A Comparison Between Aerial Mass Colors, Antibacterial Activities and RAPD Fingerprints of Soil Isolates of Streptomyces
Nor Asmara Tasrip,
Mohd Nasir Mohd Desa,
Cheah Yoke Kqueen,
Nurul Zarith Mohamad Zin,
Zainul Amiruddin Zakaria,
Chong Pei Pei
Mariana Nor Shamsudin
This study compared the distribution of aerial mass colors,
antibacterial activities and RAPD-based genomic patterns of 39 Streptomyces
isolates harvested mainly from soil at the University Agricultural Park, Universiti
Putra Malaysia. Streptomyces griseus ATCC 10137 strain was also included
as a reference strain (n = 40). Based on ISP-2 media, the aerial mass colors
observed were categorized as yellow (n = 15), grey (n = 9), brown (n = 7), white
(n = 6) and others (n = 3). Antibacterial activities were assessed on Mueller
Hinton Agar (MHA) and Tryptic Soy Agar (TSA) by perpendicular streak method
against Staphylococcus aureus ATCC 25923, Escherichia coli ATCC
25922, Salmonella sp. and Enterococcus sp. MHA demonstrated 15
isolates with broad spectrum antibacterial activities and 18 as non-broad spectrum.
TSA gave lower proportions with 15 and 9 isolates respectively. Regardless of
the test media used, a higher proportion of isolates with non-white color showed
antibacterial activities to suggest a potential correlation. RAPD dendrogram
(a composite of 3 random primers) also clustered majority of them but segregated
those of white color, which showed less antibacterial activities, in a different
cluster. Further validations involving more isolates are warranted to establish
to cite this article:
Nor Asmara Tasrip, Mohd Nasir Mohd Desa, Cheah Yoke Kqueen, Nurul Zarith Mohamad Zin, Zainul Amiruddin Zakaria, Zarizal Suhaili, Chong Pei Pei and Mariana Nor Shamsudin, 2012. A Comparison Between Aerial Mass Colors, Antibacterial Activities and RAPD Fingerprints of Soil Isolates of Streptomyces. Journal of Biological Sciences, 12: 218-224.
April 19, 2012; Accepted: June 11, 2012;
Published: July 04, 2012
Streptomycetes are filamentous Gram-positive bacteria that give the
musty odor of soil. They belong to a complex group of actinomycetes that produce
various bioactive metabolites of commercial value such as antibiotics. Streptomycetes
have been intensively isolated, leading to characterization of more than 3000
species since 1970s (Kim et al., 2004; Guo
et al., 2008). Due to such huge species diversity, traditional methods
utilizing biochemical tests and morphological examinations for identification
of these organisms are laborious and time-consuming (Cook
and Meyers, 2003; Guo et al., 2008). Polymerase
chain reaction (PCR)-based methods have been very useful to rapidly and accurately
characterize the identity of bacteria at molecular level, particularly through
the sequence homology of 16S rDNA (Taddei et al.,
2006; Arasu et al., 2009; Li
et al., 2009). PCR-Random Amplified Polymorphic DNA (RAPD) allows
the rapid differentiation of bacterial isolates based on whole genomic pattern
which may facilitate the discovery of unique genetic markers in association
with the bacterial genus and species (Welsh and McClelland,
1990; Williams et al., 1990). The technique
has been used in several studies on Streptomyces whereby the potential
of RAPD to discriminate the isolates, as well as to identify potential genetic
probes for genus and species detection was shown to be promising (Mehling
et al., 1995; Malkawi et al., 1999;
Roberts and Crawford, 2000). Later, Gharaibeh
et al. (2003) used the technique to correlate RAPD pattern with various
phenotypic features of Streptomyces. Such approach may identify evidence
at genetic level on the interrelation among the diverse phenotypes for potential
manipulation. Nevertheless, the description would be limited to the experimental
conditions used in the respective studies that may not coincide with other studies
using different cultivation approaches.
Due to the high potential of Streptomycetes as antibiotic producing
organisms, this study was undertaken to assess the phenotypic and genomic features
of our local isolates mainly from soil of the University Agricultural Park (UAP),
Universiti Putra Malaysia (UPM). In view of the lack of standardization in the
cultivation methods of Streptomyces, this study was preliminarily conducted
to characterize the morphological characteristics and antibacterial activities
of the isolates utilizing some common commercial media. For genomic typing,
the isolates were subjected to RAPD analysis and the outcomes were compared
with the phenotypic properties of the isolates for any potential correlation.
MATERIALS AND METHODS
Isolation and identification: Thirty-nine isolates of Streptomyces
strains were isolated from eight patches of soil, each collected at a different
site and labeled with an alphabet; five from various locations around UAP (center;
F, northeast; D, east; E, southeast; B and C and south; H), one from a site
further south of UAP outside universitys periphery (off-campus; I) and
one from a far off-location (off-state; A). Isolates, taken from a same patch
of soil, were selected to have at least an obvious difference in their morphological
appearances such as color of aerial mass, substrate mycelium, spore and appearance
of colony. The isolates were labeled with an alphabet representing the sampling
site followed by a number to indicate the identity of the isolate. Streptomyces
griseus ATCC 10137 was also included, giving a total of 40 strains for analysis
in this study. All isolates were confirmed as Streptomyces based on odor,
morphological characteristics and 16S rRNA gene sequence homology (Duraipandiyan
et al., 2010). Morphological characterization on aerial mass, substrate
mycelium, pigment and spore production of matured cultures on ISP-2 (International
Streptomyces Project-2) agar media (Difco, USA), were done as described
by Shirling and Gottlieb (1966) and Taddei
et al. (2006). Bacterial stocks were preserved in Actinomyces broth
(BBL, USA) with 15% glycerol and kept at -80°C. The 16 rDNA sequences of
the isolates including S. griseus ATCC 10137 strain are available at
the GenBank database (http://www.ncbi.
nlm.nih.gov) with accession numbers JN116247 to JN116248 and JN566155 to
Antimicrobial screening: Perpendicular streak method was used for rapid
antimicrobial assessment of Streptomyces cultures using Mueller Hinton
Agar (MHA) (Merck, Germany) and Tryptic Soy Agar (TSA) (Merck) as test media.
The Streptomyces isolates were inoculated in a single streak down the
middle of the test media and incubated at 28°C for seven days. Then, a single
streak of each test organism; two reference strains: Staphylococcus aureus
ATCC 25923 and Escherichia coli ATCC 25922 and two laboratory strains
of clinical origin: Salmonella sp. and Enterococcus sp., were
inoculated in perpendicular against the earlier streak on the agar medium and
incubated at room temperature for 24 h (Duraipandiyan et
al., 2010). Growth inhibition of the test bacteria was observed by absence
or presence of inhibition zone from the streak of Streptomyces culture.
Genomic DNA extraction:
Culture containing mycelia and spores was taken from single colonies on ISP-2
agar medium and suspended in tris-EDTA buffer. Genomic DNA of the isolates was
extracted using the GF-1 bacterial DNA extraction kit (Vivantis Tech., Malaysia).
Concentration and quality of DNA extract was checked by using the Biophotometer
PCR-RAPD: Three random RAPD primers (OPERON Tech., USA), 10-mer long
each, were used in this study in separate experiments; OPA-02: 5'-TGCCGAGCTG-3',
OPA-09: 5'- GGGTAACGCC-3' and OPA-10: 5'-GTGATCGCAG-3' (Gharaibeh
et al., 2003). The reaction was carried out in a 50 μL reaction
volume containing the following reagents: 10 μL of 5X i-PCR RED Master
Mix (i-DNA Biotech., Singapore), 0.2 μL of primer, 38.8 μL of nuclease
free water and 1 μL of template DNA. The thermal cycling conditions were
as follows: 3 min at 95°C for initial denaturation, 40 amplification cycles
with each comprising 30 sec at 95°C for denaturation, 30 sec at 36°C
for annealing and 1 min at 72°C for extension and a final extension at 72°C
for 7 min in TPersonal Thermocycler (Eppendorf, Germany). Upon purification,
the PCR amplification products and VC 1 kb DNA ladder (Vivantis Tech.) were
respectively mixed with EZ-Vision One DNA dye (AMRESCO, USA) at 5:1 v/v,
resolved by electrophoresis in 1.2% agarose gel and viewed under gel documentation
system. PCR reaction for each primer was repeated three times for assuring the
DNA fingerprint analysis: DNA electrophoretic patterns were analyzed
using BioNumerics gel analysis software version 6.1 (Applied Maths, Belgium)
on the basis of presence or absence of DNA band. RAPD dendrograms based on each
and composite of all primers were then generated by the software using the Dice
coefficient (Dice, 1945) and UPGMA (Unweighted Pair Group
Method of Arithmetic means) cluster analysis. The RAPD clustering patterns were
further analyzed in relation to sampling sites and other phenotypic properties
of the isolates.
Statistical analysis: The distribution of phenotypic properties of the
isolates and their potential associations were analyzed in a 2x2 contingency
table using chi-squared (χ2) and Fishers exact test with
significant level set at p<0.05.
Based on growth on ISP-2 medium,
the varieties of aerial mass color among the 40 isolates were categorized as
yellow (n = 15), grey (n = 9), brown (n = 7), white (n = 6), black (n = 1),
orange (n = 1) and peach (n = 1) with S. griseus ATCC 10137 strain having
a white color. Color of substrate mycelium, spore and soluble pigment were also
observed but not analyzed for simplicity. In the antibacterial assay, 83% of
the isolates showed antibacterial activity on MHA to at least one of the test
bacteria. However, that on TSA was only 60% of the isolates. S. griseus
ATCC 10137 strain showed antibacterial activity against all four test bacteria
on both media. Table 1 shows the detail on distribution of
the aerial mass color-categories and antibacterial activities of the isolates
based on both MHA and TSA as the test media, while Table 2
summarizes the aerial mass color-grouping to show only the significant correlation.
By classifying the test bacteria into gram-positive and gram-negative groups
respectively, with MHA as the test media, 15 isolates showed antibacterial activities
by inhibiting either one or both members in both test bacterial groups; referred
as broad spectrum, while 18 isolates inhibited either one or both in only either
one of the two bacterial groups; referred as non-broad spectrum. Those without
any antibacterial activity against the four test bacteria were referred as non-antibacterial
isolates. With TSA as the test media, 15 isolates were of broad spectrum but
only nine of non-broad. By taking the isolates with broad and non-broad antibacterial
activities as a single group in comparing with those without any antibacterial
activity, statistical analysis found a significant association between isolates
with aerial mass of non-white and white color-categories in relation to antibacterial
activities of the isolates on MHA (χ2: p = 0.001, Fishers
exact test: p = 0.005) but not on TSA (p>0.05). The analysis is stipulated
in Table 2 whereby a high percentage of isolates with antibacterial
activity on MHA is of non-white color-category (91%) as compared to isolates
with white color, which are only 33%. Although that on TSA was not found to
be significant, a high percentage is also observed for isolates with non-white
color-category (65%) to show antibacterial activities against 33% of the isolates
with white color.
In the RAPD analysis, the three random primers were able to generate consistent
DNA fingerprint pattern in the repeated experiments except primer OPA-02 on
three strains (F3, I13 and S. griseus ATCC 10137) that gave no amplification.
||Distribution of aerial mass color-categories in relation to
antibacterial activities of the isolates
|Values in brackets are percentage, others: Black, orange and
peach; n = 1 each, B: Broad spectrum, NB: Non-broad spectrum, N: Non-antibacterial
isolates, MHA: Mueller Hinton agar, TSA: Tryptic soy agar
||White and non-white color-categories in relation to antibacterial
activities of the isolates
|Values in brackets are percentage, bold figures show significant
correlation at p<0.05. B: Broad spectrum, NB: Non-broad spectrum, N:
Non-antibacterial isolates, MHA: Mueller Hinton agar, TSA: Tryptic soy agar
With the exception of the three strains, majority of RAPD patterns were different
with number of bands ranging from one to 11 for all primers and size between
350 bp and 1.5 kb for OPA-09 and OPA-10, while 450 bp and 2.5 kb for OPA-02.
RAPD pattern generated by OPA-02 had band ranging from one to three for about
50% of the isolates but that for the other two primers had more than three bands
for more than 80% of the isolates. S. griseus ATCC 10137 had three and
eight bands for primer OPA-09 and OPA-10 respectively. OPA-02 identified a similar
RAPD fingerprint pattern for two isolates, while OPA-09 and OPA-10 segregated
10 and 20% of the isolates into two and three groups, respectively sharing similar
RAPD fingerprint among members of the respective groups. None of the group member
with a common RAPD pattern as generated by one primer turned out to have a similar
pattern among the group members by the other respective primers. The isolates
also differed either in their species name as identified by the Blast analysis
based on gene 16S rRNA, or phenotypic properties (e.g. aerial mass color and
In Fig. 1, the dendrogram shows a scattered distribution
of the isolates indicating the discriminatory potential of the composite analysis
based on the three random primers.
||RAPD dendrogram based on a composite analysis in association
with isolation sites, aerial mass color-categories and antibacterial activities
of the isolates against four test bacteria on two different test media;
MHA and TSA. The indicated major clusters; A1, A2 and B are at the similarity
matrix of 42%. S. griseus ATCC 10137 is labeled with SG
|| Distribution of aerial mass color-categories and antibacterial
activities of the isolates according to RAPD clusters
|Values in brackets are percentage, Y: yellow, G: grey, B:
brown, W: white, Ot: others (black, orange and peach: n = 1 each), B: broad
spectrum, NB: Non-broad spectrum, N: Non-antibacterial isolates, MHA: Mueller
Hinton agar, TSA: Tryptic soy agar
The isolates are however clustered at 42% similarity matrix in two major clusters,
named as super cluster A and B. Cluster A is further clustered in two sub-clusters
named as A1 and A2, while cluster B stands in a single group. Sub-cluster A1
consists of 10 isolates and majority (70%) of them are from southeast of UAP.
Although two off-state isolates are present in cluster A1, both are branched
away from the rest of the clusters members. Cluster A2 contains 14 isolates
with about the same distribution of isolates from east and center of UAP. On
the other hand, majority of isolates (67%) in cluster B (n = 12) are isolates
from off-campus while the rest are from south of UAP. Four isolates from soils
of non-universitys origin are excluded from all major clusters.
The composite also identifies the members of the respective major clusters
to be in harmony in term of their aerial mass color-categories and antibacterial
activities. Table 3 shows the detail on the distribution of
the isolates by excluding fours isolates not included in the major clusters
(n = 36). A distribution pattern of aerial mass color-categories is observed
with white mostly sorted into cluster B (80%), while others mostly distributed
in cluster A; yellow (76%), grey (63%) and brown (72%). In relation to antibacterial
activity of the isolate, that on MHA and TSA were analyzed separately and patterns
of distribution were compared (Table 3). Based on MHA as the
test media, only two isolates in super cluster A (n = 24) are non-antibacterial
while the rest are either broad or non-broad antibacterial isolates. In cluster
B (n = 12), the proportion of non-antibacterial isolates is higher (42%) as
compared to that in cluster A (8%). On TSA, although the antibacterial activity
was observed to be lower than that on MHA, a similar pattern follows whereby
the proportion of non-antibacterial isolates in cluster B is higher (67%) as
compared to those in cluster A (25%). Only three isolates had uncommon color
as of the other isolates (black, orange and peach) and were grouped together
in the analysis; all are in cluster A1 and show antibacterial activities except
one with peach color.
In the assessment of antimicrobial activity, MHA and TSA have been the test
media of choice due to their wide availability and capacity to allow growth
of many types of test bacteria and diffusion of antimicrobial compounds (Sahm
and Torres, 1988; Dalsgaard, 2001). As for Streptomyces,
an earlier preliminary study had shown a good growth of some representative
Streptomyces isolates on these two media and thus were assumed to be
able to provide sufficient growth for exhibiting visible antibacterial activity
of the isolates in this study (Zin et al., 2011).
Meanwhile, the four test bacteria included in this study are those associated
with common normal flora and opportunistic pathogens that inhibitory effect
against them would be of a significant interest. Based on this experimental
design, it was observed that for some Streptomyces isolates, an inhibitory
activity was observed on one media but not on the other and a higher proportion
of isolates exhibited antibacterial activities on MHA than TSA. This suggested
that MHA may be better than TSA in assessing the isolates for antibacterial
properties. Further statistical analysis also identified a potential correlation
between aerial mass color-categories and the antibacterial activities. Thus,
to a certain extent, those with white color-category in this study could be
considered as less likely to be antibacterial isolates. The antimicrobial metabolites
could be harvested in broth culture and weighted to quantitatively establish
the impact of media on production of antimicrobial compound but such approach
would be tedious for screening purposes involving many isolates. The perpendicular
streak method used in this study, although it is rather qualitative, generated
reading based on absence and presence of the inhibition zone to rapidly justify
the antibacterial status of the isolates.
The reliability of the relationship between aerial mass color and antimicrobial
properties in this study would be affected if groups of related isolates (clone)
were present, which was possible as more than one isolate were selected from
a single patch of soil. To minimize the problem, isolates from a particular
soil sample were selected to differ in one or more in their morphological characteristics
so that the potential of having similar isolates would be reduced. The three
random RAPD primers (OPA-02, -09 and -10), citing their frequent use in discriminating
various organisms including Streptomyces (Yu et
al., 1997; Gharaibeh et al., 2003; Naffa
et al., 2006; Nomura et al., 2006;
Yaqoob et al., 2007), eventually differentiated
majority of the isolates as indicated by their different RAPD fingerprint patterns.
The reproducibility of the primers was also confirmed in repeated experiments.
Although a few isolates shared a similar RAPD fingerprint by the individual
primers, the tendency of the isolates to also show a common pattern when tested
with a different primer was not observed to rule out potential clones (Louise
and Craig, 2001). Consistently, discrepancies of the isolates with regard
to either 16S rDNA-Blasts results or phenotypic properties further indicated
that those isolates were possibly different. Nevertheless, primer OPA-02 resulted
in less number of bands for half of the total isolates as compared to the other
two primers and also failed to detect in a few isolates. Thus, a composite rather
than a single primer was used in the subsequent analysis for a better resolution
(Sigurdsson et al., 1995; Louise
and Craig, 2001; Gharaibeh et al., 2003;
Larrasa et al., 2004; Cheah
et al., 2008). In the later analysis, an obvious improvement was
then observed that each isolate was branched in its own lineage pattern.
In an attempt to correlate the genomic pattern with the aerial mass color and
antibacterial activity of the isolates, a dendrogramatic clustering pattern
based on the composite analysis was possible when the similarity level was compromised
at 42% (Fig. 1). Taking the analysis on the two phenotypes
as independent, the non-white isolates mentioned earlier to be associated with
antibacterial activities were mostly clustered in cluster A while white in cluster
B. Consistently, regardless of the test media used, cluster A happened to harbor
a higher number of isolates with antibacterial properties as compared to cluster
B. Gharaibeh et al. (2003) also conducted RAPD
analyses using the same random primers and showed that based on the composite
analysis, there was a clustering pattern according to type of sporophore and
melanin production of their isolates. This study also pointed a clustering pattern
for aerial mass color and antibacterial activity among the 40 isolates. A further
scrutiny on these linkages is however needed for potential exploitation. To
our knowledge, such comparative studies at both phenotypic and genotypic level
for Streptomyces spp. have not been widely available. It could be due
to the complexity of the spp. themselves that problems still persist to find
suitable media that can stably and simultaneously express the various phenotypes
of Streptomyces isolated from various different origins.
As a whole, UAP is a promising site to search for potential Streptomycetes,
but the issue on the media escalates the challenges in exploring the diversity
and useful traits of Streptomyces. Preliminarily, this study presents
an inference analysis for association between aerial mass color, antibacterial
and genotype but subjected to some limitations. Apparently, those observations
were only a deduction based on limited growth media and test bacteria. Moreover,
the sample size was only 40 isolates to be largely representing the diverse
Streptomyces population, particularly the color-variety of the aerial
mass that was not evenly distributed. Thus, the description and association
between aerial mass color and antibacterial property require more validations.
For a standardization purpose, this study used ISP-2 agar medium for describing
the colonies of the isolates. An earlier analysis on representative isolates
indicated the ability of this media to exhibit discrete morphological traits
in different isolates over those on MHA and TSA which were quite homogenous
(Zin et al., 2011). Thus, it was not known the
impact of using different media on the outcome of the aerial mass color and
its potential connection with the antibacterial activities among the isolates
in this study as the later was conducted on MHA and TSA. In addition, the antibacterial
status of the remaining isolates not detected to be exhibiting any antibacterial
activity was still ambiguous as it could turn out differently if other media
were used. As for the RAPD analysis, despite its rapidity, it carries the drawback
in the chemical complexities of the reaction itself that variation may be expected
when different RAPD primers, molecular reagents, PCR running protocols and thermocycler
are used (Li et al., 2009). Therefore, further
improvements are warranted involving a wider coverage of isolates, locations,
phenotypic traits and microbiological media, as well as the use of other random
primers or means of genomic typing for a better reliability and comparability
This study was funded by grants
from the Universiti Putra Malaysia (04-01-09-0715RU) and Ministry of Higher
Education, Malaysia (07-04-10-850FR).
Arasu, M.V., V. Duraipandiyan, P. Agastian and S. Ignacimuthu, 2009. In vitro antimicrobial activity of Streptomyces spp. ERI-3 isolated from Western Ghats rock soil (India). J. Mycol. Med., 19: 22-28.
CrossRef | Direct Link |
Cheah, Y.K., N.A. Salleh, L.H. Lee, S. Radu, S. Sukardi and J.H. Sim, 2008. Comparison of PCR fingerprinting techniques for the discrimination of Salmonella enterica subsp. enterica serovar Weltevreden isolated from indigenous vegetables in Malaysia. World J. Microbiol. Biotechnol., 24: 327-335.
Cook, A.E. and P.R. Meyers, 2003. Rapid identification of filamentous actinomycetes to the genus level using genus-specific 16S rRNA gene restriction fragment patterns. Int. J. Syst. Evol. Microbiol., 53: 1907-1915.
Dalsgaard, I., 2001. Selection of media for antimicrobial susceptibility testing of fish pathogenic bacteria. Aquaculture, 196: 267-275.
Dice, L.R., 1945. Measures of the amount of ecologic association between species. Ecology, 26: 297-302.
Duraipandiyan, V., A.H. Sasi, V.I.H. Islam, M. Valanarasu and S. Ignacimuthu, 2010. Antimicrobial properties of actinomycetes from the soil of Himalaya. J. Mycol. Med. J. Med. Mycol., 20: 15-20.
Gharaibeh, R., I. Saadoun and A. Mahasneh, 2003. Genotypic and phenotypic characteristics of antibiotic-producing soil Streptomyces investigated by RAPD-PCR. J. Basic Microbiol., 43: 18-27.
CrossRef | Direct Link |
Guo, Y., W. Zheng, X. Rong and Y. Huang, 2008. A multilocus phylogeny of the Streptomyces griseus 16S rRNA gene clade: Use of multilocus sequence analysis for streptomycete systematics. Int. J. Syst. Evol. Microbiol., 58: 149-159.
Kim, B.J., C.J. Kim, J. Chun, Y.H. Koh and S.H. Lee et al., 2004. Phylogenetic analysis of the genera Streptomyces and Kitasatospora based on partial RNA polymerase -subunit gene (rpoB) sequences. Int. J. Syst. Evol. Microbiol., 54: 593-598.
Larrasa, J., A. Garcia-Sanchez, N.C. Ambrose, A. Parra and J.M. Alonso et al., 2004. Evaluation of randomly amplified polymorphic DNA and pulsed field gel electrophoresis techniques for molecular typing of Dermatophilus congolensis. FEMS Microbiol. Lett., 240: 87-97.
CrossRef | Direct Link |
Li, W., D. Raoult and P.E. Fournier, 2009. Bacterial strain typing in the genomic era. FEMS Microbiol. Rev., 33: 892-916.
Louise, H.K. and H.A. Craig, 2001. Use of multiple primers in RAPD analysis of clonal organisms provides limited improvement in discrimination. BioTechniques, 30: 1262-1267.
PubMed | Direct Link |
Malkawi, H.I., I. Saadoun, F.A. Moumani and M.M. Meqdam, 1999. Use of RAPD-PCR fingerprinting to detect genetic diversity of soil Streptomyces isolates. New Microbiol., 22: 53-58.
PubMed | Direct Link |
Mehling, A., U.F. Wehmeier and W. Piepersberg, 1995. Application of Random Amplified Polymorphic DNA (RAPD) assays in identifying conserved regions of actinomycete genomes. FEMS Microbiol. Lett., 128: 119-125.
Naffa, R.G., S.M. Bdour, H.M. Migdadi and A.A. Shehabi, 2006. Enterotoxicity and genetic variation among clinical Staphylococcus aureus isolates in Jordan. J. Med. Microbiol., 55: 183-187.
Nomura, R., K. Nakano, H. Nemoto, K. Fujita and S. Inagaki et al., 2006. Isolation and characterization of Streptococcus mutans in heart valve and dental plaque specimens from a patient with infective endocarditis. J. Med. Microbiol., 55: 1135-1140.
CrossRef | Direct Link |
Roberts, M.A. and D.L. Crawford, 2000. Use of randomly amplified polymorphic DNA as a means of developing genus- and strain-specific Streptomyces DNA props. Applied Environ. Microbiol., 66: 2555-2564.
Sahm, D.F. and C. Torres, 1988. Effects of medium and inoculum variations on screening for high-level aminoglycoside resistance in Enterococcus faecalis. J. Clin. Microbiol., 26: 250-256.
Direct Link |
Shirling, E.B. and D. Gottlieb, 1966. Methods for characterization of Streptomyces species. Int. J. Syst. Evol. Microbiol., 16: 313-340.
CrossRef | Direct Link |
Sigurdsson, V., K. Anamthawat-Jonsson and A. Sigurgeirsson, 1995. DNA fingerprinting of Populus trichocarpa clones using RAPD markers. New For., 10: 197-206.
Taddei, A., M.J. Rodriguez, E. Marquez-Vilchez and C. Castelli, 2006. Isolation and identification of Streptomyces spp. from Venezuelan soils: Morphological and biochemical studies. I. Microbiol. Res., 161: 222-231.
CrossRef | Direct Link |
Welsh, J. and M. McClelland, 1990. Fingerprinting genomes using PCR with arbitrary primers. Nucleic Acids Res., 18: 7213-7218.
CrossRef | PubMed | Direct Link |
Williams, J.G.K., A.R. Kubelik, K.J. Livak, J.A. Rafalski and S.V. Tingey, 1990. DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucl. Acids Res., 18: 6531-6535.
CrossRef | PubMed |
Yaqoob, E., I. Hussain and S.U. Rahman, 2007. Molecular characterization by using Random Amplified Polymorphic DNA (RAPD) analysis of Salmonella enteritidis isolates recovered from avian and human sources. Pak. Vet. J., 27: 102-104.
Direct Link |
Yu, J.R., J.S. Chung and J.Y. Chai, 1997. Different RAPD patterns between Metagonimus yokogawai and Metagonimus Miyata type. Korean J. Parasitol., 35: 295-298.
CrossRef | Direct Link |
Zin, N.Z.M., N.A. Tasrip, M.N.M. Desa, C.Y. Kqueen, Z.A. Zakaria, R.A. Hamat and M.N. Shamsudin, 2011. Characterization and antimicrobial activities of two Streptomyces isolates from soil in the periphery of Universiti Putra Malaysia. Trop. Biomed., 28: 651-660.
Direct Link |