Anti-multi drug resistant pathogen activity of ...

Korean Journal of Microbiology (2016) Vol. 52, No. 3, pp. 327-335 pISSN 0440-2413DOI http://dx.doi.org/10.7845/kjm.2016.6033 eISSN 2383-9902Copyright ⓒ 2016, The Microbiological Society of Korea

Article

Anti-multi drug resistant pathogen activity of siderochelin A,

produced by a novel Amycolatopsis sp. KCTC 29142

Dong-Ryung Lee1†

, Jinhua Cheng1,2†

, Sung-Kwon Lee1, Hee-Jeon Hong

3, Jaekyeong Song

4, Seung Hwan Yang

5*, and

Joo-Won Suh1,2*

1Center for Nutraceutical and Pharmaceutical Materials Myongji University, Yongin 17058, Republic of Korea

2Division of Bioscience and Bioinformatics, College of Natural Science, Myongji University, Yongin 17058, Republic of Korea

3Department of Biological and Medical Sciences, Faculty of Health and Life Sciences, Oxford Brookes University, Oxford OX3

0BP, United Kingdom4Division of Agricultural Microbiology, National Academy of Agricultural Science, Rural Development Administration, Jeonju

56050, Republic of Korea5Department of Biotechnology, Chonnam National University, Gwangju 59626, Republic of Korea

Amycolatopsis sp. KCTC 29142로부터 유래된 siderochelin A의 다제 내성

균주에 대한 항균활성

이동령1† ･ 성금화

1,2† ･ 이성권

1 ･ 홍희전

3 ･ 송재경

4 ･ 양승환

5* ･ 서주원1,2*

1명지대학교 농생명바이오식의약소재개발사업단,

2명지대학교 생명과학정보학부 생명과학과,

3옥스퍼드 브룩스대학 생의과학과,

4국립농업과학원 농업미생물과,

5전남대학교 생명산업공학과

(Received June 17, 2016; Revised August 3, 2016; Accepted August 9, 2016)

ABSTRACT: A novel Amycolatopsis strain KCTC 29142 was isolated and characterized based on the polyphasic taxonomic analysis

including morphological observation, phylogenetic analysis, physiological and chemotaxonomic characteristics. The ethyl acetate extract of strain KCTC 29142 culture broth showed strong antibacterial activity and the active compound was identified as siderochelin A, a ferrous-ion chelating compound. In this study, siderochelin A showed good activity against multi-drug resistant pathogens, including

Acinetobacter baumanii, methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Staphylococcus aureus (VRSA), and Escherichia coli (E. coli ). The minimum inhibitory activity against clinical isolates was also determined.

Key words: Amycolatopsis, antibacterial activity, anti-multi drug resistant pathogens, Siderochelin A

†These authors contributed equally to this work.

*For correspondence. (S.H. Yang) E-mail: [email protected];

Tel.: +82-61-659-7306; Fax: +82-61-659-7309

(J.W. Suh) E-mail: [email protected];

Tel.: +82-31-330-6190; Fax: +82-31-321-7361

Multi drug-resistant pathogens (MDRPs) are a critical world-

wide problem because of their resistance to a bevy of commer-

cially available antibiotics and their high mortality rate. Available

evidence showed that the proportion of Gram-positive and

negative bacteria resistant to commonly used antibiotics is

increasing (Lode, 2009; Lin and Lan, 2014). The mechanisms

of this resistance are often complex, and include production of

β-lactamases, upregulated efflux pumps, and target site mutations

(Sakoulasand and Moellering, 2008). It is reported that patients

with infections due to MDRP have higher costs than do patients

with infections due to antimicrobial- susceptible organisms

(Cosgrove, 2006). So it is necessary to develop new drugs for

control of MDRP (Anderson, 2003).

Iron is an essential bioelement for most bacteria, which

involved in a variety of metabolic and informational cellular

pathways (Miethke and Marahiel, 2007). Since the importance

328 ∙ Lee et al.

미생물학회지 제52권 제3호

of iron in the growth of bacteria, many studies has assessed the

potential viability of iron chelators as therapeutic agents against

various microbes (Chan et al., 2009; Kim and Shin, 2009).

Especially in the case of multi-drug resistant bacteria, which

are no longer effective for treatment by antibiotics, iron chelators

are assessed as alternative therapeutic agents (Thomson et al.,

2012).

Siderochelins, a ferrous-ion chelating agents, were first

discovered from fermentation broth of Nocardia sp. SC11340

(Liu et al., 1981). There are six analogues of siderochelin;

siderochelin A and B (from Nocardia sp. SC11340), siderochelin

C (from Actinomycetes sp.), and siderochelin D, E, F (from

Amycolatopsis sp. LZ149) (Liu et al., 1981; Mitscher et al.,

1984; Lu et al., 2015). These siderochelins showed a broad-

spectrum of antibacterial activity, however, the inhibitory

activity of siderochelins on MDRPs have not been studied yet.

Presently we report the phenotypic, physiological, chemo-

taxonomic, and phylogenetic characterization of a novel strain

KCTC 29142 isolated from a soil sample collected from Ami

Mountain, South Korea. The culture supernatant of this strain

displayed inhibitory activities on MDRPs. The active compound

was isolated and analyzed using 1H- and

13C-nulcear magnetic

resonance (NMR) and heteronuclear multiple bond correlation

(HMBC) spectral analysis, and finally identified as the ferrous-

ion chelating compound, siderochelin A. This is the first report

of anti-MDRP activities of siderochelin A, and suggests that

siderochelin A can be developed as a supplementary agent on

the treatment of infections caused by MDRPs.

Materials and Methods

Bacterial strain

Strain KCTC 29142 was isolated from the soil sample

collected from Ami Mountain in Chungcheongnam-do province,

Republic of Korea (latitude 36°50′50.4″N, 126°39′53.2″E)

from February 23 2007. One gram of soil was suspended in

0.85% NaCl, and 100 μl of the suspension was spread on

Humic acid-Vitamin (HV) agar medium, the plates were

incubated at 28°C for 2 weeks. The single colony was picked

using a sterile toothpick and streaked on Bennett’s agar medium

(glucose 10 g/L, peptone 2 g/L, yeast extract 1 g/L, beef extract

1 g/L, and agar 15 g/L) for cultivation. The strain was finally

stored in 20% (v/v) glycerol suspensions at -80°C.

Morphological, physiological, and biochemical cha-

racteristics

Culture characteristics of strain KCTC 29142 were observed

on International Streptomyces Project (ISP), Bennett’s and S

agar media (Shiring and Gottlieb, 1966; Baker, 1990). The

physiological and biochemical features of strain KCTC 29142

were determined according to previously described procedures

(Waksman and Henrici, 1943; Becker et al., 1965). Sugar

utilization was evaluated by using ISP9 medium supplemented

with 1% (w/v) of various sugars individually as sole carbon

source. Nitrogen source utilization was assessed by degradation

of casein, xanthine, gelatin, and tyrosine as described previously

(Gordon et al., 1974).

Phylogenetic analysis, DNA G+C content, and DNA-

DNA hybridization

Genomic DNA was extracted and purified using the ExgeneTM

cell SV kit (GeneAll). The 16S rRNA gene was amplified using

the universal primer set 27F/1492R (Lane, 1991) and sequenced

by Solgent. The resulting 16S rRNA gene sequence (1.5 kb)

was aligned with available 16S rRNA gene sequences using the

EzTaxon-e server (http://eztaxon-e.ezbiocloud.net/) (Kim et

al., 2012). The 16S rRNA gene sequences of closely related

type strains were obtained from the GenBank database and

multiple alignments were conducted with the CLUSTAL_X

program, version 1.8 (Thompson et al., 1997). The phylogenetic

tree was constructed with neighbor-joining (Saitou and Nei,

1987) and Tajima-Nei (Tajima and Nei, 1984) methods using

MEGA 4 software (Tamura et al., 2007). Robustness of

individual branches was evaluated by bootstrapping of 1,000

replications. The G+C contents of the total DNA and DNA-

DNA hybridization between the strain KCTC 29142 and the

reference type strains were performed as described before

(Lechevalier et al., 1986).

Isolation and identification of antibacterial compound

The supernatant from flask fermentation was obtained

following centrifugation and solvent partitioned in a step-wise

Anti-MDRP activity of siderochelin from a novel Amycolatopsis sp. ∙ 329

Korean Journal of Microbiology, Vol. 52, No. 3

Fig. 1. The 16S rRNA gene sequence-based phylogenetic tree generated using the neighbor-joining method shows the relationships of strain KCTC 29142

with other Amycolatopsis species. Numbers at nodes indicate percentage levels of bootstrap support based on a neighbor-joining analysis of 1,000

replications. The scale bar denotes 0.02 substitutions per nucleotide position.

Fig. 1. The 16S rRNA gene sequence-based phylogenetic tree generated using the neighbor-joining method shows the relationships of strain KCTC 29142

with other Amycolatopsis species. Numbers at nodes indicate percentage levels of bootstrap support based on a neighbor-joining analysis of 1,000

replications. The scale bar denotes 0.02 substitutions per nucleotide position.

fashion to purify the antibacterial compounds. The supernatant

was extracted using ethyl acetate (EtOAc; 1:1, v/v). The solvent

layer was evaporated to dryness to yield a yellowish extract.

The extract was dissolved in methanol, applied to a Sephadex

LH-20 gel filtration column, and eluted with methanol. Fractions

displaying activity against MRSA were pooled and subjected

to semi-preparative HPLC. The reverse-phase ODS column

(YMC-Pack ODS-A column, 250 × 10 mm, 5 μm, YMC) was

eluted with 18% acetonitrile containing 1% acetic acid at a flow

rate of 2 ml/min. The compound was monitored by ultraviolet

absorbance at 254 nm. Chemical structures of purified compound

was analyzed by nuclear magnetic resonance (NMR, 1D, 2D). 1H- and

13C-NMR spectra were obtained on an Advance DPX

500 MHz NMR spectrometer (Bruker), recorded in a deuterated

chloroform (CDCl3) solution.

Evaluation of the antibacterial activity

Antibacterial activity was measured by the disc diffusion

method. Mueller-Hinton agar medium was mixed with 0.1 ml

of bacterial suspension (105 CFU/ml). Paper discs which were

soaked with 30 μg of EtOAc extract or siderochelin A were

placed on the agar surface. The plates were incubated at 37°C

for 18 h, and the antibacterial activities were assessed by

measuring the diameter of inhibition zone (mm).

Determination of the minimum inhibitory concentration

(MIC)

MIC values of the siderochelin A against clinical isolates

were determined by agar dilution method. Siderochelin A was

added into the Mueller-Hinton (MH) agar at the concentration

ranging from 0.06–128.00 μg/ml by two-fold serial dilution.

Ten microliters of each test strain was inoculated onto the agar

surface by striking, and incubated at 28°C for 18 h. MIC was

expressed as the lowest concentration of inhibited growth of

visible colony on the agar surface.

Results and Discussion

Phylogenetic analysis, determination of DNA G+C

content, and DNA-DNA hybridization

The phylogenetic tree affiliated strain KCTC 29142 with the

genus Amylcolatopsis (Fig. 1). Phylogenetic relatedness based

on the 16S rRNA gene of KCTC 29142 indicated that the closest

relatives were Amycolatopsis keratiniphila subsp. nogabecina

DSM 44586T (98%), A. decaplanina DSM 44594

T (98.3%),

and A. orientalis IMSNU 20058T (97%). The DNA G+C content

of strain KCTC 29142 was 73.9 mol%. Members of genus

Amylcolatopsis have DNA G+C contents in the range 66–73

330 ∙ Lee et al.


Fig. 2. Morphology of strain KCTC 29142 and the closely related strains.

Amycolatopsis orientalis IMSNU 20058, A. keratiniphila DSM44586, A.

decaplanina DSM 44594 and strains KCTC 29142 were grown on the

Bennett’s agar medium at 28°C for 7 days.

mol% (Tseng et al., 2006). The DNA-DNA hybridization

between strain KCTC 29142 and its nearest phylogenetic

neighbors, A. keratiniphila subsp. nogabecina DSM 44586T,

A. decaplanina DSM44594T, and A. orientalis IMSNU 20058

T,

were 28.91, 19.52, and 59.17%, respectively, which were

significantly lower than the threshold value (70%) for the

delineation of genomic species (Wayne et al., 1987). The

unique taxonomic position of strain KCTC 29142 determined

by a polyphasic taxonomic approach has prompted the present

suggestion of this strain as a new species of the genus

Amycolatopsis.

The genus Amycolatopsis was first described by Lechevalier

et al. (1986). It is a member of the family Pseudonocardiaceae,

in the phylum Actinobacteria and 65 species of the genus

Amycolatopsis have been recognized (Euzéby, 1997). Some

species of this genus were reported to produce epoxyquinomicin

C, vancomycin, rifomycin, rifamycin, and siderochelins (Sensi

et al., 1959; Matsumoto et al., 1997; Wink et al., 2003; Bala et

al., 2004; Lu et al., 2015).

Morphological, physiological, and biochemical cha-

racteristics

Strain KCTC29142 was Gram-positive, aerobic, catalase-

positive, and non-motile (data not shown). Aerial mycelia were

straight with a smooth surface. Morphology showed difference

when compared with the closely related strains on Bennett’s

medium: Aerial mycelia (front) and substrate mycelia (back)

of KCTC 29142 is white and pink, while those of A. orientalis

IMSNU 20058 are white and brownish yellow. Both A.

keratiniphila DSM 44586 and A. decaplanina DSM 44594

showed typical morphology of Amycolatopsis species, and the

colors are sand yellow (Fig. 2). Other culture characteristics of

strain KCTC 29142 on various media are summarized in

Supplementary data Table S1. Aerial mycelia were white on

most tested ISP media and vegetative mycelia were purple on

ISP3, purplish-pink on ISP4, violet on ISP7, pink on Bennett’s

medium, and beige or yellow on ISP2, ISP5, ISP6, and S

medium. Diffusible pink pigment was observed on Bennett’s

medium. The physiological and biochemical characteristics of

strain KCTC 29142 are compared with related type strains.

Strain KCTC 29142 can utilize glucose, arabinose, fructose,

mannitol, xylose and raffinose as sole carbon source, but not

cellulose, inositol, and sucrose. It can also degraded casein,

xanthine, gelatin, and tyrosine. Growth occurred at 10–37°C

(optimum, 28°C) and pH 5–10 (optimum, pH 7). The optimal

growth NaCl concentration was 0–2.5% (w/v, no growth on

5%), and was resistant to streptomycin (100 μg/ml), neomycin

(50 μg/ml), and vancomycin (50 μg/ml). These characteristics

were different with related type strains (Table 1), which

suggested it is an uncharacterized strain so far.

The phylum Actinobacteria, which includes the genus

Streptomyces, has proven particularly important as a source of

secondary metabolites include many antibiotics (Gao and Gupta,

2012). Previously, many studies have been reported on the

discovery of novel antibiotics from Actinobacteria which can

inhibit the MDRPs, but mostly are from Streptomyces species



Table 1. Physiological and biochemical characteristics of strain KCTC

29142 and related Amycolatopsis species

Characteristics 1 2 3 4

Growth on sole carbon sources (1%, w/v)

D-Glucose + + + +

D-Arabinose + + + +

D-Fructose + + + +

D-Mannitol + + + +

D-Xylose + - + -

Raffinose + - - -

Cellulose - - + -

Inositol - + + -

Sucrose - - + -

Decomposition of

Casein + + + +

Xanthine + - - -

Gelatin +/- + - +/-

Tyrosine + + + +

Growth in

NaCl (2.5%) + + + +

NaCl (5.0%) - - - +

Growth at

4°C - - - -

10°C + + + +

28°C + + + +

37°C + + + +

45°C - - - -

Antibiotics resistant on

Tobramycin 50 μg/ml - - - +

Streptomycin 100 μg/ml + - - +

Neomycin 50 μg/ml + + - +

Vancomycin 50 μg/ml + + - +

Strains: 1, strain KCTC 29142; 2, Amycolatopsis orientalis IMSNU 20058T;

3, A. keratiniphila subsp. nogabecina DSM 44586T; 4, A. decaplanina

DSM 44594T. All data are from this study.

+, positive or present; -, negative or absent

Table 2. Chemical shifts 1H (500 MHz),

13C (125 MHz), and HMBC

correlations for anti-MDRP compound (in CDCl3)

Carbon No. δH δC HMBC

1 (CH) 8.16 dd (4.5, 1.5) 140.4 C2, C3, C5

2 (CH) 7.34 dd (8.5, 4.5) 126.1 C3, C4

3 (CH) 7.41 dd (8.5, 1.5) 128.0 C1, C4, C5

4 (C) 158.5

5 (C) 134.0

6 (C) 181.4

7 (C) 85.3

8 (CH2) 2.53 dd (13.5, 10.0) 40.9 C6, C7, C9, C10, C11

2.62 dd (13.5, 2.0)

9 (CH) 4.96 dd (10.0, 2.0) 70.6 C6, C10

10 (C) 173.2

11 (CH3) 1.69 s 28.6 C6, C7, C8

Fig. 3. Chemical structure of anti-MRSA compound isolated from strain

KCTC 29142.

(Procópio et al., 2012; Cheng et al., 2013). Recently, rare

actinomycetes were recognized as a potential storehouse for

novel antibiotics, and many studies were carried out to develop

novel antibiotics from rare actinomycetes (Tiwari and Gupta,

2012).

Chemotaxonomic analysis

Whole-cell lysate of strain KCTC 29142 was determined to

contain meso-diaminopimelic acid, arabinose, and galactose.

The predominant menaquinone was determined to be MK9(H4).

Phosphatidylethanolamine, phosphatidylglycerol, and phospha-

tidylinositol were the predominant phospholipids. The cellular

fatty acid compositions of strain KCTC 29142 and the three

reference strains are listed in Supplementary data Table S2. The

major cellular fatty acids of strain KCTC29142 were found to

be iso-C16:0 (19.5%), iso-C16:0 (16.4%), iso-C15:0 (8.5%), and C18:0

(7.8%). These results also suggested that the strain KCTC 29142

belong to the genus Amycolatopsis but have difference with

closely related strains.

Isolation and structure elucidation of anti-MRSA

compound produced by strain KCTC 29142

The supernatant from flask fermentation was extracted with

EtOAc and purified by several chromatographic purification

332 ∙ Lee et al.


Table 3. Minimum inhibitory concentrations (MIC) of siderochelin A on clinical MRSA isolates

Strain Siderochelin A Norfloxacin Cephalothin Erythromycin Oxacillin Vancomycin

CCARM3040 64 4 128 >128 >128 2

CCARM3090 64 128 >128 >128 >64 4

CCARM3091 64 32 >128 >128 >64 4

CCARM3100 64 128 >128 >128 >128 1

CCARM3152 64 0.5 <0.25 16 8 0.5

CCARM3166 64 0.5 2 1 4 1

CCARM3197 64 1 0.5 0.5 0.5 0.5

CCARM3227 64 > 128 1 0.5 0.5 1

CCARM3231 64 8 16 0.5 16 1

CCARM3502 64 128 128 >128 >128 4

CCARM3568 64 > 128 128 >128 128 <1

CCARM3574 64 1 16 32 128 <1

CCARM3793 64 1 4 >128 4 2

CCARM, Culture Collection of Antimicrobial Resistant Microbes

procedures guided by anti-MRSA activity. The anti-MRSA

compound was finally obtained as a yellowish-colored crystal.

The molecular formula was determined as C11H13N3O3 according

to HR Q-TOF MS data (m/z 236.106 [M+H]+) and

1H- and

13C-NMR data (Supplementary data Fig. S1, Table 2). The

coincidence of 1H- and

13C-NMR spectroscopic properties of

the anti-MRSA compound with the data from previous reports

confirmed the compound as siderochelin A (Fig. 3). The

structure was verified by HMBC spectral data (Table 2).

Recently, 3 new siderochelins, siderochelin D, E, F were

isolated from Amycolatopsis sp. LZ149 (Lu et al., 2015). These

compounds, along with siderochelin A were reported to have

anti-mycobacterial activity. In our study, siderochelin A was

dominantly isolated from the culture broth.

Anti-MDRP activities of siderochelin A

Siderochelins, which chelate ferrous-ion, are produced from

Nocardia sp., Streptoalloteichus sp., and Amycolatopsis sp.

(Liu et al., 1981; Okuyama et al., 1982; Mitscher et al., 1984;

Lu et al., 2015). These reports also proposed that siderochelin

A has good antibacterial activity. However, the antibacterial

activity of siderochelin A on MDRPs had not been investigated

until the present study.

The MICs of siderochelin A on clinical isolates of MRSA are

summarized in Table 3. The clinical isolates were susceptible

only to vancomycin, and were resistant to either norfloxacin,

cephalothin, erythromycin, or oxacillin. However, siderochelin

A showed moderate inhibitory activity against all the clinical

isolates which is similar with previous reports (Liu et al., 1981;

Mitscher et al., 1984). The MIC for all these isolates was 64

μg/ml. This result implied that the inhibitory activity of

siderochelin A is not due to antibiotic mechanism, but may be

related with the high iron chelating activity which can limit the

growth of pathogens.

Inhibitory effects of siderochelin A on various human

infected MDRPs including Gram-positive and Gram-negative

bacteria were also investigated (Table 4). Siderochelin A

showed inhibitory activities against MDRPs, but differed in the

strains. Siderochelin A showed good activity to E. coli and

Acinetobacter baumannii. The MIC of siderochelin A to a

susceptible E. coli CCARM 230 and multi-drug resistant E. coli

CCARM 1257 are 16 and 32 μg/ml respectively. Siderochelin

A also showed good inhibitory activity against clinical isolates

of A. baumannii, which is an increasingly serious threat as a

nosocomial pathogen, and displays multi-drug resistance

(Kempf and Rolain, 2012). Even though the A. baumannii

isolates used in this study were resistant to cephalothin (MIC

128 to ≥256 μg/ml), they were susceptible to siderochelin A

(MIC 32 μg/ml). Siderochelin A showed moderate inhibitory

activity against Klebsiella pneumonia and Salmonella enterica

strains, the causative pathogen of pneumonia and salmonellosis

respectively. Klebsiella pneumoniae CCARM 10074 has extended-



Table 4. Minimum inhibitory concentrations of siderochelin A on MDRPs

Organism Characteristic MIC (μg/ml)

Enterococcus faecium CCARM 5205 VSE, Norfloxacin MIC; 4 μg/ml >128

Enterococcus faecium CCARM 5024 VRE, vanA, Norfloxacin MIC; 4 μg/ml >128

Enterococcus faecalis CCARM 5025 VSE, Norfloxacin MIC; 2 μg/ml >128

Enterococcus faecalis CCARM 5169 VRE, vanB , Norfloxacin MIC; 2 μg/ml >128

Escherichia coli CCARM 230 Susceptible, Cephalothin MIC; 8 μg/ml 16

Escherichia coli CCARM 237 Susceptible, Ampicillin MIC; 4 μg/ml 64

Escherichia coli CCARM 1256 MDR, Ampicillin MIC; ≥128 μg/ml 64

Escherichia coli CCARM 1257 MDR, Ampicillin MIC; ≥128 μg/ml 32

Klebsiella pneumoniae CCARM 10010 Susceptible, Ciprofloxacin MIC; ≥128 μg/ml 64

Klebsiella pneumoniae CCARM 10011 Susceptible Ampicillin MIC; 8 μg/ml 64

Klebsiella pneumoniae CCARM 10074 ESBL MIC; 8 μg/ml 64

Pseudomonas aeruginosa CCARM 2045 Susceptible, Norfloxacin MIC; 128 μg/ml 64

Pseudomonas aeruginosa CCARM 2046 Susceptible, Norfloxacin MIC; 128 μg/ml >128

Pseudomonas aeruginosa CCARM 2005 MDR, Norfloxacin MIC; 128 μg/ml >128

Pseudomonas aeruginosa CCARM 2006 MDR, Norfloxacin MIC; 128 μg/ml >128

Acinetobacter baumanii CCARM 12052 Cephalothin MIC; 256 μg/ml 32

Acinetobacter baumanii CCARM 12053 Cephalothin MIC; 128 μg/ml 32

Acinetobacter baumanii CCARM 12061 Cephalothin MIC; ≥256 μg/ml 32

Acinetobacter baumanii CCARM 12064 Cephalothin MIC; ≥256 μg/ml 32

Salmonella enterica CCARM 8020 Norfloxacin MIC; 0.12 μg/ml 64




VRE, vancomycin-resistant enterococci; VSE, vancomycin-susceptible enterococci; MDR, multi-drug resistant; ESBL, expanded-spectrum beta-lactamase;

CCARM, Culture Collection of Antimicrobial Resistant Microbes

spectrum beta-lactamase (ESBL) activity, and K. pneumoniae

CCARM 10010 is resistance to ciprofloxacin (MIC ≥128 μg/ml).

All of these strains were inhibited by the siderochelin A with the

MIC of 64 μg/ml. However, siderochelin A showed no inhibitory

activity against Enterococcus faecium and Pseudomonas

aeruginosa (MIC ≥128 μg/ml), which were also major nosocomial

infectious pathogens with multi-drug resistance. Different activity

may due to the different requirement for iron between pathogens.

Accurate mechanism should be investigated in the future study.

In conclusion, strain KCTC 29142 produces siderochelin A,

which have antibacterial activities on MRSA and other clinical

isolates of MDRPs. Inhibitory activity of siderochelin A against

MDRP suggested that siderochelin A can be used as a supple-

mentary agent for the treatment of infections caused by MDRPs.

적 요

본 연구에서는 신규 Amycolatopsis 균주 KCTC 29142를 분

리하여 형태학적 관찰, 계통분석 및 화학분류학적 분석 등 다

상 분류분석을 통해 분석하였다. KCTC 29142 균주의 에틸아

세테이트추출물은 강한 항균활성을 나타났고, 활성물질은 철

이온 킬레이트 물질인 siderochelin A로 동정되었다. 본 연구

에서 분리된 siderochelin A는 다제내성균인 Acinetobacter

baumanii, methicillin-resistant Staphylococcus aureus (MRSA),

vancomycin-resistant Staphylococcus aureus (VRSA), 및

Escherichia coli (E. coli)에 대해 강한 활성을 보였고, 임상에

서 분리된 다제내성균에 대한 MIC를 결정하였다.

Acknowledgements

This work was carried out with the support of “Cooperative

334 ∙ Lee et al.


Research Program for Agriculture Science & Technology

Development (Project No. PJ01128901)”, Rural Development

Administration, Republic of Korea. H.-J.H. was supported by

funding from the Royal Society, UK (51600.K5766/ROG) and

the Medical Research Council, UK (G0700141).

References

Anderson, D.I. 2003. Persistence of antibiotic resistant bacteria. Curr.

Opin. Microbiol. 6, 452–456.

Baker, D. 1990. Methods for the isolation, culture and characterization

of the Frankiaceae: soil actinomycetes and symbionts of acti-

norhizal plants. In Labeda, D.P. (eds), Isolation of Biotech-

nological Organisms from Nature, pp. 213–236. McGraw-Hill

Publishing Co., New York, USA.

Bala, S., Khanna, R., Dadhwal, M., Prabagaran, S.R., Shivaji, S.,

Cullum, J., and Lal, R. 2004. Reclassification of Amycolatopsis

mediterranei DSM 46095 as Amycolatopsis rifamycinica sp.

nov. Int. J. Syst. Evol. Microbiol. 54, 1145–1149.

Becker, B., Lechevalier, M.P., and Lechevalier, H.A. 1965. Chemical

composition of cell-wall preparations from strains of various

form-genera of aerobic actinomycetes. Appl. Microbiol. 13, 236

–243.

Chan, G.C., Chan, S., Ho, P.L., and Ha, S.Y. 2009. Effects of chelators

(deferoxamine, deferiprone and deferasirox) on the growth of

Klebsiella pneumoniae and Aeromonas hydrophila isolated

from transfusion-dependent thalassemia patients. Hemoglobin

33, 352–360.

Cheng, J., Jin, Y.Y., Yang, S.H., and Suh, J.W. 2013. Isolation and

characterization of anti-methicillin-resistant Staphylococcus

aureus/vancomycin-resistant Enterococcus compound from

Streptomyces bungoensis MJM2077. J. Korean Soc. Appl. Biol.

Chem. 56, 107–111.

Cosgrove, S.E. 2006. The relationship between antimicrobial resistance

and patient outcomes: mortality, length of hospital stay, and

health care costs. Clin. Infect. Dis. 42, 82–89.

Euzéby, J.P. 1997. List of bacterial names with standing in nomenclature:

a folder available on the internet. Int. J. Syst. Evol. Microbiol. 47,

590–592.

Gao, B. and Gupta, R.S. 2012. Phylogenetic framework and molecular

signatures for the main clades of the phylum Actinobacteria.

Microbiol. Mol. Biol. Rev. 76, 66–112.

Gordon, R.E., Barnett, D.A., Handerhan, J.E., and Pang, C.H.N. 1974.

Nocardia coeliaca, Nocardia autotrophica, and the Nocardin

strain. Int. J. Syst. Bacteriol. 24, 54–63.

Kempf, M. and Rolain, J.M. 2012. Emergence of resistance to

carbapenems in Acinetobacter baumannii in Europe, clinical

impact and therapeutic options. Int. J. Antimicrob. Agents 39,

105–114.

Kim, O.S., Cho, Y.J., Lee, K., Yoon, S.H., Kim, M., Na, H., Park, S.C.,

Jeon, Y.S., Lee, J.H., Yi, H., et al. 2012. Introducing EzTaxon-e,

a prokaryotic 16S rRNA gene sequence database with phylotypes

that represent uncultured species. Int. J. Syst. Evol. Microbiol.

62, 716–721.

Kim, C.M. and Shin, S.H. 2009. Effect of iron-chelator deferiprone on

the in vitro growth of staphylococci. J. Korean Med. Sci. 24, 289

–295.

Lane, D.J. 1991. 16S/23S rRNA sequencing. In Stackebrandt, E. and

Goodfellow, M. (eds.) Nucleic Acid Techniques in Bacterial

Systematics, pp. 115–176. Wiley, Chichester, UK.

Lechevalier, M.P., Prauser, H., Labeda, D.P., and Ruan, J.S. 1986.

Two new genera of nocardioform actinomycetes, Amycolata

gen. nov. and Amycolatopsis gen. nov. Int. J. Syst. Bacteriol. 36,

29–37.

Lin, M.F. and Lan, C.Y. 2014. Antimicrobial resistance in Acinetobacter

baumannii, from bench to bedside. World J. Clin. Cases 2, 787–

814.

Liu, W.C., Fisher, S.M., Wells, J.S. Jr., Ricca, C.S., Principe, P.A.,

Trejo, W.H., Bonner, D.P., Gougoutos, J.Z., Toeplitz, B.K., and

Sykes, R.B. 1981. Siderochelin, a new ferrous-ion chelating

agent produced by Norcardia. J. Antibiot. (Tokyo) 34, 791–799.

Lode, H.M. 2009. Clinical impact of antibiotic-resistant Gram-positive

pathogens. Clin. Microbiol. Infect. 15, 212–217.

Lu, C.H., Ye, F.W., and Shen, Y.M. 2015. Siderochelins with

anti-mycobacterial activity from Amycolatopsis sp. LZ149.

Chin. J. Nat. Med. 13, 69–72.

Matsumoto, N., Iinuma, H., Sawa, T., Takeuchi, T., Hirano, S.,

Yoshioka, T., and Ishizuka, M. 1997. Epoxyquinomicins A, B, C

and D, new antibiotics from Amycolatopsis. II. Effect on type II

collagen-induced arthritis in mice. J. Antibiot. (Tokyo) 50, 906–

911.

Miethke, M. and Marahiel, M.A. 2007. Siderophore-based iron

acquisition and pathogen control. Microbiol. Mol. Biol. Rev. 71,

413–451.

Mitscher, L.A., Högberg, T., Drake, S.D., Burgstahler, A.W., Jackson,

M., Lee, B., Sheldon, R.I., Gracey, H.E., Kohl, W., and

Theriault, R.J. 1984. Isolation and structural determination of

siderochelin C, a fermentation product of an unusual Actinomycetes

sp. J. Antibiot. (Tokyo) 37, 1260–1263.

Okuyama, D., Nakamura, H., Naganawa, H., Takita, T., Umezawa, H.,

and Iitaka, Y. 1982. Isolation, racemization and absolute

configuration of siderochelin. J. Antibiot. (Tokyo) 35, 1240–

1242.

Procópio, R.E., Silva, I.R., Martins, M.K., Azevedo, J.L., and Araújo,

J.M. 2012. Antibiotics produced by Streptomyces. Braz. J.

Infect. Dis. 16, 466–471.

Saitou, N. and Nei, M. 1987. The neighbour-joining method, a new

method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4,

406–425.

Sakoulas, G. and Moellering, R.C. Jr. 2008. Increasing antibiotic

resistance among methicillin-resistant Staphylococcus aureus



strains. Clin. Infect. Dis. 46, 360–367.

Sensi, P., Greco, A.M., and Ballotta, R. 1959. Rifomycin I. Isolation

and properties of rifomycin B and rifomycin complex. Antibiot.

Annu. 7, 262–270.

Shiring, E.B. and Gottlieb, D. 1966. Methods for characterization of

Streptomyces species. Int. J. Syst. Bacteriol. 16, 313–340.

Tajima, F. and Nei, M. 1984. Estimation of evolutionary distance

between nucleotide sequences. Mol. Biol. Evol. 1, 269–285.

Tamura, K., Dudley, J., Nei, M., and Kumar, S. 2007. MEGA4,

molecular evolutionary genetics analysis (MEGA) software

version 4.0. Mol. Biol. Evol. 24, 1596–1599.

Thompson, M.G., Corey, B.W., Si, Y., Craft, D.W., and Zurawski,

D.V. 2012. Antimicrobial activities of iron chelators against

common nosocomial pathogens. Antimicrob. Agents Chemother.

56, 5419–5421.

Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F., and

Higgins, D.G. 1997. The CLUSTAL_X windows interface,

flexible strategies for multiple sequence alignment aided by

quality analysis tools. Nucleic Acids Res. 25, 4876–4882.

Tiwari, K. and, Gupta, R.K. 2012. Rare actinomycetes, a potential

storehouse for novel antibiotics. Crit. Rev. Biotechnol. 32, 108–

132.

Tseng, M., Yang, S.F., Li, W.J., and Jiang, C.L. 2006. Amycolatopsis

taiwanensis sp. nov., from soil. Int. J. Syst. Evol. Microbiol. 56,

1811–1815.

Waksman, S.A. and Henrici, A.T. 1943. The nomenclature and

classification of the Actinomycetes. J. Bacteriol. 46, 337–341.

Wayne, L.G., Brenner, D.J., Colwell, R.R., and Truper, H.G. 1987.

Report of the Ad hoc committee on reconciliation of approaches

to bacterial systematics. Int. J. Syst. Bacteriol. 37, 463–464.

Wink, J.M., Kroppenstedt, R.M., Ganguli, B.N., Nadkarni, S.R.,

Schumann, P., Seibert, G., and Stackebrandt, E. 2003. Three

new antibiotic producing species of the genus Amycolatopsis,

Amycolatopsis balhimycina sp. nov., A. tolypomycina sp. nov.,

A. vancoresmycina sp. nov., and description of Amycolatopsis

keratiniphila subsp. Keratiniphila subsp. nov. and A. keratiniphila

subsp. Nogabecina subsp. nov. Syst. Appl. Microbiol. 26, 38–46.

Anti-multi drug resistant pathogen activity of ...

Documents

Transcript of Anti-multi drug resistant pathogen activity of ...