IJBPAS, April, 2016, 5(4): 771-789
ISSN: 2277–4998
CHARACTERIZATION AND GENOTYPING OF RESISTANT ENTERIC PATHOGEN STRAINS ISOLATED FROM FEED MILLS IN EGYPT *ZAKIA AM AHMED**; AHMED REZK** SHERIF MAROUF AND AHMED M ERFAN*** *Microbiology Department, Faculty of Vet Medicine, Cairo University **
Veterinary Hygiene and Management Department, Faculty of Veterinary Medicine, Cairo University.
***Reference Laboratory for Veterinary Quality Control on Poultry Production, Animal Health Research Institute, Dokki- Giza *Corresponding Author: Sherif Maarouf: E Mail address:
[email protected]: Dept. of bacteriology, immunology and mycology, Faculty of veterinary Medicine, Cairo University, Giza, Egypt; P.O:12211 Giza, Egypt ABSTRACT Samples were collected from feed ingredients and finished products and tested for the presence of Salmonella, E.coli and clostridium spp. Positive samples for all spp. were subjected to serotyping, antimicrobial susceptibility and gentyping of isolated resistant enteric pathogens strains. S. typhi was resistant to tested antibiotics (inhibition degrees, ID 6–16). S entiritidis was only resistant to streptomycin (ID, 12). S. typhimurium was resistant to all antibiotics (ID,6-16) except colistin sulphate. E.coli O26, H9 was resistant to all antibiotics, while un-typed strains were resistant to all antibiotics except for colistin sulphate (ID, 10). Detected aadA2 gene in all E-coli isolates indicating high virulence of these isolates. The TetA(A) and blaTEM genes for the salmonella isolates as well the virulence genes (invA, avrA, sopB and stn) were detected. The 100% positive results of the avrA, sopB and stn genes have confirmed the virulence profile of the tested salmonella isolates. The tetA(A) gene in Salmonella and aadA2 gene in E. coli isolates were matching the resistance state obtained by the tetracycline and streptomycin sensitivity tests
771 IJBPAS, April, 2016, 5(4)
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respectively. The blaTEM gene positive PCR was compatible with that of the antibiogram confirming the resistance of the isolated Salmonella to ampicillin, penicillin and amoxicillin. Alpha toxin gene of Clostridium perfringens was detected by PCR confirming the isolation of typeA Clostridium perfringens. Characterization and genotyping of resistant enteric pathogens and recognition of virulent and toxin genes are more and rapid diagnostic methods for proposed control and preventive protocol against zoonotic diseases of poultry feed origin. Keywords: feed ingredients, enteric pathogen, toxin, genotyping, antibiotic resistant. Inhibition degree INTRODUCTION Animal feed ingredients are frequently
type C strains produce alpha toxin together
contaminated with Salmonella species either
with beta toxin (6).Some strains of C.
from the source or from the processing plant.
perfringens type A produce an enterotoxin at
Steaming during the pelleting process is
the moment of sporulation, causing disease in
known
humans (7,8). Alpha toxin is a phospholipase
to
decrease
the
number
of
microorganisms in chicken feed (1). A
C
sphingomyelinase
potential and more deadly hazard has been
phospholipids
associated with the consumption of microbial
disorganization (9,10). C. perfringens type A,
toxins of bacterial and fungal origin in feed
producing the alpha toxin, and to a lesser
(2).The antimicrobial agents are of great
extent type C, producing both alpha toxin
value for devising curative measures against
and
bacterial infections. The indiscriminate use
perfringens type A produce an enterotoxin at
of antibiotics has led to the emergence of
the
antimicrobial resistance in various isolates of
responsible
bacteria (3). Multi-resistance was considered
humans.(11).Feed
the biggest threat for public health and
during
disease therapy (4). C. perfringens strains are
Contaminated
classified into five toxinotypes (A, B, C, D
zoonozes,
and E), based on the production of four major
surveillance programs for microbiolgical
toxins (a, b, o and i) (5, 6). C. perfringens
feed hazards. Microbiological quality control
typeA strains produce the chromosomal-
programs are increasingly applied throughout
encoded alpha-toxin, while C. perfringens
food chain production in order to minimize
beta
and
toxin.
moment
hydrolyzes
promotes
Some
of for
that
membrane
strains
sporulation foodborne may be
processing,storage feed
of
and disease
C.
are in
contaminated or
frequently
transport. causes
it is necessary to establish
772 IJBPAS, April, 2016, 5(4)
Sherif Maarouf et al
Research Article sulfamethoxazole˃
the risk of infection for the consumer. Even
streptomycin˃
with
gentamicin ˃ kanamycin˃
improved
methods
for
detecting
ampicillin˃
pathogens in foods, microbiologists so
amoxicillin-clavulanate˃ ceftiofur . The E.
mandated often face a “needle-in-a-haystack”
coli strain types can be divided into several
challenge (12,13, 14). Lynne et al. (15)
pathogroups,
characterized the genetic basis of Multi-
ampicillin,amoxicillin/clavulanic
Antibiotic Resistance (MAR) in Salmonella
acid,piperacillin/sulbactam,
serovar Newport isolates collected from food
tazobactam, cefuroxime . The strain carries
animals in the United States during 1997-
plasmid-borne blaCTX-M-15 and a blaTEM-
2003. They found an increase of salmonella
1 genes (18). The antibiotic susceptibility
from 4.6-10.3% and multidrug resistance to
indicated
Ampicilin, Cephalothin, Cefoxitin, Ceftifour,
frequencies for Salmonella and E. coli
Amoxicillin-clavulanic
isolates.
acid,
Cetriaxone,
resistant
alarming
Antibiotic
to
piperacillin/
multi-resistance
use
Streptomycin, Sulfisoxazole, Trimethoprim-
eliminates
Sulfamethoxazole,
increases the proportion of resistant
Chloramphenicol
and
Tetracycline. -Nthenge et al. (16) reported
nonresistant
preferentially bacteria
and
bacteria that remains (19).
that E. coli was resistant to ampicillin,
The present work was designed to
nalidixic acid and Kanamycin. Salmonella
identify and characterize and genotyping the
strains also were resistant to ampicillin,
resistant enteric pathogen of commercially
kanamycin and nalidixic acid. Most of the
available poultry and fish feed ingredients
antibiotic resistance has emerged as a result
and finished feeds sold in Egypt which
of mutation or through transfer of genetic
potentiate human health risk .
material between microorganisms. (17). The
MATERIALS AND METHODS
genetic means by which Salmonella spp has
Traditional and standardized analysis of
evolved
animal feed ingredient for the presence of
explained
to
resist
antimicrobials
either
in
bacteria relies on culture enrichment step,
chromosomally encoded genetic elements or
selective and differential plating, followed by
by
mobile
subsequent identification by morphological,
resistance genes by plasmids, integrons and
biochemical and/or immunological tests.
transposons. The greatest resistance in
These methods have low limits of detection
isolates from poultry was to tetracycline ˃
and can be used in complex food sample
acquisition
by
which
of
mutations
exogenous
773 IJBPAS, April, 2016, 5(4)
Sherif Maarouf et al
Research Article
matrices, but they are labour-intensive and
technique in accordance with the Clinical and
typically require days from initiation to
Laboratory Standards Institute (23). The
readout (13).
panel of antibiotic disks (Becton, Dickinson
1-Sample
collection: Samples
of feed
and Company) used in the panel screens
ingredients and finished feed were obtained
belonged to certain drug classes ranked by -
from different feed mills (Table 1) according
FAO, -OIE and -WHO (24). The following
to the method described by (20). The samples
antimicrobials were chosen given their
were transported in a cooler to the laboratory
common use in treating and preventing
within 1 hr.
salmonella and E. coli infection in poultry
2. Bacteriological isolation:
and human. The antimicrobial agents (13
The samples were analyzed within 2-6 hours
agents) tested and their corresponding
from collection. The different media such as
concentrations were recorded in table (2).
nutrient agar (NA), nutrient broth (NB), SS
Antibiotic discs were placed onto Mueller-
agar
(Salmonella-Shigella
(Brilliant
Green
methylene
blue)
Agar), and
Agar),
BGA
Hinton Agar (Difco), the diameters of the
EMB
(eosin
inhibition zones were measured. Resistant
McConkey
were
prepared separately.
isolates
are
defined
according
to
the
interpretation criteria to E. coli standard
Serotyping was performed according to
(ATCC No. 25922) established by CLSI.
the procedure described in Identification and
Antibacterial
Serotyping of Salmonella (21) in FDA’s
sensitivity test (25) as illustrated in table 2.
microbiological laboratories.
4- PCR virulotyping & antimicrobial
3--Antimicrobial
susceptibility
tests
according to Osman et al., (22): Each Salmonella and E.coli isolate
agents
used
in
antibiotic
resistance genes: DNA extraction:
have
DNA extracted from samples was performed
been isolated from all samples was further
using the QIAamp DNA Mini kit (Qiagen,
characterized
and
Germany, GmbH) with modifications from
The
the manufacturer’s recommendations. 200 µl
Salmonella
of the sample was added to 20 µl of
and E. coli isolates from different feed
proteinase K and 200 µl of lysis buffer and
ingredients
(unpublished data ) was
incubated at 56oC for 10 min. After
determined by standard agar disc diffusion
incubation, 200 µl of 100% ethanol was
antimicrobial
by
serotyping
susceptibility
antimicrobial susceptibility of
testing.
774 IJBPAS, April, 2016, 5(4)
Sherif Maarouf et al
Research Article
added to the lysate. The sample was then
and analyze the bands through computer
washed and centrifuged according to the
software.
manufacturer’s instructions. DNA was eluted
RESULTS AND DISCUSSION
with 100 µl of elution buffer supplied in the
Antimicrobial susceptibility tests:
kit.
Data
Oligonucleotide Primers:
susceptibility testing reveal that , Salmonella
Primers
were
(2)
the
antimicrobial
Typhi was resistant to all tested antibiotics
(Germany) and are listed in Tables (3) and
with different inhibition degrees ranged from
(4).
6 - 16, while Salmonella Entiritidis was only
PCR amplification:
resistant to streptomycin (inhibition degree
Primers were utilized in a 25- µl reaction
was 12). Salmonella Typhimurium and the
containing 12.5 µl of EmeraldAmp Max PCR
untypeable strain were resistant to all
Master Mix (Takara, Japan), 1 µl of 20 pmol
antibiotics with inhibition degrees ranged
conc. of each primer, 4.5 µl of water, and 6
from 6 - 16, except colistin sulphate (table
µl of template. The reactions were performed
2). It was found that 58.1% of the salmonella
in an applied biosystem 2720 thermal cycler.
spp. was resistant to one or more antibiotics
For multiplex PCR of different Clostridium
in Greece ( 26) or 9.2 to 11.1% in Denmark
perfringens toxins, Primers were utilized in a
by Seyfarth et al., (27) and 100% in Spain
50- µl reaction
µl of
by -Carraminana et al. (28). S. Enteritidis
EmeraldAmp Max PCR Master Mix (Takara,
showed low resistance characteristics. Low
Japan), 1 µl of 20 pmol conc. of each primer,
percentages of multiple resistance in
9 µl of water, and 8 µl of template DNA.
Enteritidis strains have been pointed out by
Analysis of the PCR Products:
other authors (29, 30,31,32,33). Salmonella
The PCR products were electrophoresed in
Typhimurium showed resistant to ampicillin,
1.5% agarose gel (Lonza) in 1x TBE buffer
tetracycline and gentamycin (34), which was
using 5V/cm gradients. 15 µl of the PCR
in contrast to the findings of Tjaniadi et
products were loaded in each gel lane. A 100
al,(4), who recorded that salmonella species
bp DNA Ladder (Fermentas) was used to
were
determine
norfloxain, while Salmonella typhi strains
documentation
from
table
Metabion
the
supplied
in
containing 25
fragment system
sizes.
(Alpha
A
gel
Innotech,
Biometra, Germany) was used to photograph
were
resistant
resistant
tetracycline.
to
ciprofloxacin
S.
and
to
streptomycin
and
The
resistance
to 775
IJBPAS, April, 2016, 5(4)
Sherif Maarouf et al
Research Article
oxytetracycline was observed in 63.3% of the
Escherichia coli strains. The effects of
isolates in USA, 46% in Senegal (35) and
supplementation with antimicrobial agents
36%
approved
in
Portugal
(36).
Resistance
to
bambermycin,
penicillin,
streptomycin was also clear and is in
salinomycin and bacitracin or a combination
conformity with other findings (37).The
of salinomycin more bacitracin. All isolates
isolate E.coli O26, H9 was resistant to all
showed some degree of resistance to multiple
antibiotics, while the isolates number 3 and 4
antibiotics and resistance to tetracycline
were resistant also to all antibiotics except
(68.5%),
for colistin sulphate, where the inhibition
(51.3%),
zone was 10. However, isolate number 2 was
sulfonamides (42%). These data demonstrate
susceptible to amoxicillin, ampicillin, colistin
that the multidrug resistance of E.coli can be
and quinolones (table 2). It was reported by
found in broilers, regardless of antimicrobial
many authors that isolate originating from
growth promoters used. (18).
different animal species, feed or environment
PCR
showed different resistance patterns (38,39).
resistance genes:
amoxicillin
(61.4%),
spectinomycin(47.2%),
virulotyping
and
ceftiofur and
antimicrobial
The antibiotic susceptibility results of E. coli
Positive amplification of the 720 bp of
isolates were in agreement with those
the phoA alkaline phosphatase housekeeping
recorded by Salehi and Bonab (40) who
gene which is usually used to detect E. coli
reported that E. coli isolates showed high
was reported confirming the bacteriological
percentage of resistance to the antibiotics.
results of isolating E. coli from the feed
Yadav et al. (41) studied the antibiogram of
samples (unpublished data). The E. coli
isolates of Escherichia coli and mentioned
isolates virulence was checked by testing
that 26.67% of the isolates were resistant to
them for iss, iutA and eaeA genes that were
norfloxacin. It was also reported that all E
detected in each of the tested isolates (table
coli isolates were susceptible for colistin
3, photo 1). There are many virulence-
sulphate and this result was agreed with
associated genes that act individually or in
Habrun et al. (42) who found a high rate of
combination in many virulence mechanisms
susceptibility (94%) of the E. coli for
as adhesion, ferric transport system and
colistin.
One study on chickens fattening
toxin-production of the pathogenic E. coli
evaluated the incidence and distribution of
have been reported (43, 44, 45). eaeA gene
antibiotic resistance in 197 commensal
encodes intimin which is an outer membrane 776
IJBPAS, April, 2016, 5(4)
Sherif Maarouf et al
Research Article
protein that mediates the attachment to the
reduced susceptibility to colistin. Resistance
intestinal epithelium (46).
rate of Salmonella to ciprofloxacin was 35%
The eaeA gene was detected in all
in USA (51), 10.2 to 16.8% in Germany (52)
isolates indicating the high virulence of these
and 9.6% in Austria (53). High level of
E. coli strains as this gene aids the
ampicillin resistance (90 to 100%) was
attachment of the bacteria to the cytoplasm
observed in almost all salmonella isolates,
membrane of the epithelial cells and leads to
which is in agreement with the findings of -
the disappearance of the brush border
Suresh et al., (54). They also observed a
microvilli.
higher proportion of ampicillin-resistant
Iss (increased serum survival) gene that was
Salmonella from eggs.
detected also in all isolates is considered as a
The salmonella inv A gene was present
promising virulence gene of the extra
in all samples as it is highly conserved
intestinal
coli
among all Salmonella serotypes (55). The
(ExPEC) such as uropathogenic E. coli
four isolated Salmonella strains showed
(UPEC), neonatal meningitis-associated E.
positive amplification the 284 bp of the inv A
coli (NMEC), and avian pathogenic E. coli
gene which is considered as an important
(APEC) (47).An outer-membrane protein
factor for the Salmonella invasion of the
(IutA) was also identified in all isolates. The
epithelial cells (56).
IutA protein is involved in the binding and
The 100% positive results of the avrA, sopB
internalisation of the bacteriocin cloacin
and stn genes (table 3, photo 2) have
DF13 and the bacteriophage 74 (48). β-
confirmed the virulence profile of the tested
lactamases
isolates. As avrA gene is an effector protein
pathogenic
of
Escherichia
Enterobacteriaceae
were
caused by plasmid-mediated β-lactamases,
of
such as TEM-1, TEM-2 and SHV-1. These β-
translocated into host cells by a type 3
lactamases are resistant to inactivation by β-
secretion system and share in the virulence of
lactamase
acid,
Salmonella spp. by rearrangement of the host
sulbactam, tazobactam) (49). Salmonella
cellular cytoskeleton through cell apoptosis
Typhimurium resistance to colistin was
of macrophages, and by the inhibition of IL-8
described by -Sun et al., (50), who assessed
and TNF-α. (57, 58, 59). The positive results
spontaneous mutations in PmrA and PmrB
of the sopB gene is a strong indicator of the
genes in S. Typhimurium LT2 that showed
Salmonella virulence because it activates the
inhibitors
(clavulanic
the
TTSS
complex
which
usually
777 IJBPAS, April, 2016, 5(4)
Sherif Maarouf et al
Research Article
host cell’s small GTPase proteins which are
obtained by the tetracycline and streptomycin
involved in the regulation of the formation of
sensitivity tests respectively. However, one
F-actin filaments, membrane ruffling and
S. Enteritidis isolate was sensitive to most of
bacterial internalization by macropinocytosis
the antibiotics, different antibiotic resistance
(60,61). Also, the positive PCR result of
genes was detected for it by PCR especially
Salmonella Stn gene proved
the blaTEM which may be due to poor
the high risk expected from this strains (62)
expression of a functional TEM-1 enzyme
as this gene encodes for the heat labile
created by a mutation in the promoter region
exotoxin which is one of the causative agents
of the gene, or due to the production of an
of diarrhea (63).he result of the blaTEM
inactive mutant TEM enzyme.
gene PCR was compatible with that of the
The PCR result for Clostridium
antibiogram confirming the resistance of the
perfringens toxins showed amplification of
isolated Salmonella to ampicillin, penicillin
the 402 bp of the alpha toxin only (table 4
and amoxicillin.
,Fig, 3), which indicates that the isolated
Although the negative qepA gene PCR
Clostridium
results for all Salmonella and E. coli isolates
Clostridium perfringens which has severe
and also the negative results of the aac(6′)-Ib-
toxigenic power and can cause myonecrosis,
cr gene of the Salmonella isolates, this don't
hemolysis,
conflict with the resistance profile reported in
permeability, and platelet aggregation (66,
the different quinolones sensitivity. As,
67). Alpha toxin is a phospholipase C
Quinolone
sphingomyelinase
resistance
in
the
was
an
related
increase
to
in
that
and
A
vascular
hydrolyzes
Enterobacteriaceae is mostly mediated by
phospholipids
point mutations in the quinolone resistance-
disorganization (9,10). Hydrolysis of lecithin
determining regions (QRDR) of the target
results in the formation of diacylglycerol,
genes (gyrA and gyrB, which encode DNA
resulting in activation of protein kinase C,
gyrase, and parC and parE, which encode
and
topoisomerase IV) (64, 65).
arachidonic acid cascade. This induces the
subsequent
promotes
type
stimulation
membrane
of
the
The current positive PCR results for the
synthesis of inflammatory mediators. These
tetA(A) gene in Salmonella isolates and
mediators cause blood vessel contraction,
aadA2 gene in E. coli isolates were
platelet
completely matching the resistance state
dysfunction, leading to acute death. (11)
aggregation
and
myocardial
778 IJBPAS, April, 2016, 5(4)
Sherif Maarouf et al
Research Article Table (1): No. of examined samples of feed ingredients and finished ones Samples
No. of sample Feed ingredients
Soya Yellow corn 44% DDGs Fish meal 65% Corn gluten meal 60% Bran mash Rice polish Poultry offal Bone meal Water
10 10 5 10 5 5 10 10 5 4 Finished feed mills
Breeder production I (mash) Breeder production I (pellet) Broiler starter (mash) Broiler finisher (pellet) Tilapia floating feed 25% Tilapia sinking feed 25%
5 5 10 10 5 5
Table (2) Antibacterial agents used in antibiotic sensitivity test (25) Antibiotic Amoxicillin (AX) Nalidixic acid (NA) Flumoquine (UB) Tetracycline (T) Gentamycin (CN) Colistin sulphate (CT) Streptomycin (S) Penicillin Enrofloxacin(ENR) Norofloxacin (NX) Danofloxacin (DO) Ciprofloxacin (CF) Ampicillin (Amp)
Concentration 25 mg 30 µG 30 µG 30 µG 10 µG 10 µG 10 µG 10 µG 5 µG 10 µG 30 mg 5 µG 10 µG
Table (3): Sequences and cycling conditions of the different PCR primers used for amplification of different E. coli and Salmonella genes. Gene
Primers sequences
eaeA
GACCCGGCACAAGCATAAGC CCACCTGCAGCAACAAGAGG CGTGTTGCTGGAGTTCTTC CTGCAGGTACTGCGTCATG CCCGCTTTCTCGTAGCA TTAGGCATCACTGCGTCTTC ATCAGCAATAAACCAGC CCCCGAAGAACGTTTTC ATGTTATTTTCTGCCGCTCTG CTATTGTGAGCAATATACCC GGCTGGACATGGGAACTGG CGTCGGGAACGGGTAGAATCG GGTTCACTCGAACGACGTCA CTGTCCGACAAGTTGCATGA TGTTGGTTACTGTGGCCGTA GATCTCGCCTTTCACAAAGC CCT GTA TTG TTG AGC GTC TGG AGA AGA GCT TCG TTG AAT GTC C TCA GAA GRC GTC TAA CCA CTC TAC CGT CCT CAT GCA CAC TC TTGTGTCGCTATCACTGGCAACC ATTCGTAACCCGCTCTCGTCC CGATTCTGGAAATGGCAAAAG CGTGATCAGCGGTGACTATGAC GTGAAATTATCGCCACGTTCGGGCAA TCATCGCACCGTCAAAGGAACC
qepA aac(6′)-Ib-cr blaTEM Iss iut A tetA(A) aadA2 avrA sopB Stn phoA invA
Amplified segment 384bp 403bp 113bp 516bp 266bp 300bp 576bp 622bp 422bp 517bp 600 bp 720 bp 284 bp
Secondary denaturation 94˚C 45 sec. 94˚C 45 sec. 94˚C 30 sec. 94˚C 45 sec 94˚C 30 sec. 94˚C 30 sec. 94˚C 45 sec. 94˚C 45 sec. 94˚C 30 sec. 94˚C 30 sec. 94˚C 1 min. 94˚C 45 sec. 94˚C 30 sec.
Annealing
Extension
References
54˚C 45 sec 50˚C 45 sec 52˚C 30 sec 54˚C 45 sec 54˚C 30 sec. 63˚C 30 sec. 50˚C 45 sec. 50˚C 45 sec. 58˚C 30 sec. 58˚C 30 sec. 59˚C 1 min. 58˚C 40 sec. 55˚C 30 sec.
72˚C 45 sec 72˚C 45 sec 72˚C 30 sec 72˚C 45 sec 72˚C 30 sec. 72˚C 30 sec. 72˚C 45 sec. 72˚C 45 sec. 72˚C 30 sec. 72˚C 30 sec. 72˚C 1 min. 72˚C 45 sec. 72˚C 30 sec.
(68) (69) (70) (71) (46)
(72) (73)
(74)
(75) (76)
(77)
779 IJBPAS, April, 2016, 5(4)
Sherif Maarouf et al
Research Article
Table (4): Sequences and cycling conditions of the different PCR primers used for detection of different Clostridium perfringens toxins Amplified segment
Gene
Primers sequences
Alpha Toxin Beta Toxin Epsilon Toxin Iota toxin
GTTGATAGCGCAGGACATGTTAAG CATGTAGTCATCTGTTCCAGCATC ACTATACAGACAGATCATTCAACC TTAGGAGCAGTTAGAACTACAGAC ACTGCAACTACTACTCATACTGTG CTGGTGCCTTAATAGAAAGACTCC GCGATGAAAAGCCTACACCACTAC GGTATATCCTCCACGCATATAGTC
5
4
Secondary denaturation
Annealing
Extension
References
94˚C 1 min.
55˚C 1 min.
72˚C 1 min.
(78)
402bp 236 bp 541 bp 317 bp
3
L
2
1
1000 500 300 100
Photo. 1 PCR results for E. coli virulence genes and quinolone resistant gene. L, 100 bp ladder (Fermentas) (100-1000 bp). 1-5 lanes are shown respectively: 1) aadA2 (622 bp), 2)phoA(720 bp), 3)iss(266 bp), 4)iutA(300 bp) and 5) eaeA(384 bp).
6
5
4
L
3
2
1
1000 1000 300 100 400 100
Photo 2. PCR results for Salmonella virulence genes and quinolone resistant genes. L, 100 bp ladder (Qiagen, Germany, GmbH) (100-600 bp). 1-6 lanes are shown respectively: 1) sopB (517 bp), 2)avrA(422 bp), 3)tetA(A) (576 bp), 4)blaTEM(516 bp), 5)stn(600 bp) and 6) invA (284 bp).
780 IJBPAS, April, 2016, 5(4)
Sherif Maarouf et al
Research Article L
1
1000
400 100
Photo 3. PCR results for Clostridium perfringens toxin. L, 100 bp ladder (Qiagen, Germany, GmbH) (100-600 bp). One lane showpositive amplification of 402 bp of alpha toxin gene only Conclusion: Presence of Salmonella strains on raw materials is a very influential factor in order to find Salmonella on meal finished feeds. Clostridial toxin is an indicator of the prospected lethal effect that may be shown in the ingesting animals
[2] Gilbert,
CONCLUSION
J
(1995):
Mycotoxins
E.coli O26, H9 and C.perferengens showed
Certification of DIN in wheat and Maize.
different levels of drug resistance. Their
American Pathology, 3 (4): 263 – 268.
their
antibiogram.
Characterization
and
Food
and
of
Salmonella typhi, Entiritidis, typhimurium,
isolated virulent gens were compatible to
in
Analysis
feed.
[3] Ghose, B., Sharda, R.D. Chhabra, D. and V.
Sharma,:
Subclinical
bacterial
genotyping of resistant strains of isolated
mastitis in cows of Malwa region of
enteric pathogens and recognition of virulent
Madhya Pradesh. Ind. Vet. J., 2003, 80.
and toxin genes are more and rapid
6. 499-501.
diagnostic methods for proposed control and
[4] Tjaniadi .P, Lesmana, M., Subekti, D.,
preventive protocol against zoonotic diseases
Machpud, N., Komalarini, S., Santoso,
of poultry feed ingredients and product
W.,
origin.
Campbell, J., Alexander, W., Beecham,
REFERENCES
I.,
[1] Quadri, S. F. and Deyoe, C.W.: Effect of
Antimicrobial
Simanjuntak,
Corwin,
A.
C.,
and
reistance
associated
Punjabi,
Oyofo,
N.,
B.A.:
of
bacterial
with
diarrheal
temperature and pelleting on Salmonella
pathogens
content of feeds. Feed stuffs London,
patients in Indonesia. J. Trop. Med. Hyg,
1975, 47, (17, 2) 65-66.
2003, 68(6). 666–670.
781 IJBPAS, April, 2016, 5(4)
Sherif Maarouf et al
Research Article
[5] Songer, J.G. and Meer, R.R. Genotyping of
Clostridium
perfringens
[10] Titball, R.W., Naylor, C.E., Miller, J.,
by
Moss, D.S. & Basak, A.K. Opening of
polymerase chain reaction is a useful
the active site of Clostridium perfringens
adjunct to diagnosis of clostridial enteric
alpha-toxin
disease in animals. Anaerobe, 2 , 1996,
membrane binding. International Journal
197 /203.
of Medical Microbiology, 2000, 290 ,
[6] Petit, L., Gilbert, M. & Popoff, M.R.
may
be
triggered
by
357 /361.
(Clostridium perfringens: toxinotype and
[11] Immerseel.F. V; Buck. J. D, Pasmans.F ,
genotype. Trends in Microbiology, 1999.
Huyghebaert.G , Haesebrouck.F and
7, 104 /110.
Ducatelle.R.
Review
article:
[7] Ridell, J., Bjorkroth, J., Eisgruber, H.,
Clostridium perfringens in poultry: an
Schalch, B., Stolle, A. & Korkeala, H.
emerging threat for animal and public
Prevalence of the enterotoxin gene and
health. Avian Pathology (December
clonality of Clostridium perfringens
2004) 33(6), 537/549 .
strains associated with food-poisoning
[12] Radonov-Pelagic,
V.:
Mikrobiologija
outbreaks..Journal of Food Protection ,
hrane za životinje. Univerzitet uNovom
1998, 61 , 240 /243.
Sadu, Poljoprivredni fakultet. Novi Sad,
[8] Sarker, M.R., Carman, R.J. & McClane, B.A. Inactivation of the gene (cpe) encoding
Clostridium
perfringens
2000. [13] Sergentet-Thevenot, D., Montet, M. and Veronzy-Rozand,
C.: Challenges
to
enterotoxin eliminates the ability of two
developing nucleic acid sequence based
cpe-positive C. perfringens type A
amplification
human gastrointestinal disease isolates to
detection of microbial pathogens in food.
affect rabbit ileal loops. Molecular
Revue Méd. Vét., 2008, 159. 10. 514-
Microbiology, 1999, 33 , 946 /958.
527.
technology
for
the
[9] Naylor, C.E., Eaton, J.T., Howells, A.,
[14] Uwaezuoke, J.C. and Ogulie, J.N.:
Justin, N., Moss, D.S., Titball, R.W. &
Microbiological quality of commercially
Basak, A.K. Structure of the key toxin in
available poultry feeds sold in parts of
gas
Eastern Nigeria. J. Appl. Sci. Environ.
gangrene.
Nature
Biology,1998, 5 , 738 /746.
Structure
Manage. 2008, Vol. 12(1). 113 – 117.
782 IJBPAS, April, 2016, 5(4)
Sherif Maarouf et al [15] Lynne,
Research Article
A.M.,
Rhodes-Clark,
B.S.,
challengein-the-new-
Bliven, K. and Zhao, S.: Antibiotic
millennium/importance-of-bacteria-
resistance
resistance-to-antibiotics-in-food
Genes
Associated
with
Salmonella enteric Serovar Newport Isolates
from
Food
[19] Srinu, B., Kumar, V., Kumar, E.
Animals.
and Madhava Rao, T.: Antimicrobial
Antimicrobial Agent and Chemotherapy,
resistance pattern of bacterial food
2008, 52 (1). 353-356.
borne
[16] Nthenge, A.K., Nahashon, S.N., Chen, F.F. and Adefope, N.N.: Poultry Science, 2008, 87. 1841–1848. [17] Huehn, S., La Ragione, R.M., Anjum,
pathogens.
Chemical
and
Journal
of
Pharmaceutical
Research, 2012, 4(7).3734-3736. [20] Solanitro,
J.P.,
Zgornicki,
Y.:
Fairchilds,
I.G.,
Isolation,
culture
Saunders, M., Woodward, M.J., Bunge,
characteristic, identification of anaerobic
C., Helmuth, R., Hauser, E., Guerra, B.,
bacteria from poultry cecum. Toxicology
Beutlich, J. Brisabois, A., Peters, T.
Program, Alabama, USA, 1986 Pp 1-40.
Svensson, L., Madajczak, G., Litrup, E.,
[21] Brenner F, McWhorter-Murlin A,
Imre, A., Herrera-Leon, S., Mevius, D.,
(1998).Identification and serotyping of
Newell,
Salmonella. Atlanta: Centers for Disease
D.G.
Virulotyping resistance
and
Malorny,
and typing
entericaserovars
B.:
antimicrobial of
Salmonella
relevant
to
human
Control and Prevention [22] Osman, K.M., Marouf, S.H. and Al Atfeehy, N.: Antimicrobial Resistance
health in Europe. Food borne Pathogens
and
Dis., 2010, 7. 523-35.
Salmonella enterica Subsp. enterica
[18] Lopez
,M.
C.
V:
Antimicrobial
Virulence-Associated
Genes
of
Serotypes Muenster, Florian, Omuna,
Resistance of Bacteria in Food – A
and
Continuous Challenge
New
Clinically Diarrheic Humans in Egypt.
Millennium. Universidad de los Andes,
Microbial Drug Resistance, 2013, 19 (5).
Colombia. Dr. Marina Pana (Ed.), 2012
370-377.
ISBN:
in
978-953-51-0472-8,
Available
the
InTech, from:
[23] CLSI:
Noya
Strains
Performance
Antimicrobial
Isolated
Standards
Susceptibility
informational
from
for
Testing;
http://www.intechopen.com/books/antibi
Ninth
supplement,
otic-resistant-bacteria-a-continuous-
NCCLS document.National committee 783
IJBPAS, April, 2016, 5(4)
Sherif Maarouf et al
Research Article
for laboratory standared, Wayne. 0 419
[28] Carraminana, J. J., Rota, C., Agustyn, I.
22320 7 (Hbk) 0 419 21730 4 (Pbk),
and Herrera, A.: High prevalence of
2008.
multiple resistance to antibiotics in
[24] FAO,
WHO,
Joint
Salmonella serovars isolated from a
FAO/OIE/WHO Expert Workshop on
poultry slaughterhouse in Spain. Vet.
Non-Human Antimicrobial Usage and
Microbiol. 2004, 104.133–139.
Antimicrobial
and
OIE.
Resistance:
:
Scientific
[29] Simango,
M.
and
Mbewe.
assessment Geneva, December Food and
Salmonella
Agriculture Organization of the United
Harare, Zimbabwe Tropical Medicine
Nations World
and International Health, 2000, 5. 503-
Health
Organization
World Organization for Animal Health, 2003
enteritidis
M.:
diarrhoea
in
506. [30] Kiessling, C.R., Cutting, J.H., Loftis, M.,
[25] Goren E.: Antibiotic sensitivity tests on
Kiessling, W.M., Datta, A.R., Sofos,
Escherichia coli isolated from poultry:
J.N.: Antimicrobial resistance of food-
significance of mixed strain inocula.
related Salmonella isolates, 1999-2000.
Avian pathology, 1979, 8(1) 13-22.
Journal of Food Protection, 2002, 65.
[26] Arvanitidou, M., Tsakris, A., Sofianou, D.
and
V.:
[31] Mammina, C., Cannova, L., Oliver, R.,
Antimicrobial resistance and R-factor
Carfi Pavia, S., Di Gaetano, V., Di
transfer of salmonellae isolated from
Piazza, F. and Nastasi, A.: Antibiotic
chicken carcasses in Greek hospitals. Int.
resistance of Salmonella isolates from
J. Food Microbiol., 1998; 14:197–201.
sewage
[27] Seyfarth,
Katsouyannopoulos,
603-608.
AM.,
Wegener,
Frimodt-Møller,
N.:
H.
serovar
humans
and
Medicine
Sicily.
Antimicrobial
Salmonellosis, proceedings 515-516. St.
production of
in
International Symposium Salmonella &
typhimurium
Journal
effluents
and
resistance in Salmonella enterica subsp. enterica
plant
from
Brieuc, France, 2002. [32] Threlfall,
E.J.:
Antimicrobial
drug
animals.
resistance in Salmonella: problems and
Antimicrobial
perspectives in food-and water-borne
Chemotherapy, 1997, Volume 40, Issue
infections.
FEMS
Microbiology
1 Pp. 67-75.
Reviews. 2002, 26. 141-148.
784 IJBPAS, April, 2016, 5(4)
Sherif Maarouf et al
Research Article
[33] Wybo, C. et al.: Antimicrobial resistance
Salmonella from poultry and their
in Salmonella from humans in Belgium:
susceptibility to antimicrobial agents.
the situation in 2000 and 2001. In:
International
International. Symposium Salmonella
Microbiology, 2003; 82:97-103.
Journal
of
Food
and Salmonellosis. St. Brieue, França:
[38] Cardoso, M.O., Ribeiro, A.R., Santos,
AFSSA: INRA: In VS: Institut Pasteur:
I.R., Pilotto, F., Moraes, H.L., Salle,
ISPAIA, 2002, 497-498.
C.T., Rocha,
S.L., Nascimento, V.P.:
[34] Kumar, S., Balakrishna, K. and Batra,
Antibiotic
resistance
H.V.: Detection of Salmonella enteric
enteritidis
isolated
Serovar Typhi ( S Typhi) by selective
carcasses.
Brazilian
amplification of invA, viaB, fliC-d and
Microbiology, 2006; 37:368-371.
prt genes by polymerase chain reaction
in
Salmonella
from
broiler
Journal
of
[39] Wasyl, D. and Hoszowski, A.: Antibiotic
in multiplex format. Letters in Applied
susceptibility
Microbiology, 2006, 42: 149-215.
isolates. Proceedings: SALINPORK, 4th
[35] Tjaniadi .P, Lesmana, M., Subekti, D.,
International
in
Salmonella
Symposium
on
swine
the
Machpud, N., Komalarini, S., Santoso,
Epidemiology and Control of Salmonella
W.,
and other food borne pathogens in Pork.
Simanjuntak,
C.,
Punjabi,
N.,
Campbell, J., Alexander, W., Beecham, I.,
Corwin,
B.A.:
[40] Pedersen, K., Hansen, H.C., Jorgensen,
bacterial
J. and Borck, B.: Serovars of Salmonella
with diarrheal
isolated from Danish turkeys between
patients in Indonesia. J. Trop. Med. Hyg,
1995 and 2000 and their antimicrobial
2003, 68(6). 666–670.
resistance. Vet Rec, 2002, 150. 471-474.
Antimicrobial pathogens
A.
and
reistance
associated
Oyofo,
2001, 432-434.
of
[36] Bada-Alambedji, F.A., Seydi, M. and
[41] Salehi, T.Z. and Bonab, S.F.: Antibiotics
Akakpo, J.A.: Antimicrobial resistance
Susceptibility Pattern of Escherichia coli
of Salmonella isolated from poultry
Strains Isolated from Chickens with
carcasses in Dakar (Senegal). Brazilian
Colisepticemia in Tabriz Province, Iran.
Journal of Microbiology, 2006; 37(4):
International Journal of Poultry Science,
510-515.
2006, 5 (7). 677-684.
[37] Antunes, P., Reu, C., Sousa, J.C., Peixe, L. and
Pestana, N.: Incidence of
[42] Yadav, M.M., Roy, A., Sharda, R. and Arya, G.: (). Detection of toxin genes 785
IJBPAS, April, 2016, 5(4)
Sherif Maarouf et al
Research Article
and antibiogram pattern in E. coli from
isolated
sheep meat on Indian market. Vet.
colisepticemia in Japan. Avian Diseases,
Arhiv., 2007, 77. 485-494
2007, 51. 656-662.
[43] Habrun, B., G. Kompes, Z. Cvetnic, S.
from
[47] Ghanbarpour,
chickens
R.
and
Oswald,
with
E.:
Špicic, M. Benic, M. and M. Mitak,:
Phylogenetic Distribution of Virulence
Antimicrobial Escherichia
susceptibility
of
Genes in Escherichia coli Isolated from
Salmonella
spp.,
Bovine Mastitis in Iran. Res. Vet. Sci.,
coli,
Pasteurella
multocida,
Streptococcus
suis
and
Actinobacillus
2010, 88, 6-10. [48] Tokano, D.V., Kawaichi, M., Venâncio,
pleuropneumoniae
isolated
from
E.J. and Vidotto, M.: Cloning and
diagnostic
from large pig
Characterization of the Iron Uptake
breeding farms in Croatia. Vet. Arhiv,
Gene IutA from Avian Escherichia coli.
2010, 80. 571-583.
Brazilian Archives Of Biology And
samples
[44] Ewers, C., Janssen, T., Kiessling, S., Philipp, Molecular
H.C.
and
Wieler,
epidemiology
of
L.H.:
Technology. 2008, Vol. 51. n. 3: pp. 473-482.
avian
[49] Chaïbi, E.B., Sirot, D., Paul G. and
pathogenic Escherichia coli (APEC)
Labia, R.: Inhibitor-resistant TEM beta-
isolated from colisepticemia in poultry.
lactamases: phenotypic, genetic and
Veterinary Microbiology. 2004, 104. 91-
biochemical
101
Antimicrob. Chemother. 1999, 43(4).
[45] Johnson, T.J., Siek, K.E. Johnson, S.J. and Nolan, L.K.: DNA sequence of a
characteristics.
J.
447-458. [50] Sun,
S.,
Negrea,
A.,
Rhen,
M.,
ColV plasmid and prevalence of selected
Andersson, D.I.: Genetic analysis of
plasmid-encoded virulence genes among
colistin resistance in Salmonella enterica
avian Escherichia coli strains. Journal of
serovar
Bacteriology, 2006, 188. 745-758
Agents and Chemotherapy. 2009, 53(6).
[46] Yaguchi, K., Ogitani, T., Osawa, R.,
typhimurium.
Antimicrobial
2298–2305.
Kawano, K., Kokumai, N., Kaneshige,
[51] Cui, S., Ge, B., Zheng, J. and Meng, J.:
T., Noro, T., Masubuchi, K. and
Prevalence and antimicrobial resistance
Shimizu, Y.,: Virulence factors of avian
of Campylobacter spp. and Salmonella
pathogenic
serovars
Escherichia
coli
strains
in organic chickens
from 786
IJBPAS, April, 2016, 5(4)
Sherif Maarouf et al
Research Article
Maryland retail stores. Applied and environmental
microbiology.
2005,
71(7). 4108-4111
[56] Dione, M.M., Ikumapayi, U., Saha, D., Mohammed,
N.I.,
Adegbola,
R.A.,
Geerts, S., Ieven, M. and Antonio, M.:
[52] Malorny, B., Schroeter, A., Guerra, B.
Antimicrobial resistance and virulence
and Helmuth, R.: Incidence of quinolone
genes
resistance in strains of Salmonella
isolates in the Gambia and Senegal. J
isolated from poultry, cattle and pigs in
Infect Dev Ctries., 2011, 5(11). 765-775
Germany
between
1998
of
non-typhoidal
Salmonella
and
[57] Ye, Z., Petrof, E., Boone, D., Claud, E.
2001.Veterinary Record, 2003, 153(21).
and Sun, J.: Salmonella Effector AvrA
643-8.
Regulation of Colonic Epithelial Cell
[53] Mayrhofer, S., Paulsen, P. Smulders, F.
Inflammation by Deubiquitination. The
and Hilbert, F.: Antimicrobial resistance
American Journal of Pathology, 2007,
profile
171 (3).
of
five
major
food-borne
pathogens isolated from beef, pork and
[58] Gan, E., Baird, F.J., Coloe, P.J. and
poultry. International Journal of Food
Smooker,
Microbiology, 2004, 97.23-29
molecular characterization of Salmonella
[54] Suresh, T., Hatha, A., Sreenivasan, D.,
P.M.:
Phenotypic
and
enterica serovar Sofia, an avirulent
Sangeetha, N., Lashmanaperumalsamy,
species
D.:
Microbiology. 2011, 157. 1056–1065.
Prevalence
and
antimicrobial
resistance of Salmonella enteritidis and
in
Australian
poultry.
[59] Borges, K.A., Furian, T.Q., Borsoi, A.,.
other Salmonellae in the eggs and egg
Moraes,
storing trays South India. Journal of
Nascimento,
Food Microbiology. 2006, 23. 294-299.
virulence-associated genes in Salmonella
[55] Upadhyay,
B.P.,
Utrarachkij,
H.L,
Salle, V.P.:
C.T.
Detection
and of
F.,
enteritidis isolates from chicken in South
Thongshoob, J., Mahakunkijcharoen, Y.,
of Brazil. Pesq. Vet. Bras, 2013; 33
Wongchinda, N., Suthienkul, O. and
(12).1416-1422.
Khusmith, S.: Detection of Salmonella
[60] Patel, J.C. and Galán, J.E.: Investigating
INVA Gene in Shrimp Enrichment
the Function of Rho Family GTPases
Culture By Polymerase Chain Reaction.
during
Southeast Asian J Trop Med Public
Interactions. Methods Enzymol, 2008,
Health. 2010, Vol 41. No. 2.
439. 145–158.
Salmonella/Host
Cell
787 IJBPAS, April, 2016, 5(4)
Sherif Maarouf et al
Research Article
[61] Nagarajan, A.G., Balasundaram, S.V.,
resistance
Janice, J., Karnam, G., Eswarappa, S.M. and
Chakravortty,
Salmonella
D.:
SopB
enterica
of
to
quinolones.
Microb
Biotechnol. 2009, 2. 40-61. [66] Hatheway, C. L.: Toxigenic clostridia.
serovar
Clin. Microbiol. Rev, 1990, 3. 66–98.
typhimurium is a potential DNA vaccine
[67] Daube, G., China, B., Simon, P., Hvala,
candidate in conjugation with live
K. and Mainil, J.: Typing of Clostridium
attenuated
perfringens by in vitro amplification of
bacteria.
Vaccine,
2009,
27(21). 2804-2811.
toxin genes. J. Appl. Bacteriol., 1994,
[62] Nakano, M., Yamasaki, E., Ichinose, A.,
77. 650–655.
Shimohata, T., Takahashi, A, Akada, J.,
[68] Wen-jie J.;Zhi-ming Z.; Yong-zhi Z.;Ai-
Nakamura, K., Moss, J., Hirayama, T.
jian Q.; Hong-xia S.; Yue-long L.; Jiao
and
Salmonella
W. and Qian-qian W. Distribution of
enterotoxin (Stn) regulates membrane
Virulence-Associated Genes of Avian
composition
Disease
Pathogenic Escherichia coli Isolates in
Models & Mechanisms, 2012, 5. 515-
China. Agricultural Sciences in China,
521
2008;7(12): 1511-1515.
Kurazono,
H.:
and
integrity
[63] Van Asten, A.J. and Van Dijk, Distribution
of
"classic"
J.E.
[69]
Cattoir V.; Poirel L. and Nordmann P.
virulence
Plasmid-mediated quinolone resistance
factors among Salmonella spp. FEMS
pump QepA2 in an Escherichia coli
Immunol
isolate from France. Antimicrob Agents
Med
Microbiol.2005;
1.
44(3).251-9.
Chemother ,2008; 52:3801-3804.
[64] Cavaco, L.M., Hasman, H., Xia S. and
[70] Lunn
A.D.;
Fàbrega
A.; Sánchez-
Aarestrup, F.M.: qnrD, a novel gene
Céspedes J. and Vila J. Prevalence of
conferring
mechanisms
resistance
transferable in
quinolone
Salmonella
enterica
decreasing
quinolone-
susceptibility among Salmonella spp.
serovar Kentucky and Bovismorbifican
clinical
strains of human origin. Antimicrob.
Microbiology. 2010; 13:15-20.
Agents Ch., 2009, 53. 603–608. [65] Fàbrega, A., Madurga, S., Giralt, E. and Vila, J.: Mechanism of action of and
[71] Colom
isolates.
K.;
Pèrez
International
J.;
Alonso
R.;
Fernández-Aranguiz A.; Lariňo E. and Cisterna
R.
Simple
and
reliable
multiplex PCR assay for detection of 788 IJBPAS, April, 2016, 5(4)
Sherif Maarouf et al
Research Article
blaTEM, blaSHV and blaOXA-1 genes
human health in Europe. Foodborne
in
Pathogens Dis 2010; 7:523-35.
Enterobacteriaceae.
FEMS
Microbiology Letters 223 .2003; 147151.
[75] Murugkar,
H.V.;Rahman,
H.
and
&Dutta, P.K. Distribution of virulence
[72] Randall, L.P.; Cooles, S.W.;
Osborn,
genes in Salmonella serovars isolated
M.K.; Piddock, L.J.V. and Woodward,
from man & animals. Indian J Med Res.,
M.J.
2003 ,117:66-70.
Antibiotic
integrons
and
resistance multiple
genes, antibiotic
[76] Hu, Q.; Tu, J.; Han, X.; Zhu, Y.; Ding,
resistance in thirty-five serotypes of
C. and Yu, S. . Development of
Salmonella
from
multiplex PCR assay for rapid detection
humans and animals in the UK. Journal
of Riemerella anatipestifer, Escherichia
of Antimicrobial Chemotherapy. 2004;
coli,
53: 208–216.
simultaneously from ducks. Journal of
enterica
isolated
[73] Walker, R. A., Lindsay, E., Woodward, M. J. et al. Variation in clonality and
and
Salmonella
enterica
Microbiological Methods ,2011. 87: 64– 69.
antibiotic-resistance genes among multi-
[77] Olivera, S.D.; Rodenbusch, C.R.; Ce,
resistant Salmonella enterica serotype
M.C.; Rocha, S.L.S. and Canal, C.W. ():
typhimurium phage-type U302 (MR
Evaluation of selective and non selective
U302) from humans, animals, and foods.
enrichment
. (): Microbiological Research ,2001: 7:
Salmonella
13–21.
Microbiol., 2003; 36: 217-221.
PCR
procedures
de-tection.
Lett.
for Appl.
[74] Huehn S, La Ragione RM, Anjum M,
[78] Yoo, H. S., S. U. Lee, K. Y. Park, and
Saunders M, Woodward MJ, Bunge C,
Y. H. Park. . Molecular typing and
Helmuth R, Hauser E, Guerra B,
epidemiological survey of prevalence
Beutlich J, Brisabois A, Peters T,
of Clostridium
Svensson L, Madajczak G, Litrup E,
multiplex PCR. J. Clin. Microbiol.;1997
Imre A, Herrera-Leon S, Mevius D,
. 35:228-232.
perfringens types
by
Newell DG, Malorny B. Virulotyping and antimicrobial resistance typing of Salmonella entericaserovars relevant to
789 IJBPAS, April, 2016, 5(4)
