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- JOURNAL OF SCIENCE OF HNUE DOI: 10.18173/2354-1059.2015-00086 Chemical and Biological Sci. 2015, Vol. 60, No. 9, pp. 112-118 This paper is available online at http://stdb.hnue.edu.vn IDENTIFICATION OF Streptomyces sp. QN63 AGAINST ANTIBIOTIC-RESISTANT Staphylococcus aureus Duong Minh Lam, Truong Thi Chien and Tran Thi Thuy Faculty of Biology, Hanoi National University of Education Abstract. Antibiotic-resistant bacteria are becoming more common around the world each day. Terrestrial actinomycetes are the major sources of known antibiotics. However, the number of new antibiotics found in terrestrial soils has fallen dramatically over the last 20 years. Marine environments and mangroves are seen as untapped environments which have high potential for new drug discovery. Actinomycetes strain QN63 was isolated from sediment collected in a mangrove environment in Yen Hung District, Quang Ninh Province, and it has been found to inhibit antibiotic-resistant Staphylococcus aureus. Morphological and molecular identification shows that QN63 is a strain of Streptomyces shenzhenensis. The identification of this strain and the published results of its antibiotic properties suggest that S. shenzhenensis QN63 might be effective in treating infections caused by antibiotic resistant Staphylococcus aureus. Keywords: Streptomyces sp. QN63, Streptomyces shenzhenensis, Staphylococcus aureus, antibiotic resistant, mangroves. 1. Introduction Methicillin Resistant Staphylococcus aureus-MRSA has become universal problem as it resists almost every antibiotic that has been used to kill it, including vancomycin. Twenty-five years ago, in 1991, more than a half of the S. aureus strains isolated in the United States of America (USA) were found to be capable of resisting penicillin, methicillin, tetracycline and erythromycin [1]. Vancomycin was the only antibiotic that was effective against S. aureus at that time. However, in 1996, a vancomycin-resistant S. aureus strain at the concentration of 4 - 8 µg/mL was found in Japan, and then in France, England and the USA [1]. In 2002, Silvestri et al. [2] found a vancomycin-resistant S. aureus strain at much higher concentration (16 µg/mL). However, treatment of severe MRSA infections continues to be vancomycin but now at trough levels of ≥ 15 mg/L [3]. Some scientists had suggested that the drug resistant bacteria were all one strain of MRSA [2]. S. aureus had infected almost 1/3 of world‟s population, and it has become a threat to public health. Traditional drugs could not combat this problem of antibiotic resistance [4]. In an attempt to control the spread of MRSA, there has been an increase in Received December 8, 2015. Accepted December 28, 2015. Contact Duong Minh Lam, e-mail address: duong.minhlam@gmail.com 112
- Identification of Streptomyces sp. QN63 against antibiotic-resistant staphylococcus aureus patient screening, decontamination, surveillance and contact isolation; an alert system has been created; hand washing in hospitals has become mandatory and the search for new antibiotics continues [5]. With integrated prevention methods, the number of healthcare associated MRSA S. aureus infections (HA-MRSA) decreased at the university hospital in Geneva, Switzerland [5, 6]. Vietnam is a country in which a high percentage of bacteria are antibiotic-resistant. Of the many patients in Hospital 175 who acquired an S. aureus infection, 55 strains were identified and 50 - 70% of those strains resisted treatment with ampicillin, co-trimoxazol, erythromycin, licomycin and methicillin. The rate of resistance to axacilin increased from 9.6% in 1989 to 20.7% in 1994 [7]. And indeed most of the antibiotics in use at that time to treat S. aureus infections had become ineffective. Looking for new and effective antibiotics to treat drug resistant S. aureus has been and is a pressing need. During our research on the screening that has been done in the search for new and valuable antibiotics from mangrove actinomycetes, strain QN63 that was isolated from soil samples in Yen Hung District, Quang Ninh Province was one which showed high activity against S. aureus which were resistant to penicillin. Antibiotics produced by the QN63 strain were purified and the chemical structure was determined to be cyclic peptide. It was a thermostable antibiotic, remaining active 30 minutes after incubation at 80 oC [8]. Research that has been done showed that antibiotics produced by the QN63 strain had high potential for application in treating drug resistant S. aureus. In order to get closer to the goal identification to species level of the studied strain is necessary and additional clinical studies on animals are also required. This paper presents a more detailed identification of the QN63 strain based on morphological and molecular characteristics. 2. Content 2.1. Materials and methods 2.1.1. Materials - Subject: The QN63 strain was isolated from soil samples collected from Yen Hung mangrove sediment, Quang Ninh Province. - Test microorganism: Staphylococcus aureus 30sd from the collection of the Department of Microbiology and Biotechnology, Hanoi National University of Education, Vietnam. - Media: Czapek-glucose (glucose 30.0 g; KCl 0.5 g; NaNO3 3.0 g; FeSO4 0.01 g; K2HPO4 1.0 g; MgSO4.7H2O 0.5 g; agar 20 g; sea water 1000 mL); Gause I (starch 20 g, FeSO4 0.01 g, K2HPO4 0.5 g, agar 20 g, MgSO4 0.5 g, KNO3 0.5 g, sea water 1000 mL, pH 7.2 - 7.4); Gause II (glucose 10 g, meat extract 3 g, pepton 5 g, agar 20 g, sea water 1000 mL, pH 7.0 - 7.2); MPA (meat extract 5 g, peptone 5 g, NaCl 5 g, agar 20 g, sea water 1000 ml, pH 7.0 -7.2). A4 (glucose 10 g, soybean powder 10 g, CaCO3 1 g, agar 20 g, sea water 1000 mL, pH 7.0); A4H (glucose 15 g, soybean powder 5 g, CaCO3 1 g, agar 20 g, sea water 1000 mL, pH 7.0); Soybean powder medium (soybean 15 g, glycerine 2.5 g, glucose 15 g, peptone 5 g, CaCO3 1 g, agar 20 g, sea water 1000 mL, pH 7.0). 2.1.2. Methods * Morphological description The strain QN63 was grown on different media for 10 days and then described morphologies of colonies, aerial and substrate hyphae, conidiophores using light microscope. 113
- Duong Minh Lam, Truong Thi Chien and Tran Thi Thuy * Electron microscopic descriptions Electron microscopic graphs were taken to analyze the characteristics of the conidiophores and spores. This was done at the National Institute of Hygiene and Epidemiology, Hanoi, Vietnam. * DNA isolation, PCR and phylogenetic analysis DNA isolation was done using the method described in Sambrookvà Russell (2001) [9]. PCR and sequencing Partial sequences of the DNA coding for 16S rRNA was amplified using the universal primer pair (forward primer 314F: 5‟-CCTACGGGAGGCAGCAG-3‟ and reverse primer 907R: 5‟-CCGTCAATTCCTTTGAGTTT-3‟) with PCR master mix produced by the Bioneer Company, Korea. The PCR products were purified using a QiaGen purification kit before sending out for sequencing by the Gentis Company, Vietnam. The consensus sequence was combined from both forward and reverse sequences. Phylogenetic analysis The sequence of the QN63 strain was used to search for similar sequences available in the Genbank through the nucleotide blast program (nBlast-NCBI) [10]. All sequences used in the analysis were annealed using ClustalX software [11] and then optimized in the BioEdit program [12]. The Neighbor-Joining analysis was applied for phylogenetic analysis of the studied sequence with 31 other selected Streptomyces sequences in the Genbank. Two sequences, Bacillus thuringiensis (EF206345) and B. gibsonii (AY737309), were used as the outgroup. A Bootstrap analysis was done using 1000 replicates. 2.2. Results 2.2.1. Morphological descriptions The QN63 strain was cultured in 7 different media, each containing a different carbon source, to assess microscopic morphological characteristics, pigments secreted into the medium (Table 1), and shapes and sizes of conidiophores and spores. Figure 1 shows the conidiophores of the QN63 strain in the seven different media. Figure 1. Conidiophores and hyphae of the QN63 strain on different media a) Czapek-glucose; b) Gause I; c) ISP 4 Based on morphological characteristics of colonies and microscopic morphological characteristics of conidiophores, the QN63 strain was found to possess typical characteristics of the Streptomyces genus, identifying the strain as of the species Streptomyces. However, more details are required to make a more precise identification. 114
- Identification of Streptomyces sp. QN63 against antibiotic-resistant staphylococcus aureus Table 1. Morphological characteristics of the QN63 strain on 7 growth media Criteria Growth Aerial Substrate Pigment Conidiospores Media rate hyphae hyphae Gause I: Conidiophores Well Pinkish - Well-developed spores visible at 5 days of Grey - (+++) Grey - Spiral conidiophores growth - Well-developed spores ISP4: Conidiophores Very - Straight-Spiral visible at 5 days of well White Grey - conidiophores growth (++++) - Poorly-developed Present, Weak spores Gause II White Grey Dark (+) - Spiral conidiophores brown - Well-developed spores Weak Czapek-dox White Grey - - Spiral conidiophores (+) - Ppoorly-developed Average Czapek-glucose White Grey - spores (++) - Spiral conidiophores - Well-developed spores Czapek-starch: Well Whitish- - Straight-Spiral conidiophores visible at Grey - (+++) grey conidiophores day 10th of growth MPA with sea water: Present, Week spores not formed after Grey Grey dark - Not yet formed (+) 15 days of growth brown 2.2.2. Morphologies under the electron microscope Figure 2. Conidiophores and spores of the strain Streptomyces sp. QN63 a) on Gause I; b) on Czapek-starch The light and electron microscopy were used to distinguish characteristics (Figure 2) of the conidiophores and spore chain of the Streptomyces sp. QN63 on different media and they showed the typical spiral spore chain of Streptomyces. The conidiophores were small (0.4 - 0.6 x 1.2 - 1.4 µm) bearing a chain of 18 - 40 spores. The spores are cylindrical, wrinkled without appendages (hair, spines...) and relatively small, 0.8 - 0.9 µm wide x 0.8 - 1.2 µm long (Figure 2). The characteristics revealed by electron microscopy confirmed that QN63 is a 115
- Duong Minh Lam, Truong Thi Chien and Tran Thi Thuy strain of Streptomyces. Streptomyces is a genera that is one of the most famous genera for antibiotic producing ability and it is one of the most abundant microorganisms in the world. However, the features revealed by culture test and electron features are not sufficient to identify which species of Streptomyces the strain is. DNA analysis provides additional information for a better identification. 2.2.3. Phylogenetic analysis Figure 3. Neighbor-Joining phylogenetic tree based on the partial 16S rRNA gene sequences of the Streptomyces sp. QN63 strain and 31 phylogenetically closely related representative species of the genus Streptomyces. Numbers at branching points indicate bootstrap percentages (based on 1,000 replications) 116
- Identification of Streptomyces sp. QN63 against antibiotic-resistant staphylococcus aureus A partial DNA sequence encoding 16S rRNA fragment, which consists of 531 nucleotides, was used for comparison purposes to search for highly similar sequences in the GenBank. The sequence KM203728 of Streptomyces shenzhenensis was found to be 99.8% similar (531/532 nucleotides). A set of 34 sequences including 32 sequences of the genus Streptomyces and the two sequences of Bacillus in which Bacillus sequences were used as out group. Neighbor- Joining analysis showed that the Streptomyces sp. QN63 strain was closest to Streptomyces shenzhenensis KM203728 with the bootstrap value of 77.9%. With this additional information, it was concluded that QN63 is of the genus Streptomyces and species shenzhenensis, denoted as S. shenzhenensis QN63. Streptomyces shenzhenensis was first presented as a new species in a publication in 2011. It was isolated from mangrove sediment in Shenzhen District, Guangdong Province, China [13]. With the finding of S. shenzhenensis in the Yen Hung mangroves, it has been suggested that mangrove actinomycetes could be dispersed in coastal water flows. The coastal mangroves of Yen Hung (Quang Ninh Province, Vietnam) and Shenzhen (Guangdong Province, China) are 900 km from each other. 3. Conclusion The actinomycete strain, QN63, which inhibited antibiotic-resistant Staphylococcus aureus, was isolated from mangrove sediment in Yen Hung District, Quang Ninh Province. It was identified as Streptomyces shenzhenensis based on light and electron microscopy revealed features along with molecular characteristics and the strain in question is now referred as S. shenzhenensis QN63. Identifying the strain to the species level is basic information needed for further practical and clinical study of S. shenzhenensis QN63. REFERENCES [1] Hiramatsu K, Hanaki H, Ino T, Yabuta K, Oguri T, Tenover FC., 1997. Methicillin- resistant Staphylococcus aureus clinical strain with reduced vancomycin susceptibility. Journal of Antimicrobial Chemotherapy Vol. 40, pp. 135-136. [2] Silvestri L, Milanese M, Oblach L, Fontana F, Gregori D, Guerra R, van Saene H., 2002. Enteral vancomycin to control methicillin-resistant Staphylococcus aureus outbreak in mechanically ventilated patients. American Journal of Infection Control 30: pp. 391-399. [3] Steinmetz T, Eliakim-Raz N, Goldberg E, Leibovici L, Yahav D, 2015. Association of vancomycin serum concentrations with efficacy in patients with MRSA infections: a systematic review and meta-analysis. Clinical Microbiology and Infection Vol. 21, No.7, pp. 665-67. [4] Richardson AR, Libby SJ, Fang FC, 2015. A Nitric Oxide–Inducible Lactate Dehydrogenase Enables Staphylococcus aureus to Resist Innate Immunity. Science 319: pp. 1672-1676. [5] Fankhauser C, Schrenzel J, Francois P, Pittet D, Harbarth S. 2015. Secular trends of methicillin-resistant Staphylococcus aureus (MRSA) at Geneva University Hospitals (HUG) over a 14-year period. Antimicrobial Resistance and Infection Control 2015, 4(Suppl 1):O9 http://www.aricjournal.com/content/4/S1/O9. 117
- Duong Minh Lam, Truong Thi Chien and Tran Thi Thuy [6] Olearo F, Albrich W, Harbarth S, Kronenberg A. 2015. Ten years of MRSA surveillance in Switzerland: similarities and differences with Europe. Antimicrobial Resistance and Infection Control 2015, 4(Suppl 1):O8 http://www.aricjournal.com/content/4/S1/O8. [7] Vu Bao Chau, 2000. Antibiotic resistant level of some bacteria isolated from infected surgeries at hospital 175. http://203.162.1.204/tapchi/sottyd/nam2000/bai 9-11- 2000.htm. [8] Duong Minh Lam, Dang Ngoc Quang, Nguyen Thi Ha. 2013. Isolation, purification and characterization of drug resistant Staphylococcus aureus antibiotics by Streptomyces sp. QN63. Journal of Sciences and Technology Vol. 51, No. 5: pp. 555-563. [9] Sambrook J, Russell D., 2001. Molecular Cloning: A Laboratory Manual, 3rd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. [10] http://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch &LINK_LOC=blasthome. [11] Thompson JD, Gibson TJ, Plewniak F, Higgins DG, 1997. The Clustal X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nuc. Acid. Res., 24: pp. 4876-4882. [12] Hall TA., 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nuc. Acid. Symp. Ser., Vol. 41, pp. 95-98. [13] Lin HP, Xie Q, Li L, Xie XQ, Sun M, Hong K, 2011. Streptomyces shenzhenensis sp. nov., a novel actinomycete isolated from mangrove sediment. Antonie Van Leeuwenhoek Vol. 100, pp. 631-637. 118
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