Investigation of Novel pmrB and eptA Mutations in Isogenic Acinetobacter baumannii Isolates Associated with Colistin Resistance and Increased Virulence In Vivo.

Colistin resistance in Acinetobacter baumannii is of great concern and is a threat to human health. In this study, we investigate the mechanisms of colistin resistance in four isogenic pairs of A. baumannii isolates displaying an increase in colistin MICs. A mutation in pmrB was detected in each colistin-resistant isolate, three of which were novel (A28V, I232T, and ΔL9-G12). Increased expression of pmrC was shown by semi-quantitative reverse transcription-PCR (qRT-PCR) for three colistin-resistant isolates, and the addition of phosphoethanolamine (PEtN) to lipid A by PmrC was revealed by mass spectrometry. Interestingly, PEtN addition was also observed in some colistin-susceptible isolates, indicating that this resistance mechanism might be strain specific and that other factors could contribute to colistin resistance. Furthermore, the introduction of pmrAB carrying the short amino acid deletion ΔL9-G12 into a pmrAB knockout strain resulted in increased pmrC expression and lipid A modification, but colistin MICs remained unchanged, further supporting the strain specificity of this colistin resistance mechanism. Of note, a mutation in the pmrC homologue eptA and a point mutation in ISAba1 upstream of eptA were associated with colistin resistance and increased eptA expression, which is a hitherto undescribed resistance mechanism. Moreover, no cost of fitness was observed for colistin-resistant isolates, while the virulence of these isolates was increased in a Galleria mellonella infection model. Although the mutations in pmrB were associated with colistin resistance, PEtN addition appears not to be the sole factor leading to colistin resistance, indicating that the mechanism of colistin resistance is far more complex than previously suspected and is potentially strain specific.

Evaluation of an Immunochromatographic Lateral Flow Assay (OXA-48 K-SeT) for Rapid Detection of OXA-48-Like Carbapenemases in Enterobacteriaceae.

We evaluated an immunochromatographic lateral flow assay to detect OXA-48-like carbapenemases (OXA-48 K-SeT) in Enterobacteriaceae (n = 82). One hundred percent sensitivity and specificity were observed using bacteria recovered from both solid medium and spiked blood culture bottles, and the results were obtained in <10 min.

In Vitro Antibacterial Activity of Curcumin-Polymyxin B Combinations against Multidrug-Resistant Bacteria Associated with Traumatic Wound Infections.

Bacterial infections resulting from nonsurgical traumatic wounds can be life threatening, especially those caused by multidrug-resistant (MDR) bacteria with limited therapeutic options. The antimicrobial activity of polymyxin B (1) and curcumin (2) alone and in combination was determined versus MDR bacterial isolates associated with traumatic wound infections. Cytotoxicity assays for 1 and 2 were undertaken in keratinocyte cell lines. Minimum inhibitory concentrations of 1 were significantly reduced in the presence of 2 (3- to 10-fold reduction), with synergy observed. Time-kill assays showed the combinations produced bactericidal activity. Cytotoxicity assays indicate the toxicity of 2 was reduced in the presence of 1.

Colistin and Fusidic Acid, a Novel Potent Synergistic Combination for Treatment of Multidrug-Resistant Acinetobacter baumannii Infections.

The spread of multidrug-resistant Acinetobacter baumannii (MDRAB) has led to the renaissance of colistin (COL), often the only agent to which MDRAB remains susceptible. Effective therapy with COL is beset with problems due to unpredictable pharmacokinetics, toxicity, and the rapid selection of resistance. Here, we describe a potent synergistic interaction when COL was combined with fusidic acid (FD) against A. baumannii. Synergy in vitro was assessed against 11 MDRAB isolates using disc diffusion, checkerboard methodology (fractional inhibitory concentration index [FICI] of ≤ 0.5, susceptibility breakpoint index [SBPI] of >2), and time-kill methodology (≥2 log10 CFU/ml reduction). The ability of FD to limit the emergence of COL resistance was assessed in the presence and absence of each drug alone and in combination. Synergy was demonstrated against all strains, with an average FICI and SBPI of 0.064 and 78.85, respectively. In time-kill assays, COL-FD was synergistic and rapidly bactericidal, including against COL-resistant strains. Fusidic acid prevented the emergence of COL resistance, which was readily selected with COL alone. This is the first description of a novel COL-FD regimen for the treatment of MDRAB. The combination was effective at low concentrations, which should be therapeutically achievable while limiting toxicity. Further studies are warranted to determine the mechanism underlying the interaction and the suitability of COL-FD as an unorthodox therapy for the treatment of multidrug-resistant Gram-negative infections.

In Vitro Activity of Epigallocatechin Gallate and Quercetin Alone and in Combination versus Clinical Isolates of Methicillin-Resistant Staphylococcus aureus.

Topical infections can become life threatening in immunocompromised patients. However, fewer treatments are available as multi-drug-resistant bacteria become more common. The natural compounds epigallocatechin gallate (1) and quercetin (2) alone and in combination were tested as potential antimicrobial clinical therapies. Strong antimicrobial activity was produced by 1 alone against methicillin-resistant Staphylococcus aureus, and activity was significantly increased in the presence of 2. A synergistic interaction was observed between the two compounds. Kill kinetics indicate the combination is bactericidal over 24 h.

In vitro and in vivo activities of tigecycline-colistin combination therapies against carbapenem-resistant Enterobacteriaceae.

We assessed the activity of tigecycline (TGC) combined with colistin (COL) against carbapenem-resistant enterobacteria. Synergy occurred in vitro against the majority of isolates, with the exception of Serratia marcescens. In a simple animal model (Galleria mellonella), TGC-COL was superior (P < 0.01) in treating Escherichia coli, Klebsiella pneumoniae, and Enterobacter infections, including those with TGC-COL resistance. Clinical studies are needed to determine whether TGC-COL regimens may be a viable option.

In vitro activity of curcumin in combination with epigallocatechin gallate (EGCG) versus multidrug-resistant Acinetobacter baumannii.

BACKGROUND: Acinetobacter baumannii is an opportunistic human pathogen often associated with life-threatening infections in the immunocompromised and the critically ill. Strains are often multidrug-resistant (MDR) and due to the lack of new synthetic antimicrobials in development for treatment, attention is increasingly focused on natural compounds either as stand-alone or adjunctive agents. Curcumin (CCM) is a natural polyphenol found in turmeric and isolated from the plant, Curcuma longa. Curcumin has been found to possess many biological properties, including antibacterial activity. In this study the antimicrobial activity of CCM and synergistic effects with epigallocatechin gallate (EGCG) against multidrug-resistant strains of A. baumannii were investigated and assessed via checkerboard and time-kill assays. RESULTS: The MIC of CCM was >256 µg/mL against all strains of A. baumannii whilst those for EGCG ranged from 128-1024 µg/mL. In checkerboard studies synergy was observed against 5/9 isolates, with an additive effect noted in the remaining 4. The addition of EGCG reduced the MIC of CCM by 3- to 7-fold, with the greatest interaction resulting in a CCM MIC of 4 µg/mL. Time-kill curves indicated that a CCM-EGCG (1:8 and 1:4) combination was bactericidal with a 4 to 5-log reduction in viable counts after 24 h compared to the most effective polyphenol alone. CONCLUSIONS: This study demonstrates that despite little antibacterial activity alone, CCM activity is greatly enhanced in the presence of EGCG resulting in antibacterial activity against MDR A. baumannii. The combination may have a potential use in medicine as a topical agent to prevent or treat A. baumannii infections.

The manganese carbonyl complex [Mn(CO)3(tqa-κ3N)]Br: A novel antimicrobial agent with the potential to treat avian pathogenic Escherichia coli (APEC) infections.

The development of alternatives to antibiotics is essential for the treatment of animal infections and as a measure to reduce the selective pressure on antibiotics that are critical for human medicine. Metal complexes have been highlighted for their antimicrobial activity against several bacterial pathogens. In particular, manganese carbonyl complexes have shown efficacy against multidrug-resistant Gram-negative pathogens, and relatively low cytotoxicity against avian macrophages and in wax moth larval models. They are thus potential candidates for deployment against Avian Pathogenic Escherichia coli (APEC), the aetiological agent of avian colibacillosis, which results in severe animal welfare issues and financial losses worldwide. This study aimed to determine the efficacy of [Mn(CO)3(tqa-κ3N)]Br in Galleria mellonella and chick models of infection against APEC. The results demonstrated in vitro and in vivo antibacterial activity against all antibiotic-resistant APEC test isolates screened in the study.

Antibacterial green tea catechins from a molecular perspective: mechanisms of action and structure-activity relationships.

This review summarizes the mechanisms of antibacterial action of green tea catechins, discussing the structure-activity relationship (SAR) studies for each mechanism. The antibacterial activity of green tea catechins results from a variety of mechanisms that can be broadly classified into the following groups: (1) inhibition of virulence factors (toxins and extracellular matrix); (2) cell wall and cell membrane disruption; (3) inhibition of intracellular enzymes; (4) oxidative stress; (5) DNA damage; and (6) iron chelation. These mechanisms operate simultaneously with relative importance differing among bacterial strains. In all SAR studies, the highest antibacterial activity is observed for galloylated compounds (EGCG, ECG, and theaflavin digallate). This observation, combined with numerous experimental and theoretical evidence, suggests that catechins share a common binding mode, characterized by the formation of hydrogen bonds and hydrophobic interactions with their target.

Manganese complex [Mn(CO)3(tpa-κ3N)]Br increases antibiotic sensitivity in multidrug resistant Streptococcus pneumoniae.

OBJECTIVES: The emergence of multidrug-resistance (MDR) in Streptococcus pneumoniae clones and non-vaccine serotypes necessitate the development of novel treatment strategies. This work aimed to determine the efficacy of the Mn complex [Mn(CO)3(tpa-κ3N)]Br against clinically important MDR strains of S. pneumoniae. METHODS: Twenty MDR clinicalS. pneumoniae strains were included in this study. Minimum inhibitory concentrations (MICs) of [Mn(CO)3(tpa-κ3N)]Br were determined via broth microdilution alone and in combination with other antimicrobial agents using checkerboard assays and/or disc diffusion tests. In vitro efficacy was assessed by time-kill assays while in vivo efficacy was tested using the insect model Galleria mellonella. RESULTS: [Mn(CO)3(tpa-κ3N)]Br showed moderate in vitro efficacy against S. pneumoniae coupled with bactericidal activity. Checkerboard and disc diffusion assays showed synergy between [Mn(CO)3(tpa-κ3N)]Br and tetracycline, and the combination of both agents caused rapid kill-kinetics and reduced the MIC below the susceptibility breakpoint of 1 mg/L even for tetracycline-resistant strains of S. pneumoniae. Similar results were observed for the erythromycin- and the co-trimoxazole-Mn complex combination. In the G. mellonella infection model, mortality and morbidity rates at 96 h were significantly lower in larvae treated with [Mn(CO)3(tpa-κ3N)]Br than phosphate buffered saline, while treatment with the tetracycline-Mn complex combination was superior to monotherapy, resulting in significantly lower mortality and morbidity rates (p < 0.049). CONCLUSIONS: We show that [Mn(CO)3(tpa-κ3N)]Br has in vitro and in vivo antibacterial activity against clinically relevant strains of S. pneumoniae and has the potential to be used in combination with currently available antibiotics to increase their effectiveness against MDR S. pneumoniae.

Diversity of amino acid substitutions in PmrCAB associated with colistin resistance in clinical isolates of Acinetobacter baumannii.

This study aimed to investigate the mechanisms of colistin resistance in 64 Acinetobacter baumannii isolates obtained from patients with ventilator-associated pneumonia hospitalised in Greece, Italy and Spain. In total, 31 A. baumannii isolates were colistin-resistant. Several novel amino acid substitutions in PmrCAB were found in 27 colistin-resistant A. baumannii. Most substitutions were detected in PmrB, indicating the importance of the histidine kinase for colistin resistance. In two colistin-resistant isolates, 93 amino acid changes were observed in PmrCAB compared with A. baumannii ACICU, and homologous recombination across different clonal lineages was suggested. Analysis of gene expression revealed increased pmrC expression in isolates harbouring pmrCAB mutations. Complementation of A. baumannii ATCC 19606 and ATCC 17978 with a pmrAB variant revealed increased pmrC expression but unchanged colistin MICs, indicating additional unknown factors associated with colistin resistance. Moreover, a combination of PmrB and PmrC alterations was associated with very high colistin MICs, suggesting accumulation of mutations as the mechanism for high-level resistance. The pmrC homologue eptA was detected in 29 colistin-susceptible and 26 colistin-resistant isolates. ISAba1 was found upstream of eptA in eight colistin-susceptible and one colistin-resistant isolate and eptA was disrupted by ISAba125 in two colistin-resistant isolates. Whilst in most isolates an association of eptA with colistin resistance was excluded, in one isolate an amino acid substitution in EptA (R127L) combined with a point mutation in ISAba1 upstream of eptA contributed to elevated colistin MICs. This study helps to gain an insight into the diversity and complexity of colistin resistance in A. baumannii.

Restoring the activity of the antibiotic aztreonam using the polyphenol epigallocatechin gallate (EGCG) against multidrug-resistant clinical isolates of Pseudomonas aeruginosa.

Introductio n. Pseudomonas aeruginosa is an important Gram-negative pathogen that is intrinsically multidrug-resistant (MDR) and frequently associated with healthcare-associated outbreaks. With increasing resistance to antibiotics and with very few novel drugs under development, clinicians often use combinations to treat critically ill patients.Aim. The aim of this study was to evaluate the ability of epigallocatechin (EGCG) to restore the activity of aztreonam against clinical MDR strains of P. aeruginosa.Methodology. Checkerboard and time-kill kinetic assays were performed to assess synergy in vitro and the Galleria mellonella model of infection was used to test the efficacy of the combination in vivo. Accumulation assays were performed to gain insight into the mechanism of action.Results. The results demonstrate that synergy between aztreonam and EGCG exists [fractional inhibitory concentration indices (FICIs) 0.02-0.5], with the combination affording significantly (P=<0.05) enhanced bacterial killing, with a >3 log10 reduction in colony-forming units ml-1 at 24 h. EGCG was able to restore susceptibility to aztreonam to a level equal to or below the breakpoint set by the European Committee for Antimicrobial Susceptibility Testing. In G. mellonella, the combination was superior to monotherapy, with increased larval survival observed (94 % vs ≤63 %). We also demonstrated the relatively low toxicity of EGCG to human keratinocytes and G. mellonella larvae. Accumulation assay data suggest that the mechanism of synergy may be due to EGCG increasing the uptake of aztreonam.Conclusion. EGCG was able to restore the activity of aztreonam against MDR P. aeruginosa. The data presented support further evaluation of the aztreonam-EGCG combination and highlight its potential for use in clinical medicine.

Characterization of a colistin-resistant Avian Pathogenic Escherichia coli ST69 isolate recovered from a broiler chicken in Germany.

In recent years, several plasmids harbouring genes encoding phosphoethanolamine transferases conferring colistin resistance have been described in multiple Enterobacteriaceae species. Avian Pathogenic E. coli (APEC) causes colibacillosis and is responsible for a considerable proportion of the disease burden in commercial poultry flocks, and may be linked to zoonotic infections in humans. Here, we describe the genotypic and phenotypic characteristics of a multidrug-resistant APEC ST69 isolate (APECA2), recovered in 2016 from a diseased broiler at post-mortem examination in Germany. The isolate was resistant to several antibiotics of human and veterinary importance, including colistin. The mcr-1 gene was detected on a mobile genetic element located on an IncHI2/ST4 plasmid, which was characterized using long-read Nanopore and short-read Illumina sequencing of purified plasmid. Isolate APECA2 displayed resistance to chicken serum and harbours numerous virulence genes. This study highlights the public health importance of enhanced antimicrobial resistance surveillance and strict antimicrobial stewardship in human and veterinary healthcare.

Novel Antibacterials: Alternatives to Traditional Antibiotics.

With the advent of the global antimicrobial resistance (AMR) crisis, our arsenal of effective antibiotics is diminishing. The widespread use and misuse of antibiotics in human and veterinary medicine, compounded by the lack of novel classes of antibiotic in the pharmaceutical pipeline, has left a hole in our antibiotic armamentarium. Thus, alternatives to traditional antibiotics are being investigated, including two major groups of antibacterial agents, which have been extensively studied, phytochemicals and metals. Within these groups, there are several subclasses of compound/elements, including polyphenols and metal nanoparticles, which could be used to complement traditional antibiotics, either to increase their potency or extend their spectrum of activity. Alone or in combination, these antibacterial agents have been shown to be effective against a vast array of human and animal bacterial pathogens, including those resistant to licensed antibacterials. These alternative antibacterial agents could be a key element in our fight against AMR and provide desperately needed options, to veterinary and medical clinicians alike.

In vitro and In vivo Activity of Theaflavin-Epicatechin Combinations versus Multidrug-Resistant Acinetobacter baumannii.

INTRODUCTION: Acinetobacter baumannii is an important human nosocomial pathogen; most clinical isolates are multidrug-resistant (MDR). Infections caused by A. baumannii often lead to high morbidity and mortality, with limited treatment options. Owing to the small number of anti-Gram-negative antibiotics in the development pipeline, researchers are looking to other natural compounds. The aim of this study was to determine the in vitro kill kinetics, in vivo efficacy and toxicity of theaflavin-epicatechin combinations against MDR A. baumannii. METHODS: Kill-kinetic assays were performed in Mueller-Hinton 2 broth over 24 h. Toxicity of the compound in the insect model, Galleria mellonella was investigated. The effect of theaflavin-epicatechin combinations on mortality and morbidity were assessed in Acinetobacter baumannii-infected G. mellonella. Larvae were scored for morbidity (melanisation: scale; 0-4) and mortality over 96 h. RESULTS: Kill-kinetic assays revealed that monotherapy had bacteriostatic activity over 24 h, whereas theaflavin-epicatechin combinations were bactericidal (a >3 log reduction in bacterial numbers at 24 h compared with the starting inoculum). Both polyphenols were non-toxic to G. mellonella at concentrations of up to 1000 mg/kg. In vivo treatment assays showed that the combination significantly increased (t test; p ≤ 0.05) larval survival at 96 h to 86% [±17 standard deviation percentage points (pp)] compared to monotherapy with theaflavin (52% ± 14 pp), epicatechin (44% ± 25 pp) or PBS (31% ± 13 pp). Morbidity was also lower in larvae treated with the combination, compared with monotherapy. CONCLUSION: Polyphenol combinations produce effective antibacterial action against A. baumannii and show great potential for the treatment of infections caused by MDR A. baumannii.

Green synthesis of highly concentrated aqueous colloidal solutions of large starch-stabilised silver nanoplatelets.

A simple, environmentally friendly and cost-effective method has been developed to prepare a range of aqueous silver colloidal solutions, using ascorbic acid as a reducing agent, water-soluble starch as a combined crystallising, stabilising and solubilising agent, and water as the solvent. The diameter of silver nanoplatelets increases with higher concentrations of AgNO3 and starch. The silver nanoparticles are also more uniform in shape the greater the diameter of the nanoparticles. Colloidal solutions with a very high concentration of large, flat, hexagonal silver nanoplatelets (~230 nm in breadth) have been used to deposit and fix an antibacterial coating of these large starch-stabilised silver nanoplates on commercial cotton fibres, using a simple dip-coating process using water as the solvent, in order to study the dependence of the antibacterial properties of these nanoplatelets on their size.

Whiter, brighter, and more stable cellulose paper coated with antibacterial carboxymethyl starch stabilized ZnO nanoparticles.

Small, carboxymethyl-starch-stabilised zinc oxide nanoparticles with a defined shape, size and morphology were prepared in situ in water at relatively low reaction temperatures using soluble carboxymethyl starch (CMS) as a combined crystallising, stabilising and solubilising agent and triethanolamine as the reducing agent. Aqueous colloidal solutions of these CMS-stabilised ZnO nanoparticles were used to deposit a coating of ZnO nanoparticles on cellulose paper by a wet-chemistry, polyelectrolyte, layer-by-layer approach using water as the only solvent. Such cellulose paper samples, coated with these CMS-stabilised ZnO nanoparticles, show higher brightness and whiteness than that of blank reference paper and are more stable to UV-radiation than the paper reference as well as demonstrating good antibacterial activity against MRSA and A. baumannii.

Antifungal synergy of theaflavin and epicatechin combinations against Candida albicans.

New antifungal agents are required to compensate for the increase in resistance to standard antifungal agents of Candida albicans, which is an important opportunistic fungal pathogen that causes minor infections in many individuals but very serious infections in those who are immune-compromised. In this study, combinations of theaflavin and epicatechin are investigated as potential antifungal agents and also to establish whether antifungal synergy exists between these two readily accessible and cost-effective polyphenols isolated from black and green tea. The results of disc diffusion assays showed stronger antibacterial activity of theaflavin:epicatechin combinations against C. albicans NCTC 3255 and NCTC 3179, than that of theaflavin alone. Minimum inhibitory concentrations (MICs) of 1,024 µg/ml with theaflavin and 128-256 µg/ml with theaflavin:epicatechin combinations were found. The fractional inhibitory concentration indexes were calculated, and the synergy between theaflavin and epicatechin against both isolates of C. albicans was confirmed. Theaflavin:epicatechin combinations show real potential for future use as a treatment for infections caused by C. albicans.