World Journal of Microbiology and Biotechnology

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World Journal of Microbiology and Biotechnology

July 2005
Volume 21, Number 5


Prevalence and antibiotic resistance of Escherichia coli in
tropical seafood
(619 - 623)
H. S. Kumar, A. Parvathi, I. Karunasagar, I. Karunasagar
DOI: 10.1007/s11274-004-3555-8

Rapid detection of Salmonella typhi in foods by combination of
immunomagnetic separation and polymerase chain reaction
(625 - 628)
S Kumar, K Balakrishna, G.P. Singh, H.V. Batra
DOI: 10.1007/s11274-004-3553-x

DNA amplification fingerprinting as a tool for checking genetic
purity of strains in the cyanobacterial inoculum
(629 - 634)
B. Jeberlin Prabina, K. Kumar, S. Kannaiyan
DOI: 10.1007/s11274-004-3566-5

Utilization of a polyphasic approach in the taxonomic
reassessment of antibiotic- and enzyme-producing Bacillus spp.
isolated from the Philippines
(635 - 644)
Marie Antonette Ruth V. Guerra-Cantera and Asuncion K. Raymundo
DOI: 10.1007/s11274-004-3567-4

Relationship among acidophilic bacteria from diverse
environments as determined by randomly amplified polymorphic
DNA analysis (RAPD)
(645 - 648)
Tanveer Akbar, Kalsoom Akhtar, Muhammad A. Ghauri, Munir A. Anwar, Moazur
Rehman, Mehboobur Rehman, Yusuf Zafar, Ahmad M. Khalid
DOI: 10.1007/s11274-004-3568-3

Purification and characterization of a thermoalkalophilic
xylanase from Bacillus sp.
(649 - 654)
M. P. Sapre, H. Jha, M. B. Patil
DOI: 10.1007/s11274-004-3569-2

Liquid–liquid extraction of an extracellular alkaline protease
from fermentation broth using aqueous two-phase and reversed
micelles systems
(655 - 659)
T. S. Porto, T. I. R. Monteiro, K. A. Moreira, J. L. Lima-Filho, M. P. C. Silva, A. L. F.
Porto, M. G. Carneiro-da-Cunha
DOI: 10.1007/s11274-004-3570-9

Bioleaching of nickel from equilibrium fluid catalytic cracking
catalysts
(661 - 665)
Oguz Bayraktar
DOI: 10.1007/s11274-004-3573-6


Decolourization of textile dye Reactive Violet 5 by a newly
isolated bacterial consortium RVM 11.1
(667 - 672)
Safia Moosvi, Haresh Keharia, Datta Madamwar
DOI: 10.1007/s11274-004-3612-3

Exopolysaccharide production by Lactobacillus delbruckii
subsp. bulgaricus and Streptococcus thermophilus strains
under different growth conditions
(673 - 677)
Belma Aslim, Zehra Nur Yüksekdag˘, Yavuz Beyatli, Nazime Mercan
DOI: 10.1007/s11274-004-3613-2

Isolation and characterization of actinomycetes antagonistic to
pathogenic Vibrio spp. from nearshore marine sediments
(679 - 682)
J. L. You, L. X. Cao, G. F. Liu, S. N. Zhou, H. M. Tan, Y. C. Lin
DOI: 10.1007/s11274-004-3851-3

Mathematical description of bikaverin production in a fluidized
bed bioreactor
(683 - 688)
Ma. del Carmen Chávez-Parga, Omar González-Ortega, Guadalupe Sánchez-
Cornejo, Ma. de la Luz X. Negrete-Rodríguez, Guillermo González-Alatorre,
Eleazar M. Escamilla-Silva
DOI: 10.1007/s11274-004-3854-0

Adsorption-elution purification of chimeric Bacillus
stearothermophilus leucine aminopeptidase II with raw-starch-
binding activity
(689 - 694)
Yu-Wen Hua, Meng-Chun Chi, Huei-Fen Lo, Lih-Ying Kuo, Kuo-Lung Ku, Long-Liu
Lin
DOI: 10.1007/s11274-004-3853-1

Continuous citric acid fermentation by Candida oleophila under
nitrogen limitation at constant C/N ratio
(695 - 705)
S. Anastassiadis, C. Wandrey, H. -J. Rehm
DOI: 10.1007/s11274-004-3850-4

The influence of different yeasts on the fermentation,
composition and sensory quality of cachaça
(707 - 715)
Evelyn Souza Oliveira, Helena Maria André Bolini Cardello, Elisangela Marques
Jeronimo, Elson Luiz Rocha Souza, Gil Eduardo Serra
DOI: 10.1007/s11274-004-4490-4

Biological control of sheep parasite nematodes by nematode-
trapping fungi: in vitro activity and after passage through the
gastrointestinal tract
(717 - 722)
Érika B. N. Graminha, Alvimar J. Costa, Gilson P. Oliveira, Antonio C. Monteiro,
Solange B. S. Palmeira
DOI: 10.1007/s11274-004-4045-8

Micro-encapsulation of Bifidobacterium lactis for incorporation
into soft foods
(723 - 728)
L. D. McMaster and S. A. Kokott
DOI: 10.1007/s11274-004-4798-0

Evaluation of fluorescent Pseudomonads and Bacillus isolates
for the biocontrol of a wilt disease complex of pigeonpea
(729 - 732)
Shazia Siddiqui, Zaki A. Siddiqui, Iqbal Ahmad
DOI: 10.1007/s11274-004-4799-z

Purification and characterization of a thermostable chitinase
from Bacillus licheniformis Mb-2
(733 - 738)
Aris Toharisman, Maggy Thenawidjaja Suhartono, Margarethe Spindler-Barth,
Jae-Kwan Hwang, Yu-Ryang Pyun
DOI: 10.1007/s11274-004-4797-1

Use of alginate and cryo-protective sugars to improve the
viability of lactic acid bacteria after freezing and freeze-drying
(739 - 746)
B. De Giulio, P. Orlando, G. Barba, R. Coppola, M. De Rosa, A. Sada, P. P. De
Prisco, F. Nazzaro
DOI: 10.1007/s11274-004-4735-2

Bio-production of compost with low pH and high soluble
phosphorus from sugar cane bagasse enriched with rock
phosphate
(747 - 752)
Gaber Zayed and Heba Abdel-Motaal
DOI: 10.1007/s11274-004-5407-y

Purification and characterization of naringinase from a newly
isolated strain of Aspergillus niger 1344 for the transformation of
flavonoids
(753 - 758)
Munish Puri and Sukirti Kalra
DOI: 10.1007/s11274-004-5488-7

Galacto-oligosaccharide production by a thermostable
recombinant β-galactosidase from Thermotoga maritima
(759 - 764)
Eun-Su Ji, Nyun-Ho Park, Deok-Kun Oh
DOI: 10.1007/s11274-004-5487-8

Production of poly(3-hydroxybutyrate) and poly(3-
hydroxybutyrate-co-3-hydroxyvalerate) by Rhodopseudomonas
palustris SP5212
(765 - 769)
Mahuya Mukhopadhyay, A. Patra, A. K. Paul
DOI: 10.1007/s11274-004-5565-y

Effect of sugar-feeding strategies on astaxanthin production by
Xanthophyllomyces dendrorhous
(771 - 775)
Zhong-Ce Hu, Yu-Guo Zheng, Zhao Wang, Yin-Chu Shen
DOI: 10.1007/s11274-004-5566-x




Prevalence and antibiotic resistance of
Escherichia coli
in tropical seafood
H.S.Kumar,A.Parvathi,I.Karunasagar and I.Karunasagar*
Department of Fishery Microbiology,University of Agricultural Sciences,College of Fisheries,Mangalore 575 002,
India
*Author for correspondence:Tel.:+91-824-2246384,Fax:91-824-2246384,E-mail:karuna8sagar@yahoo.com
Keywords:
antibiotic resistance,
Escherichia coli
,faecal coliforms,plasmid,seafood
Summary
The occurrence and antibiotic resistance of
Escherichia coli
in tropical seafood was studied.A 3-tube MPN
method was used for determining the level of faecal contamination of fresh and processed seafood.Of the 188
samples tested which included finfish,shellfish,water and ice,155 were positive for the presence of faecal
coliforms following incubation at 44.5
￿
C.However,
E.coli
was isolated from only 47% of the samples positive
for faecal coliforms.The antibiotic resistance of 116 strains isolated from seafood was tested using 14 different
antibiotics including ampicillin,cephalothin,chloramphenicol,ciprofloxacin,gentamycin,nalidixic acid,strep-
tomycin and vancomycin.Seven strains were resistant to more than five antibiotics of which one was resistant to
eight antibiotics.The multiple drug resistant strains harboured plasmids of varying sizes.Antibiotic susceptibility
studies revealed that seafood from India contains multiple antibiotic resistant strains of
E.coli
which may serve
as a reservoir for antibiotic resistance genes in the aquatic environment.All the strains used in this study did not
harbour any virulence genes commonly associated with pathogenic
E.coli,
when tested by polymerase chain
reaction (PCR).
Introduction
Seafood is a major vehicle for transmission of several
bacterial diseases.The faecal contamination of natural
water bodies has emerged as a major challenge in
developing and densely populated countries like India.
Estuaries and coastal water bodies,which are the
major sources of seafood in India,are often contam-
inated by the activities of adjoining populations and
partially treated or untreated sewage from the town-
ships is released into these water bodies.The fish
harvested from such areas often contain human path-
ogenic microorganisms.In addition,poor sanitation in
landing centers and the open fish markets exacerbates
the situation.
Escherichia coli
has been traditionally
recognized as an indicator organism of faecal contam-
ination of water and seafood (Geldreich 1997).Testing
of seafood for the presence of
E.coli
is still a gold
standard used to assess the faecal contamination in
seafood processing plants in India and elsewhere.
E.coli
is a normal inhabitant of the intestinal tracts
of all warm blooded animals.However,strains of
human pathogenic
E.coli
have evolved that are
recorded as causative agents of a broad range of
human diseases compared to any other pathogenic
bacteria (Nataro & Kaper 1998;Paton & Paton 1998).
Antimicrobial resistance in bacteria associated with
food and water has been a global concern.It is now
widely accepted that there is an association between the
use of antimicrobial agents and the occurrence of
resistance.Antimicrobials exert a selective pressure on
microorganisms and therefore their use is considered a
key issue in epidemiological studies (McGeer 1998).
The disease threat from antibiotic resistant strains of
pathogens has increased in recent years (Williams &
Heymann 1998).Antimicrobial resistance can spread
through horizontal transfer of resistance genes from
one type of bacteria to another.The presence of
resistance,together with the acquisition of virulence
genes can lead to clonal expansion and spread of a
particular disease-causing agent.Hence it is considered
important to study the antimicrobial resistance in
pathogenic as well as indicator bacteria associated
with food animals (OIE 1999).
The study reported here was undertaken to determine
the prevalence of
E.coli
in seafood,the occurrence of
any pathogenic genotypes in seafood isolates of
E.coli
and the distribution of antibiotic resistance in them.The
results demonstrate the widespread occurrence of
E.coli
in tropical seafood and highlight the associated health
risks due to the distribution of antibiotic resistance in
seafood-associated strains of
E.coli
in India.
World Journal of Microbiology & Biotechnology (2005) 21:619–623
￿
Springer 2005
DOI 10.1007/s11274-004-3555-8
Materials and methods
Samples
A total of 188 samples were analysed which included
finfish,shellfish,ice and water (Table 1) for total and
faecal coliforms using the 3-tube MPN method (FDA
1998).Samples of fish and shellfish included both fresh
(
n
¼
128) and frozen (
n
¼
23),collected from different
fish markets,landing centres and seafood processing
plants,in and around Mangalore.Twelve water samples
and 10 ice samples were collected from the fresh fish
market and 15 ice samples were collected from shrimp-
processing plants.The samples were transported to the
laboratory immediately and analysed within an hour of
collection.All dehydrated media used in this study for
microbiological analysis were obtained from Himedia,
Mumbai,India and prepared according to manufac-
turer’s instructions.Briefly,the samples were homoge-
nized in a sterile blender and inoculated into lauryl
sulphate tryptose broth (LSTB) and incubated at 37
￿
C
for 24–48 h.The LSTB tubes showing turbidity and gas
in Durham tubes were recorded as positive.Two
loopfuls from positive LSTB tubes were inoculated into
corresponding labelled tubes with 5 ml of EC (
E.coli
)
broth medium.EC broth tubes exhibiting turbidity and
gas production following 24 h incubation at 44.5
￿
Cina
water bath were considered positive for the presence of
faecal coliforms.For isolation of
E.coli,
two loopfuls
from positive EC broth tubes were streaked onto eosin
methylene blue (EMB) agar plates.A minimum of five
typical colonies were picked up,purified on tryptone
soya agar (TSA) plates and subjected to standard
biochemical tests for the identification of
E.coli
.
Antimicrobial susceptibility testing
A total of 116
E.coli
strains were tested for antibiotic
resistance by standard agar disc diffusion technique
(Bauer
et al
.1966) on Mueller Hinton agar using
commercial discs (HiMedia,Mumbai,India).The
following antibiotics with the disc strength in parenthe-
ses were used:ampicillin (10 mcg),amoxycillin
(30 mcg),ceftriaxone (30 mcg),cephalothin (30 mcg),
chloramphenicol (30 mcg),ciprofloxacin (5 mcg),gen-
tamycin (10 mcg),kanamycin (30 mcg),nalidixic acid
(30 mcg),penicillin G (10 U),rifampicin (5 mcg),strep-
tomycin (10 mcg),tetracycline (30 mcg) and vancomy-
cin (30 mcg).
Characterization of
E.coli
strains
The four STEC strains used in this study were those
previously described (Kumar
et al
.2001).These in-
cluded one each of O76:H21 (
stx
1
+
,
stx
2
+
,
ehxA
+
) and
ONT:H21 (
stx
1
+
,
stx
2
+
,
ehxA
+
) and two O110:NM
(
stx
1
+
,
ehxA
+
).The remaining 112 strains were tested
for the presence of virulence genes by polymerase chain
reaction (PCR) using primers previously described for
labile toxin (
lt
) and stable toxin (
st
) genes of enterotox-
igenic
E.coli
(Olsvik & Strockbine 1990),shiga toxin
genes
stx
1
and
stx
2
(Pal
et al.
1999),
E.coli
attachment
and effacement (
eae)
gene (Paton & Paton 1998) and
enterohaemorrhagic
E.coli
haemolysin A (
ehxA
) gene
(Fratamico
et al
.1995).
Extraction of plasmids
Plasmids were extracted by the alkaline lysis method
(Sambrook
et al
.1989) from 10 strains exhibiting
multiple resistance to the antimicrobials tested.Briefly,
1.5 ml of the overnight culture in Luria Bertani (LB)
broth was centrifuged at 10,000 rev/min for 10 min in a
biofuge (Heraeus,Germany) and the pellet resuspended
in 100
l
l of solution I (50 mMglucose;25 mMTris–Cl,
pH 8.0;10 mMEDTA,pH 8.0).The cells were lysed by
the addition of 200
l
l of solution II (0.2 N NaOH;
1% SDS).The genomic DNA was precipitated by the
addition of solution III (5 Mpotassiumacetate and 5 M
glacial acetic acid) and pelleted by centrifugation at
10,000 rev/min for 10 min.The supernatant containing
plasmid DNA was extracted twice with phenol:chloro-
form,ethanol precipitated,treated with RNAase (20
l
l/ml) and finally dissolved in 50
l
l TE buffer (10 mM
Tris,pH 8.0;1 mM EDTA).The extracted plasmids
were electrophoresed on 0.8%agarose gel,stained with
ethidium bromide and photographed using gel docu-
mentation system (Herolab,Weisloch,Germany).
Table 1
.Prevalence of faecal coliforms and
E.coli
in seafood,ice and water samples collected from different sources.
Sample type Source No.of samples No.positive
for faecal coliforms
No.positive
for
E.coli
%Prevalence
of
E.coli
Fin fish Fresh fish market 21 21 8 38
Fin fish Landing centre 20 20 5 25
Shrimp Fresh fish market 20 20 3 15
Shrimp Landing centre 25 20 4 16
Shrimp Processing plants 23 8 2 8.6
Clams Fresh fish market 32 32 25 78
Oysters Estuaries 10 10 10 100
Water Fresh fish market 12 12 8 66
Ice Fresh fish market 10 7 7 70
Ice Processing plants 15 5 1 6.66
Total 188 155 73 38.8
620
H.S.Kumar
et al.
Results
Incidence of faecal coliforms and
E.coli
in fish
and shellfish
Of the total 188 samples tested,155 were found to be
positive for the presence of faecal coliforms in EC broth
when incubated at 44.5
o
C suggesting a prevalence of
82.4% but interestingly only 73 of 155 (47%) samples
positive for faecal coliforms were in fact positive for
E.coli
(Table 1).Overall,38.8% (73 of 188) samples
were positive for
E.coli
after isolation and biochemical
characterization.
Though all the samples of fresh finfish collected from
landing centre and fresh fish market were positive for the
presence of faecal coliforms by MPN method,only 8/21
(38.8%) finfish samples from fresh fish market and 5/20
(25%) fromlanding centre were confirmed as
E.coli.
All
of the 20 shrimp samples collected frommarket and 20 of
25 samples from landing centre were positive for the
presence of faecal coliforms (Table 1) but the prevalence
of
E.coli
in these samples was just 15% and 16%,
respectively.In the case of frozen shrimp samples
collected from processing plants in Mangalore,8/23
(34.78%) were found to be positive for the presence of
faecal coliforms of which only 2 (8.6%) contained
E.coli
.
Results in Table 2 show that among freshly caught
shrimp samples,
P.monodon
and
P.indicus
were negative
for the organism while
E.coli
was detected in
Metapenaeus
spp.and
Parapenaeopsis
spp.While none
of the frozen
P.monodon
samples were positive for
E.coli
,
2%of frozen
P.indicus
was found to harbour
E.coli.
All of the 32 clam samples examined in the present
study were positive for the presence of faecal coliforms
and 25 (78%) of these were positive for
E.coli.
Both the
species of clams used in this study,
Meretrix meretrix
and
M.casta
harboured
E.coli
as seen from Table 2.In
the case of the oyster sample,
Crassostrea madrasensis,
all of the 10 samples examined in this study were
positive for faecal coliforms and
E.coli
,recording a cent
percent level of prevalence in the samples undertaken in
this study.
Prevalence of
E.coli
in ice and water
All of the 12 samples of water collected from fresh fish
market were faecal coliform positive,of which 8
contained
E.coli
(Table 1).Seven of 10 ice samples
from fresh fish market were positive for faecal coliforms
and
E.coli
.Ice samples fromprocessing plants were also
analysed and the results showed 5 of 15 samples positive
for faecal coliforms and interestingly only one positive
for
E.coli
.
Antibiotic susceptibility testing
Forty two of the 116 strains of
E.coli
analysed during
this study were resistant to at least two antimicrobials
tested and no strain was sensitive to all the antimicro-
bials tested.However,29 were resistant to one antibi-
otic,25 to 3 antibiotics,14 to 4 antibiotics,one was
resistant to 5,3 were resistant to 6,and 1 was resistant to
8 antibiotics used.The 8 antibiotics to which one of the
strains was resistant included ampicillin,cephalothin,
kanamycin,nalidixic acid,penicillin,streptomycin,tri-
methoprim,and vancomycin.Among 6 STEC strains
tested,one was resistant to 6 antibiotics (ampicillin,
amoxycillin,cephalothin,ceftriaxone,penicillin,and
vancomycin).Significantly 3 of the 6 strains tested were
resistant to penicillin and cephalothin while two were
resistant to amoxycillin.
Table 3 describes the percentage of strains resistant to
individual antibiotic types.Resistance to vancomycin
Table 2
.Incidence of
E.coli
in different species of finfish and shellfish.
Sample types No.tested No.positive
for
E.coli
Finfish
Sardines 8 3
Mackerles 7 2
White bait 5 2
Soles 5 –
Pink perch 7 2
Scianids 5 2
Fresh water fish 4 2
Total 41 13
Shell fish
Fresh shrimps
P.monodon
10 –
P.indicus
15 –
Metapenaeus
spp.15 5
Parapenaeopsis
spp
.
52
Frozen shrimps
P.monodon
8–
P.indicus
15 2
Total 68 9
Clams
Meretrix meretrix
22 18
M.casta
10 7
Total 32 25
Oysters
Crassostrea madrasensis
10 10
Total 10 10
Table 3
.Percentage of
E.coli
strains resistant to antibiotics tested in
this study.
Antibiotic used %Resistant
Amoxycillin 2.5
Ampicillin 4.3
Ceftriaxone 0.86
Cephalothin 67.2
Chloramphenicol 0
Ciprofloxacin 0
Gentamycin 0
Kanamycin 2.5
Nalidixic acid 0.86
Penicillin G 42
Rifampicin 0
Streptomycin 6
Tetracycline 0.86
Vancomycin 94
E.coli
in tropical seafood
621
was observed in 94% of the strains followed by
cephalothin (67.2%) and penicillin 42%.No resistance
was observed to chloramphenicol,ciprofloxacin,rifam-
picin and gentamycin.Six percent of the strains were
resistant to streptomycin while 4.3% were resistant to
ampicillin.Resistance to amoxycillin and kanamycin
was observed in 2.5%of the strains.A small percentage
(0.86) of the strains was resistant to tetracycline and
nalidixic acid.The resistant strains of
E.coli
were found
to harbour plasmids of varying sizes (Figure 1).
Discussion
A large percentage of samples tested (82%) were
positive for the presence of faecal coliforms by the
MPN method (Table 1) as indicated by the production
of acid and gas in EC broth incubated at an elevated
temperature of 44.5
o
C but only 38.8% of the samples
positive for faecal coliforms contained
E.coli,
strength-
ening the earlier observation by Jeyasekaran
et al.
(1990).The prevalence of
E.coli
was the highest
(78%) in clams from the fresh fish market.This is to
be expected since these shellfish are present in the
estuarine environment where contamination with faecal
matter is often recorded.It is of significance that water
and ice from the fish market had a high prevalence of
E.coli
(66%and 70%,respectively) which is in contrast
to ice from processing plants where the prevalence was
only 6.6%.This shows that water and ice used in the
fresh fish markets and landing centre in Mangalore is of
poor quality and are thus important sources of con-
tamination for fish
.E.coli
does not survive in the
marine environment for long and therefore this organ-
ism cannot be expected in fish harvested off-shore.
E.coli
was isolated from pelagic fish such as sardines,
mackerels,whitebaits and scianids,while the soles,a
demersal fish group,were negative for the presence of
E.coli
(Table 2).However,the presence of
E.coli
detected in some marine fish in the present study might
represent post-harvest contamination such as in the
landing centre and fish market through use of unpotable
ice and water.Interestingly,fresh shrimps such as
P.monodon
and
P.indicus
were free from
E.coli.
In
view of their high market value,it is possible that these
items are carefully handled to prevent any contamina-
tion.In line with the above observation,low value
shrimps such as
Metapenaeus
spp.and
Parapenaeopsis
spp.showed contamination by
E.coli
,perhaps due to
poor handling.Among frozen shrimp samples,
P.monodon
was free from
E.coli
while 13% of
P.indicus
had this organism.From the results presented
in Table 1 where 8 of 23 frozen shrimp samples from
processing plants were positive for faecal coliforms but
the recovery of
E.coli
from only 2 samples (3.6%),it is
inferred that the low temperature sensitivity of
E.coli
is
responsible for its low positivity.However when it is
recovered fromprocessed seafood,it could be attributed
to contamination during post-process handling and
transportation.
In spite of the observations that the faecal coliform
test often leads to overestimation of the contamination
in tropical seafood (Knutton
et al.
1977),it remains the
most convenient method in the absence of any other
reliable indicator organism.Results of our study indi-
cate that it is necessary to isolate
E.coli
from faecal
coliform-positive samples to confirm faecal contamina-
tion.Kumar
et al
.(2001) have reported the occurrence
of pathogenic
E.coli
strains in Indian seafood.No
separate enrichment methods are available to selectively
isolate various pathogenic groups of
E.coli
.Isolation of
E.coli
will help further characterization of such strains.
The prevalence of various pathogenic strains in Indian
seafood needs to be studied to estimate the disease
burden from
E.coli
contamination.
Antibiotic resistance in
E.coli
isolated from seafood
The results of the antibiotic resistance study (Tables 3
and 4) indicate that
E.coli
strains resistant to more than
one drug are widespread in seafood included in this
study.Resistant strains of
E.coli
were recorded to 9 out
of 13 antibiotics tested.Apart froma large percentage of
strains (67%) that were resistant to the cephalosporin
antibiotic,cephalothin,low levels of resistance was
observed against ampicillin and the fluoroquinolone
antibiotic,nalidixic acid.
The presence of multiple antibiotic resistance in
seafood strains of
E.coli
is of grave concern.In
Mangalore,the untreated or partially treated domestic
sewage is released into open estuaries.The presence of
antibiotic residues in such wastes cannot be ruled out
since these also contain hospital wastes,though no
comprehensive studies have been conducted to support
this hypothesis.In addition to the sources of contam-
ination already mentioned,the surface runoff during the
monsoon might introduce resistant strains of
E.coli
into
the estuarine environment.It is therefore to be expected
Figure 1.
Agarose gel electrophoresis analysis of plasmids extracted
from multiple antibiotic resistant
E.coli
strains.Lane 1,DNA marker
(
k
DNA digested with
Eco
RI &
Hin
d III);lanes 2,3,4 & 5,plasmids
from different
E.coli
strains;lane 6,100 bp DNA ladder.
622
H.S.Kumar
et al.
that the fish and shellfish harvested from such environ-
ments would be contaminated with
E.coli
.On the other
hand,introduction of
E.coli
may also take place away
from the areas of seafood harvest such as in the open
market by use of contaminated ice and water used for
preserving or washing the fish.
There is a paucity of data regarding the presence of
antibiotic resistance either in clinical or environmental
strains of
E.coli
fromIndia.The presence of cephalothin
and penicillin resistance among the seafood strains of
E.coli
might suggest a hospital or veterinary origin for
such strains.
E.coli
in aquatic environment is exposed to
sublethal levels of antibiotics in the aquatic environments
brought in by the discharged wastes.The use of antibi-
otics is also widespread in animal industry and agricul-
ture.It is estimatedthat the use of antibiotics in animals is
100–1000 times that in human population (Feimen 1998;
Levy 1998;Witte 1998).No study so far has been
conducted to understand the effects of antibiotic use in
animals,agriculture and aquaculture in India.The
unregulated use of antibiotics in such systems in India
contributes significantly to the antibiotic residues in meat
and aquaculture products.The antibiotic resistance
patterns of seafood strains of
E.coli
observed in this
study suggests a greater risk in the form of transfer of
resistance to other pathogenic bacteria.The frequent
exchange of plasmids between
E.coli
and other coliform
bacteria has been previously reported (Grabow
et al
.
1973,1976).Our previous study reports the presence of
Salmonella
sp.in a significant percentage of fish and
shellfish (Kumar
et al.
2001).The antibiotic resistance in
non-pathogenic
E.coli
reported here cannot be ruled out
as insignificant since the transfer of resistance genes can
take place between closely related bacteria such as
members of Enterobacteriaceae family.The resistant
strains in seafood can formcommensal flora via the food
chain (Van den Bogaard & Stobberingh 1999;Witte
1998).The source and routes of contamination is difficult
to predict as the aquatic system receives bacterial
population from diverse sources.A comprehensive sur-
veillance is required to determine the presence and
distribution of antibiotic resistant
E.coli
in foods,
animals and agriculture in India.
References
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method.
American Journal of Clinical Pathology
45
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Edberg,S.C.,Patterson,J.E.& Smith,D.B.1994 Differentiation of
distribution system,source water and clinical coliforms by DNA
analysis.
Journal of Clinical Microbiology
32
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Bacteriological Analytical Manual
,
Food and Drug Admin-
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64
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Escherichia coli
O157:H7 by multiplex PCR.
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of Clinical Microbiology
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(STEC) in fresh
seafood and meat marketed in Mangalore,India by PCR.
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shiga toxigenic
Escherichia coli
by using multiplex PCR assays for
stx
1
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stx
2
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eaeA
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E.coli
in tropical seafood
623
Rapid detection of
Salmonella typhi
in foods by combination of immunomagnetic
separation and polymerase chain reaction
S.Kumar*,K.Balakrishna,G.P.Singh and H.V.Batra
Division of Microbiology,Defence R&D Establishment,Jhansi Road,Gwalior 474 002,India
*Author for correspondence:Tel.:91-751-2340245/354,Fax:91-751-2341148,E-mail:subodh_kumar@email.com
Keywords:
Immunomagnetic separation,IMS-PCR,meat,milk,polymerase chain reaction,
Salmonella
Summary
A combination of immunomagnetic separation and polymerase chain reaction (IMS-PCR) was used to detect
Salmonella typhi
in food and water samples.IMS was found to be an effective method for specific capture of
S.typhi
from artificially inoculated meat rinse samples.The bacteria could be detected within 6 h by IMS-PCR with a
sensitivity of 10
5
cells.However,when tested in milk samples,the method was less effective.In comparison to
conventional culture method,IMS-PCR is a rapid and specific method for detection of
S.typhi
and could be useful
in outbreak situations for tracing the source of infection.
Introduction
Salmonella typhi
is the primary aetiological agent of
typhoid fever which is responsible for significant mor-
bidity and mortality,particularly in the developing
countries.The incidence of typhoid fever has been
estimated to be between 17 and 33 million cases
annually with 600,000 associated deaths (Pang 1998;
Hoiseth 2000).
S.typhi
is an obligate human pathogen.
It causes infection by the faecal-oral route.Typhoid
fever is typically acquired by ingesting food or
water that has been contaminated by faeces of
typhoid-infected individuals.Many outbreaks,caused
either by consumption of contaminated food (Usera
et al.
1993;Cote
et al.
1995) or water (Nishina
et al.
1989;Mermin
et al.
1999) have been reported.
The standard procedure for isolation of
S.typhi
from
food,water or any other environmental sample usually
requires recovery by three steps:pre-enrichment,enrich-
ment and selective plating that take minimum of 3 days
and several additional days for confirmation of pre-
sumptive positive results.Polymerase chain reaction
(PCR) is a sensitive and rapid technique.But it can be
inhibited by several factors present in food and other
complex environmental samples (Lantz
et al.
1994).The
removal of these inhibitory substances like food com-
ponents,humic acid,bile salts and lipids etc.is critical
step in the preparation of samples of template DNA for
PCR-based detection of food pathogens.Immunomag-
netic separation (IMS) is a powerful technique that can
specifically capture the target bacteria from food sam-
ples for template DNA preparation with little or no
non-specific bacteria or interfering factors.The present
work aimed at the concentration of
S.typhi
bacteria by
IMS fromartificially inoculated food and water samples
and their subsequent detection by PCR.
Materials and methods
Preparation of antibodies to flagellin
Salmonella typhi
(strain SKST),obtained from the
Christian Medical College,Vellore,India was grown in
nutrient broth or LB broth (Difco) for 18–24 h at 37

C.
Flagellin was purified as described by Ibrahim
et al.
(1985).Antiserum to flagellin was raised in New
Zealand white rabbits (Kumar
et al.
2003);which gave
weak cross-reaction with
S.weltevreden,S.infantis
and
S.paratyphi
A.The antiserum was made monospecific
by adsorbing 1 ml antiserum with a mixture of cultures
obtained by growing each cross-reacting serotype on
two nutrient agar plates.Adsorption was carried out at
50

C for 1 h.The antiserum was separated by
centrifugation at 10,000
·
g
for 15 min and checked
for cross-reactivity again.Adsorption was repeated
against cross-reactive antigens.Immunoglobulins G
(IgG) were purified from the adsorbed antiserum by
gel exclusion chromatography using Bio-Rad matrix Bio
A1.5 (henceforth called polyclonal antibodies).
Coating of magnetic beads
Epoxy superparamagnetic beads (Dynal,Oslo,2.8
l
m
dia.) were coated with anti-flagellar polyclonal antibod-
ies (PoAb) as per manufacturer’s instructions.Briefly,
World Journal of Microbiology & Biotechnology (2005) 21:625–628

Springer 2005
DOI 10.1007/s11274-004-3553-x
5 mg beads were washed twice in 0.1 M phosphate
buffer (PB),pH 7.2,before resuspending in 100
l
lof
same buffer.Antiflagellar PoAb (5
l
l,20 mg ml
)
1
) was
mixed with the beads along with 95
l
l PB.This was
followed by the addition of 100
l
l of 3M ammonium
sulphate solution.The mixture was mixed in mixer
(Dynal,Oslo) overnight at roomtemperature.The beads
were washed 4 times with PBST (0.1 M phosphate-
buffered saline,pH7.2,0.5%Tween 20) and were stored
in 300
l
l of PB with 0.1% BSA (w/v) at 4

C.
Polymerase chain reaction and IMS-PCR
A pair of oligonucleotide primers targeting the
fliC
-d
gene was used to detect
S.typhi
.The primers (Forward
5
¢
TGGGCGACGATT TCT ATGCC 3
¢
and Reverse
5
¢
TTT GCGGAACCT GGT TCGCC3
¢
) are reported
to pick H=d positive salmonellae specifically
(Chaudhry
et al.
1997).PCR was carried out in 25
l
l
reaction containing 30 pmol of each primer,200
l
Mof
each dNTP,0.75 units of Taq polymerase,1.5 mM
MgCl
2
in 1
·
PCR buffer (MBI Fermentas).PCR was
taken through 30 cycles of 94

C for 1 min (denatur-
ation),57

C for 1 min (annealing),and 72

C for 1 min
(extension).The DNA was denatured for 4 min in the
beginning and finally extended for 5 min at 72

C.PCR
products were analysed on 1.0% (w/v) agarose gel.To
determine the detection sensitivity of PCR for pure
culture,DNA was prepared fromserial 10-fold dilutions
(ca.10
7
–10
2
c.f.u.ml
)
1
) of overnight growth.Each
dilution (1 ml) was centrifuged at 8000
·
g
for 10 min.
The cell pellet thus obtained was resuspended in 50
l
lof
distilled water (DW) and boiled for 10 min.This was
immediately placed on ice for 5 min.Particulate mate-
rial was removed by centrifugation at 12,000
·
g
for
5 min.The lysate was removed and 2.5
l
l was used as
the template in PCR assay immediately or following
storage at
)
20

C.Viable count was determined by plate
count.
S.typhi
bacteria were captured with immuno-
magnetic beads from the second set of dilutions.Coated
beads (10
l
l,10
7
beads) were added to each dilution
(1 ml).After mixing for 1 h in Dynal mixer,the beads
were washed twice with PBST followed by concentration
by magnet (MPL1,Dynal).The beads were suspended
in 50
l
l of DW and boiled for 10 min.The rest of the
procedure was same as described above.Supernatant
was separated and used as template DNA.In mixed
culture experiments,
E.coli
MTCC 732 or
Salmonella
paratyphi
A (clinical isolate) were added to the above
mentioned dilutions of
S.typhi
and the process repeated.
Artificial inoculation of food and water samples
Samples of milk,meat and vegetables were procured
from the local market.Meat and green leafy vegetable
rinses were prepared by suspending 150 g of each food
sample in 150 ml of buffered peptone water (BPW) and
rinsing thoroughly with the medium.Aliquots of 10 ml
food rinse were made and stored at
)
20

C for further
use.Water samples were collected from laboratory and
household supply.10 ml each of food or water sample
were inoculated with 100
l
lof
S.typhi
culture to achieve
a concentration of 10
7
–10
3
c.f.u.ml
)
1
.An uninoculated
control was prepared by seeding samples with 100
l
lof
BPW.After inoculation each sample was incubated for
10 min at roomtemperature.The inoculated sample was
added in 90 ml of BPW and mixed well.One millilitre
sample was taken and processed for DNA extraction by
boiling after centrifugation or IMS.The DNA was used
as template in PCR assay.
Results and discussion
Typhoid fever continues to be a major problem of the
developing countries.
S.typhi
,the causative agent is
transmitted by consumption of contaminated food or
water.In the event of an outbreak,it is essential to
locate the source of infection in order to take timely
control measures.Owing to the complex nature of food
and other environmental matrices,the detection of any
bacteria including
Salmonella
from these matrices,is a
difficult task.The procedure of
Salmonella
isolation and
identification generally takes 3–5 days.The sensitivity
and rapidity of PCR as a detection method is unques-
tionable.The only problem it suffers from is that high
quality of DNA is required.
In this study,PCR generated the expected product of
489 bp.The detection sensitivity of PCR assay was
found to be 1.0
·
10
3
c.f.u.When bacteria were cap-
tured by IMS for subsequent detection of extracted
DNA by PCR,there was a visible decrease in the
intensity of the band at this concentration;however,
there was no apparent change in the sensitivity
(Figure 1a).The low intensity of the band may be due
to the fact that recovery of bacteria with beads is never
100% and there is additional loss of bacteria during
washing steps.We observed by plate counts that at least
40% of the bacteria are captured by the beads and
another 6–8% are lost during each washing step
(unpublished data).It was further observed that the
presence of
E.coli
or
S.paratyphi
A does not make any
difference in detection sensitivity of IMS-PCR
(Figure 1b).We also found that two washings can be
used during IMS procedure.Three washing were
reported to give extremely low level of recoveries and
were found unsuitable for detection of
Listeria mono-
cytogenes
by IMS-PCR (Hudson
et al.
2001).
The results of IMS-PCR in spiked food samples are
shown in Table 1.The meat and milk samples tested in
this study had a background bacterial count of about
10
6
and 10
8
c.f.u.ml
)
1
respectively.The background
bacterial count in vegetable rinse was however much
lower,of the order of 10
2
c.f.u.ml
)
1
.BPWwas added to
the spiked samples to dilute the PCR inhibitory sub-
stances.PCR performed with DNA samples extracted
by direct boiling of spiked meat rinse samples after
centrifugation always gave unequivocal results.On most
626
S.Kumar
et al.
occasions,a smear was observed.The IMS method was
able to capture the
S.typhi
bacteria in sufficient purity
so as to enable their subsequent detection by PCR;
however there was a drop in detection sensitivity by two
logs when compared to pure culture.But the advantage
of IMS-PCR is that it took only about 5 h to complete
the assay and could be used directly on meat rinse
samples.IMS was partially successful with milk sam-
ples.Only three out of five samples were positive ca.
10
6
c.f.u.ml
)
1
.It is possible that the magnetic beads got
entrapped in the fat particles present in the milk.Fats
and other substances have been reported to interfere
with the process of DNA extraction from milk samples
(Ramesh
et al.
2002).None of the milk samples tested
positive by direct PCR.As calcium ions present in milk
may inhibit the PCR reaction,we tried different con-
centrations of MgCl
2
(1.5,2.0 and 2.5 mM) in PCR
reaction without any success.All the tested vegetable
rinse and water samples tested positive both by direct
PCRand IMS-PCRto same level of detection sensitivity
(1.2
·
10
3
c.f.u.).Therefore,there was no added advan-
tage of IMS over direct extraction of DNA in these two
samples.This is possibly due to the simple nature of the
matrix of water and vegetable rinse samples that may
not have much PCR-interfering material.
Superparamagnetic beads coated with specific anti-
bodies specifically capture the target bacteria from
complex food samples with the background of inhibi-
tory substances and competing bacteria.The utility of
immunomagnetic separation for subsequent culture or
PCR has been shown for capture of
Listeria
monocytogenes
,
E.coli
and other bacteria (Hudson
et al.
2001;Kerr
et al.
2001).In this study,IMS led to a drop
in sensitivity of order of 1–2 log from food samples.We
believe that this would still not affect the utility of IMS-
PCR as a rapid detection method in identifying the
source of infection in disease outbreak situation.How-
ever,the detection sensitivity of IMS-PCR,most likely
can be increased by short pre-enrichment of the cap-
tured bacteria.
Acknowledgement
Authors are thankful to Director,DRDE for providing
the necessary facilities and encouragement.
References
Chaudhry,R.,Laxmi,B.V.,Nisar,N.,Ray,K.& Kumar,D.1997
Standardisation of polymerase chain reaction for detection of
Salmonella typhi
in typhoid fever.
Journal of Clinical Pathology
50,
437–439.
Cote,T.R.,Convery,H.,Robinson,D.,Ries,A.,Barrett,T.,
Frank,L.,Furlong,W.,Horan,J.& Dwyer,D.1995 Typhoid
fever in the park:epidemiology of an outbreak at a cultural
interface.
Journal of Community Health
20,
451–458.
Hoiseth,S.K.2000 Vaccines,Bacterial.In
Encyclopedia of Microbiol-
ogy,
2nd edn.,Vol 4,ed.Lederberg,J.pp 767–778.New York:
Academic Press.ISBN 0-12-226800-8.
Hudson,J.A.,Lake,R.J.,Savill,M.G.,Scholes,P.&McCormick,R.E.
2001 Rapid detection of
Listeria monocytogenes
in ham samples
using immunomagnetic separation followed by polymerase chain
reaction.
Journal of Applied Microbiology
90
,614–621.
Figure 1.
PCRamplification of the
fliC
-d gene of
S.typhi
bacteria in pure culture (panel a) or mixed culture (panel b).DNA was extracted from
the cells ca.10
6
(A:lanes 1,4),10
5
(a:lanes 2,5) or 10
4
(a:lanes 3,6) by boiling after centrifugation (a:lanes 1–3,7) or IMS (a:lanes 4–6;b:lanes
1–8).
S.typhi
bacteria ca.10
8
or 10
5
mixed with (b:lanes 2 & 6,4 & 8) or without (b:lanes 1 & 5,3 & 7) equal number of
E.coli
bacteria were
processed during IMS by one (b:lanes 1–4) or two (b:lanes 5–8).washing steps.Positive control and 100 bp ladder are shown in lane 7 and 8 in
panel a.
Table 1
.Detection of
S.typhi
in various food and water samples by
IMS-PCR.
Samples Inoculation level c.f.u.ml
)
1
number tested
positive/total tested
2.5
·
10
7
2.5
·
10
6
2.5
·
10
5
2.5
·
10
4
2.5
·
10
3
Meat rinse 5/5 4/5 0/5 0/5 0/5
Milk 4/5 3/5 0/5 0/5 0/5
Vegetable
rinse
5/5 5/5 5/5 3/5 0/5
Water 5/5 5/5 5/5 4/5 0/5
IMS-PCR for
S.typhi
627
Ibrahim,G.F.,Fleet,G.H.,Lyons,M.J.& Walker,R.A.1985 Method
for isolation of highly purified
Salmonella flagellins.Journal of
Clinical Microbiology
22
,1040–1044.
Kerr,P.,Finlay,D.,Thomson-Carter,F.& Ball,H.J.2001 A
comparison of monoclonal antibody-based sandwich ELISA and
immunomagnetic bead selective enrichment for the detection of
Escherichia coli
O157 from bovine faeces.
Journal of Applied
Microbiology
91
,1–4.
Kumar,S.,Balakrishna,K.,Tuteja,U.& Batra,H.V.2003 Applica-
tion of monoclonal antibodies to flagellin of
Salmonella typhi
for
its detection in foods.
Indian Journal of Microbiology
43
,193–197.
Lantz,P.G.,Hahn-Hagerdal,B.& Radstrom,P.1994 Sample
preparation methods in PCR detection of Food pathogens.
Trends
in Food Science and Technology
5
,384–389.
Mermin,J.H.,Villar,R.,Carpenter,J.,Roberts,L.,Samaridden,A.,
Gasanova,L.,Lomakina,S.,Bopp,C.,Hutwagner,L.,Mead,
P.,Ross,B.& Mintz,E.D.1999 A massive epidemic of
multidrug-resistant typhoid fever in Tajikistan associated with
consumption of municipal water.
Journal of Infectious Diseases
179
,1416–1422.
Nishina,T.,Shiozawa,K.,Hayashi,M.,Akiyama,M.,Sahara,K.,
Miwa,N.,Nakatsugawa,S.,Murakami,M.&Nakamura,A.1989
Supply system in Fuji city.
Kansenshogaku Zasshi
63
,240–247.
Pang,T.1998 Genetic dynamics of
Salmonella typhi
– diversity in
clonality.
Trends in Microbiology
6
,339–342.
Ramesh,A.,Padmapriya,B.P.,Chandrashekar,A.&Varadaraj,M.C.
2002 Application of convenient DNA extraction method and
mutiplex PCR for the direct detection of
Staphylococcus aureus
and
Yersinia enterocolitica
in milk samples.
Molecular and Cellular
Probes
16
,307–314.
Usera,M.A.,Aladuena,A.,Echeita,A.,Amor,E.,Gomez-Garces,
J.L.,Ibanez,C.,Mendez,I.,Sanz,J.C.& Lopez-Brea,M.1993
Investigation of an outbreak of
Salmonella typhi
in a public school
in Madrid.
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9,
251–54.
628
S.Kumar
et al.
DNA amplification fingerprinting as a tool for checking genetic purity
of strains in the cyanobacterial inoculum
B.Jeberlin Prabina,K.Kumar* and S.Kannaiyan
Department of Agricultural Microbiology,Tamil Nadu Agricultural University,Coimbatore 641 003,Tamil Nadu,
India
*Author for correspondence:Tel.:+91-0422-2431222 ext.303,E-mail:azollakumar@rediffmail.com
Keywords
:Cyanobacteria,cluster analysis,genetic purity,RAPD–PCR profile,strain identification
Summary
The electrophoretic patterns for 17 different cyanobacterial cultures derived from 6 different decamer primers were
analysed to provide diagnostic fingerprints for each culture and their genetic distances based on RAPDmarkers.The
primer OPB 09 produced a maximum of 24 amplified products.The primers OPB 09,OPG 04 and OPAH 02
generated markers specific for
Nostoc
cultures.
Westiellopsis
was found to be distinct from other cyanobacterial
cultures in the RAPDprofile obtained with the primer OPAH02.The primer OPF 03 generated specific markers for
Tolypothrix tenuis
.
Fischerella
cultures could be identified with the primers OPB 09,OPAG 03 and OPF 05.The
study revealed that these RAPD markers could be further used to identify and establish the genetic purity of the
strains in the cyanobacterial inoculum.There was a similarity of 60–90% within
Westiellopsis
cultures.
Nostoc
cultures shared 50–80% similarity with
Westiellopsis
cultures.
Anabaena
cultures were similar to
Westiellopsis
cultures by 60–70%.The markers produced for each culture were also applied to phylogenetic analysis to infer
genetic relatedness in this group of prokaryotes.The dendrogram analysis clearly revealed that free-living
cyanobacterial cultures are closely related to each other and are distinct from the symbiotic forms.
Introduction
Cyanobacteria are unique among the prokaryotes due to
their capacity for oxygenic photosynthesis.An impor-
tant feature of many cyanobacteria is their ability to fix
atmospheric nitrogen both under free-living and symbi-
otic conditions.The species of cyanobacteria which are
known to fix atmospheric nitrogen are classified into
three groups
viz.,
heterocystous-aerobic forms,aerobic
unicellular forms and non-heterocystous filamentous
microaerophilic forms.Nitrogen-fixing cyanobacteria
can use sunlight as the sole energy source for the fixation
of carbon and nitrogen and therefore have potential as
biofertilizers (Kannaiyan 1985).The composite inocu-
lum consisting of cyanobacterial cultures
viz.,Nostoc
,
Anabaena
,
Calothrix,Tolypothrix
,
Plectonema,Apha-
notheca,Gleocapsa,Cylindrospermum,Oscillatoria,
Aulosira
and
Scytonema
has been used for inoculation
in rice (Kannaiyan 1993).
Mass production of high quality inoculum of the
desirable strains is an important stage when considering
the inoculation of nitrogen-fixing cyanobacteria as
biofertilizer for rice crops (Watanabe & Roger 1984).
The biofertilizer potential of cyanobacteria is limited,
mainly due to the non-availability of good quality
inoculants and the difficulty in re-establishing the
inoculated strains varied in the rice field (Roger &
Watanabe 1986).Rapid and reliable quality control
methods have not yet been developed for cyanobacterial
biofertilizers.Current cyanobacterial taxonomy is based
primarily on observed morphological characteristics,
which is often confusing and does not identify poten-
tially different strains.More importantly,the morphol-
ogy of cyanobacteria in laboratory cultures is often
considerably altered from the original morphology of
environmental isolates and the diversity of strains within
a culture may be reduced because of selective culturing
conditions (Doers & Parker 1998).Analyses of photo-
synthetic pigment content,isoenzyme variation or dif-
ferentiated cell culture may also be misleading because
of the variable expression of cyanobacterial gene prod-
ucts in culture (Rippka
et al
.1979;Kato
et al
.1991).
Taxonomic characters change so drastically that reliable
identification of species becomes difficult or even impos-
sible (Kumar
et al.
2000).A number of new valuable
molecular biological tools for taxonomic purpose have
been developed which could be effectively utilized for
checking the genetic purity of strains in the cyanobac-
terial inoculants.
Nelissen
et al.
(1996) developed a cyanobacterial-
specific oligonucleotide which could identify the PCR
products from 16S rRNA after separation by agarose
World Journal of Microbiology & Biotechnology (2005) 21:629–634
￿
Springer 2005
DOI 10.1007/s11274-004-3566-5
gel electrophoresis.DNA/DNA hybridization has been
used successfully to resolve taxonomic problems at the
generic and species level in cyanobacteria (Kelly &
Cowie 1972).Restriction Fragment Length Polymor-
phisms have been used extensively as an efficient DNA
fingerprinting method to identify symbiotic cyanobac-
teria (Nierzwicki-Bauer & Haselkorn 1986).
Polymerase Chain Reaction (PCR) technology had a
significant impact in almost all the areas of molecular
biology and the modification of this basic procedure has
allowed a number of assays for detecting variation at the
nucleotide level.Among them,Random Amplified
Polymorphic DNA (RAPD) developed by Williams
et al
.(1990) has been widely used in various fields of
molecular biology.The presence of large numbers
of decamer sequences in a large genome forms the basis
of this technique,which utilizes random primers so that
just by chance at several unpredictable locations,two
primers will anneal sufficiently close to one another on
opposite strands of the template to amplify the inter-
vening regions.The RAPD technique in conjunction
with PCR has been employed to identify many organ-
isms to the strain level (Welsh & McClelland 1990).This
technique is sensitive and specific because the entire
genome of an organism is used as the basis for
generating the DNA profile.Eskew
et al
.(1993) used
DNA fingerprinting to differentiate the isolates in the
Azolla

Anabaena
symbiosis.In this investigation,our
objective was to develop an easy and reliable method for
checking the genetic purity of strains in the cyanobac-
terial inoculum using RAPD analysis.We have demon-
strated that a PCR fingerprinting method could be used
as a genetic tool for identification of species and strains
analysis of genetic relatedness of cyanobacteria.The
method was found to be accurate in distinguishing and
classifying even closely related strains.
Materials and methods
Cyanobacterial cultures and culture conditions
The cyanobacterial cultures used in this study were
collectedfromdifferent biofertilizer productioncentres of
India
.
The cultures
Anabaena azollae
-SK-SL-TNAU1,
A.
azollae-
MPK-SK-AM-24,
A.azollae
-MPK-SK-AM-25,
A.azollae-
MPK-SK-AF-38,
Anabaena
-TR 52-ST 1,
An-
abaena
sp.,
Nostoc muscorum
–DOH,
Westiellopsis
-4A
2,
Westiellopsis
–C
100
-TR
5
-ST
3
-PA-SK,
Anabaena variabi-
lis
-SA
0,
Nostoc
sp.and
Westiellopsis
sp.were selected
from the Germplasm collections at the
Azolla
Labora-
tory,Department of Agricultural Microbiology
,
Tamil
Nadu Agricultural University (TNAU),Coimbatore,
while
Westiellopsis–
ARM48 was obtained from the
Department of Microbiology,PSG College of Arts and
Science,Coimbatore and
Aulosira pseudoramosa,To-
lypothrix tenuis,Westiellopsis prolifica
and
Fischerella
sp.
were collectedfromthe National Centre for Conservation
and Utilization of BGA (NCCUBGA),Indian Agricul-
tural Research Institute (IARI),New Delhi.All the
cyanobacterial cultures were grown in BG-11 mediumin
conical flasks.The flasks were incubatedunder controlled
conditions in culture room with a light intensity of
3000 lux (16 h/8 hdayandnight cycle) andatemperature
of 25 ± 1
￿
C.Thirty days old cultures were used for the
study.
Extraction of total genomic DNA for RAPD analysis
The total genomic DNA from the cyanobacterial cul-
tures was isolated by the 2%cetyl trimethyl ammonium
bromide (CTAB) procedure (Clark 1997) with little
modification.Approximately 2 g of blot-dried cyano-
bacterial culture was ground in an autoclaved and pre-
chilled mortar using liquid nitrogen.The macerated
samples along with 2 ml of CTAB buffer were trans-
ferred to eppendorf tubes and incubated at 65
￿
C for
30 min.Protein was removed by treating with equal
volumes of a 1:1 chloroform-isoamyl alcohol mixture.
DNA was precipitated with the addition of ice-cold
isopropanol.The precipitated DNA was hooked out
with a bent Pasteur pipette and dissolved in 500
l
lof
10 mM Tris–1 mM EDTA buffer.Then the DNA was
concentrated by precipitation with ethanol and sodium
acetate.The pellet was dissolved in 50
l
l of Tris-EDTA
buffer and stored at
)
20
￿
C for further use.
Oligonucleotide primers and PCR amplification
The primers were obtained from OPERON Techniques
Inc.,USA.The DNA amplification was performed in
a PTC-100 TM Programmable Thermocycler (MJ
Research Inc.,Water Town,Mass).The reaction mix-
ture consisted of 3
l
lof25ng
l
l
)
1
DNA,1
l
1of
10 mMdNTPs,1.2
l
l primer,2
l
l of 10 x Assay buffer,
0.2
l
l
Taq
polymerase (Bangalore Genei Inc.,),12.6
l
l
sterile distilled water.The incubation cycle was initial
denaturation (95
￿
C for 2 min),denaturation (94
￿
C for
1 min),annealing (37
￿
C for 1 min),extension (72
￿
C
for 2 min) and final extension (72
￿
C for 5 min).Thirty
five incubation cycles were followed for denaturation,
annealing and extension steps.After the reaction,15
l
l
of the PCRproduct was separated by 1.5%agarose gels,
stained with ethidium bromide,viewed and photo-
graphed using alpha imager TM 1200 documentation
and analysis system.
Dendrogram analysis
Each band visualized on a gel was considered a RAPD
marker and part of the total RAPD fingerprint gener-
ated for a particular genera.Bands bisected by similar
perpendicular lines drawn across the gel were consid-
ered homologous characteristics.The assumption that
these bands contained homologous primer recognition
sequences and identical intervening sequence was made.
Therefore,the presence or absence of a band at any
position on the gel was used to construct a binary
630
B.J.Prabina
et al.
matrix for cyanobacterial RAPD markers.Each lane of
the PCR product for different samples with different
primers was scored and cluster analysis was carried out
using NTSYS pc version 1.7 (Rohlf 1992).
Results and discussion
RAPD–PCR analysis of cyanobacterial cultures
A total of 12 primers were initially chosen to generate
RAPD patterns for 17 different cyanobacterial cultures.
The criteria for choosing these primers were their
availability and the generally accepted bias towards
oligonucleotides of high G+C content.All the 12
primers were tried and 6 primers (Table 1) were found
to produce informative and reproducible genetic mark-
ers for different cyanobacterial cultures.
Total genomic DNA extracted from the 17 cyano-
bacterial cultures were used as templates.The primer
OPB 09 produced totally 24 amplified DNA fragments
of size ranging from 9000 bp to 125 bp (Figure 1).
There was one monomorphic band and 23 polymorphic
bands.The percentage of polymorphism observed was
95.8% (Table 2).An amplified fragment of molecular
size 1636 bp was unique for
Fischerella
sp.The primer
OPG 04 produced a unique fingerprinting for all the
cyanobacterial cultures (Figure 2).The primer amplified
a DNA fragment of molecular size 8144 bp exclusively
in
Westiellopsis
-C
100
-TR
5
-ST
3
-PA-SK.
Nostoc musco-
rum
had an amplified fragment of size around 3054 bp.
A band of molecular size 5090 bp was found in
Anabaena
sp.and
Anabaena
TR 52 ST1.The primer
OPAH 02 generated distinct pattern for
Westiellopsis
cultures and it could be used to distinguish
Westiellopsis
from other cultures (Figure 3).The primer OPF 03
(Figure 4) produced a total of 12 amplified DNA
fragments of molecular size ranging from 7000 bp to
1000 bp.It produced a single band of molecular size
around 6800 bp for
Tolypothrix tenuis
.Considerable
variation in banding pattern was observed between and
among the different genera of cyanobacteria investi-
gated.The total number of amplified DNA fragments
produced by the primer OPAG 03 was 16 (Figure 5).
Among the
Westiellopsis
cultures,the fragment separa-
tion was different for the acid tolerant culture
Westiell-
opsis
-4A
2
.This primer differentiated
Fischerella
sp.from
the other cyanobacterial cultures by the presence of a
single fragment of size around 4500 bp.The primer OPF
05 produced a DNA fragment of molecular size around
3052 bp in most of the cultures (Figure 6).For
Fische-
rella
sp.it produced two bands of size 7000 bp and
1636 bp,whereas in
A.pseudoramosa,
it produced a
distinct pattern at 6000 bp which was not found in other
cultures.RAPD–PCR was used to generate unique and
identifying DNA profiles for members of the cyanobac-
terial genera
Anabaena
and
Microcystis
by Neilan
(1995)
.
Rasmussen & Svenning (1998) identified the
symbiotic and free-living cyanobacterial cultures using
RAPD technique.Also it was used for discriminating
genotypes of
Microcystis
(Nishihara
et al.
1997).The
results of the present study indicated the potential use of
RAPD markers as a rapid method to detect genetic
variation and the genetic relatedness of the cyanobac-
terial strains at the level of DNA.The primers OPB 09,
OPG 04 and OPAH 02 generated markers specific for
Nostoc
cultures.
Westiellopsis
was found to be distinct
from other cyanobacterial cultures in the RAPD profile
obtained with the primer OPAH02.The primer OPF 03
generated specific markers for
Tolypothrix tenuis
.The
primers OPB 09,OPAG 03 and OPF 05 could be used
for the identification of
Fischerella
cultures.
Table 1.
Primers used for RAPD–PCR analysis of cyanobacterial
cultures.
Primer Sequence %G +C
OPAH 02 CACTTCCGCT 60
OPAG 03 TGCGGGAGTG 70
OPB 09 TGGGGGACTC 70
OPG 04 AGCGTGTCTG 60
OPF 05 CCGAATTCCC 60
OPF 03 CCTGATCACC 60
Figure 1
.RAPDprofile of cyanobacterial cultures for primer OPB 09.
See Table 3 for key to the numbers.
Table 2.
Number of PCR products generated in RAPD analysis of cyanobacterial cultures with different primers.
Primers No.of monomorphic
bands
No.of polymorphic
bands
Total no.of
bands
Percentage of
polymorphism
OPAH 02 0 13 13 100
OPAG 03 0 16 16 100
OPB 09 1 23 24 95.8
OPG 04 0 18 18 100
OPF 05 0 13 13 100
OPF 03 0 12 12 100
Checking genetic purity of strains
631
It should be noted that RAPD profiles may be altered
by the presence of transposable elements and plasmids
in prokaryotes as well as by the contaminating bacteria
in a sample.The RAPD technique does not require
previous knowledge of an organism’s gene sequences
and requires only 1/1000 of the amount of DNA
required for RFLP-Southern hybridization experiments
which makes it more advantageous over traditional
methods.The RAPDmarkers developed may be used as
molecular markers for identification of the standard
strains used in the preparation of cyanobacterial inoc-
ulum which will serve as a valuable tool for checking
their genetic purity in order to ensure the supply of
quality cyanobacterial inoculants to the farmers at an
economic rate.
Phylogenetic analysis
Genetic relatedness among the cyanobacterial genera
was determined with banding pattern from the RAPD
reaction.The dendrogram analysis of the seventeen
cyanobacterial cultures revealed three major clusters
(Figure 7).In the first major cluster,all the
Westiell-
opsis
cultures are clustered together and show similar-
ity of 71%.The relatedness between different
Anabaena
sp.ranged between 74.0 and 87.4%.
Nostoc muscorum
and
Nostoc
sp.are related by 85.5%.Neilan,1995
determined the genetic diversity among strains of
Anabaena
and
Microcystis
sp.With banding patterns
from the multiplex RAPD reaction.The multiplexing
of primers CRA 22(CCGCAGCCAA) and CRA 23
(GCGATCCCCA) generated a total of 33 RAPD
markers and 29 RAPD markers when analysed on
Figure 2
.RAPDprofile of cyanobacterial cultures for primer OPG04.
See Table 3 for key to the numbers.
Figure 3
.RAPD profile of cyanobacterial cultures for primer OPAH
02.See Table 3 for key to the numbers.
Figure 4
.RAPDprofile of cyanobacterial cultures for primer OPF 03.
See Table 3 for key to the numbers.
Figure 5
.RAPD profile of cyanobacterial cultures for primer OPAG
03.See Table 3 for key to the numbers.
Figure 6
.RAPDprofile of cyanobacterial cultures for primer OPF 05.
See Table 3 for key to the numbers.
Figure 7
.Phylogenetic relationship between the cyanobacterial cul-
tures by RAPD analysis.
632
B.J.Prabina
et al.
agarose gels.The phenogram clearly delineated the
genera
Anabaena
and
Microcystis.
Giovannoni
et al
.
(1988) studied the phylogeny of fresh-water cyanobac-
teria obtained with 16S rRNA gene sequences.It has
been shown that as few as three primers used sepa-
rately provided enough polymorphic information to
identify species of the symbiotic genes of
Anabaena
and
to create a phylogenetic tree with a topology similar to
that derived with 22 primers (Neilan 1995).The result
from the present study also analysed the genetic
relatedness of different cyanobacterial genera with 6
primers which revealed that free-living cyanobacteria
are closely related to each other and are distinct from
symbiotic forms.
Acknowledgement
The authors are grateful to the Indian Council of
Agricultural Research,New Delhi for the financial
support to carry out this study under National Agricul-
tural Technology Project-Team of Excellence on Bio-
fertilizers for rice-based cropping systems.
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Table 3.
Relevant characteristics and specific RAPD marker for the cyanobacterial cultures.
Sl.No.Cyanobacterial cultures used Relevant characteristics Specific RAPD marker
1.
Westiellopsis
sp.The cells are dark green in colour and are larger
and compact compared to other cultures.The vegetative
cells are cylindrical and true branching is present.
1.2 kb&0.7 kb-OPAH02
4.00 kb-OPB09
2.
Westiellopsis
-C
100
TR
5
ST
3
PA-SK As 1 Saline-tolerant culture.8.14 kb-OPG04
4.
Westiellopsis
4A
2
As 1 Acid-tolerant culture.1.2 kb&0.7 kb-OPAH02
6.
Nostoc muscorum
The trichomes are non motile,wavy and are tightly
coiled.Vegetative cells are ovoid and are distinct.
Heterocysts intercalary or terminal.Constrictions at
the cross - walls are conspicuous.
3.05 kb-OPG04
7.
Nostoc
sp.As 6.–
8.
Anabaena variabilis
Trichomes are straight.Vegetative cells are spherical,
barrel shaped and separated by conspicuous constrictions
at the cross wall.Heterocysts are intercalary or terminal.
5.00 kb-OPB09
9.
A.azollae
-SK-SL-TNAU 1 As 7.5.00 kb-OPB09
10.
A.azollae
MPK-SK-AM-24 As 7.5.00 kb-OPB09
11.
A.azollae
MPK-SK-AM-25 As 7.5.00 kb-OPB09
12.
A.azollae
MPK-SK-AF-38 As 7.5.00 kb-OPB09
13.
Anabaena
sp.As 7.5.09 kb-OPGO4
14.
Anabaena-
TR52ST1 As 7.5.09 kb-OPGO4
15.
Tolypothrix tenuis
Basal to apical tapering degree is low.Cells shorter at
terminal end.Heterocysts are terminal.The cells are
short,cylindrical and filamentous.The cells are enclosed
in a sheath.
0.56 kb-OPAH02
6.8 kb-OPF03
16.
Aulosira pseudoramosa
The cells are cylindrical.The heterocysts are elongated
and are intercalary or terminal.Trichomes are straight
and filamentous.
6.00 kb-OPF05
17.
Fischerella
sp.The cells are spherical and thick walled.True branching
is present.Heterocysts are elongate,spherical and are
terminal or intercalary.
7.00 kb,1.63 kb-OPF05
1.63 kb-OPB09
Checking genetic purity of strains
633
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Phylogenetic relationships of non-axenic filamentous cyanobacte-
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42
,194–200.
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Anabaena
living freely or in symbiotic association with
Azolla
.
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5
,29–35.
Nishihara,H.,Miwa,H.,Watanabe,M.,Nagashima,M.,Yagi,O.&
Takamura,Y.1997 Random Amplified Polymorphic DNA
(RAPD) analysis for discriminating genotypes of
Microcystis
cyanobacteria.
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tandemly repeated repetitive sequences.
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R.Y.1979 Genetic assignments,strain histories and properties of
pure culture of cyanobacteria.
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111
,1–61.
Roger,P.A.& Watanabe,I.1986 Technologies for utilizing biological
nitrogen fixation in Wetland rice.Potentialities,current usage and
limiting factors.In
Nitrogen Economy in Flooded Rice Soils
.Dutta,
S.K.& Patrick,N.H Jr.eds.pp.37–77.Dordrecht:Martinus
Nijhoff Publishers,The Netherlands.ISBN 90-2473361-8.
Rohlf,F.G.1992 NTSYS-pc Numerical taxonomic and multivariate
analysis system version 1.7 Owner manual.
Watanabe,I.& Roger,P.A.1984 Nitrogen fixation in wetland rice
field.In
Current Developments in Biological Nitrogen Fixation
.
Subba Rao,N.S.ed.pp.237–236 New.Delhi:Oxford and IBH
Publishing Co,.India ISBN 0-713112877-1.
Welsh,J.& McClelland,M.1990 Finger printing of genomes using
PCR with arbitrary primers.
Nucleic Acids Research
18
,7213–
7218.
Williams,J.G.K.,Kubleik,A.R.,Livak,K.J.,Raflaski,J.A.& Tingey,
S.V.1990 DNA polymorphisms amplified by arbitrary primers are
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,6531–6534.
634
B.J.Prabina
et al.
Utilization of a polyphasic approach in the taxonomic reassessment of antibiotic-
and enzyme-producing
Bacillus
spp.isolated from the Philippines
Marie Antonette Ruth V.Guerra-Cantera
1
and Asuncion K.Raymundo
2,
*
1
National Institute of Molecular Biology and Biotechnology (BIOTECH),University of the Philippines Los Ban
˜
os,
College,Laguna 4031,Philippines
2
Institute of Biological Sciences,UP Los Ban
˜
os,College,Laguna 4031,Philippines
*
Author for correspondence:Tel.:+6349-536-2893,Fax:+6349-536-2517,Email:akr@laguna.net.
Keywords:Bacillus
,chemotaxonomy,molecular fingerprint,phenotype,taxonomy
Summary
The taxonomy of 58 locally isolated antibiotic- and enzyme-producing
Bacillus
isolates deposited at the Philippine
National Collection of Microorganisms (PNCM)-BIOTECH was reassessed in this study using a polyphasic
approach since they had been only partially identified prior to deposition in the culture collection.The isolates had
41.1–69% G + C,and possessed the characteristic diaminopimelic acid (DAP) and fatty acid methyl ester
(FAMEs) properties of
Bacillus
species.Molecular analysis using specific PCR primers differentiated the isolates
into two major groups,the
Bacillus cereus
group and the
Bacillus subtilis
group.To further differentiate these
isolates,they were subjected to 39 phenotypic tests.Using the dichotomous key constructed for
Bacillus,
45 isolates
maintained their original identities,five were named at the species level,and 12 were re-identified and renamed.
These results showed that the classical phenotypic tests allowed the reclassification of the isolates,while modern
techniques of chemotaxonomy and the molecular approach led only to genus and cluster classification and
confirmation.
Introduction
The genus
Bacillus
contains a systematically diverse
assemblage of Gram-positive,aerobic or facultatively
anaerobic spore-forming organisms (Claus & Berkeley
1986).Members of this genus exhibit a wide range of
nutritional requirements,growth conditions,DNA base
compositions (Slepecky & Hemphill 1991),and major
amino acid compositions of the cell wall.In addition to
phenotypic heterogeneity,they also appear to be phy-
logenetically diverse (Ash
et al.
1991),with five phylo-
genetically distinct clusters emerging from a
comparative analysis of the small subunit rRNA.For
instance,the
Bacillus cereus
group includes the following
species:
Bacillus anthracis
,
Bacillus mycoides,Bacillus
thuringiensis
and
Bacillus cereus
(Priest
et al.
1988).In
contrast,five genotypic groups belonging to the
Bacillus
subtilis
cluster were separated on the basis of Southern
blot analyses done by Shida (2001),namely,
Bacillus
amyloliquefaciens
,
Bacillus mojavensis
,
Bacillus licheni-
formis
,
Bacillus subtilis
subsp.
subtilis
,
Bacillus subtilis
spizizenii
,
Bacillus vallis mortis,Bacillus pumilus
and
Bacillus atrophaeus
.To date,the genus
Bacillus
has been
subjected to numerous taxonomic reclassifications
resulting in the proposal of new genera and species
(Niimura
et al.
1990;Wisotzkey
et al.
1992;Ash
et al.
1993;Nakamura 1993;Takagi
et al.
1993;Shida
et al.
1994,1995,1996,1997;Heyndrickx
et al.
1996,1998;
Spring
et al.
1996;Waino
et al.
1999;Fortina
et al.
2001;Nazina
et al.
2001;Schlesner
et al.
2001),using
polyphasic identification.
The locally isolated antibiotic- and enzyme-producing
Bacillus
isolates deposited at the Philippine National
Collection of Microorganisms (PNCM),National Insti-
tute of Molecular Biology and Biotechnology (BIO-
TECH) had only been partially identified prior to
deposition in the culture collection.With the current
status of
Bacillus
taxonomy brought about by these
taxonomic rearrangements and a continuous search for
novel species,the taxonomy of these industrially
important
Bacillus
isolates needed to be reassessed to
give them a more valid description and nomenclature.
Due to the high cost and difficulty in obtaining complete
rRNA sequences from an adequate number of isolates
to infer a reliable outline of phylogenetic relationships,a
rapid and less costly technique such as molecular
fingerprinting using specific primers must be used in
classification and identification.This requirement is an
especially important consideration in the developing
countries,where taxonomic work must be done under
tight budgetary constraints,without the state-of-the art
equipment available in Western laboratories.This study
World Journal of Microbiology & Biotechnology (2005) 21:635–644

Springer 2005
DOI 10.1007/s11274-004-3567-4
established the utility of the different approaches to
Bacillus
taxonomy and ascertained the appropriate
taxonomic position of these local
Bacillus
isolates using
a composite analysis of phenotypic,genotypic and
chemotaxonomic characteristics.
Materials and methods
Bacterial strains and culture conditions
Fifty-eight local
Bacillus
isolates and seven reference
strains of
Bacillus
were used (Table 1).The cultures
were grown in Nutrient Agar (NA),Luria–Bertani (LB)
Agar or Tryptone Glucose Yeast Extract Agar (TGYA)
at 30

C.Active and glycerol stocks were maintained at
4 and
)
70

C,respectively.
Chemotaxonomic characterization
Determination of cellular fatty acid composition
Approximately 40 mg of bacterial cells from 24-h old
cultures grown in trypticase soy agar (TSA) at 28–30

C
were harvested by centrifugation and placed in a sterile
screw-capped tube.Fatty-acid methyl esters (FAMEs)
were prepared as described by Sasser (2001).The
resulting extracts were sprayed with nitrogen gas to
avoid oxidation and sent to the Japan Collection of
Microorganisms (JCM) in Saitama,Japan for gas
chromatography (GC) analysis.
Determination of the diaminopimelic acid (DAP)
isomer composition of the cell wall
DAP isomers were separated by TLC as described by
Staneck & Roberts (1974).Standard DL-DAP (Sigma
Chemical Co.) was used to confirm the respective
R
f
values.
Genotypic characterization
DNA preparation
The genomic DNA of the isolates was extracted using
the procedure as described by Ausubel
et al.
(1995).
Determination of DNA base composition
The DNA G+C content was determined by HPLC
(Mesbah
et al.
1989) using a Cosmosil 5C18
(4.6 mm
·
150 mm) column.HPLC conditions included
a flow rate of 1.0 ml/min at 40

C,with detection
wavelength of 260 or 270 nm.Peak areas were cali-
brated using the DNA GC Kit (Seikagaku-Kogyo).
16S rRNA amplification using species-specific primers
16S rRNA was PCR-amplified using either forward
primer Bsub-F (5
¢
-CGG ATG GTT GTT TGA ACC
GCA TGG TTG A-3
¢
) or Bcer-F (5
¢
-GTT AGG GAA
GAA CAA GTG CTA GTT G-3
¢
) (Shida 2001) and
reverse primer 1377R (5
¢
-GGC ATG CTG ATC CGC
GAT TAC TAG C-3
¢
) (Shida
et al.
1996).The PCR
reaction mixture contained 10 ng of total DNA,
50 pmol each of the primers,1.2 U of
Taq
DNA
polymerase (Roche),1