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.

Selective pressures for public antibiotic resistance.

The rapid increase of antibiotic-resistant pathogens is severely limiting our current treatment possibilities. An important subset of the resistance mechanisms conferring antibiotic resistance have public effects, allowing otherwise susceptible bacteria to also survive antibiotic treatment. As susceptible bacteria can survive treatment without bearing the metabolic cost of producing the resistance mechanism, there is potential to increase their relative frequency in the population and, as such, select against resistant bacteria. Multiple studies showed that this altered selection for resistance is dependent on various environmental and treatment parameters. In this review, we provide a comprehensive overview of their most important findings and describe the main factors impacting the selection for resistance. In-depth understanding of the driving forces behind selection can aid in the design and implementation of alternative treatments which limit the risk of 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.

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.

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.

Identification and characterization of an anti-pseudomonal dichlorocarbazol derivative displaying anti-biofilm activity.

Pseudomonas aeruginosa strains resistant towards all currently available antibiotics are increasingly encountered, raising the need for new anti-pseudomonal drugs. We therefore conducted a medium-throughput screen of a small-molecule collection resulting in the identification of the N-alkylated 3,6-dihalogenocarbazol 1-(sec-butylamino)-3-(3,6-dichloro-9H-carbazol-9-yl)propan-2-ol (MIC = 18.5 µg mL⁻¹). This compound, compound 1, is bacteriostatic towards a broad spectrum of Gram-positive and Gram-negative pathogens, including P. aeruginosa. Importantly, 1 also eradicates mature biofilms of P. aeruginosa. 1 displays no cytotoxicity against various human cell types, pointing to its potential for further development as a novel antibacterial drug.

Competition quenching strategies reduce antibiotic tolerance in polymicrobial biofilms.

Bacteria typically live in dense communities where they are surrounded by other species and compete for a limited amount of resources. These competitive interactions can induce defensive responses that also protect against antimicrobials, potentially complicating the antimicrobial treatment of pathogens residing in polymicrobial consortia. Therefore, we evaluate the potential of alternative antivirulence strategies that quench this response to competition. We test three competition quenching approaches: (i) interference with the attack mechanism of surrounding competitors, (ii) inhibition of the stress response systems that detect competition, and (iii) reduction of the overall level of competition in the community by lowering the population density. We show that either strategy can prevent the induction of antimicrobial tolerance of Salmonella Typhimurium in response to competitors. Competition quenching strategies can thus reduce tolerance of pathogens residing in polymicrobial communities and could contribute to the improved eradication of these pathogens via traditional methods.

Small RNAs regulating biofilm formation and outer membrane homeostasis.

Nowadays, the identification of small non-coding RNAs takes a prominent role in deciphering complex bacterial phenotypes. Evidences are given that the post-transcriptional layer of regulation mediated by sRNAs plays an important role in the formation of bacterial biofilms. These sRNAs exert their activity on various targets, be it directly or indirectly linked to biofilm formation. First, and best described, are the sRNAs that act in core regulatory pathways of biofilm formation, such as those regulating motility and matrix production. Second, overlaps between the regulation of biofilm formation and the outer membrane (OM) are becoming obvious. Additionally, different studies indicate that defects in the OM itself affect biofilm formation through this shared cascade, thereby forming a feedback mechanism. Interestingly, it is known that the OM itself is extensively regulated by different sRNAs. Third, biofilms are also linked to global metabolic changes. There is also evidence that metabolic pathways and the process of biofilm formation share sRNAs.

Anomalous diffusion of nanoparticles in the spatially heterogeneous biofilm environment.

Biofilms contain extracellular polymeric substances (EPS) that provide structural support and restrict penetration of antimicrobial treatment. To overcome limited penetration, functionalized nanoparticles (NPs) have been suggested as carriers for antimicrobial delivery. Using microscopy, we evaluate the diffusion of nanoparticles in function of the structure of Salmonella biofilms. We observe anomalous diffusion and heterogeneous mobility of NPs resulting in distinct NPs distribution that depended on biofilm structure. Through Brownian dynamics modeling with spatially varying viscosity around bacteria, we demonstrated that spatial gradients in diffusivity generate viscous sinks that trap NPs near bacteria. This model replicates the characteristic diffusion signature and vertical distribution of NPs in the biofilm. From a treatment perspective, our work indicates that both biofilm structure and the level of EPS can impact NP drug delivery, where low levels of EPS might benefit delivery by immobilizing NPs closer to bacteria and higher levels hamper delivery due to shielding effects.

Evolution-proof inhibitors of public good cooperation: a screening strategy inspired by social evolution theory.

Interference with public good cooperation provides a promising novel antimicrobial strategy since social evolution theory predicts that resistant mutants will be counter-selected if they share the public benefits of their resistance with sensitive cells in the population. Although this hypothesis is supported by a limited number of pioneering studies, an extensive body of more fundamental work on social evolution describes a multitude of mechanisms and conditions that can stabilize public behaviour, thus potentially allowing resistant mutants to thrive. In this paper we theorize on how these different mechanisms can influence the evolution of resistance against public good inhibitors. Based hereon, we propose an innovative 5-step screening strategy to identify novel evolution-proof public good inhibitors, which involves a systematic evaluation of the exploitability of public goods under the most relevant experimental conditions, as well as a careful assessment of the most optimal way to interfere with their action. Overall, this opinion paper is aimed to contribute to long-term solutions to fight bacterial infections.

Origanum vulgare ethanolic extracts as a promising source of compounds with antimicrobial, anti-biofilm, and anti-virulence activity against dental plaque bacteria.

Dental caries and periodontal diseases remain a challenge for oral health, especially given the lack of effective and safe treatment options that are currently available. Against the backdrop of an ongoing antimicrobial resistance crisis, a renewed interest in traditional medicinal plants as a potential source of new bioactive compounds has surfaced. In this context, we systematically screened the antimicrobial and anti-biofilm activities of both ethanolic and aqueous extracts of nine Algerian medicinal plants (Artemisia herba alba, Centaurium erythraea, Juglans regia, Laurus nobilis, Matricaria recutita, Mentha pulegium, Mentha piperita, Origanum vulgare and Taraxacum officinale). To evaluate the activity spectrum of the extracts, the screening was carried out against an extensive collection of Streptococcus, Enterococcus and Lacticaseibacillus isolates recovered from dental plaques of Algerian patients. Broad-spectrum antimicrobial and anti-biofilm properties were observed, especially among ethanolic extracts, which marks them as a promising source for bioactive compounds to control oral biofilms. The ethanolic extract of O. vulgare, which showed the most promising effects in the initial screening, was further characterized. We first verified the biocompatibility of this extract using human oral keratinocytes and selected a range of non-cytotoxic concentrations (0.195-0.781 mg/ml) to further validate its anti-biofilm and anti-virulence potential. At these concentrations, the extract not only prevented biofilm formation (10.04 ± 0.75-87.91 ± 9.08% of reduction) of most dental plaque isolates on a polystyrene surface, but also significantly reduced their adherence to hydroxyapatite (34.58 ± 9.09-62.77 ± 0.95%). Moreover, the extract showed curative potential against mature biofilms grown under conditions mimicking the oral niche. In addition to its anti-biofilm properties, we observed an inhibition of glucosyltransferase activity, a reduction in acidogenesis and a downregulation in the expression of multiple virulence-associated genes for extract-treated samples. Since anti-virulence properties are more robust to the development of resistance, they provide an attractive complementation to the antimicrobial activities of the extract. Thymol was identified as an important active compound of the extract using GC-MS analysis, but synergy with other compounds was also detected, suggesting a potential advantage of using the whole extract over purified thymol. Further research into the bioactive compounds of the O. vulgare ethanolic extract could yield novel products to fight dental caries.

Effect of chemical modifications of tannins on their antimicrobial and antibiofilm effect against Gram-negative and Gram-positive bacteria.

Background: Tannins have demonstrated antibacterial and antibiofilm activity, but there are still unknown aspects on how the chemical properties of tannins affect their biological properties. We are interested in understanding how to modulate the antibiofilm activity of tannins and in delineating the relationship between chemical determinants and antibiofilm activity. Materials and methods: The effect of five different naturally acquired tannins and their chemical derivatives on biofilm formation and planktonic growth of Salmonella Typhimurium, Pseudomonas aeruginosa, Escherichia coli and Staphylococcus aureus was determined in the Calgary biofilm device. Results: Most of the unmodified tannins exhibited specific antibiofilm activity against the assayed bacteria. The chemical modifications were found to alter the antibiofilm activity level and spectrum of the tannins. A positive charge introduced by derivatization with higher amounts of ammonium groups shifted the anti-biofilm spectrum toward Gram-negative bacteria, and derivatization with lower amounts of ammonium groups and acidifying derivatization shifted the spectrum toward Gram-positive bacteria. Furthermore, the quantity of phenolic OH-groups per molecule was found to have a weak impact on the anti-biofilm activity of the tannins. Conclusion: We were able to modulate the antibiofilm activity of several tannins by specific chemical modifications, providing a first approach for fine tuning of their activity and antibacterial spectrum.