Restoring colistin sensitivity in colistin-resistant Salmonella and Escherichia coli: combinatorial use of berberine and EDTA with colistin.

The appearance and prevalence of multidrug-resistance (MDR) Gram-negative bacteria (GNB) have limited our antibiotic capacity to control bacterial infections. The clinical efficacy of colistin (COL), considered as the "last resort" for treating GNB infections, has been severely hindered by its increased use as well as the emergence and prevalence of mobile colistin resistance (MCR)-mediated acquired drug resistance. Identifying promising compounds to restore antibiotic activity is becoming an effective strategy to alleviate the crisis of increasing MDR. We first demonstrated that the combination of berberine (BBR) and EDTA substantially restored COL sensitivity against COL-resistant Salmonella and Escherichia coli. Molecular docking indicated that BBR can interact with MCR-1 and the efflux pump system AcrAB-TolC, and BBR combined with EDTA downregulated the expression level of mcr-1 and tolC. Mechanically, BBR combined with EDTA could increase bacterial membrane damage, inhibit the function of multidrug efflux pump, and promote oxidative damage, thereby boosting the action of COL. In addition, transcriptome analysis found that the combination of BBR and EDTA can accelerate the tricarboxylic acid cycle, inhibit cationic antimicrobial peptide (CAMP) resistance, and attenuate Salmonella virulence. Notably, the combination of BBR and EDTA with COL significantly reduced the bacterial load in the liver and spleen of a mice model infected with Salmonella. Our findings revealed that BBR and EDTA can be used as adjuvants collectively with COL to synergistically reverse the COL resistance of bacteria. IMPORTANCE: Colistin is last-resort antibiotic used to treat serious clinical infections caused by MDR bacterial pathogens. The recent emergence of transferable plasmid-mediated COL resistance gene mcr-1 has raised the specter of a rapid worldwide spread of COL resistance. Coupled with the fact of barren antibiotic development pipeline nowadays, a critical approach is to revitalize existing antibiotics using antibiotic adjuvants. Our research showed that berberine combined with EDTA effectively reversed COL resistance both in vivo and in vitro through multiple modes of action. The discovery of berberine in combination with EDTA as a new and safe COL adjuvant provides a therapeutic regimen for combating Gram-negative bacteria infections. Our findings provide a potential therapeutic option using existing antibiotics in combination with antibiotic adjuvants and address the prevalent infections caused by MDR Gram-negative pathogens worldwide.

Analysis of Regulatory Mechanism of AcrB and CpxR on Colistin Susceptibility Based on Transcriptome and Metabolome of Salmonella Typhimurium.

With the increasing and inappropriate use of colistin, the emerging colistin-resistant isolates have been frequently reported during the last few decades. Therefore, new potential targets and adjuvants to reverse colistin resistance are urgently needed. Our previous study has confirmed a marked increase of colistin susceptibility (16-fold compared to the wild-type Salmonella strain) of cpxR overexpression strain JSΔacrBΔcpxR::kan/pcpxR (simplified as JSΔΔ/pR). To searching for potential new drug targets, the transcriptome and metabolome analysis were carried out in this study. We found that the more susceptible strain JSΔΔ/pR displayed striking perturbations at both the transcriptomics and metabolomics levels. The virulence-related genes and colistin resistance-related genes (CRRGs) were significantly downregulated in JSΔΔ/pR. There were significant accumulation of citrate, α-ketoglutaric acid, and agmatine sulfate in JSΔΔ/pR, and exogenous supplement of them could synergistically enhance the bactericidal effect of colistin, indicating that these metabolites may serve as potential adjuvants for colistin therapy. Additionally, we also demonstrated that AcrB and CpxR could target the ATP and reactive oxygen species (ROS) generation, but not proton motive force (PMF) production pathway to potentiate antibacterial activity of colistin. Collectively, these findings have revealed several previously unknown mechanisms contributing to increased colistin susceptibility and identified potential targets and adjuvants for potentiating colistin treatment of Salmonella infections. IMPORTANCE Emergence of multidrug-resistant (MDR) Gram-negative (G-) bacteria have led to the reconsideration of colistin as the last-resort therapeutic option for health care-associated infections. Finding new drug targets and strategies against the spread of MDR G- bacteria are global challenges for the life sciences community and public health. In this paper, we demonstrated the more susceptibility strain JSΔΔ/pR displayed striking perturbations at both the transcriptomics and metabolomics levels and revealed several previously unknown regulatory mechanisms of AcrB and CpxR on the colistin susceptibility. Importantly, we found that exogenous supplement of citrate, α-ketoglutaric acid, and agmatine sulfate could synergistically enhance the bactericidal effect of colistin, indicating that these metabolites may serve as potential adjuvants for colistin therapy. These results provide a theoretical basis for finding potential new drug targets and adjuvants.

Pannexin1 Channel-Mediated Inflammation in Acute Ischemic Stroke.

Emerging evidence suggests that inflammation mediated by the pannexin1 channel contributes significantly to acute ischemic stroke. It is believed that the pannexin1 channel is key in initiating central system inflammation during the early stages of acute ischemic stroke. Moreover, the pannexin1 channel is involved in the inflammatory cascade to maintain the inflammation levels. Specifically, the interaction of pannexin1 channels with ATP-sensitive P2X7 purinoceptors or promotion of potassium efflux mediates the activation of the NLRP3 inflammasome, triggering the release of pro-inflammatory factors such as IL-1 and IL-18, exacerbating and sustaining inflammation of brain. Also, increased release of ATP induced by cerebrovascular injury activates pannexin1 in vascular endothelial cells. This signal directs peripheral leukocytes to migrate into ischemic brain tissue, leading to an expansion of the inflammatory zone. Intervention strategies targeting pannexin1 channels may greatly alleviate inflammation after acute ischemic stroke to improve this patient population's clinical outcomes. In this review, we sought to summarize relevant studies on inflammation mediated by the pannexin1 channel in acute ischemic stroke and discussed the possibility of using brain organoid-on-a-chip technology to screen miRNAs that exclusively target the pannexin1 channel to provide new therapeutic measures for targeted regulation of pannexin1 channel to reduce inflammation in acute ischemic stroke.

Prevalence of the optrA gene among Streptococcus suis isolates from diseased pigs and identification of a novel integrative conjugative element ICESsu988S.

Aim of this study was to investigate the prevalence and genetic environment of the oxazolidinone resistance gene optrA in Streptococcus suis (S. suis) isolates from diseased pigs in China. A total of 178 S. suis isolates were screened for the optrA gene by PCR. The phenotypes and genotypes of optrA-positive isolates were investigated by antimicrobial susceptibility testing, core genome Multilocus Sequence Typing (cgMLST), capsular serotypes determination and whole-genome sequencing (WGS). Fifty-one (28.7%) S. suis isolates were positive for optrA. Phylogenetic analysis indicated that the spread of the optrA among S. suis isolates was primarily due to horizontal transfer. Analysis of S. suis serotypes from diseased pigs revealed substantial diversity. The genetic environment of optrA was complex and diverse and could be divided into 12 different types. Interestingly, we identified a novel integrative and conjugative element ICESsu988S, carrying optrA and erm(T) genes. This is to the best of our knowledge the first report of the optrA and erm(T) co-located on an ICE in S. suis. Our results showed a high prevalence of optrA gene in S. suis isolates in China. Further research is needed to evaluate the importance of ICEs, as they horizontally propagate important clinical resistance genes.

Synergistic antibacterial activity of baicalin and EDTA in combination with colistin against colistin-resistant Salmonella.

The emergence and rapid spread of multidrug resistant (MDR) Gram-negative bacteria have posed a serious threat to global health and security. Because of the time-consuming, high cost and high risk of developing new antibiotics, a significant method is to use antibiotic adjuvants to revitalize the existing antibiotics. The purpose of the study is to research the traditional Chinese medicine baicalin with the function of inhibiting the efflux pump and EDTA whether their single or combination can increase the activity of colistin against colistin-resistant Salmonella in vitro and in vivo, and to explore its molecular mechanisms. In vitro antibacterial experiments, we have observed that baicalin and EDTA alone could enhance the antibacterial activity of colistin. At the same time, the combination of baicalin and EDTA also showed a stronger synergistic effect on colistin, reversing the colistin resistance of all Salmonella strains. Molecular docking and RT-PCR results showed that the combination of baicalin and EDTA not only affected the expression of mcr-1, but also was an effective inhibitor of MCR-1. In-depth synergistic mechanism analysis revealed that baicalin and EDTA enhanced colistin activity through multiple pathways, including accelerating the tricarboxylic acid cycle (TCA cycle), inhibiting the bacterial antioxidant system and lipopolysaccharide (LPS) modification, depriving multidrug efflux pump functions and attenuating bacterial virulence. In addition, the combinational therapy of colistin, baicalin and EDTA displayed an obvious reduction in bacterial loads cfus of liver and spleen compared with monotherapy and 2-drug combination therapy. In conclusion, our study indicates that the combination of baicalin and EDTA as a novel colistin adjuvant can provide a reliable basis for formulating the therapeutic regimen for colistin resistant bacterial infection.

Molecular Characterization of a Novel Integrative Conjugative Element ICEHpa1 in Haemophilus parasuis.

ICEHpa1 was identified in the genome of a serovar 8 Haemophilus parasuis ST288 isolate YHP170504 from a case of swine lower respiratory tract infection. The aim of the present study was to characterize the integrative conjugative element ICEHpa1 and its multiresistance region. Susceptibility testing was determined by broth microdilution and the complete ICEHpa1 was identified by WGS analysis. The full sequence of ICEHpa1 was analyzed with bioinformatic tools. The presence of ICEHpa1, its circular intermediate and integration site were confirmed by PCR and sequence analysis. Transfer of ICEHpa1 was confirmed by conjugation. ICEHpa1 has a size of 68,922 bp with 37.42% GC content and harbors 81 genes responsible for replication and stabilization, transfer, integration, and accessory functions, as well as seven different resistance genes [bla Rob- 3, tet(B), aphA1, strA, strB, aac(6)'-Ie-aph(2')-Ia, and sul2]. Conjugation experiments showed that ICEHpa1 could be transferred to H. parasuis V43 with frequencies of 6.1 × 10-6. This is the first time a multidrug-resistance ICE has been reported in H. parasuis. Seven different resistance genes were located on a novel integrative conjugative element ICEHpa1, which suggests that the ICEHpa1 is capable of acquiring foreign genes and serving as a carrier for various resistance genes.

Molecular characterization of a novel bla CTX-M-3-carrying Tn6741 transposon in Morganella morganii isolated from swine.

Introduction. The bla CTX-M-3 gene has rarely been reported in Morganella morganii strains and its genetic environment has not yet been investigated.Aim. To identify the bla CTX-M-3 gene in M. morganii isolated from swine and characterize its genetic environment.Methodology. A M. morganii isolate (named MM1L5) from a deceased swine was identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and subjected to antimicrobial susceptibility testing. The bla genes were detected and then the genetic location and environment of bla CTX-M-3 were investigated by Southern blot and PCR mapping, respectively. The M. morganii bla CTX-M-3 gene was cloned and expressed in Escherichia coli.Results. Isolate MM1L5 harboured the bla CTX-M-3 and bla TEM-1 genes. The bla CTX-M-3 gene, located on the chromosome, was co-carried with an IS26 and bla TEM-1 gene by a novel 6361 bp IS26-flanked composite transposon, designated Tn6741. This transposon consisted of a novel bla CTX-M-3-containing module, IS26-ΔISEcp1-bla CTX-M-3-Δorf477-IS26 (named Tn6710), and a bla TEM-1-containing module, IS26-Δorf477-bla TEM-1-tnpR-IS26, differing from previous reports. Phylogenetic analysis showed a significant variation based on the sequence of Tn6741, as compared to those of other related transposons. Interestingly, although the cloned bla CTX-M-3 gene could confer resistance to ceftiofur, cefquinome, ceftriaxone and cefotaxime, one amino acid substitution (Ile-142-Thr) resulted in a significant reduction of resistance to these antimicrobials.Conclusion. This is the first time that bla CTX-M-3 has been identified on a chromosome from a M. morganii isolate. Furthermore, the bla CTX-M-3 gene was located with an IS26 element and bla TEM-1 gene on a novel IS26-flanked composite transposon, Tn6741, suggesting that Tn6741 might act as a reservoir for the bla CTX-M-3 and bla TEM-1 genes and may become an important vehicle for their dissemination among M. morganii.

Characterization of pTS14, an IncF2:A1:B1 Plasmid Carrying tet(M) in a Salmonella enterica Isolate.

The objective of this study was to explore the genetic and biological features of the tet(M)-harboring plasmid pTS14 in Salmonella enterica strain S14 isolated from a chicken fecal sample. Plasmid pTS14 was identified by conjugation, S1-pulsed-field gel electrophoresis (PFGE), Southern hybridization, and plasmid sequencing. The biological characteristics of pTS14 were assessed via stability, growth kinetics, and starvation survival experiments. Strain S14, belonging to ST3007, harbored a 119-kb tet(M)-bearing IncF2:A1:B1 conjugative plasmid pTS14. The plasmid pTS14 contained a novel transposon Tn6709 with the genetic structure IS26-tnpA1-tnpA2-Δorf13-LP-tet(M)-tnpX-ΔtnpR-IS26, and the resistance genes tet(B), tet(D), strAB, sul2, and bla TEM-1b. In addition, pTS14 was found to be highly stable in the recipient strain E. coli J53. The transconjugant TS14 exhibited a higher survival ratio than E. coli J53 under permanent starvation-induced stress. The tet(M)-bearing IncF2 epidemic plasmid lineage may accelerate the dissemination of tet(M) and other genes by coselection, which could constitute a potentially serious threat to clinical treatment regimens.