Lindsay Kalan

Antibiotic resistance is ancient

The discovery of antibiotics more than 70 years ago initiated a period of drug innovation and implementation in human and animal health and agriculture. These discoveries were tempered in all cases by the emergence of resistant microbes. This history has been interpreted to mean that antibiotic resistance in pathogenic bacteria is a modern phenomenon; this view is reinforced by the fact that collections of microbes that predate the antibiotic era are highly susceptible to antibiotics. Here we report targeted metagenomic analyses of rigorously authenticated ancient DNA from 30,000-year-old Beringian permafrost sediments and the identification of a highly diverse collection of genes encoding resistance to β-lactam, tetracycline and glycopeptide antibiotics. Structure and function studies on the complete vancomycin resistance element VanA confirmed its similarity to modern variants. These results show conclusively that antibiotic resistance is a natural phenomenon that predates the modern selective pressure of clinical antibiotic use.

The Comprehensive Antibiotic Resistance Database

ABSTRACT The field of antibiotic drug discovery and the monitoring of new antibiotic resistance elements have yet to fully exploit the power of the genome revolution. Despite the fact that the first genomes sequenced of free living organisms were those of bacteria, there have been few specialized bioinformatic tools developed to mine the growing amount of genomic data associated with pathogens. In particular, there are few tools to study the genetics and genomics of antibiotic resistance and how it impacts bacterial populations, ecology, and the clinic. We have initiated development of such tools in the form of the Comprehensive Antibiotic Research Database (CARD; http://arpcard.mcmaster.ca). The CARD integrates disparate molecular and sequence data, provides a unique organizing principle in the form of the Antibiotic Resistance Ontology (ARO), and can quickly identify putative antibiotic resistance genes in new unannotated genome sequences. This unique platform provides an informatic tool that bridges antibiotic resistance concerns in health care, agriculture, and the environment.

Antibiotic adjuvants: multicomponent anti-infective strategies

The unremitting emergence of multidrug-resistant bacterial pathogens highlights a matching need for new therapeutic options. For example, new carbapenemases such as KPC (class A Klebsiella pneumoniae) and NDM-1 (New Delhi metallo-β-lactamase 1) are surfacing, resulting in almost total resistance to β-lactam antibiotics. Furthermore, resistance is quickly disseminated, not only in the healthcare sector, but also within the community at large, because many resistance determinants are carried on mobile genetic elements readily shared among pathogens. The absence of new antibiotics has led to a growing reliance on older, more toxic drugs such as colistin, but resistance to these is already arising. One approach to combat this growing problem is the use of combination drug antibiotic adjuvant therapy, which potentiates the activity of antibiotics. Here, we review the current situation and discuss potential drug combinations that may increase the potency of antibiotics in the future. Adjuvant therapies include antibiotic combinations, synergy between antibiotics and nonantibiotics, inhibition of resistance and molecules that alter the physiology of antibiotic-insensitive cells, such as those in biofilms. We provide a rationale for these multicomponent strategies, highlighting current research and important considerations for their clinical use and pharmacological properties.

Redefining the Chronic-Wound Microbiome: Fungal Communities Are Prevalent, Dynamic, and Associated with Delayed Healing

ABSTRACT Chronic nonhealing wounds have been heralded as a silent epidemic, causing significant morbidity and mortality especially in elderly, diabetic, and obese populations. Polymicrobial biofilms in the wound bed are hypothesized to disrupt the highly coordinated and sequential events of cutaneous healing. Both culture-dependent and -independent studies of the chronic-wound microbiome have almost exclusively focused on bacteria, omitting what we hypothesize are important fungal contributions to impaired healing and the development of complications. Here we show for the first time that fungal communities (the mycobiome) in chronic wounds are predictive of healing time, associated with poor outcomes, and form mixed fungal-bacterial biofilms. We longitudinally profiled 100, nonhealing diabetic-foot ulcers with high-throughput sequencing of the pan-fungal internal transcribed spacer 1 (ITS1) locus, estimating that up to 80% of wounds contain fungi, whereas cultures performed in parallel captured only 5% of colonized wounds. The “mycobiome” was highly heterogeneous over time and between subjects. Fungal diversity increased with antibiotic administration and onset of a clinical complication. The proportions of the phylum Ascomycota were significantly greater (P = 0.015) at the beginning of the study in wounds that took >8 weeks to heal. Wound necrosis was distinctly associated with pathogenic fungal species, while taxa identified as allergenic filamentous fungi were associated with low levels of systemic inflammation. Directed culturing of wounds stably colonized by pathogens revealed that interkingdom biofilms formed between yeasts and coisolated bacteria. Combined, our analyses provide enhanced resolution of the mycobiome during impaired wound healing, its role in chronic disease, and impact on clinical outcomes. IMPORTANCE Wounds are an underappreciated but serious complication for a diverse spectrum of diseases. High-risk groups, such as persons with diabetes, have a 25% lifetime risk of developing a wound that can become chronic. The majority of microbiome research related to chronic wounds is focused on bacteria, but the association of fungi with clinical outcomes remains to be elucidated. Here we describe the dynamic fungal communities in 100 diabetic patients with foot ulcers. We found that communities are unstable over time, but at the first clinical presentation, the relative proportions of different phyla predict healing times. Pathogenic fungi not identified by culture reside in necrotic wounds and are associated with a poor prognosis. In wounds stably colonized by fungi, we identified yeasts capable of forming biofilms in concert with bacteria. Our findings illuminate the associations of the fungal mycobiome with wound prognosis and healing. Wounds are an underappreciated but serious complication for a diverse spectrum of diseases. High-risk groups, such as persons with diabetes, have a 25% lifetime risk of developing a wound that can become chronic. The majority of microbiome research related to chronic wounds is focused on bacteria, but the association of fungi with clinical outcomes remains to be elucidated. Here we describe the dynamic fungal communities in 100 diabetic patients with foot ulcers. We found that communities are unstable over time, but at the first clinical presentation, the relative proportions of different phyla predict healing times. Pathogenic fungi not identified by culture reside in necrotic wounds and are associated with a poor prognosis. In wounds stably colonized by fungi, we identified yeasts capable of forming biofilms in concert with bacteria. Our findings illuminate the associations of the fungal mycobiome with wound prognosis and healing.

Temporal Stability in Chronic Wound Microbiota Is Associated With Poor Healing

Microbial burden of chronic wounds is believed to play an important role in impaired healing and the development of infection-related complications. However, clinical cultures have little predictive value of wound outcomes, and culture-independent studies have been limited by cross-sectional design and small cohort size. We systematically evaluated the temporal dynamics of the microbiota colonizing diabetic foot ulcers, a common and costly complication of diabetes, and its association with healing and clinical complications. Dirichlet multinomial mixture modeling, Markov chain analysis, and mixed-effect models were used to investigate shifts in the microbiota over time and their associations with healing. Here we show, to our knowledge, previously unreported temporal dynamics of the chronic wound microbiome. Microbiota community instability was associated with faster healing and improved outcomes. Diabetic foot ulcer microbiota were found to exist in one of four community types that experienced frequent and nonrandom transitions. Transition patterns and frequencies were associated with healing time. Exposure to systemic antibiotics destabilized the wound microbiota, rather than altering overall diversity or relative abundance of specific taxa. This study provides evidence that the dynamic wound microbiome is indicative of clinical outcomes and may be a valuable guide for personalized management and treatment of chronic wounds.

Outbreak of Vancomycin-Susceptible Enterococcus faecium Containing the Wild-Type vanA Gene

ABSTRACT Accurate detection of vancomycin-resistant enterococci (VRE) is essential in preventing transmission in health care settings. Chromogenic media are widely used for screening VRE because of fast turnaround times (TAT) and high sensitivity. We report an outbreak of Enterococcus faecium bearing vanA yet susceptible to vancomycin (vancomycin-variable Enterococcus [VVE]). Between October 2009 to March 2011, clinical and screening specimens (n = 14,747) were screened for VRE using VRE-selective medium and/or PCR. VVE isolates were genotyped to determine relatedness. Plasmids from these isolates were characterized by sequencing. Overall, 52 VVE isolates were identified, comprising 15% of all VRE isolates identified. Isolates demonstrated growth on Brilliance VRE agar (Oxoid) at 24 h of incubation but did not grow on brain heart infusion agar with 6 μg/ml vancomycin (Oxoid) or bile esculin azide agar with 6 μg/ml vancomycin (Oxoid) and were susceptible to vancomycin. Genotyping of 20 randomly selected VVE isolates revealed that 15/20 were identical, while 5 were highly related. PCR of the VVE transposon confirmed the presence of vanHAXY gene cluster; however, vanS (sensor) and vanR (regulator) genes were absent. The outbreak was controlled through routine infection control measures. We report an emergence of a fit strain of E. faecium containing vanA yet susceptible to vancomycin. Whether this new strain represents VRE has yet to be determined; however, unique testing procedures are required for reliable identification of VVE.

Silver oxynitrate: An Unexplored Silver Compound with Antimicrobial and Antibiofilm Activity

ABSTRACT Historically it has been accepted, and recent research has established, that silver (Ag) is an efficacious antimicrobial agent. A dwindling pipeline of new antibiotics, combined with an increase in the number of antibiotic-resistant infections, is bringing Ag to the fore as a therapeutic compound to treat infectious diseases. Currently, many formulations of Ag are being deployed for commercial and medical purposes, with various degrees of effectiveness at killing microbial cells. Here, we evaluated the antimicrobial and antibiofilm capacity of our lead compound, silver oxynitrate [Ag(Ag3O4)2NO3 or Ag7NO11], against other metal compounds with documented antimicrobial activity, including Ag2SO4, AgNO3, silver sulfadiazine (AgSD), AgO, Ag2O, and CuSO4. Our findings reveal that Ag7NO11 eradicates biofilm and planktonic populations of Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, uropathogenic Escherichia coli (UPEC), fluoroquinolone-resistant Pseudomonas aeruginosa (FQRP), and methicillin-resistant Staphylococcus aureus (MRSA) at lower concentrations than those of the other tested metal salts. Altogether, our results demonstrate that Ag7NO11 has an enhanced efficacy for the treatment of biofilm-forming pathogens.

Harnessing the Synthetic Capabilities of Glycopeptide Antibiotic Tailoring Enzymes: Characterization of the UK-68,597 Biosynthetic Cluster

In this study, a draft genome sequence of Actinoplanes sp. ATCC 53533 was assembled, and an 81‐kb biosynthetic cluster for the unusual sulfated glycopeptide UK‐68,597 was identified. Glycopeptide antibiotics are important in the treatment of infections caused by Gram‐positive bacteria. Glycopeptides contain heptapeptide backbones that are modified by many tailoring enzymes, including glycosyltransferases, sulfotransferases, methyltransferases, and halogenases, generating extensive chemical and functional diversity. Several tailoring enzymes in the cluster were examined in vitro for their ability to modify glycopeptides, resulting in the synthesis of novel molecules. Tailoring enzymes were also expressed in the producer of the glycopeptide aglycone A47934, generating additional chemical diversity. This work characterizes the biosynthetic program of UK‐68,597 and demonstrates the capacity to expand glycopeptide chemical diversity by harnessing the unique chemistry of tailoring enzymes.

Vancomycin-Variable Enterococci Can Give Rise to Constitutive Resistance during Antibiotic Therapy

ABSTRACT Vancomycin-resistant enterococci (VRE) are notorious clinical pathogens restricting the use of glycopeptide antibiotics in the clinic setting. Routine surveillance to detect VRE isolated from patients relies on PCR bioassays and chromogenic agar-based test methods. In recent years, we and others have reported the emergence of enterococcal strains harboring a “silent” copy of vancomycin resistance genes that confer a vancomycin-susceptible phenotype (vancomycin-susceptible enterococci [VSE]) and thus escape detection using drug sensitivity screening tests. Alarmingly, these strains are able to convert to a resistance phenotype (VSE→VRE) during antibiotic treatment, severely compromising the success of therapy. Such strains have been termed vancomycin-variable enterococci (VVE). We have investigated the molecular mechanisms leading to the restoration of resistance in VVE isolates through the whole-genome sequencing of resistant isolates, measurement of resistance gene expression, and quantification of the accumulation of drug-resistant peptidoglycan precursors. The results demonstrate that VVE strains can revert to a VRE phenotype through the constitutive expression of the vancomycin resistance cassette. This is accomplished through a variety of changes in the DNA region upstream of the resistance genes that includes both a deletion of a likely transcription inhibitory secondary structure and the introduction of a new unregulated promoter. The VSE→VRE transition of VVE can occur in patients during the course of antibiotic therapy, resulting in treatment failure. These VVE strains therefore pose a new challenge to the current regimen of diagnostic tests used for VRE detection in the clinic setting.

Glycopeptide Sulfation Evades Resistance

The incidence of antibiotic resistance among pathogenic microorganisms is increasing at an alarming rate. Resistance against front-line therapeutics such as the glycopeptide antibiotic vancomycin has emerged and has spread to highly virulent pathogens, including Staphylococcus aureus. Glycopeptide antibiotics are natural products from the Actinomycetes that have a characteristic heptapeptide core. The chemical diversity of the class is achieved through glycosylation, halogenation, methylation, and acylation of the core, modifications that are implicated in improved solubility, stability, or activity of the molecule. Sulfation is yet another modification observed infrequently in glycopeptides, but its role is not known. Although glycopeptide sulfotransferases are found in the environmental metagenome and must therefore serve an evolutionary purpose, all previous studies have reported decreased antibiotic activity with sulfation. We report that sulfation of glycopeptides has little effect on the compounds ability to bind its target, the d-Ala-d-Ala peptidoglycan precursors of the bacterial cell wall. However, sulfation does impact glycopeptide dimerization, and importantly, sulfated glycopeptides are significantly less potent inducers of the resistance gene cluster vanHAX in actinomycetes. Our results begin to unravel the mystery of the biological role of glycopeptide sulfation and offer a potential new strategy for the development of new antibiotics that avoid resistance.