Jonathan W. Betts

In Vitro and In Vivo Activities of Tigecycline-Colistin Combination Therapies against Carbapenem-Resistant Enterobacteriaceae

ABSTRACT 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

BackgroundAcinetobacter 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.ResultsThe MIC of CCM was >256xa0μg/mL against all strains of A. baumannii whilst those for EGCG ranged from 128-1024xa0μg/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 4xa0μg/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 24xa0h compared to the most effective polyphenol alone.ConclusionsThis 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.

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

ABSTRACT 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.

Activity of colistin in combination with tigecycline or rifampicin against multidrug-resistant Stenotrophomonas maltophilia

The antimicrobial treatment of Stenotrophomonas maltophilia infections is complicated by intrinsic multidrug resistance and a lack of reliable susceptibility data. We assessed the activity of colistin (COL), rifampicin (RIF) and tigecycline (TGC) alone and in combination using a range of in vitro susceptibility testing methodologies and a simple invertebrate model of S. maltophilia infection (Galleria mellonella). Synergy [fractional inhibitory concentration indices (FICIs) ≤0.5] between COL and either RIF or TGC was observed against 92xa0% and 88xa0% of 25xa0S. maltophilia isolates, respectively, despite resistance to one or another of the single agents alone. In time–kill assays, COL combined with either RIF or TGC was superior to single agents, but only the COL/RIF regimen was reliably bactericidal. The in vitro findings correlated with treatment outcomes in G. mellonella, with heightened survival observed for larvae treated with COL/RIF or COL/TGC compared with COL, RIF or TGC alone. COL combined with RIF was the most effective combination overall in both in vitro and in vivo (pu2009<u20090.05) assays. Given the difficulty in selecting appropriate therapy for S. maltophilia infections, regimens consisting of COL combined with RIF or TGC could be considered for clinical use.

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

ABSTRACT 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.

Lack of AcrB efflux function confers loss of virulence on Salmonella enterica serovar typhimurium

ABSTRACT AcrAB-TolC is the paradigm resistance-nodulation-division (RND) multidrug resistance efflux system in Gram-negative bacteria, with AcrB being the pump protein in this complex. We constructed a nonfunctional AcrB mutant by replacing D408, a highly conserved residue essential for proton translocation. Western blotting confirmed that the AcrB D408A mutant had the same native level of expression of AcrB as the parental strain. The mutant had no growth deficiencies in rich or minimal medium. However, compared with wild-type SL1344, the mutant had increased accumulation of Hoechst 33342 dye and decreased efflux of ethidium bromide and was multidrug hypersusceptible. The D408A mutant was attenuated in vivo in mouse and Galleria mellonella models and showed significantly reduced invasion into intestinal epithelial cells and macrophages in vitro. A dose-dependent inhibition of invasion was also observed when two different efflux pump inhibitors were added to the wild-type strain during infection of epithelial cells. RNA sequencing (RNA-seq) revealed downregulation of bacterial factors necessary for infection, including those in the Salmonella pathogenicity islands 1, 2, and 4; quorum sensing genes; and phoPQ. Several general stress response genes were upregulated, probably due to retention of noxious molecules inside the bacterium. Unlike loss of AcrB protein, loss of efflux function did not induce overexpression of other RND efflux pumps. Our data suggest that gene deletion mutants are unsuitable for studying membrane transporters and, importantly, that inhibitors of AcrB efflux function will not induce expression of other RND pumps. IMPORTANCE Antibiotic resistance is a major public health concern. In Gram-negative bacteria, overexpression of the AcrAB-TolC multidrug efflux system confers resistance to clinically useful drugs. Here, we show that loss of AcrB efflux function causes loss of virulence in Salmonella enterica serovar Typhimurium. This is due to the reduction of bacterial factors necessary for infection, which is likely to be caused by the retention of noxious molecules inside the bacterium. We also show that, in contrast to loss of AcrB protein, loss of efflux does not induce overexpression of other efflux pumps from the same family. This indicates that there are differences between loss of efflux protein and loss of efflux that make gene deletion mutants unsuitable for studying the biological function of membrane transporters. Understanding the biological role of AcrB will help to assess the risks of targeting efflux pumps as a strategy to combat antibiotic resistance. Antibiotic resistance is a major public health concern. In Gram-negative bacteria, overexpression of the AcrAB-TolC multidrug efflux system confers resistance to clinically useful drugs. Here, we show that loss of AcrB efflux function causes loss of virulence in Salmonella enterica serovar Typhimurium. This is due to the reduction of bacterial factors necessary for infection, which is likely to be caused by the retention of noxious molecules inside the bacterium. We also show that, in contrast to loss of AcrB protein, loss of efflux does not induce overexpression of other efflux pumps from the same family. This indicates that there are differences between loss of efflux protein and loss of efflux that make gene deletion mutants unsuitable for studying the biological function of membrane transporters. Understanding the biological role of AcrB will help to assess the risks of targeting efflux pumps as a strategy to combat antibiotic resistance.

In vitro and in vivo activity of ML302F: a thioenolate inhibitor of VIM-subfamily metallo β-lactamases

The thioenol ML302F was recently identified as an inhibitor of class B metallo-β-lactamases (MBLs). We assessed the activity of ML302F when combined with meropenem (MEM) against 31 carbapenem resistant Gram-negative clinical isolates. Minimum inhibitory concentrations of MEMu2006:u2006ML302F were determined at fixed ratios of 1u2006:u20064 and 1u2006:u20068 using strains producing variants of the clinically relevant VIM-like MBL. Toxicity and efficacy in vivo was assessed in a Galleria mellonella invertebrate model against strains producing VIM-1, VIM-2 and VIM-4 variants. At a fixed MEMu2006:u2006ML302F ratio of 1u2006:u20068, 22/31 isolates were rendered either susceptible (MIC ≤ 2 mg L−1), or intermediate (MIC 4–8 mg L−1) to MEM. ML302F alone was not toxic at up to 80 mg kg−1 in G. mellonella and treatment with MEM 0.6 mg kg−1u2006:u2006ML302F 4.8 mg kg−1 significantly improved the survival of infected larvae. As ML302F was able to successfully restore susceptibility to resistant strains both in vitro and in vivo it represents a structurally interesting inhibitor in the search for new MBL inhibitors.

Occurrence and characterization of extended-spectrum cephalosporin-resistant Enterobacteriaceae in healthy household dogs in Greece

Extended-spectrum cephalosporin- and/or carbapenem-resistant (ESCR and/or CarbR) Enterobacteriaceae constitute a public health hazard because of limited treatment options and are endemic among humans in Greece. Recently, ESCR and CarbREnterobacteriaceae have been increasingly isolated from companion animals, stressing their potential role as a reservoir for humans. However, the presence of ESCR bacteria in companion animals within Greek households has not been determined yet. Genes conferring the ESCR and CarbR phenotype were detected among canine isolates and their chromosomal or plasmid location was determined. Standard methods were applied for plasmid characterization. The clonal relatedness of the recovered isolates was examined by multilocus sequence typing (MLST). Here, we report the first findings on the presence of ESCREnterobacteriaceae in healthy Greek dogs. ESCREscherichia coli isolates were associated with different sequence types (STs), including the human pandemic ST131 clone. The occurrence of human-related ESBL/pAmpC genes, plasmid types and/or strain STS in this animal reservoir suggests possible bilateral transmission.

Genomic and functional characterisation of IncX3 plasmids encoding bla SHV-12 in Escherichia coli from human and animal origin

The blaSHV-12 β-lactamase gene is one of the most prevalent genes conferring resistance to extended-spectrum β-lactams in Enterobacteriaceae disseminating within and between reservoirs, mostly via plasmid-mediated horizontal gene transfer. Yet, studies regarding the biology of plasmids encoding blaSHV-12 are very limited. In this study, we revealed the emergence of IncX3 plasmids alongside IncI1α/γ in blaSHV-12 in animal-related Escherichia coli isolates. Four representative blaSHV-12-encoding IncX3 plasmids were selected for genome sequencing and further genetic and functional characterization. We report here the first complete sequences of IncX3 plasmids of animal origin and show that IncX3 plasmids exhibit remarkable synteny in their backbone, while the major differences lie in their blaSHV-12-flanking region. Our findings indicate that plasmids of this subgroup are conjugative and highly stable, while they exert no fitness cost on their bacterial host. These favourable features might have contributed to the emergence of IncX3 amongst SHV-12-producing E. coli in the Netherlands, highlighting the epidemic potential of these plasmids.

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.