The Battle Against Microbial Pathogens: Basic Science, Technological Advances and Educational Programs (A. Mndez-Vilas, Ed.

Daptomycin: Discovery, Development and Perspectives

Ipsita Chakravarty1, Kanika Kundu2 and Subir Kundu*,1
1
School of Biochemical Engineering, Indian Institute of Technology (Banaras Hindu University), Varanasi - 221005, India
2
Chemistry Section, MMV, Banaras Hindu University, Varanasi - 221005, India
* Corresponding author: email: [email protected]; Tel.: +91 7080185543

The emergence of antimicrobial drugs is a breakthrough health intervention that has helped save millions of lives and
reduces the morbidity of several infectious diseases. However, the concrete advances in control of infectious diseases need
to tackle the parallel upsurge in resistance to antimicrobials. There has been an alarming situation in the healthcare
industry today, since antibiotic resistance has become a serious clinical issue. The armory of antibiotics is becoming less
efficient leaving only few dependable replacements. The healthcare sector is intimidated by the labor and costs of putting
an entirely new molecule in the market. The reliability of developing new anti-infective molecules is being questioned.
Methicillin-resistant Staphylococcus aureus (MRSA) has challenged many potential antibiotics. MRSA may lead to
deactivation and inefficacy of antibiotics. The increased use of vancomycin and linezolid, effectual antibiotics against
Methicillin-resistant Staphylococcus aureus (MRSA) has resulted in resistant isolates. Amongst few novel antibiotics,
lipopeptide antibiotics have emerged as stars of the recent times. Daptomycin is a promising member of the lipopeptide
antibiotic family, which has displayed a broad spectrum of activity in vitro against a wide range of gram-positive
bacteria, including Methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococci. Daptomycin is not
affected by mechanisms that confer specific resistance to beta-lactam agents (including methicillin), glycopeptides (such
as vancomycin), quinupristin/dalfopristin, linezolid or other agents potentially useful against Gram-positive bacteria
species. The unique mechanism of action and low resistance profile, together with rapid bactericidal action make
Daptomycin a promising alternative in future to act against resistant organisms.

Keywords: Daptomycin; multi-drug resistance; methicillin-resistant Staphylococcus aureus (MRSA); cyclic lipopeptide
antibiotic

1. Introduction to Daptomycin
Antibiotic resistance has become a serious clinical issue in todays world. The failure of generations of antibiotics to
combat Superbugs has threatened the healthcare sector. Though several antimicrobials are being worked upon but still
there is an uncertainty regarding their efficacy and cost-effectiveness [1]. Methicillin-resistant Staphylococcus aureus
(MRSA) has challenged many established antibiotics [2]. The increased use of popular antibiotic against Methicillin-
resistant Staphylococcus aureus (MRSA) like Vancomycin has resulted in resistant isolates. The smart antibiotic
resistant strains have prevented vancomycin from reaching the target nascent cell wall precursors through thickening of
their cell walls [3]. There is a need for potential antibiotics against MRSA activity with a modified mechanism of action
[4]. Lipopeptide antibiotics are novel antibiotics against MRSA with unique mechanism of action [5]. They are
produced by soil actinomycetes via non-ribosomal biosynthetic pathways. Their structure consists of an acyl chain
conjugated to a linear or cyclic peptide sequence; the peptide portion can either have cationic or anionic residues [6].
Daptomycin is a promising member of the lipopeptide antibiotic family, which has displayed a broad spectrum of
activity in vitro against a wide range of gram-positive bacteria, including Methicillin-resistant Staphylococcus aureus
and vancomycin-resistant Enterococci. The mechanism of its action involves calcium-dependent dissipation of
membrane potential leading to the release of intracellular ions from the cell and bacterial death [7].

1.1 The Challenge of Methicillin Resistant Microorganisms

Methicillin resistant microorganisms have posed threat to the life-saving drugs from a long time. The advent of the first
penicillin-resistant cocci in 1944 came as a serious challenge [8]. Methicillin came into existence in 1956 and only two
years later the first Methicillin-resistant strain of Staphylococcus aureus (MRSA) had been isolated [9]. Since then,
there has been a tremendous increase in the number of antibiotic resistant strains. Studies suggest the prevalence of
resistant isolates in the US where 59% from skin and soft tissue infections were resistant to Methicillin [10]. In France ,
3.6%, while in Greece- 75% of MRSA strains were reported [11, 12] .Vancomycin, a tricyclic glycopeptides produced
by Streptomyces orientalis was discovered in 1956. It has been a popular drug for a long time in the treatment of MRSA
infections [13]. Vancomycin has side-effects, like nephrotoxicity and ototoxicity [14]. In 1996 the first vancomycin-
resistant S. aureus (VRSA) was detected. The thickening in the bacterial cell wall has hampered the efficacy of
vancomycin [15]. In order to combat such problems, proper drug monitoring is required [16]. Linezolid is another
valuable drug which is the first marketed antibiotic of the oxazolidinone class demonstrated activity against antibiotic-
susceptible and antibiotic-resistant aerobic Gram-positive cocci. Though, Linezolid can be orally administered but it has
few known side effects, e.g., bone marrow depression and poses higher risks to patients in case of solid organ

FORMATEX 2015 895

 The Battle Against Microbial Pathogens: Basic Science, Technological Advances and Educational Programs (A. Mndez-Vilas, Ed.)

transplantation. In this context, there is a need for a drug that is effective on MRSA and has minimal side-effects [17].
Owing to these conditions, Daptomycin has received much attention as a potential anti-infective drug.
Table 1 A comparative study of Daptomycin with other popular anti-MRSA drugs [18].
Properties Vancomycin Daptomycin Linezolid
Class of Glycopeptide Lipopeptide oxazolidinone
antibiotics
Mode of Action Inhibits cell-wall synthesis Calcium-dependent dissipation of Inhibits protein synthesis
membrane potential
Applications Severe infections caused by Complicated skin and skin structure Complicated skin and skin
susceptible strains of infections. S.aureus bloodstream structure infections including
methicillin-resistant S.aureus. infections (bacteremia),including those diabetic foot ulcers caused by
As a combinatorial drug with with right-sided infective endocarditis, MSSA. Treats Vancomycin
aminoglycoside for endocarditis. caused by MRSA isolates Resistant E.faecium infections
Bioavailibility Incomplete absorption Complete absorption Incomplete absorption
Clearance 0.06L/h/kg 0.10L/h/kg 0.01L/h/kg
Volume of 0.3 to 0.43 L/kg 0.7-0.8 L/kg 0.1L/kg
Distribution
Half Life 4-6h 4-5h 8h
Protein Binding 55% 31% 93%
Tissue Average High Low
penetaration and
effects
Dosing Therapy 1000mg IV over 60 min. Q12h 600mg IV over 30-120 min. Q12H 400- Increased dose interval
with monitoring 600 mg PO Q12h

1.2 Discovery of Daptomycin

Daptomycin is the first approved member of the A21978C family of the cyclic anionic 13-amino acid lipopeptide
antibiotics, produced as a secondary metabolite by Streptomyces roseosporus. It was initially developed in the late
1980s and early 1990s, at Eli Lilly and Company, by supplying decanoic acid to the growth media of Streptomyces
roseosporus during fermentation, but was held over due to concerns regarding myopathy. Cubist Pharmaceuticals Inc.
licensed worldwide rights from Eli Lilly and Company in 1997[5]. Daptomycin is a cyclic lipopeptide which consists of
a 13-member amino acid cyclic lipopeptide (hydrophilic core) with a decanoyl side chain (lipophilic tail) as shown in
Figure 1. The empirical formula of Daptomycin is C72H101N17O26; the molecular weight is 1620.67, while its
empirical name is N-decanoyl-L tryptophyl-D-asparaginyl-L-aspartyl-L-threonylglycyl-L-ornithyl-L-aspartyl-D-alanyl-
L aspartylglycyl-D-seryl-threo-3-methyl-L-glutamyl-3-anthraniloyl-L-alanine 1-lactone[18].The initial trials dealt with
a dose of 4 mg twice daily and had to be stopped in 1991 because of frequent elevations of serum levels of creatine
kinase (CK) probably related to skeletal muscle toxicity[18]. In 1999 the drug was re-introduced into clinical trials. The
FDA approved Daptomycin for the treatment of cSSSIs at a dose of 4 mg/kg daily (preceded by a starting dose of 6
mg/kg/ day) at last in 2003. In 2006 an additional FDA approval was granted for the treatment of bloodstream
infections and right-sided endocarditis caused by methicillin-sensitive S. aureus (MSSA) and MRSA. The approval was
confirmed in Europe in 2007[19].

Fig. 1 Chemical Structure of Daptomycin[19].

2. Development of Daptomycin
The mechanism of biological synthesis of Daptomycin has always remained an inquisitive and interesting phenomenon.
Various possible strategies have been applied to increase the production and activity of this useful antibiotic. Literatures
suggest that Daptomycin has a peculiar structure consisting of 13 amino acids and shares a ten-membered macrolactone
ring and three exocyclic residues. The factors can be distinguished by the fatty acyl-moiety attached to the N-terminal
Trp1, which ranges from 10-13 carbon atoms. These fatty acyl moieties comprise of n-decanoyl, anteiso-undecanoyl,
iso-dodecanoyl and anteiso-tridecanoyl, respectively. The peptide core is composed of a set of nonproteinogenic amino

896 FORMATEX 2015

 The Battle Against Microbial Pathogens: Basic Science, Technological Advances and Educational Programs (A. Mndez-Vilas, Ed.)

acids including D-Asn2, Orn6, D-Ala8, D-Ser11, (2S, 3R)-methyl glutamate (MeGlu) and kynurenine (Kyn13), that forms
an ester bond with Thr4 and builds up the macrolactone ring[20] . Daptomycin inherits a specific EF-hand motif
(DXDG) initially found in the ribosomally assembled calmodulin that is supposed to be involved in Ca2 +-binding [21].
The relative position of D-configured amino acids is conserved within this family as is the long chain fatty acid attached
to the cyclic core. The typical biosynthesis of Daptomycin by S. roseosporus is governed by three nonribosomal peptide
synthetases (NRPS), DptA, DptBC and DptD and in trans-acting enzymes [20].

Fig. 2 Representing the Biosynthesis of Daptomycin [19].

2.1 The multidomain organization of Nonribosomal peptide synthetases (NRPS)

Nonribosomal peptides are synthesized by nonribosomal peptide-synthetase (NRPS) enzymes which are independent
of messenger RNA. Each module is responsible for the specific recognition, activation, covalent binding and
incorporation of a building block into the oligopeptide chain and can be furthermore dissected into catalytic domains
[22]. A set of gene is involved in the acylation of the N-terminal amino acid. The initiation mechanism consists of an
acyl-CoA ligase, that activates the fatty acid, and an acyl-carrier-protein (ACP) to which the FA is covalently bound
[20]. The N-terminal C-domain (type CIII) of the initiation module subsequently catalyzes the condensation between the
FA and Trp1 and chain elongation is commenced[24]. Initiation of daptomycin biosynthesis is mediated by the action of
the two distinct enzymes DptE and DptF, encoded upstream of dptD[25]. DptE, which shares a high degree of
homology to the acyl-CoA ligase superfamily, was shown to activate the fatty acid moiety attached to the N-terminus of
daptomycin in an ATP-dependent manner. The activated FA is subsequently transferred onto the 4-Ppan group of
DptF, the cognate acyl-carrier-protein (ACP). The N-terminal C-domain of the DptA initiation module is predicted to
catalyze the condensation of the ACP-bound FA and tryptophan. The broad substrate tolerance of DptE towards the
length and type of fatty acids is believed to reflect in the composition of the A21978C factors [7]. After the initiation,
chain elongation is mediated by the linearly operating NRPSs DptA, DptBC and DptD. The three epimerization
domains present in the synthetases correlate with the D-configured amino acids D-Asn2, D-Ala8 and D-Ser11,
respectively [20]. Apart from the nonproteinogenic amino acids Orn6 and Kyn13, a -methylated glutamate residue
(MeGlu12) is located within the ten-membered macrolactone ring [26]. Deacylation was achieved by a highly efficient
deacylase derived from Actinoplanes utahensis[28]. Reacylation was performed chemically with activated acyl esters
after protection of side-chain nucleophiles. The generated derivatives varied not only in the acyl functionality but also
in the number of exocyclic amino acids (e.g. -N-acylated-Phe)[29].

2.2 Combinatorial Approach for Biosynthesis

Combinatorial approach of biosynthesis of Daptomycin has gained much importance as it involves an entire
reprogramming of genes responsible for the enzymatic machinery of the product [32]. S. roseosporus can easily accept
genetic manipulations and the responsible gene cluster has been sequenced, cloned and heterologously expressed [33]
.Combinatorial biosynthesis of daptomycin and hybrid molecules in S. roseosporus has already been extensively carried
out [27,34-35]. The modular NRPS assembly line offers substitutions and manipulations through tailoring from a single
module to multi modules. Initially, dptA and dptD were deleted from the original locus[36]. These genes were
subsequently introduced into S. roseosporus to trans-complement deletions of dptA and dptD by construction of plasmid
cloning vectors allowing conjugal transfer of genetic information from Escherichia coli to the target strain [36-37].

FORMATEX 2015 897

 The Battle Against Microbial Pathogens: Basic Science, Technological Advances and Educational Programs (A. Mndez-Vilas, Ed.)

3. Mode of action of Daptomycin

Linezolid is protein synthesis inhibitor which stops the bacterial growth by disrupting translation of messenger
RNA (mRNA) into proteins in the ribosome. It prevents the formation of the initiation complexes as it binds to
the 23S portion of the 50S subunit[38]. The intrinsic resistance of most Gram-negative bacteria to linezolid is due to the
activity of efflux pumps, which actively "pump" linezolid out of the cell faster than it can accumulate. Vancomycin acts
by inhibiting proper cell wall synthesis in gram-positive bacteria[39]. Vancomycin is not active against gram-negative
bacteria. The large hydrophilic molecule is able to form hydrogen bond interactions with the terminal D-alanyl-D-
alanine moieties of the NAM/NAG-peptides. This binding of vancomycin to the D-Ala-D-Ala prevents cell wall
synthesis of the long polymers of N-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG) that form the
backbone strands of the bacterial cell wall, and it prevents the backbone polymers that do manage to form from cross-
linking with each other. In resistant bacteria, the last D-ala residue has been replaced by a D-lactate, so vancomycin
cannot bind. In the nonresistant bacteria, the vancomycin bound to the peptide chains prevents them from interacting
properly with the cell wall cross-linking enzyme. However, in the resistant bacteria, stable cross links are formed[40].
The distinctive action Daptomycin in antibacterial activities can be better understood by its mode of action. The present
mode of action of Daptomycin suggests the discrete role of calcium ions in the antimicrobial activities . The initial
studies by Jung et al. proposed a two-step mechanism of action derived from structural changes observed in NMR-
experiments, CD-measurements and fluorescence spectroscopy. In the first step Ca2+ binds to daptomycin in solution
and induces a conformational change, increasing amphipathicity and decreasing its charge [41]. This process facilitates
oligomerization and leads to micelle formation which allows daptomycin to interact with neutral or acidic membranes.
In a second step, Ca2+ bridges the gap between daptomycin and the acidic phospholipids. As indicated by CD-
measurements, daptomycin undergoes a second structural transition allowing a deeper insertion into the membrane
bilayer.

Fig. 3 The mode of action of Daptomycin [41].

In contrast to the study conducted by Silverman et al., it was proposed that cytoplasmic membrane depolarization is
not the main cause of cell death as it occurred subsequently [42]. The recent studies negate this mode of action on the
basis of an NMR-structure of Daptomycin in the presence of Mg2+[43]. It was found that Mg2+ also promotes micelle
formation, but does not induce a conformational change. Daptomycin was found to form oligomers consisting of 14-16
monomers upon addition of Ca2+ in a 1:1-ratio. In accordance to Jung et al. it was proposed that divalent cations mask
the negatively charged residues and enable micelle formation either by -stacking interactions between aromatic residues
or by arrangement of the lipid tails towards the interior of the micelle [43]. Scott et al. [44] confirm that Daptomycin
experiences only a minor conformational rearrangement upon binding to DHPC in the presence of Ca2+.

4. Production Strategies for Daptomycin

The increased demand of Daptomycin has led to different processing strategies to improve its production. The wild type
strain of Streptomyces roseosporus NRRL11379 has been preferentially used for Daptomycin production [45]. Also, S.
roseosporus LC-51, a mutant of S. roseosporus NRRL11379 has shown improved ability for Daptomycin production.
Rational screening based on metabolic engineering is being considered for improvement of the selection process. Low-
power laser irradiation technology is being used for high yielding strains, nowadays [46]. Chemical mutation by N-
methyl-N-nitro-N nitrosoguanidine (NTG) has been reported as a successful method for mutation and screening of high-
yield strains, which can be adapted by any in Pharmaceutical Industry for Antibiotic Production.[47] Various strategies,
such as medium optimization, fermentative strategy, genetic engineering modification and metabolic flux analysis have
been established to achieve higher production of Daptomycin.

898 FORMATEX 2015

 The Battle Against Microbial Pathogens: Basic Science, Technological Advances and Educational Programs (A. Mndez-Vilas, Ed.)

Table 2 Various strains producing substantial amount of Daptomycin.

Strains Daptomycin or A21978C Working volume Culture time References
(mg/L) (L) (h)
Streptomyces lividans TK23 and 55 (A21978C) 168240 Penn et al. [49]
TK64
Streptomyces roseosporus 812 3.6 282 I-Son Ng et al.
NRRL11379 [45]
Streptomyces roseosporus LC-51 632 7.5 132 Yu et al. [48]

Streptomyces roseosporus 296 (A21978C) 7.0 144 Lu et al. [46]

NRRL11379

4.1 Role of manipulation of Cofactors for enhanced Daptomycin Production

Biochemical reactions are governed by various cofactors of enzymes which play a key role in the production of various
fermentation products.Thus, the manipulation of cofactors concentration in the fermentation culture could be crucial in
order to increase overall process yield of the secondary metabolites [48]. The effects of eight cofactors of enzymes on
Daptomycin production were usually investigated through many researches, which included Nicotinic acid (VPP),
Riboflavin (VB2), Heme, Thiamine (VB1), Biotin (VH), Cyanocobalamin (VB12), Tetrahydrofolic acid (THF) and
Pyridoxal 5-phosphate (VB6). The effects of Heme, THF, VB12 and VB6 on Daptomycin production were especially
notable. Daptomycin yield increased to 632 mg/l, which is over 4.5-fold higher than that of the control (without
cofactors), at 132 h in a 7.5 Litre fermenter, by supplementation all of the eight cofactors at optimized concentrations
(VPP 4 mg/l, VB2 0.5 mg/l, Heme 9 mg/l, VB1 0.4 mg/l, VH 0.1 mg/l, VB12 0.04 mg/l, THF 6 mg/l and VB6 0.4
mg/l). This strategy used for for increasing the Daptomycin production in Streptomyces roseosporus LC-51 by
manipulation of cofactors concentration in the fermentation culture may provide an alternative approach to enhance the
production of metabolites in other Streptomyces [48, 52].

4.2 Precursor Utilization in Daptomycin Production

Decanoic acid is the essential precursor for Daptomycin production. Almost no daptomycin is produced from S.
roseosporus without decanoic derivative chemicals addition. Decanoic acid, sodium decanoate and Cuphea oil
[49]which is also a rich source of decanoic acid have been used to improve the Daptomycin fermentation. The two
major problems of precursor inhibition and product inhibition of S.roseosporus need to be addressed. Many research
from literature indicated that the growth of cells would be inhibited by the toxicity of n-decanoic acid by overfeeding of
n-decanoic acid during fermentation.[48] Decanoic acid, Sodium decanoate and Cuphea oil were compared for
Daptomycin production. However, the decanoic acid (a type of organic acid) might be a kind of the toxic element in
culture of S. roseosporus.
Table 3 Comparitive Daptomycin production using various precursors.
S.No. Precursors Daptomycin Production(mg/L) Culture Time(h) Working Volume(L) References
1 Without any Precursor Not defined 282 3.6 [45]
2 Decanoic acid 296 144 7 [46]
3 Sodium Decanoate 812 282 3.6 [45]
4 Cuphea oil 600 186 20 [50]

As the precursor, sodium decanoate was in favor of the daptomycin production compared with the aliphatic fatty
acids which were examined for enhancement of other lipopeptide antibiotics. The addition time of precursor with
maximum daptomycin production was achieved when sodium decanoate was added at the second day after inoculation.
Delaying the addition time of the precursor also prevented inhibition of cell growth [51].

4.3 Fed Batch Strategy for Daptomycin Production

Substrate inhibition and product inhibition are two major problems that hamper antibiotic production. In order to have
large scale production of antibiotic without losing much product to substrate inhibition, improved fed batch strategy is
established as a feedback control. Daptomycin was produced in shake-flasks, batch, and fed-batch fermentation.
However, the production of Daptomycin was effectively increased in by intermittent feeding of dextrin upto 812.0 mg/L
from 217.5 mg/L in batch fermentation. [45]. Fed-batch strategy developed in fermentation is often applicable in the
improvement of antimicrobial products [52].

4.4 Flux Analysis to improve media utilization

A comprehensive metabolic flux analysis model was set up and used to evaluate Daptomycin metabolism, amino acid
utilization, and simultaneous consumption of nutrient sources. Stoichiometric model were used to demonstrate the

FORMATEX 2015 899

 The Battle Against Microbial Pathogens: Basic Science, Technological Advances and Educational Programs (A. Mndez-Vilas, Ed.)

intracellular carbon flux distribution. This was an innovative approach which compared the flux changes for different
fermentation conditions and helped to interpret of the dependency of Daptomycin yield on environmental perturbations
(e.g. pH) and principal pathways. Experimental and calculated values for both the specific growth rate of Streptomyces
roseosporus LC-54-20 (mutated strain) and Daptomycin production rate indicated that the in silico model proved a
powerful tool to analyze the metabolic behaviors. Flux distribution variations revealed that the daptomycin production
could be significantly influenced by the branch nodes of glucose 6-phosphate, 3-phosphoglycerate,
phosphoenolpyruvate, pyruvate, and oxaloacetate. Unlike the previous studies [29], where decanoic acid was the sole
precursor which controlled Daptomycin production, the yield of Daptomycin was enhanced by adding five precursors in
batch fermentation. [53] The strategy embellished conventional medium design. Many recent researches also suggest
the role of metabolic engineering of the genes involved in biosynthesis of Daptomycin in enhancement of carbon uptake
and precursor tolerance.

4.5 Total Chemical synthesis of Daptomycin

The first total chemical synthesis of Daptomycin was recently illustrated. Instead of biosynthesis, a complex strategy
was developed using a combination of solution-phase synthesis and SPPS to successfully assemble the linear
Daptomycin peptide precursor. An efficient and scalable ozonolysis strategy was introduced to synthesize Kyn-
containing peptide fragments from the correspondingTrp-containing peptides. The key macrocyclization step in the
synthesis of Daptomycin was achieved via a chemoselective serine ligation, which enabled the large-scale synthesis of
daptomycin since the cyclization could be accomplished at concentrations of up to 50 mM. Two important conclusions
were drawn from this methodology -serine/threonine ligation could be a general tool for peptide cyclization;[54] and
ozonolysis of a Trp-containing peptide fragment can lead to the formation of the corresponding Kyn-containing peptide
fragment in a highly efficient manner if the Trp moiety is protected. This success suggests that a Kyn-containing cyclic
peptide could be readily synthesized via serine/threonine ligation and ozonolysis from a suitable linear Trp-containing
peptide precursor.[55]

4.6 Future Considerations for bulk production

The chemical synthesis of key intermediates involves multiple steps and hazardous reaction conditions. To develop
high-valued antibiotics at large-scale like Daptomycin, various strategies should be taken up to improve mass transfer
characteristics [56-57]. The fermentation process is highly aerobic and involves filamentous microorganism
(actinomycetes). Free and immobilized microbial cells can be cultivated using various cultivation modes of batch and
continuous strategy using different bioreactors (stirred tank bioreactor, air lift bioreactor and packed bed column)[58-
60].

5. Clinical Significance of Daptomycin-Case Studies

5.1 Deep sternal wound infections post cardiac operation

Papov et.al. reported the incidence of deep sternal wound infection (DSWI) after cardiac surgery, 0.4-5% with
Staphylococcus aureus as the most common pathogen isolated from infected wound sternotomies and bacteraemic
blood cultures. This infection is associated with a higher morbidity and mortality than other known causations. The
application of Daptomycin was quantified for the treatment of deep sternal wound infection due to gram-positive
organisms post cardiac surgery. An observational analysis was carried out for 23 cases of post sternotomy deep sternal
wound infection I with gram-positive organisms in February 2009 and September 2010. The incidence of deep sternal
wound infection was 1.46%. The mean dose of Daptomycin application was 4.4 0.9 mg/kg/d and the average duration
of the Daptomycin application was 14.47 7.33 days. Treatment of deep sternal wound infection due to gram-positive
organisms with a Daptomycin-containing antibiotic regimen was found to be safe, effective for local wound infections.
[61]

5.2 Diabetic foot ulcers and infections

Total103 patients diagnosed with diabetic foot ulcer infection were clinically evaluated; 47 were treated with
Daptomycin and 56 received a comparator. Infections were predominantly due to Staphylococcus aureus. Success rates
for patients treated with Daptomycin or the comparators were not statistically different for clinical (66% versus 70%,
respectively; 95% CI, 14.4, 21.8) or microbiological outcomes. Both treatments were generally well tolerated, with
most adverse events of mild to moderate severity. [62]

5.3 Combination drugs containing Daptomycin for Vancomycin-Resistant Enterococcus faecium

Fosfomycin is an effective drug for vancomycin-resistant enterococcus (VRE) infections. 32 VRE isolates from renal
transplant patients with urinary stent infections were susceptible to fosfomycin, daptomycin, and linezolid and resistant

900 FORMATEX 2015

 The Battle Against Microbial Pathogens: Basic Science, Technological Advances and Educational Programs (A. Mndez-Vilas, Ed.)

to amoxicillin, minocycline, and nitrofurantoin based on their MIC50s and MIC90s. Fosfomycin was bacteriostatic at
0.5 to 16_the MIC (32 to 2,048 _g/ml); synergy occurred when fosfomycin was combined with daptomycin (2.8 to 3.9
log10 CFU/ml kill; P<0.001) or amoxicillin (2.6 to 3.4; P<0.05). These combinations were found to be potent options
to treat VRE urinary infections pending investigation of clinical efficacy [63].

5.4 Daptomycin for osteomyelitis caused by MRSA in a renal transplant recipient with FabryAnderson
disease
Daptomycin is licensed in adults for the management of Staphylococcus aureus methicillinresistantinfections, including
bone and skin complicated infections. We describe for the first time its use in a renal transplant recipient for
FabryAnderson Disease with right heel osteomyelitis. The patient was unresponsive to firstline Teicoplanin and
secondline Tigecycline, whereas he was successfully treated with thirdline Daptomycin monotherapy at 4 mg/Kg/qd for
4 weeks. Local debridement was performed in advance of each line of treatment [64].

5.5 Brain Damage and Hearing Loss in Infant Rat Pneumococcal Meningitis
Aggravation of cerebrospinal fluid (CSF) inflammation in response to bacteriolysis by beta-lactam antibiotics
contributes to brain damage and neurological sequelae in bacterial meningitis. Daptomycin, a non-lytic antibiotic acting
on Gram-positive bacteria, lessens inflammation and brain injury compared to ceftriaxone. With a view to a clinical
application for pediatric bacterial meningitis, the effect of combining daptomycin or rifampin with ceftriaxone was
evaluated in an infant rat pneumococcal meningitis model. CSF was sampled at 6 and 22 h after the initiation of
therapy and was assessed for concentrations of defined chemokines and cytokines. Brain damage was quantified by
histomorphometry at 40 h after infection and hearing loss was assessed at 3 weeks afterinfection. Daptomycin plus
ceftriaxone versus ceftriaxone significantly (P<0.04) lowered CSF concentrations of monocyte chemoattractant protein
1 (MCP-1), MIP-1_, and interleukin 6 (IL-6) at 6 h and MIP-1_, IL-6, and IL-10 at 22 h after initiation of therapy, led
to significantly (P<0.01) less apoptosis, and significantly (P<0.01) improved hearing capacity. While rifampin plus
ceftriaxone versus ceftriaxone also led to lower CSF inflammation (P<0.02 for IL-6 at 6 h), it had no significant effect
on apoptosis and hearing capacity. Adjuvant daptomycin could therefore offer added benefits for the treatment of
pediatric pneumococcal meningitis [65].

5.6 Immunomodulatory effects of Daptomycin.

After vancomycin and linezolid, dapto-mycin gains increasing importance in treatment of wound infections after
cardiac surgery. Daptomycin showed immunomodulatory properties, resulting in the suppression of cytokine expression
after host immune response stimulation by MRSA. Experimental studies revealed an improved efficacy of daptomycin
in combination with administration of vitamin E before infecting wounds by MRSA[66].

5.7 Prosthetic joint infection by Enterococcus faecalis

Enterococci are implicated in less than 2.3% of prosthetic joint infections. These infections can be difficult to treat and
therapeutic failures are not uncommon. In these situations, Daptomycin is a safe and effective alternative. This clinical
case was presented with a successful response to the prolonged use of high dose Daptomycin[67].

6. Conclusion
The prominence of bactericidal effect of Daptomycin against Gram-positive organisms has opened up new scopes to
combat MRSA and fight drug resistance. The future prospects of this outstanding drug are quite bright and promising.
It is indeed a miraculous drug that can effectively deal with life-threatening infections, especially drug-resistant
staphylococcal infections. Though much endeavor has been made to bring forth the biosynthetic mechanisms of
Daptomycin through various literary reports. Yet, the yield of Daptomycin needs to be further enhanced in a cost-
effective manner. In order to avail the benefits of this high-value secondary metabolite, new strategies should be
incorporated for its large-scale production. The amalgamation of metabolic engineering as well as biochemical
engineering would open up new paths for this valuable product. Also, an awareness about this drug and efforts to
improve its bulk production would help to serve a larger population.

FORMATEX 2015 901

 The Battle Against Microbial Pathogens: Basic Science, Technological Advances and Educational Programs (A. Mndez-Vilas, Ed.)

References
[1] Stefani, S. & Varaldo, P. E. Epidemiology of Methicillin resistant staphylococci in Europe. Clinical Microbiology and
Infection. 2003; 9: 117986.
[2] Centres for Disease Control and Prevention Staphylococcus aureus resistant to VancomycinUnited States, 2002. Morbidity
and Mortality Weekly Report, 2002; 5657.
[3] Cui, L., Ma, X., Sato, K. et al, Cell wall thickening is a common feature of vancomycin resistance in Staphylococcus aureus.
Journal of Clinical Microbiology 2003; 41: 514.
[4] Judith N. Steenbergen, Jeff Alder, Grace M. Thorne and Francis P. Tally Cubist Pharmaceuticals, Inc., 65 Hayden Avenue,
Lexington, MA 02421.Daptomycin: a lipopeptide antibiotic for the treatment of serious Gram-positive infections. Journal of
Antimicrobial Chemotherapy. 2005; 283288.
[5] Dohmen et.al. Methicillin-resistant staphylococcus aureus infective endocarditis ; Mendez-Vilas A: Microbial pathogens and
strategies for combating them: science, technology and education Formatex Research Center, Badajoz, Spain.2013 ; 1765-1769,
ISBN 978-84-942134-1-0
[6] Lars Robbel and Mohamed A. Marahiel. Daptomycin, a Bacterial Lipopeptide Synthesized by Non-ribosomal Machinery. J.
Biol. Chem. 2010; 285:27501-27508.
[7] Silverman, J., Harris, B., Cotroneo, N., et al. Daptomycin (DAP) treatment induces membrane and cell wall alterations in
Staphylococcus aureus, In Inter science Conference on Antimicrobial Agents and Chemotherapy, Chicago, IL.. American
Society for Microbiology, Washington, DC, USA, Abstract C12135, 2003: 103.
[8] Gots JS. The detection of penicillinases-producing properties of microorganisms. Science. 1945; 102:309.
[9] Smith JT, Hamilton-miller JM, Knox R. Isoxazolyl penicillins and penicillinases. Nature. 1962; 195:13001301.
[10] Moran GJ, Krishnadasan A, Gorwitz RJ, et al. Methicillin-resistant S. aureus infections among patients in the emergency
department. N Engl J Med. 2006; 355:666674.
[11] Dauwalder O, Lina G, Durand G, et al. Epidemiology of invasive Methicillin-resistant Staphylococcus aureus clones collected
in France in 2006 and 2007. J Clin Microbiol. 2008; 46:34543458.
[12] Chini V, Petinaki E, Foka A, Paratiras S, Dimitracopoulos G, Spiliopoulou I. Spread of Staphylococcus aureus clinical isolates
carrying Panton-Valentine leukocidin genes during a 3-year period in Greece. Clin Microbiol Infect. 2006; 12:2934.
[13] Centers of disease control and prevention national nosocomial infections Surveillance system: MRSA among ICU patients,
19952004.
[14] Sivagnanam S, Deleu D. Red man syndrome. Crit Care. 2003; 7: 119120.
[15] Tenover FC, Lancaster MV, Hill BC, et al. Characterization of staphylococci with reduced susceptibilities to vancomycin and
other glycopeptides. J Clin Microbiol. 1998; 36:10201027.
[16] Steinkraus G, White R, Friedrich L. Vancomycin MIC creep in non-vancomycin intermediate Staphylococcus aureus (VISA),
vancomycin-susceptible clinical methicillin-resistant S.aureus (MRSA) blood isolates from 200105. J Antimicrob Chemother.
2007.
[17] Kerry S. Estes, Hartmut Derendorf .Comparison of the Pharmacokinetic Properties of Vancomycin, Linezolid, Tigecyclin,
And Daptomycin. European Journal of Medical Research. 2010; 15:533-543
[18] Dvorchik, B.H., d. Brazier, M.f. deBruin, and R.d. arbeit, Daptomycin pharmacokinetics and safety following administration
of escalating doses once daily to healthy subjects. Antimicrob Agents Chemother. 2003 Apr;47(4):1318-1323
[19] Tally FP, DeBruin MF. Development of Daptomycin for gram-positive infections. J Antimicrob Chemother.2000; 46:523526
[20] Miao, V., Coeffet-LeGal, M. F., Brian, P., Brost, R., Penn, J., Whiting, A., Martin, S., Ford, R., Parr, I., Bouchard, M., Silva,
C. J., Wrigley, S. K., and Baltz, R. H. Microbiology 2005;151: 15071523
[21] Yazawa, M., and Yagi, K. Biochem. Biophys. Res. Commun. 1980; 96: 377381
[22] Fischbach, M. A., and Walsh, C. T. Chem. Rev. 2006;106: 34683496
[23] Sieber, S. A., and Marahiel, M. A. Chem. Rev. 2005;105:715738
[24] Rausch, C., Hoof, I., Weber, T., Wohlleben, W., and Huson, D. H. BMC Evol. Biol.2007; 7:78
[25] Wittmann, M., Linne, U., Pohlmann, V., and Marahiel, M. A. FEBS J.2008; 275: 53435354
[26] Nguyen, K. T., Kau, D., Gu, J. Q., Brian, P., Wrigley, S. K., Baltz, R. H., and Miao, V. Mol.Microbiol. 2006;61: 12941307
[27] Debono, M., Abbott, B. J., Molloy, R. M., Fukuda, D. S., Hunt, A. H., Daupert, V. M., Counter, F. T., Ott, J. L., Carrell, C. B.,
Howard, L. C., Boeck, L. V., and Hamill, R. L. J. Antibiot.1988; 41: 10931105
[28] Boeck, L. D., Fukuda, D. S., Abbott, B. J., and Debono, M. J. Antibiot.1988; 41: 10851092
[29] Huber, F. M., Pieper, R. L., and Tietz, A. J. J. Biotechnol.1988; 7: 283292
[30] Grunewald, J., Sieber, S. A., Mahlert, C., Linne, U., and Marahiel, M. A. J. Am. Chem. Soc. 2004;126: 1702517031
[31] Nguyen, K. T., Ritz, D., Gu, J. Q., Alexander, D., Chu, M., Miao, V., Brian, P., and Baltz, R. H. Proc. Natl. Acad. Sci. U.S.A.
2006;103: 1746217467
[32] Walsh, C. T. ChemBioChem .2002;3: 125134
[33] Penn, J., Li, X., Whiting, A., Latif, M., Gibson, T., Silva, C. J., Brian, P., Davies, J., Miao, V., Wrigley, S. K., and Baltz, R. H.
J. Ind. Microbiol. Biotechnol. 2006;33: 121128
[34] Baltz, R. H., Brian, P., Miao, V., and Wrigley, S. K. J. Ind. Microbiol. Biotechnol. 2006;33: 6674
[35] Miao, V., Coeffet-Le Gal, M. F., Nguyen, K., Brian, P., Penn, J., Whiting, A., Steele, J., Kau, D., Martin, S., Ford, R.,
Gibson, T., Bouchard, M., Wrigley, S. K., and Baltz, R. H. Chem. Biol. 2006;13: 269276
[36] Coeffet-Le Gal, M. F., Thurston, L., Rich, P., Miao, V., and Baltz, R. H. Microbiology 2006;152: 29933001
[37] McHenney, M. A., and Baltz, R. H. Microbiology.1996; 142: 23632373
[38] Ament PW, Jamshed N, Horne JP (February 2002). "Linezolid: its role in tAment PW, Jamshed N, Horne JP (February
2002). "Linezolid: its role in the treatment of gram-positive, drug-resistant bacterial infections". American Family
Physician 65 (4): 663

902 FORMATEX 2015

 The Battle Against Microbial Pathogens: Basic Science, Technological Advances and Educational Programs (A. Mndez-Vilas, Ed.)

[39] Schumacher A, Trittler R, Bohnert JA, Kmmerer K, Pags JM, Kern WV (June 2007). "Intracellular accumulation of linezolid
in Escherichia coli, Citrobacter freundii and Enterobacter aerogenes: role of enhanced efflux pump activity and
inactivation" (PDF). Journal of Antimicrobial Chemotherapy 59 (6): 12614
[40] J Clin Invest. 2014 Jul; 124(7):2836-40. Epub 2014 Jul 1.Mechanisms of vancomycin resistance in Staphylococcus aureus.
[41] Jung, D., Rozek, A., Okon, M., and Hancock, R. E. Chem. Biol. 2004;11: 949957
[42] Silverman, J. A., Perlmutter, N. G., and Shapiro, H. M. Antimicrob. Agents Chemother. 2003;47: 25382544
[43] Jung, D., Rozek, A., Okon, M., and Hancock, R. E. (2004) Chem. Biol. 11, 949957
[44] Scott, W. R., Baek, S. B., Jung, D., Hancock, R. E., and Straus, S. K. (2007) Biochim. Biophys. Acta 1768, 31163126
[45] I-Son Ng, Chiming Ye, Zhixiang Zhang, Yinghua Lu,Keju Jing,Daptomycin antibiotic production processes in fed-batch
fermentation by Streptomyces roseosporus NRRL 11379 with precursor effect and medium optimization. Bioprocess
Biosystems Eng,2013,doi:10.1007/s00449-013-1007-2
[46] Lu W, Fan J, Wen J, Xia Z, Caiyin Q (2011) Kinetic analysis and modeling of daptomycin batch fermentation by Streptomyces
roseosporus. Appl Biochem Biotechnol 163:453462
[47] Yu G, Jia X, Wen J, Lu W, Wang G, Caiyin Q, Chen Y (2011) Strain Improvement of Streptomyces roseosporus for
Daptomycin production by rational screening of HeNe laser and NTG induced mutants and kinetic modeling. Appl Biochem
Biotechnol163:729743
[48] Yu G, Jia X, Wen J, Wang G, Chen Y (2011) Enhancement of daptomycin production in Streptomyces roseosporus LC-51 by
manipulation of cofactors concentration in the fermentation culture. World J Microbiol Biotechnol 27:18591868
[49] Penn J, Li X, Whiting A, Latif M, Gibson T, Silva CJ, Brian P, Davies J, Miao V, Wrigley SK, Baltz RH (2006) Heterologous
production of daptomycin in Streptomyces lividans. J Ind Microbiol Biotechnol 33:12112857.
[50] Gianluca Bertetti et.al. , Process for the production of Daptomycin ,US 20100047873 A1,issued on February 25,2010
[51] Boeck LD, Wetzel RW (1990) A54145, a new lipopeptide antibiotic complex: factor control through precursor directed
biosynthesis.J Antibiot 43:607615
[52] Muller JM, Risse JM, Jussen D, Flaschel E (2013) Development of fed-batch strategies for the production of streptavidin
by Streptomyces avidinii based on power input and oxygen supply studies. J Biotechnol 163:325332
[53] Di Huang & Xiaoqiang Jia & Jianping Wen & Guoying Wang & Guanghai Yu & Qinggele Caiyin & Yunlin Chen Metabolic
Flux Analysis and Principal Nodes Identification for Daptomycin Production Improvement by Streptomyces roseosporus ,
Appl Biochem Biotechnol, 2011; 7251739. 61.
[54] C.T.T.Wong, H. Y. Lam, X. Li, Org. Biomol. Chem. 2013, 11,76167620
[55] Hiu Yung Lam, Rannveig Ingebrigtsen Gaarden, and Xuechen Li 2014 A Journey to the Total Synthesis of Daptomycin Chem.
Rec. , 14, 10861099
[56] Mahapatra A.C., Kundu K., Nigam V.K., Mandava M.V.P., Kundu, S. Comparative studies of CPC production by free and
immobilized cells of Cephalosporium acremonium in different modes of bioreactors. Indian J. Microbiol. 2002; 42: 319-322
[57] Srivastava, P. and Kundu, S. A laboratory air lift reactor for cephalosporin-C. J. Ind.Chem Engg. 1995; 37: 138-139.
[58] Srivastava, P. and Kundu, S. Studies on cephalosporin-C production in an air lift reactor using different growth modes of
Cephalosporium acremonium. Process Biochem. 1999; 34: 329-333.
[59] Kundu, S., Mahapatra A.C. Microbial Production of cephalosporin C using co cultures of Cephalosporium acremonium and
Chlorella pyrenoidosa in a packed bed reactor. Avanna C. Recent trends in Biotechnology. India: Tata McGraw Hill; 1993:31-
35.
[60] Gaurav K., Kundu S., Srivastava R. Synthesis and in vitro antibacterial activity of some novel cephem antibiotics. International
Journal of Pharmacy and Pharmaceutical Sciences. 2012; 4(3): 659-667.
[61] Popov et al. 2011Treatment of gram-positive deep sternal wound infections in cardiac surgery -experiences with Daptomycin
Journal of Cardiothoracic Surgery , 6:112
[62] Lipsky BA, Stoutenburgh U. Daptomycin for treating infected diabetic foot ulcers: evidence from a randomized, controlled trial
comparing daptomycin with vancomycin or semi-synthetic penicillins for complicated skin and skin-structure infections. J
Antimicrob Chemother 2005
[63] Descourouez et al.2013 Fosfomycin Synergy In Vitro with Amoxicillin, Daptomycin, and Linezolid against Vancomycin-
Resistant Enterococcus faecium from Renal Transplant Patients with Infected Urinary Stents Antimicrobial Agents and
Chemotherapy 57(3):1518-1520
[64] Polilli et.al. Successful salvage therapy with Daptomycin for osteomyelitis caused by methicillinresistant Staphylococcus
aureus in a renal transplant recipient with FabryAnderson disease Annals of Clinical Microbiology and Antimicrobials 2012,
11:6,1-4
[65] Denis Grandgirard, Melchior Burri, Philipp Agyeman, and Stephen L. Leiba 2012, Adjunctive Daptomycin Attenuates Brain
Damage and Hearing Loss More Efficiently than Rifampin in Infant Rat Pneumococcal Meningitis , Antimicrobial Agents and
Chemotherapy 56(8): 42894295
[66] Theodor Trilomis Daptomycin and its immunomodulatory effect: consequences for antibiotic treatment of methicillin-resistant
Staphylococcus aureus wound infections after heart surgery. Frontiers in Immunology 2014,5(97),1-5
[67] Rafael Canton, Patricia Ruiz-Garbajosa, Ricardo L. Chaves and Alan P. Johnson.A potential role for daptomycin in
enterococcal infections: what is the evidence? J Antimicrob Chemother 2010; 65: 11261136

FORMATEX 2015 903