Article

Halogenated quinolines bearing polar functionality at the 2-position: Identification of new antibacterial agents with enhanced activity against Staphylococcus epidermidis

Authors:
To read the full-text of this research, you can request a copy directly from the authors.

Abstract

Antibiotic-resistant bacteria and surface-attached biofilms continue to play a significant role in human health and disease. Innovative strategies are needed to identify new therapeutic leads to tackle infections of drug-resistant and tolerant bacteria. We synthesized a focused library of 14 new halogenated quinolines to investigate the impact of ClogP values on antibacterial and biofilm-eradication activities. During these investigations, we found select polar appendages at the 2-position of the HQ scaffold were more well-tolerated than others. We were delighted to see multiple compounds display enhanced activities against the major human pathogen S. epidermidis. In particular, HQ 2 (ClogP = 3.44) demonstrated enhanced activities against MRSE 35984 planktonic cells (MIC = 0.59 μM) compared to MRSA and VRE strains in addition to potent MRSE biofilm eradication activities (MBEC = 2.35 μM). Several of the halogenated quinolines identified here reported low cytotoxicity against HeLa cells with minimal hemolytic activity against red blood cells. We believe that halogenated quinoline small molecules could play an important role in the development of next-generation antibacterial therapeutics capable of targeting and eradicating biofilm-associated infections.

No full-text available

Request Full-text Paper PDF

To read the full-text of this research,
you can request a copy directly from the authors.

... Synthetically tunable antibacterial agents that operate through unique modes of action to target antibiotic-resistant and tolerant bacteria are of importance to human health. [1][2][3][4] Pathogenic bacteria present 2 distinct clinical problems: (1) acquired antibiotic resistance and (2) innate antibiotic tolerance. Bacteria acquire resistance to conventional antibiotics during therapy using one or more well-defined mechanism(s), including point mutations in antibiotic targets, enzyme-mediated antibiotic inactivation/degradation, and changes in membrane chemistry to impede drug penetration. ...
... 2,14 Our third-generation HQ series was motivated to probe the effects that water-solubilizing moieties at the 2-position of the HQ scaffold had on antibacterial activities. 4 The most potent second-generation HQ analogues had ClogP values of ⩾4.6 and we wanted to design analogues with reduced ClogP values to improve the therapeutic potential of these compounds. With that, we designed a series of focused HQ analogues to incorporate chemical functionality to allow more favorable hydrogenbond interactions with water. ...
... Following the chemical synthesis of 14 new HQs bearing focused appendages at the 2-position to enhance water solubility, we evaluated these analogues against a panel of drugresistant strains, including MRSA ATCC 1707, MRSE ATCC 35984, and VRE ATCC 700221. 4 We were interested to find 4 new HQ analogues from this collection (1, From these investigations, we have identified new HQ analogues with potent and enhanced antibacterial activities against methicillin-resistant S epidermidis 35984. These findings are indeed timely, as S epidermidis has emerged as an opportunistic pathogen in health care-associated infections in patients with indwelling medical devices. ...
Article
Full-text available
Antibiotic-resistant bacteria and surface-attached bacterial biofilms play a significant role in human disease. Conventional antibiotics target actively replicating free-floating, planktonic cells. Unfortunately, biofilm communities are endowed with nonreplicating persister cells that are tolerant to antibiotics. Innovative approaches are necessary to identify new molecules able to eradicate resistant and tolerant bacterial cells. Our group has discovered that select halogenated quinolines (HQs) can eradicate drug-resistant, gram-positive bacterial pathogens and their corresponding biofilms. Interestingly, the HQ scaffold is synthetically tunable and we have discovered unique antibacterial profiles through extensive analogue synthesis and microbiologic studies. We recently reported the synthesis of 14 new HQs to investigate the impact of ClogP values on antibacterial and biofilm eradication activities. We conducted diverse synthetic modifications at the 2-position of the HQ scaffold in an attempt to enhance water solubility and found new compounds that display enhanced activities against Staphylococcus epidermidis. In particular, HQ 2 (ClogP = 3.44) demonstrated more potent antibacterial activities against methicillin-resistant S epidermidis (MRSE) 35984 planktonic cells (minimum inhibitory concentration = 0.59 µM) compared with methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus isolates while demonstrating potent MRSE biofilm eradication activities (minimum biofilm eradication concentration = 2.35 µM). We believe that HQ could play a critical role in the development of next-generation antibacterial therapeutics.
... The short alkyl chains like ethyl and propyl have also performed well in comparison with long or heteroalkyl chains. 96 Interestingly, the PBT2 compound (18k), which has been studied for the treatment of Alzheimer's 97 and Huntington's diseases, 98 has shown activity against erythromycin-resistant group A Streptococcus (GAS), MRSA, and VRE in the presence of zinc, and this combination did not show cytotoxicity in human primary tonsil epithelial cells. In Streptococcus uberis, 18k seems to act in two different ways: altering the metal ion homeostasis and impairing the redox system. ...
... In general, dihalogenated 8HQ derivatives substituted at position 2 exhibited excellent activity against MRSA, MRSE, and VRE, with MIC values in the low micromolar range. 93,94,96 Vancomycin resistant Enterococcus and methicillin resistant S. aureus are present in the WHO priority list for research and development of new antibiotics. 1,3 Interestingly, these derivatives were also capable of eradicating the biofilm associated with these bacterial strains, which represents a great advance since biofilms are inherently resistant to conventional antimicrobial agents and are involved in a large number of chronic infections. ...
... One major group of biofilm inhibitors, inspired by scaffolds found in natural products with reported activity, are a diverse range of heterocyclic containing families, which include indoles [64][65][66][67][68][69][70][71], 2-aminoimidazoles [72][73][74][75][76][77][78][79][80][81][82][83][84][85][86][87][88][89], phenazines [90][91][92][93][94][95][96][97][98], and quinolines [99][100][101][102][103][104][105][106]. These lead molecules are the results of significant medicinal chemistry effort to improve potency and other drug-like properties. ...
Article
Full-text available
Biofilms, the predominant growth mode of microorganisms, pose a significant risk to human health. The protective biofilm matrix, typically composed of exopolysaccharides, proteins, nucleic acids, and lipids, combined with biofilm-grown bacteria’s heterogenous physiology, leads to enhanced fitness and tolerance to traditional methods for treatment. There is a need to identify biofilm inhibitors using diverse approaches and targeting different stages of biofilm formation. This review discusses discovery strategies that successfully identified a wide range of inhibitors and the processes used to characterize their inhibition mechanism and further improvement. Additionally, we examine the structure–activity relationship (SAR) for some of these inhibitors to optimize inhibitor activity.
... However, substituting the nitrite with a bromine coupled with addition of a bromine at the 7-position proved effective only when a methyl group was stationed at the 2-position. Interestingly, previous studies evaluating halogenated quinoline against MRSE also identified the 2-position as a key component of the quinoline scaffold for enhanced activity [44]. However, it appears that compound 15, bearing a chlorinated phenol group attached via a nitrogen group, did not show effectiveness. ...
Article
Full-text available
Background: Mycoplasmas primarily cause respiratory or urogenital tract infections impacting avian, bovine, canine, caprine, murine, and reptilian hosts. In animal husbandry, mycoplasmas cause reduced feed-conversion, decreased egg production, arthritis, hypogalactia or agalactia, increased condemnations, culling, and mortality in some cases. Antibiotics reduce transmission and mitigate clinical signs; however, concerning levels of antibiotic resistance in Mycoplasma gallisepticum and M. capricolum isolates exist. To address these issues, we evaluated the minimum inhibitory concentrations (MICs) of halogenated phenazine and quinoline compounds, an N-arylated NH125 analogue, and triclosan against six representative veterinary mycoplasmas via microbroth or agar dilution methods. Thereafter, we evaluated the minimum bactericidal concentration (MBC) of efficacious drugs. Results: We identified several compounds with MICs ≤25 μM against M. pulmonis (n = 5), M. capricolum (n = 4), M. gallisepticum (n = 3), M. alligatoris (n = 3), M. agassizii (n = 2), and M. canis (n = 1). An N-arylated NH125 analogue, compound 21, served as the most efficacious, having a MIC ≤25 μM against all mycoplasmas tested, followed by two quinolines, nitroxoline (compound 12) and compound 20, which were effective against four and three mycoplasma type strains, respectively. Nitroxoline exhibited bactericidal activity among all susceptible mycoplasmas, and compound 21 exhibited bactericidal activity when the MBC was able to be determined. Conclusions: These findings highlight a number of promising agents from novel drug classes with potential applications to treat veterinary mycoplasma infections and present the opportunity to evaluate preliminary pharmacokinetic indices using M. pulmonis in rodents as an animal model of human infection.
... These studies demonstrated great potentials of numerous HQ derivatives as antibacterial agents and biofilm eradicators, so further investigations have been focused on development of the HQ analogues with improved water solubility while maintaining potent biofilm eradication properties against major human pathogens (Basak et al. 2018;Huigens 2018). Conducting diverse synthetic modification at the C(2)-position of the HQ scaffold in order to enhanced water solubility, a new compound that had lower ClogP value (3.44) than parent compound 7; ClogP ¼ 4.19) was developed. ...
Article
Infective diseases have become health threat of a global proportion due to appearance and spread of microorganisms resistant to majority of therapeutics currently used for their treatment. Therefore, there is a constant need for development of new antimicrobial agents, as well as novel therapeutic strategies. Quinolines and quinolones, isolated from plants, animals, and microorganisms, have demonstrated numerous biological activities such as antimicrobial, insecticidal, antiinflammatory, antiplatelet, and antitumor. For more than two centuries quinoline/quinolone moiety has been used as a scaffold for drug development and even today it represents an inexhaustible inspiration for design and development of novel semi-synthetic or synthetic agents exhibiting broad spectrum of bioactivities. The structural diversity of synthetized compounds provides high and selective activity attained through different mechanisms of action, as well as low toxicity on human cells. This review describes quinoline and quinolone derivatives with antibacterial, antifungal, anti-virulent, antiviral, and anti-parasitic activities with the focus on the last 10 years literature.
... It has been proposed that biofilm inhibitors should include a hydrophilic group with hydrogen bond donor (HBD) atoms and a lipophilic alkyl chain ( Figure 2). Among the hydrophilic heterocyclic templates that have been studied for the design of biofilm inhibitors [21], some quinolones are described to possess high bioactivity [22][23][24][25][26][27]. We have previously reported a direct route for the synthesis of 2-amino-4-quinolone (compound 1, Figure 2) and 2,3-diamino-4-quinoline (compound 2) as key synthons for the preparation of tricyclic compounds [28]. ...
Article
Full-text available
Infection from multidrug resistant bacteria has become a growing health concern worldwide, increasing the need for developing new antibacterial agents. Among the strategies that have been studied, biofilm inhibitors have acquired relevance as a potential source of drugs that could act as a complement for current and new antibacterial therapies. Based on the structure of 2-alkyl-3-hydroxy-4-quinolone and N-acylhomoserine lactone, molecules that act as mediators of quorum sensing and biofilm formation in Pseudomonas aeruginosa, we designed, prepared, and evaluated the biofilm inhibition properties of long chain amide derivatives of 2-amino-4-quinolone in Staphylococcus aureus and P. aeruginosa. All compounds had higher biofilm inhibition activity in P. aeruginosa than in S. aureus. Particularly, compounds with an alkyl chain of 12 carbons exhibited the highest inhibition of biofilm formation. Docking scores and molecular dynamics simulations of the complexes of the tested compounds within the active sites of proteins related to quorum sensing had good correlation with the experimental results, suggesting the diminution of biofilm formation induced by these compounds could be related to the inhibition of these proteins.
Article
Drug resistant bacteria pose a major health concern and affect a large section of global population. Antibacterial drug discovery has stagnated owing to multiple factors including unattractive returns for major pharmaceutical companies. Thus, discovery of effective antibacterial drugs against drug-resistant bacteria is an urgent unmet need affecting healthcare systems globally. In this study, fluorine-containing 2,3-diarylquinolines (4a-l) and non-fluorinated analog 4m were synthesized utilizing environmentally benign chemistry of arenediazonium salts and arynes for regioselective installation of aryl groups at C-2 and C-3 positions, respectively. In vitro antibacterial evaluation against various Gram-negative and Gram-positive bacteria revealed inhibitory activity of majority of these compounds against Gram-positive S. aureus ATCC 29213. Compounds 4e, 4i, 4j and 4l were most potent inhibitors with MIC values of 10.95−24.0 µM. None of the compounds inhibited Gram-negative bacteria. 4e, 4i and 4l also displayed low levels of cytotoxicity against Vero cells, therefore, offering high safety profiles. Importantly, 4e, 4i and 4l exhibited equipotent inhibition of Methicillin and Vancomycin-resistant S. aureus, rendering them potential hits for further development. Molecular docking studies with topoisomerase II DNA gyrase demonstrated significant interactions of these inhibitors with target protein, which provided valuable insights into their potent antibacterial activity.
Article
Natural products have historically been a rich source of diverse chemical matter with numerous biological activities, and have played an important role in drug discovery in many areas including infectious disease. Synthetic and medicinal chemistry have been, and continue to be, important tools to realize the potential of natural products as therapeutics and as chemical probes. The formation of biofilms by bacteria in an infection setting is a significant factor in the recalcitrance of many bacterial infections, conferring increased tolerance to many antibiotics and to the host immune response, and as yet there are no approved therapeutics for combatting biofilm-based bacterial infections. Small molecules that interfere with the ability of bacteria to form and maintain biofilms can overcome antibiotic tolerance conferred by the biofilm phenotype, and have the potential to form combination therapies with conventional antibiotics. Many natural products with anti-biofilm activity have been identified from plants, microbes, and marine life, including: elligic acid glycosides, hamamelitannin, carolacton, skyllamycins, promysalin, phenazines, bromoageliferin, flustramine C, meridianin D, and brominated furanones. Total synthesis and medicinal chemistry programs have facilitated structure confirmation, identification of critical structural motifs, better understanding of mechanistic pathways, and the development of more potent, more accessible, or more pharmacologically favorable derivatives of anti-biofilm natural products.
Article
Antibiotic‐resistant bacteria continue to play an important role in human health and disease. Inventive strategies are necessary to develop new therapeutic leads to challenge drug‐resistance problems. From this perception, new quinoline hybrids bearing bioactive pharmacophores were synthesized. The newly synthesized compounds were evaluated for their in vitro antibacterial activity against nine bacterial pathogenic strains. The results revealed that most compounds exhibited good antibacterial activities. Seven compounds (2b, 3b, 4, 6, 8b, and 9c,d) displayed enhanced activity against methicillin‐resistant Staphylococcus aureus compared to ampicillin. These compounds were subjected to an in vitro S. aureus DNA gyrase ATPase inhibition study, which revealed that compounds 8b, 9c, and 9d showed the highest inhibitory activity with IC50 values of 1.89, 2.73, and 2.14 μM, respectively, comparable to novobiocin (IC50, 1.636 μM). Compounds 2a–c, 3a, 7c, 9c,d, and 10a,b revealed half the potency of levofloxacin in inhibiting the growth of Pseudomonas aeruginosa. As an attempt to rationalize the observed antibacterial activity for the most active compounds 8b, 9c, and 9d, molecular docking in the ATP binding site of S. aureus gyrase B was performed using Glide. Such compounds could be considered as promising scaffolds for the development of new potent antibacterial agents. Compounds 8b, 9c, and 9d displayed promising activity against the Gram‐positive bacterium methicillin‐resistant Staphylococcus aureus (MRSA) in addition to S. aureus DNA gyrase inhibition activity. Among them, 2‐(6‐bromo‐2‐methylquinoline‐4‐carbonyl)‐N‐(p‐tolyl)hydrazine‐1‐carbothioamide (8b) showed the highest DNA gyrase inhibition (IC50 = 1.89 µM), whereas compounds 9c and 9d showed lower inhibitory activity (IC50 = 2.73 and 2.14 µM, respectively).
Article
Bacterial biofilms are surface‐attached communities of slow‐growing and non‐replicating persister cells that demonstrate high levels of antibiotic tolerance. Biofilms occur in nearly 80% of infections and present unique challenges to our current arsenal of antibiotic therapies, all of which were initially discovered for their abilities to target rapidly‐dividing, free‐floating planktonic bacteria. Bacterial biofilms are credited as the underlying cause of chronic and recurring bacterial infections. Innovative approaches are required to identify new small molecules that operate through bacterial growth‐independent mechanisms to effectively eradicate biofilms. One source of inspiration comes from within the lungs of young Cystic Fibrosis (CF) patients, who often endure persistent Staphylococcus aureus infections. As these CF patients age, Pseudomonas aeruginosa co‐infects the lungs and utilize phenazine antibiotics to eradicate the established S. aureus infection. Our group has taken a special interest in this microbial competition strategy and we are investigating the potential of phenazine antibiotic‐inspired compounds and synthetic analogues thereof to eradicate persistent bacterial biofilms. To discover new biofilm‐eradicating agents, we have established an interdisciplinary research program involving synthetic medicinal chemistry, microbiology and molecular biology. From these efforts, we have identified a series of halogenated phenazines (HP) that potently eradicate bacterial biofilms and future work aims to translate these preliminary findings into groundbreaking clinical advances for the treatment of persistent biofilm infections.
Article
A microwave‐assisted method for the synthesis of 8‐methylquinoline derivatives via Rh‐catalyzed C(sp3)‐H activation under solvent‐free condition is reported. In comparison with traditional method, this reaction proceeded more efficiently with excellent yield, a broad range substrate scope and good functional group tolerance. A microwave‐assisted method for the synthesis of 8‐methylquinoline derivatives via Rh‐catalyzed C(sp3)‐H activation under solvent‐free condition is reported. In comparison with traditional method, this reaction proceeded more efficiently with excellent yield (up to 95%), a broad range substrate scope and good functional group tolerance. Mechanistic studies revealed that the present reaction might involve an ortho‐C‐H bond cleavage which is likely the rate‐limiting step in this transformation.
Article
Full-text available
Bacterial cells are constantly exposed to environmental stress; for example, almost all cells must endure starvation, and antimicrobials, of course, are administered to kill bacteria. These stressed cells enter a resting state known as persistence in which they become tolerant to nearly all antibiotics without undergoing genetic change. These dormant cells survive courses of antibiotics, as antibiotics are most effective against actively metabolizing cells, and reconstitute infections. In humans, most of these bacterial infections occur in biofilms in which bacteria attach to one another via secreted proteins, polysaccharides and even DNA. Herein, biotechnological methods are described to combat persister cells and to eradicate biofilms by understanding the genetic basis of both phenomena.
Article
Full-text available
During microbial infection, antimicrobial peptides are utilized by the immune response to rapidly eradicate microbial pathogens through the destruction of cellular membranes. Inspired by antimicrobial peptides, quaternary ammonium cationic (QAC) compounds have emerged as agents capable of destroying bacterial membranes leading to rapid bacterial death, including the eradication of persistent, surface-attached bacterial biofilms. NH125, an imidazolium cation with a sixteen membered fatty tail, was recently reported to eradicate persister cells and was our starting point for the development of novel antimicrobial agents. Here, we describe the design, chemical synthesis and biological investigations of a collection of 30 diverse NH125 analogues which provided critical insights into structural features that are important for antimicrobial activities in this class. From these studies, multiple NH125 analogues were identified to possess potent antibacterial and antifungal activities, eradicate both bacterial and fungal biofilms and rapidly eradicate MRSA persister cells in stationary phase. NH125 analogues also demonstrated more rapid persister cell killing activities against MRSA when tested alongside a panel of diverse membrane-active agents, including BAC-16 and daptomycin. NH125 analogues could have a significant impact on persister- and biofilm-related problems in numerous biomedical applications.
Article
Full-text available
Unlike individual, free-floating planktonic bacteria, biofilms are surface-attached communities of slow- or non-replicating bacteria encased within a protective extracellular polymeric matrix enabling persistent bacterial populations to tolerate high concentrations of antimicrobials. Our current antibacterial arsenal is composed of growth-inhibiting agents that target rapidly-dividing planktonic bacteria but not metabolically dormant biofilm cells. We report the first modular synthesis of a library of 20 halogenated phenazines (HP), utilizing the Wohl-Aue reaction, that targets both planktonic and biofilm cells. New HPs, including 6-substituted analogues, demonstrate potent antibacterial activities against MRSA, MRSE and VRE (MIC = 0.003–0.78 µM). HPs bind metal(II) cations and demonstrate interesting activity profiles when co-treated in a panel of metal(II) cations in MIC assays. HP 1 inhibited RNA and protein biosynthesis while not inhibiting DNA biosynthesis using ³H-radiolabeled precursors in macromolecular synthesis inhibition assays against MRSA. New HPs reported here demonstrate potent eradication activities (MBEC = 0.59–9.38 µM) against MRSA, MRSE and VRE biofilms while showing minimal red blood cell lysis or cytotoxicity against HeLa cells. PEG-carbonate HPs 24 and 25 were found to have potent antibacterial activities with significantly improved water solubility. HP small molecules could have a dramatic impact on persistent, biofilm-associated bacterial infection treatments.
Article
Full-text available
Bacterial biofilms are surface-attached communities of slow- or non-replicating bacterial cells that display high levels of tolerance toward conventional antibiotic therapies. It is important to know that our entire arsenal of conventional antibiotics originated from screens used to identify inhibitors of bacterial growth, so it should be little surprise that our arsenal of growth-inhibiting agents have little effect on persistent biofilms. Despite this current state, a diverse collection of natural products and their related or inspired synthetic analogues are emerging that have the ability to kill persistent bacterial biofilms and persister cells in stationary cultures. Unlike conventional antibiotics that hit bacterial targets critical for rapidly-dividing bacteria (i.e., cell wall machinery, bacterial ribosomes), biofilm-eradicating agents operate through unique growth-independent mechanisms. These naturally occurring agents continue to inspire discovery efforts aimed at effectively treating chronic and recurring bacterial infections due to persistent bacterial biofilms.
Article
Full-text available
Herein, we disclose the development of a catalyst- and protecting-group-free microwave-enhanced Friedländer synthesis which permits the single-step, convergent assembly of diverse 8-hydroxyquinolines with greatly improved reaction yields over traditional oil bath heating (increased from 34% to 72%). This rapid synthesis permitted the discovery of novel biofilm-eradicating halogenated quinolines (MBECs = 1.0-23.5 μM) active against MRSA, MRSE, and VRE. These small molecules exhibit activity through mechanisms independent of membrane lysis, further demonstrating their potential as a clinically useful treatment option against persistent biofilm-associated infections.
Article
Full-text available
Persistent bacteria, including persister cells within surface-attached biofilms and slow-growing pathogens lead to chronic infections that are tolerant to antibiotics. Here, we describe the structure-activity relationships of a series of halogenated phenazines (HP) inspired by 2-bromo-1-hydroxyphenazine 1. Using multiple synthetic pathways, we probed diverse substitutions of the HP scaffold in the 2-, 4-, 7- and 8-positions providing critical information regarding their antibacterial and bacterial eradication profiles. Halogenated phenazine 14 proved to be the most potent biofilm-eradicating agent (≥99.9% persister cell killing) against MRSA (MBEC < 10 µM), MRSE (MBEC = 2.35 µM) and VRE (MBEC = 0.20 µM) biofilms while 11 and 12 demonstrated excellent antibacterial activity against M. tuberculosis (MIC = 3.13 µM). Unlike antimicrobial peptide mimics that eradicate biofilms through the general lysing of membranes, HPs do not lyse red blood cells. HPs are promising agents that effectively target persistent bacteria while demonstrating negligible toxicity against mammalian cells.
Article
Full-text available
Small molecules capable of eradicating non-replicating bacterial biofilms are of great importance to human health as conventional antibiotics are ineffective against these surface-attached bacterial communities. Here, we report the discovery of several halogenated quinolines (HQs) identified through a reductive amination reaction that demonstrated potent eradication of MRSA (; MBEC = 125 μM), MRSE (; MBEC = 3.0 μM) and VRE (, and ; MBEC = 1.0 μM) biofilms. HQs were evaluated using the Calgary Biofilm Device (CBD) and demonstrated near equipotent killing activities against planktonic and biofilm cells based on MBC and MBEC values. When tested against red blood cells, these HQ analogues demonstrated low haemolytic activity (3 to 21% at 200 μM) thus we conclude that these HQ analogues do not operate primarily through the destruction of bacterial membranes, typical of other biofilm-eradicating agents (i.e., antimicrobial peptides). HQ antibacterial agents are potent biofilm-eradicating compounds and could lead to useful treatments for biofilm-associated bacterial infections.
Article
Full-text available
Batch culture of biofilms on peg lids is a versatile method that can be used for microtiter determinations of biofilm antimicrobial susceptibility. In this paper, we describe a core protocol and a set of parameters (surface composition, the rate of rocking or orbital motion, temperature, cultivation time, inoculum size, atmospheric gases and nutritional medium) that can be adjusted to grow single- or multispecies biofilms on peg surfaces. Mature biofilms formed on peg lids can then be fitted into microtiter plates containing test agents. After a suitable exposure time, biofilm cells are disrupted into a recovery medium using sonication. Microbicidal endpoints can be determined qualitatively using optical density measurements or quantitatively using viable cell counting. Once equipment is calibrated and growth conditions are at an optimum, the procedure requires approximately 5 h of work over 4-6 d. This efficient method allows antimicrobial agents and exposure conditions to be tested against biofilms on a high-throughput scale.
Article
Full-text available
Determination of the MIC, based on the activities of antibiotics against planktonic bacteria, is the standard assay for antibiotic susceptibility testing. Adherent bacterial populations (biofilms) present with an innate lack of antibiotic susceptibility not seen in the same bacteria grown as planktonic populations. The Calgary Biofilm Device (CBD) is described as a new technology for the rapid and reproducible assay of biofilm susceptibilities to antibiotics. The CBD produces 96 equivalent biofilms for the assay of antibiotic susceptibilities by the standard 96-well technology. Biofilm formation was followed by quantitative microbiology and scanning electron microscopy. Susceptibility to a standard group of antibiotics was determined for National Committee for Clinical Laboratory Standards (NCCLS) reference strains: Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, and Staphylococcus aureus ATCC 29213. Growth curves demonstrated that biofilms of a predetermined size could be formed on the CBD at specific time points and, furthermore, that no significant difference (P > 0.1) was seen between biofilms formed on each of the 96 pegs. The antibiotic susceptibilities for planktonic populations obtained by the NCCLS method or from the CBD were similar. Minimal biofilm eradication concentrations, derived by using the CBD, demonstrated that for biofilms of the same organisms, 100 to 1,000 times the concentration of a certain antibiotic were often required for the antibiotic to be effective, while other antibiotics were found to be effective at the MICs. The CBD offers a new technology for the rational selection of antibiotics effective against microbial biofilms and for the screening of new effective antibiotic compounds.
Article
Full-text available
Biofilms--matrix-enclosed microbial accretions that adhere to biological or non-biological surfaces--represent a significant and incompletely understood mode of growth for bacteria. Biofilm formation appears early in the fossil record (approximately 3.25 billion years ago) and is common throughout a diverse range of organisms in both the Archaea and Bacteria lineages, including the 'living fossils' in the most deeply dividing branches of the phylogenetic tree. It is evident that biofilm formation is an ancient and integral component of the prokaryotic life cycle, and is a key factor for survival in diverse environments. Recent advances show that biofilms are structurally complex, dynamic systems with attributes of both primordial multicellular organisms and multifaceted ecosystems. Biofilm formation represents a protected mode of growth that allows cells to survive in hostile environments and also disperse to colonize new niches. The implications of these survival and propagative mechanisms in the context of both the natural environment and infectious diseases are discussed in this review.
Article
Full-text available
DNA microarrays were used to probe the transcriptional response of Escherichia coli to N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN). Fifty-five transcripts were significantly up-regulated, including all of the genes that are regulated by Zur and many that are regulated by Fur. In the same TPEN-treated cells, 46 transcripts were significantly down-regulated.
Article
Full-text available
The sequencing of the first complete bacterial genome in 1995 heralded a new era of hope for antibacterial drug discoverers, who now had the tools to search entire genomes for new antibacterial targets. Several companies, including GlaxoSmithKline, moved back into the antibacterials area and embraced a genomics-derived, target-based approach to screen for new classes of drugs with novel modes of action. Here, we share our experience of evaluating more than 300 genes and 70 high-throughput screening campaigns over a period of 7 years, and look at what we learned and how that has influenced GlaxoSmithKline's antibacterials strategy going forward.
Article
Microbial biofilms, which are elaborate and highly resistant microbial aggregates formed on surfaces or medical devices, cause two-thirds of infections and constitute a serious threat to public health. Immunocompromised patients, individuals who require implanted devices, artificial limbs, organ transplants, or external life support and those with major injuries or burns, are particularly prone to become infected. Antibiotics, the mainstay treatments of bacterial infections, have often proven ineffective in the fight against microbes when growing as biofilms, and to date, no antibiotic has been developed for use against biofilm infections. Antibiotic resistance is rising, but biofilm-mediated multidrug resistance transcends this in being adaptive and broad spectrum and dependent on the biofilm growth state of organisms. Therefore, the treatment of biofilms requires drug developers to start thinking outside the constricted “antibiotics” box and to find alternative ways to target biofilm infections. Here, we highlight recent approaches for combating biofilms focusing on the eradication of preformed biofilms, including electrochemical methods, promising antibiofilm compounds and the recent progress in drug delivery strategies to enhance the bioavailability and potency of antibiofilm agents.
Article
The cover picture shows the destruction of a bacterial membrane by a new NH125 analogue. Through chemical synthesis and biological evaluation, the authors have identified potent N-arylated compounds that rapidly kill MRSA persister cells (99.99 % in 30 minutes) and eradicate surface-attached bacterial biofilms of multiple Gram-positive pathogens through the destructive lysis of bacterial membranes. These N-arylated NH125 analogues demonstrated enhanced activity in killing persister cells and eradicating biofilm when tested alongside a structurally diverse panel of membrane-active agents.More information can be found in the communication by R. W. Huigens III et al. (DOI: 10.1002/cbic.201600622).
Article
The 2-position of the halogenated quinoline (HQ) scaffold can be dramatically tuned through diverse and practical synthetic pathways, including reductive amination and alkylation reactions. New HQs discovered during these investigations potently eradicate methicillin-resistant Staphylococcus aureus (MRSA), methicillin-resistant Staphylococcus epidermidis (MRSE) and vancomycin-resistant Enterococcus faecium (VRE) biofilms, which display high levels of tolerance towards conventional antibiotic therapies. The background image is reproduced with permission and copyright© of the British Editorial Society of Bone and Joint Surgery (Bone Joint J. 2013, 95B, 678–682; Figure 2 b). More information can be found in the Full Paper by R. W. Huigens III et al. (DOI: 10.1002/chem.201600926).
Article
Bacteria that attach to surfaces aggregate in a hydrated polymeric matrix of their own synthesis to form biofilms. Formation of these sessile communities and their inherent resistance to antimicrobial agents are at the root of many persistent and chronic bacterial infections. Studies of biofilms have revealed differentiated, structured groups of cells with community properties. Recent advances in our understanding of the genetic and molecular basis of bacterial community behavior point to therapeutic targets that may provide a means for the control of biofilm infections.
Article
Conventional antibiotics are ineffective against non-replicating bacteria (for example, bacteria within biofilms). We report a series of halogenated phenazines (HP), inspired by marine antibiotic 1, that targets persistent bacteria. HP 14 demonstrated the most potent biofilm eradication activities to date against MRSA, MRSE, and VRE biofilms (MBEC=0.2-12.5 μM), as well as the effective killing of MRSA persister cells in non-biofilm cultures. Frontline MRSA treatments, vancomycin and daptomycin, were unable to eradicate MRSA biofilms or non-biofilm persisters alongside 14. HP 13 displayed potent antibacterial activity against slow-growing M. tuberculosis (MIC=3.13 μM), the leading cause of death by bacterial infection around the world. HP analogues effectively target persistent bacteria through a mechanism that is non-toxic to mammalian cells and could have a significant impact on treatments for chronic bacterial infections.
Article
Most bacterial cells lead lives of quiet desperation in biofilms, combatting stress; yet, their prevalence attests to their ability to alter gene regulation to cope with myriad insults. Since biofilm bacteria are faced with starvation and other environmental stress (e.g., antibiotics from competitors, oxidative stress from host immune systems), it behooves them to be able to ramp down their metabolism in a highly regulated manner and enter a resting state known as persistence to weather stress. Hence, persister cells are metabolically dormant cells that arise predominantly as a response to stress through elegant gene regulation that includes toxin/antitoxin systems. In this review, an analysis is made of the genetic pathways that lead to persistence, of cell signaling via the interspecies and interkingdom signal indole that leads to persistence, and the means found to date for combatting these cells which are frequently tolerant to a range of antibiotics. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
Article
Bacterial biofilms are difficult to eradicate because of reduced antibiotic sensitivity and altered metabolic processes; thus, the development of new approaches to biofilm eradication is urgently needed. Antimicrobial peptides (AMPs) and quaternary ammonium cations (QACs) are distinct, yet well-known, classes of antibacterial compounds. By mapping the general regions of charge and hydrophobicity of QACs onto AMP structures, we designed a small library of QACs to serve as simple AMP mimics. In order to explore the role that cationic charge plays in biofilm eradication, structures were varied with respect to cationic character, distribution of charge, and alkyl side chain. The reported compounds possess minimum biofilm eradication concentrations (MBEC) as low as 25 μM against Gram-positive biofilms, making them the most active anti-biofilm structures reported to date. These potent AMP mimics were synthesized in 1–2 steps and hint at the minimal structural requirements for biofilm destruction.
Article
Bacterial cells may escape the effects of antibiotics without undergoing genetic change; these cells are known as persisters. Unlike resistant cells that grow in the presence of antibiotics, persister cells do not grow in the presence of antibiotics. These persister cells are a small fraction of exponentially growing cells (due to carryover from the inoculum) but become a significant fraction in the stationary phase and in biofilms (up to 1%). Critically, persister cells may be a major cause of chronic infections. The mechanism of persister cell formation is not well understood, and even the metabolic state of these cells is debated. Here, we review studies relevant to the formation of persister cells and their metabolic state and conclude that the best model for persister cells is still dormancy, with the latest mechanistic studies shedding light on how cells reach this dormant state.
Article
The spread of resistant bacteria, leading to untreatable infections, is a major public health threat but the pace of antibiotic discovery to combat these pathogens has slowed down. Most antibiotics were originally isolated by screening soil-derived actinomycetes during the golden era of antibiotic discovery in the 1940s to 1960s. However, diminishing returns from this discovery platform led to its collapse, and efforts to create a new platform based on target-focused screening of large libraries of synthetic compounds failed, in part owing to the lack of penetration of such compounds through the bacterial envelope. This article considers strategies to re-establish viable platforms for antibiotic discovery. These include investigating untapped natural product sources such as uncultured bacteria, establishing rules of compound penetration to enable the development of synthetic antibiotics, developing species-specific antibiotics and identifying prodrugs that have the potential to eradicate dormant persisters, which are often responsible for hard-to-treat infections.
Article
Chronic infections affect 17 million people yearly, and approximately 550,000 people die each year from, or with, their chronic infections. Acute and chornic infection differences are well known to clinicians, but the role of bacteria in producing these clinical differences remains poorly understood. This review relies on basic science, clinical studies, and a general review of the medical biofilm literature. The basic science studies are level A and B quality of evidence. The clinical studies are mainly retrospective cohort (level B) and case studies (level C). The biofilm literature includes reviews with varying levels of evidence. All articles have been peer reviewed and meet the standard of evidence-based medicine. Acute infections are associated with planktonic bacteria and must be diagnosed rapidly and accurately to prevent tissue damage and/or death. In contrast, biofilm behavior pursues a more parasitic course by producing sustained host hyperinflammation, with the biofilm feeding on plasma exudate. Chronic infections vacillate over long periods of time, responding only partially to antibiotics and reemerging once the antibiotics are withdrawn. Chronic wounds exhibit similar clinical behavior seen in other chronic infections and are associated with biofilm phenotype bacteria on their surface. Biofilm infections, such as chronic wounds, cannot be adequately diagnosed with current clinical cultures; therefore, molecular methods are necessary. Biofilm phenotype bacteria require multiple concurrent strategies, including débridement and targeted antibiofilm agents. Biofilm phenotype bacteria predominate on the surface of wounds, and biofilm-based management improves wound healing outcomes, indicating that biofilm is the right target for managing the bioburden barrier of chronic wounds.
Article
Though biofilms were first described by Antonie van Leeuwenhoek, the theory describing the biofilm process was not developed until 1978. We now understand that biofilms are universal, occurring in aquatic and industrial water systems as well as a large number of environments and medical devices relevant for public health. Using tools such as the scanning electron microscope and, more recently, the confocal laser scanning microscope, biofilm researchers now understand that biofilms are not unstructured, homogeneous deposits of cells and accumulated slime, but complex communities of surface-associated cells enclosed in a polymer matrix containing open water channels. Further studies have shown that the biofilm phenotype can be described in terms of the genes expressed by biofilm-associated cells. Microorganisms growing in a biofilm are highly resistant to antimicrobial agents by one or more mechanisms. Biofilm-associated microorganisms have been shown to be associated with several human diseases, such as native valve endocarditis and cystic fibrosis, and to colonize a wide variety of medical devices. Though epidemiologic evidence points to biofilms as a source of several infectious diseases, the exact mechanisms by which biofilm-associated microorganisms elicit disease are poorly understood. Detachment of cells or cell aggregates, production of endotoxin, increased resistance to the host immune system, and provision of a niche for the generation of resistant organisms are all biofilm processes which could initiate the disease process. Effective strategies to prevent or control biofilms on medical devices must take into consideration the unique and tenacious nature of biofilms. Current intervention strategies are designed to prevent initial device colonization, minimize microbial cell attachment to the device, penetrate the biofilm matrix and kill the associated cells, or remove the device from the patient. In the future, treatments may be based on inhibition of genes involved in cell attachment and biofilm formation.
Article
Modern medicine is facing the spread of biofilm-related infections. Bacterial biofilms are difficult to detect in routine diagnostics and are inherently tolerant to host defenses and antibiotic therapies. In addition, biofilms facilitate the spread of antibiotic resistance by promoting horizontal gene transfer. We review current concepts of biofilm tolerance with special emphasis on the role of the biofilm matrix and the physiology of biofilm-embedded cells. The heterogeneity in metabolic and reproductive activity within a biofilm correlates with a non-uniform susceptibility of enclosed bacteria. Recent studies have documented similar heterogeneity in planktonic cultures. Nutritional starvation and high cell density, two key characteristics of biofilm physiology, also mediate antimicrobial tolerance in stationary-phase planktonic cultures. Advances in characterizing the role of stress response genes, quorum sensing and phase variation in stationary-phase planktonic cultures have shed new light on tolerance mechanisms within biofilm communities.
Article
Several well-recognized puzzles in microbiology have remained unsolved for decades. These include latent bacterial infections, unculturable microorganisms, persister cells and biofilm multidrug tolerance. Accumulating evidence suggests that these seemingly disparate phenomena result from the ability of bacteria to enter into a dormant (non-dividing) state. The molecular mechanisms that underlie the formation of dormant persister cells are now being unravelled and are the focus of this Review.
Article
Biofilms contain bacterial cells that are in a wide range of physiological states. Within a biofilm population, cells with diverse genotypes and phenotypes that express distinct metabolic pathways, stress responses and other specific biological activities are juxtaposed. The mechanisms that contribute to this genetic and physiological heterogeneity include microscale chemical gradients, adaptation to local environmental conditions, stochastic gene expression and the genotypic variation that occurs through mutation and selection. Here, we discuss the processes that generate chemical gradients in biofilms, the genetic and physiological responses of the bacteria as they adapt to these gradients and the techniques that can be used to visualize and measure the microscale physiological heterogeneities of bacteria in biofilms.