL has the potential to be a new antibacterial adjuvant to vancomycin and could provide a promising therapeutic option in combating VRE.

Evaluation of Agar Dilution Method in Susceptibility Testing of Polymyxins for Enterobacteriaceae and Non-Fermentative Rods: Advantages Compared to Broth Microdilution and Broth Macrodilution.

An accurate and reliable susceptibility testing method for polymyxins is urgently needed not only for the clinical laboratory but also for new polymyxin-like lipopeptide development. Reference broth microdilution (rBMD), which was the recommended method by CLSI-EUCAST in clinics, has been proven not to be ideal, while the agar dilution (AD) method that was widely used in new antibiotics discovery has been neglected. In the present study, the AD method was compared with rBMD and broth macrodilution (BMAD) in susceptibility testing of polymyxin B and colistin against >200 Gram-negative isolates. AD showed strong agreement with BMAD for colistin (except for Klebsiella aerogenes and Pseudomonas aeruginosa); however, its performance was poor for polymyxin B or compared to rBMD. MICs of AD method were not affected when different types of Petri dishes were used, while glass-bottom microtiter plates could lower the MIC of polymyxins 2−8 times compared to tissue-culture-treated polystyrene plates when using rBMD, which demonstrated that tissue-culture-treated plates were not suitable. It was then validated with non-tissue-culture-treated plates. The culture volume was another influencing factor of accuracy for rBMD, and 200 µL seemed to be the most suitable volume for MIC detection of polymyxins. Additionally, no lack of growth phenomenon (skipped well) was observed for AD when it frequently occurred for both BMAD and rBMD. As for strains carrying mcr-1 gene, 100% of AD results were in essential agreement (EA) and categorical agreement (CA) with both rBMD and BMAD. Overall, rBMD is convenient and widely accepted for susceptibility testing of polymyxins. Although it may be too early to say that AD is superior compared to rBMD and BMAD, it did show some advantages in repeatability and anti-interference ability.

Roles of cysteine in the structure and metabolic function of Mycobacterium tuberculosis CYP142A1.

CYP142A1 is a cytochrome P450 (CYP) enzyme expressed in Mycobacterium tuberculosis (Mtb), which supports the growth of Mtb H37Rv relying on cholesterol, in the absence of CYP125A1. Since cysteine residues usually play a fundamental role in maintaining the structure and function of CYP enzymes, in this study, we aimed to determine the potential biochemical functions of six cysteine residues except for the heme-binding cysteine in the amino acid sequence of recombinant Mtb CYP142A1 by replacing each one using site-directed mutagenesis. Recombinant CYP142A1 mutants were heterologously expressed, purified, and analyzed using ESI-MS, far-UV CD spectroscopy, UV-vis spectrophotometric titration, and metabolic function assays. Substitution of the cysteine residues caused various effects on the structure and function of CYP142A1. Separate substitution of the six cysteine residues resulted in numerous changes in the secondary structure, expression level, substrate-binding ability, inhibitor-binding ability, thermal stability and oxidation efficiency of the enzyme. These results contribute to our understanding of the biochemical roles of cysteine residues in the structure and function of Mtb CYP enzymes, especially their effects on the structure and function of CYP142A1.

Effect of Different Tolerable Levels of Constitutive mcr-1 Expression on Escherichia coli.

To study the effect of different tolerable levels of constitutive mcr-1 expression on Escherichia coli, and to provide direct evidence for moderate resistance mediated by mcr-1, construction of E. coli strains carrying mcr-1 on the chromosome with promoters of different strengths was conducted using λ-red recombination. Our results demonstrated that over-high expression of mcr-1 cannot be tolerated, and seven constructs with more than 200-fold mcr-1 transcriptional expression differences were obtained. The colistin MICs of the seven strains increased with the increase of MCR-1 levels, and the highest MIC was 8 µg/mL. Lower expression of mcr-1 didn't demonstrate many effects on bacteria, while higher tolerable expression of mcr-1 tended to show fitness costs in growth rate, competitive ability, and cell structures, but no obvious change of virulence was observed in mice. Bacteria demonstrated colistin MICs of 4-8 µg/mL at mcr-1 expression levels similar to clinical isolates, which were the mcr-1 expression levels with relatively lower fitness costs. IMPORTANCE The effects of relatively lower tolerable levels of mcr-1 were not evaluated thoroughly, and direct evidence for moderate resistance mediated by mcr-1 was lacking. In the present study, we made constructs carrying mcr-1 on the E. coli K12 chromosome under the control of serial constitutive promoters of different strengths and studied the effects of different tolerable levels of mcr-1 expression in vitro and in vivo. The results demonstrated that generally, except QH0007 (the construct with the highest mcr-1 expression that showed some extent of cell death), the fitness costs of tolerable mcr-1 expression on bacteria were not apparent or low. Bacteria demonstrated colistin MICs of 4-8 µg/mL at mcr-1 expression levels similar to clinical isolates, which corresponded to the lower levels of mcr-1 expression that can lead to colistin resistance, indicating the cleverness of bacteria to balance the benefit and cost of MCR-1-mediated colistin resistance.

Parallel Reaction Monitoring Mass Spectrometry for Rapid and Accurate Identification of ß-Lactamases Produced by Enterobacteriaceae.

The increasing spread of drug-resistant bacterial strains presents great challenges to clinical antibacterial treatment and public health, particularly with regard to ß-lactamase-producing Enterobacteriaceae. A rapid and accurate detection method that can expedite precise clinical diagnostics and rational administration of antibiotics is urgently needed. Targeted proteomics, a technique involving selected reaction monitoring or multiple reaction monitoring, has been developed for detecting specific peptides. In the present study, a rapid single-colony-processing procedure combined with an improved parallel reaction monitoring (PRM) workflow based on HRAM Orbitrap MS was developed to detect carbapenemases (Klebsiella pneumoniae carbapenemase, KPC; imipenemase, IMP; Verona integron-encoded metallo-ß-lactamase, VIM; New Delhi metallo-ß-lactamase, NDM; and oxacillinase, OXA), extended spectrum ß-lactamases (TEM and CTX-M), and AmpC (CMY-2) produced by Enterobacteriaceae. Specific peptides were selected and validated, and their coefficients of variation and stability were evaluated. In total, 188 Enterobacteriaceae strains were screened using the workflow. Fourteen out of total 19 peptides have 100% specificity; three peptides have specificity >95% and two peptides have specificity ranged from 74∼85%. On the sensitivity, only nine peptides have 95∼100% sensitivity. The other 10 peptides have sensitivity ranged from 27∼94%. Thus, a screening method based on peptide groups was developed for the first time. Taken together, this study described a rapid extraction and detection workflow for widespread ß-lactamases, including KPC, IMP, VIM, NDM, OXA, CMY, CTX-M, and TEM, using single colonies of Enterobacteriaceae strains. PRM-targeted proteomics was proven to be a promising approach for the detection of drug-resistant enzymes.

Recent Advances in Histidine Kinase-Targeted Antimicrobial Agents.

The prevalence of antimicrobial-resistant pathogens significantly limited the number of effective antibiotics available clinically, which urgently requires new drug targets to screen, design, and develop novel antibacterial drugs. Two-component system (TCS), which is comprised of a histidine kinase (HK) and a response regulator (RR), is a common mechanism whereby bacteria can sense a range of stimuli and make an appropriate adaptive response. HKs as the sensor part of the bacterial TCS can regulate various processes such as growth, vitality, antibiotic resistance, and virulence, and have been considered as a promising target for antibacterial drugs. In the current review, we highlighted the structural basis and functional importance of bacterial TCS especially HKs as a target in the discovery of new antimicrobials, and summarize the latest research progress of small-molecule HK-inhibitors as potential novel antimicrobial drugs reported in the past decade.

The discovery of 1, 3-diamino-7H-pyrrol[3, 2-f]quinazoline compounds as potent antimicrobial antifolates.

The shortage of new antibiotics makes infections caused by gram-negative (G-) bacteria a significant clinical problem. The key enzymes involved in folate biosynthesis represent important targets for drug discovery, and new antifolates with novel mechanisms are urgently needed. By targeting to dihydrofolate reductase (DHFR), a series of 1,3-diamino-7H-pyrrol[3,2-f]quinazoline (PQZ) compounds were designed, and exhibited potent antibacterial activities in vitro, especially against multi-drug resistant G- strains. Multiple experiments indicated that PQZ compounds contain a different molecular mechanism against the typical DHFR inhibitor, trimethoprim (TMP), and the thymidylate synthase (TS) was identified as another potential but a relatively weak target. A significant synergism between the representative compound, OYYF-175, and sulfamethoxazole (SMZ) was observed with a strong cumulative and significantly bactericidal effect at extremely low concentrations (2 µg/mL for SMZ and 0.03 pg/mL for OYYF-175), which could be resulted from the simultaneous inhibition of dihydropteroate synthase (DHPS), DHFR and TS. PQZ compounds exhibited therapeutic effects in a mouse model of intraperitoneal infections caused by Escherichia coli (E. coli). The co-crystal structure of OYYF-175-DHFR was solved and the detailed interactions were provided. The inhibitors reported represent innovative chemical structures with novel molecular mechanism of action, which will benefit the generation of new, efficacious bactericidal compounds.

Strategies for Rapid Identification of Acinetobacter baumannii Membrane Proteins and Polymyxin B's Effects.

Acinetobacter baumannii, especially multidrug resistant Acinetobacter baumannii, is a notable source of pressure in the areas of public health and antibiotic development. To overcome this problem, attention has been focused on membrane proteins. Different digestion methods and extraction detergents were examined for membrane proteome sample preparation, and label-free quantitative and targeted proteome analyses of the polymyxin B-induced Acinetobacter baumannii ATCC 19606 membrane proteome were performed based on nano LC-MS/MS. Ultracentrifugation of proteins at a speed of 150,000×g, digestion by trypsin, filter-aided sample preparation, and detergents such as lauryldimethylamine-N-oxide were proved as a fast and effective way for identification of membrane proteome by nano LC-MS/MS. Upon treatment with polymyxin B, expression levels of 15 proteins related to membrane structure, transporters, cell surface, and periplasmic space were found to be significantly changed. Furthermore, targeted proteome was also used to confirm these changes. A relatively rapid membrane proteome preparation method was developed, and a more comprehensive view of changes in the Acinetobacter baumannii membrane proteome under polymyxin B pressure was obtained.

Ostarine attenuates pyocyanin in Pseudomonas aeruginosa by interfering with quorum sensing systems.

Antimicrobial resistance has been an increasingly serious threat to global public health. Anti-virulence strategies are being developed to manage antibiotic resistance because they apply a lower selective pressure for antimicrobial-resistant pathogens than that created using traditional bactericides. We aimed to discover novel small molecules that can reduce the production of virulence factors in Pseudomonas aeruginosa and determine the mechanism of action underlying these effects. A clinical compound library was screened, and ostarine was identified as a potential anti-virulence agent. The effects of ostarine were studied via antimicrobial susceptibility testing, bacterial growth assays, pyocyanin quantitation assays, transcriptomic analysis, quorum sensing signal molecule quantification, and real-time PCR assays. Ostarine treatment significantly decreased the synthesis of pyocyanin without any bactericidal action. Besides, ostarine treatment did not affect the relative growth rate and cell morphology of bacteria. Treatment with ostarine interfered with quorum sensing by decreasing the transcription of genes associated with quorum sensing systems and the production of signalling molecules. The inhibition of ostarine on pyocyanin production and gene expression can be alleviated when signalling molecules were supplemented externally. Overall, ostarine may act as a novel anti-virulence agent that can attenuate P. aeruginosa pyocyanin by interfering with quorum sensing systems.

The Attenuated Protective Effect of Outer Membrane Vesicles Produced by a mcr-1 Positive Strain on Colistin Sensitive Escherichia coli.

Resistance to colistin, especially mobilized colistin resistance (mcr), is a serious threat to public health since it may catalyze a return of the "pre-antibiotic era". Outer membrane vesicles (OMVs) play a role in antibiotic resistance in various ways. Currently, how OMVs participate in mcr-1-mediated colistin resistance has not been established. In this study, we showed that both OMVs from the mcr-1 negative and positive Escherichia coli (E. coli) strains conferred dose-dependent protection from colistin. However, OMVs from the mcr-1 positive strain conferred attenuated protection when compared to the OMVs of a mcr-1 negative strain at the same concentration. The attenuated protective effect of OMVs was related to the reduced ability to absorb colistin from the environment, thus promoting the killing of colistin sensitive E. coli strains. Lipid A modified with phosphoethanolamine was presented in the OMVs of the mcr-1 positive E. coli strain and resulted in decreased affinity to colistin and less protection. Meanwhile, E. coli strain carrying the mcr-1 gene packed more unmodified lipid A in OMVs and kept more phosphoethanolamine modified lipid A in the bacterial cells. Our study provides a first glimpse of the role of OMVs in mcr-1 -mediated colistin resistance.

Low Prevalence of mcr-1 Among Clinical Enterobacteriaceae Isolates and Co-transfer of mcr-1 and blaNDM-1 from Separate Donors.

Aims: mcr-1 and blaNDM-1 co-harboring isolates have been reported, usually reside on different plasmids, suggesting co-transfer possibility of the two genes from separate donors to the same recipient strain. This study aims at screening and characterization of mcr-1 carrying Enterobacteriaceae in Northern China, and studying the transfer ability of mcr-1 alone and in company with blaNDM-1 from a second donor. Results: Three Escherichia coli strains and one Klebsiella pneumoniae strain carrying mcr-1 gene were screened out from 1992 isolates in our study. Co-existence of multiple resistance genes was found in the mcr-1-carrying strains, but none of them carried blaNDM-1. One E. coli demonstrated an single nucleotide polymorphism (SNP) (A-G) at -10 region of mcr-1, and one E. coli showed 2 SNPs (G-T and G-A) in the Shine-Dalgarno sequence-like region of mcr-1. The mcr-1 gene was located on plasmids of about 33-276 kb, and capable of transferring alone in three out of four mcr-1-positive isolates by conjugation. Co-transfer ability analysis demonstrated that mcr-1 from E. coli 13-68, which could not be transferred alone to E. coli C600, was successfully transferred in company with blaNDM-1 from K. pneumoniae ATCC BAA-2146. Conclusions: mcr-1 showed low incidence in our Enterobacteriaceae isolates. Co-transfer ability of mcr-1 and blaNDM-1 from separate donors provides direct evidence for the emergence of the mcr-1 and blaNDM-1 co-harboring isolates.

Quantitative proteomics approach to investigate the antibacterial response of Helicobacter pylori to daphnetin, a traditional Chinese medicine monomer.

Helicobacter pylori is a Gram-negative bacterium related to the development of peptic ulcers and stomach cancer. An increasing number of infected individuals are found to harbor antibiotic-resistant H. pylori, which results in treatment failure. Daphnetin, a traditional Chinese medicine, has a broad spectrum of antibacterial activity without the development of bacterial resistance. However, the antibacterial mechanisms of daphnetin have not been elucidated entirely. To better understand the mechanisms of daphnetin's effect on H. pylori, a label-free quantitative proteomics approach based on an EASY-nLC 1200 system coupled with an Orbitrap Fusion Lumos mass spectrometer was established to investigate the key protein differences between daphnetin- and non-daphnetin-treated H. pylori. Using the criteria of greater than 1.5-fold changes and adjusted p value <0.05, proteins related to metabolism, membrane structure, nucleic acid and protein synthesis, ion binding, H. pylori colonization and infection, stress reaction, flagellar assembly and so on were found to be changed under daphnetin pressure. And the changes of selected proteins in expression level were confirmed by targeted proteomics. These new data provide us a more comprehensive horizon of the proteome changes in H. pylori that occur in response to daphnetin.

The combined antibacterial effects of sodium new houttuyfonate and berberine chloride against growing and persistent methicillin-resistant and vancomycin-intermediate Staphylococcus aureus.

BACKGROUND: Infections caused by drug-resistant Staphylococcus aureus, especially vancomycin-intermediate Staphylococcus aureus (VISA), leave clinicians with limited therapeutic options for treatment. Persister cells is a leading cause of recalcitrant infection and antibiotic treatment failure, and there is no drug in clinical use that specifically targets persister cells currently. Here, we report a promising combination therapy of sodium new houttuyfonate (SNH) and berberine chloride (BBR) which is able to eradicate both growing and persistent drug-resistant Staphylococcus aureus. RESULTS: The susceptibility test showed SNH exhibited anti-MRSA activity with MIC90 at 64 µg/mL, while BBR showed weak anti-MRSA activity with MIC90 at 512 µg/mL. MICs of BBR in combination with 1/2 MIC SNH decreased by 4 to 64 folds compared with MICs of BBR alone. The results of time-killing assays revealed that the combined use of sub-MIC SNH and BBR offered an in vitro synergistic action against growing MRSA (including pathogenic MRSA) and VISA strains. More importantly, the combination of SNH and BBR was able to eradicate VISA Mu50 and pathogenic MRSA persister cells. The synergistic effect is likely related to the interruption of the cell membrane caused by SNH, which is confirmed by scanning electron microscope and membrane potential and permeability analysis. CONCLUSIONS: Our study provide a promising clinical curative strategy for combating drug-resistant S. aureus infections, especially for recalcitrant infections caused by persister cells.

Clinical and microbiological characteristics of hypervirulent Klebsiella pneumoniae (hvKp) in a hospital from North China.

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.