J. Mark Sutton

Mechanisms of increased resistance to chlorhexidine and cross-resistance to colistin following exposure of Klebsiella pneumoniae clinical isolates to chlorhexidine

ABSTRACT Klebsiella pneumoniae is an opportunistic pathogen that is often difficult to treat due to its multidrug resistance (MDR). We have previously shown that K. pneumoniae strains are able to “adapt” (become more resistant) to the widely used bisbiguanide antiseptic chlorhexidine. Here, we investigated the mechanisms responsible for and the phenotypic consequences of chlorhexidine adaptation, with particular reference to antibiotic cross-resistance. In five of six strains, adaptation to chlorhexidine also led to resistance to the last-resort antibiotic colistin. Here, we show that chlorhexidine adaptation is associated with mutations in the two-component regulator phoPQ and a putative Tet repressor gene (smvR) adjacent to the major facilitator superfamily (MFS) efflux pump gene, smvA. Upregulation of smvA (10- to 27-fold) was confirmed in smvR mutant strains, and this effect and the associated phenotype were suppressed when a wild-type copy of smvR was introduced on plasmid pACYC. Upregulation of phoPQ (5- to 15-fold) and phoPQ-regulated genes, pmrD (6- to 19-fold) and pmrK (18- to 64-fold), was confirmed in phoPQ mutant strains. In contrast, adaptation of K. pneumoniae to colistin did not result in increased chlorhexidine resistance despite the presence of mutations in phoQ and elevated phoPQ, pmrD, and pmrK transcript levels. Insertion of a plasmid containing phoPQ from chlorhexidine-adapted strains into wild-type K. pneumoniae resulted in elevated expression levels of phoPQ, pmrD, and pmrK and increased resistance to colistin, but not chlorhexidine. The potential risk of colistin resistance emerging in K. pneumoniae as a consequence of exposure to chlorhexidine has important clinical implications for infection prevention procedures.

Retention of virulence following adaptation to colistin in Acinetobacter baumannii reflects the mechanism of resistance

OBJECTIVESnColistin resistance in Acinetobacter baumannii has been associated with loss of virulence and a negative impact on isolate selection. In this study, exposure of clinical isolates to suboptimal concentrations of colistin was used to explore the capacity to develop resistance by diverse mechanisms, and whether acquired resistance always reduces fitness and virulence.nnnMETHODSnTwelve colistin-susceptible clinical A. baumannii isolates were exposed to a sub-MIC concentration of colistin over 6 weeks with weekly increases in concentration. Stable resistance was then phenotypically investigated with respect to antibiotic/biocide resistance, virulence in Galleria mellonella and growth rate. Putative mechanisms of resistance were identified by targeted sequencing of known resistance loci.nnnRESULTSnEight A. baumannii isolates acquired resistance to colistin within 1 week with MICs ranging from 2 to >512 mg/L. By 6 weeks 11 isolates were resistant to colistin; this was linked to the development of mutations in pmr or lpx genes. Strains that developed mutations in lpxACD showed a loss of virulence and increased susceptibility to several antibiotics/disinfectants tested. Two of the colistin-resistant strains with mutations in pmrB retained similar virulence levels to their respective parental strains in G. mellonella.nnnCONCLUSIONSnAcquisition of colistin resistance does not always lead to a loss of virulence, especially when this is linked to mutations in pmrB. This underlines the importance of understanding the mechanism of colistin resistance as well as the phenotype.

Evaluation of antibiotic efficacy against infections caused by planktonic or biofilm cultures of Pseudomonas aeruginosa and Klebsiella pneumoniae in Galleria mellonella

The lack of novel antibiotics for more than a decade has placed increased pressure on existing therapies to combat the emergence of multidrug-resistant (MDR) bacterial pathogens. This study evaluated the Galleria mellonella insect model in determining the efficacy of available antibiotics against planktonic and biofilm infections of MDR Pseudomonas aeruginosa and Klebsiella pneumoniae strains in comparison with in vitro minimum inhibitory concentration (MIC) determination. In general, in vitro analysis agreed with the G. mellonella studies, and susceptibility in Galleria identified different drug resistance mechanisms. However, the carbapenems tested appeared to perform better in vivo than in vitro, with meropenem and imipenem able to clear K. pneumoniae and P. aeruginosa infections with strains that had bla(NDM-1) and bla(VIM) carbapenemases. This study also established an implant model in G. mellonella to allow testing of antibiotic efficacy against biofilm-derived infections. A reduction in antibiotic efficacy of amikacin against K. pneumoniae and P. aeruginosa biofilms was observed compared with a planktonic infection. Ciprofloxacin was found to be less effective at clearing a P. aeruginosa biofilm infection compared with a planktonic infection, but no statistical difference was seen between K. pneumoniae biofilm and planktonic infections treated with this antibiotic (P>0.05). This study provides important information regarding the suitability of Galleria as a model for antibiotic efficacy testing both against planktonic and biofilm-derived MDR infections.

Poly(N-isopropylacrylamide-co-allylamine) (PNIPAM-co-ALA) nanospheres for the thermally triggered release of Bacteriophage K.

Due to the increased prevalence of resistant bacterial isolates which are no longer susceptible to antibiotic treatment, recent emphasis has been placed on finding alternative modes of treatment of wound infections. Bacteriophage have long been investigated for their antimicrobial properties, yet the utilization of phage therapy for the treatment of wound infections relies on a suitable delivery system. Poly(N-isopropylacrylamide) (PNIPAM) is a thermally responsive polymer which undergoes a temperature dependent phase transition at a critical solution temperature. Bacteriophage K has been successfully formulated with PNIPAM nanospheres copolymerized with allylamine (PNIPAM-co-ALA). By utilizing a temperature responsive polymer it has been possible to engineer the nanospheres to collapse at an elevated temperature associated with a bacterial skin infection. The nanogels were reacted with surface deposited maleic anhydride in order to anchor the nanogels to non-woven fabric. Bacteriophage incorporated PNIPAM-co-ALA nanospheres demonstrated successful bacterial lysis of a clinically relevant bacterial isolate - Staphylococcus aureus ST228 at 37°C, whilst bacterial growth was unaffected at 25°C, thus providing a thermally triggered release of bacteriophage.

Thermally triggered release of the bacteriophage endolysin CHAPK and the bacteriocin lysostaphin for the control of methicillin resistant Staphylococcus aureus (MRSA)

&NA; Staphylococcus aureus infections of the skin and soft tissue pose a major concern to public health, largely owing to the steadily increasing prevalence of drug resistant isolates. As an alternative mode of treatment both bacteriophage endolysins and bacteriocins have been shown to possess antimicrobial efficacy against multiple species of bacteria including otherwise drug resistant strains. Despite this, the administration and exposure of such antimicrobials should be restricted until required in order to discourage the continued evolution of bacterial resistance, whilst maintaining the activity and stability of such proteinaceous structures. Utilising the increase in skin temperature during infection, the truncated bacteriophage endolysin CHAPK and the staphylococcal bacteriocin lysostaphin have been co‐administered in a thermally triggered manner from Poly(N‐isopropylacrylamide) (PNIPAM) nanoparticles. The thermoresponsive nature of the PNIPAM polymer has been employed in order to achieve the controlled expulsion of a synergistic enzybiotic cocktail consisting of CHAPK and lysostaphin. The point at which this occurs is modifiable, in this case corresponding to the threshold temperature associated with an infected wound. Consequently, bacterial lysis was observed at 37 °C, whilst growth was maintained at the uninfected skin temperature of 32 °C. Graphical abstract Figure. No caption available.

Current Advances in Developing Inhibitors of Bacterial Multidrug Efflux Pumps

Antimicrobial resistance represents a significant challenge to future healthcare provision. An acronym ESKAPEE has been derived from the names of the organisms recognised as the major threats although there are a number of other organisms, notably Neisseria gonorrhoeae, that have become equally challenging to treat in the clinic. These pathogens are characterised by the ability to rapidly develop and/or acquire resistance mechanisms in response to exposure to different antimicrobial agents. A key part of the armoury of these pathogens is a series of efflux pumps, which effectively exclude or reduce the intracellular concentration of a large number of antibiotics, making the pathogens significantly more resistant. These efflux pumps are the topic of considerable interest, both from the perspective of basic understanding of efflux pump function, and its role in drug resistance but also as targets for the development of novel adjunct therapies. The necessity to overcome antimicrobial resistance has encouraged investigations into the characterisation of resistance-modifying efflux pump inhibitors to block the mechanisms of drug extrusion, thereby restoring antibacterial susceptibility and returning existing antibiotics into the clinic. A greater understanding of drug recognition and transport by multidrug efflux pumps is needed to develop clinically useful inhibitors, given the breadth of molecules that can be effluxed by these systems. This review discusses different bacterial EPIs originating from both natural source and chemical synthesis and examines the challenges to designing successful EPIs that can be useful against multidrug resistant bacteria.

Simple and rapid sample preparation system for the molecular detection of antibiotic resistant pathogens in human urine

Antibiotic resistance in urinary tract infections (UTIs) can cause significant complications without quick detection and appropriate treatment. We describe a new approach to capture, concentrate and prepare amplification-ready DNA from antibiotic resistant bacteria in human urine samples. Klebsiella pneumoniae NCTC13443 (blaCTX-M-15 positive) spiked into filtered human urine was used as a model system. Bacteria were captured using anion exchange diaethylaminoethyl (DEAE) magnetic microparticles and concentrated 200-fold within ~3.5xa0min using a custom, valve-less microfluidic chip. Eight samples were processed in parallel, and DNA was released using heat lysis from an integrated resistive heater. The crude cell lysate was used for real time Recombinase Polymerase Amplification (RPA) of the blaCTX-M-15 gene. The end to end processing time was approximately 15xa0min with a limit of detection of 1000 bacteria in 1xa0mL urine.

Ultra-fast electronic detection of antimicrobial resistance genes using isothermal amplification and thin film transistor sensors

A low cost thin-film transistor (TFT) nanoribbon (NR) sensor has been developed for rapid real-time detection of DNA amplification using an isothermal Recombinase Polymerase Amplification (RPA) method. The semiconductor chip measures DNA amplification through a pH change, rather than via fluorescence. The utility of the method was demonstrated by amplifying CTX-M and NDM, two genes that confer bacterial resistance to cephalosporins and carbapenems, respectively. It is shown that this approach provides extremely fast and sensitive detection. It can detect <10 copies of the gene in genomic DNA extracted from E. coli or K. pneumoniae clinical isolates within a few minutes. A differential readout system was developed to minimize the effect of primer-dimer amplification on the assay. The simple device has the potential for low cost, portable and real-time nucleic acid analysis as a Point of Care device.

Recent advances in therapeutic delivery systems of bacteriophage and bacteriophage-encoded endolysins

Antibiotics have been the cornerstone of clinical management of bacterial infection since their discovery in the early 20th century. However, their widespread and often indiscriminate use has now led to reports of multidrug resistance becoming globally commonplace. Bacteriophage therapy has undergone a recent revival in battle against pathogenic bacteria, as the self-replicating and co-evolutionary features of these predatory virions offer several advantages over conventional therapeutic agents. In particular, the use of targeted bacteriophage therapy from specialized delivery platforms has shown particular promise owing to the control of delivery location, administration conditions and dosage of the therapeutic cargo. This review presents an overview of the recent formulations and applications of such delivery vehicles as an innovative and elegant tool for bacterial control.

Cold atmospheric pressure plasma elimination of clinically important single- and mixed-species biofilms

Mixed-species biofilms reflect the natural environment of many pathogens in clinical settings and are highly resistant to disinfection methods. An indirect cold atmospheric-pressure air-plasma system was evaluated under two different discharge conditions for its ability to kill representative Gram-positive (Staphylococcus aureus) and Gram-negative (Pseudomonas aeruginosa) pathogens. Plasma treatment of individual 24-h-old biofilms and mixed-species biofilms that contained additional species (Enterococcus faecalis and Klebsiella pneumoniae) was considered. Under plasma conditions that favoured the production of reactive nitrogen species (RNS), individual P. aeruginosa biofilms containing ca. 5.0u2009×u2009106 CFU were killed extremely rapidly, with no bacterial survival detected at 15u2009s of exposure. Staphylococcus aureus survived longer under these conditions, with no detectable growth after 60u2009s of exposure. In mixed-species biofilms, P. aeruginosa survived longer but all species were killed with no detectable growth at 60u2009s. Under plasma conditions that favoured the production of reactive oxygen species (ROS), P. aeruginosa showed increased survival, with the lower limit of detection reached by 120u2009s, and S. aureus was killed in a similar time frame. In the mixed-species model, bacterial kill was biphasic but all pathogens showed viable cells after 240u2009s of exposure, with P. aeruginosa showing significant survival (ca. 3.6u2009±u20090.6u2009×u2009106 CFU). Overall, this study shows the potential of indirect air plasma treatment to achieve significant bacterial kill, but highlights aspects that might affect performance against key pathogens, especially in real-life settings within mixed populations.