British Microbiology Research Journ

British Microbiology Research Journal
3(3): 280-294, 2013
SCIENCEDOMAIN international
www.sciencedomain.org
Emergence of Antibiotic-Producing
Microorganisms in Residential Versus
Recreational Microenvironments
Yvon Woappi
1
, Prashant Gabani
1
and Om V. Singh
1*
1
Division of Biological and Health Sciences, University of Pittsburgh, Bradford, PA-16701,
USA.
Authors’ contributions
This work was carried out in collaboration between all authors. Author YW designed the
performed studies and wrote the first draft of the manuscript. Author PG managed and
assisted in the data analyses. Author OVS designed, advised, evaluated the data, and
finalized the manuscript for publication. All authors read and approved the final manuscript.
Received 8
th
February 2013
Accepted 4
th
April 2013
Published 23
rd
April 2013
ABSTRACT
Aims: To identify novel antibiotic-producing microbial strains with unprecedented
pertinence. We hypothesize that site-specific soil samples will contain a variety of antibioticproducing species (APS) with diverse specificity of molecular elements.
Place and Duration of Study: Laboratory of Microbiology, Division of Biological and Health
Sciences, University of Pittsburgh, Bradford, PA-16701, USA, between August 2010 and
May 2011.
Methodology: The environmental soil samples were collected from residential and
recreational sites in Southern, PA, USA at longitude: -76 42 21.7116, latitude: 39 56
35.7252; approximately 201 meters above sea level. Over 70 natural antibiotic-producing
soil bacteria were screened against 19 pathogenic microorganisms. Agar-plug assay was
established to identify the antibiotics’ potency and pathogenic inhibitory index calculations
were employed to measure the inhibitory potential of each isolate; 16S rRNA sequencing
was used for microbial classification.
Results: A total of 71 microorganisms from residential soil demonstrated zones of inhibition
(ZOI), followed by 9 organisms from recreational soil sample. A total of 15 bioactive strains
demonstrated convincing growth inhibitory properties against 16 clinically relevant
pathogens; 40% revealed pDNA presence, of which 67% exhibited stringent potencies
against S. aureus. We observed a highly bioactive residential soil microbiota compared to
recreational soil.
Conclusion: 16S rRNA sequence analysis corroborated several of the species belonging
to Enterobacteriaceae, Xanthomonadaceae, and Bacillaceae. These findings may indicate
a co-evolutionary biosynthesis of novel antibiotics driven by the increase of bioactive
microbiota in residential environments.
Keywords: 16S rRNA; antibiotics; microorganisms; antibiotic potency; zone of inhibition;
pathogens; antibiotic resistance.
1. INTRODUCTION
The microbial products of secondary metabolism carry an important role in human health,
providing roadmaps for the biosynthesis of many synthetic and semi-synthetic drugs. In
nature, microbial bioactive products are present as mycotoxins or bacteriosins derived from
filamentous and non-filamentous bacteria and fungi. The soil-based actinomycetes have
been the source of countless drugs, from streptomycin and actinomycin, to erythromycin and
vancomycin [1]. Natural soil harbors over 109 microorganisms/ gram and provides an ideal
reservoir for bioactive microbiota, which springs virtually all clinical antibiotics used today
[2,3]. Today nearly 500 antibiotics are found each year and over 80% of antibiotics in clinical
use are obtained from soil isolates [1]. These bioactive microorganisms are most abundantly
present at the top few inches of the soil, in soil containing straw and agricultural products
[2,4]. Studies have also suggested that soil from areas containing residential-derived
materials, such as human fecal matter, contains 10-20 times more antibiotic resistant strains
than recreational or industrial soils [5]. Consequently and in addition to the elevated use of
therapeutic drugs in urban or residential environments, residential soils contain exceptionally
high bioactivity and are abundant reservoirs of antibiotic-resistant microorganisms [5,6].
Plasmid-mediated antibiotic producing genes can be acquired environmentally by
transposition from one microorganism to another [2]. The phenomenon of intracellular
plasmid acquisition enables antibiotic-producing bacteria to co-exist with antibiotic-resistant
strains through the synthesis of novel bioactive compounds, which allows them to combat
their continuously evolving antibiotic resistant counterparts [7,8]. This sophisticated coevolutionary adaptation is perpetual within microbial-rich soil and represents a great
reservoir for novel, natural (non-synthesized) antibiotics. The successful advancement of
non-synthetic and synthetic antibiotic therapeutic applications therefore requires constant
identification and characterization of natural antibiotic-producing microorganisms.
Due to their topographical inconvenience, little research has been thoroughly conducted on
residential microenvironments. We aimed to explore the natural antibiotic biosynthesis
potentials of microorganisms isolated from soil collected at residential and recreational sites
and hypothesized that site-specific soil samples will contain a diversity of antibioticproducing strains (APS) with a variety of molecular elements. The molecular characterization
of several isolated growth-inhibitory species revealed plasmid DNA (pDNA) presence. We
observed a higher antibiotic-producing community in residential soil compared to
recreational soil. The diverse microbial population, approximately 105 microorganisms per
gram of soil, displayed copious amounts of soil-dwelling APS against 16 clinically relevant
pathogens; 16S rRNA sequencing revealed unique heterogeneity traits in these APS.
British Microbiology Research Journal, 3(3): 280-294, 2013
282
2. MATERIALS AND METHODS
2.1 Strains Isolation
Environmental soil samples were collected in Southern, PA, USA from 2010-2011 at
longitude: -76 42 21.7116, latitude: 39 56 35.7252, approximately 201 meters above sea
level. Two primary locations were selected for sample collection: a recreational area with
high human traffic (i.e. 94 yard of an industrial site) and an authorized residential property
with an existent human populace. Collected samples were placed in polyethylene bags and
stored in dark at 4
0
C until analyzed for antimicrobial properties. The soil type obtained at
both locations was MT. Airy-Glenelg-Linganore soil (PA087; soilmap.psu.edu).
Due to the heterogeneous nature of soil samples, three different growth medium, Tryptic Soy
Agar (TSA), Potato Dextrose Agar (PDA), and Nutrient Agar (NA), were utilized for the
emergence and isolation of microorganisms. The antibiotic-producing abilities of the
collected microbiota was tested in triplicates and assayed by growth inhibition zone against
19 clinically relevant pathogenic microorganisms obtained from American Type Culture
Collection (ATCC): Streptococcus pneumoniae, Staphylococcus aureus, Streptococcus
thermophilus, Listeria monocytogenes, Ochrobae anthropic, Staphylococcus sciuri, Kocuria
kristinae, Klebsiella pneumoniae, Escherichia coli, Enterococcus saccharolyticus, Klebsiella
oxytoca, Streptococcus zooepidemicus, Candida albicans, Enterococcus faecalis, Serratia
marcescens, Saccharomyces cerevisiae, Micrococcus luteus, Bacillus cereus, and
Pseudomonas fluorescens.
2.2 Primary and Secondary Screening
All pathogenic microorganisms were spread on 90-mm polystyrene petri dishes filled with
TSA, PDA, and NA medium, respectively. The collected bioactive soil samples (1-5g) from
each 112 location were dried at 37ºC and sprinkled (~ 80-100 particles) on dishes spread
with each respective ATCC pathogen. Control plates were merely spread with ATCC
pathogens and incubated at 37ºC. After 48 h incubation period, microorganisms displaying
clear zones of inhibition against the pathogens were sequestered onto plates of respective
medium and assigned numbers. Repeated streaking purified the isolated microorganisms
and single-cell colonies were picked up for further screening and temporary storage at 4ºC in
medium-agar-filled slants. Microorganisms displaying little or no pathogenic inhibitory
properties were retested and discarded if scarce antibiotic biosynthesis persisted. Purified
microorganisms obtained from this primary screening were further tested for growthinhibitory properties by using the perpendicular cross-plate method (PCPM) [9] against all 19
pathogenic microorganisms. Antibiotic-producing isolates were considered as any
microorganisms which inhibited the growth of another microbe within its perimeters [10].
Plates were incubated at 37ºC and analyzed after regular time intervals (12 h, 16 h, 24 h,
and 32h) for zones of inhibition (ZOI) across the junctions. Microorganisms displaying little or
no growth inhibitory properties were retested and discarded if insufficient antibiotic
biosynthesis persisted. APS revealing inhibitory zones against one or more pathogen were
further purified and maintained on agar slants at 4ºC in respective growth medium. The APS
were grown in an orbital shaker incubator at 37ºC overnight in respective liquid culture
medium and rescreened against all 19 ATCC microorganisms using PCPM as
aforementioned.
British Microbiology Research Journal, 3(3): 280-294, 2013
283
2.1.1 Antibiotic production assay
Due to their potential industrial significance, we aimed to thoroughly investigate antibiotic
secreting abilities among the screened APSs. Secondarily screened APSs and blank
controls were

Hasil (Bahasa Indonesia) 1:

Inggris Mikrobiologi penelitian Journal
3(3): 280-294, 2013
SCIENCEDOMAIN internasional
www.sciencedomain.org
memproduksi munculnya antibiotik
mikroorganisme dalam perumahan Versus
rekreasi Microenvironments
Yvon Woappi
1
, Prashant Gabani
1
dan Om V. Singh
1 *
1
Divisi biologi dan Ilmu Kesehatan, Universitas Pittsburgh, Bradford,-Bearing,
USA.
kontribusi penulis
Pekerjaan ini dilaksanakan dalam kerjasama antara semua penulis. Penulis YW dirancang
dilakukan penelitian dan menulis draft pertama naskah. Penulis PG dikelola dan
dibantu dalam analisis data. Penulis OVS dirancang, menyarankan, dievaluasi data, dan
selesai naskah untuk publikasi. Semua penulis membaca dan menyetujui naskah akhir.
menerima 8
th
February 2013
4 diterima
th
April 2013
diterbitkan 23
rd
April 2013
abstrak
tujuan: untuk mengidentifikasi novel antibiotik yang menghasilkan strain mikroba dengan belum pernah terjadi sebelumnya
kebenaran. Kami berhipotesis bahwa contoh spesifik lokasi tanah akan berisi berbagai spesies antibioticproducing (APS) dengan beragam kekhususan dari molekul elemen.
tempat dan durasi studi: Laboratorium mikrobiologi, Divisi biologis dan kesehatan
Sciences, Universitas Pittsburgh, Bradford, Bearing, USA, antara Agustus 2010 dan
Mei 2011.
metodologi: sampel tanah lingkungan dikumpulkan dari perumahan dan
rekreasi situs di Selatan, PA, Amerika Serikat di bujur:-76 42 21.7116, lintang: 39 56
35.7252; sekitar 201 meter di atas permukaan laut. Lebih dari 70 alam antibiotik memproduksi
bakteri-bakteri tanah diputar melawan mikroorganisme patogen 19. Agar-plug assay adalah
didirikan untuk mengidentifikasi potensi antibiotik dan perhitungan indeks penghambatan patogen
dipekerjakan untuk mengukur potensi penghambatan isolat masing-masing; 16S rRNA Sekuensing
digunakan untuk klasifikasi mikroba.
hasil: Total 71 mikroorganisme dari tanah perumahan menunjukkan zona inhibition
(ZOI), diikuti oleh organisme 9 dari sampel tanah rekreasi. Total 15 bioaktif strain
menunjukkan meyakinkan pertumbuhan properti penghambatan terhadap 16 secara klinis relevan
patogen; 40% mengungkapkan kehadiran pDNA, yang 67% dipamerkan ketat potensi
terhadap S. aureus. Kami mengamati mikrobiota tanah residensial yang sangat bioactive dibandingkan
rekreasi tanah.
kesimpulan: 16S rRNA urutan analisis diperkuat beberapa spesies milik
Enterobacteriaceae, Xanthomonadaceae dan Bacillaceae. Temuan ini dapat menunjukkan
biosintesis co-evolusi novel antibiotik yang didorong oleh peningkatan bioaktif
mikrobiota di lingkungan perumahan.
kata kunci: 16S rRNA; antibiotik; mikroorganisme; antibiotik potensi; zona hambat;
patogen; antibiotik perlawanan.
1. PENGENALAN
produk mikroba sekunder metabolisme melakukan peran penting dalam kesehatan manusia,
memberikan peta jalan untuk biosintesis banyak obat sintetis dan semi sintetis. Di
alam, produk mikroba bioaktif hadir sebagai mycotoxins atau bacteriosins berasal dari
berserabut dan bebas-berserabut bakteri dan jamur. Tanah berbasis aktinomycetes memiliki
menjadi sumber banyak obat-obatan, dari Streptomisin dan actinomycin, Eritromisin dan
Vankomisin [1]. Alam tanah pelabuhan mikroorganisme lebih dari 109 / gram dan menyediakan ideal
reservoir untuk mikrobiota bioaktif, yang muncul hampir semua antibiotik klinis yang digunakan hari ini
[2,3]. Hari ini hampir 500 antibiotik yang ditemukan setiap tahun dan lebih dari 80% dari antibiotik dalam klinis
penggunaan yang Diperoleh dari tanah isolat [1]. Mikroorganisme ini bioaktif yang paling berlimpah
hadir di atas beberapa inci tanah, di tanah yang mengandung jerami dan pertanian Produk
[2,4]. Studi juga menyarankan bahwa tanah dari daerah yang mengandung perumahan yang diturunkan
bahan, seperti manusia tinja, mengandung strain 10 - 20 kali lebih resisten antibiotik
daripada rekreasi atau industri tanah [5]. Akibatnya dan menggunakan ditinggikan dari
terapi obat di lingkungan perkotaan atau perumahan, perumahan tanah mengandung sangat
tinggi bioactivity dan yang berlimpah waduk resisten antibiotik mikroorganisme [5,6].
Plasmid-dimediasi antibiotik memproduksi gen dapat diperoleh lingkungan oleh
transposisi dari mikroorganisme satu untuk lain [2]. Fenomena intraseluler
akuisisi plasmid memungkinkan memproduksi antibiotik bakteri untuk hidup berdampingan dengan resisten antibiotik
strain melalui sintesis novel senyawa bioaktif, yang memungkinkan mereka untuk memerangi
terus menerus berkembang resisten terhadap antibiotik rekan-rekan mereka [7,8]. Adaptasi coevolutionary canggih ini abadi dalam tanah mikroba-kaya dan mewakili besar
reservoir untuk novel, alam antibiotik (non-disintesis). Kemajuan sukses
aplikasi terapi antibiotik non sintetis dan sintetis karena itu memerlukan konstan
identifikasi dan karakterisasi alami memproduksi antibiotik mikroorganisme.
karena ketidaknyamanan mereka topografi, penelitian kecil telah benar-benar dilakukan pada
microenvironments perumahan. Kami bertujuan untuk mengeksplorasi biosintesis antibiotik alami
potensi mikroorganisme yang terisolasi dari tanah yang dikumpulkan di situs tempat tinggal dan rekreasional
dan hipotesis bahwa contoh spesifik lokasi tanah akan mengandung keragaman antibioticproducing strain (APS) dengan berbagai elemen molekul. Karakterisasi molekul
beberapa spesies penghambatan pertumbuhan terisolasi mengungkapkan plasmid kehadiran DNA (pDNA). Kami
diamati memproduksi antibiotik komunitas yang lebih tinggi di tanah perumahan dibandingkan
rekreasi tanah. Beragam populasi mikroba, sekitar 105 mikroorganisme per
gram tanah, ditampilkan jumlah berlebihan tinggal di tanah APS terhadap 16 secara klinis relevan
patogen; 16S rRNA Sekuensing mengungkapkan sifat-sifat unik heterogenitas dalam ini APS
British Journal riset mikrobiologi, 3(3): 280-294, 2013
282
2. BAHAN dan metode
2.1 strain isolasi
Sampel tanah lingkungan dikumpulkan di Selatan, PA, Amerika Serikat dari 2010-2011 di
bujur:-76 42 21.7116, lintang: 39 35.7252 56, sekitar 201 meter di atas laut
tingkat. Dua utama lokasi yang dipilih untuk pengambilan sampel: area rekreasi dengan
tinggi lalu lintas manusia (yaitu 94 halaman situs industri) dan properti residensial resmi
dengan penduduk manusia yang ada. Sampel dikumpulkan ditempatkan di polietilena tas dan
disimpan dalam gelap di 4
0
C sampai dianalisis untuk sifat antimikroba. Jenis tanah yang diperoleh di
kedua lokasi adalah tanah MT. Airy-Glenelg-Linganore (PA087; soilmap.psu.edu).
Due sifat heterogen tanah sampel, tiga berbeda pertumbuhan media, kedelai Tryptic
Agar (TSA), kentang dekstrosa Agar (PDA), dan gizi Agar (NA), digunakan untuk
munculnya dan isolasi mikroorganisme. Antibiotik yang menghasilkan kemampuan
mikrobiota dikumpulkan diuji di triplicates dan diuji oleh pertumbuhan zona hambat terhadap
19 mikroorganisme patogen secara klinis relevan Diperoleh dari jenis budaya Amerika
koleksi (ATCC): Streptococcus pneumoniae, Staphylococcus aureus, Streptococcus
thermophilus, Listeria monocytogenes, Ochrobae antropik, Staphylococcus sciuri, Kocuria
kristinae, Klebsiella pneumoniae, Escherichia coli, Enterococcus saccharolyticus, Klebsiella
oxytoca, Streptococcus zooepidemicus, Candida albicans, Enterococcus faecalis, Serratia
marcescens, Saccharomyces cerevisiae, Micrococcus luteus, Bacillus cereus, dan
Pseudomonas fluorescens.
2.2 primer dan sekunder penyaringan
Semua mikroorganisme patogen yang tersebar pada 90-mm piring petri polystyrene dipenuhi
TSA, PDA dan NA media, masing-masing. Sampel mengumpulkan bioaktif tanah (1-5g) dari
masing-masing 112 dikeringkan di 37ºC dan ditaburi (~ 80-100 partikel) pada hidangan menyebar
dengan patogen ATCC masing-masing masing-masing. Kontrol piring hanya tersebar dengan ATCC
patogen dan burung di 37ºC. Setelah periode inkubasi 48 h, mikroorganisme yang menampilkan
jelas zona hambat terhadap patogen yang diasingkan ke piring dari masing-masing
menengah dan ditugaskan nomor. Diulang melesat dimurnikan mikroorganisme terisolasi
dan single-sel koloni dijemput untuk pemeriksaan lebih lanjut dan penyimpanan sementara di 4ºC di
media-agar-penuh miring. Mikroorganisme yang menampilkan sedikit atau tidak ada patogen penghambatan
properti diuji ulang dan dibuang jika langka antibiotik biosintesis bertahan. Dimurnikan
mikroorganisme yang Diperoleh dari pemeriksaan utama ini lebih lanjut diuji untuk sifat-sifat growthinhibitory dengan menggunakan metode tegak lurus salib-piring (PCPM) [9] terhadap semua 19
patogen mikroorganisme. Memproduksi antibiotik isolat tersebut dianggap sebagai salah satu
mikroorganisme yang menghambat pertumbuhan lain mikroba dalam nya strategis [10].
piring diinkubasi di 37ºC dan dianalisis setelah interval waktu yang teratur (12 h 16 h, 24 h,
dan 32h) untuk zona inhibisi (ZOI) di persimpangan. Mikroorganisme yang menampilkan sedikit atau
Properti penghambatan pertumbuhan tidak diuji ulang dan dibuang jika tidak mencukupi antibiotik
biosintesis bertahan. APS mengungkapkan zona penghambatan terhadap satu atau lebih patogen yang
lebih lanjut dimurnikan dan dikelola di agar-agar miring di 4ºC di media pertumbuhan masing-masing. APS
yang ditanam di inkubator orbital shaker di 37ºC semalam di masing-masing cair budaya
menengah dan rescreened terhadap semua 19 ATCC mikroorganisme menggunakan PCPM sebagai
tersebut.
British Journal riset mikrobiologi, 3(3): 280-294, 2013
283
2.1.1 antibiotik produksi assay
karena signifikansi industri yang potensial, kami bertujuan untuk melakukan penyidikan menyeluruh antibiotik
mensekresi kemampuan antara APSs disaring. Sekunder disaring APSs dan kosong
kontrol yang