The Anthelmintic Drug Niclosamide Synergizes with Colistin and Reverses Colistin Resistance in Gram-Negative Bacilli.
ABSTRACT: There is an urgent need for new therapies to overcome antimicrobial resistance especially in Gram-negative bacilli (GNB). Repurposing old U.S. Food and Drug Administration-approved drugs as complementary agents to existing antibiotics in a synergistic combination presents an attractive strategy. Here, we demonstrate that the anthelmintic drug niclosamide selectively synergized with the lipopeptide antibiotic colistin against colistin-susceptible but more importantly against colistin-resistant GNB, including clinical isolates that harbor the mcr-1 gene. Breakpoints for colistin susceptibility in resistant Gram-negative bacilli were reached in the presence of 1 μg/ml (3 μM) niclosamide. Reversal of colistin resistance was also observed in combinations of niclosamide and polymyxin B. Enhanced bacterial killing was evident for the combination, in comparison to colistin monotherapy, against resistant Pseudomonas aeruginosa, Acinetobacter baumannii, Klebsiella pneumoniae, Escherichia coli, and Enterobacter cloacae Accumulating evidence in the literature, along with our results, strongly suggests the potential for the combination of niclosamide and colistin to treat colistin-resistant Gram-negative bacillary infections. Our finding is significant since colistin is an antibiotic of last resort for multidrug-resistant Gram-negative bacterial infections that are nonresponsive to conventional treatments. With the recent global dissemination of plasmid-encoded colistin resistance, the addition of niclosamide to colistin therapy may hold the key to overcome colistin resistance.
Project description:Increasing antibiotic resistance in multidrug-resistant (MDR) Gram-negative bacteria (MDR-GNB) presents significant health problems worldwide, since the vital available and effective antibiotics, including; broad-spectrum penicillins, fluoroquinolones, aminoglycosides, and ?-lactams, such as; carbapenems, monobactam, and cephalosporins; often fail to fight MDR Gram-negative pathogens as well as the absence of new antibiotics that can defeat these "superbugs". All of these has prompted the reconsideration of old drugs such as polymyxins that were reckoned too toxic for clinical use. Only two polymyxins, polymyxin E (colistin) and polymyxin B, are currently commercially available. Colistin has re-emerged as a last-hope treatment in the mid-1990s against MDR Gram-negative pathogens due to the development of extensively drug-resistant GNB. Unfortunately, rapid global resistance towards colistin has emerged following its resurgence. Different mechanisms of colistin resistance have been characterized, including intrinsic, mutational, and transferable mechanisms.In this review, we intend to discuss the progress over the last two decades in understanding the alternative colistin mechanisms of action and different strategies used by bacteria to develop resistance against colistin, besides providing an update about what is previously recognized and what is novel concerning colistin resistance.
Project description:BACKGROUND:Cases of ventilator-associated pneumonia (VAP) due to multidrug-resistant (MDR) gram-negative bacilli (GNB) mainly Acinetobacter baumannii, Pseudomonas aeruginosa and enterobacteria are common in hospitalised patients of Tunisian intensive care units (ICUs). Parenteral colistin has been used for the therapy of VAP caused by MDR GNB at Tunisian hospitals over the past few years with a favourable clinical response. However, its use fell out of favour because of the reported drug-related nephrotoxicity and neurotoxicity. OBJECTIVES:To determine whether aerosolised (AS) colistin was beneficial and safe in therapy of gram-negative VAP. METHODS:This was a randomised, single-blind study, in 149 critically ill adults who developed gram-negative VAP. Included patients were divided into two groups whether they received AS colistin (intervention group; n = 73) or intravenous (IV) colistin (control group; n = 76). AS colistin was given as 4 million units (MU) by nebulisation three times per 24 h. IV colistin was given as a loading dose of 9 MU followed by 4.5 MU two times per 24 h. Patients were followed during 28 days. Primary outcome was cure of VAP assessed at day 14 of therapy and defined as resolution of clinical signs of VAP and bacteriological eradication. Secondary outcomes were incidence of acute renal failure (ARF), mechanical ventilation length, ICU length of stay and 28-day mortality. Results were analysed based on intention-to-treat concept. RESULTS:The patient's baseline characteristics and distribution of pathogens VAP in both groups were similar. The clinical cure rate was 67.1 % in AS group and 72 % in IV group (p = 0.59). When administered in monotherapy or in combination, the AS regimen was as effective as IV regimen. Patients in AS group had significantly lower incidence of ARF (17.8 vs 39.4 %, p = 0.004), more favourable improvement of P/F ratio (349 vs 316 at day 14, p = 0.012), shortened time to bacterial eradication (TBE) (9.89 vs 11.26 days, p = 0.023) and earlier weaning from ventilator in ICU survivors with a mean gain in ventilator-free days of 5 days. No difference was shown in the length of stay and the 28-day mortality. CONCLUSION:Aerosolised colistin seems to be beneficial. It provided a therapeutic effectiveness non-inferior to parenteral colistin in therapy of MDR bacilli VAP with a lower nephrotoxicity, a better improvement of P/F ratio, a shortened bacterial eradication time and earlier weaning from ventilator in ICU survivors. Trial registration ClinicalTrials.gov Identifier: NCT02683603.
Project description:Plasmid-borne colistin resistance mediated by mcr-1 may contribute to the dissemination of pan-resistant Gram-negative bacteria. Here, we show that mcr-1 confers resistance to colistin-induced lysis and bacterial cell death, but provides minimal protection from the ability of colistin to disrupt the Gram-negative outer membrane. Indeed, for colistin-resistant strains of Enterobacteriaceae expressing plasmid-borne mcr-1, clinically relevant concentrations of colistin potentiate the action of antibiotics that, by themselves, are not active against Gram-negative bacteria. The result is that several antibiotics, in combination with colistin, display growth-inhibition at levels below their corresponding clinical breakpoints. Furthermore, colistin and clarithromycin combination therapy displays efficacy against mcr-1-positive Klebsiella pneumoniae in murine thigh and bacteremia infection models at clinically relevant doses. Altogether, these data suggest that the use of colistin in combination with antibiotics that are typically active against Gram-positive bacteria poses a viable therapeutic alternative for highly drug-resistant Gram-negative pathogens expressing mcr-1.
Project description:Colistin is a member of cationic polypeptide antibiotics known as polymyxins. It is widely used in animal husbandry, plant cultivation, animal and human medicine and is increasingly used as one of the last available treatment options for patients with severe infections with carbapenem-resistant Gram-negative bacilli. Due to the increased use of colistin in treating infections caused by multidrug-resistant (MDR) bacteria, the resistance to this antibiotic ought to be monitored. Bacterial resistance to colistin may be encoded on transposable genetic elements (e.g. plasmids with the mcr genes). Thus far, nine variants of the mcr gene, mcr-1 - mcr-9, have been identified. Chromosomal resistance to colistin is associated with the modification of lipopolysaccharide (LPS). Various methods, from classical microbiology to molecular biology methods, are used to detect the colistin-resistant bacterial strains and to identify resistance mechanisms. The broth dilution method is recommended for susceptibility testing of bacteria to colistin.Colistin is a member of cationic polypeptide antibiotics known as polymyxins. It is widely used in animal husbandry, plant cultivation, animal and human medicine and is increasingly used as one of the last available treatment options for patients with severe infections with carbapenem-resistant Gram-negative bacilli. Due to the increased use of colistin in treating infections caused by multidrug-resistant (MDR) bacteria, the resistance to this antibiotic ought to be monitored. Bacterial resistance to colistin may be encoded on transposable genetic elements (e.g. plasmids with the mcr genes). Thus far, nine variants of the mcr gene, mcr-1 – mcr-9, have been identified. Chromosomal resistance to colistin is associated with the modification of lipopolysaccharide (LPS). Various methods, from classical microbiology to molecular biology methods, are used to detect the colistin-resistant bacterial strains and to identify resistance mechanisms. The broth dilution method is recommended for susceptibility testing of bacteria to colistin.
Project description:Resistance to colistin, a polypeptide drug used as an agent of last resort for the treatment of infections caused by multidrug-resistant (MDR) and extensively drug-resistant (XDR) Gram-negative bacteria, including carbapenem-resistant Enterobacteriaceae (CRE), severely limits treatment options and may even transform an XDR organism into one that is pan-resistant. We investigated the synergistic activity of colistin in combination with 19 antibiotics against a collection of 20 colistin-resistant Enterobacteriaceae isolates, 15 of which were also CRE. All combinations were tested against all strains using an inkjet printer-assisted digital dispensing checkerboard array, and the activities of those that demonstrated synergy by this method were evaluated against a single isolate in a time-kill synergy study. Eighteen of 19 combinations demonstrated synergy against two or more isolates, and the 4 most highly synergistic combinations (colistin combined with linezolid, rifampin, azithromycin, and fusidic acid) were synergistic against ≥90% of strains. Sixteen of 18 combinations (88.9%) that were synergistic in the checkerboard array were also synergistic in a time-kill study. Our findings demonstrate that colistin in combination with a range of antibiotics, particularly protein and RNA synthesis inhibitors, exhibits synergy against colistin-resistant strains, suggesting that colistin may exert a subinhibitory permeabilizing effect on the Gram-negative bacterial outer membrane even in isolates that are resistant to it. These findings suggest that colistin combination therapy may have promise as a treatment approach for patients infected with colistin-resistant XDR Gram-negative pathogens.
Project description:Colistin is one of the last-resort therapeutic agents to combat multidrug-resistant Gram-negative bacteria (GNB) including Klebsiella pneumoniae. Although it happens rarely, resistance to colistin has been reported for several GNB. A total of 20 colistin resistant (col-R) and three colistin susceptible (col-S) clinical isolates of K. pneumoniae were studied to explore the underlying mechanisms of colistin resistance. The presence of plasmid encoded resistance genes, mcr-1, mcr-2, mcr-3, and mcr-4 genes were examined by PCR. The nucleotide sequences of pmrA, pmrB, phoP, phoQ, and mgrB genes were determined. To evaluate the association between colistin resistance and upregulation of pmrHFIJKLM and pmrCAB operons, transcriptional level of the pmrK and pmrC genes encoding for lipopolysaccharide target modifying enzymes was quantified by RT-qPCR analysis. None of the plasmid encoded resistance genes were detected in the studied isolates. Inactivation of MgrB due to nonsense mutations and insertion of IS elements was observed in 15 col-R isolates (75%). IS elements (IS5-like and IS1-like families) most commonly targeted the coding region and in one case the promoter region of the mgrB. Complementation with wild-type MgrB restored colistin susceptibility in isolates with altered mgrB. All col-R isolates lacked any genetic alterations in the pmrA, phoP, and phoQ genes and substitutions identified in the pmrB were not found to be involved in resistance conferring determined by complementation assay. Colistin resistance linked with upregulation of pmrHFIJKLM and pmrCAB operons with the pmrK and pmrC being overexpressed in 20 and 11 col-R isolates, respectively. Our results demonstrated that MgrB alterations are the major mechanisms contributing to colistin resistance in the tested K. pneumoniae isolates from Iran.
Project description:One avenue to combat multidrug-resistant Gram-negative bacteria is the coadministration of multiple drugs (combination therapy), which can be particularly promising if drugs synergize. The identification of synergistic drug combinations, however, is challenging. Detailed understanding of antibiotic mechanisms can address this issue by facilitating the rational design of improved combination therapies. Here, using diverse biochemical and genetic assays, we examine the molecular mechanisms of niclosamide, a clinically approved salicylanilide compound, and demonstrate its potential for Gram-negative combination therapies. We discovered that Gram-negative bacteria possess two innate resistance mechanisms that reduce their niclosamide susceptibility: a primary mechanism mediated by multidrug efflux pumps and a secondary mechanism of nitroreduction. When efflux was compromised, niclosamide became a potent antibiotic, dissipating the proton motive force (PMF), increasing oxidative stress, and reducing ATP production to cause cell death. These insights guided the identification of diverse compounds that synergized with salicylanilides when coadministered (efflux inhibitors, membrane permeabilizers, and antibiotics that are expelled by PMF-dependent efflux), thus suggesting that salicylanilide compounds may have broad utility in combination therapies. We validate these findings in vivo using a murine abscess model, where we show that niclosamide synergizes with the membrane permeabilizing antibiotic colistin against high-density infections of multidrug-resistant Gram-negative clinical isolates. We further demonstrate that enhanced nitroreductase activity is a potential route to adaptive niclosamide resistance but show that this causes collateral susceptibility to clinical nitro-prodrug antibiotics. Thus, we highlight how mechanistic understanding of mode of action, innate/adaptive resistance, and synergy can rationally guide the discovery, development, and stewardship of novel combination therapies.IMPORTANCE There is a critical need for more-effective treatments to combat multidrug-resistant Gram-negative infections. Combination therapies are a promising strategy, especially when these enable existing clinical drugs to be repurposed as antibiotics. We examined the mechanisms of action and basis of innate Gram-negative resistance for the anthelmintic drug niclosamide and subsequently exploited this information to demonstrate that niclosamide and analogs kill Gram-negative bacteria when combined with antibiotics that inhibit drug efflux or permeabilize membranes. We confirm the synergistic potential of niclosamide in vitro against a diverse range of recalcitrant Gram-negative clinical isolates and in vivo in a mouse abscess model. We also demonstrate that nitroreductases can confer resistance to niclosamide but show that evolution of these enzymes for enhanced niclosamide resistance confers a collateral sensitivity to other clinical antibiotics. Our results highlight how detailed mechanistic understanding can accelerate the evaluation and implementation of new combination therapies.
Project description:BACKGROUND:Colistin is recommended in the empirical treatment of ventilator-associated pneumonia (VAP) with a high prevalence of carbapenem-resistant gram-negative bacilli (CR-GNB). However, the efficacy and safety of colistin are not well defined. METHODS:A multicenter prospective randomized trial conducted in 32 European centers compared the efficacy and safety of colistin (4.5 million unit loading dose followed by a maintenance dose of 3 million units every 8?h) versus meropenem (2?g every 8?h), both in combination with levofloxacin (500?mg every 12?h) for 7-14?days in patients with late VAP. Between May 2012 and October 2015, 232 patients were randomly assigned to the 2 treatment groups. The primary endpoint was mortality at 28?days after randomization in the microbiologically modified intention-to-treat (mMITT) population. Secondary outcomes included clinical and microbiological cure, renal function at the end of the treatment, and serious adverse events. The study was interrupted after the interim analysis due to excessive nephrotoxicity in the colistin group; therefore, the sample size was not achieved. RESULTS:A total of 157 (67.7%) patients were included in the mMITT population, 36 of whom (22.9%) had VAP caused by CR-GNB. In the mMITT population, no significant difference in mortality between the colistin group (19/82, 23.2%) and the meropenem group (19/75, 25.3%) was observed, with a risk difference of -?2.16 (-?15.59 to 11.26, p?=?0.377); the noninferiority of colistin was not demonstrated due to early termination and limited number of patients infected by carbapenem-resistant pathogens. Colistin plus levofloxacin increased the incidence of renal failure (40/120, 33.3%, versus 21/112, 18.8%; p?=?0.012) and renal replacement therapy (11/120, 9.1%, versus 2/112, 1.8%; p?=?0.015). CONCLUSIONS:This study did not demonstrate the noninferiority of colistin compared with meropenem, both combined with levofloxacin, in terms of efficacy in the empirical treatment of late VAP but demonstrated the greater nephrotoxicity of colistin. These findings do not support the empirical use of colistin for the treatment of late VAP due to early termination. TRIAL REGISTRATION:ClinicalTrials.gov, NCT01292031. Registered 9 February 2011.
Project description:Post-weaning diarrhea (PWD) is one of the most serious threats for the swine industry worldwide. It is commonly associated with the proliferation of enterotoxigenic Escherichia coli in the pig intestine. Colistin, a cationic antibiotic, is widely used in swine for the oral treatment of intestinal infections caused by E. coli, and particularly of PWD. However, despite the effectiveness of this antibiotic in the treatment of PWD, several studies have reported high rates of colistin resistant E. coli in swine. Furthermore, this antibiotic is considered of very high importance in humans, being used for the treatment of infections due to multidrug-resistant (MDR) Gram-negative bacteria (GNB). Moreover, the recent discovery of the mcr-1 gene encoding for colistin resistance in Enterobacteriaceae on a conjugative stable plasmid has raised great concern about the possible loss of colistin effectiveness for the treatment of MDR-GNB in humans. Consequently, it has been proposed that the use of colistin in animal production should be considered as a last resort treatment only. Thus, to overcome the economic losses, which would result from the restriction of use of colistin, especially for prophylactic purposes in PWD control, we believe that an understanding of the factors contributing to the development of this disease and the putting in place of practical alternative strategies for the control of PWD in swine is crucial. Such alternatives should improve animal gut health and reduce economic losses in pigs without promoting bacterial resistance. The present review begins with an overview of risk factors of PWD and an update of colistin use in PWD control worldwide in terms of quantities and microbiological outcomes. Subsequently, alternative strategies to the use of colistin for the control of this disease are described and discussed. Finally, a practical approach for the control of PWD in its various phases is proposed.
Project description:<b>Background:</b> Emergence, prevalence and widely spread of plasmid-mediated colistin resistance in Enterobacteriaceae strongly impairs the clinical efficacy of colistin against life-threatening bacterial infections. Combinations of antibiotics and FDA-approved non-antibiotic agents represent a promising means to address the widespread emergence of antibiotic-resistant pathogens. <b>Methods:</b> Herein, we investigated the synergistic activity between melatonin and antibiotics against MCR (mobilized colistin resistance)-positive Gram-negative pathogens through checkerboard assay and time-killing curve. Molecular mechanisms underlying its mode of action were elucidated. Finally, we assessed the <i>in vivo</i> efficacy of melatonin in combination with colistin against drug-resistant Gram-negative bacteria. <b>Results:</b> Melatonin, which has been approved for treating sleep disturbances and circadian disorders, substantially potentiates the activity of three antibiotics, particularly colistin, against MCR-expressing pathogens without enhancing its toxicity. This is evidence that the combination of colistin with melatonin enhances bacterial outer membrane permeability, promotes oxidative damage and inhibits the effect of efflux pumps. In three animal models infected by <i>mcr-1</i>-carrying <i>E. coli</i>, melatonin dramatically rescues colistin efficacy. <b>Conclusion:</b> Our findings revealed that melatonin serves as a promising colistin adjuvant against MCR-positive Gram-negative pathogens.