Download Isolation, Identification, and Enumeration of Pathogenic Salmonella

Isolation, Identification, and Enumeration of Pathogenic Salmonella Serovars
from Environmental Waters
Timothy M. Smith Jr.
Senior Honors Research Proposal
Cook-Cole College of Arts and Sciences
Department of Biological and Environmental Sciences
Sponsored by Dr. David W. Buckalew
Proposal Submitted on January 3, 2012
Spring 2012-Fall 2012
Thesis supervisor: ___________________________________________
(Dr. David W. Buckalew)
BES Department Chair: _______________________________________
(Dr. Mark L. Fink)
Introduction
Since the acceptance of the Germ Theory of Disease, society has been interested in water
quality and its influence on public health. Awareness of causal links to contaminated drinking
water and disease led to questions about the best gauge or indicators of water quality. As early
as 1880, Von Fritsch suggested the use of Klebsiella pneumoniae and K. rhinoscleromatis as
suitable indicators due to their presence in human feces (Geldreich, 1978). Five years later,
Theodor Escherich discovered Bacillus coli (now Escherichia coli) whose presence was also
observed in high numbers in the feces of warm blooded animals (Escherich, 1885). Several
workers at that time argued for the use of total coliform bacteria as the gauge of fecal
contamination in water (Hutchinson and Ridgway 1977). However, in 1905, Alfred MacConkey
proclaimed that many coliforms in water were not of fecal origin (MacConkey, 1905). This led
scientists to focus their attention mainly on E. coli while using total coliforms as a very loose
guideline, although coliform bacteria are still the focus of many current water quality assays
depending on location.
Water quality is of as much interest now as it was in the mid-to-late 1800’s when Pasteur and
Koch were helping postulate the Germ Theory of Disease. Since then, there have been many
discoveries of pathogens that are transmitted via the fecal-oral route. Some of these
pathogens include: hemorrhagic E. coli O157:H7, Campylobacter jejuni, Salmonella enterica,
enteric viruses, and certain protozoans, to name a few -- all of which cause some form of
gastroenteritis which, in some cases, can be fatal. This understanding has increased both the
interest and the need to ensure safe water quality for human contact.
Of these potential human pathogens, this study will mainly focus on the clinically relevant
serovars of Salmonella. The taxonomy of Salmonella is somewhat complex. The current
taxonomic scheme consists of the genus Salmonela containing two species being S. enterica and
S. bongori (Bhaduri et al. 2009). Within the species S. enterica, there are seven groups or
subspecies (ssp.) which are further composed of serovars. The main pathogens for humans are
found in group 1 which is Salmonella enterica ssp. enterica (Miljkovid-Selimovid et al. 2010).
Within this subspecies, there are many serovars, but the two serovars that are clinically most
important are serovars Typhimurium and Enteritidis (Madigan et al. 2006). According to the
World Health Organization (2005), 61% of reported worldwide cases of salmonellosis are
contributed to the serovar Enteritidis while serovar Typhimurium contributes to 18% of
worldwide cases.
The primary question posed by this study is: To what degree can potentially pathogenic
Salmonella serovars be found in our local waterways? Most of the available environmental
literature focuses on the entire genus of Salmonella without specificity towards pathogenic
serovars. This study will attempt to identify the presence of S. enterica ssp. enterica serovars
Typhimurium, Enteritidis, Hadar, and Heidelberg. To accomplish this task, molecular (nuclearacid based) characteristics are to be assessed by multiplex polymerase chain reaction (MPCR)
schemes to distinguish amongst the serovars (Sanchez 2006).
A second question posed by this study is: How effective is the presence of indicator bacteria,
specifically E. coli, at predicting the simultaneous presence of pathogenic Salmonella? This is a
particularly interesting question due to the seemingly contradictory findings in the literature.
Ahmed et al. (2008) and Schriewer et al. (2010) correlated fecal indicators, including E. coli,
with potential pathogens, including Salmonella spp., concluding that there was poor correlation
between the two groups while others such as Krometis et al. (2004) have shown a significant
correlation between the two groups.
Project Overview
Hypotheses
The primary goal of this research study is to isolate and enumerate potentially pathogenic
Salmonella serovars from environmental water samples to examine the proportion of them to
all Salmonella found. The primary hypothesis I propose to test is:
H01: There are no pathogenic Salmonella serovars in the local waterways or the
numbers of these pathogenic serovars are too low to be detected.
The secondary goal of this study is to examine the correlation between an indicator bacterium,
such as E. coli, and pathogenic Salmonella serovars. The secondary hypothesis I propose to test
is:
H02: There is no correlation between the amount of specific pathogenic
Salmonella serovars and the amount of E. coli in the three stream sample
locations of this study.
Preliminary Work
Beginning in January 2011, work began on determining how to best isolate, enumerate, and
confirm Salmonella spp. from environmental waters. Water samples were collected from three
water sources around the Farmville area: Appomattox River at the Rt. 45 bridge (APP2), Sayler’s
Creek at the Rt. 620 bridge (SAY5), and Green Creek at the Rt. 600 (GRE16). After raw water
samples were processed by membrane filtration, different primary enrichment media were
tried to determine which worked best in selecting for Salmonella spp before deciding on
tetrathionate (Oxoid) enrichment broth enhanced with novobiocin (TT-n) for 8 hours followed
by a differential incubation using brilliant green bile (BGB) broth (Merck).
The proceeding months focused on gaining experience in identifying the Salmonella colonies,
enumeration techniques to improve identification accuracy, as well as performing and analyzing
confirmatory tests. Data was continuously analyzed to best discern which colonial phenotypes
typically test positive for being Salmonella.
This experience has been utilized to begin a library of serologically confirmed Salmonella
isolates for future serotyping. New isolates will continue to be added to the library throughout
the study as they are detected.
Generalized Process
In order to better appreciate the methodology of this study, a general overview of the steps
from sample collection to serotype identification is given below:
Obtain a confirmed Salmonella isolate
o Raw water samples are collected from 3 environmental water sources and
returned to the lab according to published standardized protocols
o One mL aliquots of raw water samples are diluted with sterile, buffered matrix
water and vacuum filtered, onto filter membranes with 0.45 um pore size
o The filter membrane is placed on selective TT-n enrichment media and incubated
for 8 hrs
o The membrane is then transferred to BGB diagnostic broth and incubated 24 hrs
o Colonies with physical characteristics of Salmonella are enumerated and
transferred to Triple Sugar Iron (TSI) agar slants for confirmatory testing
o After 48 hrs of incubation, the TSI’s are subjected to a polyclonal
immunoglobulin (antibody) agglutination test to serologically determine identity
o TSI cultures confirmed as being Salmonella are transferred to sterile nutrient
broth (NB) tubes and added to the isolate library
Determine if the Salmonella isolate is one of the group 1 serotypes being identified
o Isolates are cultured in Luria-Bertani (LB) broth, centrifuged, then subjected to a
a DNA extraction kit in order to obtain the genomic DNA (gDNA)
o Extracted gDNA is then combined with a PCR kit and primers and subjected to a
Multiplex Polymerase Chain Reaction (MPCR) in a thermocycler to amplify
known DNA sequences that differ amongst Salmonella group 1 serovars
o Amplified DNA sequences are then subjected to a Pulsed-Field Gel
Electrophoresis (PFGE) containing both the amplified product and 100 base pair
(bp) molecular weight marker to determine the length of the amplicons
o Completed gels are stained further if necessary and then viewed under Ultraviolet (UV) irradiation to detect bands
o Banding patterns are analyzed to identify if the Salmonella sample is one of the
serovars of interest
Methodology
Creating the Salmonella Isolate Library
Water samples are collected from three locations: Appomattox River (APP2), Saylor’s Creek
(SAY5), and Green Creek (GRE16). Each sampling location was chosen as they reveal a variable
record of high indicator bacterial presence. The samples are collected by lowering a sterile
container mid-column into the streams, avoiding the uptake of autochthonous debris. Samples
are then placed on ice and transported back to the laboratory for processing.
In the laboratory, the samples are assayed via membrane filtration. One milliliter (mL) of
sample is diluted with sterile, buffered water and filtered through a 0.45 um pore size filter
membrane (Millipore, Bedford, MA) and transferred to a 50 mm petri plate containing 1.5 mL
of tetrathionate enrichment broth (Oxoid, United Kingdom) enhanced with novobiocin (Merck,
Whitehouse Station, NJ) (40 mg/L) and incubated at 35oC for 6-8 hours. The sample is then
transferred to a 50 mm petri plate containing 1.5 mL of sterile BGB broth (Remel, Lanexa, KS)
and incubated an additional 24 hrs at 35oC.
The plates are then examined for all colony forming units (CFU) that are thought to be
Salmonella spp. based upon colonial phenotype (i.e. color and morphology) -- all presumed
colonies are enumerated. Representative CFU’s of each different colonial phenotype are
photographed and then aseptically transferred to TSI agar slants for further diagnostic testing
and for later serological confirmation. TSI agar slants are incubated at 35oC for 48 hrs.
After the incubation period, acid reaction, CO2 production, H2S production, and growth
morphology are recorded from the TSI tubes. Aseptically obtained samples from the TSI tubes
are then subjected to serologic confirmation using Oxoid Rapid Salmonella Antibody Beads™.
Oxoid Rapid Salmonella Antibody agglutination is performed by mixing a loop of suspect
bacteria into a polyclonal antibody mixture for ten seconds on a card, which is then tilted back
and forth for an additional minute or so as per manufacturer’s instructions. Agglutination of the
beads signifies a positive test for Salmonella spp.
A bacterial isolate from TSI agar slants revealing positive agglutination results is transferred to
NB to be added to the isolate library.
Enumeration of E. coli
The same water samples are split for the assessment of total coliforms and E. coli via Defined
Substrate Test using the Colilert (Idexx, Westbrook, ME) Quanti-tray 2000 system. Twenty-five
mL of water sample is diluted with 75 mL of sterile, buffered water and then processed based
on the manufacturer’s instructions and incubated at 44.5° ± 0.5° C for 24 ± 2 hours.
After incubation, the numbers of wells presenting chromogenicity (yellow coloration) are
counted for total coliform enumeration. The trays are then subjected to long wavelength UV
light (365 nm) to count the fluorescing wells for E. coli enumeration. The results are compared
to a Most Probable Number based system with a quantification range of <1 to 9,680 CFU per
100 mL when using a 25 mL sample dilution. According to Edberg et al. (1990) and Buckalew et
al. (2006), confirmatory testing of coliform bacteria and E. coli using Colilert media is not
necessary.
Extraction of gDNA from Isolates
An isolate is inoculated in LB broth and incubated at 37 oC for at least 18 hrs. Genomic DNA of
the isolate is then extracted using a DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany), or
a similar kit, according to the manufacturer’s instructions (modified from Si Hong et al. 2009).
The cell lysate is then centrifuged at full speed (13,000 rpm for 5 min) to pellet the cellular
debris. Two uL of the supernatant is used as a template for the MPCR modified from Jamshidi et
al. (2009).
MPCR Primer Design
In order to distinguish between different bacteria or groups of bacteria, researchers often look
for unique differences between the genomes of the bacteria. To differentiate between the
serogroups of Salmonella, the genetic code within each serogroup that codes for certain
antigens (a foreign structure that invokes an immune response) is often chosen as the
differentiation factor (DF). To view genetic differences, a variety of synthesized primers are
utilized in the MPCR. This study will adopt the serogroup-specific primer sequences that were
used by Hong et al. (2008).
Hong et al. (2008) chose the O antigen (part of the lipopolysaccaride on the surface of gram
negative bacteria), H1 antigen, and H2 antigen (H antigen proteins are associated with the
flagella of bacteria) as DF’s for pathogenic Salmonella serogroupings. The genetic differences
between the alleles that code for the O, H1, and H2 antigens amongst the serogroups may be
exploited to differentiate them because the O, H1, and H2 antigens all have several possible
alleles (Joys, 1985; Samuel and Reeves, 2003). For the antigenic formulas for each S. enterica
serovar see Table 1.
To develop the specific primers needed to isolate the sequence of nucleotides that code for
these DF’s, the serogroup-specific wba operon (related to the O antigen), fliC allele (related to
H1 antigen), and fliB allele (related to H2 antigen) were compared amongst serogroups to
identify portions of sequences that consistently differed between groups. The specific primer
sequence along with its recognized antigenic allele can be seen in Table 2. Primers for this study
are to be synthesized by Integrated DNA Technologies®, Inc. (US) or a similar company.
Table 1: Antigenic allele formula key for S. enterica serovars identifying O alleles A,B,C1,C2,D1,E1; H1 alleles
i,gm,r,z10; H2 alleles 1,2,enx – Adapted from Hong et al. (2008)
O
H1
H2
S. enterica serovar
A
B
B
B
B
B
B
B
B
B
B
B
B
B
B
C1
C1
C1
C1
C1
C1
C1
C1
C1
C1
C1
C1
C2
C2
C2
C2
D1
D1
D1
D1
D1
D1
D1
D1
E1
E1
E1
E1
a
b
e,h
e,h
f,g
i
l,v
l,v
b
e,h
f,g,s
r
z
z
z10
b
c
c
d
g,m,s
k
m,t
z29
e,h
r
z10
z28
d
e,h
i
z10
a
a
g,m
g,p
l,v
f,g,t
l,z28
e,h
l,v
e,h
l,v
l,5
l,2
l,2
l,5
l,2
l,7
e,n,z15
e,n,x
l,2
l,5
l,7
l,2
l,w
l,5
l,5
l,w
l,5
e,n,z15
l,5
e,n,z15
l,2
l,2
z6
e,n,x
l,5
l,5
l,5
l,5
l,5
l,7
l,6
l,6
Paratyphi A
Paratyphi B
Saintpaul
Reading
Derby
Typhimurium
Bredeney
Brandenburg
Java
Chester
Agona
Heidelburg
Kiambu
Indiana
Haifa
Ohio
Choleraesuis
Paratyphi C
Livingstone
Montevideo
Thompson
Oranienburg
Tennessee
Braenderup
Infantis
Mbandaka
Lille
Muenchen
Newport
Kentucky
Hadar
Miami
Sendai
Enteritidis
Dublin
Panama
Gallinarum
Berta
Javiana
Meunster
Give
Anatum
London
Table 2: Forward (F) and reverse (R) primer sequences used in MPCR – Adapted from Hong et al. (2008)
Target gene
O-antigen multiplex
abe1 (B)
Primer sequence
F: GGCTTCCGGCTTTATTGG
Expected amplicon size
561
R: TCTCTTATCTGTTCGCCTGTTG
wbaD-manC (C1)
F: ATTTGCCCAGTTCGGTTTG
341
R: CCATAACCGACTTCCATTTCC
abe2 (C2)
F: CGTCCTATAACCGAGCCAAC
397
R: CTGCTTTATCCCTCTCACCG
prt (A/D1)
F: ATGGGAGCGTTTGGGTTC
624
R: CGCCTCTCCACTACCAACTTC
wzx –
wzy (E1)
F: GATAGCAACGTTCGGAAATTC
281
R: CCCAATAGCAATAAACCAAGC
H1-1 Multiplex
fliC (i)
F:
AACGAAATCAACAACAACCTGC
508
R: TAGCCATCTTTACCAGTTCCC
fliC (g,m)
F: GCAGCAGCACCGGATAAAG
309
R: CATTAACATCCGTCGCGCTAG
H1-2 Multiplex
fliC (r)
F: CCTGCTATTACTGGTGATC
169
R: GTTGAAGGGAAGCCAGCAG
fliC (z10)
F: GCACTGGCGTTACTCAATCTC
363
R: GCATCAGCAATACCACTCGC
H2 Multiplex
fljB (I:
1,2; 1,5; 1,6; 1,7)
F: AGAAAGCGTATGATGTGAAA
294
R: ATTGTGGTTTTAGTTGCGCC
fljB (II:
e,n,x; e,n,z15)
F: TAACTGGCGATACATTGACTG
152
R: TAGCACCGAATGATACAGCC
MPCR and PFGE
While it may be possible to allelotype many of the serotypes listed in Table 1, this study will
focus on allelotyping the four serovars discussed in the introduction: Typhimurium, Enteritidis,
Hadar, and Heidelberg. Due to logistical conflicts regarding the different primers, a single MPCR
with all three major types of primers is not possible. Instead, an isolate will first undergo an
MPCR containing the O antigen primers to determine which O allele the isolate possesses. Once
the O antigen allele is established, if it matches one of the four serovars for the putative O
antigen allele (see Table 1) then a second allelotyping is performed using the appropriate H1
primer set. Again, if this matches one of the four serovars above then a third and final MPCR
using the H2 primer set is completed.
The determination of allelic presence after the MPCR’s compete is by analyzing the amplicon
banding patterns via PFGE.
For the MPCR reactions, 2 uL of extracted gDNA is added to a PCR kit such as the QIAGEN
Multiplex PCR Kit (Qiagen, Hilden, Germany) along with the appropriate primer set, all of which
is contained in a sterilized 0.5 mL PCR tube (modified from Hassanein et al. 2011). PCR kits
typically contain Taq polymerase (an enzyme that anneals nucleotides to DNA strands; capable
of surviving the high temperatures needed for DNA denaturation), free deoxynucleoside
triphosphates (used as the building blocks of amplicons), and buffers. The enzyme, nucleotides,
and buffers are collectively known as the master mix. The 0.5 mL PCR tube containing the
extracted gDNA, master mix, and appropriate primer set are placed into a thermocycler
programmed to raise and lower its internal temperature over prescribed times in a series of
cycles. The changes in temperature are to promote three major steps in MPCR: denaturation
(strands of the gDNA disassociate with one another at high temperatures), annealing (the
primers associate with the complement strand of gDNA at lower temperatures), and extension
(Taq polymerase builds the remainder of the strand containing the primer using the free
nucleotides at medium temperatures. The specific MPCR conditions for this experiment are 30
cycles of 94oC for 1 min (denaturation), 55oC for 1 min (annealing), and 72oC for 1 min
(extension) according to Hong et al. (2008).
Once the MPCR is completed, 5 uL of the product containing the amplicons has 1 uL of loading
dye added to visualize the amplicons on the agarose gel. The 6 uL of solution, 6 uL of 100bp
marker, and 6 uL of a non-template control is loaded onto a 1.5% agarose gel submerged in 1X
Tris-acetate-EDTA buffer stained with ethidium bromide and subjected to electrophoresis for
30 min at 100 volts per centimeter. The gels are then further stained with ethidium bromide (if
needed) and photographed on a UV transilluminator (modified from Nashwa et al. 2009).
Final Analysis of Data
The first null hypothesis will be accepted or rejected based on whether or not clinically relevant
S. enterica serovars are detected via the MPCR and PFGE protocols described above. If such
serovars are indeed confirmed to be present, the colonial phenotype of that isolate will be
noted and enumerated on all collection events. A final average percentage of a pathogenic
isolate per total Salmonella (pathogenic serovar/genus) will be reported. The final average
percentage will be calculated by finding the arithmetic mean of the number of pathogenic
serovar colonies per total Salmonella colonies times 100 for each collection event. An example
would be that on 4 separate collection events 6, 8, 19, and 15 pathogenic serovar colonies were
enumerated out of 45, 59, 82, and 61 Salmonella spp. colonies respectively.
This is to say that of the Salmonella spp., roughly 6.2% are the particular pathogenic serovar.
Additional statistical tests such as Q-tests, student t-tests, and correlative analysis may also be
completed if the data permits. If applicable, the second null hypothesis will be accepted or
rejected by analyzing the correlations between the serovars and E. coli using the least squares
method of regression analysis.
Estimated Cost
Materials
Colilert envelopes and media
DNA extraction kit
Primer synthesis
MPCR kit
PFGE gels and reagents
Total:
Est. Cost ($)
350
200
250
300
200
1300
Tentative Timeline
Spring 2011
Fall 2011
December 2011/Early 2012
Spring 2012
Summer 2012
Fall 2012
December 2012
Isolate Salmonella spp. from
environmental waters
Increase accuracy of colonial phenotype
recognition for Salmonella spp.
Literature review
Proposal preparation
Continued work on accuracy
Edit/complete proposal
Oral presentation of proposal to SHR
Committee
Secure funds for the study
Order necessary materials and reagents
Isolate and serotype environmental
Salmonella spp.
Possibly begin enumerations of E. coli and
Salmonella strains
Begin outline of thesis manuscript
Continue/finish enumerations
Data compilation
Statistical analysis of data
Defend thesis
Proposed Committee Members
Dr. Dale Beach
o Molecular Biologist-Department of Biological and Environmental Sciences
 Longwood University
Farmville, VA
Dr. Amorette Barber
o Microbiologist/Immunologist-Department of Biological and Environmental
Sciences
 Longwood University
Farmville, VA
Mr. Dennis Jones
o Total Maximum Daily Load (TMDL) specialist
 U.S. Dept. of Agriculture/Natural Resource Conservation Service (NRCS)
Farmville, VA
Literature Cited
Ahmed W, Huygens F, Goonetilleke A, and T Gardner. Real-Time PCR Detection of Pathogenic
Microorganisms in Roof-Harvested Rainwater in Southeast Queensland, Australia. Appl.
Environ. Microbiol. 74(2008): 5490-5496.
Bhaduri A, S Kalaimathy, and R Sowdhamini. "Conservation And Divergence Among Salmonella
Enterica Subspecies." Infectious Disorders - Drug Targets 9.3 (2009): 248-256.
Buckalew D W, Hartman L J, Grimsley G A, Martin A E, and K M Register. "A Long-Term Study
Comparing Membrane Filtration With Colilert® Defined Substrates In Detecting Fecal Coliforms
And Escherichia Coli In Natural Waters." Journal Of Environmental Management 80.(2006): 191197.
Edberg S C, Allen M J, Smith D B, and N J Kriz. Enumeration of total coliforms and Escherichia
coli from source water by the defined substrate technology. Appl. Environ. Microbiol.
56(1990):366–369.
Escherich, T. Die Darmbakterien des Neugeborenen und Säuglings. Fortschr.
Med. 3.(1885):515-522, 547-554. (In German.)
Geldreich, E E. Bacterial populations and indicator concepts in feces, sewage, stormwater and
solid wastes. In Indicators of Viruses in Water and Food (ed. G. Berg) 1978, p. 51-97. Ann Arbor
Science, Ann Arbor, MI.
Hassanein R, Ali S, Abd El-Malek A, Mohamed M, and K. Elsayh. "Detection And Identification
Of Salmonella Species In Minced Beef And Chicken Meats By Using Multiplex PCR In Assiut
City." Veterinary World 4.1 (2011): 5-11.
Hong Y, Liu T, Lee M, Hofacre C, Maier M, White D, Ayers S, Wang L, Berghaus R, & J. Maurer.
"Rapid Screening Of Salmonella Enterica Serovars Enteritidis, Hadar, Heidelberg And
Typhimurium Using A Serologically-Correlative Allelotyping PCR Targeting The O And H Antigen
Alleles." BMC Microbiology 8.(2008): 178.
Hutchinson, M and J W Ridgway. Microbiological Aspects of Drinking Water
Supplies 1977, p. 180. Academic Press, London.
Jamshidi A, G A Kalidari, and M Hedayati. "Isolation And Identification Of Salmonella
Enteritidis And Salmonella Typhimurium From The Eggs Of Retail Stores In Mashhad, Iran Using
Conventional Culture Method And Multiplex Pcr Assay." Journal Of Food Safety 30.3 (2010):
558-568.
Joys, T M. "The Covalent Structure Of The Phase-1 Flagellar Filament Protein Of Salmonella
Typhimurium And Its Comparison With Other Flagellins." The Journal Of Biological Chemistry
260.29 (1985): 15758-15761.
Krometis L, Characklis G, Drummey P, and Sobsey, M. "Comparison Of The Presence And
Partitioning Behavior Of Indicator Organisms And Salmonella Spp. In An Urban Watershed."
Journal Of Water & Health 8.1 (2010): 44-59.
MacConkey, A T. Lactose-fermenting bacteria in faeces. J. Hyg. 5.(1905):333.
Madigan, M. T, J.M. Martinko, D.A. Stahl, and D.P. Clark. Brock Biology of Microorganisms
13.(2006), p. 1036. Pearson Education, Inc. San Francisco, CA.
Miljković-Selimović B, T Babić, and P Stojanović. "Salmonella Enterica Subspecies Enterica
Serovar Enteritidis - Actualities And Importance." Acta Medica Medianae 49.3 (2010): 71-75.
Nashwa, M H, A H Mahmoud, and S A Sami. "Application Of Multiplex Polymerase Chain
Reaction (MPCR) For Identification And Characterization Of Salmonella Enteritidis And
Salmonella Typhimurium." Journal Of Applied Sciences Research 5.12 (2009): 2343-2348.
Samuel, G and P Reeves. "Perspective/Review: Biosynthesis Of O-Antigens: Genes And
Pathways Involved In Nucleotide Sugar Precursor Synthesis And O-Antigen Assembly."
Carbohydrate Research 338.Bacterial Antigens and Vaccines (2003): 2503-2519.
Sanchez S. Making PCR a normal routine of the food microbiology lab. PCR Methods in Foods
2006, p. 51-68. Accessed by Google books on Dec. 19, 2011: http://books.google.com/books
Schriewer A, et al. Presence of Bacteroidales as a predictor of pathogens in surface waters of
the central California coast. Appl. Environ. Microbiol. 76(2010):5802-5814.
Si Hong P, Hyun Joong K, Woo Hee C, Jae Hwan K, Mi Hwa O, Sung Hun K, Bok Kwon L, Ricke
S, and K. Hae Yeong. "Identification Of Salmonella Enterica Subspecies I, Salmonella Enterica
Serovars Typhimurium, Enteritidis And Typhi Using Multiplex PCR." FEMS Microbiology Letters
301.1 (2009): 137-146.
WHO. Global Salmonella Survey, Progress Report. 2005. Accessed on Dec. 16, 2011:
http://www.who.int/salmsurv/links/GSSProgressReport2005.pdf