# data[].attributes.attachment_link.readable_text
Claudio O. Gualerzi, Academic Editor

Abstract

Macrolides, as a class of natural or semisynthetic products, express their antibacterial activity primarily by reversible binding to the bacterial 50S ribosomal subunits and by blocking nascent proteins' progression through their exit tunnel in bacterial protein biosynthesis. Generally considered to be bacteriostatic, they may also be bactericidal at higher doses. The discovery of azithromycin from the class of macrolides, as one of the most important new drugs of the 20th century, is presented as an example of a rational medicinal chemistry approach to drug design, applying classical structure-activity relationship that will illustrate an impressive drug discovery success story. However, the microorganisms have developed several mechanisms to acquire resistance to antibiotics, including macrolide antibiotics. The primary mechanism for acquiring bacterial resistance to macrolides is a mutation of one or more nucleotides from the binding site. Although azithromycin is reported to show different, two-step process of the inhibition of ribosome function of some species, more detailed elaboration of that specific mode of action is needed. New macrocyclic derivatives, which could be more potent and less prone to escape bacterial resistance mechanisms, are also continuously evaluated. A novel class of antibiotic compounds-macrolones, which are derived from macrolides and comprise macrocyclic moiety, linker, and either free or esterified quinolone 3-carboxylic group, show excellent antibacterial potency towards key erythromycin-resistant Gram-positive and Gram-negative bacterial strains, with possibly decreased potential of bacterial resistance to macrolides.

Keywords: macrocycles, macrolides, quinolones, ribosome binding, dual-binding inhibition, azithromycin, erythromycin

1. Introduction

It is well known that macrocyclic compounds have great potential for broad use in the treatment of different diseases and therefore make very interesting molecules. Most of macrocyclic drugs are predominantly used for the treatment of infectious diseases, but they have also been used in the treatment of cancer, auto-immune, and inflammatory diseases [ 1, 2, 3, 4, 5, 6, 7, 8], since they possess significant anti-inflammatory and immunomodulatory properties [ 9, 10, 11, 12, 13], as well as tuberculostatic, antifungal, antiparasitic, antimalarial, antiviral, and antitumor properties [ 5, 6, 7, 14, 15, 16, 17]. The best known macrocyclic structures are macrolides, natural compounds produced by Streptomyces species that are the most commonly used class of antibiotics, and newly-synthesized macrocycles that also belong to the macrolide or cyclic peptide class. The first 14-membered macrolide, erythromycin A, has been in clinical use since 1952. Erythromycin is active against Gram-positive and some Gram-negative microorganisms and is used in treatment of respiratory, gastrointestinal, and genital tract infections, as well as skin and soft tissue infections [ 18]. To improve acidic stability and oral bioavailability of erythromycin A, the first generation of natural or semisynthetic macrolides such as spiramycin [ 19], roxithromycin [ 20], dirithromycin [ 21], and clarithromycin [ 22] were prepared and introduced to medical practice. Discovery of the first 15-membered macrolide-azithromycin, characterized by a basic nitrogen atom inserted into the macrocyclic ring, represented a breakthrough in the macrolide antibiotic era. Azithromycin became one of the best-selling branded antibiotics worldwide.

2. Macrolides and Their Mode of Action as Anti-Infectives

The era of modern anti-infective drug discovery started in 1928 when Alexander Fleming discovered (by chance) the first antibiotic from mold: penicillin from Penicillium notatum. Together with the discovery of the cephalosporins (from Cephalosporium acremonium), the penicillins are part of a large group of beta-lactams, the first generation of antibiotics. Another significant group of antibiotics were the tetracyclines, developed initially from a product of Streptomyces aureofaciens (chlorotetracycline). Oxytetracycline, a product of Streptomyces rimosus, was discovered in 1950. The macrolides represent a third family of well-known oral antibiotics. Medically-important macrolide antibiotics were originally characterized by a 12-membered (methymycin-like), 14-membered (erythromycin-like), or 16-membered (josamycin-like) lactone ring to which amino and neutral side-sugars are attached A.

Originally applied to compounds originally extracted from natural sources, macrolides, as a broader chemical term, now encompasses all macrocyclic ring lactones varying in size from eight-membered up to 62-membered rings ().

Actinomycetes are very effective in production of bioactive compounds, such as macrolides (antibiotics), rapamycin (immunosuppressant) [ 13], avermectin (antiparastic) [ 15], nystatin (antifungal) [ 14], and especially with respect to antitumor compounds (doxorubicin, bleomycin) [ 16, 17]. The importance and use of macrocyclic compounds in immunosuppression, inflammation, cancer, and infection are rapidly growing. The chemical modifications of existing macrocyclic drugs are in progress and their new therapeutic characteristic have improved further. Inflammatory cells bioaccumulate macrolides and transport them to the infected tissues. During the treatment of bacterial infections, macrolide accumulation in inflammatory cells plays an important role, since inflamed tissue releases a whole range of chemoattractant molecules, and polymorphonuclear cells, which are loaded with the antibacterial agent are, therefore, concentrated in the inflamed tissue. Bacterial components activate and degranulate inflammatory cells, and the macrolide is released into the surrounding tissue, contributing to faster clearance of the infectious pathogen [ 30].

The medically most important macrolide antibiotics are those structured from 12- to 16-membered large lactone rings with one or more sugar moieties, generally desosamine and cladinose, linked to the macrocyclic core [ 32]. Macrolides express their antibacterial activity by binding themselves to the bacterial 50S ribosomal subunits and inhibiting protein synthesis. More specifically, they have interactions with the region of the structure of the 50S subunit defining the catalytic core and the ribosomal exit tunnel (peptidyl transferase-associated region) [ 26]. They bind to the 23S rRNA at the nascent peptide exit tunnel and inhibit the growth of nascent peptides [ 33].

3. From Erythromycin to Azithromycin

The first 14-membered macrolide, erythromycin A, isolated from the actinomycete Streptomyces erythreus ( Saccharopolyspora erythraea), has been in human use since 1952. Erythromycin has an antimicrobial spectrum similar to that of penicillin, and was widely used for patients who are allergic to penicillin. It exerts bacteriostatic and bactericidal properties, depending on the type of microorganism and the antibiotic concentration used. It is most effective against Staphylococcus aureus cocci, streptococcal group A, enterococci, and pneumococci. It inhibits the Neisseriae strain, and some strains of Haemophilus influenzae, Pasteurellae multocidae, Brucellae, Rickettsiae, and Treponemae. It is also effective against Mycoplasma pneumoniae, Chlamydiae, Legionellae pneumophilae, and some other atypical mycobacteria. The bioavailability of erythromycin is 30%-65%, and it is distributed in most tissues and body fluids. Plasma protein binding of erythromycin is 70%-90% and it is metabolized in the liver, partly with the formation of inactive metabolites with a t 1/2 of about 1.4-2 h [ 42].

Erythromycin, with its ten chiral centers and two sugar substituents (l-cladinose and d-desosamine, ), was a good starting point for numerous medicinal chemistry efforts for improvement of its biological profile (better activity, higher stability, and improved bioavailability) since the first generation of macrolides, which had low toxicity and good tolerability, were unstable in acidic media, had low toxicity and good tolerability. In the acidic environment of the stomach, erythromycin A is metabolized to its inactive 8,9-anhydroerythromycin-6,9-hemiketal and anhydroerythromycin-6,9:9,12-spiroketal. To improve the acidic stability and oral bioavailability of erythromycin A, the first generation of semisynthetic macrolides were prepared and introduced to medical practice. The main goal was to avoid ketal formation, initially by modifying the keto group or reactive hydroxyl. This approach resulted in various new chemical entities [ 43, 44, 45]. The most important erythromycin derivative from this group of new derivatives was erythromycin A oxime, prepared from erythromycin A and hydroxylamine hydrochloride in the presence of a weak base and buffer () [ 46, 47, 48]. The same was identified by chemists at PLIVA (the largest pharmaceutical company in Croatia and one of the leading companies in Southeast Europe and, today, a member of the Teva Group) and the work continued for the further synthesis of new, more active compounds, such as roxithromycin, dirithromycin, and clarithromycin. The roxithromycin enriched with an N-oxime side chain attached to the macrolactone ring [ 20], dirithromycin, in which the 9-keto group of the macrolactone ring was converted to an amino group [ 21], and clarithromycin, in which an additional methyl group in the 6- O-position, in comparison to erythromycin these modifications significantly improved acid-stability [ 22] ().

The first 15-membered macrolide antibiotic on the market-azithromycin (9a-methyl-9-deoxo-9-dihydro-9a-aza-9a-homoerythromycin) (), characterized by a basic nitrogen atom inserted into the macrocyclic ring, was synthesized in 1980 by a team of researchers at PLIVA Laboratories [ 49, 50]. Twenty years later, in 2000, for their outstanding contribution to chemistry they received the medal of highest honor, "Heroes of Chemistry", awarded by the American Chemical Society. The discovery of the 15-membered imino-ether, produced by Beckmann's rearrangement of 9(E)-erythromycin A oxime to the amide, led to the production of a qualitatively new group of macrolide antibiotics, named the azalides [ 50, 51, 52, 53]. The key reaction in the formation of azalides was established during the synthesis of O-sulfonyl derivatives of 9(E)-erythromycin A oxime [ 50, 51]. Treatment of 9(E)-erythromycin A oxime with benzenesulfonyl chloride in an acetone-water mixture with sodium bicarbonate yielded an unexpected product, erythromycin-6,9-imino-ether () [ 51, 52, 53]. A breakthrough was the discovery of a way to open the smaller ring by hydration, and it was confirmed that a 15-membered macrocyclic ring with an "incorporated" amino group had been formed [ 51]. At that moment, the dogma that the macrocyclic erythromycin ring was responsible for antibiotic activity of macrolides was shattered. A new substance had similar activity against Gram-positive bacteria and significantly better activity against Gram-negative bacteria in comparison to erythromycin A [ 31]. The stability in an acid environment was improved, and acute toxicity decreased, but slightly less than erythromycin. However, this reaction was just one innovative step towards a much more important discovery. Reductive methylation of 9-dihydro-9-deoxo-9a-aza-9a-homoerythromycin A with formaldehyde and formic acid yielded a novel product, a significantly more potent and bioavailable macrolide antibiotic named azithromycin () [ 51, 54]. Its broad spectrum of activity covered all relevant bacteria causing respiratory tract infections, including Haemophilus influenzae and Moraxella catarrhalis. Azithromycin was up to four times more potent than erythromycin against Haemophilus influenzae and Neisseria gonorrhoeae, and two-fold more potent against Branhamella catarrhalis, Campylobacter and Legionella sp., and significantly more potent in comparison to many genera of the family Enterobacteriaceae. Its minimal inhibitory concentration (MIC) for 90% of strains of Escherichia, Salmonella, Shigella, and Yersinia was ≤4 μg/mL, compared with 16 to 128 μg/mL for erythromycin [ 54]. Tests in animal model has shown that azithromycin treatment is able to clear chlamydial genital infection but is unable to eliminate chlamydial infection in the cecum within the same animal in doses which were effective in clearing the genital infection [ 55].

The in vivo tests showed that azithromycin is less than half as toxic as erythromycin (erythromycin i.v./p.o. LD50 is 360/4000 mg/kg, while azithromycin i.v./p.o. LD50 is 825/10,000 mg/kg) [ 52]. Furthermore, in all studies for in vivo efficacy against systemic infections in mice (by S. aureus, S. typhimurium, S. pyogenes, and S. peneumoniae), significant superiority of azithromycin was demonstrated, regardless of the administration route (subcutaneous or oral) [ 52, 56]. Pharmacokinetic studies in mice, rats, rabbits, and dogs showed the unique properties of the new molecule, quite different than those of erythromycin. While serum levels of erythromycin oxime and amine, and levels of azithromycin were similar (several times higher than that of erythromycin), retention time was significantly longer, and urine levels were up to a hundred times higher. High and prolonged levels were observed in tissues of some organs, even 24 h after injection (liver, lung, intestine, and kidney). Brain tissue levels were low, indicating that it does not pass the brain-blood barrier [ 52, 57, 58]. Chronic toxicity was monitored in rats and dogs after 15 days and after one, three, and six months of daily dosing with 25, 50, 100, and 200 mg/kg azithromycin. Equal or small reversible adverse changes were observed with azithromycin than with erythromycin in animals given the same doses. Mutagenic, carcinogenic, and teratogenic tests were negative [ 52].

Simultaneously with the testing for anti-infective properties, the physicochemical, pharmacological, and structural properties of azithromycin were investigated [ 61]. Azithromycin proved to be highly superior to that of erythromycin, relative to stability in acidic conditions [ 62]. With the determination of the crystal structure of the bacterial ribosome, various scientists tried to prepare binding complexes of ribosome and macrolide antibiotics. Analysis of the crystal structure of the large ribosomal subunit (50S) from Deinococcus radiodurans, complexed with azithromycin, showed that azithromycin exerts its antimicrobial activity by blocking the protein exit tunnel [ 26].

In 1988, azithromycin was introduced to the market by joint collaborative agreement between PLIVA (Sumamed ®), and Pfizer (Zithromax ®). After the year 2000, azithromycin became the market leader among antibiotics for respiratory tract infections. Pfizer's Zithromax was one of the best-selling branded antibiotics in the United States and worldwide, with total sales peaking at US $2 billion in 2005 before starting to decline with the loss of patent protection in 2006 and the resulting generic competition. Azithromycin, one of the leading drugs of the late 20th century, represents an excellent example of a unique and rational medicinal chemistry and classical structure-activity relationship approach towards drug design. As a result, azithromycin is a drug discovery success story [ 31]. The discovery of azithromycin was not a typical example of a drug discovery project, since the drug was developed on the basis of its anti-infective activity, and its precise mechanism of action and interaction with the ribosome-RNA complex were only discovered later, subsequent to its successful marketing [ 63].

4. New Macrocyclic Ribosome Inhibitors as Possible Way to Avoid Resistance Problem

The microorganisms have developed several mechanisms to acquire resistance to antibiotics, including macrolides. One of these mechanisms responsible for the lower binding and deceasing of anti-infective activity of macrolides is the change in the structure of the ribosome target either by methylation or mutation of the 23S rRNA, or mutation and consequent structural changes in the L4 and L22 ribosomal proteins in 50S ribosomal subunit [ 64]. The resistance to macrolide antibiotics is due to modification of the ribosomal target by methylation or mutation, decreased uptake of the molecules, and active efflux of the drug.

A family of rRNA methyltransferases designated as the Erm enzymes (more than 30 proteins have been reported) in bacteria are responsible for the development of the bacterial resistance to the antibiotics due the methylation of the 23S rRNA, in particular at adenine 2058 [ 65]. Methylation at A2058 significantly influences the binding of macrolides to the ribosome target and it is responsible for development of a cross-resistance to macrolides [ 66]. The expression of erm genes of bacteria is inducible, regulated by silencing of gene expression and constitutive erm gene translation, or constitutive, and it has been reported that the ability of macrolides to induce the expression of the erm genes depends on their structure. Macrolides with a 14-member lactone ring are strong inducers of the erm genes and these include erythromycin [ 67]. In addition to the structural changes of the ribosome target in the resistance development, an important role in the resistance development is played by the efflux pumps. For example, the msrA gene encodes an ABC transporter in staphylococci and the mefA gene encodes the expression of a MFS pump in streptococci [ 68, 69] which transport the antibiotics out of the bacteria.

Azithromycin exerts its antimicrobial activity by blocking the protein exit tunnel, but in contrast to other macrolides, this effect is possibly linked with the distinct binding sites, since an additional binding site has been also recognized within the large ribosomal subunit of D. radiodurans [ 26]. Nitrogen inserted into the lactone ring does not directly contribute to the binding of azithromycin to the ribosome, but this modification alters the conformation of the lactone ring sufficiently to induce novel contacts. One azithromycin molecule interacts with domains IV and V of 23S rRNA, whereas the second azithromycin interacts with two ribosomal proteins L4 and L22 and domain II of 23S rRNA [ 26], so azithromycin can be considered as a dual-binding ribosome inhibitor. Additionally, in an earlier example of binding of azithromycin on the Escherichia coli ribosome, it is shown that azithromycin binds in a two-step process-placing of the drug in a low-affinity site located in the upper part of the exit tunnel, and slow formation of a final complex that is much stronger and more potent in preventing the synthesis of the nascent peptide through the exit tunnel [ 24].

In order to rationally design better drugs which would be able to overcome the existing resistance mechanisms, new directions in drug discovery have been evaluated, and rational approach to drug design, based on already-known mechanisms of drug actions has been applied. Multiple new macrolides were synthesized with the aim to inhibit the growth or to kill resistant bacterial strains. Concept of combining active macrolide scaffold and (hetero) aromatic unit via a flexible linker resulted with compounds showing remarkable antibacterial activity. This effect was first recognized in the ketolide group of macrolide antibiotics, where the cladinose sugar is substituted with a keto-group, with a cyclic carbamate group attached in the lactone ring, with telithromycin () [ 29, 70, 71, 72, 73]. These modifications enable ketolides to become more active on a much broader spectrum than other macrolides [ 70]. Furthermore, another potential proof of concept (at least in anti-bacterial activity) has been done on azitromycin 4″ derivatives (on cladinose sugar), where quinolone moieties have been linked with macrolactone ring, and activities toward strains which were not sensitive toward classical anti-infective macrolides have been significantly improved () [ 27].

A novel class of macrolide antibiotics-macrolones, which are derived from macrolides are comprised of a macrocyclic moiety, linker and either free or esterified quinolone 3-carboxylic group. Macrolones showed excellent antibacterial potency towards key erythromycin-resistant Gram-positive and Gram-negative bacterial strains. Compared to azithromycin or to quinolones, most of the compounds exhibited improved and superior in vitro potency against the key erythromycin-resistant respiratory pathogens [ 27, 28, 29]. They show activity on eryS- and MLSb-resistant S. pneumoniae (ribosome methylation as the major mechanisms of erythromycin resistance) in the murine pneumonia model [ 74]. For macrolones no demonstration has confirmed yet that their functional target is the ribosome or that they inhibit protein synthesis. However, due to flexibility of their structure, the macrolones are good candidates for possible multiple interactions with the ribosome, and the macrolide part of the macrolone should have a major role in this binding. As is the case with "classical" macrolides, which are known to possess favorable pharmacokinetic properties by accumulating in inflammatory cells, the macrolone class of compounds, with its distinct structural features, shows equal or better accumulation in inflammatory cells [ 75]. Unlike macrolides and ketolides, macrolones showed rapid bactericidal effects against H. influenzae, and they exhibited equal or lower in vitro resistance development potential than azithromycin and telithromycin in S. pneumoniae, H. influenzae, S. aureus, and M. catarrhalis [ 74]. Macrolones have a low clearance, large volume of distribution, and long half-life, complying with once-daily dosing potential [ 74]. However, even after considerable data about its in vitro activity, mode of action, in vivo efficacy, and recognizing macrolones as superior in comparison to the known macrolide antibiotics, true potential of that compound class should still be evaluated after obtaining more detailed structural insight into binding mode to the ribosome. Finally, a full pre-clinical package of ADME/Tox properties (in vitro/in vivo) of this, and similar, classes of conjugates of macrolides and other active moieties, such as quinolones, should be done before we can make a final conclusion about macrolones as a promising new class of compounds.

5. Concluding Remarks

The continuous increase of resistant bacterial strains causes a significant problem in modern health care and drug discovery. Discovery of first macrolide antibiotic erythromycin, and development of semisynthetic macrolides prepared and introduced to medical practice, such as roxithromycin, dirithromycin, spiramycin, clarithromycin, josamycin, and especially azithromycin, significantly changed anti-infective drug picture of 20th century. Azithromycin provided a considerable boost to the worldwide therapy of bacterial infections. Yet now, well into the 21st century, the ketolide group of antibiotics, such as telithromycin and solithromycin as its most important members, together with azithromycin and some of the other macrolide antibiotics, continue to reveal unexpected activities. These factors have maintained the intense research interest and may well spawn new derivatives with therapeutic effects well beyond the field of antibiotics. However, the microorganisms have developed several mechanisms to acquire resistance to all antibiotics, among them also to macrolides. Therefore, in order to design better drugs which would be able to overcome the existing resistance mechanisms, new rational directions in drug discovery and drug design have been and should be evaluated. In addition to the abovementioned ketolides, and azithromycin's possible dual-binding mode of action, where one azithromycin molecule interacts with rRNA domains, and the second azithromycin interacts with two ribosomal proteins (in a two-step process), the concept of combining active macrolide scaffold and (hetero)aromatic unit via a flexible linker has yielded compounds showing remarkable antibacterial activity. The novel class of macrolide antibiotics-macrolones, which are derived from macrolides, and which comprise macrocyclic moiety, linker, and either free or esterified quinolone group, could be great examples of combining excellent antibacterial potency towards key erythromycin-resistant pathogens. The potential novel concept of dual-binding ribosome inhibition is a possible mechanism for preventing growing resistance development.

Acknowledgments

Publishing of this article in open access is supported by the Department of Biotechnology University of Rijeka.

Abbreviations

The following abbreviations are used in this manuscript:

rRNA

ribosomal Ribonucleic acid

tRNA

transfer Ribonucleic acid

MIC

Minimal inhibitory concentration

i.v

Intravenous injection, a route of administration of a drug

p.o

Per oral, a route of administration of a drug

LD50

The median lethal dose

EryS

Erythromycin susceptible

EryR

Erythromycin resistant

MLSb

Macrolide-Lincosamide-Streptogramin b

Conflicts of Interest

The authors declare no conflict of interest.

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From Erythromycin to Azithromycin and New Potential Ribosome-Binding Antimicrobials

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Claudio O. Gualerzi, Academic Editor Macrolides, as a class of natural or semisynthetic products, express their antibacterial activity primarily by reversible binding to the bacterial 50S ribosomal subunits and by blocking nascent proteins' progression through their exit tunnel in bacterial protein biosynthesis. Generally considered to be bacteriostatic, they may also be bactericidal at higher doses.

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New student co-creators team building for Antibiotics Undersea 💊🌊

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New student co-creators team building for Antibiotics Undersea 💊🌊

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Review on Antibacterial Compounds from Marine Bacteria, 2010–2015

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Review on Antibacterial Compounds from Marine Bacteria, 2010–2015

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Antimicrobial Susceptibility of Vibrio vulnificus and Vibrio parahaemolyticus 

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Antimicrobial Susceptibility of Vibrio vulnificus and Vibrio parahaemolyticus 

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Kristi S. Shaw,

Rachel E. Rosenberg Goldstein,

Xin He,

John M. Jacobs,

Byron C. Crump,

Amy R. Sapkota

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Abstract

Vibrio vulnificus and V. parahaemolyticus in the estuarine-marine environment are of human health significance and may be increasing in pathogenicity and abundance. Vibrio illness originating from dermal contact with Vibrio laden waters or through ingestion of seafood originating from such waters can cause deleterious health effects, particularly if the strains involved are resistant to clinically important antibiotics. The purpose of this study was to evaluate antimicrobial susceptibility among these pathogens. Surface-water samples were collected from three sites of recreational and commercial importance from July to September 2009. Samples were plated onto species-specific media and resulting V. vulnificus and V. parahaemolyticus strains were confirmed using polymerase chain reaction assays and tested for antimicrobial susceptibility using the Sensititre® microbroth dilution system. Descriptive statistics, Friedman two-way Analysis of Variance (ANOVA) and Kruskal-Wallis one-way ANOVA were used to analyze the data. Vibrio vulnificus (n = 120) and V. parahaemolyticus (n = 77) were isolated from all sampling sites. Most isolates were susceptible to antibiotics recommended for treating Vibrio infections, although the majority of isolates expressed intermediate resistance to chloramphenicol (78% of V. vulnificus, 96% of V. parahaemolyticus). Vibrio parahaemolyticus also demonstrated resistance to penicillin (68%). Sampling location or month did not significantly impact V. parahaemolyticus resistance patterns, but V. vulnificus isolates from St. Martin's River had lower overall intermediate resistance than that of the other two sampling sites during the month of July ( p = 0.0166). Antibiotics recommended to treat adult Vibrio infections were effective in suppressing bacterial growth, while some antibiotics recommended for pediatric treatment were not effective against some of the recovered isolates. To our knowledge, these are the first antimicrobial susceptibility data of V. vulnificus and V. parahaemolyticus recovered from the Chesapeake Bay. These data can serve as a baseline against which future studies can be compared to evaluate whether susceptibilities change over time.

Citation: Shaw KS, Rosenberg Goldstein RE, He X, Jacobs JM, Crump BC, Sapkota AR (2014) Antimicrobial Susceptibility of Vibrio vulnificus and Vibrio parahaemolyticus Recovered from Recreational and Commercial Areas of Chesapeake Bay and Maryland Coastal Bays. PLoS ONE 9(2): e89616. https://doi.org/10.1371/journal.pone.0089616

Editor: Raymond Schuch, Rockefeller University, United States of America

Received: October 16, 2013; Accepted: January 21, 2014; Published: February 25, 2014

This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

Funding: Financial support for this research was graciously provided by National Oceanic and Atmospheric Administration award EA133C07CN0163 ( www.noaa.gov). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript, but funder's employee, Dr. John Jacobs, was a collaborator in all portions of this study.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Bacterial antimicrobial resistance is a critical public health issue of increasing importance for those who recreate and work in coastal regions. Pathogenic bacteria and antimicrobial resistance genes are often released with wastewater discharges into aquatic environments [1]. Naturally occurring bacteria produce antibiotics in the environment for signaling and regulatory purposes in microbial communities [2]. Bacteria protect themselves from the toxicity of these antibiotics by acquiring and expressing antibiotic resistance genes [3]. As a result, naturally-occurring aquatic bacteria are capable of serving as reservoirs of resistance genes and those genes, coupled with the introduction and accumulation of antimicrobial agents, detergents, disinfectants, and residues from industrial processes, may play an important role in the evolution and spread of antibiotic resistance in aquatic environments [1].

Vibrio bacteria in the estuarine-marine environment are of particular concern for human health and may be increasing in pathogenicity and abundance [4]. Cases of vibriosis are rising in the United States, with Vibrio vulnificus and V. parahaemolyticus being two of the three most commonly reported sources of Vibrio infection [5]. V. parahaemolyticus is implicated as the primary source of escalation in vibriosis incidence [5] and highly pathogenic serotypes of this species are emerging on a global scale, including the Atlantic coasts of the United States and Spain [6]. It is estimated that only 1 in 142 cases of V. parahaemolyticus illness is detected [7]. Calculations based upon probable incidence of vibriosis have estimated that V. vulnificus and V. parahaemolyticus are the first and third most costly marine-borne pathogens, costing $233 and $20 million, respectively[8].

Antimicrobial susceptibility patterns among Vibrio spp. inhabiting estuarine-marine environments may have implications for recreational and commercial users of these environments, and for those who consume Vibrio-contaminated seafood. Previous studies exploring antimicrobial susceptibility of Vibrio vulnificus and V. parahaemolyticus have been conducted in South Carolina, the United States Gulf region and Italy [9], [10], [11], [12]. However, to our knowledge, no similar studies have been completed in the Chesapeake Bay, the largest estuary in the U.S., which lies in a watershed where 17 million people work, live and play.

The work of our group and others has demonstrated that concentrations of V. vulnificus and V. parahaemolyticus in the Chesapeake Bay are high enough to result in possible illnesses among exposed recreationists, particularly among those who are immunocompromised [13], [14], [15], [16], [17], [18]. Moreover, current models predict that total tissue loading of shellfish and finfish with V. vulnificus and V. parahaemolyticus is associated not only with surface water concentrations but also with the risk of illness for those consuming contaminated seafood products [19], [20], [21]. Given these data, along with the knowledge that environmental conditions may be increasingly more favorable for Vibrio growth [22], it is not surprising that rates of Vibrio infections are increasing in Maryland and other U.S. states [23]. In this context, it is critical to gain a better understanding of the antimicrobial susceptibility patterns of V. vulnificus and V. parahaemolyticus originating from estuarine-marine environments.

This study evaluated antimicrobial susceptibility patterns of V. vulnificus and V. parahaemolyticus recovered from the Chesapeake Bay and Maryland Coastal Bays. Our findings provide the first antimicrobial susceptibility data among Vibrio bacteria isolated from this region. These data will be helpful in short and long-term predictions of human health risks associated with exposures to Vibrio populations in the Chesapeake Bay area.

Materials and Methods

Sampling sites

Three sampling sites were selected based on their importance for human use in the Chesapeake Bay, Maryland Coastal Bays region. Two sites, Sandy Point State Park and St. Martin's River, were characterized by frequent recreational use; and one site, the Pocomoke Sound, was characterized by heavy commercial fishing use ( Figure 1). Sandy Point State Park includes an artificial beach on the western shore of the Chesapeake mid-Bay region, at the base of the Chesapeake Bay Bridge. It is open year round and frequented by approximately 768,000 visitors annually, many of whom visit the park's beach during the summer (Sandy Point Park staff, Maryland Department of Natural Resources, personal communication). St. Martin's River is a tributary of the Maryland Coastal Bays with approximately 10,000 residents. Land-use in the St. Martin's River watershed is approximately 10% residential, 48% agricultural, and 34% forested [24]. The Pocomoke Sound is a major embayment of the Chesapeake Bay's Eastern Shore. It is influenced by agricultural practices, including high-density concentrated poultry feeding operations, and is a popular destination for commercial and recreational fishing. No specific permissions were required for each sampling location, as they are public access waterways, and no endangered or protected species were involved in sampling activities.

(Tracey Saxby, Kate Boicourt, Integration and Application Network, University of Maryland Center for Environmental Science (ian.umces.edu/imagelibrary/displayimage-127-5815.html).

Sample collection

Sampling dates were chosen to coincide with times of high recreational and/or commercial use. Surface water samples (n = 9) were collected during Summer 2009, once a month, at each site, for three consecutive months (July, August, September) within two hours of high tide and on approximately the same date each month. Water samples were collected just below the surface in sterile wide mouth polypropylene 1 L environmental sampling bottles (Nalgene Thermo Scientific, Waltham, MA). Bottles were rinsed three times with surface water and then dipped below the surface for a final 1 L collection volume. Samples collected for Vibrio culture were kept in insulated coolers, while water samples for enterococci culture were stored in an insulated container on ice (4°C) upon collection, returned to the laboratory within four hours and processed immediately upon arrival.

Physical and chemical water quality measurements

Water-column depth and surface-water salinity, temperature, dissolved oxygen, conductivity, and pH were measured on every sampling date and at each location with a YSI 556 Multi-probe system (YSI Incorporated, Yellow Springs, OH) in accordance with the manufacturer's instructions.

Fecal indicator measurements

Fecal indicator measurements were conducted following the standard methods as described for enterococci in Standard Methods for the Examination of Water and Wastewater [25]. Briefly, surface-water samples were filtered in triplicate onto sterile 0.45 µm pore size, 47 mm diameter, nitrocellulose Fisherbrand water-testing membrane filters (Fisher Scientific, Pittsburgh, PA), and plated onto Difco m Enterococcus (BD, Franklin Lakes, NJ) agar. According to manufacturer's instructions, plates were incubated for 48 hours at 35°C. All light to dark red colonies were recorded as presumptive enterococci.

Vibrio isolation

Surface water samples (100 µL) were spread plated in triplicate onto Chromagar Vibrio media (DRG International, Mountainside, NJ) and incubated for 24 hours at 37°C. After incubation, each plate was observed for characteristically colored bacterial colonies associated with V. vulnificus (turquoise) or V. parahaemolyticus (mauve). As V. vulnificus and V. cholerae both appear as turquoise colonies on Chromagar Vibrio media, all turquoise colonies were replated onto cellobiose-collistin (CC) agar (FDA 2004) media to confirm V. vulnificus species. The CC agar cultures were incubated for 24 hours at 37°C and yellow-colored colonies were considered presumptive V. vulnificus. Tryptic soy broth (TSB), supplemented with 5% sodium chloride, was then inoculated with individual presumptive colonies of V. vulnificus or V. parahaemolyticus and incubated at 37°C for 24 hours and stored with 30% glycerol at −80°C.

Vibrio species confirmation

Vibrio DNA template was obtained by producing crude cell lysates by boiling 1 mL aliquots of TSB cultures in 2 mL micro-centrifuge tubes at 100°C for 10 minutes. A Bio-rad CFX96 Touch Real-Time PCR Detection System (Bio-rad, Hercules, CA, USA) was used to confirm the species of isolates with primers designed to detect Vibrio vulnificus [26] or V. parahaemolyticus [27]. Following initial confirmation, samples testing positive for either species were subjected to further testing for virulence genes ( V. vulnificus: virulence correlated gene clinical variant ( vcgC) [28]; V. parahaemolyticus: thermostable direct hemolysin ( tdh), and thermostable related hemolysin ( trh) genes [27]) using real-time PCR.

Real-time PCR was performed by using 1X PCR Buffer (Qiagen, Valencia, CA), (Qiagen), 0.2 mM dNTP's solution (Qiagen), 1X Q solution (Qiagen), 2.25U TopTaq DNA polymerase (Qiagen), 75 nM internal control primers (each), 150 nM internal control probe, 2 µL internal control DNA, target primer and probe concentrations as detailed in Table 1, and 3 µL DNA template per reaction, with the exception of the Vv vcgC assay, where 5 µL of DNA template was used and the internal control components were absent. DNase-RNase free water was added in a quantity sufficient for a 25 uL total reaction volume. Two-stage qPCR cycling parameters are presented in Table 1. A linear synthetic exogenous DNA internal control, including a primer set, probe and internal control DNA, was incorporated simultaneously into each assay (excluding the assays for the V. vulnificus vcgC target) to test for the presence and influence of inhibitors (Nordstrom et al., 2007). The following positive controls were used in each qPCR: Vibrio parahaemolyticus USFDA TX2103 and Vibrio vulnificus ATCC 27562.

A randomly chosen subset of isolates were taxonomically identified with 16 S rRNA gene sequences. DNA extracted from cultures was PCR-amplified with bacteria-specific primers 27f (5′-AGAGTTTGATCCTGGCTCAG-3′) and 907r (5′-CCGTCAATTCCTTTRAGTTT-3′) using the following conditions: 94°C for 2 min, followed by 25 cycles of 55°C for 30 s, 72°C for 30 s, and 94°C for 2 min, followed by 72°C for 5 min. The PCR products were sequenced bi-directionally using the same primers on an ABI 3730 XL Genetic Analyzer in the BioAnalytical Services Laboratory at the University of Maryland Center for Environmental Science. Paired reads for each organism were analyzed and assembled with Phred and Phrap [29], [30], manually edited with Consed [31], and aligned and analyzed with the ARB sequence alignment program [32]. DNA sequences were deposited in the GenBank database under accession numbers KF990336 to KF990363.

Clinical isolates

Clinical isolates of V. parahaemolyticus (n = 8) were graciously provided by the State of Maryland's Department of Health and Mental Hygiene for comparison purposes with our environmental isolates. Sample type and source of infection are presented in Table 2.

Antimicrobial susceptibility testing

Antimicrobial susceptibility testing was performed using the Sensititre® microbroth dilution system (Trek Diagnostic Systems, Westlake, Ohio) in accordance with the manufacturer's instructions on all PCR-confirmed V. vulnificus (n = 120 (3 vcgC+)) and V. parahaemolyticus (n = 77 (1 tdh+, 1 trh+)). Cultures were grown overnight on tryptic soy agar (TSA)+2.5% NaCl plates at 37°C. Vibrio cultures were transferred to sterile demineralized 2.5% saline solution to achieve a 0.5 McFarland standard. Then, 100 µL of each suspension was transferred to sterile cation-adjusted Mueller Hinton broth (Trek Diagnostic Systems, Westlake, Ohio), and 50 µL of the broth solution was dispensed into CML1FMAR custom minimal inhibitory concentration (MIC) plates (Trek Diagnostic Systems Inc.) with the following 26 antibiotics (range of concentrations in μg/ml): amikacin (AMI; 8-64), ampicillin (AMP; 4-32), ampicillin-sulbactam 2∶1 (A/S2; 8/4-32/16), apramycin (APR; 8-32), cefoxitin (FOX; 8-32), ceftriaxone (AXO; 8-64), cephalothin (CEP; 8-128), chloramphenicol (CHL; 8-32), ciprofloxacin (CIP; 1-4), oflaxacin (OFL; 1-8), ceftazidime (TAZ; 8-32), cefepime (FEP; 8-32), cefotaxime (FOT; 8-64), meropenem (MERO; 2-16), doxycycline (DOX; 2-16), imipenem (IMI; 2-16), levofloxacin (LEVO; 2-8), cefuroxime (FUR; 8-32), trimethoprim-sulfamethoxazole (SXT; 2/38-4/76), penicillin (PEN; 16-128), piperacillin (PIP; 16-128); piperacillin-tazobactam (P/T4; 16/4-128/4), streptomycin (STR; 8-128), tetracycline (TET; 4-32), gentamicin (GEN; 2-16), and amox/clav 2∶1(AUG2; 8/4-32/16). Escherichia coli ATCC 25922 and E.coli ATCC 35218 were used as quality control strains. MICs were recorded as the lowest concentration of an antimicrobial that completely inhibited bacterial growth [33]. Resistance breakpoints published by the Clinical and Laboratory Standards Institute were used [33]. Breakpoints not available from CLSI (streptomycin, apramycin, penicillin) were derived from ranges used in similar studies [9], [10], [34], [35]. Multidrug resistance (MDR) was defined as resistance to two or more antibiotics.

Statistical analyses

Descriptive statistics were used to compare the percentage of isolates demonstrating intermediate resistance or resistance to tested antibiotics at each sampling site and sampled month, as well as the average number of antibiotics that V. vulnificus and V. parahaemolyticus isolates were resistant to at each sampling location and during each month. Nonparametric Friedman two-way Analysis of Variance (ANOVA) was used to determine effects related to sampling site and month sampled. For samples for which month influenced percent resistance, stratified Kruskal-Wallis one-way ANOVA and pairwise post-hoc tests were conducted for each month separately to evaluate differences in the occurrence of antimicrobial susceptibility between strains that carried or did not carry virulence genes. All statistical analyses were performed using StataIC 12 and p-values of ≤0.05 were defined as statistically significant. (StatCorp LP, College Station, TX).

Results

Physical, chemical and bacterial water quality

Water temperature, pH, and dissolved oxygen (DO) were uniform across the three sampling locations ( Table 3). Average salinity (± standard deviation) in St. Martin's River (24.5 ppt (±1.07)) was approximately double that of the Pocomoke Sound (10.5 ppt (±0.54)) and Sandy Point State Park (9.4 ppt (±0.72)) sampling sites. Water depth at the Pocomoke Sound was approximately double that of Sandy Point State Park and three to four-fold that of St. Martin's River.

Enterococci counts (colony forming units (CFU)) per 100 ml −1 were uniformly low at Sandy Point during each sampling time point and below the single sample regulatory closure level of 10 ⁴ CFU per 100 ml −1 [36]. On one sampling occasion in St. Martin's River (August) enterococci counts exceeded closure levels ( Table 3).

Presumptive Vibrio colonies isolated during this study indicated that V. vulnificus and V. parahaemolyticus were present in all tested water samples ( Table 3). One-hundred twenty V. vulnificus and 77 V. parahaemolyticus isolates were purified, confirmed via PCR and tested for antimicrobial susceptibility.

Vibrio species and virulence identification

Sequence analysis (16S rRNA) of a selected subset of tested Vibrio isolates confirmed all isolates ( Figure 2), except for two isolates with sequences similar to Photobacterium damselae. Virulence testing of all isolates identified three V. vulnificus isolates positive for vcgC, one V. parahaemolyticus isolate positive for tdh, and one V. parahaemolyticus isolate positive for trh.

Prevalence of antimicrobial resistance in V. vulnificus

All tested V. vulnificus isolates (n = 120) were susceptible to 14 of the 26 antibiotics tested, including the following drug classes that are recommended by the Centers for Disease Control and Prevention (CDC) for the treatment of V. vulnificus infections: tetracyclines, quinolones, and folate pathway inhibitors ( Table 4, Figure 3). With regard to CDC recommended antimicrobial agents, 2% of the tested isolates exhibited intermediate resistance against ceftazidime, a third generation cephalosporin. Within the aminoglycoside class of antibiotics, isolates exhibited resistance to apramycin (1%) and streptomycin (4%). Intermediate resistance was expressed against amikacin (1%), apramycin (5%) and streptomycin (8%). Gentamicin was the only tested aminoglycoside to which all V. vulnificus isolates were completely susceptible. The aminoglycoside streptomycin was associated with the highest percentage of resistance (7% of all tested isolates) and second highest percentage of intermediate resistance (17% of all tested isolates) out of all of the antimicrobials tested. Isolates displayed the highest percentage of intermediate resistance (78% of all isolates) to chloramphenicol.

Antimicrobial resistance in vcgC+ V. vulnificus

Of the three isolates positive for the virulence correlated gene clinical variant ( vcgC), none displayed resistance to any of the tested antibiotics, but all three expressed intermediate resistance (100%) to chloramphenicol.

Prevalence of antimicrobial resistance in V. parahaemolyticus

All tested V. parahaemolyticus isolates were susceptible to 11 of the 26 tested antibiotics and four (carbapenems, tetracyclines, quinolones and folate pathway inhibitors) of the eight tested antimicrobial classes ( Table 4). Conversely, 96% of isolates were characterized by intermediate resistance to chloramphenicol, followed by ampicillin (25%), cephalothin (17%), penicillin (16%) and cefuroxime sodium (14%). A high percentage of resistance was observed against some of the penicillins (penicillin (68%); ampicillin (53%)), while a low percentage of resistance was seen against piperacillin (4%) and streptomycin (4%).

Antimicrobial resistance in tdh/trh+ V. parahaemolyticus

One V. parahaemolyticus isolate was tdh+ and one isolate was trh+. The trh+ V. parahaemolyticus isolate was resistant to ampicillin and penicillin and expressed intermediate resistance to chloramphenicol. The tdh+ V. parahaemolyticus isolate was resistant to ampicillin, ampicillin-sulbactam, penicillin, piperacillin-tazobactam, and amoxicillin-clavulanic acid and expressed intermediate resistance to chloramphenicol.

Impact of sampling site and month on antimicrobial resistance

Friedman two-way ANOVA:.

The month when sampling occurred significantly influenced rates of antibiotic resistance and intermediate resistance among V. parahaemolyticus ( p<0.0001, p<0.0001, respectively), as well as resistance and intermediate resistance among V. vulnificus ( p = 0.0008, p = 0.0098, respectively). After adjusting for the repeated measures over time (month), sampling site also significantly influenced resistance and intermediate resistance among V. vulnificus ( p = 0.0321, p = 0.0029, respectively), but not among V. parahaemolyticus ( p = 0.6133, p  = 0.7660, respectively).

Kruskal-Wallis one-way ANOVA:.

As there was a significant month effect in the Friedman two-way ANOVA for both V. vulnificus and V. parahaemolyticus isolates expressing antibiotic resistance and intermediate resistance, stratified Kruskal-Wallis one-way ANOVA and pairwise post-hoc tests were conducted on the sampling site differences for each month separately. Results showed no significant difference between sampling sites by month for V. vulnificus or V. parahaemolyticus expressing resistance (July, August, September; ( p = 0.5340, 0.2801, 0.4966); ( p = 0.7246, 0.9448, 0.6809), respectively) or V. parahaemolyticus expressing intermediate resistance ( p = 0.5959, 0.8046, 0.2135). After testing V. vulnificus expressing intermediate resistance for sampling site differences by each month separately, it was determined that there was a significant sampling site effect only in July ( p = 0.035). Post-hoc testing clarified that the site, St. Martin's River, was different from Sandy Point during the month of July, with reduced intermediate resistance among V. vulnificus isolates recovered from St. Martin's River ( p  = 0.0166).

Kruskal-Wallis one-way ANOVA further elucidated that there was no significant difference in the median intermediate resistance or resistance patterns during the sampling period when St. Martin's River (August) ( p  = 0.44) had higher levels of bacterial-indicator species.

Clinical V. parahaemolyticus

Clinical isolates tested displayed comparable resistance profiles to environmental isolates tested ( Table 5). However, environmental isolates demonstrated intermediate resistance and resistance to a greater range of antibiotics (15 antibiotics in four classes) when compared to clinical isolates (five antibiotics in three classes). Yet, based on analyses with two-sample proportion tests, the overall percentage of resistance and intermediate resistance (% =  number of antimicrobials to which there was demonstrated resistance/number of total antimicrobials tested) among clinical isolates was not statistically different ( p  = 0.511, 0.430; respectively) from that of environmental isolates.

Overall resistance profiles

The percentage of isolate resistance, defined as resistance to any one antibiotic (AR) or resistance to two or more classes of antibiotics is depicted in Table 6. Resistance profiles were comparable for isolates with and without detected virulence genes (6A), isolates from varying sampling locations (6B), and isolates recovered in different sampling months (6C) for both V. parahaemolyticus and V. vulnificus.

Discussion

Treatability of Chesapeake Bay related Vibrio illness

Vibrio vulnificus and V. parahaemolyticus are the causative agents for wound infections, primary septicemia, and gastroenteritis related to seafood and seawater exposure [37]. While antibiotic treatment is not typically necessary for gastroenteritis, it is required for wound infection and primary septicemia caused by both Vibrio species analyzed in this study. Most isolates tested in this study were susceptible to the antimicrobial agents recommended by the CDC for clinical treatment. Treatment recommendations for Vibrio infections include tetracyclines (doxycycline, tetracycline), flouroquinolones (ciprofloxacin, levofloxacin), third-generation cephalosporins (cefotaxime, ceftazidime, ceftriaxone), aminoglycosides (amikacin, apramycin, gentamicin, streptomycin) and folate pathway inhibitors (trimethoprim-sulfamethoxazole) [38], [39]. The CDC recommends a treatment course of doxycycline (100 mg PO/IV twice a day for 7-14 days) and a third-generation cephalosporin (e.g.,ceftazidime 1-2 g IV/IM every eight hours), although they state that single agent regimens employing a fluoroquinolone have been reported to be at least as effective in an animal model as combination drug regimens with doxycycline and a cephalosporin [39].

All tested V. vulnificus isolates were susceptible to third and fourth generation cephalosporins, although two V. parahaemolyticus isolates (3%) demonstrated intermediate resistance to cefotaxime, a third-generation cephalosporin, and two isolates demonstrated a degree of resistance to cefepime, a fourth-generation cephalosporin. While the percentage of isolates expressing intermediate resistance and resistance to the newer generation cephalosporins was relatively low, these antibiotics are considered to be some of the best defenses against the severe infections that these organisms can elicit, so even a small percentage of resistant isolates could be cause for concern [39].

Due to the contraindication of doxycycline and fluoroquinolones in children, a combination of trimethoprim-sulfamethoxazole and an aminoglycoside antibiotic is recommended [39]. Given that three of the four tested aminoglycosides (amikacin, apramycin, streptomycin) were associated with intermediate resistance or resistance (e.g., streptomycin intermediate resistance and resistance in V. vulnificus: 17%, 7%, respectively; V. parahaemolyticus: 8%, 4%, respectively) in a subset of isolates, this may be a resistance pattern of concern. Conversely, for the aminoglycoside, gentamicin, all tested isolates were fully susceptible. Based on these data, physicians in the Bay region may consider focusing on gentamicin as the aminoglycoside of choice in multi-drug treatment regimens for Vibrio infections contracted by children recreating in the Chesapeake Bay.

Comparison to other studies of V. vulnificus and V. parahaemolyticus antimicrobial susceptibility

The percent resistance among Vibrios, in this study was comparable to a similar study conducted on Vibrios isolated from Gulf Coast oysters in Louisiana [11]. Han et al. (2007) also found higher levels of resistance among V. parahaemolyticus compared to V. vulnificus isolates. In addition, ampicillin was the only tested antimicrobial in the Gulf Coast study to which a large percentage of V. parahaemolyticus isolates demonstrated intermediate resistance to resistance (∼81% of all tested isolates). This trend was seen as early as the 1970s in a study that tested resistance of V. parahaemolyticus to ampicillin and β-lactamase inhibitors [40], where over 90% of isolates were found to be resistant to ampicillin. In contrast to the present study, Han et al. (2007) found no resistance in either Vibrio species to chloramphenicol, cefotaxime, or ceftazidime, while we observed intermediate resistance against these three antimicrobial agents among a subset of V. vulnificus (78%, 0%, 2%, respectively) and V. parahaemolyticus (96%, 3%, 0%, respectively).

Our findings are also in partial agreement with two large studies of V. vulnificus and V. parahaemolyticus isolates originating from the Georgia and South Carolina coastline of the United States [10]. While our Chesapeake Bay isolates did not show the same high prevalence of antimicrobial resistance, the antimicrobial agents to which isolates displayed resistance were similar (i.e., amoxicillin, apramycin, penicillin and streptomycin for V. parahaemolyticus). V. vulnificus isolates demonstrated similar resistance profiles, particularly with regard to percent intermediate resistance and resistance to the penicillin class and cefoxitin. Baker-Austin et al. (2009) reported higher percent intermediate resistance and resistance among V. vulnificus against apramycin and streptomycin compared to that of the isolates reported in our study. In addition, key antimicrobials to which V. parahaemolyticus isolates from Georgia/South Carolina displayed susceptibility were also found to be susceptibile in our study (i.e., ceftriaxone, ciprofloxacin, imipenem, ofloxacin, meropenem, tetracycline), except in the case of chloramphenicol, for which no or low ( V. vulnificus) resistance was observed in the Georgia and South Carolina study. In contrast to this study, Baker-Austin et al. (2009) found only one (<0.01%) V. vulnificus isolate to be completely susceptible to all antimicrobials tested, while the present study found 15 (12.5%) isolates to be susceptible to all tested antimicrobials.

A recent study of antimicrobial susceptibility in toxigenic and non-toxigenic V. parahaemolyticus isolates from shellfish and clinical samples in Italy [12] produced interesting comparisons to our findings. Similar to other studies, no intermediate resistance or resistance to chloramphenicol was found in Italian V. parahaemolyticus samples, whereas our study found high levels of intermediate resistance to this antibiotic. The Italian study found isolates to be 100% (n = 170) resistant to ampicillin, while our study detected 53% (n = 40) resistance and 25% (n = 25) intermediate resistance. Resistance to cefotaxime was found in approximately 20% (n = 21) of Italian samples, compared to 0% resistance and 4% (n = 3) intermediate resistance in this study. In contrast to our study, which detected full susceptibility to ciprofloxacin, the Italian study found resistance (9%, n = 10), particularly in clinical samples. Comparable susceptibility patterns are reported in these studies for trimethoprim-sulfamethoxazole, doxycycline and tetracycline, as all V. parahaemolyticus tested in this study were fully susceptible to these three antibiotics, while Italian isolates displayed intermediate resistance for trimethoprim-sulfamethoxazole (4%, n = 4) and tetracycline (11%, n = 12).

Sampling sites and influences of pollution

Each sampling site included in this study has a history of water pollution. Sandy Point State Park has historically been a site of low bacteriological water quality and is adjacent to the Magothy River, a site where there have been numerous wastewater treatment overflows. The Pocomoke River is located adjacent to many agricultural operations, including poultry concentrated animal feeding operations (CAFOs), which may increase the introduction of antimicrobial residues into the waterway due to runoff of fecal matter contaminated with antimicrobials used in poultry production [41]. Finally, St. Martin's River is adjacent to many homes on septic systems, notorious for leakage [42]. While each of the sampling sites has a history of contamination that may increase the incidence of antimicrobial residues and associated changes in resident bacteria in the estuarine environment, this study only detected a small difference in levels of antibiotic resistance between sites. Specifically, in the month of July, V. vulnificus recovered from St. Martin's River expressed higher percentages of intermediate resistance compared to isolates recovered from Sandy Point.

However, it should be noted that this study was limited by the inability to culture V. vulnificus and V. parahaemolyticus from areas presumed to be void of contamination by human or animal sewage or industrial pollution. Due to this limitation, resistance levels detected at each of the three studied sites could not be compared to that of a local "pristine" site and this likely reduced our ability to differentiate pollution-related resistance from naturally occurring resistance among tested isolates.

Antimicrobial susceptibility as compared to enterococci concentrations

In this study, we also tested for enterococci as an indicator of fecal contamination in order to specifically evaluate whether areas that were characterized by higher levels of possible fecal contamination were also marked by higher levels of antibiotic resistance. We observed a range of enterococci concentrations over the course of the study, although most sampling sites were within the range of acceptable water quality for recreation on each sampling date. Interestingly, concentrations of enterococci were not correlated with percentages of antibiotic resistance in the studied environments. During the one instance that the geometric mean of enterococci was higher than regulation limits, there was no discernible difference in levels of resistance among isolates originating from that site (St. Martin's River - August). This is counter to previous observations where percent antimicrobial resistance was elevated at sites contaminated with higher levels of enterococci that may have originated from fecal waste of humans [43] and animals [44].

Conclusions

This study represents the first investigation of antimicrobial susceptibility of Vibrio species recovered from the Chesapeake Bay and provides a baseline against which future studies can be compared to determine whether susceptibilities change over time. Isolates tested in this study displayed high intermediate resistance to chloramphenicol, when compared to similar studies. Isolates' intermediate resistance and resistance to some aminoglycosides should be noted because these antibiotics are used to treat pediatric Vibrio illnesses originating from the Chesapeake waters or seafood. Low-level intermediate resistance and resistance to third and fourth generation cephalosporins may also limit treatment effectiveness and should be monitored. As most of the antimicrobial agents recommended for treatment of Vibrio illnesses by CDC were fully effective against V. vulnificus and V. parahaemolyticus isolated from the Chesapeake Bay, treating infections contracted from the Bay, at least in adults, is not likely to be problematic. Based on our data, treatment of pediatric illnesses may benefit from the use of trimethoprim-sulfamethoxazole and the aminoglycoside, gentamicin, which was the only aminoglycoside that was 100% effective against Vibrios recovered in this study.

Acknowledgments

We would like to thank Lara Kleinfelter and Bryan Shaw for laboratory assistance, Brian Sturgis and Eric Sherry (National Park Service at Assateague Island) and Dr. Eric May (University of Maryland Eastern Shore) for assistance in water collection and boat operation, Sabeena Nazar for assistance in sequencing isolates (BioAnalytical Services Laboratory, Institute of Marine and Environmental Technology), Matt Rhodes for technical assistance (Cooperative Oxford Laboratory), Dr. Craig Baker-Austin (Centre for Environment, Fisheries and Aquaculture Science, United Kingdom) for useful conversations regarding antimicrobial susceptibility testing, and Maryland Department of Health and Mental Hygiene scientists (Alvina Chu, Emily Ricotta, Dr. Robert Myers, Dr. Jafar Razeq, Celere Leonard) for sharing clinical isolates and associated metadata.

Author Contributions

Conceived and designed the experiments: KSS JMJ BCC ARS. Performed the experiments: KSS RERG ARS. Analyzed the data: KSS XH. Contributed reagents/materials/analysis tools: KSS RERG JMJ BCC ARS. Wrote the paper: KSS ARS BCC.

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Antimicrobial Susceptibility of Vibrio vulnificus and Vibrio parahaemolyticus Recovered from Recreational and Commercial Areas of Chesapeake Bay and Maryland Coastal Bays

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Vibrio vulnificus and V. parahaemolyticus in the estuarine-marine environment are of human health significance and may be increasing in pathogenicity and abundance. Vibrio illness originating from dermal contact with Vibrio laden waters or through ingestion of seafood originating from such waters can cause deleterious health effects, particularly if the strains involved are resistant to clinically important antibiotics.

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Incidence and prevalence of Vibrio parahaemolyticus in seafood

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Incidence and prevalence of Vibrio parahaemolyticus in seafood

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Incidence and prevalence of Vibrio parahaemolyticus in seafood: a systematic review and meta-analysis

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Vibrio parahaemolyticus is an important seafood borne human pathogen worldwide due to it occurrence, prevalence and ability to cause gastrointestinal infections. This current study aim at investigating the incidence and prevalence of V. parahaemolyticus in seafood using systematic review-meta-analysis by exploring heterogeneity among primary studies.

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Vibrio parahaemolyticus - an overview | ScienceDirect Topics

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Vibrio parahaemolyticus is the most commonly isolated noncholera vibrio. Like V. cholerae, it is found in marine environments. Vibrio parahaemolyticus causes gastroenteritis as a result of consumption of raw or partially cooked food, particularly seafood. V. parahaemolyticus accounts for a large proportion of food-borne illness in Japan, where consumption of seafood is common.

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The graph shows how the antibiotic resistance of common infections has been rising exponentially.

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Vibrio bacteria in Jacksonville Florida oysters and water (Science on the St.  Johns)

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MIcrobiology Society publication on antibiotic resistance

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MIcrobiology Society publication on antibiotic resistance

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02 June 2017 publication

Increasing levels of antimicrobial resistance (AMR) mean that conditions such as tuberculosis, HIV and malaria are again becoming increasingly difficult to treat. Antimicrobial resistance also compromises the success of surgery, organ transplantation, childbirth and chemotherapy, making many treatments more risky. Antimicrobial-resistant infections cause over 700,000 deaths worldwide annually, and this number is set to grow. This briefing looks at the threat posed by AMR, and the human activities that are making the problem worse - such as the overuse of antibiotics in medicine, the proliferation of resistant microbes in care homes and hospitals, and the use of medically vital antimicrobials as growth promoters or prophylactics in livestock and crop production. The briefing also considers the actions needed to tackle the spread of AMR, including reducing non-essential usage of antimicrobials and investment in research to develop new antimicrobials and alternative antimicrobial therapies, and the steps that are already being taken by international bodies such as the WHO.

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02 June 2017 publication Increasing levels of antimicrobial resistance (AMR) mean that conditions such as tuberculosis, HIV and malaria are again becoming increasingly difficult to treat. Antimicrobial resistance also compromises the success of surgery, organ transplantation, childbirth and chemotherapy, making many treatments more risky.

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World Antibiotic Awareness Week 2017: Always seek the advice of a healthcare professional before taking antibiotics.

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An undergraduate collaborative project with the Microbiology Society to search for new antibiotic compounds from aquatic sediments
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Each November, World Antibiotic Awareness Week (WAAW) aims to increase global awareness of antibiotic resistance and to encourage best practices among the general public, health workers and policy makers to avoid the further emergence and spread of antibiotic resistance.

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Bacteria can be resistant to new antibiotics..

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Bacteria can be resistant to new antibiotics..

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Perhaps the chief poster child of antibiotic resistance is methicillin-resistant Staphylococcus aureus, or MRSA. The bacterium is impervious to a suite of antibiotics, and can cause blood infections, pneumonia...even death. And you'd assume that it developed its namesake resistance to methicillin...by being exposed to methicillin.

But that doesn't seem to be the case. Instead, the culprit for resistance appears to be an earlier, and chemically related antibiotic: penicillin. "We think it's a very early use of penicillin that forced the strains to pick up these mechanisms." Matthew Holden, a molecular microbiologist at the University of Saint Andrews in Scotland.

Holden and his team analyzed the genomes of freeze-dried strains of MRSA bacteria, from the 1960s through the '80s. "In effect what we were doing was sort of genomic archaeology, in looking at the genomes, and comparing the variation and using that information to effectively reconstruct the evolutionary histories."

What they found was that the Staph bacteria seem to have acquired the methicillin-resistance gene in the mid-1940s-about 15 years before methicillin even hit the market. And they determined that it was the widespread use of penicillin that led to that adaptation. The results are in the journal Genome Biology. [Catriona P. Harkins et al., Methicillin-resistant Staphylococcus aureus emerged long before the introduction of methicillin into clinical practice]

Methicillin was introduced in the U.K. in 1959. Less than a year later, resistance was first reported-resistance that it appears now was already baked into the staph strains. Looking ahead, Holden says we'd do well to vigilantly monitor the genetics of circulating strains-to find out which bugs may be armed to battle our newest antibiotics as soon as they're developed.

-Christopher Intagliata

[ The above text is a transcript of this podcast.]

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Bacteria Can Be Resistant to Brand-New Antibiotics

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Perhaps the chief poster child of antibiotic resistance is methicillin-resistant Staphylococcus aureus, or MRSA. The bacterium is impervious to a suite of antibiotics, and can cause blood infections, pneumonia...even death. And you'd assume that it developed its namesake resistance to methicillin...by being exposed to methicillin. But that doesn't seem to be the case.

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Interesting information on how bacteria work together!

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It's one of the underwater world's classic partnerships: clownfish and [ Finding Nemo clip: "Anemonenene. Anemonem. Monemone."] Nemo hides out in the anemone, which helps keep predatory fish at bay.

"But at the same time there are other fish species that will kind of nibble on the sea anemones, and the clownfish will actually go out and scare them away." Jeff Gore, a biophysicist at MIT. "So in this case there's a cross-protection mutualism between these two species in which they help to avoid predation."

Such cross-protection is usually seen between two animals. But Gore studies the same sort of mutualism in microbes. He and his team demonstrated the first experimental example of that cross-protective relationship in drug-resistant microbes, using two strains of antibiotic-resistant E. coli bacteria: one resistant to ampicillin, the other to chloramphenicol.

The researchers grew the bacteria together in a test tube, in the presence of both antibiotics. And rather than succumbing to the drugs, each bacterial strain deactivated one of the two antibiotics-thus protecting the other strain. That activity led to a stable coexistence over time. Which Gore says could in theory give the bugs an opportunity to swap resistance genes, through what's called horizontal gene transfer-one bacterium donates genetic material to another. Any such transfer could make either or both strains individually resistant to both types of antibiotics. The findings are in the Proceedings of the National Academy of Sciences. [Eugene Anatoly Yurtsev, Oscillatory dynamics in a bacterial cross-protection mutualism]

The same story might play out in our guts, too-but it's too early to translate it into medical guidance. "It's always important to remember that just because we see a particular dynamic in the laboratory doesn't mean we should immediately go and change our clinical dosing practices. But then those sorts of results are then used as a guide for the kinds of phenomena that clinicians may want to watch out for in their circumstances." 'Cause the more we know about this bacterial buddy system, the better we may be at breaking it up if they team up against us.

-Christopher Intagliata

[ The above text is a transcript of this podcast.]

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Alliance of Bacterial Strains Disables Antibiotics

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It's one of the underwater world's classic partnerships: clownfish and [ Finding Nemo clip: "Anemonenene. Anemonem. Monemone."] Nemo hides out in the anemone, which helps keep predatory fish at bay. "But at the same time there are other fish species that will kind of nibble on the sea anemones, and the clownfish will actually go out and scare them away."

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Questions and answers on antibiotic resistance

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Questions and answers on antibiotic resistance

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Received: 13 September 2010

Accepted: 14 September 2010

Published: 20 September 2010

Is antibiotic resistance inevitable?

Yes. Historically, the discovery of the sulfa drugs in the 1930 s and the subsequent development of penicillin during World War II ushered in a new era in the treatment of infectious diseases. Infections that were common causes of death and disease in the pre-antibiotic era - rheumatic fever, syphilis, cellulitis and bacterial pneumonia - became treatable, and over the next 20 years most of the classes of antibiotics that find clinical use today were discovered and changed medicine in a profound way. The availability of antibiotics enabled revolutionary medical interventions such as cancer chemotherapy, organ transplants and essentially all major invasive surgeries from joint replacements to coronary bypass. Antibiotics, though, are unique among drugs in that their use precipitates their obsolescence. Paradoxically, these cures select for organisms that can evade them, fueling an arms race between microbes, clinicians and drug discoverers.

How do successful antibiotics work and what is the basis of resistance to them?

Antibiotics target essential bacterial physiology and biochemistry, causing microbial cell death or the cessation of growth. There are five major antibiotic targets: the bacterial cell wall, the cell membrane, protein synthesis, DNA and RNA synthesis, and folic acid (vitamin B9) metabolism (Figure 1). These bacterial targets are different or nonexistent in eukaryotic cells (including those of humans), which means that antibiotics are relatively nontoxic drugs. For example, the β-lactam antibiotics such as penicillins, cephalosporins and carbapenems block the synthesis of the bacterial cell wall. This structure is absent in higher organisms but is essential for bacterial survival. The bacterial ribosome is the target of the tetracycline, aminoglycoside, macrolide and other antibiotics, and is sufficiently different from the eukaryotic ribosome that cross-inhibition does not occur.

Resistance to antibiotics occurs through four general mechanisms: target modification; efflux; immunity and bypass; and enzyme-catalyzed destruction (Figure 1). Target modification can occur through mutation of the targets themselves - for example, the topoisomerases that are the target of the fluoroquinolone antibiotics - or by the production of enzymes that modify antibiotic targets, as, for example, in ribosomal methylation. Vancomycin resistance is a version of target modification where new biosynthetic machinery is engaged to alter cell-wall structure. Efflux occurs through a large family of protein pumps that eject antibiotics from inside the cell. In immunity, antibiotics or their targets are bound by proteins that prevent the antibiotic binding to its target. Arguably, the most specific and evolved mechanism of antibiotic resistance are enzymes that recognize antibiotics and modify them in such a way as to eliminate the functional characteristics that enable them to interact with their targets. For example, β-lactamases hydrolytically cleave the core β-lactam ring that is characteristic of the class and essential to antibiotic action.

Has the problem of antibiotic resistance worsened over time?

Resistance to antibiotics was recorded even before the first clinical use of penicillin in the early 1940 s. In the intervening years, resistance to all classes of antibiotics has emerged, and there are no antibiotics for which resistance does not exist. There are two general strategies for resistance. One comprises mechanisms that transfer resistance vertically from a bacterium to its progeny. Examples are mutations in chromosomal genes that give rise to drug-insensitive products, such as the point mutations in the genes encoding DNA gyrase or topoisomerase IV that result in resistance to fluoroquinolone antibiotics such as ciprofloxacin. The second strategy includes the actions of genes that can be transmitted both vertically to progeny and horizontally to other bacteria, even those of different genera. These genes are located on mobile genetic elements such as plasmids, which can carry one or more resistance genes. Many of the β-lactamase genes that confer resistance to the penicillin, cephalosporin, penem and monobactam antibiotics are located on such elements, as is the glycopeptide-resistance gene cluster vanHAX, which provides resistance to vancomycin. The prevalence and mobility of resistance genes in previously sensitive pathogenic bacteria has now reached crisis levels in many cases because new antibiotics are no longer being developed at a rate that can keep pace with microbial evolution.

In the past two decades we have witnessed:

* the rise of so-called extended spectrum β-lactamases (ESBLs), which are mutants of enzymes that previously could only inactivate penicillins but now have gained activity against many cephalosporins;

* carbapenemases such as KPC and NDM-1 that inactivate all β-lactam antibiotics;

* plasmid-mediated (and thus horizontally disseminated) resistance to fluoroquinolone antibiotics;

* the spread of virulent MRSA (methicillin-resistant Staphylococcus aureus) in the community;

* the rise of multi-drug resistant Neisseria gonorrhoea;

* the emergence and global dissemination of multi-drug resistant Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae and Enterobacteriaceae;

* the spread of extensively drug resistant Mycobacterium tuberculosis;

* the development of resistance to the two newest antibiotics to be approved for clinical use - daptomycin and linezolid.

Resistance is relentless and unavoidable as long as we use antibiotics.

Where does resistance come from?

Antibiotic resistance is the evolutionary response to the strong selective pressure that results from exposure to these compounds. The horizontal dissemination of resistance genes into bacterial species and genera that are not themselves intrinsically resistant, as well as the maintenance of resistance mutations vertically through populations is likely to be the result of contemporary use of these drugs in the clinic and on the farm. Support for this hypothesis is the infrequency of antibiotic resistance in collections of pathogenic bacteria that pre-date the antibiotic era.

Nevertheless, antibiotic resistance is a natural phenomenon. It has been recognized for decades that the resistance mechanisms that have emerged in the clinic parallel those that are intrinsic to the bacteria that produce antibiotics. Recent studies of non-pathogenic soil bacteria have revealed that the majority of environmental bacteria tested are multi-drug resistant. This reflects the fact that these microbes live and have evolved in an environment where small bioactive molecules, some toxic, some benign, are plentiful and diverse. Bacteria have simply evolved to interact with them and control their biological effects. Pathogens, on the other hand, are often more virulent forms of our commensal bacteria and simply have not been exposed to the diversity and types of small molecules found in the environment; as a result, they have not required the gamut of resistance genes found in some environmental bacteria.

Furthermore, the genes and proteins responsible for resistance in environmental bacteria are homologous to those found circulating in pathogens, strongly suggesting contemporary horizontal gene transfer. Opportunistic pathogens with environmental reservoirs - for example, P. aeruginosa and A. baumannii - are highly drug resistant and have a remarkable capacity to acquire new resistance genes. The environment is therefore a large reservoir of potential resistance genes: the environmental 'resistome'.

Given the vast numbers of bacteria on the planet and the massive selection pressure provided by antibiotics, the movement of antibiotic-resistance elements from benign, but resistant, microbes into previously susceptible pathogens is simply a matter of time and opportunity.

Can anything be done to slow down the emergence of resistance?

Antibiotics themselves are the source of the evolutionary pressure that eventually renders them obsolete. Limiting exposure of microbes to antibiotics therefore makes good sense to reduce the opportunity for the selection and dissemination of resistance. The inappropriate use of antibiotics by clinicians and the agricultural community needs to be curtailed. Over the past several years, the medical community in particular has made concrete efforts to curb the improper use of antibiotics. The European Union has taken the lead in limiting the non-therapeutic use of antibiotics in food animals. Robust surveillance networks that span the clinic and the farm need to be supported in order to monitor the impact of resistance and the emergence of new threats in real time. In North America, efforts such as the Strategies to Address Antimicrobial Resistance Act seek to diminish antibiotic use in agriculture and improve surveillance. Furthermore, there have been several successful campaigns to educate the public on the importance of antibiotics and the proper use of these drugs. While none of these efforts is perfect, there is much to be celebrated and encouraged.

These measures all serve to reduce antibiotic use and, as a result, delay the emergence of resistance. Furthermore, by decreasing selection pressure, the opportunity for the rise of particularly clinically challenging or virulent organisms should be lessened. All strategies that reduce the incorrect use of antibiotics are welcome, but in the end new drugs will always be needed because of the inevitability of resistance.

Unfortunately, in the developing world, access to antibiotics is frequently not regulated and their use in agriculture is often rampant. These facts make antibiotic stewardship especially challenging. In an era of rapid intercontinental travel, pathogens are no longer geographically contained and can move from country to country with ease. The recent examples of transcontinental spread of the severe acute respiratory syndrome (SARS) virus from Guangdong province in China to Hong Kong and then Canada in 2003, and the NDM-1 carbapenemase, which inactivates all β-lactam antibiotics and appears to have originated in the Indian subcontinent but is now found in North America, the UK and Europe, make the point.

What about new antibiotics?

The growing problem of resistance fuels a continuous need for new antibiotic drugs. The enterobacteria that produce carbapenemase are just one example of antibiotic-resistant enterobacteria. Other Gram-negative pathogens resistant to virtually all antibiotics include multi-drug resistant A. baumannii and P. aeruginosa. The expanding problem of MRSA, and the global challenge of extensively drug-resistant M. tuberculosis (also called extreme drug-resistant M. tuberculosis), require new therapies.

There are some promising new candidates on the horizon, especially for the treatment of infections caused by Gram-positive pathogens such as MRSA and enterococci. As already mentioned, two new drugs active against this microbial spectrum - daptomycin and linezolid - have been introduced in the past decade. Tigecycline, a third-generation semi-synthetic tetracycline antibiotic approved in 2005, also has activity against MRSA. The semi-synthetic glycopeptide antibiotic telavancin recently received approval in the United States and the fifth-generation cephalosporin ceftobiprole is available in some European countries and Canada. However, there are few candidates in late-stage clinical trials suitable for the problem of Gram-negative pathogens. Here, often the choice of last resort is colistin, an antibiotic discovered more than 50 years ago and seldom used in the past because of adverse affects, including kidney toxicity; however, it is now increasingly used.

Why are there so few new drugs?

There are a number of reasons, some economic, for the paucity of new antibiotics. They include challenging and shifting processes of government regulatory approval that add to the risk for the pharmaceutical industry. Furthermore, considerations of return on investment favor drugs for chronic diseases, which are taken by patients over long periods of time, often decades. In contrast, antibiotics cure disease and are taken for short periods of time.

Other reasons for the decline in antibiotic discovery and development are scientific. The first wave of antibiotics discovered five decades ago have been termed the 'low hanging fruit'. Despite the discovery of numerous compounds with antibiotic properties in the years since, few have had the requisite properties to become drugs. Most antibiotics are natural products or their derivatives that have been isolated from soil bacteria. Some researchers have suggested that this source might now be exhausted.

Furthermore, the promise of the genomic era and the reality of hundreds of available bacterial genomes have so far failed to deliver the hoped-for new molecular targets for antibiotics. Other new technologies, such as high-throughput screening of libraries of synthetic molecules, have not resulted in new drugs, although this may reflect a poor choice of chemical classes in the screens, emphasizing molecules more active in human biology than as antibiotics. Test compounds were often skewed in favor of small lipophilic molecules with physical properties meeting the criteria of Lipinski's Rule of 5. However, though helpful in assessing the prospect of a compound to be an orally active drug for human disease, this strategy has been shown to fail when searching for antibiotics.

So what would be suitable chemical matter for leads?

Well, natural chemicals have significant advantages. Although the first antibiotics introduced into the clinic were the synthetic sulfonamides, the majority of antibiotics in current clinical use are bacterially produced natural products or their derivatives; only a few are completely synthetic in origin. The reasons for this in part reflect the history of antibiotic discovery post-penicillin, and the relative ease of discovery of suitable molecules through screening the products of soil microbes compared with libraries of synthetic compounds. Many of these 'natural' antibiotics have desirable drug-like qualities, such as good bioavailability, the ability to cross the cell membrane (and outer membrane in the case of compounds with Gram-negative activity) and the ability to evade efflux systems, and chemical structures that favor binding to vital cellular targets, supporting the idea that natural products encompass privileged structures in antibiotic drug discovery. However, the increasing difficulty of identifying new chemical compounds with equally suitable drug-like characteristics from natural sources has caused natural-product-based screening programs to fall out of favor in many pharmaceutical firms over the past few decades.

Instead, the ability of parallel synthesis methods to generate hundreds of thousands of synthetic molecules suitable for modern high-throughput screening has shifted the focus in favor of synthetic molecules in commercial antibacterial drug discovery. The advantages of synthetic compounds are not insignificant: pure lead molecules can easily be produced in quantity and quality suitable for clinical trials, and are relatively easily modified to improve target affinity. However, after two to three decades of emphasis on such molecules and millions of dollars spent on high-throughput in vitro and cell-based screens, no new synthetic antibiotics have emerged. Linezolid, the one synthetic antibiotic to be brought to market in the past decade, was discovered using traditional medicinal chemistry in a research program with a plant-disease focus in the early 1980 s.

So does that mean natural products are best after all?

They do have great advantages, although a direct comparison of the success and failure of synthetic as against natural product libraries is unfair. Microbial natural products have evolved over millennia to interact with biological molecules, whereas the synthetic chemical libraries used in antibiotic drug-discovery screens were generally developed with a focus on eukaryotic drug-discovery campaigns, as noted earlier. Efforts to develop physical-property rules for antibiotics and to incorporate natural-product-like chemical complexity in libraries of synthetic chemicals will no doubt improve success in identifying new synthetic antibiotic leads.

Ironically, at the same time that the pharmaceutical industry was abandoning natural-product libraries, university researchers were making remarkable advances in understanding the molecular details of natural-product biosynthesis by bacteria. Many bacteria, especially the actinomycete group of common environmental bacteria, are prodigious producers of natural products. These are termed secondary metabolites to contrast with molecules of primary metabolism, such as carbohydrates, amino acids and so on. Secondary metabolites range in molecular weight from around 100 daltons (Da) to up to 5,000 Da and they have diverse biological activities, including induction of cell death (antibiotics such as tetracycline, vancomycin and daptomycin, and anticancer agents such as adriamycin), iron sequestration (for example, enterobactin), facilitation of cell-cell communication (γ-butyrolactones), protection from oxidizing agents (phenazines), and a host of others.

The bacterial natural products that are most important as antibiotics include polyketides, such as the macrolides and tetracyclines, and non-ribosomal peptides - that is, peptides that are not synthesized on ribosomes - which include β-lactams and glycopeptides such as vancomycin. These are produced in the cell in assembly-line fashion on large dedicated enzyme platforms called, respectively, polyketide synthases and non-ribosomal peptide synthetases. Following assembly the compounds are then 'decorated' by a series of modifying enzymes, such as glycosyltransferases. The end result is a molecule of often complex structure, with multiple chiral centers and functional groups such as sugars, halogens, sulfates, acyl groups and others.

In general, bacterial genes that encode the production of natural products are clustered together in the genome, greatly facilitating analysis and prediction of biosynthetic pathways and structures. Indeed, several software packages (for example, NP. searcher) have been developed based on rules-based understanding of natural-product biosynthesis. The availability of cheap, rapid genome sequencing means that time-consuming construction and screening of gene libraries for natural-product clusters can now be bypassed. Genome sequencing has also revealed a hitherto unrealized richness in the quantity and variability of natural-product biosynthetic clusters. Sequenced genomes of bacteria of the actinomycetes class reveal 20 to 30 biosynthetic clusters in each organism. Furthermore, natural-product producing bacteria from non-soil environments are being investigated and these have already resulted in new chemical matter, suggesting that there is a fantastic wealth of untapped chemical diversity waiting to be discovered. Perhaps some of this diversity will include new antibiotic chemical scaffolds.

We are in a remarkably productive time for natural-product research that is serving to reinvigorate interest in this sector. At the same time, the application of synthetic biology approaches to this field could serve to improve issues of yield and expand chemical diversity.

Are there alternatives to new antibiotics?

Yes. First, existing discarded antibiotics can be re-examined. The development of daptomycin is instructive. Daptomycin was discovered by the antibiotic group at Eli Lilly in the 1980 s, but was not fully developed because of toxicity concerns. The antibiotic was obtained by researchers at Cubist in 1997 and by altering the dosing, this group was able to bring the antibiotic to market in 2003, since when it has proved highly successful in treating infections caused by Gram-positive pathogens. Certainly, there are other 'old' antibiotics discovered by the pharmaceutical industry but not developed at the time that could be resurrected as leads for new drugs.

A second option is the combination of antibiotics with each other and with other drugs to improve efficacy. Infectious-disease physicians often combine antibiotics in an effort to achieve synergy, and this well-established practice has resulted in formulated drug combinations, such as co-trimoxazole (trimethoprim and sulfamethoxazole). Combination of antibiotics with non-antibiotics deserves investigation as well. Several natural products have been discovered by Satoshi Omura's group that potentiate the activities of antibiotics such as imipenem in S. aureus by unknown mechanisms.

Other antibiotic adjuvants are inhibitors of resistance mechanisms. The tremendous commercial and clinical success of Augmentin (ampicillin together with the β-lactamase inhibitor clavulanic acid) and other similar combinations speaks to the power of such combinations. Our growing understanding of the mechanisms of resistance should fuel such approaches. Inhibitors of efflux pumps, for example, have been discovered, and though challenging to implement in organisms with multiple redundant systems, are worthy of continued investigation.

Finally, other strategies orthogonal to antibiotics must be on the table. We should never forget vaccines as proven and outstanding protective agents against infectious diseases. Bacterial viruses (bacteriophages) were used extensively to treat bacterial infections in the former Soviet Union and could find new application in the face of outbreaks of multi-drug resistant bacteria, especially in settings such as hospital infections. The use of enhancers of innate immunity, such as cationic antimicrobial peptides, is also an approach worth investigating.

What is the outlook for new drugs and further resistance?

We need antibiotics to maintain our current standard of health care. As already stated, resistance is a natural evolutionary phenomenon that cannot be stopped. Through judicious use of current drugs and the development of new ones, the pace of resistance development can be controlled without impairing our ability to control disease. The need for new drugs is, however, acute. Antimicrobial stewardship alone cannot fulfill our requirement for new antibiotics.

We are in a remarkably exciting time for fundamental research in antibiotics. The rapidity of genome sequencing, the maturing of our knowledge of natural product biosynthesis, a growing understanding of the physical properties of ideal antibiotics, the development of new strategies to develop synthetic compounds with improved antibiotic properties, and the possibilities of synthetic biology combine to suggest that we are entering a highly productive period of antibiotic discovery. The challenges of moving these advances into the clinic fast enough to keep pace with resistance are significant, but with concerted effort between scientists, funders, industry, regulators and clinicians, I believe they can be overcome.

Where can I go for more information?

Declarations

Acknowledgements

Research in the author's lab on antibiotic resistance is supported by a Canada Research Chair, the Canadian Institutes of Health Research and the Natural Sciences and Engineering Research Council.

Authors' original submitted files for images

Below are the links to the authors' original submitted files for images.

Authors' Affiliations

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Michael G DeGroote Institute for Infectious Disease Research and the Department of Biochemistry and Biomedical Sciences, McMaster University

References

1. Baquero F, Alvarez-Ortega C, Martinez JL: Ecology and evolution of antibiotic resistance. Env Microbiol Rep. 2009, 1: 469-476. 10.1111/j.1758-2229.2009.00053.x.

2. Fischbach MA, Walsh CT: Antibiotics for emerging pathogens. Science. 2009, 325: 1089-1093. 10.1126/science.1176667.

3. Kumarasamy KK, Toleman MA, Walsh TR, Bagaria J, Butt F, Balakrishnan R, Chaudhary U, Doumith M, Giske CG, Irfan S, Krishnan P, Kumar AV, Maharjan S, Mushtaq S, Noorie T, Paterson DL, Pearson A, Perry C, Pike R, Rao B, Ray U, Sarma JB, Sharma M, Sheridan E, Thirunarayan MA, Turton J, Upadhyay S, Warner M, Welfare W, Livermore DM, Woodford N: Emergence of a new antibiotic resistance mechanism in India, Pakistan, and the UK: a molecular, biological, and epidemiological study. Lancet Infect Dis. 2010, 10: 597-602. 10.1016/S1473-3099(10)70143-2.

4. Yamazaki H, Nonaka K, Masuma R, Omura S, Tomoda H: Xanthoradones, new potentiators of imipenem activity against methicillin-resistant Staphylococcus aureus, produced by Penicillium radicum FKI-3765-2: I. Taxonomy, fermentation, isolation and biological properties. J Antibiot (Tokyo). 2009, 62: 431-434. 10.1038/ja.2009.69.

© 2017 BioMed Central Ltd unless otherwise stated. Part of Springer Nature.

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Q&A: Antibiotic resistance: where does it come from and what can we do about it?

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Received: 13 September 2010 Accepted: 14 September 2010 Published: 20 September 2010 Yes. Historically, the discovery of the sulfa drugs in the 1930 s and the subsequent development of penicillin during World War II ushered in a new era in the treatment of infectious diseases.

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World Health Organisation - Global Action Plan

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World Health Organisation - Global Action Plan

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The goal of the draft global action plan is to ensure, for as long as possible, continuity of successful treatment and prevention of infectious diseases with effective and safe medicines that are quality-assured, used in a responsible way, and accessible to all who need them.

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A Radio 4 play  "Resistance" has been written about antibiotic resistance

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A Radio 4 play  "Resistance" has been written about antibiotic resistance

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Currently not available free to download.. but available to purchase from Amazon or from Audible

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val mcdermid talks about writing resistance

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Val McDermid talks about writing Resistance, Resistance, Dangerous Visions - BBC Radio 4

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"There's nothing more dramatic than the end of the world as we know it"

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Public Health England video on Antibiotic Resistance

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Public Health England video on Antibiotic Resistance

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What is antibiotic resistance, and why should we care? Watch this video then visit http://antibioticguardian.com/ for more information and to make a pledge to save some of our most precious medicines. Image credits: NHS (Crown Copyright), CDC, Wellcome Library London

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Letter sent to Universities to promote antibiotic awareness

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Letter sent to Universities to promote antibiotic awareness

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How does antimicrobial resistance spread?

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This animation is based on the following infographic, available in all EU/EEA languages: http://ecdc.europa.eu/en/eaad/antibiotics-get-informed/infographics/Pages/antibiotic-resistance-spread.aspx Transcript in English How does antibiotic resistance spread? Antibiotic resistance is the ability of bacteria to combat the action of one or more antibiotics. Humans and animals do not become resistant to antibiotic treatments, but bacteria carried by humans and animals can.

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To enable subtitles for the video, click on cc (lower right section of the video screen)

Antibiotic resistance is one of the biggest threats facing us today.
Antibiotic Guardian supports the UK Antimicrobial Resistance strategy, European Antibiotic Awareness Day (18 November) and World Antibiotic Awareness Week (13-19 November 2017)

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To enable subtitles for the video, click on cc (lower right section of the video screen) Antibiotic Guardian supports the UK Antimicrobial Resistance strategy, European Antibiotic Awareness Day (18 November) and World Antibiotic Awareness Week (13-19 November 2017)

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Antibiotic  Research programme for Antibiotic research UK

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Antibiotic  Research programme for Antibiotic research UK

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Developing a new antibiotic therapy by the early 2020's

Antibiotic Research UK's research programmes
Typically it can take up to 15 years and expenditure of many hundreds of millions of pounds to develop a new drug. ANTRUK aims to significantly reduce these timelines and costs by using smart approaches which will be very different to conventional drug development procedures. The Charity's research focus initially will be on preserving our existing antibiotics rather than trying to find new ones.

Multi-drug resistant (MDR) Gram-negative bacteria are the major risk to modern medicine.

About 50% of all hospital infections are due to Gram-negative bacteria

Of particular concern are antibiotic resistant bacteria associated with four different species, Klebsiella pneuomoniae, Pseudomonas aeruginosa, Escherichia coli and Acinetobacter baumanii. All three species can be found in the mouth, skin or gut

A Multi Drug Resistant bacterium can produce specific metallobeta-lactamases which are resistant to all penicillins and cephalosporins and all beta-lactamase inhibitors such as clavulanic acid and avibactam. These bacteria are also resistant to virtually all other antibiotics, with the exception of colistin

If resistance arises to colistin, which it has, then there are no treatments left.

[/column]
Diagram courtesy of www.knowabouthealth.com showing the difference between non-resistant bacteria and drug resistant bacteria. Non-resistant bacteria multiply, and upon drug treatment, the bacteria die. Drug resistant bacteria multiply as well, but upon drug treatment, the bacteria continue to spread.

A number of authorities globally have spoken about the rapidly-emerging crisis. There is an urgent need to invent new antibiotics, though it may already be too late for this to happen in time and at the scale required. Therefore it is essential that we protect current antibiotics, particularly 'last line of defence' drugs, from resistance, and develop 'Antibiotic Resistance Breakers' (ARBs) that break the resistance that is sure to emerge.

In particular the charity is focussing on repurposing or repositioning existing drugs.

This involves

1. taking drugs that are currently used for one indication such as cancer treatment or heart disease and examining them for antibiotic activity

2. combining two existing antibiotics to see if they can overcome antibiotic resistance or

3. combining one or more drugs together with the antibiotic to see if the antibiotic resistance can be overcome (Antibiotic Resistance Breakers).

The first three of the Charity's research programmes will focus on Antibiotic Resistance Breakers against five widely used antibiotic classes viz. meropenem, ciprofloxacin, aztreonam, gentamicin, colistin and tigecycline to which resistance in Gram-negative bacteria has developed. Resistance can occur in a number of ways. One of these involves the loss of pores in the bacterial cell wall so that the antibiotic cannot penetrate the bacteria. If new pores could be created by the ARB then resistance might be overcome (see football analogy diagram)

The Programmes

Research programmes and costs

Test tube screening studies have been undertaken with pan-resistant Gram-negative antibiotic resistant bacteria. As these bacteria are hazardous, handling them requires specialist laboratory facilities in the United Kingdom that can handle these antibiotic resistant bacteria.

The research programme has been overseen by Dr Lloyd Czaplewski as project manager under the overall control of the Science Committee.

The project will focus on overcoming resistance to the fluoroquinolone class of antibiotics in the Gram-negative bacteria Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeroginosa and Acinetobacter baumanni. The test tube studies will take the resistant bacteria and expose them to fluoroquinolone and another drug(s). The latter will be drawn from any drug that has been approved for any human disease ever.

The research programme will be overseen by a project manager under the overall control of STAC.

This project will focus on overcoming resistance to colistin in the Gram-negative bacteria Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter baumannii. The test tube studies will take the resistant bacteria and expose them to colistin and another drug(s). The latter drugs will be drawn from any drug that has been approved for any human disease ever.

The research programme will be overseen by a project manager under the overall control of STAC.

This project will focus on finding a new antibiotic from a chemical compound library, natural products or any other source. Gram-negative bacteria will be the target species and the library will either be obtained from commercial sources, provided by a pharmaceutical company, university or any other source of interesting compounds.

As this programme will examine novel compounds not hitherto used as drugs for any therapeutic area, by its nature it will be more expensive and take a longer time to complete than Projects 1-3.

This project will focus on finding a new antibiotic from a chemical compound library, natural products or any other source.. The focus of this project will be different from Project 4, either by examining a different library or using alternative screening systems and / or endpoints. Gram-negative bacteria will be the target species and the library will either be obtained from commercial sources, provided by a pharmaceutical company, university or any other source of interesting compounds.

As this programme will examine novel compounds not hitherto used as drugs for any therapeutic area, by its nature it will be more expensive and take a longer time to complete than Projects 1-3.

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ANTRUK's Research Programmes - Antibiotic Research UK

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Developing a new antibiotic therapy by the early 2020's Antibiotic Research UK's research programmes Typically it can take up to 15 years and expenditure of many hundreds of millions of pounds to develop a new drug. ANTRUK aims to significantly reduce these timelines and costs by using smart approaches which will be very different to conventional drug development procedures.

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This review was commissioned in July 2014 by the UK Prime Minister to analyse drug resistance and ways to tackle this problem

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A UK charity for the development of antibiotic research

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A charity committed to developing new antibiotic therapies in the fight against antibiotic resistant bacteria. Help us save modern medicine!

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Download the game to your iPad ... How long can you hold up against the superbugs?

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Read reviews, compare customer ratings, see screenshots and learn more about Superbugs: The game. Download Superbugs: The game and enjoy it on your iPhone, iPad and iPod touch.

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World Health Organisation - priority pathogens list for Research and Development of new antibiotics

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World Health Organisation - priority pathogens list for Research and Development of new antibiotics

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WHO today published its first ever list of antibiotic-resistant "priority pathogens" - a catalogue of 12 families of bacteria that pose the greatest threat to human health.

The list was drawn up in a bid to guide and promote research and development (R&D) of new antibiotics, as part of WHO's efforts to address growing global resistance to antimicrobial medicines.

The list highlights in particular the threat of gram-negative bacteria that are resistant to multiple antibiotics. These bacteria have built-in abilities to find new ways to resist treatment and can pass along genetic material that allows other bacteria to become drug-resistant as well.

"This list is a new tool to ensure R&D responds to urgent public health needs," says Dr Marie-Paule Kieny, WHO's Assistant Director-General for Health Systems and Innovation. "Antibiotic resistance is growing, and we are fast running out of treatment options. If we leave it to market forces alone, the new antibiotics we most urgently need are not going to be developed in time."

The WHO list is divided into three categories according to the urgency of need for new antibiotics: critical, high and medium priority.

The most critical group of all includes multidrug resistant bacteria that pose a particular threat in hospitals, nursing homes, and among patients whose care requires devices such as ventilators and blood catheters. They include Acinetobacter, Pseudomonas and various Enterobacteriaceae (including Klebsiella, E. coli, Serratia, and Proteus). They can cause severe and often deadly infections such as bloodstream infections and pneumonia.

These bacteria have become resistant to a large number of antibiotics, including carbapenems and third generation cephalosporins - the best available antibiotics for treating multi-drug resistant bacteria.

The second and third tiers in the list - the high and medium priority categories - contain other increasingly drug-resistant bacteria that cause more common diseases such as gonorrhoea and food poisoning caused by salmonella.

G20 health experts will meet this week in Berlin. Mr Hermann Gröhe, Federal Minister of Health, Germany says "We need effective antibiotics for our health systems. We have to take joint action today for a healthier tomorrow. Therefore, we will discuss and bring the attention of the G20 to the fight against antimicrobial resistance. WHO's first global priority pathogen list is an important new tool to secure and guide research and development related to new antibiotics."

The list is intended to spur governments to put in place policies that incentivize basic science and advanced R&D by both publicly funded agencies and the private sector investing in new antibiotic discovery. It will provide guidance to new R&D initiatives such as the WHO/Drugs for Neglected Diseases initiative (DNDi) Global Antibiotic R&D Partnership that is engaging in not-for-profit development of new antibiotics.

Tuberculosis - whose resistance to traditional treatment has been growing in recent years - was not included in the list because it is targeted by other, dedicated programmes. Other bacteria that were not included, such as streptococcus A and B and chlamydia, have low levels of resistance to existing treatments and do not currently pose a significant public health threat.

The list was developed in collaboration with the Division of Infectious Diseases at the University of Tübingen, Germany, using a multi-criteria decision analysis technique vetted by a group of international experts. The criteria for selecting pathogens on the list were: how deadly the infections they cause are; whether their treatment requires long hospital stays; how frequently they are resistant to existing antibiotics when people in communities catch them; how easily they spread between animals, from animals to humans, and from person to person; whether they can be prevented (e.g. through good hygiene and vaccination); how many treatment options remain; and whether new antibiotics to treat them are already in the R&D pipeline.

"New antibiotics targeting this priority list of pathogens will help to reduce deaths due to resistant infections around the world," says Prof Evelina Tacconelli, Head of the Division of Infectious Diseases at the University of Tübingen and a major contributor to the development of the list. "Waiting any longer will cause further public health problems and dramatically impact on patient care."

While more R&D is vital, alone, it cannot solve the problem. To address resistance, there must also be better prevention of infections and appropriate use of existing antibiotics in humans and animals, as well as rational use of any new antibiotics that are developed in future.

WHO priority pathogens list for R&D of new antibiotics

Priority 1: CRITICAL

Acinetobacter baumannii, carbapenem-resistant

Pseudomonas aeruginosa, carbapenem-resistant

Enterobacteriaceae, carbapenem-resistant, ESBL-producing

Priority 2: HIGH

Enterococcus faecium, vancomycin-resistant

Staphylococcus aureus, methicillin-resistant, vancomycin-intermediate and resistant

Helicobacter pylori, clarithromycin-resistant

Campylobacter spp., fluoroquinolone-resistant

Salmonellae, fluoroquinolone-resistant

Neisseria gonorrhoeae, cephalosporin-resistant, fluoroquinolone-resistant

Priority 3: MEDIUM

Streptococcus pneumoniae, penicillin-non-susceptible

Haemophilus influenzae, ampicillin-resistant

Shigella spp., fluoroquinolone-resistant

Media contacts

Olivia Lawe-Davies
WHO Department of Communications
Mobile: +41 79 475 5545
Office: +41 22 791 1209
Email: lawedavieso@who.int

Simeon Bennett
WHO Department of Communications
Mobile: +41 79 472 7429
Office: +41 22 791 4621
E-mail: simeonb@who.int

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WHO publishes list of bacteria for which new antibiotics are urgently needed

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WHO today published its first ever list of antibiotic-resistant "priority pathogens" - a catalogue of 12 families of bacteria that pose the greatest threat to human health. The list was drawn up in a bid to guide and promote research and development (R&D) of new antibiotics, as part of WHO's efforts to address growing global resistance to antimicrobial medicines.

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World Health Organisation -Antibacterial compounds in clinical development

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World Health Organisation -Antibacterial compounds in clinical development

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The 10 most dangerous antibiotic resistant bacteria

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The 10 most dangerous antibiotic resistant bacteria

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An excellent Podcast on Antibiotic Resistance

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Why choose to use soil from aquatic environments?

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Why choose to use soil from aquatic environments?

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Microbes from marine (sea/ ocean/coastal) environments are notoriously difficult to culture, although new methods are identifying these organisms as having great potential for the discovery of natural product compounds.
For our past 'Antibiotics Unearthed project' we have generated over 40 different marine bacteria cultures from sites around the UK.  We will use these bacteria to screen for potential antibiotics to Vibrio parahaemolyticus

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Antibiotics Undersea is a students as co-creator project which the University of Westminster has joined for 2018-2019. After the exciting experience from last year, the Antibiotics Undersea is coming back with the aim to find new Antibiotics from the soil bacteria found in Antibiotics Unearthed. Particularly, we will focus on Vibrio parahaemolyticus a rising threat that is affecting more and more the seafood and the human health. Read through the documents below, to find out more about the project.

WHO CAN JOIN THE PROJECT?
We are looking for enthusiastic and motivated undergraduate students from the University of Westminster who would be willing to commit to working on the project every Wednesday afternoon in semester 2  (full day Wednesday in the student engagement week)  starting from January 2019.  There will also be the opportunity to participate in outreach activities to promote the project and the topic of antibiotic resistance. You do not necessarily need a background in science - we are also looking for students with skills in website creation, communication and social media.  Read through the documents below, to find out more about the project.

HOW TO APPLY TO JOIN THE PROJECT
To apply to join the project,  please submit EITHER a short paragraph (max 300 words) or a short video (max 3 mins) outlining why you would like to participate and the skills you could bring to the  project to Linda Percy  by email at  percyl@westminster.ac.uk by Friday 7th December 2018.
The project will start in January 2019.

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Antibiotics are life-saving drugs used to treat bacterial infections. But there's a problem - some bacteria are becoming resistant to antibiotics, meaning our drugs no longer work against them. By 2050, 10 million people could die every single year from antibiotic resistance - unless we act now.

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Hypha Discovery - a microbial technology company have joined with us for this project and will support us in the analysis of potential antimicrobials we discover.

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Sign up for our newsletter Come and meet us at the following events:
Bio Japan 2017, 11th-13th October, Pacifico Yokohama, Japan

Hypha will join the Mayor's International Business Programme Trade Mission to Bio Japan.

Request a meeting or drop by the UK Pavilion
ありがとうございます Download a copy of our brochure on drug metabolites and C-H activated derivatives
Our new brochure on drug metabolites and C-H activated derivatives is now available. Inside we feature interesting case studies and client projects, including the following topics:

Phase l CYP and non-CYP metabolites

Phase ll conjugated metabolites

Isotopically labelled metabolites

Lead diversification and late stage functionalization

Polar / C-H activated derivatives

Download a copy here Download a copy of our latest newsletter
In our latest newsletter we look at off-target activity of some drug metabolites, exemplified with a case study looking at metabolism of valsartan.
Download a copy here

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Sign up for our newsletter Come and meet us at the following events: Bio Japan 2017, 11th-13th October, Pacifico Yokohama, Japan Hypha will join the Mayor's International Business Programme Trade Mission to Bio Japan.

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We will be working with environmental soil samples and some experiments will involve the use of class 2 micro-organisms.  This means that new or expectant mothers,  or those who are immunosuppressed due to individual health status could be more susceptible to infection, and would be unable to partake in these lab sessions.  There are plenty of other tasks in the project which are less hazardous, in which you could be involved.  Please email Linda  percyl@westminster.ac.uk if you have any questions regarding this.

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What skills will you learn in the project?

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What skills will you learn in the project?

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The project will give you the opportunity to work in the laboratroy environment on a team - based real - life project.   We will provide essential Health and Safety  training and training in the necessary microbiology techniques, including DNA analysis of microbes and chemical analysis.  BUT how the project develops over time will be very much depend on the commitment of  the student groups. You will learn lab-based skills, team-working skills and develop communication skills.  You will be able to add this research project to your Higher Education Achievement Record (HEAR).   You need to bring enthusiasm, commitment and ideas for communication, outreach, and novel ways to develop the project further!

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Availability of new antibiotics has reduced  over time

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Availability of new antibiotics has reduced  over time

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Timeline of discovery, introduction and resistance to antibiotics

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Timeline of discovery, introduction and resistance to antibiotics

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This timeline shows the time frame between some antibiotic discovery and usage, and antibiotic resistance developing in bacteria.

Kim Lewis Nature Reviews Drug Discovery 12, 371–387 (2013)

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Timeline of the discovery and introduction of antibiotics : Platforms for antibiotic discovery : Nature Reviews Drug Discovery : Nature Research

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Multidrug-resistant (MDR) efflux; an active transport system for the removal of several structurally non-related antibiotics from cells. The major facilitator (MF) family of MDRs are drug or proton antiporters present in all bacteria that are primarily responsible for efflux of hydrophobic cations and have some role in protecting bacteria from disinfectants, but not from systemically used antibiotics.

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Antibiotic resistance is one of the biggest threats to global health, food security, and development today.
Antibiotic resistance can affect anyone, of any age, in any country.

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Fact sheet: Antibiotics are medicines used to prevent and treat bacterial infections. Antibiotic resistance occurs when bacteria change in response to the use of these medicines. Bacteria, not humans, become antibiotic resistant. These bacteria may then infect humans and are harder to treat than non-resistant bacteria.

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WHY USE SOIL TO SEARCH FOR NEW ANTIBIOTICS?

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WHY USE SOIL TO SEARCH FOR NEW ANTIBIOTICS?

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The most recent antibiotics have been found from naturally occurring soil bacteria.

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Antibiotics- wonders of the microbial world

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Antibiotics- wonders of the microbial world

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Antibiotics - Seven Wonders of the Microbe World (5/7)

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TELL US WHAT YOU THINK and help us improve our Free Educational Resources https://www.surveymonkey.co.uk/r/YT2017_descr For more like this subscribe to the Open University channel https://www.youtube.com/channel/UCXsH4hSV_kEdAOsupMMm4Qw Free learning from The Open University http://www.open.edu/openlearn/science-maths-technology/science/biology --- In this video, experts reveal how the natural processes of microbes are used to fight disease.

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In 2017, a new antibiotic pseudouridimycin was discovered from a soil microbe

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Infections that are resistant to the drugs used to treat them, also known as superbugs, are a growing problem worldwide. If nothing is done, 10 million people are estimated to die each year from antibiotic resistance infections by 2050.

Promoting the judicious use of antibiotics in medicine and agriculture is an important way to combat superbugs, but developing new antibiotics to treat infections is also critical. Although few pharmaceutical companies are currently developing new antibiotics, scientists from Rutgers University-New Brunswick report an important finding that could help lead to a new drug down the line.

In the journal Cell, the researchers report they've discovered a new antibiotic called pseudouridimycin that appears to work well in animal models. The antibiotic comes from a microbe that was discovered in Italian soil as part of an effort to discover new drug compounds.
MORE: Why Bacteria Are More Threatening Than Ever
Beyond the discovery, the researchers were able to test the antibiotic and show that it acts against drug-resistant bacteria in test tubes and appears to combat infections in mice models. The way the antibiotic targets bacteria is different from similar drugs on the market and makes it less prone to developing resistance, the researchers say. "I think pharma made a mistake in leaving this space," says study author Richard H. Ebright, Board of Governors professor of chemistry and chemical biology at Rutgers-New Brunswick.

Ebright says that he and his research team will likely spend 1-3 more years trying to make it even more effective. "We can make small tweaks to the chemical structure that would make it a bit more potent," says Ebright.

Much more research needs to be done on the new compound before it's available. Should the new antibiotic make it to market, it would be one of the first new antibiotic drugs made available in more than a decade.

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There's a New Antibiotic You Should Know About

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Infections that are resistant to the drugs used to treat them, also known as superbugs, are a growing problem worldwide. If nothing is done, 10 million people are estimated to die each year from antibiotic resistance infections by 2050.

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In 2015, the newest class of antibiotic to be discovered in 30 years .. (yes 30 years!) was discovered from an undescribed soil microorganism
(provisionally named Eleftheria terrae).

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The Microbiology Society is a membership charity for scientists interested in microbes, their effects and their practical uses.

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Microbiology Society - Antibiotics Unearthed

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Microbiology Society - Antibiotics Unearthed

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Antibiotics Unearthed will give the general public, students and educators in the UK and Ireland the opportunity to work with scientists as part of a global initiative to discover new antibiotics from soil bacteria.

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In order to tackle growing levels of antimicrobial resistance, the challenge set for the Longitude Prize is to invent an affordable, accurate, fast and easy-to-use test for bacterial infections that will allow health professionals worldwide to administer the right antibiotics at the right time.

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Antibiotics Unearthed will give the general public, students and educators in the UK and Ireland the opportunity to work with scientists as part of a global initiative to discover new antibiotics from soil bacteria.