Improving the Management and Treatment of Diabetic Foot Infection: Challenges and Research Opportunities.

Diabetic foot infection (DFI) management requires complex multidisciplinary care pathways with off-loading, debridement and targeted antibiotic treatment central to positive clinical outcomes. Local administration of topical treatments and advanced wound dressings are often used for more superficial infections, and in combination with systemic antibiotics for more advanced infections. In practice, the choice of such topical approaches, whether alone or as adjuncts, is rarely evidence-based, and there does not appear to be a single market leader. There are several reasons for this, including a lack of clear evidence-based guidelines on their efficacy and a paucity of robust clinical trials. Nonetheless, with a growing number of people living with diabetes, preventing the progression of chronic foot infections to amputation is critical. Topical agents may increasingly play a role, especially as they have potential to limit the use of systemic antibiotics in an environment of increasing antibiotic resistance. While a number of advanced dressings are currently marketed for DFI, here we review the literature describing promising future-focused approaches for topical treatment of DFI that may overcome some of the current hurdles. Specifically, we focus on antibiotic-impregnated biomaterials, novel antimicrobial peptides and photodynamic therapy.

Modified poly(L-lysine)-based structures as novel antimicrobials for diabetic foot infections, an in-vitro study.

Background: Wound infections occur as sequelae to skin trauma and cause significant hospitalizations, morbidity and mortality. Skin traumas arise more frequently in those with diabetes or cardiovascular disease and in these settings, may be chronic with poorer outcomes including lower limb amputation. Treatment of chronic wound infection is challenging due to antibiotic resistance and biofilm formation by bacteria including S. aureus and P. aeruginosa, which are among the most frequent causative pathogens. Managing these challenging infections requires new molecules and modalities. Methods: We evaluated antimicrobial and anti-biofilm activity of star-shaped poly(L-lysine) (PLL) polymers against S. aureus and P. aeruginosa strains and clinical isolates recovered from wounds including diabetic foot wounds (DFW) in a Dublin Hospital in 2019. A star-shaped PLL polypeptide series, specifically G2(8)PLL 20, G3(16)PLL 10, G4(32)PLL 5 with variation in polypeptide chain length and arm-multiplicity, were compared to a linear peptide, PLL 160 with equivalent number of lysine residues. Results: All PLLs, including the linear polypeptide, were bactericidal at 1µM against S. aureus 25923 and P. aeruginosa PAO1, with log reduction in colony forming units/ml between 2.7-3.6. PLL 160 demonstrated similar killing potency against 20 S. aureus and five P. aeruginosa clinical isolates from DFW, mean log reductions: 3.04 ± 0.16 and 3.96 ± 0.82 respectively after 1 hour incubation. Potent anti-biofilm activity was demonstrated against S. aureus 25923 but for clinical isolates, low to moderate loss of biofilm viability was shown using PLL 160 and G3(16)PLL 10 at 50 µM ( S. aureus) and 200 µM ( P. aeruginosa) with high inter-isolate variability . In the star-shaped architecture, antimicrobial activity was retained with incorporation of 5-mer hydrophobic amino-acid modifications to the arms of the polypeptides (series G3(16)PLL 20-coPLT 5, G3(16)PLL 20-coPLI 5, G3(16)PLL 20-coPLP 5). Conclusion: These polypeptides offer structural flexibility for clinical applications and have potential for further development, particularly in the setting of diabetic foot and other chronic wound infections.

Three-dimensionally printable shear-thinning triblock copolypeptide hydrogels with antimicrobial potency.

Through rational design, block sequence controlled triblock copolypeptides comprising cysteine and tyrosine as well as a lysine or glutamic acid central block are devised. In these copolypeptides, each block contributes a specific property to the hydrogels to render them extrusion printable and antimicrobial. Three-dimensional (3D) printing of complex hydrogel structures with high shape retention is demonstrated. Moreover, composition dependent potent antimicrobial activity in contact-killing assays is elucidated.

Antimicrobial and degradable triazolinedione (TAD) crosslinked polypeptide hydrogels.

Hydrogels are perfectly suited to support cell and tissue growth in advanced tissue engineering applications as well as classical wound treatment scenarios. Ideal hydrogel materials for these applications should be easy to produce, biocompatible, resorbable and antimicrobial. Here we report the fabrication of degradable covalent antimicrobial lysine and tryptophan containing copolypeptide hydrogels, whereby the hydrogel properties can be independently modulated by the copolypeptide monomer ratio and chiral composition. Well-defined statistical copolypeptides comprising different overall molecular weights as well as ratios of l- and d-lysine and tryptophan at ratios of 35 : 15, 70 : 30 and 80 : 20 were obtained by N-carboxyanhydride (NCA) polymerisation and subsequently crosslinked by the selective reaction of bifunctional triazolinedione (TAD) with tryptophan. Real-time rheology was used to monitor the crosslinking reaction recording the fastest increase and overall modulus for copolypeptides with the higher tryptophan ratio. Water uptake of cylindrical hydrogel samples was dependent on crosslinking ratio but found independent of chiral composition, while enzymatic degradation proceeded significantly faster for samples containing more l-amino acids. Antimicrobial activity on a range of hydrogels containing different polypeptide chain lengths, lysine/tryptophan composition and l/d enantiomers was tested against reference laboratory strains of Gram-negative Escherichia coli (E. coli; ATCC25922) and Gram-positive, Staphylococcus aureus (S. aureus; ATCC25923). log reductions of 2.8-3.4 were recorded for the most potent hydrogels. In vitro leachable cytotoxicity tests confirmed non-cytotoxicity as per ISO guidelines.

Exploiting a Neutral BODIPY Copolymer as an Effective Agent for Photodynamic Antimicrobial Inactivation.

We report the synthesis and photophysical properties of a neutral BODIPY photosensitizing copolymer (poly-8-(4-hydroxymethylphenyl)-4,4-difluoro-2,6-diethynyl-4-bora-3a,4a-diaza-s-indacene) containing ethynylbenzene links between the BODIPY units. The copolymer absorbs further towards the red in the UV-vis spectrum compared to the BODIPY precursor. Photolysis of the polymer produces a singlet excited state which crosses to the triplet surface in less than 300 ps. This triplet state was used to form singlet oxygen with a quantum yield of 0.34. The steps leading to population of the triplet state were studied using time-resolved spectroscopic techniques spanning the pico- to nanosecond timescales. The ability of the BODIPY polymer to generate a biocidal species for bactericidal activity in both solution- and coating-based studies was assessed. When the BODIPY copolymer was dropcast onto a surface, 4 log and 6 log reductions in colony forming units/ml representative of Gram-positive and Gram-negative bacteria, respectively, under illumination at 525 nm were observed. The potent broad-spectrum antimicrobial activity of a neutral metal-free copolymer when exposed to visible light conditions may have potential clinical applications in infection management.

Investigation of the Physical, Chemical and Microbiological Stability of Losartan Potassium 5 mg/mL Extemporaneous Oral Liquid Suspension.

Extemporaneous oral liquid preparations are commonly used when there is no commercially available dosage form for adjustable dosing. In most cases, there is a lack of stability data to allow for an accurately assigned shelf life and storage conditions to give greater confidence of product safety and efficacy over its shelf life. The aim of this study was to evaluate the physical, chemical and microbiological stability of an extemporaneous oral liquid suspension of losartan potassium, 5 mg/mL, used to treat paediatric hypertension in Our Lady's Children's Hospital Crumlin, Ireland. The losartan content of extemporaneous oral suspensions, prepared with and without addition of water, was measured by UV and confirmed by HPLC analysis. Suspensions were stored at 4 °C and room temperature (RT) and were monitored for changes in; pH, colour, odour, re-dispersibility, Total Aerobic Microbial Count, Total Yeast and Mould Count and absence of E. coli. Results showed that suspensions prepared by both methods, stored at 4 °C and RT, were physically and microbiologically stable over 28 days. Initial losartan content of all suspensions was lower than expected at 80-81% and did not change significantly over the 28 days. HPLC and NMR did not detect degradation of losartan in the samples. Suspensions prepared in water showed 100% losartan content. The reduced initial losartan content was confirmed by HPLC and was related to the acidic pH of the suspension vehicle. Physiochemical properties of the drug are important factors for consideration in the selection of suspension vehicle for extemporaneous compounding of oral suspensions as they can influence the quality, homogeneity and efficacy of these preparations.

Screening the nose, throat and the naso-pharynx for methicillin-resistant Staphylococcus aureus: a pilot study.

Patients who carry nasal methicillin-resistant Staphylococcus aureus (MRSA) may also harbour MRSA in the oro-pharyngeal cavity. However, the naso-oro-pharyngeal co-carriage is infrequently assessed. The incidence of concurrent MRSA carriage of the naso-oro-pharynx was ascertained, and the sensitivity of two methods, a throat swab and a phosphate buffered saline (PBS) oral rinse, for MRSA detection was investigated. Among nasal MRSA carriers, 80% harboured MRSA in the oro-pharynx. Among these patients, 15% had MRSA detected in the oro-pharynx and not in the throat. Oro-pharyngeal colonisation represents a significant reservoir to persistence as well as nasal recolonisation. Decolonisation methods effective in reducing oro-pharyngeal MRSA in addition to nasal carriage should be investigated.

Photo-activated CO-release in the amino tungsten Fischer carbene complex, [(CO)5WC(NC4H8)Me], picosecond time resolved infrared spectroscopy, time-dependent density functional theory, and an antimicrobial study.

Picosecond time-resolved infrared spectroscopy was used to probe the photo-induced early state dynamics preceding CO loss in the Fischer carbene complex, [(CO)5WC(NC4H8)CH3]. Time-dependent density functional theory calculations were employed to help in understanding the photochemical and photophysical processes leading to CO-loss. Electrochemical initiated CO release was quantified using gas chromatography. The potential of [(CO)5WC(NC4H8)CH3], as an antimicrobial agent under irradiation conditions was studied using a Staphylococcus aureus strain.

Synthesis and characterisation of Co(iii) complexes of N-formyl hydroxylamines and antibacterial activity of a Co(iii) peptide deformylase inhibitor complex.

The X-ray crystal structure and pKa values of GSK322, a well-known and effective peptide deformylase inhibitor and antibacterial drug candidate, are reported. The first examples of Co(iii) complexes of N-formyl hydroxylamines are reported. Reaction of N-hydroxy-N-phenylformamide (HFA) with [Co(tren)Cl2]Cl and [Co(tpa)Cl2]Cl (where tren = tris(2-aminoethyl)amine, tpa = tris(2-pyridylmethyl)amine) with one equivalent of NaOH in H2O afforded [Co(tren)(HFA-1H)](PF6)1.5Cl0.5 (1) and [Co(tpa)(HFA-1H)]Cl2 (2), respectively. X-ray crystal structures of both complexes revealed that the N-formyl hydroxylamine group acts as a bidentate ligand, coordinating the Co(iii) centres via the carbonyl oxygen and deprotonated hydroxy group (O,O'), a coordination mode typically observed for closely related mono-deprotonated hydroxamic acids. Reaction of the N-formyl hydroxylamine-based GSK322 with [Co(tpa)Cl2]Cl afforded the corresponding Co(iii) chaperone complex of the peptide deformylase inhibitor, [Co(tpa)(GSK322-1H)](PF6)2. GSK322 and [Co(tpa)(GSK322-1H)](PF6)2 exhibited better Gram-positive activity than Gram-negative, where low MICs (1.56-6.25 µM) were determined for S. aureus strains, independent of their antibiotic susceptibility.

Vibrating Mesh Nebulisation of Pro-Antimicrobial Peptides for Use in Cystic Fibrosis.

BACKGROUND: There has been considerable interest in the use of antimicrobial peptides (AMPs) as antimicrobial therapeutics in many conditions including cystic fibrosis (CF). The aim of this study is to determine if the prodrugs of AMPs (pro-AMPs) can be delivered to the lung by a vibrating mesh nebuliser (VMN) and whether the pro-AMP modification has any effect on delivery. METHODS: Physical characteristics of the peptides (AMP and pro-AMP) and antimicrobial activity were compared before and after nebulisation. Droplet size distribution was determined by laser diffraction and cascade impaction. Delivery to a model lung was determined in models of spontaneously-breathing and mechanically-ventilated patients. RESULTS: The physical characteristics and antimicrobial activities were unchanged after nebulisation. Mean droplet size diameters were below 5 µm in both determinations, with the fine particle fraction approximately 67% for both peptides. Approximately 25% of the nominal dose was delivered in the spontaneously-breathing model for both peptides, with higher deliveries observed in the mechanically-ventilated model. Delivery times were approximately 170 s per mL for both peptides and the residual volume in the nebuliser was below 10% in nearly all cases. CONCLUSIONS: These results demonstrate that the delivery of (pro-)AMPs to the lung using a VMN is feasible and that the prodrug modification is not detrimental. They support the further development of pro-AMPs as therapeutics in CF.

Molecular Characterization of Nasal Methicillin-Resistant Staphylococcus aureus Isolates Showing Increasing Prevalence of Mupirocin Resistance and Associated Multidrug Resistance following Attempted Decolonization.

Sequential methicillin-resistant Staphylococcus aureus (MRSA) isolates from patients following attempted mupirocin nasal decolonization showed an increase in mupirocin resistance (MR) from 6.6% to 20%. MR isolates from patients who failed decolonization yielded indistinguishable spa types and carried multiple antimicrobial and antiseptic resistance genes, which may guide infection control and prevention.

Action of antimicrobial peptides and their prodrugs on model and biological membranes.

Antimicrobial peptides (AMPs) are promising broad-spectrum antibiotic candidates in the wake of multi-drug resistant pathogens. Their clinical use still requires a solution based on lead optimisation and/or formulation to overcome certain limitations, such as unwanted cytotoxicity. A prodrug approach could overcome this safety barrier and can be achieved through reversible reduction or neutralisation of the AMPs' net cationic charge. By prodrug activation through pathogen associated enzymes, this approach could increase the therapeutic index of membrane active peptides. P18, a cecropin/magainin hybrid, and WMR, a myxinidin analogue from hagfish, were used as templates for the design strategy. The membrane permeabilizing activities of these AMPs and their prodrugs are reported here for liposomes of either Escherichia coli polar lipid extract or a human model lipid system of phosphatidylcholine and cholesterol. These results are compared with their antibacterial and haemolytic activities. Overall, correlation between liposome permeabilization and the corresponding bioactivity is observed and indicate that the broad-spectrum antibacterial effect exerted by these peptides is associated with membrane disruption. Furthermore, the prodrug modification had a general negative influence on membrane disruption and bioactivity, notably as much on bacterial as on human membranes. This prodrug strategy is particularly successful when complete neutralisation of the AMP's net charge occurs. Thus, on-target selectivity between bacterial and human membranes can be improved, which may be used to prevent the unnecessary exposure of host cells and commensal bacteria to active AMPs.

Observational cross-sectional study of nasal staphylococcal species of medical students of diverse geographical origin, prior to healthcare exposure: prevalence of SCCmec, fusC, fusB and the arginine catabolite mobile element (ACME) in the absence of selective antibiotic pressure.

OBJECTIVE: The aim of this study was to investigate co-located nasal Staphylococcus aureus and coagulase-negative staphylococci (CoNS) (mainly Staphylococcus epidermidis), recovered from healthy medical students in their preclinical year, prior to exposure to the healthcare environment, for the carriage of genes and genetic elements common to both species and that may contribute to S. aureus and methicillin-resistant S. aureus (MRSA) evolution. DESIGN: Prospective observational cross-sectional study. Carriage of antimicrobial resistance and virulence-associated genes in the absence of significant antibiotic selective pressure was investigated among healthy medical students from geographically diverse origins who were nasally co-colonised with S. aureus and CoNS. Clonal lineages of S. aureus isolates were determined. SETTING/PARTICIPANTS: Dublin-based international undergraduate medical students. RESULTS: Nasal S. aureus carriage was identified in 137/444 (30.8%) students of whom nine (6.6%) carried MRSA (ST59-MRSA-IV (6/9), CC1-MRSA-V-SCCfus (3/9)). The genes mecA, fusB, ileS2, qacA/qacC and the arginine catabolic mobile element-arc were detected among colonising nasal staphylococci and had a significantly greater association with CoNS than S. aureus. The rate of co-carriage of any of these genes in S. aureus/CoNS pairs recovered from the same individual was <1%. CONCLUSIONS: The relatively high prevalence of these genes among CoNS of the healthy human flora in the absence of significant antibiotic selective pressure is of interest. Further research is required to determine what factors are involved and whether these are modifiable to help prevent the emergence and spread of antibiotic resistance among staphylococci.

Vancomycin-Resistant Enterococci (VRE) in The Intensive Care Unit in a Nonoutbreak Setting: Identification of Potential Reservoirs and Epidemiological Associations Between Patient and Environmental VRE.

OBJECTIVE Among nosocomial bloodstream infections caused by enterococcal species, Ireland has the highest proportion caused by vancomycin-resistant enterococci (VRE) in Europe at 45.8%. The contribution of the near-patient environment to VRE transmission outside of outbreaks was investigated. DESIGN A prospective observational study was conducted during 7 sampling periods. METHODS Recovery of VRE isolates by swabbing the near-patient environment and patients in the intensive care unit (ICU) was conducted to identify reservoirs, clinical and molecular epidemiological associations, and the success of active surveillance cultures (ASCs). RESULTS Of 289 sampling occasions involving 157 patients and their bed spaces, VRE isolates were recovered from patient bed spaces, clinical samples, or both on 114 of 289 sampling occasions (39.4%). The patient and their bed space were positive for VRE on 34 of 114 VRE-associated sampling occasions (29.8%). Of 1,647 environment samples, 107 sites (6.5%) were VRE positive, with significantly greater VRE recovery from isolation rooms than from the open-plan area (9.1% vs 4.1%; P < .0001). The most frequently VRE-contaminated sites were the drip stand, bed control panel, and chart holders, which together accounted for 61% of contaminated sites. The use of ASCs resulted in a 172% increase in identification of VRE-colonized patients. Molecular typing revealed 2 environmental clusters, 1 cluster involving 3 patients and generally greater heterogeneity of patient isolates compared to environmental isolates. CONCLUSION Even outside of outbreaks, near-patient ICU environmental contamination with VRE is common. Better infection control policies that limit environmental transmission of VRE in the ICU and that are supported by molecular epidemiological studies, in real time, are needed. Infect Control Hosp Epidemiol 2018;39:40-45.

Eradication of Staphylococcus aureus Biofilm Infections Using Synthetic Antimicrobial Peptides.

Here, we demonstrate that antimicrobial peptides (AMPs) are an effective antibiofilm treatment when applied as catheter lock solutions (CLSs) against S. aureus biofilm infections. The activity of synthetic AMPs (Bac8c, HB43, P18, Omiganan, WMR, Ranalexin, and Polyphemusin) was measured against early and mature biofilms produced by methicillin-resistant S. aureus and methicillin-susceptible S. aureus isolates from patients with device-related infections grown under in vivo-relevant biofilm conditions. The cytotoxic and hemolytic activities of the AMPs against human cells and their immunomodulatory potential in human blood were also characterized. The D-Bac8c2,5Leu variant emerged as the most effective AMP during in vitro studies and was also highly effective in eradicating S. aureus biofilm infection when used in a CLS rat central venous catheter infection model. These data support the potential use of D-Bac8c2,5Leu, alone or in combination with other AMPs, in the treatment of S. aureus intravenous catheter infections.

Using Disease-Associated Enzymes to Activate Antimicrobial Peptide Prodrugs.

Prodrugs of antimicrobial peptides can be generated by modifying their sequences at their N-termini with a linker and a negatively charged promoiety. These modifications can be selectively reversed by a disease-associated enzyme, thereby confining the activity of the peptide to pathologically affected body parts. A general method for the generation of prodrug candidates, based on a linker constituting the substrate of a disease-associated protease and an oligo-glutamic acid promoiety, as well as a protocol to validate the activation of the prodrug, are described herein.

The Molecular Epidemiology of Resistance in Cefotaximase-Producing Escherichia coli Clinical Isolates from Dublin, Ireland.

In view of continued high clinical prevalence of infections involving extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli, this study sought to characterise the blaCTX-M genes, their associated mobile genetic elements and the integrons present in 100 ESBL-producing E. coli isolates collected in a Dublin hospital and associated community healthcare facilities. Polymerase chain reaction (PCR) mapping and sequencing was used to detect blaCTX-M alleles, their associated insertion sequences (ISs) and class 1 and 2 integrons in the collection. ESBL plasmids were characterised by PCR-based replicon typing and replicon sequence typing (RST). Cefotaximases were harboured by 94% of isolates (66 blaCTX-M-15, 8 blaCTX-M-14, 7 blaCTX-M-1, 4 blaCTX-M-3, 3 blaCTX-M-9, 2 blaCTX-M-27, 2 blaCTX-M-55, 1 blaCTX-M-32 and 1 blaCTX-M-2). An ISEcp1 promoter was linked to a group 1 blaCTX-M gene in 45% of isolates. A further 34% of isolates contained blaCTX-M-15 downstream of IS26, an arrangement typical of epidemic UK strain A. Class 1 integrons were found in 66% of isolates, most carrying trimethoprim/aminoglycoside resistance genes. CTX-M plasmids were primarily of multireplicon IncF or IncI1 type, but IncN and unidentified types were also found. Novel IncF RSTs F1:A-:B-, F45:A1:B-, F45:A4:B- and a novel IncI1 sequence type, ST159, were identified. CTX-M plasmids and integrons resembled those identified recently in animal isolates from Ireland and Western Europe. The molecular epidemiology of CTX-M-producing E. coli in Dublin suggests that horizontal spread of mobile genetic elements contributes to antimicrobial resistant human infections. Further investigations into whether animals or animal products represent an important local reservoir for these elements are warranted.

A novel dual-functioning ruthenium(II)-arene complex of an anti-microbial ciprofloxacin derivative - Anti-proliferative and anti-microbial activity.

7-(4-(Decanoyl)piperazin-1-yl)-ciprofloxacin, CipA, (1) which is an analogue of the antibiotic ciprofloxacin, and its ruthenium(II) complex [Ru(η(6)-p-cymene)(CipA-H)Cl], (2) have been synthesised and the x-ray crystal structures of 1·1.3H2O·0.6CH3OH and 2·CH3OH·0.5H2O determined. The complex adopts a typical pseudo-octahedral 'piano-stool' geometry, with Ru(II) π-bonded to the p-cymene ring and σ-bonded to a chloride and two oxygen atoms of the chelated fluoroquinolone ligand. The complex is highly cytotoxic in the low µM range and is as potent as the clinical drug cisplatin against the human cancer cell lines A2780, A549, HCT116, and PC3. It is also highly cytotoxic against cisplatin- and oxaliplatin-resistant cell lines suggesting a different mechanism of action. The complex also retained low µM cytotoxicity against the human colon cancer cell line HCT116p53 in which the tumour suppressor p53 had been knocked out, suggesting that the potent anti-proliferative properties associated with this complex are independent of the status of p53 (in contrast to cisplatin). The complex also retained moderate anti-bacterial activity in two Escherichia coli, a laboratory strain and a clinical isolate resistant to first, second and third generation ß-lactam antibiotics.

Differential In Vitro and In Vivo Toxicities of Antimicrobial Peptide Prodrugs for Potential Use in Cystic Fibrosis.

There has been considerable interest in the use of antimicrobial peptides (AMPs) as antimicrobial agents for the treatment of many conditions, including cystic fibrosis (CF). The challenging conditions of the CF patient lung require robust AMPs that are active in an environment of high proteolytic activity but that also have low cytotoxicity and immunogenicity. Previously, we developed prodrugs of AMPs that limited the cytotoxic effects of AMP treatment by rendering the antimicrobial activity dependent on the host enzyme neutrophil elastase (NE). However, cytotoxicity remained an issue. Here, we describe the further optimization of the AMP prodrug (pro-AMP) model for CF to produce pro-WMR, a peptide with greatly reduced cytotoxicity (50% inhibitory concentration against CFBE41o- cells, >300 µM) compared to that of the previous group of pro-AMPs. The bactericidal activity of pro-WMR was increased in NE-rich bronchoalveolar lavage (BAL) fluid from CF patients (range, 8.4% ± 6.9% alone to 91.5% ± 5.8% with BAL fluid; P = 0.0004), an activity differential greater than that of previous pro-AMPs. In a murine model of lung delivery, the pro-AMP modification reduced host toxicity, with pro-WMR being less toxic than the active peptide. Previously, host toxicity issues have hampered the clinical application of AMPs. However, the development of application-specific AMPs with modifications that minimize toxicity similar to those described here can significantly advance their potential use in patients. The combination of this prodrug strategy with a highly active AMP has the potential to produce new therapeutics for the challenging conditions of the CF patient lung.