Isolation and molecular characterization of Salmonella enterica serovar
Enteritidis from poultry house and clinical samples during 2010
Ezat H. Mezala,b,f, Ashley Sabolc, Mariam A. Khand, Nawab Alib, Rossina Stefanovae,
Ashraf A. Khana,*
aMicrobiology Division, National Center for Toxicological Research, U.S. Food and Drug Administration, Jefferson, AR 72079, USA
bUniversity of Arkansas at Little Rock, Little Rock, AR 72205, USA
cCenters for Disease Control and Prevention, Atlanta, GA 30333, USA
dUniversity of Central Arkansas, Conway, AR, USA
eThe Arkansas Department of Health, Little Rock, AR, USA
fUniversity of Thi-Qar, Science of College, Biology of Dept., Thi-Qar, Iraq
a r t i c l e i n f o
Received 18 May 2012
Received in revised form
12 July 2013
Accepted 9 August 2013
Available online 27 August 2013
Pulsed-field gel electrophoresis
a b s t r a c t
A total of 60 Salmonella enterica serovar (ser.) Enteritidis isolates, 28 from poultry houses and 32 from
clinical samples, were isolated during 2010. These isolates were subjected to testing and analyzed for
antibiotic resistance, virulence genes, plasmids and plasmid replicon types. To assess genetic diversity,
pulsed-field gel electrophoresis (PFGE) fingerprinting, using the XbaI restriction enzyme, Multiple-Locus
Variable-Number Tandem Repeat Analysis (MLVA) and plasmid profiles were performed. All isolates from
poultry, and 10 out of 32 clinical isolates were sensitive to ampicillin, chloramphenicol, gentamicin,
kanamycin, nalidixic acid, sulfisoxazole, streptomycin, and tetracycline. Twenty-one of thirty-two clinical
isolates were resistant to ampicillin and tetracycline, and one isolate was resistant to nalidixic acid. PFGE
typing of sixty ser. Enteritidis isolates by XbaI resulted in 10e12 bands and grouped into six clusters each
with similarity from 95% to 81%. The MLVA analysis of sixty isolates gave 18 allele profiles with the
majority of isolates displayed in three groups, and two clinical isolates found to be new in the PulseNet
national MLVA database. All isolates were positive for 12 or more of the 17 virulence genes mostly found
in S. enterica (spvB, spiA, pagC, msgA, invA, sipB, prgH, spaN, orgA, tolC, iroN, sitC, IpfC, sifA, sopB, and pefA)
and negative for one gene (cdtB). All isolates carried a typical 58 kb plasmid, type Inc/FIIA. Three poultry
isolates and one clinical isolate carried small plasmids with 3.8, 6, 7.6 and 11.5 kb. Ten of the clinical
isolates carried plasmids, with sizes 36 and 38 kb, types IncL/M and IncN, and one isolate carried an 81 kb
plasmid, type IncI. Southern hybridization of a plasmid with an Inc/FIIA gene probe hybridized one large
58 kb plasmid in all isolates. Several large and small plasmids from poultry isolates were not typed by our
PCR-based method. These results confirmed that PFGE fingerprinting has limited discriminatory power
for ser. Enteritidis in both poultry and clinical sources. However, the plasmid and MLVA allele profiles
were a useful and important epidemiology tool to discriminate outbreak strains of ser. Enteritidis from
poultry and clinical samples.
Published by Elsevier Ltd.
Salmonella is one of the leading causes of foodborne illnesses
worldwide. The Centers for Disease Control and Prevention (CDC)
has estimated that 9.4 million foodborne illnesses, 55,961 hospi-
talizations and 1351 deaths occur in the United State each year
(Scallan et al., 2011). Non-typhoidal Salmonella causes an estimated
1 million illnesses with approximately 20,000 hospitalizations and
approximately 378 deaths each year (Scallan et al., 2011). Further-
more Salmonella infections are usually associated with the con-
sumption of contaminated food products from poultry, pigs and
ruminants, contaminated drinking water or direct contact with
infected animals (Matsuoka et al., 2004; Mullneret al., 2009). So far,
more than 2610 serovars of Salmonella enterica have been recog-
nized from all over the world, and almost all are able to cause
illness in humans and animals (Guibourdenche et al., 2010). One of
the most important Salmonella serovars is ser. Enteritidis, which is
* Corresponding author. Tel.: þ1 870 543 7601; fax: þ1 870 543 7307.
Contents lists available at ScienceDirect
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Food Microbiology 38 (2014) 67e74
an important cause of human illness with symptoms typically
including fever, vomiting, diarrhea and abdominal cramps 12e72 h
after ingestion of the bacterium (CDC, 2010). The risk groups of
infection with ser. Enteritidis are infants (under 3 months of age),
the elderly, and the immunocompromised (CDC, 1990).
Eggs, egg-containing food products and inadequately cooked
poultry have been the most common foods source for ser. Enter-
itidis (Abdullah et al., 2010; Bichleret al.,1994). In the UnitedStates,
the outbreaks of ser. Enteritidis from 1985 to 1999 were identified
as egg-associated (Patrick et al., 2004). There are two routes which
can cause contamination of eggs by Salmonella; the first route is
horizontal transmission where the infected feces penetrates
eggshell pores, or contaminates eggs via cracks on eggs shells (CDC,
2010). The second route is vertical transmission inside the infected
hen, where the eggshell membranes, albumen and yolk are
contaminated before oviposition (Messens et al., 2005; Gantois
et al., 2009). From May 1 to November 30, 2010, approximately
1939 illnesses were reported in the United States that were likely to
be associated with ser. Enteritidis (CDC, 2010). Poultry houses are
the likely sources of the contaminated eggs with ser. Enteritidis
through cracks in the shell (Gantois et al., 2009). Salmonella can
survive and persist in poultry houses for a long time. Salmonella has
also the ability to spread between hosts, for example bacteria can
passage from infected farm animals to vegetables as a result of field
fertilization with raw, contaminated manure and Salmonella can
infiltrate, colonize and persist on plants (Davies and Wray, 1995;
Winfield and Groisman, 2003).
PFGE has been proven to be important for establishing genetic
relatedness of different bacterial strains and is commonly used for
investigation of outbreaks associated with a particular pathogen
(Akiyama et al., 2011; Foley et al., 2009; Ponce et al., 2008; Khan
et al., 2002, 2007). PFGE is the current gold standard subtyping
method for foodborne bacterial pathogens used by PulseNet, the
national molecular subtyping network for foodborne disease sur-
veillance in the United States (Swaminathan et al., 2001). MLVA is
another high discriminatory subtyping method that is based on the
detection of short sequence repeats in the microbial genome
(Seongbeom et al., 2007). Recently, MLVA has been proposed as an
alternative to PFGE for subtyping of ser. Enteritidis and a number of
other pathogenic bacteria (Boxrud et al., 2007; Ramisse et al., 2004;
Svraka et al., 2006). Plasmid profile analysishas been helpful for the
characterization of many Salmonella serovars including ser. Enter-
itidis (Mezal et al., 2013; Ridley et al., 1998). Many strains of Sal-
monella spp. carry plasmids that play an important role in invasion
In this study, ser. Enteritidis isolates cultured from poultry
houses and clinical specimens isolated during 2010 were examined
for PFGE profiles, MLVA typing, plasmid analysis, antibiotic sus-
ceptibility and PCR for virulence genes to assess the relatedness
among clinical and poultry isolates.
2. Materials and methods
2.1. Bacterial strains
Sixty isolates of ser. Enteritidis were selected for this study.
These strains were isolated from poultry houses during 2010, and
clinical samples from Arkansas Department of Health. Twenty-
eight of these isolates were from poultry houses from the FDA
-Arkansas Regional Laboratory (ARL) and thirty-two isolates were
of clinical origin and were obtained from the Arkansas Department
of Health (ADH). All isolateswerestored in LuriaeBertani (LB) broth
containing 20% glycerol at ?70?C. Organisms were grown over-
night at 37?C in LB broth or on tryptic soy agar plates supple-
mented with 5% blood agar.
2.2. Antimicrobial susceptibility testing by disk diffusion
All isolates of ser. Enteritidis used in this study were tested for
resistance to eight antimicrobials on Mueller-Hinton agar (Difco
Laboratories, Detroit, MI) by a disk agar diffusion method (Khan
et al., 2006). The following antimicrobials were used: ampicillin
(10 mg), chloramphenicol (30 mg), gentamicin (10 mg), kanamycin
(30 mg), streptomycin (10 mg), sulfisoxazole (0.25 mg), tetracycline
(30 mg), and nalidixic acid (30 mg). Sensitivity and resistance were
determined by the criteria of the Clinical and Laboratory Standard
Institute (CLSI, 2006). Escherichia coli ATCC 25922, which is sus-
ceptible to all of the antibiotics, was used for quality control.
2.3. Pulsed-field gel electrophoresis (PFGE)
Ser. Enteritidis cells were grown overnight on blood agar plates
(Thermo Fisher Scientific, Remel products, Lenexa, KS) at 37?C.
PFGE was performed following a procedure described byRibotet al.
(2006) with some modifications. Each culture was suspended in TE
buffer (100 mM TriseHCl, 100 mM EDTA, pH 8.0) to a turbidity of
2.0 and 2.2 OD610as measured using an Ultraspec 3100 pro Spec-
trophotometer (Pharmacia Biochem Ltd. Cambridge, UK). To pre-
pare the agarose plugs, 20 ml of Proteinase K (20 mg/ml stock) was
added to 380 ml of the adjusted cell suspension. Then 400 ml of
melted 1% SeaKem Gold agarose: 1% SDS was added to the 400 ml
cell suspension/Proteinase K mixture and mixed gently. The
mixture was immediately dispensed into wells of plug molds. The
bacterial cells in the agarose plugs were lysed by treatment with a
lysis solution containing 0.1 mg/ml Proteinase K (GIBCO-BRL, Gai-
thersburg, MD), 50 mM TriseHCl (pH 8.0), 50 mM EDTA, and 1% N-
lauroylsarcosine, for 2 h at 54??C.
The plugs were washed two times by adding 10e15 ml sterile
water that has been pre-heated to 54e55?C and tubes were shaked
in a 54?C waterbath for 15 min. The plugs werewashed three times
for 15 min with pre-heated sterile TE Buffer (10 mM Tris:1 mM
EDTA, pH 8.0) in a 54?C water bath. The plugs were digested with
12 U of restriction enzyme XbaI (Promega Corp., Madison, WI) for
5 h at 37?C. Digested fragments were resolved in a 1% SeaKem Gold
agarose (Cambrex Bio Science Rockland, Inc., Rockland, Maine) gel
in 0.5? TriseBorateeEDTA (TBE) buffer using a contour-clamped
homogeneous electric field (CHEF) apparatus (CHEF-DR III, Bio-
Rad Laboratories, Richmond, CA). Electrophoresis was performed
at 6 V/cm with 2.2e54.2 s linear ramp time for 19 h. Gels were
cooled at 14?C throughout the run and then stained with ethidium
bromide and destained with distilled water. Banding patterns were
visualized by UV and photographed. The Salmonella Braenderup
strain H9812 PulseNet standard was used as a molecular weight
marker after digestion with XbaI. Fingerprinting profiles were
examined by using the BioNumerics software version 6.x (Applied
Maths, Austin TX) and confirmed visually. Clustering was done by
the unweighted pair group average method (UPGMA) using the
2.4. PCR detection of virulence genes
All isolates of ser. Enteritidis were screened for 17 virulence
genes (spvB, spiA, pagC, msgA, invA, sipB, prgH, spaN, orgA, tolC, iroN,
sitC, IpfC, sifA, sopB, cdtB and pefA) by a simplex PCR method
(Skyberg et al., 2006). Primers used for this study are listed in
Table 1. Total genomic DNA from the isolates was extracted from
overnight cultures by using the DNeasy? Blood and Tissue kit
(Qiagen, Valencia, CA, USA). The composition of the PCR mixture
was: 1? PCR buffer, 200 mM of each dNTP, 0.25 mM of forward and
reverse primers, 2.5 units of Taq DNA polymerase (Qiagen) and 1 ml
of template DNA. The PCR cycling conditions were 5 min at 95?C;
E.H. Mezal et al. / Food Microbiology 38 (2014) 67e74
30 cycles of 40 s at 94?C, 60 s at 66.5?C, and 90 s at 72?C, with an
additional extension for 10 min at 72?C. The PCR products were
visualized by electrophoresis on 1.2% agarose gels in 1? TAE buffer
at 50 V for 85 min.
2.5. Multiple-Locus Variable-Number Tandem Repeat Analysis
(MLVA) was performed according to the procedures described in
the Laboratory Standard Operating Procedure for PulseNet MLVA of
S. enterica serotype Enteritidis e Beckman Coulter CEQ and Pulse-
Net Standard Operating Procedure for Analysis of MLVA data of S.
enterica serotype Enteritidis in BioNumerics e Beckman Coulter
CEQ 8000 data (http://www.pulsenetinternational.org/protocols/
Pages/mlva.aspx). The composite analysis was based on equal
weighting of XbaI and MLVA data and unweighted pair group
method with arithmetic mean (UPGMA) clustering.
2.6. PCR detection of plasmid replicon typing
All 60 Salmonella isolates were screened for 15 plasmid repli-
cons by a simplex PCR method (Carattoli et al., 2005). Primers are
listed inTable 2. The final volume of PCR reaction mixture was 20 ml
that included 1 ml of template DNA, 1? PCR buffer, 200 mM of each
dNTP, 0.25 mM of forward and reverse primers, and 2.5 units of Taq
DNA polymerase. The PCR amplification conditions were included
5 min at 94?C, 30 s at 94?C; 30 cycles of 30 s at 60?C, and 90 s at
72?C, and a final extension for 5 min at 72?C. Products of PCR were
electrophoresed on 1.2% agarose gel containing ethidium bromide
Primers used in PCR for detection of plasmid replicon typing in S. Enteritidis.
Replicon Sequence of nucleotidesSize (bp) Target site
rep A, B, C
Primers used in PCR for detection of virulence genes in S. Enteritidis.
Gene Sequence of nucleotidesSize (bp)Function of gene
717Growth within host
550Survival within macrophage
454Survival within macrophage
189 Survival within macrophage
1070 Host recognition/invasion
875 Entry into nonphagocytic cells
756 Host recognition/invasion
504Entry into nonphagocytic cells
449 Filamentous structure formation
E.H. Mezal et al. / Food Microbiology 38 (2014) 67e74
in 1? TAE buffer at 50 V for 85 min, and visualized by using BIO-
RAD Gel DOC XR imaging system.
2.7. Plasmid profiling
Plasmid DNA of the strains was isolated by using the alkaline
lysis method following the protocol of Ponce et al. (2008). 1.5 ml
from overnight cultures of bacterial growth in Luria broth (LB,
Difco) was centrifuged at 12,000 ? g for 1 min. The pellet was
resuspended in 1 ml of SET buffer (20% sucrose, 50 mM EDTA, and
50 mM TriseHCl, pH 7.6), centrifuged for 1 min at 12,000 ? g and
resuspended in 150 ml of SET buffer. Cells were lysed by mixing
with 350 ml lysis buffer (1% SDS and 0.2 M NaOH) and incubated
for 30 min in ice. Then, 250 ml of acetate buffer (3.0 M sodium
acetate, pH 4.8) was added. Tubes were mixed by inversion and
incubated for 20 min in ice. After centrifugation at 12,000 ? g at
4?C, 700 ml of the upper aqueous phase was transferred to a clean
tube and DNA was precipitated by one volume of isopropyl
alcohol. The pellets were washed with 1 ml ethanol and dissolved
in 50 ml of TE buffer (50 mM Tris, 1 mM EDTA, pH 8.0). The
plasmids were separated on 1.0% agarose gels in 1? Triseacetatee
EDTA (TAE) buffer at 64 V for 2 h. The supercoiled DNA ladder
(Invitrogen Corporation, Carlsbad, CA) was used as a molecular
marker. The molecular sizes of plasmids were determined by
Fig. 1. Dendrograms of XbaI-PFGE analysis (a) and Dendrograms of MLVA analysis (b) of clinical and poultry house isolates of S. Enteritidis generated by BioNumerics software
version-6. For the antibiotic susceptibility test (AST), the red color indicates resistance to the corresponding antimicrobials, the green color indicates intermediate susceptibility and
light blue color indicates susceptibility. For the detection of virulence genes, the dark blue indicates the presence of the gene while yellow indicates the absence of the gene. The size
of any detected plasmids and their identified replicon types are indicated. (For interpretation of the references to color in this figure legend, the reader is referred to the web version
of this article.)
E.H. Mezal et al. / Food Microbiology 38 (2014) 67e74
using S1 nuclease (Promega, Madison, WI), following a procedure
described by Akiyama et al. (2011).
2.8. Southern hybridization of plasmid DNA
A 270-bp PCR product was amplified using IncFIIA primers
(Carattoli et al., 2005) from a Salmonella plasmid with the IncFIIA
gene, which was PCR labeled by PCR (DIG) Probe Synthesis Kit
(Roche Diagnostics, Mannheim, Germany). The plasmids were
separated on 0.8% agarose gels and stained with ethidium bro-
mide for visualization, then transferred and cross linked to posi-
tively charged nylon membranes (Roche, Indianapolis, IN). The
resulting blot was incubated at 65?C for 30 min in DIG Easy Hyb
(Roche) prior to addition of digoxigenin (DIG)-labeled DNA probe
(denatured at 95?C for 10 min). The nylon membranes were
hybridized with an IncFIIA gene probe at 45?C overnight in a DIG
Hyb Solution (Roche) according to the manufacturer’s instructions
to detect probe target hybrids. Briefly, the membrane was washed
in 2? SSC (1? SSC is comprised of 0.015 M sodium citrate and
0.15 M NaCl ?0.1% SDS solution twice for 5 min each time at room
temperature. The membrane was then washed twice for 15 min in
0.1? SSCe0.1% SDS solutions at 68?C .The membrane was blocked
in 1% blocking reagent (DIG) for 30 min at room temperature.
conjugated anti-DIG antibody (Roche) and the chemilumines-
cent substrate, disodium 3-(4-methoxyspiro (1, 2-dioxetane-3, 20-
(50-chloro) tricyclo [188.8.131.52.7] decan)-4-yl) phenylphosphate
Fig. 1. (continued).
E.H. Mezal et al. / Food Microbiology 38 (2014) 67e74
Antimicrobial susceptibility testing of 60 ser. Enteritidis strains
showed that all twenty-eight poultry isolates and ten of the 32
clinical isolates were sensitive to all eight antimicrobials (Fig. 1)
Eleven clinical isolates were resistant to the ampicillin, while ten
clinical isolates showed resistance to tetracycline. Only one clinical
strain was resistant to nalidixic acid. Eleven of the isolates (five
poultry isolates and six clinical isolates) showed intermediate to
one or two of the antimicrobials tested (Figs. 1a).
All sixty isolates were positive for twelve or more of the viru-
lence genes tested (Figs.1a, 2 and 3). Only cdtB was not found in any
of the isolates. These results suggest that ser. Enteritidis from
poultry is virulent, similar to the clinical isolates that may be
capable of causing salmonellosis in humans.
The PFGE typing of sixty ser. Enteritidis isolates by XbaI resulted
in 10e12 bands and grouped into six clusters each with similarity
from 95% to 81% (Fig. 1a). The PFGE pattern with XbaI restriction
enzyme of isolates from poultry and clinical samples showed
considerable overlap. Eighteen MLVA allele profiles were detected
among the 60 clinical and environmental isolates, with the majority
of isolates displaying patterns A, B, and C (Table 3). MLVA pattern
names were assigned only to groups consisting of 3 or more iso-
lates. Most of the MLVA allele profiles had been seen before in the
PulseNet national MLVA database; however, patterns for two of the
clinical isolates (10000442 and 9001995) were new. Additionally,
23 isolates with 5 different MLVA patterns were rare in the data-
base (less than 0.6%). Analysis of the PFGE and MLVA data as a
composite dataset improved the discrimination between isolates
significantly compared to either dataset alone, dividing the isolates
into 25 different genotypes (Fig. 1b). The largest cluster of isolates
in the combined dataset consisted of 11 isolates, which was the
only genotype to contain both clinical and environmental isolates.
Several large and small plasmids were isolated from ser. Enter-
itidis. All isolates carried one or more large plasmids of approxi-
mately 58 kb and 38 kb, and one clinical isolate carried a mega
plasmid (81 kb). Three poultry isolates and one clinical isolate
carried small plasmids with sizes ranging from 3.8 to 11.7 kb
(Fig. 1a, 3a). The incompatibility (Inc) groups of plasmids were
determined by PCR-based replicon typing, which showed that most
of the clinical and poultry isolates carried a 58 kb plasmid, type
IncFIIA. The Southern hybridization of plasmids with the IncFIIA
probe showed that the 58 kb plasmid belongs to the IncFIIA type.
One of the isolates, 2481110, did not have the 58 kb plasmid (Fig. 3a,
3b) indicating that this isolate might have lost this plasmid. On the
other hand, IncL/M and IncN types were detected in ten clinical
isolates, and most of themwereresistant totwo antibiotics (Fig.1a).
The PCR methods used in this study for replicon typing did not
identify the incompatibility group of all plasmids (Carattoli et al.,
2005). These data suggest that MLVA pattern and plasmid profile
analysis can be useful in discriminating the isolates from different
Ser. Enteritidis is one of the most common serovars of Salmo-
nella that cause foodborne outbreaks in the U.S. Furthermore, Sal-
environment and the animal reservoir. Certain unspecified host
factors make humans particularly susceptible to infection (CDC,
2006). The environment, e.g., surfaces in and around poultry
houses, feed mills and egg water is the likely sources of the infec-
tion of layers and contamination of shell eggs, which are a source of
ser. Enteritidis infections in humans (Abdullah et al., 2010). Ser.
Enteritidis can survive in the environment such as dry materials
(dust), feces and animal feed and water for a long time in a dormant
state, but can multiply rapidly if a suitable environment is present
(Akhtar et al., 2010). Our study shows that ser. Enteritidis isolated
from poultry houses carried the same sixteen virulence genes
present in clinical isolates, which might play an important role in
invasion and survival in the host (Skyberg et al., 2006). These
findings confirm that the poultry house isolates are capable of
contributing to human infection. Recently, Akiyama et al. (2011)
and Mezal et al. (2013) indicated that the isolates from the envi-
ronment carried thesame virulence genesasclinical isolates, which
are capable of causing human infections.
Of the 60 ser. Enteritidis isolates examined in this study from
poultry and clinical sources, twenty one were resistant to either
ampicillin or tetracycline and one was resistant to nalidixic acid.
distribution in the
Fig. 2. Agarose gel electrophoresis of amplified DNA in the simplex PCR protocol from
S. Enteritidis strain 247110 using specific primers (Table 1) for virulence genes. Lane, 1
and 18,100 bp ladder, Lane 2 to 17 PCR products of (from left to right) pefA, tolC, msgA,
sopB, orgA, sifA, pagC, spaN, spiA, lpfC, spvB, prgH, sitC, sipB, invA, iroN virulence genes.
Fig. 3. Plasmid analysis (a) and Southern hybridization of S. Enteritidis after hybridization with a nonradioactive IncFIIA(FIIs) gene probe (b). Lane STD, size marker (supercoiled DNA
molecular weight marker, Invitrogen, Carlsbad, CA); lane 1, strain 247110; lane 2, strain 2481110; lane 3, strain 2491110; lane 4, strain 2501110; lane 5, strain 2731110; lane 6, strain
2741110; lane 7, strain 2771110; lane 8, strain 2781110; lane 9, strain 2791110; lane 10, strain 2801110; lane 11, strain 2811110; lane 12, strain 2821110; lane 13, strain 2871110.
E.H. Mezal et al. / Food Microbiology 38 (2014) 67e74
These resistant isolates were all of clinical origin whereas all
poultry isolates were pan-susceptible to antimicrobials, although
most of the isolates harbored one or more plasmids. Lower rates of
resistance in this study are in agreement with other studies that
have reported a low prevalence of antimicrobial resistance among
ser. Enteritidis isolates from different sources. Yang et al. (2002)
examined 14 and 22 strains each of S. Enteritidis and S. Typhimu-
rium from sources in South Korea and found that S. Typhimurium
isolates were extremely high (100%) compared to S. Enteritidis
PFGE is the current gold standard method used to assess relat-
edness among Salmonella isolates from different sources (Lynne
et al., 2009; Ponce et al., 2008) and for outbreak investigations
(CDC, 2010). However, PFGE exhibits limited discriminatory power
for some serotypes, including ser. Enteritidis (Boxrud et al., 2007).
The PFGE pattern of the ser. Enteritidis isolates from poultry and
clinical samples showed considerable overlap, although six main
clusters were observed. When PFGE results of poultry isolates were
compared to isolates from clinical samples, there was considerable
overlap. These results correspond with other observations of
limited discriminatory power of PFGE for ser. Enteritidis (CDC,
2010). In this situation, MLVA may add to the discrimination be-
tween isolates. This was confirmed in this study where in total 18
different profiles were detected compared to six different XbaI
PFGE restriction patterns (Fig. 1b). The 41 isolates that comprised
the most common PFGE genotype were distributed among 14
different MLVA patterns. However, even better discrimination was
achieved by combining the two methods (Fig.1b). For example, the
15 isolates that comprised the most common MLVA pattern
exhibited two different PFGE types. Additionally, some of the more
common PFGE-MLVA composite genotypes were further discrimi-
nated by the antimicrobial resistance and plasmid profiles. It can
therefore be concluded that for highly clonal organisms, such as
serovar Enteritidis, the best resolution is achieved by using a
combination of different typing methods.
The high MLVA diversity among the environmental isolates il-
lustrates the complexity of the ecology in a contaminated poultry
house. When assessing the interrelationships of the MLVA patterns
two key observations can be made: first, many of the patterns are
highly divergent from each other, such as patterns A and F, indi-
cating that multiple different strains co-exist in the poultry house;
this suggests multiple contamination events and sources; second,
some of the patterns, such as patterns D and F, can be considered
close variants of each other differing at a single highly variable
locus by one to two repeats. This suggests that the strains, once
established in the poultry house, evolve over time.
Plasmid profiling has proved to be helpful fordifferentiation and
characterization of Salmonella serovars (Olsen et al., 1994). In our
study most isolates carried one or more large plasmids of approx-
imately 58 kb and 38 kb, with four isolates possessing small plas-
mids. These results correspond closely to those previously reported
(Liebisch and Schwarz,1996). Additionally, Bichleret al. (1994) have
reported the presence of a 54e57 kb plasmid in ser. Enteritidis.
The Southern hybridization experiments revealed that the 58 kb
plasmid belongs to type IncFIIA in all of the tested ser. Enteritidis
isolates. These results correlate with those of the previous study by
Rychlik et al. (2006). Ten of the clinical isolates were resistant to
ampicillin and tetracycline, which belong to two incompatibility
groups (plasmids L/M and N). Several investigators have shown that
Salmonella strains resistant to antimicrobials (blaCTX-M-3 and
blaSHV-5 genes) have plasmids of these replicon types (Preston
et al., 2003; Carattoli, 2009). These results suggest plasmid profile
analysis and typing is a useful and reliable tool for discriminating
isolates during outbreaks caused by ser. Enteritidis.
The similarities in virulence genotypes between isolates poultry
and clinical indicates that the isolates of poultry are capable of
causing human infection through contaminated egg shells, which
are the most common source for ser. Enteritidis (Patrick et al.,
2004). Most clinical and poultry isolates of ser. Enteritidis belong
to one of very few PFGE patterns, and MLVA is a powerful com-
plementary technique to PFGE to further discriminate these iso-
lates. Furthermore, the plasmid profilesareuseful and an important
epidemiological tool to discriminate ser. Enteritidis strains from
various sources. This report and national antimicrobial resistance
monitoring system (NARMS) shows that there is a low prevalence
of antibiotic resistance among ser. Enteritidis isolates from poultry
and clinical sources.
We thank Drs. John B. Sutherland, M. S. Nawaz, and Carl E.
Cerniglia for critical review of the manuscript. The authors would
like to thank Christine Summage-West for technical help, Gwen-
dolyn Anderson and Stephanie Horton for Salmonella isolates. The
authors would like to acknowledge Alessandra Carattoli, Steven
Foley, and Rebecca L. Lindseyfor replicon typing control strains. The
views presented in this article do notnecessarily reflect those of the
US Food and Drug Administration.
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