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Antimicrobial nanocoatings may be a means of preventing nosocomial infections, which account for significant morbidity and mortality. The role of hospital sink traps in these infections is also increasingly appreciated. We describe the preparation, material characterization and antibacterial activity of a pipe cement-based silver nanocoating applied to unplasticized polyvinyl chloride, a material widely used in wastewater plumbing. Three-dimensional surface topography imaging and scanning electron microscopy showed increased roughness in all surface finishes versus control, with grinding producing the roughest surfaces. Silver stability within nanocoatings was >99.89% in deionized water and bacteriological media seeded with bacteria. The nanocoating exhibited potent antibiofilm (99.82-100% inhibition) and antiplanktonic (99.59-99.99% killing) activity against three representative bacterial species and a microbial community recovered from hospital sink traps. Hospital sink trap microbiota were characterized by sequencing the 16S rRNA gene, revealing the presence of opportunistic pathogens from genera including Pseudomonas , Enterobacter and Clostridioides . In a benchtop model sink trap system, nanocoating antibiofilm activity against this community remained significant after 11 days but waned following 25 days. Silver nanocoated disks in real-world sink traps in two university buildings had a limited antibiofilm effect, even though in vitro experiments using microbial communities recovered from the same traps demonstrated that the nanocoating was effective, reducing biofilm formation by >99.6% and killing >98% of planktonic bacteria. We propose that conditioning films forming in the complex conditions of real-world sink traps negatively impact nanocoating performance, which may have wider relevance to development of antimicrobial nanocoatings that are not tested in the real-world.

Antimicrobial resistance has been a global health challenge that threatens our ability to control and treat life-threatening bacterial infections. Despite ongoing efforts to identify new drugs or alternatives to antibiotics, no new classes of antibiotic or their alternatives have been clinically approved in the last three decades. A combination of antibiotics and non-antibiotic compounds that could inhibit bacterial resistance determinants or enhance antibiotic activity offers a sustainable and effective strategy to confront multidrug-resistant bacteria. In this review, we provide a brief overview of the co-evolution of antibiotic discovery and the development of bacterial resistance. We summarize drug-drug interactions and uncover the art of repurposing non-antibiotic drugs as potential antibiotic adjuvants, including discussing classification and mechanisms of action, as well as reporting novel screening platforms. A pathogen-by-pathogen approach is then proposed to highlight the critical value of drug repurposing and its therapeutic potential. Finally, general advantages, challenges and development trends of drug combination strategy are discussed., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Objectives: Epidermicin is a novel antimicrobial peptide that has potent activity against Gram-positive pathogens, including methicillin-resistant Staphylococcus aureus, and it may have potential for use in therapy for infections caused by these bacteria, though in vivo efficacy needs to be demonstrated. Galleria mellonella larvae have recently been introduced as an alternative in vivo model to mammalian systems and here we examined the ability of a synthetic version of epidermicin to protect G. mellonella larvae from infection with S. aureus strains., Methods: G. mellonella larvae were infected with ∼2.5 × 10(6) cells per larva and then treated with vancomycin or epidermicin and survival recorded over a 120 h period. Vancomycin was used at up to 50 mg/kg and epidermicin at up to 200 mg/kg with administration of treatments occurring 0-2 h post-infection., Results: Epidermicin was shown to be non-toxic and did not stimulate the G. mellonella immune system. When administered 2 h post-infection at a maximum dose of 200 mg/kg, epidermicin significantly increased survival in larvae; however, altering the dosage regimen by reducing the time to administration to 30 min post-infection increased the potency of the peptide., Conclusions: This is the first report of antimicrobial activity of an artificially synthesized peptide from the type IIc bacteriocin family. The novel peptide protects G. mellonella larvae from infection with both methicillin-susceptible and -resistant S. aureus, although the pharmacodynamic properties are not yet optimal.

We describe the discovery, purification, characterization, and expression of an antimicrobial peptide, epidermicin NI01, which is an unmodified bacteriocin produced by Staphylococcus epidermidis strain 224. It is a highly cationic, hydrophobic, plasmid-encoded peptide that exhibits potent antimicrobial activity toward a wide range of pathogenic Gram-positive bacteria including methicillin-resistant Staphylococcus aureus (MRSA), enterococci, and biofilm-forming S. epidermidis strains. Purification of the peptide was achieved using a combination of hydrophobic interaction, cation exchange, and high-performance liquid chromatography (HPLC). Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analysis yielded a molecular mass of 6,074 Da, and partial sequence data of the peptide were elucidated using a combination of tandem mass spectrometry (MS/MS) and de novo sequencing. The draft genome sequence of the producing strain was obtained using 454 pyrosequencing technology, thus enabling the identification of the structural gene using the de novo peptide sequence data previously obtained. Epidermicin NI01 contains 51 residues with four tryptophan and nine lysine residues, and the sequence showed approximately 50% identity to peptides lacticin Z, lacticin Q, and aureocin A53, all of which belong to a new family of unmodified type II-like bacteriocins. The peptide is active in the nanomolar range against S. epidermidis, MRSA isolates, and vancomycin-resistant enterococci. Other unique features displayed by epidermicin include a high degree of protease stability and the ability to retain antimicrobial activity over a pH range of 2 to 10, and exposure to the peptide does not result in development of resistance in susceptible isolates. In this study we also show the structural gene alone can be cloned into Escherichia coli strain BL21(DE3), and expression yields active peptide.

Objectives: Multilocus sequence typing (MLST) has been used to characterize diverse pathogens, including uropathogenic Escherichia coli (UPEC). There has been significant interest in the contribution of the O25b:H4-ST131 lineage to UPEC disease, as these isolates are often highly virulent and exhibit multidrug resistance. To reveal the wider impact of sequence type (ST) 131, we have examined its contribution to the overall population structure of UPEC isolates that were not selected on the basis of virulence or antibiotic resistance., Methods: Three hundred UPEC isolates were recovered from community and hospital urine samples examined by clinical microbiology laboratories in the Northwest region of England in June 2007 and June 2009. They were characterized by susceptibility profiling, MLST and virulence gene PCR. PFGE was used to examine isolates from key clones., Results: The most common lineage was ST73 (16.6%) followed by ST131 (13.3%), ST69 (9%), ST95 (6.3%), ST10 (4.3%) and ST127 (3.6%). ST131 isolates were significantly more likely to exhibit high levels of antibiotic resistance (35% being CTX-M-15 PCR positive) and those of ST127 were the most widely susceptible but carried the highest number of virulence genes. Only when the CTX-M-15-O25b-positive strains were examined was a high level of virulence observed for ST131 isolates. PFGE indicated ongoing local evolution in ST131., Conclusions: ST131 isolates are well established in the wider UPEC population. This clone is still evolving and we further support suggestions that it represents a real threat to health. We suggest that ST127 is a recently emerged, community-associated, virulent clone that warrants further study.

Stenotrophomonas maltophilia is an emerging nosocomial pathogen capable of causing healthcare-associated infections, including pneumonia and bacteremia. Intrinsic resistance in S. maltophilia is exhibited towards many broad-spectrum antibiotics, and treatment recommendations are controversial. One of the major causes of antimicrobial resistance is attributed to a robust array of efflux pumps that extrude drug compounds from the cell. Using checkerboard and growth kinetic assays, we evaluated the in vitro activity of a polyclonal antibody raised against an ATP-binding cassette efflux protein in S. maltophilia. Six clinical strains of S. maltophilia and one type strain were challenged with co-trimoxazole, ticarcillin-clavulanate, and ciprofloxacin, alone and in combination with antibody. One clinical strain was tested by growth curve experiments for each antibiotic-antibody combination. The use of antibody resulted in significantly increased susceptibility in 71.4% (15/21) of treatments tested, with 33.3% displaying synergy and 38.1% an additive effect. In growth kinetic studies, synergy was obtained for each antibiotic-antibody combination. Thus, the use of antibody raised against multidrug efflux pumps for the treatment of multidrug-resistant organisms warrants further investigation. Antibody targeting substrate recognition sites, or other functionally important epitopes, may lead to inhibition of multiple efflux pumps that share the same substrate and is an attractive area that should be explored.

Our interest in Staphylococcus epidermidis strain A487 was prompted by the unusual nature of its inhibitory activity in screening tests against methicillin-resistant Staphylococcus aureus isolates. The inhibitory activity was detected in deferred antagonism tests only if the agar plate was preheated for at least 35 min at ≥ 55 °C before inoculation of the indicator bacteria, this phenomenon indicating possible involvement of a heat-labile immunity agent or protease. The inhibitor was purified to homogeneity by ammonium sulphate precipitation, followed by cation-exchange and reversed-phase chromatography. Tandem MS revealed a novel peptide of molecular weight 2588.4 Da. The draft genome sequence of strain A487 was determined using 454 GS FLX technology, allowing the identification of the structural gene (hlp) encoding the mature peptide MQFITDLIKKAVDFFKGLFGNK. The deduced amino acid sequence of peptide 487 exhibited 70.8% similarity to that of a putative haemolysin from Staphylococcus cohnii. Analysis of the genome of strain A487 showed several additional inhibitor-encoding genes, including hld, the determinant for staphylococcal δ-lysin. This work indicates that potentially useful inhibitors could be overlooked in agar-based inhibitor screening programmes lacking a heat pretreatment step and also highlights the utility of draft genome sequence examination in antibacterial agent discovery., (© 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

Salivaricin 9 (Sal9) is a 2560 Da lantibiotic having just 46 % amino acid identity with its closest known homologue, the Streptococcus pyogenes lantibiotic SA-FF22. The Sal9 locus (designated siv) in Streptococcus salivarius strain 9 was partially sequenced and localized to an approximately 170 kb megaplasmid, which also harbours the locus for the lantibiotic salivaricin A4. The entire locus was fully characterized in the draft genome sequence of S. salivarius strain JIM8780 and shown to consist of eight genes, having the following putative functions: sivK, sensor kinase; sivR, response regulator; sivA, Sal9 precursor peptide; sivM, lantibiotic modification enzyme; sivT, ABC transporter involved in the export of Sal9 and concomitant cleavage of its leader peptide; and sivFEG, encoding lantibiotic self-immunity. Intriguingly, in contrast to strain 9, the siv locus was chromosomally located in strain JIM8780--the first lantibiotic locus shown not to be exclusively plasmid-associated in S. salivarius. Sal9-containing extracts specifically induced lantibiotic production in both strain 9 and strain JIM8780, indicating that Sal9 functions as a signal peptide for upregulation of its own biosynthesis. Screening representative strains of three streptococcal species (S. salivarius, S. pyogenes and S. mitis) for sivA indicated that it was present only in S. salivarius, with 12 of 28 tested S. salivarius positive. Since Sal9 was inhibitory to all tested S. pyogenes strains it appears to have potential as an important component of the bacteriocin armoury of S. salivarius probiotics intended to control S. pyogenes infections of the human oral cavity.

Delta-lysins (DL) that are produced by various species of staphylococci are not widely known for their antimicrobial activity. We have purified and characterized a novel DL variant, E229DL and examined its spectrum of inhibitory activity. The biological activity of E229DL, produced by Staphylococcus epidermidis strain E229, shows relatively broad-spectrum activity against Gram-positive pathogens, including representatives of MRSA and epidemic MRSA type 15. E229DL was purified to homogeneity from 95% acidified-methanol extracts of cell cultures by using a series of reversed-phase chromatographic separations. The fully processed form of E229DL is a 25-amino-acid peptide with a predicted mass of 2841.4 Da, but the purified biologically active molecule appears to be N-formylated (mass 2867.33 Da). The DL gene (hld) resembles that of other types of DL, but differs in five codons with hld in Staphylococcus aureus (26 residues) and one codon with the closest homolog, the hld-II in S. warneri (25 residues). The characterization of E229DL showed that its activity is stable in agar exposed to high temperatures (80 degrees C/45 min). In addition, biological testing of the native and synthetic peptides against a range of human and animal erythrocytes and Vero cells indicated that E229DL is an antibacterial agent with no detectable cytopathic effects at concentrations equivalent to the minimum inhibitory concentration for EMRSA15-A208. Initial investigation of the mode of action of E229DL indicated that it is rapidly lytic for target cells. This is the first description of a native form of DL having only limited cytotoxic activity for eukaryotic cells at concentrations that are inhibitory to staphylococci., (Copyright 2010 Elsevier Inc. All rights reserved.)

Aims: To examine the response of Staphylococcus epidermidis isolates from prosthetic orthopaedic infections to vancomycin concentrations below the minimum inhibitory concentration., Methods: Staphylococcal biofilms were grown in 96-well flat-bottomed cell culture plates under a variety of culture conditions and stained using an ammonium crystal violet solution. Optical density (450 nm wavelength) was recorded to estimate the biofilm density for each strain. Population analysis and time-kill studies were also performed on selected isolates., Results: A range of responses were observed, including increased biofilm density at drug concentrations approaching the minimum inhibitory concentration. This increased density was associated with the presence of a more resistant population identified on population analysis but without an apparent effect on the time-kill curves., Conclusions: The ability of some strains to show increased biofilm density could be a factor in the failure of vancomycin therapy reported in some cases. The demonstration that low concentrations of vancomycin may increase the density of newly forming S epidermidis biofilms may indicate an area of potential concern in the use of vancomycin in orthopaedic implants and intravascular catheter locks, and may partially account for some cases of treatment failure.

Objectives: The aac(6')-Ib-cr gene has been described in plasmids from CTX-M-15-producing Escherichia coli in the worldwide ST131 lineage, but has not been systematically sought in other quinolone-resistant strains in the UK. A rise in quinolone resistance in bacteraemia isolates in the UK preceded the increased prevalence of CTX-M-producing strains. This study aimed to describe the presence of plasmid-encoded quinolone resistance genes in historical and current strains of E. coli not producing extended-spectrum beta-lactamases (ESBLs)., Methods: Ciprofloxacin-resistant, non-ESBL-producing E. coli isolates included nationally distributed isolates from the BSAC UK bacteraemia surveillance programme between 2001 and 2005, urinary isolates from a regional project in 2000 and local strains in 2006. The aac(6')-Ib-cr gene was detected using PCR followed by restriction fragment length polymorphism analysis. Multiplex PCR was used to detect qnr genes. Isolates with aac(6')-Ib-cr were assessed for aminoglycoside susceptibilities and were serotyped., Results: The prevalence of the aac(6')-Ib-cr gene was 3% and 9% in current local urinary and historic national bacteraemia quinolone-resistant non-ESBL-producing E. coli, respectively. Of 521 regional urinary E. coli isolates from 2000, 14 were norfloxacin-resistant, none of which carried the aac(6')-Ib-cr gene. National positive bacteraemia isolates from 2001/2 were type O102-ST405 and, in 2004/5, types O1-ST645 and O25-ST131. Positive local urinary isolates from 2006 included serotypes O1 and O25., Conclusions: In the UK, aac(6')-Ib-cr occurs in E. coli in the absence of CTX-M-15, but with a restricted serotype distribution. Its presence in widespread bacteraemia isolates of a single type from 2001 to 2002, prior to the spread of CTX-M-15 in Britain, might suggest a lineage from which plasmid recombination occurred in man or other species.

Streptococcus salivarius 20P3 produces a 22-amino-acid residue lantibiotic, designated salivaricin A (SalA), that inhibits the growth of a range of streptococci, including all strains of Streptococcus pyogenes. Lantibiotic production is associated with the sal genetic locus comprising salA, the lantibiotic structural gene; salBCTX genes encoding peptide modification and export machinery proteins; and salYKR genes encoding a putative immunity protein and two-component sensor-regulator system. Insertional inactivation of salB in S. salivarius 20P3 resulted in abrogation of SalA peptide production, of immunity to SalA, and of salA transcription. Addition of exogenous SalA peptide to salB mutant cultures induced dose-dependent expression of salA mRNA (0.2 kb), demonstrating that SalA production was normally autoregulated. Inactivation of salR encoding the response regulator of the SalKR two-component system led to reduced production of, and immunity to, SalA. The sal genetic locus was also present in S. pyogenes SF370 (M type 1), but because of a deletion across the salBCT genes, the corresponding lantibiotic peptide, designated SalA1, was not produced. However, in S. pyogenes T11 (M type 4) the sal locus gene complement was apparently complete, and active SalA1 peptide was synthesized. Exogenously added SalA1 peptide from S. pyogenes T11 induced salA1 transcription in S. pyogenes SF370 and in an isogenic S. pyogenes T11 salB mutant and salA transcription in S. salivarius 20P3 salB. Thus, SalA and SalA1 are examples of streptococcal lantibiotics whose production is autoregulated. These peptides act as intra- and interspecies signaling molecules, modulating lantibiotic production and possibly influencing streptococcal population ecology in the oral cavity.