Microbial Interspecies Interactions and Their Impact on the Emergence and Spread of Antimicrobial Resistance.

Bacteria are social organisms that commonly live in dense communities surrounded by a multitude of other species. The competitive and cooperative interactions between these species not only shape the bacterial communities but also influence their susceptibility to antimicrobials. While several studies have shown that mixed-species communities are more tolerant toward antimicrobials than their monospecies counterparts, only limited empirical data are currently available on how interspecies interactions influence resistance development. We here propose a theoretic framework outlining the potential impact of interspecies social behavior on different aspects of resistance development. We identify factors by which interspecies interactions might influence resistance evolution and distinguish between their effect on (a) the emergence of a resistant mutant and (b) the spread of this resistance throughout the population. Our analysis indicates that considering the social life of bacteria is imperative to the rational design of more effective antibiotic treatment strategies with a minimal hazard for resistance development.

Antibiotic Cycling Affects Resistance Evolution Independently of Collateral Sensitivity.

Antibiotic cycling has been proposed as a promising approach to slow down resistance evolution against currently employed antibiotics. It remains unclear, however, to which extent the decreased resistance evolution is the result of collateral sensitivity, an evolutionary trade-off where resistance to one antibiotic enhances the sensitivity to the second, or due to additional effects of the evolved genetic background, in which mutations accumulated during treatment with a first antibiotic alter the emergence and spread of resistance against a second antibiotic via other mechanisms. Also, the influence of antibiotic exposure patterns on the outcome of drug cycling is unknown. Here, we systematically assessed the effects of the evolved genetic background by focusing on the first switch between two antibiotics against Salmonella Typhimurium, with cefotaxime fixed as the first and a broad variety of other drugs as the second antibiotic. By normalizing the antibiotic concentrations to eliminate the effects of collateral sensitivity, we demonstrated a clear contribution of the evolved genetic background beyond collateral sensitivity, which either enhanced or reduced the adaptive potential depending on the specific drug combination. We further demonstrated that the gradient strength with which cefotaxime was applied affected both cefotaxime resistance evolution and adaptation to second antibiotics, an effect that was associated with higher levels of clonal interference and reduced cost of resistance in populations evolved under weaker cefotaxime gradients. Overall, our work highlights that drug cycling can affect resistance evolution independently of collateral sensitivity, in a manner that is contingent on the antibiotic exposure pattern.

Competitive interactions facilitate resistance development against antimicrobials.

While the evolution of antimicrobial resistance is well studied in free-living bacteria, information on resistance development in dense and diverse biofilm communities is largely lacking. Therefore, we explored how the social interactions in a duo-species biofilm composed of the brewery isolates Pseudomonas rhodesiae and Raoultella terrigena influence the adaptation to the broad-spectrum antimicrobial sulfathiazole. Previously, we showed that the competition between these brewery isolates enhances the antimicrobial tolerance of P. rhodesiae. Here, we found that this enhanced tolerance in duo-species biofilms is associated with a strongly increased antimicrobial resistance development in P. rhodesiae. Whereas P. rhodesiae was not able to evolve resistance against sulfathiazole in monospecies conditions, it rapidly evolved resistance in the majority of the duo-species communities. Although the initial presence of R. terrigena was thus required for P. rhodesiae to acquire resistance, the resistance mechanisms did not depend on the presence of R. terrigena. Whole genome sequencing of resistant P. rhodesiae clones showed no clear mutational hot spots. This indicates that the acquired resistance phenotype depends on complex interactions between low-frequency mutations in the genetic background of the strains. We hypothesize that the increased tolerance in duo-species conditions promotes resistance by enhancing the selection of partially resistant mutants and opening up novel evolutionary trajectories that enable such genetic interactions. This hypothesis is reinforced by experimentally excluding potential effects of increased initial population size, enhanced mutation rate, and horizontal gene transfer. Altogether, our observations suggest that the community mode of life and the social interactions therein strongly affect the accessible evolutionary pathways toward antimicrobial resistance.IMPORTANCEAntimicrobial resistance is one of the most studied bacterial properties due to its enormous clinical and industrial relevance; however, most research focuses on resistance development of a single species in isolation. In the present study, we showed that resistance evolution of brewery isolates can differ greatly between single- and mixed-species conditions. Specifically, we observed that the development of antimicrobial resistance in certain species can be significantly enhanced in co-culture as compared to the single-species conditions. Overall, the current study emphasizes the need of considering the within bacterial interactions in microbial communities when evaluating antimicrobial treatments and resistance evolution.

Pre-clinical In Vitro Models of Vascular Graft Coating in the Prevention of Vascular Graft Infection: A Systematic Review.

OBJECTIVE: Vascular graft infection (VGI) is a feared complication. Prevention is of the utmost importance and vascular graft coatings (VGCs) could offer a potential to do this, with in vitro research a first crucial step. The aim of this study was to summarise key features of in vitro models investigating coating strategies to prevent VGI in order to provide guidance for the setup of future translational research. DATA SOURCES: A comprehensive search was performed in MEDLINE, Embase, and Web of Science. METHODS: A systematic review was conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-analyses guidelines. For each database, a specific search strategy was developed. Quality was assessed with the Toxicological data Reliability Assessment Tool (ToxRTool). In vitro models using a VGC and inoculation of the graft with a pathogen were included. The type of graft, coating, and pathogen were summarised. The outcome assessment in each study was evaluated. RESULTS: In total, 4 667 studies were identified, of which 45 papers met the inclusion criteria. The majority used polyester grafts (68.2%). Thirty-one studies (68.9%) included antibiotics, and nine studies (20%) used a commercial silver graft in their protocol. New antibacterial strategies (e.g., proteolytic enzymes) were investigated. A variety of testing methods was found and focused mainly on bacterial adherence, coating adherence and dilution, biofilm formation, and cytotoxicity. Ninety-three per cent of the studies (n = 41) were considered unreliable. CONCLUSION: Polyester is the preferred type of graft to coat on. The majority of coating studies are based on antibiotics; however, new coating strategies (e.g., antibiofilm coating) are coming. Many in vitro setups are available. In vitro studies have great potential, they can limit the use, but cannot replace in vivo studies completely. This paper can be used as a guidance document for future in vitro research.

A systematic review of preclinical data regarding commercial silver-coated vascular grafts.

OBJECTIVE: Vascular graft infection (VGI) is a serious complication with high mortality and morbidity rates. Several measures could be taken to decrease this risk, including the use of silver-containing vascular grafts. However, to date, no clinical advantages have been reported. This study reviews the outcome of preclinical studies focusing on the role of commercially available silver-coated grafts in the prevention of VGI. METHODS: A systematic review was performed with a focus on the preclinical role of commercially available silver-coated vascular grafts in the prevention and treatment of VGI. A comprehensive search was conducted in Medline, Embase, and Web of Science. RESULTS: Nine in vitro and five in vivo studies were included. Two commercial grafts were used (INTERGARD SILVER and Silver Graft). In vitro studies used both gram-positive and gram-negative strains. A positive antimicrobial effect was observed in seven of nine studies (77.8%). A delayed antifungal effect against Candida species was observed in vitro, but disappeared when adding serum proteins. In vivo studies witnessed a microbicidal effect in two out of five studies (40%), but only tested a single causative pathogen (ie, Staphylococcus aureus). CONCLUSIONS: Both in vitro and in vivo studies demonstrated conflicting and mixed results concerning the antimicrobial efficacy of commercially available silver-containing grafts in the prevention of VGI. In general, the study setup was heterogeneous in the different articles. Given the lack of convincing preclinical evidence and their poor performance in clinical studies, more data are needed at this time to guide the appropriate use of silver grafts.

Pre-clinical in vivo Models of Vascular Graft Coating in the Prevention of Vascular Graft Infection: A Systematic Review.

OBJECTIVE: Vascular graft infection (VGI) remains an important complication with a high mortality and morbidity rate. Currently, studies focusing on the role of vascular graft coatings in the prevention of VGI are scarce. Therefore, the aims of this study were to survey and summarise key features of pre-clinical in vivo models that have been used to investigate coating strategies to prevent VGI and to set up an ideal model that can be used in future preclinical research. DATA SOURCES: A systematic review was conducted in accordance with the Preferred reporting items for Systematic Reviews and Meta-Analysis guidelines. A comprehensive search was performed in MEDLINE (PubMed), Embase, and Web of Science. REVIEW METHODS: For each database, a specific search strategy was developed. Quality was assessed with the Toxicological data Reliability Assessment Tool (ToxRTool). The type of animal model, graft, coating, and pathogen were summarised. The outcome assessment in each study was evaluated. RESULTS: In total, 4 667 studies were identified, of which 94 papers focusing on in vivo testing were included. Staphylococcus aureus was the organism most used (n = 65; 67.7%). Most of the graft types were polyester grafts. Rifampicin was the most frequently used antibiotic coating (n = 43, 48.3%). In the outcome assessment, most studies mentioned colony forming unit count (n = 88; 91.7%) and clinical outcome (n = 72; 75%). According to the ToxRTool, 21 (22.3%, n = 21/94) studies were considered to be not reliable. CONCLUSION: Currently published in vivo models are very miscellaneous. More attention should be paid to the methodology of these pre-clinical reports when transferring novel graft coatings into clinical practice. Variables used in pre-clinical reports (bacterial strain, duration of activity coating) do not correspond well to current clinical studies. Based on the results of this review, a proposal for a complete and comprehensive set up for pre-clinical invivo testing of anti-infectious properties of vascular graft coatings was defined.

2-Aminoimidazoles as potent inhibitors of contaminating brewery biofilms.

Cleaning and disinfection protocols are not always able to remove biofilm microbes present in breweries, indicating that novel anti-biofilm strategies are needed. The preventive activities of three in-house synthesized members of the 2-aminoimidazole class of anti-biofilm molecules were studied against 17 natural brewery biofilms and benchmarked against 18 known inhibitors. Two 2-aminoimidazoles belonged to the top six inhibitors, which were retested against 12 defined brewery biofilm models. For the three best inhibitors, tannic acid (n° 1), 2-aminoimidazole imi-AAC-5 (n° 2), and baicalein (n° 3), the effect on the microbial metabolic activity was evaluated. Here, the top three inhibitors showed similar effectiveness, with baicalein possessing a slightly higher efficacy. Even though the 2-aminoimidazole was the second-best inhibitor, it showed a lower biocidal activity than tannic acid, making it less prone to resistance evolution. Overall, this study supports the potential of 2-aminoimidazoles as a preventive anti-biofilm strategy.

Drug repurposing: phosphate prodrugs of anticancer and antiviral FDA-approved nucleosides as novel antimicrobials.

OBJECTIVES: Following a drug repurposing approach, we aimed to investigate and compare the antibacterial and antibiofilm activities of different classes of phosphate prodrugs (HepDirect, cycloSal, SATE and mix SATE) of antiviral and anticancer FDA-approved nucleoside drugs [zidovudine (AZT), floxouridine (FUDR) and gemcitabine (GEM)] against a variety of pathogenic Gram-positive and -negative bacteria. METHODS: Ten prodrugs were synthesized and screened for antibacterial activity against seven Gram-negative and two Gram-positive isolates fully susceptible to traditional antibiotics, alongside six Gram-negative and five Gram-positive isolates with resistance mechanisms. Their ability to prevent and eradicate biofilms of different bacterial pathogens in relation to planktonic growth inhibition was also evaluated, together with their effect on proliferation, viability and apoptosis of different eukaryotic cells. RESULTS: The prodrugs showed decreased antibacterial activity compared with the parent nucleosides. cycloSal-GEM-monophosphate (MP) prodrugs 20a and 20b were the most active agents against Gram-positive bacteria (Enterococcus faecalis and Staphylococcus aureus) and retained their activity against antibiotic-resistant isolates. cycloSal-FUDR-MP 21a partially retained good activity against the Gram-positive bacteria E. faecalis, Enterococcus faecium and S. aureus. Most of the prodrugs tested displayed very potent preventive antibiofilm specific activity, but not curative. In terms of cytotoxicity, AZT prodrugs did not affect apoptosis or cell viability at the highest concentration tested, and only weak effects on apoptosis and/or cell viability were observed for GEM and FUDR prodrugs. CONCLUSIONS: Among the different prodrug approaches, the cycloSal prodrugs appeared the most effective. In particular, cycloSal (17a) and mix SATE (26) AZT prodrugs combine the lowest cytotoxicity with high and broad antibacterial and antibiofilm activity against Gram-negative bacteria.

Pseudomonas putida as a potential biocontrol agent against Salmonella Java biofilm formation in the drinking water system of broiler houses.

BACKGROUND: Environmental biofilms can induce attachment and protection of other microorganisms including pathogens, but can also prevent them from invasion and colonization. This opens the possibility for so-called biocontrol strategies, wherein microorganisms are applied to control the presence of other microbes. The potential for both positive and negative interactions between microbes, however, raises the need for in depth characterization of the sociobiology of candidate biocontrol agents (BCAs). The inside of the drinking water system (DWS) of broiler houses is an interesting niche to apply BCAs, because contamination of these systems with pathogens plays an important role in the infection of broiler chickens and consequently humans. In this study, Pseudomonas putida, which is part of the natural microbiota in the DWS of broiler houses, was evaluated as BCA against the broiler pathogen Salmonella Java. RESULTS: To study the interaction between these species, an in vitro model was developed simulating biofilm formation in the drinking water system of broilers. Dual-species biofilms of P. putida strains P1, P2, and P3 with S. Java were characterized by competitive interactions, independent of P. putida strain, S. Java inoculum density and application order. When equal inocula of S. Java and P. putida strains P1 or P3 were simultaneously applied, the interaction was characterized by mutual inhibition, whereas P. putida strain P2 showed an exploitation of S. Java. Lowering the inoculum density of S. Java changed the interaction with P. putida strain P3 also into an exploitation of S. Java. A further increase in S. Java inhibition was established by P. putida strain P3 forming a mature biofilm before applying S. Java. CONCLUSIONS: This study provides the first results showing the potential of P. putida as BCA against S. Java in the broiler environment. Future work should include more complex microbial communities residing in the DWS, additional Salmonella strains as well as chemicals typically used to clean and disinfect the system.

Occurrence and characterisation of biofilms in drinking water systems of broiler houses.

BACKGROUND: Water quality in the drinking water system (DWS) plays an important role in the general health and performance of broiler chickens. Conditions in the DWS of broilers are ideal for microbial biofilm formation. Since pathogens might reside within these biofilms, they serve as potential source of waterborne transmission of pathogens to livestock and humans. Knowledge about the presence, importance and composition of biofilms in the DWS of broilers is largely missing. In this study, we therefore aim to monitor the occurrence, and chemically and microbiologically characterise biofilms in the DWS of five broiler farms. RESULTS: The bacterial load after disinfection in DWSs was assessed by sampling with a flocked swab followed by enumerations of total aerobic flora (TAC) and Pseudomonas spp. The dominant flora was identified and their biofilm-forming capacity was evaluated. Also, proteins, carbohydrates and uronic acids were quantified to analyse the presence of extracellular polymeric substances of biofilms. Despite disinfection of the water and the DWS, average TAC was 6.03 ± 1.53 log CFU/20cm2. Enumerations for Pseudomonas spp. were on average 0.88 log CFU/20cm2 lower. The most identified dominant species from TAC were Stenotrophomonas maltophilia, Pseudomonas geniculata and Pseudomonas aeruginosa. However at species level, most of the identified microorganisms were farm specific. Almost all the isolates belonging to the three most abundant species were strong biofilm producers. Overall, 92% of all tested microorganisms were able to form biofilm under lab conditions. Furthermore, 63% of the DWS surfaces appeared to be contaminated with microorganisms combined with at least one of the analysed chemical components, which is indicative for the presence of biofilm. CONCLUSIONS: Stenotrophomonas maltophilia, Pseudomonas geniculata and Pseudomonas aeruginosa are considered as opportunistic pathogens and could consequently be a potential risk for animal health. Additionally, the biofilm-forming capacity of these organisms could promote attachment of other pathogens such as Campylobacter spp. and Salmonella spp.

Rational design of small molecules that modulate the transcriptional function of the response regulator PhoP.

The response regulator PhoP, which is part of the PhoP/PhoQ two-component system, regulates the expression of multiple genes involved in controlling virulence in Salmonella enterica serovar Typhimurium and other species of Gram-negative bacteria. Modulating the phosphorylation-mediated dimerization in the receiver domain may interfere with the transcriptional function of PhoP. In this study, we analyzed the therapeutic potential of the PhoP receiver domain by exploring it as a potential target for drug design. The structural information was then applied to identify the first hit compounds from commercial chemical libraries by combining pharmacophore modelling and docking methods with a GFP (Green Fluorescent Protein)-based promoter-fusion bioassay. In total, one hundred and forty compounds were selected, purchased, and tested for biological activity. Several novel scaffolds showed acceptable potency to modulate the transcriptional function of PhoP, either by enhancing or inhibiting the expression of PhoP-dependent genes. These compounds may be used as the starting point for developing modulators that target the protein-protein interface of the PhoP protein as an alternative strategy against antibiotic resistance.

Enhancing the anti-biofilm activity of 5-aryl-2-aminoimidazoles through nature inspired dimerisation.

The increased tolerance of biofilms against disinfectants and antibiotics has stimulated research into new methods of biofilm prevention and eradication. In our previous work, we have identified the 5-aryl-2-aminoimidazole core as a scaffold that demonstrates preventive activity against biofilm formation of a broad range of bacterial and fungal species. Inspired by the dimeric nature of natural 2-aminoimidazoles of the oroidin family, we investigated the potential of dimers of our decorated 5-aryl-2-aminoimidazoles as biofilm inhibitors. A synthetic approach towards 2-aminoimidazole dimers linked by an alkyl chain was developed and a total of 48 dimers were synthesized. The linkers were introduced at two different positions, the N1-position or the N2-position, and the linker length and the substitution of the 5-phenyl ring (H, F, Cl, Br) were varied. Although, no clear correlation between linker length and biofilm inhibition was observed, a strong increase in anti-biofilm activity for almost all N1,N1'-linked dimers was obtained, compared to the respective monomers against Salmonella Typhimurium, Escherichia coli and Staphylococcus aureus. The N2,N2'-linked dimers, having a H- or F-substitution, were also found to show a strong increase in anti-biofilm activity compared to the respective monomers against these three bacterial species and against Pseudomonas aeruginosa. In addition, the obtained growth measurements suggest a broad concentration range with specific biofilm inhibition and no effect on the planktonic growth against Salmonella Typhimurium and Pseudomonas aeruginosa.

Meeting report on the ASM Conference on Mechanisms of Interbacterial Cooperation and Competition.

The ASM Conference on Mechanisms of Interbacterial Cooperation and Competition was held in Washington DC, from 1 to 4 March 2017. The conference provided an international forum for sociomicrobiologists from different disciplines to present and discuss new findings. The meeting covered a wide range of topics, spanning molecular mechanisms, ecology, evolution, computation and manipulation of interbacterial interactions, and encompassed social communities in medicine, the natural environment, and industry. This report summarizes the presentations and emerging themes.

Frequency-based haplotype reconstruction from deep sequencing data of bacterial populations.

Clonal populations accumulate mutations over time, resulting in different haplotypes. Deep sequencing of such a population in principle provides information to reconstruct these haplotypes and the frequency at which the haplotypes occur. However, this reconstruction is technically not trivial, especially not in clonal systems with a relatively low mutation frequency. The low number of segregating sites in those systems adds ambiguity to the haplotype phasing and thus obviates the reconstruction of genome-wide haplotypes based on sequence overlap information.Therefore, we present EVORhA, a haplotype reconstruction method that complements phasing information in the non-empty read overlap with the frequency estimations of inferred local haplotypes. As was shown with simulated data, as soon as read lengths and/or mutation rates become restrictive for state-of-the-art methods, the use of this additional frequency information allows EVORhA to still reliably reconstruct genome-wide haplotypes. On real data, we show the applicability of the method in reconstructing the population composition of evolved bacterial populations and in decomposing mixed bacterial infections from clinical samples.

FabR regulates Salmonella biofilm formation via its direct target FabB.

BACKGROUND: Biofilm formation is an important survival strategy of Salmonella in all environments. By mutant screening, we showed a knock-out mutant of fabR, encoding a repressor of unsaturated fatty acid biosynthesis (UFA), to have impaired biofilm formation. In order to unravel how this regulator impinges on Salmonella biofilm formation, we aimed at elucidating the S. Typhimurium FabR regulon. Hereto, we applied a combinatorial high-throughput approach, combining ChIP-chip with transcriptomics. RESULTS: All the previously identified E. coli FabR transcriptional target genes (fabA, fabB and yqfA) were shown to be direct S. Typhimurium FabR targets as well. As we found a fabB overexpressing strain to partly mimic the biofilm defect of the fabR mutant, the effect of FabR on biofilms can be attributed at least partly to FabB, which plays a key role in UFA biosynthesis. Additionally, ChIP-chip identified a number of novel direct FabR targets (the intergenic regions between hpaR/hpaG and ddg/ydfZ) and yet putative direct targets (i.a. genes involved in tRNA metabolism, ribosome synthesis and translation). Next to UFA biosynthesis, a number of these direct targets and other indirect targets identified by transcriptomics (e.g. ribosomal genes, ompA, ompC, ompX, osmB, osmC, sseI), could possibly contribute to the effect of FabR on biofilm formation. CONCLUSION: Overall, our results point at the importance of FabR and UFA biosynthesis in Salmonella biofilm formation and their role as potential targets for biofilm inhibitory strategies.

Modulation of the Substitution Pattern of 5-Aryl-2-Aminoimidazoles Allows Fine-Tuning of Their Antibiofilm Activity Spectrum and Toxicity.

We previously synthesized several series of compounds, based on the 5-aryl-2-aminoimidazole scaffold, that showed activity preventing the formation of Salmonella enterica serovar Typhimurium and Pseudomonas aeruginosa biofilms. Here, we further studied the activity spectrum of a number of the most active N1- and 2N-substituted 5-aryl-2-aminoimidazoles against a broad panel of biofilms formed by monospecies and mixed species of bacteria and fungi. An N1-substituted compound showed very strong activity against the biofilms formed by Gram-negative and Gram-positive bacteria and the fungus Candida albicans but was previously shown to be toxic against various eukaryotic cell lines. In contrast, 2N-substituted compounds were nontoxic and active against biofilms formed by Gram-negative bacteria and C. albicans but had reduced activity against biofilms formed by Gram-positive bacteria. In an attempt to develop nontoxic compounds with potent activity against biofilms formed by Gram-positive bacteria for application in antibiofilm coatings for medical implants, we synthesized novel compounds with substituents at both the N1 and 2N positions and tested these compounds for antibiofilm activity and toxicity. Interestingly, most of these N1-,2N-disubstituted 5-aryl-2-aminoimidazoles showed very strong activity against biofilms formed by Gram-positive bacteria and C. albicans in various setups with biofilms formed by monospecies and mixed species but lost activity against biofilms formed by Gram-negative bacteria. In light of application of these compounds as anti-infective coatings on orthopedic implants, toxicity against two bone cell lines and the functionality of these cells were tested. The N1-,2N-disubstituted 5-aryl-2-aminoimidazoles in general did not affect the viability of bone cells and even induced calcium deposition. This indicates that modulating the substitution pattern on positions N1 and 2N of the 5-aryl-2-aminoimidazole scaffold allows fine-tuning of both the antibiofilm activity spectrum and toxicity.

Gene expression variability in clonal populations: Causes and consequences.

During the last decade it has been shown that among cell variation in gene expression plays an important role within clonal populations. Here, we provide an overview of the different mechanisms contributing to gene expression variability in clonal populations. These are ranging from inherent variations in the biochemical process of gene expression itself, such as intrinsic noise, extrinsic noise and bistability to individual responses to variations in the local micro-environment, a phenomenon called phenotypic plasticity. Also genotypic variations caused by clonal evolution and phase variation can contribute to gene expression variability. Consequently, gene expression studies need to take these fluctuations in expression into account. However, frequently used techniques for expression quantification, such as microarrays, RNA sequencing, quantitative PCR and gene reporter fusions classically determine the population average of gene expression. Here, we discuss how these techniques can be adapted towards single cell analysis by integration with single cell isolation, RNA amplification and microscopy. Alternatively more qualitative selection-based techniques, such as mutant screenings, in vivo expression technology (IVET) and recombination-based IVET (RIVET) can be applied for detection of genes expressed only within a subpopulation. Finally, differential fluorescence induction (DFI), a protocol specially designed for single cell expression is discussed.

Evaluation of the antibacterial and antibiofilm activities of novel CRAMP-vancomycin conjugates with diverse linkers.

We report the design, synthesis and antibacterial activity analysis of conjugates of vancomycin and cathelicidin-related antimicrobial peptides (CRAMP). Vancomycin inhibits the nascent peptidoglycan synthesis and is highly active against Gram-positive bacteria, whereas Gram-negative bacteria are generally insensitive due to a protective outer membrane. CRAMP is known to translocate across the Gram-negative outer membrane by a self-promoted uptake mechanism. Vancomycin-CRAMP conjugates were synthesized using click chemistry with diverse hydrophilic and hydrophobic linkers, with CRAMP functioning as a carrier peptide for the transfer of vancomycin through the outer membrane. Small hydrophobic linkers with an aromatic group result in the most active conjugates against planktonic Gram-negative bacteria, while maintaining the high activity of vancomycin against Gram-positive bacteria. These conjugates thus show a broad-spectrum activity, which is absent in CRAMP or vancomycin alone, and which is strongly improved compared to an equimolar mixture of CRAMP and vancomycin. In addition, these conjugates also show a strong inhibitory activity against S. Typhimurium biofilm formation.

Derivatives of the mouse cathelicidin-related antimicrobial peptide (CRAMP) inhibit fungal and bacterial biofilm formation.

We identified a 26-amino-acid truncated form of the 34-amino-acid cathelicidin-related antimicrobial peptide (CRAMP) in the islets of Langerhans of the murine pancreas. This peptide, P318, shares 67% identity with the LL-37 human antimicrobial peptide. As LL-37 displays antimicrobial and antibiofilm activity, we tested antifungal and antibiofilm activity of P318 against the fungal pathogen Candida albicans. P318 shows biofilm-specific activity as it inhibits C. albicans biofilm formation at 0.15 µM without affecting planktonic survival at that concentration. Next, we tested the C. albicans biofilm-inhibitory activity of a series of truncated and alanine-substituted derivatives of P318. Based on the biofilm-inhibitory activity of these derivatives and the length of the peptides, we decided to synthesize the shortened alanine-substituted peptide at position 10 (AS10; KLKKIAQKIKNFFQKLVP). AS10 inhibited C. albicans biofilm formation at 0.22 µM and acted synergistically with amphotericin B and caspofungin against mature biofilms. AS10 also inhibited biofilm formation of different bacteria as well as of fungi and bacteria in a mixed biofilm. In addition, AS10 does not affect the viability or functionality of different cell types involved in osseointegration of an implant, pointing to the potential of AS10 for further development as a lead peptide to coat implants.