Antibiotics produced by Streptomyces

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The Brazilian Journal of

INFECTIOUS DISEASESwww.elsev ier .com/ locate /b j id

Review article

Antibiotics produced by Streptomyces

Rudi Emerson de Lima Procópioa,∗, Ingrid Reis da Silvaa, Mayra Kassawara Martinsa,João Lúcio de Azevedoa, Janete Magali de Araújob

a Microbiology Laboratory, Centro de Biotecnologia da Amazônia (CBA), Manaus, AM, Brazilb Antibiotics Department, Centro de Ciências Biológicas, Universidade Federal de Pernambuco (UFPE), Recife, PE, Brazil

a r t i c l e i n f o

Article history:

Received 21 March 2012

Accepted 6 May 2012

Available online 11 September 2012

Keywords:

Streptomyces

Antibiotics

Resistance

Infection

a b s t r a c t

Streptomyces is a genus of Gram-positive bacteria that grows in various environments, and

its shape resembles filamentous fungi. The morphological differentiation of Streptomyces

involves the formation of a layer of hyphae that can differentiate into a chain of spores.

The most interesting property of Streptomyces is the ability to produce bioactive secondary

metabolites, such as antifungals, antivirals, antitumorals, anti-hypertensives, immunosup-

pressants, and especially antibiotics. The production of most antibiotics is species specific,

and these secondary metabolites are important for Streptomyces species in order to compete

with other microorganisms that come in contact, even within the same genre. Despite the

success of the discovery of antibiotics, and advances in the techniques of their production,

infectious diseases still remain the second leading cause of death worldwide, and bacterial

infections cause approximately 17 million deaths annually, affecting mainly children and

the elderly. Self-medication and overuse of antibiotics is another important factor that con-

tributes to resistance, reducing the lifetime of the antibiotic, thus causing the constant need

for research and development of new antibiotics.

© 2012 Elsevier Editora Ltda. All rights reserved.

Streptomyces

Streptomyces is a genus of Gram-positive bacteria that growsin various environments, with a filamentous form similarto fungi. The morphological differentiation of Streptomycesinvolves the formation of a layer of hyphae that can differ-entiate into a chain of spores. This process is unique amongGram-positives, requiring a specialized and coordinated

metabolism. The most interesting property of Streptomyces isthe ability to produce bioactive secondary metabolites suchas antifungals, antivirals, antitumoral, anti-hypertensives,

∗ Corresponding author at: Centro de Biotecnologia da Amazônia (CBA),Brazil.

E-mail address: [email protected] (R.E. de Lima Procópio).1413-8670/$ – see front matter © 2012 Elsevier Editora Ltda. All rights rhttp://dx.doi.org/10.1016/j.bjid.2012.08.014

and mainly antibiotics and immunosuppressives.1–3 Anothercharacteristic is of the genus is complex multicellular devel-opment, in which their germinating spores form hyphae, withmultinuclear aerial mycelium, which forms septa at regularintervals, creating a chain of uninucleated spores.4

When a spore finds favorable conditions of temperature,nutrients, and moisture, the germ tube is formed and thehyphae develops. The aerial hyphae follows, and a stage setinitiates the organization of various processes such as growth

Av. Danilo Areosa 690, Distrito Industrial, Manaus, AM, 69075-351,

and cell cycle. Esporogenic cell may contain 50 or more copiesof the chromosome; the order, position, and segregation ofchromosomes during sporulation is linear, which involves at

eserved.

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Table 1 – Streptomyces with their available genome sequence.

Organism GenBank Size % CG Protein Importance References

S. celicolor AL645882.2 9.05 Mb 72.0 7.825 Genetic studies Bentley et al.10

S. avermitilis BA000030.3 9.11 Mb 70.7 7.583 Antibiotic (Avermictin) Omura et al.1

S. griseus AP009493.1 8.54 Mb 72.2 7.138 Antibiotic (Streptomicin) Ohnishi et al.4

S. bingchenggensis CP002047 11,93 Mb 70,8 10.023 Antihelmintic (Milbemicin) Wang et al.13

8.746 Phytopathogen Bignell et al.14

Antibiotics and fluorometabolites Barbe et al.15

ltohgttwi

ralAspaamwwiagtibmSogg

n1tim

tmciiaetser

Platensimycin2006 S. platensisDaptomycin2003 S. roseosporusLinezolid2000 SyntheticMupirocin1985 Pseudomonas fluorescensRibostamycin1970 S. ribosidificusFosfomycin1969 S. fradiaeTrimethoprim1968 SyntheticGentamicin1963 Micromonospora purpureaFusidic acid1963 Fusidium coccineumNalidixic acid1962 SyntheticTinidazole1959 SyntheticKanamycin1957 S. kanamyceticusRifamycin1957 Amycolatopsis mediterraneiNoviobiocin1956 S. niveusVancomycin1956 S. orientalisCycloserine1955 S. garyphalusLincomycin1952 S. lincolnensisErithromycin1952 Saccharopolyspora erythraeaVirginiamycin1952 in S. pristinaespiralis S. virginiaeIsoniazid1951 SyntheticViomycin1951 S. vinaceus e S. capreolusIsoniazid1951 SyntheticViomycin1951 S. vinaceus e S. capreolusNystatin1950 S. nourseiTetracycline1950 S. aureofaciensNeomycin1949 S. fradiaeChloramphenicol1949 S. venezuelaePolymyxin1947 Bacillus polymyxaNitrofurantoin1947 SyntheticCephalosporins1945 S. clavuligerusBacitracin1945 Bacillus licheniformisCephalosporins1945 S. clavuligerusStreptomycin1944 S. griseusPenicillin1941 Penicillium chrysogenum

2000

1970

1960

1950

1940

Fig. 1 – Key findings and dates of antibiotics. Highlights of

S. scabiei FN554889.1 10 MbS. cattleya NC 016111 8.1 Mb

east two systems (ParAB and FtsK), which lead to differentia-ion and septation of apical cells into chains of spores. Severalther genes that are essential for the sporulation of aerialyphae have been reported in S. coelicolor, for example, theenes whiG, whiH, whiI, whiA, whiB, and whiD. The explana-ion for the presence of spores in Streptomyces is probably thathese fragments appeared mycelial under selective pressure,hich might involve the need to survive outside of plants and

nvertebrates, or in extreme environments.The ability of the spores to survive in these hostile envi-

onments must have been increased due to the pigment androma present in the spores in some species,5 which stimu-ates cell development and secondary metabolite production.6

nother important point is the tip of the hypha, which is con-idered to be the most important region where membraneroteins and lipids may be secreted, especially in the apicalrea of growth.7 In some Streptomyces, secondary metabolismnd differentiation can be related.8,9 Phylogenetically, Strepto-yces are a part of Actinobacteria, a group of Gram-positiveshose genetic material (DNA) is GC-rich (70%) when comparedith other bacteria such as Escherichia coli (50%). The great

mportance given to Streptomyces is partly because these aremong the most numerous and most versatile soil microor-anisms, given their large metabolite production rate andheir biotransformation processes, their capability of degrad-ng lignocellulose and chitin, and their fundamental role iniological cycles of organic matter.10 Two species of Strepto-yces have been particularly well studied: S. griseus, the first

treptomyces to be used for industrial production of an antibi-tic - streptomycin, and S. coelicolor, the most widely used inenetic studies. Various strains have been sequenced and theirenomes have been mapped (Table 1).

The genome of S. coelicolor, for example, encodes a largeumber of secreted proteins (819), including 60 proteases,3 chitinases/chitosanases, eight cellulases/endoglucanases,hree amylases, and two pactato lyases. Streptomyces are alsomportant in the initial decomposition of organic material,

ostly saprophytic species.11

The production of most antibiotics is species specific, andhese secondary metabolites are important so the Strepto-yces spp. can compete with other microorganisms that may

ome in contact, or even within the same genus. Anothermportant process involving the production of antibioticss the symbiosis between Streptomyces and plants, as thentibiotic protects the plant against pathogens, and plantxudates allows the development of Streptomyces.12 Data inhe literature suggest that some antibiotics originated as

ignal molecules, which are able to induce changes in thexpression of some genes that are not related to a stressesponse.11

the Streptomyces.

Antibiotics

Despite the success of the discovery of antibiotics, andadvances in the process of their production, infectiousdiseases still remain the second leading cause of death world-wide, and bacterial infections cause approximately 17 milliondeaths annually, affecting mainly children and the elderly. Thehistory of antibiotics derived from Streptomyces began with thediscovery of streptothricin in 1942, and with the discovery ofstreptomycin two years later, scientists intensified the searchfor antibiotics within the genus. Today, 80% of the antibioticsare sourced from the genus Streptomyces, actinomycetes beingthe most important.16 This can be seen in Fig. 1.

Mechanism of action of antibiotics

The molecular basis of this action is well understood andthe main targets are well known. They are classified by the

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Cytoplasmicmembrane

Daptomycinpolymyxin

platensimycin

Protein

mRNAR

ibos

ome

Cel

lula

r w

all

30S

50S

50S

TetracyclinStreptomycinKanamycinGentamycin

ErithomycinClindamycinChloramphenicol

PenicillinCarbapenemsCephalosporinVancomycinFosfomycinBacitracinDaptomycin

RifampicinRNA polymeraseDNA-dependent

DNA gyrase

DNA

CiprofloxacinNovobiocin

Fig. 2 – Schematic representation of the target andmechanism of action of certain antibiotics.

are frequent in this environment. Many resistance genes

interaction of antibiotics targeting essential cellular functions,the fundamental principle to inhibit bacterial growth.17 This isa complex process that starts with the physical interaction ofthe molecule and its specific targets and involves biochemical,molecular, and structural changes, acting on multiple cellulartargets such as: 1) DNA replication, 2) RNA synthesis, 3) cellwall synthesis, and 4) protein synthesis (Fig. 2).

DNA replication

DNA gyrase (topoisomerase) controls the topology of the DNAby catalyzing the cleavage pattern and DNA binding. This reac-tion is important for DNA synthesis and mRNA transcription,and the complex-quinolone topoisomerase-DNA cleavage pre-vents replication, leading to death of the bacteria.18–20

Synthesis of RNA

The DNA-dependent RNA polymerase mediates the transcrip-tion process and is the main regulator of gene expressionin prokaryotes. The enzymatic process is essential for cellgrowth, making it an attractive target for antibiotics. Oneexample is rifamycin, which inhibits the synthesis of RNA byusing a stable connection with high affinity to the �-subunitin the RNA/DNA channel, separating the active site by inhib-iting the initiation of transcription and blocking the path ofribonucleotide chain growth.18–20

Cell wall synthesis

The bacterial cell wall consists of peptidoglycan, which helpsmaintain the osmotic pressure, conferring ability to sur-vive in diverse environments. The peptidoglycan biosynthesisinvolves three stages: the first stage occurs in the cytoplasm,where low molecular weight precursors are synthesized. In

the second stage, the cell wall synthesis is catalyzed bymembrane-bound enzymes; and in the third stage the antibi-otic acts by preventing the �-lactams and polymerization

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of the glycan synthesis of cell wall enzymes, acting ontranspetidades.18–20

Protein synthesis

The translation process of mRNA occurs in three phases:initiation, elongation, and termination involving cytoplasmicribosomes and other components. The ribosome is composedof two subunits (50S and 30S), which are targets of the mainantibiotic that inhibits protein synthesis. Macrolides act byblocking the 50S subunit, preventing the formation of the pep-tide chain: tetracycline in the 30S subunit acts by blocking theaccess of the aminoacyl tRNA-ribosome; spectinomycin inter-feres with the stability of the peptidyl-tRNA binding to theribosome; and streptomycin, kanamycin, and gentamicin actin the 16S rRNA that is part of the 30S ribosome subunit.18–20

Cytoplasmic membrane

The cytoplasmic membrane acts as a diffusion barrierto water, ions, and nutrients. The transport systems arecomposed primarily of lipids, proteins, and lipoproteins. Dap-tomycin inserts into the cytoplasmic membrane of bacteria ina calcium-dependent fashion, forming ion channels, trigger-ing the release of intracellular potassium. Several antibioticscan cause disruption of the membrane. These agents canbe divided into cationic, anionic, and neutral agents. Thebest known compounds are polymyxin B and colistemethate(polymyxin E). The polymyxins are not widely used becausethey are toxic to the kidney and to the nervous system.18–20

The latest antibiotic launched in 2006 by Merck (platen-simycin) has different mechanism of action from the previousones, since it acts by inhibiting the beta-ketoacyl synthases I /II (FabF / B), which are key enzymes in the production of fattyacids, necessary for bacterial cell membrane.13

Resistance

According to Nikaido20 100,000 tons of antibiotics are pro-duced annually, which are used in agriculture, food, andhealth. Their use has impacted populations of bacteria, induc-ing antibiotics resistance. This resistance may be due togenetic changes such as mutation or acquisition of resis-tance genes through horizontal transfer, which most oftenoccurs in organisms of different taxonomy.21,22 Mutationscan cause changes at the site of drug action, hindering theaction of the antibiotic.23 Most of the resistance genes arein the same cluster as the antibiotic biosynthesis gene.24 Innature, the main function of antibiotics is to inhibit competi-tors, which are induced to inactivate these compounds bychemical modification (hydrolysis), and changes in the siteof action and membrane permeability.25 A study carried outwith Streptomyces from urban soil showed that most strains areresistant to multiple antibiotics, suggesting that these genes

20

are located on plasmids (plasmid A), which can be passed byconjugation to a susceptible strain; these plasmids are stableand can express the resistance gene.26 The susceptibility to a

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articular antibiotic can be affected by the physiological statef the bacteria, and the concentration of the antibiotic; thisay be observed in biofilms through a mechanism known

s persister formation - small subpopulations of bacteria sur-ive the lethal concentration of antibiotic without any specificesistance mechanisms, although this mechanism does notroduce high-level resistance.27

Microorganisms growing in a biofilm are associated withhronic and recurrent human infections and are resistanto antimicrobial agents.28 The spread of resistant strains isot only linked to antibiotic use, but also to the migrationf people, who disperse resistant strains among people inemote communities where the use of antibiotics is veryimited.24 Due to the difficulty of obtaining new antibiotics,he drug industry has made changes to existing antibiotics;hese semi-synthetics are more efficient and less susceptibleo inactivation by enzymes that cause resistance. This practiceas become the strategy for the current antibiotics used todaynd is known as the second, third, and fourth generation ofntibiotics.29,30

enome and new antibiotics

ith the availability of genomes from a large number ofathogens, hundreds of genes have been evaluated as tar-ets for new antibiotics. A gene is recognized as essentialhen the bacterium can not survive while the gene is inac-

ive, and can become a target when a small molecule canlter its activity.31 Genetic analysis has shown that a geneay encode a function that is important in one bacteria but

ot in another.32 167 genes have been determined as essen-ial for bacterial growth and are potential targets for newntibiotics.33,34 GlaxoSmithKline has conducted studies withhe antibiotic GKS299423 acting on topoisomerase II, in ordero prevent the bacteria from developing resistance.35

se

he world’s demand for antibacterials (antibiotics) is steadilyrowing. Since their discovery in the 20th century, antibioticsave substantially reduced the threat of infectious diseases.he use of these “miracle drugs”, combined with improve-ents in sanitation, housing, food, and the advent of mass

mmunization programs, led to a dramatic drop in deaths fromiseases that were once widespread and often fatal. Over theears, antibiotics have saved lives and eased the suffering ofillions. By keeping many serious infectious diseases under

ontrol, these drugs also contributed to the increase in lifexpectancy during the latter part of the 20th century.

The increasing resistance of pathogenic organisms, lead-ng to severe forms of infection that are difficult to treat,as further complicated the situation, as in the case ofarbapenem-resistant Klebsiella pneumoniae,36,37 and othericroorganisms.38 Infections caused by resistant bacteria do

ot respond to treatment, resulting in prolonged illness andreater risk of death. Treatment failures also lead to long peri-ds of infectivity with high rates of resistance, which increasehe number of infected people circulating in the community

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and thus expose the population to the risk of contracting amultidrug-resistant strain.39

As bacteria become resistant to first generation antibiotics,treatment has to be changed to second or third generationdrugs, which are often much more expensive and sometimestoxic. For example, the drug needed to treat multi-drug resis-tant Streptococcus pneumoniae, Staphylococcus aureus, Klebsiellapneumoniae, and Mycobacterium tuberculosis, can cost 100 timesmore than first generation drugs used to treat non-resistantforms. Most worrisome is that resistance to virtually all antibi-otics has increased.

Even though the pharmaceutical industry has intensifiedefforts to develop new drugs to replace those in use, currenttrends suggest that some infections will have no effective ther-apies within the next ten years. The use of antibiotics is thecritical factor in the selection of resistance.40,41 Paradoxically,underuse through lack of access and inadequate treatmentmay play a role as important as overuse. For these reasons,proper use is a priority to prevent the emergence and spreadof bacterial resistance. Patient-related factors are the maincauses of inappropriate use of antibiotics. For example, manypatients believe that new and expensive drugs are more effec-tive than older drugs.

In addition to causing unnecessary expenditure, this per-ception encourages the selection of resistance to these newdrugs, as well as to the older drugs in their class.42 Self-medication with antibiotics is another important factor thatcontributes to resistance, because patients may not take ade-quate amounts of the drug. In many developing countries,antibiotics are purchased in single doses and taken only untilthe patient feels better, which may occur before the bacteriais eliminated.43

Physicians can be pressured to prescribe antibiotics tomeet patient expectations, even in the absence of appropri-ate indications, or by manufacturers’ influence. Some doctorstend to prescribe antibiotics to cure viral infections, makingthem ineffective against other infections. In some culturalcontexts, antibiotics administered by injection are consid-ered more effective than oral formulations. Hospitals are acritical component of the problem of antimicrobial resis-tance worldwide.14,44 The combination of highly susceptiblepatients, patients with serious infections, and intense andprolonged use of antibiotics has resulted in highly resistantnosocomial infections, which are difficult to control, makingeradication of the pathogen expensive.

In September 2001, the World Health Organization (WHO)launched the first global strategy to combat the serious prob-lems caused by the emergence and spread of antimicrobialresistance. Known as the WHO Global Strategy for the Contain-ment of Antimicrobial Resistance,45 the strategy recognizesthat antimicrobial resistance is a global problem that mustbe addressed in all countries. No nation, however effective,can close its border to resistant bacteria, thus proper controlis required in all places. Much of the responsibility lies withnational governments, with a strategy and particular attentionto interventions that involve the introduction of legislation

and policies governing the development, licensing, distribu-tion, and sale of antibiotics.46

Finding new antibiotics that are effective against bacte-rial resistance is not impossible, but it is a complex and

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challenging area of research. It is also an area that has not beenthe primary focus of the pharmaceutical industry in recentyears, because antibiotics generally represent a relatively lowreturn of investment, and the high standards for drug devel-opment are also factors that influence this lack of interest.

Despite the expected growth trends for the global marketof antibiotics, their long-term success is primarily influencedby two main factors - resistance and generic competition.Antibiotic resistance forces reduction in use. The increasein antibiotic resistance makes curing infections difficult. Amajor disadvantage is the difficulty of the industry to findnew antibiotics - those in use are generally ongoing modifi-cations to produce new forms. Despite the advantages largecompanies have in the development of new antibiotics: a)well-defined targets, b) mode of research effectively estab-lished, c) biomarkers for monitoring, d) sophisticated tools tostudy dosing, and e) faster approval by regulatory agencies,they have given priority to other diseases, because the returnon investment for antibiotics is low, despite representing amarket of $ 45 billion, second only to drugs for cardiovascu-lar problems and central nervous system.47 Another problemis competition from generics at far lower prices.48 In somecases the large companies have transferred the responsibil-ity to small businesses to develop new antibiotics, such asdaptomycin, developed by Cubist and licensed to Lilly.49

Perspectives

Despite this scenario, some companies have established asocial position and responsibility to maintain the develop-ment of new antibiotics. An example is the potential for suchpartnerships in the fight against tuberculosis (TB). Today, mul-tidrug resistant TB affects half a million people annually, takestwo years to treat, is cured in only half of the cases, and occursmainly in areas where the human development index is low.

To accelerate the development of new treatments, animportant collaboration, the TB Alliance, is exploring cre-ative funding mechanisms and support for the final phase ofclinical trials. Another important action is the collection ofmicroorganisms in different environments, such as marineenvironments, for the isolation of new substances; thesestudies have achieved important results evaluating these envi-ronment actinomycetes.30,50 Another initiative is the AmazonBiotechnology Center-CBA, that has been studying microor-ganisms in the Amazon region, since this region, with itshigh diversity of microorganisms, has the capacity to producenew antibiotics; excellent results have been achieved mainlyregarding Mycobacterium tuberculosis.

There is still a need for regulation of the use of antibioticsto encourage pharmaceutical companies to invest in devel-oping new antibiotics. The main challenge remains at theregulatory level, in order to find a solution that ensures thecommercial viability of antibiotic development. The mergerof these companies has an immediate impact, reducing thenumber of competing research and development groups; such

changes often cause a strategic review of the therapeutic areasof research and development, where development of newantibiotics must compete with other areas that may be morecommercially attractive.

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In contrast to the first antibiotic, where the molecular modeof action was unknown until after it was introduced intothe market, technologies have evolved (functional genomics),allowing the evaluation of the interaction between the mecha-nism of action of the antibiotic target and the development ofspecific resistance of the bacteria.51,52 Despite the sequencingprojects of pathogenic organisms and the study of new targets,little success has been achieved.53,54 From a technical perspec-tive, companies that remain committed to research into newantibiotics using the new technologies will be successful; thechallenges are great, but not insurmountable.

Conflict of interest

All authors declare to have no conflict of interest.

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