O26 Polysaccharides as Key Players in Enteropathogenic E. coli Immune Evasion and Vaccine Development.

Enteropathogenic Escherichia coli (EPEC) produce a capsule of polysaccharides identical to those composing the O-antigen polysaccharide of its LPS (lipopolysaccharide) molecules. In light of this, the impact of O26 polysaccharides on the immune evasion mechanisms of capsulated O26 EPEC compared to non-capsulated enterohemorrhagic Escherichia coli (EHEC) was investigated. Our findings reveal that there was no significant difference between the levels in EPEC and EHEC of rhamnose (2.8:2.5), a molecule considered to be a PAMP (Pathogen Associated Molecular Patterns). However, the levels of glucose (10:1.69), heptose (3.6:0.89) and N-acetylglucosamine (4.5:2.10), were significantly higher in EPEC than EHEC, respectively. It was also observed that the presence of a capsule in EPEC inhibited the deposition of C3b on the bacterial surface and protected the pathogen against lysis by the complement system. In addition, the presence of a capsule also protected EPEC against phagocytosis by macrophages. However, the immune evasion provided by the capsule was overcome in the presence of anti-O26 polysaccharide antibodies, and additionally, these antibodies were able to inhibit O26 EPEC adhesion to human epithelial cells. Finally, the results indicate that O26 polysaccharides can generate an effective humoral immune response, making them promising antigens for the development of a vaccine against capsulated O26 E. coli.

Pyridylpiperazine efflux pump inhibitor boosts in vivo antibiotic efficacy against K. pneumoniae.

Antimicrobial resistance is a global problem, rendering conventional treatments less effective and requiring innovative strategies to combat this growing threat. The tripartite AcrAB-TolC efflux pump is the dominant constitutive system by which Enterobacterales like Escherichia coli and Klebsiella pneumoniae extrude antibiotics. Here, we describe the medicinal chemistry development and drug-like properties of BDM91288, a pyridylpiperazine-based AcrB efflux pump inhibitor. In vitro evaluation of BDM91288 confirmed it to potentiate the activity of a panel of antibiotics against K. pneumoniae as well as revert clinically relevant antibiotic resistance mediated by acrAB-tolC overexpression. Using cryo-EM, BDM91288 binding to the transmembrane region of K. pneumoniae AcrB was confirmed, further validating the mechanism of action of this inhibitor. Finally, proof of concept studies demonstrated that oral administration of BDM91288 significantly potentiated the in vivo efficacy of levofloxacin treatment in a murine model of K. pneumoniae lung infection.

Prevalence of colistin resistance and antibacterial resistance in commensal Escherichia coli from chickens: An assessment of the impact of regulatory intervention in South Africa.

BACKGROUND: Antimicrobial resistance (AMR) is a global health problem largely due to the overuse of antimicrobials. In recognition of this, the World Health Assembly in 2015 agreed on a global action plan to tackle AMR. Following the global emergence of the mcr-1-associated colistin resistance gene in the livestock industry in 2016, several countries including South Africa restricted the veterinary use of colistin as the gene threatens the clinical utility of the drug. This study is a follow-up to the restriction in place in order to evaluate the impact of such policy adoption. OBJECTIVE: To assess the prevalence of antibacterial resistance (ABR), and the mcr-1 colistin resistance gene in broiler chicken over a 2-year period, as a follow-up to the veterinary ban on colistin use in South Africa. METHODS: A total of 520 swab samples were obtained during 2019 (March-April) and 2020 (February-March), from healthy broiler chicken carcasses (n = 20) and chicken droppings in transport crates (n = 20) at various poultry abattoirs (N = 7) in the Gauteng province of South Africa. Escherichia coli organisms were isolated and subjected to a panel of 24 antibacterials using the MicroScan machine. Screening for mcr-1 colistin resistance gene was undertaken using PCR. RESULT: Four hundred and thirty-eight (438) E. coli strains were recovered and none demonstrated phenotypic resistance towards colistin, amikacin, carbapenems, tigecycline and piperacillin/tazobactam. The mcr-1 gene was not detected in any of the isolates tested. Resistances to the aminoglycosides (0%-9.8%) and fluoroquinolones (0%-18.9%) were generally low. Resistances to ampicillin (32%-39.3%) and trimethoprim/sulphamethoxazole (30.6%-3.6%) were fairly high. A significant (p < 0.05) increase in cephalosporins and cephamycin resistance was noted in the year 2020 (February-March) when compared with the year 2019 (March-April). CONCLUSION: The absence of mcr-1 gene and colistin resistance suggests that mitigation strategies adopted were effective and clearly demonstrated the significance of regulatory interventions in reducing resistance to critical drugs. Despite the drawback in regulatory framework such as free farmers access to antimicrobials OTC and a dual registration system in place, there is a general decline in the prevalence of ABR when the present data are compared with the last national veterinary surveillance on AMR (SANVAD 2007). To further drive resistance down, mitigation strategies should focus on strengthening regulatory framework, the withdrawal of OTC dispensing of antimicrobials, capping volumes of antimicrobials, banning growth promoters and investing on routine surveillance/monitoring of AMR and antimicrobial consumption.

An effective antimicrobial strategy of colistin combined with the Chinese herbal medicine shikonin against colistin-resistant Escherichia coli.

IMPORTANCE: Infections caused by multidrug-resistant Escherichia coli (MDR E. coli) have become a major global healthcare problem due to the lack of effective antibiotics today. The emergence of colistin-resistant E. coli strains makes the situation even worse. Therefore, new antimicrobial strategies are urgently needed to combat colistin-resistant E. coli. Combining traditional antibiotics with non-antibacterial drugs has proved to be an effective approach of combating MDR bacteria. This study investigated the combination of colistin and shikonin, a Chinese herbal medicine, against colistin-resistant E. coli. This combination showed good synergistic antibacterial both in vivo and in vitro experiments. Under the background of daily increasing colistin resistance in E. coli, this research points to an effective antimicrobial strategy of using colistin and shikonin in combination against colistin-resistant E. coli.

Detection of Extended-spectrum ß-lactamase-producing Escherichia coli isolates by isothermal amplification and association of their virulence genes and phylogroups with extraintestinal infection.

Extraintestinal pathogenic Escherichia coli (ExPEC) producing extended-spectrum ß-lactamases (ESBL) cause serious human infections due to their virulence and multidrug resistance (MDR) profiles. We characterized 144 ExPEC strains (collected from a tertiary cancer institute) in terms of antimicrobial susceptibility spectrum, ESBL variants, virulence factors (VF) patterns, and Clermont's phylogroup classification. The developed multiplex recombinase polymerase amplification and thermophilic helicase-dependent amplification (tHDA) assays for blaCTX-M, blaOXA, blaSHV, and blaTEM detection, respectively, were validated using PCR-sequencing results. All ESBL-ExPEC isolates carried blaCTX-M genes with following prevalence frequency of variants: blaCTX-M-15 (50.5%) > blaCTX-M-55 (17.9%) > blaCTX-M-27 (16.8%) > blaCTX-M-14 (14.7%). The multiplex recombinase polymerase amplification assay had 100% sensitivity, and specificity for blaCTX-M, blaOXA, blaSHV, while tHDA had 86.89% sensitivity, and 100% specificity for blaTEM. The VF genes showed the following prevalence frequency: traT (67.4%) > ompT (52.6%) > iutA (50.5%) > fimH (47.4%) > iha (33.7%) > hlyA (26.3%) > papC (12.6%) > cvaC (3.2%), in ESBL-ExPEC isolates which belonged to phylogroups A (28.4%), B2 (28.4%), and F (22.1%). The distribution of traT, ompT, and hlyA and phylogroup B2 were significantly different (P < 0.05) between ESBL-ExPEC and non-ESBL-ExPEC isolates. Thus, these equipment-free isothermal resistance gene amplification assays contribute to effective treatment and control of virulent ExPEC, especially antimicrobial resistance strains.

Gigantol restores the sensitivity of mcr carrying multidrug-resistant bacteria to colistin.

BACKGROUND: The emergence and wide spread of plasmid-mediated colistin resistance gene (mcr-1) and its mutants have immensely limited the efficacy of colistin in treating multidrug-resistant (MDR) Gram-negative bacterial infections. The development of synergistic combinations of antibiotics with a natural product that coped with the resistance of MDR bacteria was an economic strategy to restore antibiotics activity. Herein, we investigated gigantol, a bibenzyl phytocompound, for restoring in vitro and in vivo, the sensitivity of mcr-positive bacteria to colistin. METHODS: The synergistic activity of gigantol and colistin against multidrug-resistant Enterobacterales was studied via checkerboard assay and time-killing curve. Subsequently, the transcription and protein expression levels of mcr-1 gene were determined by RT-PCR and Western blots. The interaction of gigantol and MCR-1 was simulated via molecular docking and verified via site-directed mutagenesis of MCR-1. Hemolytic activity and cytotoxicity assay were used to evaluate the safety of gigantol. Finally, the in vivo synergistic effect was evaluated via two animal infection models. RESULTS: Gigantol restored the activity of colistin against mcr-positive bacteria E.coli B2 (MIC from 4 µg/ml to 0.25 µg/ml), Salmonella 15E343 (MIC from 8 µg/ml to 1 µg/ml), K. pneumoniae 19-2-1 (MIC from 32 µg/ml to 2 µg/ml) carrying mcr-1, mcr-3, mcr-8, respectively. Mechanistic studies revealed that gigantol down-regulated the expression of genes involved in LPS-modification, reduced the MCR-1 products and inhibited the activity of MCR-1 by binding to amino acid residues Tyr287 and Pro481 in its D-glucose-binding pocket. Safety evaluation showed that the addition of gigantol relieves the hemolysis caused by colistin. Compared with monotherapy, the combination of gigantol and colistin significantly improved the survival rate of Gallgallella mellonella larvae and mice infected by E.coli B2. Moreover, there was a considerable decrease in the bacterial load present in the viscera of mice. CONCLUSION: Our results confirmed that gigantol was a potential colistin adjuvant, and could be used to tackle multi-drug resistant Gram-negative pathogen infections combined with colistin.

Effects of the Rho GTPase-activating toxin CNF1 on fibroblasts derived from Rett syndrome patients: A pilot study.

The bacterial product CNF1, through its action on the Rho GTPases, is emerging as a modulator of crucial signalling pathways involved in selected neurological diseases characterized by mitochondrial dysfunctions. Mitochondrial impairment has been hypothesized to have a key role in paramount mechanisms underlying Rett syndrome (RTT), a severe neurologic rare disorder. CNF1 has been already reported to have beneficial effects in mouse models of RTT. Using human RTT fibroblasts from four patients carrying different mutations, as a reliable disease-in-a-dish model, we explored the cellular and molecular mechanisms, which can underlie the CNF1-induced amelioration of RTT deficits. We found that CNF1 treatment modulates the Rho GTPases activity of RTT fibroblasts and induces a considerable re-organization of the actin cytoskeleton, mainly in stress fibres. Mitochondria of RTT fibroblasts show a hyperfused morphology and CNF1 decreases the mitochondrial mass leaving substantially unaltered the mitochondrial dynamic. From a functional perspective, CNF1 induces mitochondrial membrane potential depolarization and activation of AKT in RTT fibroblasts. Given that mitochondrial quality control is altered in RTT, our results are suggestive of a reactivation of the damaged mitochondria removal via mitophagy restoration. These effects can be at the basis of the beneficial effects of CNF1 in RTT.

Effect of propolis on Th2 and Th17 cells: interplay with EtxB- and LPS-treated dendritic cells.

Dendritic cells (DCs) are antigen-presenting cells that drive the differentiation of T CD4+ cells into different profiles according to the nature of the antigen or immunomodulator. Propolis is a resinous product made by bees that has numerous pharmacological properties, including an immunomodulatory action. To assess whether propolis can modulate the activation of CD4+ T cells by stimulating DCs with heat-labile enterotoxin B subunit (EtxB) or lipopolysaccharide (LPS), we aimed to elucidate the mechanisms affected by propolis in the differential activation of T lymphocytes. Cell viability, lymphocyte proliferation, gene expression (GATA-3 and RORc), and cytokine production (interleukin (IL)-4 and IL-17A) were analyzed. Propolis, EtxB, and LPS induced a higher lymphoproliferation compared with the control. Propolis induced GATA-3 expression and, in combination with EtxB, maintained the baseline levels. Propolis alone or in combination with LPS inhibited RORc expression. EtxB alone and in combination with propolis increased IL-4 production. Propolis in combination with LPS prevented LPS-induced IL-17A production. These results opened perspectives for the study of biological events that may be favored by propolis by promoting Th2 activation or helping in the treatment of inflammatory conditions mediated by Th17 cells.

Cajanin stilbene acid: A direct inhibitor of colistin resistance protein MCR-1 that restores the efficacy of polymyxin B against resistant Gram-negative bacteria.

BACKGROUND: The resistance of Gram-negative bacteria to polymyxin B, caused by the plasmid-mediated colistin resistance gene mcr-1, which encodes a phosphoethanolamine transferase (MCR-1), is a serious threat to global public health. Therefore, it is urgent to find new drugs that can effectively alleviate polymyxin B resistance. Through the screening of 78 natural compounds, we found that cajanin stilbene acid (CSA) can significantly restore the susceptibility of polymyxin B to mcr-1 positive Escherichia coli (E. coli). PURPOSE: In this study, we tried to evaluate the ability of CSA to restore the susceptibility of polymyxin B towards the E. coli, and explore the mechanism of sensitivity recovery. STUDY DESIGN AND METHODS: Checkerboard MICs, time-killing curves, scanning electron microscope, lethal and semi-lethal models of infection in mice were used to assess the ability of CSA to restore the susceptibility of polymyxyn to E. coli. The interaction between CSA and MCR-1 was evaluated using surface plasmon resonance (SPR), and molecular docking experiments. RESULTS: Here, we find that CSA, a potential direct inhibitor of MCR-1, effectively restores the sensitivity of E. coli to polymyxin B. CSA can restore the sensitivity of polymyxin B to drug-resistant E. coli, and the MIC value can be reduced to 1 µg/ml. The time killing curve and scanning electron microscopy results also showed that CSA can effectively restore polymyxin B sensitivity. In vivo experiments showed that the simultaneous use of CSA and polymyxin B can effectively reduce the infection of drug-resistant E. coli in mice. SPR and molecular docking experiments confirmed that CSA strongly bound to MCR-1. The 17-carbonyl oxygen and 12- and 18­hydroxyl oxygens of CSA were the key sites binding to MCR-1. CONCLUSION: CSA is able to significantly restore the sensitivity of polymyxin B to E. coli in vivo and in vitro. CSA inhibits the enzymatic activity of the MCR-1 protein by binding to key amino acids at the active center of the MCR-1 protein.

International manufacturing and trade in colistin, its implications in colistin resistance and One Health global policies: a microbiological, economic, and anthropological study.

BACKGROUND: The emergence of colistin-resistant Enterobacterales is a global public health concern, yet colistin is still widely used in animals that are used for food as treatment, metaphylaxis, prophylaxis, and growth promotion. Herein, we investigate the effect of colistin-resistant Enterobacterales in Pakistan, global trade of colistin, colistin use at the farm level, and relevant socioeconomic factors. METHODS: We conducted a microbiological, economic, and anthropological study of colistin-resistant Escherichia coli in humans, animals, and the environment and international trade and knowledge of colistin in Pakistan, Bangladesh, Nigeria, China, India, and Viet Nam. We collected backyard poultry cloacal swabs, commercial broiler cloacal swabs, cattle and buffalo rectal swabs, human rectal swabs, wild bird droppings, cattle and buffalo meat, sewage water, poultry flies, chicken meat, and canal water from 131 sites across Faisalabad, Pakistan, to be tested for mcr-1-positive and mcr-3-positive Escherichia coli. We recruited new patients admitted to Allied Hospital, Faisalabad, Pakistan, with abdominal pain and diarrhoea for rectal swabs. Patients with dysentery and those who were already on antibiotic treatment were excluded. Data for colistin trade between 2017 and 2020, including importation, manufacturing, and usage, were accessed from online databases and government sources in Pakistan, Bangladesh, and Nigeria. We recruited participants from poultry farms and veterinary drug stores in Pakistan and Nigeria to be interviewed using a structured questionnaire. International manufacturing, import, and export data; value analysis; and trade routes of colistin pharmaceutical raw material (PRM), feed additive, and finished pharmaceutical products (FPPs) were accessed from 2017-21 export data sets. FINDINGS: We collected 1131 samples between May 12, 2018, and July 1, 2019: backyard poultry cloacal swabs (n=100), commercial broiler cloacal swabs (n=102), cattle and buffalo rectal swabs (n=188), human rectal swabs (n=200), wild bird droppings (n=100), cattle and buffalo meat (n=100), sewage water (n=90), poultry flies (n=100), chicken meat (n=100), and canal water (n=51). We recruited 200 inpatients at Allied Hospital, Faisalabad, Pakistan, between Nov 15, 2018, and Dec 14, 2018, for rectal swabs. We recruited 21 participants between Jan 1, 2020, and Dec 31, 2020, from poultry farms and drug stores in Pakistan and Nigeria to be interviewed. 75 (7%) of 1131 samples contained mcr-1-positive E coli, including wild bird droppings (25 [25%] of 100), commercial broiler cloacal swabs (17 [17%] of 100), backyard poultry cloacal swabs (one [1%] of 100), chicken meat (13 [13%] of 100), cattle and buffalo meat (two [2%] of 100), poultry flies (eight [8%] of 100), sewage water (six [7%] of 90), and human rectal swabs (three [2%] of 200). During 2017-20, Pakistan imported 275·5 tonnes (68·9 tonnes per year, 95% CI 41·2-96·6) of colistin as PRM, all sourced from China, 701·9 tonnes (175·5 tonnes per year, 140·9-210·1) of colistin as feed additives from China and Viet Nam, and 63·0 tonnes (15·8 tonnes per year, 10·4-21·1) of colistin as FPPs from various countries in Asia and Europe. For Bangladesh and Nigeria, colistin PRM and FPPs were imported from China and Europe. Colistin knowledge and usage practices in Pakistan and Nigeria were unsatisfactory in terms of understanding of the effects on human medicine and usage other than for treatment purposes. China is the major manufacturer of PRM and feed additive colistin and exported a total of 2664·8 tonnes (666·2 tonnes per year, 95% CI 262·1 to 1070·2) of PRM and 2570·2 tonnes (642·6 tonnes per year, -89·4 to 1374·5) of feed additive in 1330 shipments during 2018-21 to 21 countries. INTERPRETATION: Regardless of 193 countries signing the UN agreement to tackle antimicrobial resistance, trading of colistin as PRM, FPPs, and feed additive or growth promoter in low-income and middle-income countries continues unabated. Robust national and international laws are urgently required to mitigate the international trade of this antimicrobial listed on WHO Critically Important Antimicrobials for Human Medicine. FUNDING: Pakistan Agricultural Research Council and INEOS Oxford Institute for Antimicrobial Research TRANSLATION: For the Urdu translation of the abstract see Supplementary Materials section.

Effect of Changes in the Redox Status on Biofilm Formation in Escherichia coli.

Changes in the redox balance in the medium and in Escherichia coli cells significantly affect the ability of bacteria to form biofilms. An increase in the level of aeration in the culture of wild-type bacteria led to a 3-fold decrease in the mass of biofilms. Mutants lacking components of the glutathione and thioredoxin redox systems, as well as transporters involved in the transmembrane cycling of glutathione, demonstrated increased biofilm formation ability. The effect of exogenous glutathione on biofilm formation depended on the culturing conditions. The addition of 0.1-1 mM Trolox (a water-soluble analog of vitamin E) was accompanied by a 30-40% reduction in biofilm formation.

Evolution of the proportion of colistin-resistant isolates in animal clinical Escherichia coli over time - A hierarchical mixture model approach.

Colistin resistance has been the subject of much attention since mcr genes encoding plasmid-mediated colistin resistance description in 2015. To date, surveillance data about resistance levels encountered in food-producing animals are scarce. In France, the Resapath dataset, consisting in a large collection of disk diffusion antibiogram results transmitted by a network of laboratories. It offers a unique opportunity to study the evolution of resistance towards colistin over the past 15 years in Escherichia coli isolated from diseased food-producing animals. This study used a Bayesian hierarchical Gaussian mixture model to estimate the resistant proportions from those data. This non-classical approach deals with the colistin-specific problem of overlapping distributions of diameters measured for susceptible and resistant isolates that makes the definition of epidemiological cut-off very hard. This model also considers the variability observed between the measurements performed by different laboratories. Proportion of resistant isolates has been calculated for several food-producing animals and most encountered diseases. From those estimations, a marked evolution of the proportions of resistant isolates is noticeable, for swine suffering from digestive disorders. In this group, an increase over the 2006-2011 period from 0.1% [ 0.0%, 1.2%] in 2006-28.6% [25.1%, 32.3%] in 2011 was followed by a decrease to reach 3.6% [2.3%;5.3%] in 2018. For isolates related to digestive disorders in calves, percentages increased and reached 7% in 2009 then decreased as for swine. In contrast, for poultry productions, estimated proportions and credibility intervals were constantly very close to zero.

Deep mutational scanning of essential bacterial proteins can guide antibiotic development.

Deep mutational scanning is a powerful approach to investigate a wide variety of research questions including protein function and stability. Here, we perform deep mutational scanning on three essential E. coli proteins (FabZ, LpxC and MurA) involved in cell envelope synthesis using high-throughput CRISPR genome editing, and study the effect of the mutations in their original genomic context. We use more than 17,000 variants of the proteins to interrogate protein function and the importance of individual amino acids in supporting viability. Additionally, we exploit these libraries to study resistance development against antimicrobial compounds that target the selected proteins. Among the three proteins studied, MurA seems to be the superior antimicrobial target due to its low mutational flexibility, which decreases the chance of acquiring resistance-conferring mutations that simultaneously preserve MurA function. Additionally, we rank anti-LpxC lead compounds for further development, guided by the number of resistance-conferring mutations against each compound. Our results show that deep mutational scanning studies can be used to guide drug development, which we hope will contribute towards the development of novel antimicrobial therapies.

2-Aminoimidazole Analogs Target PhoP Altering DNA Binding Activity and Affect Outer Membrane Stability in Gram-Negative Bacteria.

Multidrug-resistant bacteria cause immense public health concerns as once effective antibiotics no longer work against even common infections. Concomitantly, there has been a decline in the discovery of new antibiotics, and the current global clinical pipeline is woefully inadequate, especially against resistant Gram-negative bacteria. One major contribution to Gram-negative resistance is the presence of a protective outer membrane. Consequently, an appealing option for tackling resistance is to adversely affect that outer membrane. With that in mind, we define the response regulator PhoP as a target for new 2-aminoimidazole compounds and show that they affect the integrity of the outer membrane in resistant strains of Escherichia coli and Klebsiella pneumoniae. We also provide empirical evidence for the 2-aminoimidazole mechanism of action.

Worldwide Disseminated IncX4 Plasmid Carrying mcr-1 Arrives to Wild Mammal in Portugal.

The mcr-1 gene spread is worldwide recognized as a public health threat at multidrug-resistant infections therapy level. Here, we report for the first time, to the best of our knowledge, the detection of the globally distributed IncX4 plasmid carrying mcr-1 (mcr-1/IncX4) in Escherichia coli isolated from a wild mammal in Portugal and Europe. This plasmid was found in a ST533 E. coli isolate with a multidrug-resistant profile, virulence potential, and possibly phylogenetically related to human isolates. Our work contributes to highlight the importance of antimicrobial resistance (AMR) surveillance in wildlife, an important compartment of the whole ecosystem often overlooked in the fight against AMR. IMPORTANCE Colistin resistance mediated by plasmids is recognized worldwide as an emergency problem connected with the whole ecosystem, since is well described in the interface of the human-animal-environment. The plasmid IncX4 is reported as one of the most prevalent plasmids harboring the gene mcr-1. On an European scale the plasmid IncX4 carrying mcr-1 has been described in humans, the environment, and animals, including wildlife, but only in wild birds. This study shows the first report of the plasmid IncX4 harboring mcr-1 in a wild mammal in Portugal and Europe, identified in a ST533 E. coli commensal that is, curiously, more related to isolates from humans than from livestock. Our findings show that the plasmid IncX4 harboring mcr-1 is well established in a colistin resistance drive embracing the whole ecosystem.

Global epidemiology, genetic environment, risk factors and therapeutic prospects of mcr genes: A current and emerging update.

Background: Mobile colistin resistance (mcr) genes modify Lipid A molecules of the lipopolysaccharide, changing the overall charge of the outer membrane. Results and discussion: Ten mcr genes have been described to date within eleven Enterobacteriaceae species, with Escherichia coli, Klebsiella pneumoniae, and Salmonella species being the most predominant. They are present worldwide in 72 countries, with animal specimens currently having the highest incidence, due to the use of colistin in poultry for promoting growth and treating intestinal infections. The wide dissemination of mcr from food animals to meat, manure, the environment, and wastewater samples has increased the risk of transmission to humans via foodborne and vector-borne routes. The stability and spread of mcr genes were mediated by mobile genetic elements such as the IncHI2 conjugative plasmid, which is associated with multiple mcr genes and other antibiotic resistance genes. The cost of acquiring mcr is reduced by compensatory adaptation mechanisms. MCR proteins are well conserved structurally and via enzymatic action. Thus, therapeutics found effective against MCR-1 should be tested against the remaining MCR proteins. Conclusion: The dissemination of mcr genes into the clinical setting, is threatening public health by limiting therapeutics options available. Combination therapies are a promising option for managing and treating colistin-resistant Enterobacteriaceae infections whilst reducing the toxic effects of colistin.

Novel Antibiofilm Inhibitor Ginkgetin as an Antibacterial Synergist against Escherichia coli.

As an opportunistic pathogen, Escherichia coli (E. coli) forms biofilm that increases the virulence of bacteria and antibiotic resistance, posing a serious threat to human and animal health. Recently, ginkgetin (Gin) has been discovered to have antiinflammatory, antioxidant, and antitumor properties. In the present study, we evaluated the antibiofilm and antibacterial synergist of Gin against E. coli. Additionally, Alamar Blue assay combined with confocal laser scanning microscope (CLSM) and crystal violet (CV) staining was used to evaluate the effect of antibiofilm and antibacterial synergist against E. coli. Results showed that Gin reduces biofilm formation, exopolysaccharide (EPS) production, and motility against E. coli without limiting its growth and metabolic activity. Furthermore, we identified the inhibitory effect of Gin on AI-2 signaling molecule production, which showed apparent anti-quorum sensing (QS) properties. The qRT-PCR also indicated that Gin reduced the transcription of curli-related genes (csgA, csgD), flagella-formation genes (flhC, flhD, fliC, fliM), and QS-related genes (luxS, lsrB, lsrK, lsrR). Moreover, Gin showed obvious antibacterial synergism to overcome antibiotic resistance in E. coli with marketed antibiotics, including gentamicin, colistin B, and colistin E. These results suggested the potent antibiofilm and novel antibacterial synergist effect of Gin for treating E. coli infections.

Acridinium-conjugated aromatic heterocycles as highly potent FtsZ inhibitors: Design, synthesis, and biological evaluation.

The epidemic of multidrug resistance (MDR) is a serious threat to public health, and new classes of antibiotics with novel mechanisms of action are in critical need. We rationally designed and efficiently synthesized three series of new chemical entities with potential antibacterial activity targeting filamenting temperature-sensitive mutant Z (FtsZ). Evaluation of these compounds against a panel of Gram-positive bacteria including MDR and vancomycin-resistant Enterococcus strains indicated that most compounds showed enhanced antibacterial efficacy, comparable or even superior to the reference drugs. The newly synthesized compounds proved to be substrates of the Escherichia coli efflux pump AcrB, thus affecting the activity. Their structure-activity relationships were summarized in detail. The most potent compound 10f quickly eliminated bacteria in a bactericidal mode, with low susceptibility to induce bacterial resistance. Further mechanistic studies with the BsFtsZ protein revealed that 10f functioned as an effective FtsZ inhibitor through altering the dynamics of FtsZ self-polymerization via a stimulatory mechanism, which leads to inhibition of cell division and cell death. Besides, 10f not only displayed no obvious cytotoxicity to mammalian cells but also had a high efficacy in a murine model of bacteremia in vivo. Regarded as a whole, our findings highlight 10f as a promising new FtsZ-targeting bactericidal agent.

Interactive Dynamics of Cell Volume and Cell Death in Human Erythrocytes Exposed to α-Hemolysin from Escherichia coli.

α-hemolysin (HlyA) of E. coli binds irreversibly to human erythrocytes and induces cell swelling, ultimately leading to hemolysis. We characterized the mechanism involved in water transport induced by HlyA and analyzed how swelling and hemolysis might be coupled. Osmotic water permeability (Pf) was assessed by stopped-flow light scattering. Preincubation with HlyA strongly reduced Pf in control- and aquaporin 1-null red blood cells, although the relative Pf decrease was similar in both cell types. The dynamics of cell volume and hemolysis on RBCs was assessed by electrical impedance, light dispersion and hemoglobin release. Results show that HlyA induced erythrocyte swelling, which is enhanced by purinergic signaling, and is coupled to osmotic hemolysis. We propose a mathematical model of HlyA activity where the kinetics of cell volume and hemolysis in human erythrocytes depend on the flux of osmolytes across the membrane, and on the maximum volume that these cells can tolerate. Our results provide new insights for understanding signaling and cytotoxicity mediated by HlyA in erythrocytes.

Plant-derived human recombinant growth factors and serum albumin maintain stemness of human-induced pluripotent stem cells.

Stem cells are an important therapeutic source for recovery and regeneration, as their ability of self-renewal and differentiation offers an unlimited supply of highly specialized cells for therapeutic transplantation. Growth factors and serum are essential for maintaining the characteristics of stem cells in culture and for inducing differentiation. Because growth factors are produced mainly in bacterial (Escherichia coli) or animal cells, the use of such growth factors raises safety concerns that need to be resolved for the commercialization of stem cell therapeutics. To overcome this problem, studies on proteins produced in plants have been conducted. Here, we describe the functions of plant-derived fibroblast growth factor 2 (FGF2) and human serum albumin in the maintenance and differentiation of human-induced pluripotent stem cells (hiPSCs). Plant-derived FGF2 and human epidermal growth factor EGF were able to differentiate hiPSCs into neural stem cells (NSCs). These NSCs could differentiate into neuronal and glial cells. Our results imply that culturing stem cells in animal-free culture medium, which is composed of plant-derived proteins, would facilitate stem cell application research, for example, for cell therapy, by reducing contamination risk.