afab volume 5 issue 2
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This journal is a peer reviewed scientific forum for the latest advancements in bacteriology research on a wide range of topics including food safety, food microbiology, gut microbiology, biofuels, bioremediation, environmental microbiology, fermentation, probiotics, and veterinary microbiology.TRANSCRIPT
Volume 5 Issue 22015
ISSN: 2159-8967www.AFABjournal.com
52 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 5, Issue 2 - 2015
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 5, Issue 2 - 2015 53
Sooyoun Ahn University of Florida, USA
Walid Q. Alali University of Georgia, USA
Kenneth M. Bischoff NCAUR, USDA-ARS, USA
Debabrata Biswas University of Maryland, USA
Claudia S. Dunkley University of Georgia, USA
Michael Flythe USDA, Agricultural Research Service
Lawrence Goodridge McGill University, Canada
Leluo Guan University of Alberta, Canada
Joshua Gurtler ERRC, USDA-ARS, USA
Yong D. Hang Cornell University, USA
Armitra Jackson-Davis Alabama A&M University, USA
Divya Jaroni Oklahoma State University, USA
Weihong Jiang Shanghai Institute for Biol. Sciences, P.R. China
Michael Johnson University of Arkansas, USA
Timothy Kelly East Carolina University, USA
William R. Kenealy Mascoma Corporation, USA
Hae-Yeong Kim Kyung Hee University, South Korea
Woo-Kyun Kim University of Georgia, USA
M.B. Kirkham Kansas State University, USA
Todd Kostman University of Wisconsin, Oshkosh, USA
Y. M. Kwon University of Arkansas, USA
Maria Luz Sanz Murias Instituto de Quimica Organic General, Spain
Byeng R. Min Tuskegee University in Tuskegee, AL
Melanie R. Mormile Missouri University of Science and Tech., USA
Rama Nannapaneni Mississippi State University, USA
Jack A. Neal, Jr. University of Houston, USA
Benedict Okeke Auburn University at Montgomery, USA
John Patterson Purdue University, USA
Toni Poole FFSRU, USDA-ARS, USA
Marcos Rostagno LBRU, USDA-ARS, USA
Roni Shapira Hebrew University of Jerusalem, Israel
Kalidas Shetty North Dakota State University, USA
EDITORIAL BOARD
54 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 5, Issue 2 - 2015
EDITOR-IN-CHIEFSteven C. RickeUniversity of Arkansas, USA
EDITORSTodd R. CallawayFFSRU, USADA-ARS, USA
Philip G. CrandallUniversity of Arkansas, USA
Janet Donaldson Mississippi State University, USA
Ok-Kyung KooKorea Food Research Institute, South Korea
MANAGING and LAYOUT EDITOREllen J. Van LooGhent, Belgium
TECHNICAL EDITORJessica C. ShabaturaFayetteville, USA
ONLINE EDITION EDITORC.S. ShabaturaFayetteville, USA
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EDITORIAL STAFF
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 5, Issue 2 - 2015 55
A Surveillance of Cantaloupe Genotypes for the Prevalence of Listeria and Salmonella
G. Dev Kumar, K. Crosby, D. Leskovar, H. Bang, G.K. Jayaprakasha, B. Patil, and S. Ravishankar
73
The Efficacy of a Commercial Antimicrobial for Inhibiting Salmonella in Pet Food C. A. O’Bryan, C. L. Hemminger, P. M. Rubinelli, O. Kyung Koo, R. S. Story, P. G. Crandall, and S. C. Ricke
65
ARTICLES
The Mutating Gastrointestinal Flora, Multidrug Resistant Enterococcus faeciumA. Limayem
56
BRIEF COMMUNICATIONS
Instructions for Authors87
Introduction to Authors
The publishers do not warrant the accuracy of the articles in this journal, nor any views or opinions by their authors.
TABLE OF CONTENTS
56 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 5, Issue 2 - 2015
www.afabjournal.comCopyright © 2015
Agriculture, Food and Analytical Bacteriology
ABSTRACT
Currently, 80% of the antibiotics used in the United States (U.S.) are dedicated to agricultural systems,
primarily to promote animal livestock growth and control microbial contaminant load at slaughter. More-
over, 87% of drug resistant microorganisms including Enterococcus faecium were detected in ground tur-
key products due to antibiotic agent usage namely, virginiamycin. Given that quinupristin/dalfopristin (QD),
like virginiamycin (VIR), another mixture of streptogramin has been used in hospitals as a last alternative to
treat immunocompromised population infected by vancomycin-resistant E. faecium (VRE). Consequently,
understanding the epidemiology and the antibiotic resistance in some E. faecium strains from food ani-
mals’ to humans is a matter of great concern that urges effective strategies to intervention. This review
encompasses the most prominent knowledge on the ecology and dissemination of the multidrug resistant
E. faecium (MEF). Beneficial attributes of some E. faecium strains are also reviewed. Future directions in-
cluding mitigation strategies through systemic and molecular approaches are suggested.
Keywords: Multidrug resistant E. faecium, Fecal indicators, Farm animals, Food chain, Hospitals,
Antimicrobials, Virginiamycin, Vancomycin, Immunocompromised
INTRODUCTION
Increase in multi-drug resistance jeopardizes hu-
man and animal health in the U.S. and worldwide
(Centers for Disease Control (CDC), 2013). Vanco-
mycin resistant strains of Enterococcus including pri-
Correspondence: Alya Limayem, [email protected]: +1 -813-974-7404
marily E. faecium causes hospital-acquired infections
of approximately 20,000 and the death of 1,300 per
year in the U.S (CDC, 2013). Some strains of Entero-
coccus faecium, previously known as Streptococcus
faecium started to emerge as a nosocomial VRE in
1984 (Nannini and Murray, 2006). Clonal Complex
strains (CCs), predominantly (CC17) cause urinary
tract and bloodstream infections among patients
in hospitals (CDC, 2013). Resistant (CC17) could
The mutating gastrointestinal flora, multidrug resistant Enterococcus faecium
A. Limayem1
1 Department of Cell Biology, Microbiology and Molecular Biology, University of South Florida, 4202 East Fowler Avenue, Tampa, FL 33620, USA
Agric. Food Anal. Bacteriol. 5: 56-64, 2015
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 5, Issue 2 - 2015 57
further lead to endocarditis and death in immuno-
compromised populations (Lebreton et al., 2013).
Enterococci can be carried on the hands of health
care workers, transferred from one patient to anoth-
er and can persist for up to 60 minutes (Gilmore et
al., 2002). Transmission from a health care worker’s
hands to the patient could take place upon contact
with the patient’s intravenous or urinary catheters.
This can result in colonization of the patient’s GI
tract with the acquired strain, which then becomes
part of the patient’s endogenous flora. The acquired
strain, carrying antibiotic resistance genes, is able to
live in the GI tract. Infections then arise from these
newly acquired strains, most commonly of the uri-
nary tract producing cystisis, prostatitis, and epididy-
mitis (Gilmore et al., 2002). This study demonstrated
that Enterococci are also found in intra-abdominal,
pelvic, and soft tissue infections and can cause noso-
comial bacteremia. However, endocarditis is consid-
ered the most serious enterococcal infection, as it
causes inflammation of the heart valves. In many cas-
es of endocarditis, antibiotic treatment fails and sur-
gery to remove the infected valve is necessary. Due
to the substantial multidrug resistance of the leading
nosocomial E. faecium, treatment of these infections
at an early stage is difficult. An estimated of 10,000
of vancomycin resistant E. faecium infections and
650 deaths occur in the U.S. each year (CDC, 2013).
Over the past 20 years, the incidence of multidrug
resistant E. faecium has significantly increased; 77%
of enterococcal bloodstream infections involve this
microorganism (CDC, 2013). The immune-deficient
populations including patients affected by hema-
tologic malignancies are considered at high-risk of
MEF exposure. Patients subjected to Intensive Care
Unit (ICU), organ transplants and prolonged stays
in hospitals are also of prime concern (Zhou et al.,
2013). MEF infections have also been associated
with surgical wounds from indwelling catheter use
(Ryan, 2004). In the U.S., the level of VRE has dramat-
ically climbed in the last 27 years to achieve an un-
precedented increase of 80% (CDC, 2013). As early
as 1980, the VRE epidemic began in Europe and was
partially correlated to the extensive use of avoparcin
in farm animals (Arias and Murray, 2012). Despite the
ban on the usage of avoparcin in livestock, there has
been a noticeable increase of VRE infections in some
European hospitals (Lebreton et al., 2013; Top et al.,
2008). In the U.S., in spite of focused awareness from
federal administrations for controlling drug use in
livestock, there is no action for similar ban in agricul-
ture use. Virginiamycin has been used in agricultural
system for growth promotion and bacterial control of
farm animals (Barton et al., 2003; Claycamp and Hoo-
berman, 2004). As such, without a similar strepto-
gramin ban, MEF strains would disseminate in food
animal products (Barton et al., 2003). As such, anti-
biotic resistance would disseminate in farming ani-
mals and subsequently pass through the food chain
to humans (Busani et al., 2004; Hayes et al., 2004;
Tejedor-Junco et al., 2005). Recently Limayem et al.
(2015) reported that out of 30 ground turkey samples
collected from multiple grocery stores, there were
27.7% multidrug resistant E. faecium strains. All the
isolated MEF strains were resistant to QD and its
homologous virginiamycin. Several factors impact
this threat of further dissemination of resistant E.
faecium. This strain has an unrelenting ability to mu-
tate and generate intrinsic and extrinsic mechanisms
of resistance to a broad continuum of antibiotics.
(Tremblay et al., 2011). Moreover, MEF has a tremen-
dous capability to gain drug resistance via conjuga-
tion mechanisms or gene transfer from other micro-
organisms (Werner et al., 2000). Aside from its gene
permutation properties, the phenotypic versatility of
MEF enables the gene to occupy different locations
in the cell including plasmids, integrons and operons
(Tremblay et al., 2011).
Such alarming evidence for antibiotic resistance
spread, propelled the need to ensure greater knowl-
edge on the ecology, epidemiology and antibiotic
resistance of some E. faecium strains from food ani-
mals to clinical settings. Beneficial attributes of some
E. faecium strains are also discussed. Further stud-
ies on the risk from resistant E. faecium strains are
suggested to ensure greater preventive actions and
public health safety.
58 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 5, Issue 2 - 2015
ECOLOGY AND SPECIFIC CHARACTER-ISTICS
E. faecium has a spherical shape and is a Gram-
positive facultative anaerobic microbe that grows
over a large temperature range, from 10 to over
45°C, where the optimal temperature for growth
is 42.7°C. Some strains of E. faecium survive harsh
environmental conditions that include acidic en-
vironments and exposure to detergents (Jackson
et al., 2005). Enterococci in general exhibit similar
physiology to Streptococci with the discrimination
established primarily by the Lancefield group D
antigen and secondarily by growth in high salt con-
centrations (Fisher and Phillips, 2009). E. faecium’s
remarkable phenotypic elasticity and intrinsic ability
to generate resistance or virulence genes through
chromosomal exchanges, plasmids transfers, and
mutations, give rise to the possibility of pathoge-
nicity (Davies et al., 2010). Moreover, E. faecium is
able to survive the heating process used during the
making of foods such as sausage and cheese. With
its high resistance to salt (6.5%), the ubiquitous E.
faecium strain can survive in marine environments for
a long period of time (Hardwood et al., 2000). It has
been reported by Fisher and Phillips (2009) that sub-
stantial multidrug resistant E. faecium strains have
been observed in numerous aquatic environment
and pristine waters (Rice et al., 1995; Valenzuela et
al., 2010). Consequently, several strains of E. faecium
have been found in seafood including shellfish along
with fishes and brined nordic shrimps (Mejlholm et
al., 2008; Thapa et al., 2006; Valenzuela et al., 2010).
Although most of the E. faecium strains inhabit the
warm-blooded animal gut, there are some strains
that have been isolated from oral cavities and vagi-
nal tracts of humans and animals (Rice et al., 1995).
E. faecium was also isolated from a wide range of
surfaces including water, soil, and mechanical equip-
ment such as medical and agricultural devices (klein,
2003). There are some strains of E. faecium that can
survive on inanimate objects for up to four months
(Kramer et al., 2006; Lebreton et al., 2013). In Europe,
E. faecium strains have been commonly isolated
from pets, farm animals, and food products (Frei-
tas et al., 2011; Garcia-Migura et al., 2005; Garcia-
Migura et al., 2007). Furthermore, Enterococci have
been isolated from the runoff of farms that used pig
manure as well as urban sewage (Khun et al., 2003).
E. faecium can be found in concentrations of 104 to
105 per gram of human fecal material, making it a key
indicator for fecal contamination (Franz et al., 1999).
While E. faecium can be used to indicate contamina-
tion of water supplies from farmland, its isolation is
less prevalent from livestock than from human feces
(Franz et al., 1999).
BENEFICIAL ATTRIBUTES
Some strains of E. faecium are also lactic acid
bacteria and are known for their probiotic attributes.
They have been extensively added in food for their
fermentative ability and health benefits. It has been
shown that rabbits in animal husbandries that were
given water containing E. faecium as a probiotic had
higher average weight gains as well as a healthier
natural intestinal flora (Laukova et. al., 2012). While
E. faecium helps prevent antibiotic-associated diar-
rhea, enhance the immune system, and lower the
cholesterol level (Franz et al., 2011), other strains are
used for their food safety attributes in limiting zoo-
notic pathogens from food animals through bacte-
riocin production (Franz et al., 2011). As evidenced
by De Kwaadsteniet et al. (2005), E. faecium P21 iso-
lated from sausage produces both enterocins A and
B. Enterocins proved to be active against a substan-
tial range of Gram-positive bacteria including pri-
marily Listeria species and Staphylococcus aureus.
E. faecium, RZS C5 strain, has been isolated from
natural cheese and also demonstrates antilisterial
properties without exhibiting virulence factors (Le-
roy et al., 2003). Nonvirulent strains of E. faecium
have been suggested as a possible probiotic against
microbes possessing antimicrobial resistances (Franz
et al., 2011; Lund and Edlund, 2001). A substantial
review on the antibacterial properties of bacteriocins
has been implemented by Fisher and Phillips (2009).
However, the tremendous ability of some strains to
acquire virulence genes from other strains and con-
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 5, Issue 2 - 2015 59
vert into pathogenic strains would hinder the benefi-
cial attributes of E. faecium. This is increasingly more
problematic due to the considerable ability of E. fae-
cium to mutate and acquire virulent genes in mul-
tiple types of environment (Arias and Murray, 2012).
EPIDEMIOLOGY
The previously named Streptococcus faecium
started to evolve as a nosocomial vancomycin resis-
tant E. faecium in 1984. In the U.S., the level of VRE
has substantially increased from approximately 1 % to
80% throughout the past three decades (CDC, 2013).
Currently, the opportunistic strains of E. faecium are
being considered among the second leading causes
of hospital-associated infections (Arias and Murray,
2012). The transmission of E. faecium is via fecal-oral
route with transfer primarily through contaminated
food or water and catheter-related E. faecium in-
oculation and colonization in hospitals (Austin et al.,
1999; Sydnor and Perl, 2011). The E. faecium strain
can also form a niche in the human gut and constitute
a fetal reservoir for the susceptible immunocompro-
mised human population that have a hematological
malignancy (Zhou et al., 2013). E. faecium can cause
serious health outcomes related to wound infections,
bacteremia, and urinary tract infections that can fur-
ther lead to septicemia and endocarditis in some
cases (Teixeira et al., 2007). Symptoms, depending on
the infective dose and ranging from mild to severe
causes, most commonly involve fever and chills along
with flank pain and shortness of breath for the patient
who is affected by endocarditis (Chan et al., 2012).
As previously mentioned, in Europe, the VRE epi-
demic began in the late 1980s and was partially as-
sociated with the extensive use of avoparcin in farm-
ing animals (Arias and Murray, 2012). Wegener et al
(1999) have reported that glycopeptide avoparcin
contained high levels of vanA encoding resistance
to glycopeptide agent found on the Tn1546 trans-
poson (Wegener, 1999; Biavasco et al., 2007). The
spread of VRE infections within human populations
led to avoparcin prohibition in European countries
(Lebreton et al., 2013).
Currently the MEF strains are proliferating at a
considerable rate thus impacting the downstream
food chain as well as surrounding pristine aquatics
systems (Rice et al., 1995; Valenzuela et al., 2010).
E faecium strains that are multi-drug resistant are
evolving as the leading cause of nosocomial patho-
gens constituting a serious level of threat that has
caused almost 10,000 infections and 650 deaths
each year in the U.S. (CDC, 2013).
Typically, there are two subpopulations of E. fae-
cium (Freitas et al., 2011): (1) the commensal/com-
munity-associated (CA) strains and (2) the hospital-
associated (HA) strains, Clonal Complex 17 (CC17).
The CC17 strains gave rise to the worldwide noso-
comial VRE as the most prevalent source of entero-
coccus bloodstream and urinary tract infections. It
has putative pathogenicity island-carrying putative
glycoside hydrolase (hyl) and enterococcal surface
protein (esp) genes that enables it to adhere to the
host tissue, aggregate, form persistent biofilms, and
cause infections (Freitas et al., 2010). Additionally,
Arias and Murray (2012) extensively reviewed the
main enterococcal E. faecium’s pathogenesis and
the components causing virulence phenotypes in
vivo. It is also worth mentioning that the virulence
expression depends on both strains and the host cell
tissue. Currently, there are an increasing number of
the MEF strains beyond VRE that are growing at a
rapid pace and cause severe complications primarily
for the immune-deficient human population. It could
also lead to sudden death if the appropriate choice
of antibiotic is not determined at an early stage.
ANTIBIOTIC RESISTANCE
The high intrinsic capability and phenotypic
elasticity of the MEF strain enable it to acquire ex-
ogenous genes from the environment, mutate
continuously, and transfer resistant genes to other
pathogens within harsh environmental conditions
(Lund and Edlund, 2001). Several MEF strains are
intrinsically resistant to a wide range of antibiotics
including the aminoglycosides. As thoroughly re-
viewed by Arias and Murray (2012), the main antibi-
60 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 5, Issue 2 - 2015
otic resistance mechanisms of enterococcal strains
encompass the alteration of the target binding site
and the reduction of binding affinity (Franz et al.,
2011). Furthermore, as being homologous to QD,
virginiamycin is the last alternative used to treat VRE
in hospitals. Virginiamycin is used in large scale to
promote animal growth in farm animals and to sup-
press microbial contaminant load at slaughter (Do-
nabedian et al., 2006; Kasimoglu-Dogru et al., 2010).
The mechanisms of resistance that are used by MEF
strains against streptogramin agents involve mainly
the alteration of ribosomal sites, active efflux and the
inactivation of enzymes or drug modification (Soltani
et al., 2001). The most common enzyme inactivation
mechanism against streptogramin type A includes
O-acetyltransferase. Acetyltransferase of virginiamy-
cin agent is encoded by the gene vat. Among the
most prevalent vat genes (Simjee et al., 2006) encod-
ing streptogramin A in E. faecium are vat (D) and vat
(E) (Werner and Witte, 1999). Changes in vat (E) gene
in streptogramin resistant E. faecium due to single
base replacement, has been reported by Soltani et
al. (2001). The active efflux is noted to extrude strep-
togramins via ABC porters (Sletvold et al., 2008) en-
coded by vga (A) or vgb (B) alleles. Several studies
have reported that the erm (B) genes in enterococci
have already been found widely disseminated in the
environment (De Leener et al., 2005; Hayes et al.,
2005; Werner et al., 2000).
CONCLUSIONS-FUTURE DIRECTIONS
Extensive research studies have elucidated the
antibiotic resistance profile of VRE in human popu-
lations and hospitals (Acharya et al., 2007; Haris-
berger et al., 2011; Getachew et al., 2013; Ghidán
et al., 2008a ; Ghidán et al., 2008b ; González et al.,
2009; Jung et al., 2007; Novais et al., 2006; Poeta et
al., 2006). Additional studies have also evidenced
the resistance profile of Enterococcus genera in
food animals (Persoons et al., 2010; Pesavento et al.,
2014; Simjee et al., 2007) primarily in poultry (Fra-
calanzza et al., 2007; Ghidán et al., 2008a ; Ghidán et
al., 2008b; Schwaiger et al., 2010; Usui et al., 2014).
While numerous molecular investigations are con-
ducted on Enterococcus strains, (Aslam et al., 2012;
Cha et al., 2012; Garnier et al., 2004; Getachew et
al., 2013) including their genetic relatedness (Frei-
tas et al., 2010; González et al., 2009; Hwang et al.,
2010; Oh et al., 2007; Tremblay et al., 2011), there is
an exigency for a deeper genomic analysis to trace
connections between drug resistance profile shared
between humans and food animals in the U.S. Fur-
thermore, the rising MEF strain of its type beyond
VRE from food animals to hospitals (Getachew et al.,
2013; Pesavento et al., 2014) broad continuum is of
paramount threat level. Hence, there is an urgent
need to trace the genetic profile of MEF strains from
the source to hospitals within a comprehensive mod-
eling approach.
Further investigations including a complete char-
acterization of the MEF strain revealing rapid detec-
tion and quantification within a comprehensive risk
model will enable the development of effective miti-
gation strategies for the emerging drug resistance
in food. It thus, offers a clear insight to managers to
track the contamination pathways and set preventive
actions to ensure food and public health safety.
ACKNOWLEDGEMENTS
The author thanks the Moffitt Cancer Center
(MCC) in Florida State for their kind donation of the
multidrug resistant clinical isolates of E. faecium. The
author also extends her thanks to the National Sani-
tary Foundation Laboratory for their support in pro-
viding the poultry samples and the clinical isolates
from Michigan State.
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Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 5, Issue 2 - 2015 65
www.afabjournal.comCopyright © 2015
Agriculture, Food and Analytical Bacteriology
ABSTRACT
A commercially available antimicrobial consisting of a proprietary mixture of 5-25% (wt/vol) nonanoic
acid, 1-25% (wt/vol) butyric acid, 1-50% (wt/vol) trans-2-hexenal and water was tested for efficacy against
Gram-negative and Gram-positive bacteria, some isolates of Salmonella spp in vitro and activity against
Salmonella in pet food. The in vitro efficacy of the antimicrobial was found to be generally effective against
both Gram-positive and Gram-negative bacteria. Minimal inhibitory concentrations (MICs) were deter-
mined for isolates of Salmonella serotypes. Isolates of Heidelberg, Montevideo and Enteritidis had MICs
of 1.5 μl/ml while the other five tested isolates had MICs of 2.0 μl/ml. The effectiveness of the antimicrobial
in ground pet food artificially contaminated with a high level of Salmonella was assessed at 0, 1.0, 1.5, or
2.0 ml/kg of feed. Contaminated feed was sampled on days 0, 1, 4, 7 and 14 after treatment. All levels of
antimicrobial resulted in nearly a 1.0 log CFU/g reduction of Salmonella numbers at time of treatment, and
Salmonella levels were 2.0 log CFU/g lower at day 14 as compared to the untreated control. This antimicro-
bial would be useful in extending the shelf life of dried pet foods as well as limiting survival and growth of
Salmonella.
Keywords: Salmonella; pet food; organic acids; butyric acid; nonanoic acid; trans-2-hexenal; anti-
microbial; food safety; foodborne illness; companion animals
INTRODUCTION
Companion animals have become an increasing
aspect of the family unit in most societies. In the US
alone there are an estimated 37% of households with
at least one dog and 30% of households with a cat
Correspondence: Steven C. Ricke, [email protected]: +1 -479-575-4678
(AVMA, 2014). Most of these households feed their
pets dry food for at least part of the diet (Buchanan
et al., 2011). Many of these dry pet foods contain in-
gredients of animal origin, and thus are at risk for
contamination with Salmonella spp. Dry pet foods
are made using extrusion manufacturing in which the
combined ingredients are heated and formed into
the final product of various shapes and sizes. The
extrusion process takes place at very high tempera-
The Efficacy of a Commercial Antimicrobial for Inhibiting Salmonella in Pet Food
C. A. O’Bryan1, C. L. Hemminger1, P. M. Rubinelli1, O. Kyung Koo2, R. S. Story1, P. G. Crandall1, and S. C. Ricke1
1 Center for Food Safety and Department of Food Science, University of Arkansas, Fayetteville, AR 727042 Current address: Food Safety Research Group, Korea Food Research Institute, Seongnam-si,
Gyeonggi-do, Republic of Korea.
Agric. Food Anal. Bacteriol. 5: 65-72, 2015
66 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 5, Issue 2 - 2015
tures which acts as a kill step for pathogens. How-
ever, high temperatures also destroy some of the nu-
trients present in the food, so flavor enhancers and
fat, both of animal origin, are then sprayed on after
extrusion. However there is no additional kill step for
pathogens after this process (Thompson, 2008).
White et al. (2003) sampled randomly collected
dog treats derived from pig ears and other animal
parts in the United States and cultured them for the
presence of Salmonella. Forty-one percent of the
samples were found to be positive for Salmonella
and 24 different serotypes were isolated from the
positive samples. They isolated S. Infantis with PFGE
patterns indistinguishable from the strains respon-
sible for the 1999 Canadian outbreak from several
products. Li et al. (2012) reported on the prevalence
of Salmonella spp. in animal feeds. They isolated
Salmonella from 6.1% of pet foods and treats, and
from 7.1% of supplement-type pet products. More
recently, Nemser et al. (2014) found only 1 of 670
dry pet foods or treats were positive for Salmonella
spp. Nevertheless, Salmonella infections have been
found both in pets and in humans, and were deter-
mined to be linked to contaminated pet foods and
treats (Clark et al. 2001; CDC 2005; Behravesh et al.
2010; Imanishi et al. 2014).
In 1999 in Canada, an outbreak of Salmonella se-
rotype Infantis infections in humans was found to be
associated with pet treats for dogs produced from
processed pig ears. Phage typing and pulsed-field
gel electrophoresis (PFGE) determined that Salmo-
nella enterica serotype Infantis isolated from pig ear
pet treats as well as isolates from humans exposed
to the pig ears were the same (Clark et al., 2001).
Schotte et al. (2007) reported on a large outbreak of
canine salmonellosis in German military watch dogs.
The outbreak was recognized by a monitoring pro-
gram and was found to be due to 2 serotypes of Sal-
monella, Montevideo and Give. Dogs in 4 kennels
were exposed and 63.8% of the dogs had positive
fecal samples, although only 9 dogs exhibited clini-
cal disease. Two commercial dehydrated dog foods
were implicated by risk analysis as the suspected
infectious sources and S. Montevideo and S. Give
with similar plasmid profiles and PFGE-restriction
patterns were isolated from the suspected foods
and fecal samples. In 2012 in the United States a
routine sample collected of dry dog food was found
to be positive for S. Infantis (Imanishi et al., 2014).
The Centers for Disease Control and Prevention was
able to link the genetic fingerprint of this isolate with
humans with infections caused by S. Infantis. The
subsequent outbreak investigation identified 53 ill
humans infected with the outbreak strain in 21 states
and 2 provinces in Canada. Traceback investigations
identified one production plant as the source of the
contaminated food, and the outbreak strain was iso-
lated from unopened bags of dry dog food and fecal
specimens from dogs that had eaten the food and
lived with ill people.
These outbreaks confirm that large outbreaks of
salmonellosis occur after feeding contaminated dry
pet foods and pet treats. This also puts pet owners
and vulnerable members of their households at risk
as they often live in close contact with their animals.
These highly publicized salmonellosis outbreaks and
recalls of dry pet foods due to contamination with S.
enterica have caused a major review of microbiologi-
cal control programs, and have reinforced the idea
that food safety should extend beyond traditional
factory quality management processes. As in food
for human consumption, ensuring the microbiologi-
cal integrity of pet foods must cover the entire pro-
duction pipeline (‘farm-to-fork approach’). The study
reported in this paper was developed to determine
whether a commercial antimicrobial would restrict
the survival and growth of Salmonella in dry dog
food. The antimicrobial contains butyric acid, nona-
noic acid and trans-2-hexenal.
MATERIALS AND METHODS
Determination of antimicrobial spec-trum
Lyophilized cultures of test organisms (Salmonella
Typhimurium, Escherichia coli, Staphylococcus aure-
us, Clostridium perfringens, Lactobacillus plantarum,
Streptococcus agalactiae and Campylobacter jejuni)
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 5, Issue 2 - 2015 67
were obtained from the American Type Culture Col-
lection (ATCC; Manassas, VA). Cultures were resus-
citated according to ATCC recommended methods
and transferred to Standard Methods Agar (SMA; BD
Diagnostics, Franklin Lakes, NJ). Agar plates were in-
cubated for 24 hours at 35°C.
After incubation, bacterial colonies were trans-
ferred to individual test tubes containing 10 mL of
Trypticase Soy Broth (TSB; BD Diagnostics). Test
tubes were incubated at 35°C for 18 to 24 hours. The
level of bacteria in the broth culture was determined
by serial dilution and plating on SMA. Cultures were
diluted to a final concentration of 105 cfu ml-1 with
Butterfield’s phosphate buffer.
One mL of CO-60 surfactant and 1 mL of the an-
timicrobial (Preserv-8®; Anitox Corp., Lawrenceville,
GA) were mixed together (the surfactant was used to
allow the antimicrobial to be soluble in water for test
purposes). A 0.2 ml aliquot of the mixture was added
to 9.8 mL of sterile, deionized water to prepare a 1%
stock solution (10 ml kg-1). The stock solution was di-
luted with sterile deionized water to the equivalent of
5, 1, 0.5, 0.1 and 0.05 ml kg-1.
A 100 μL aliquot of the 105 cfu ml-1 inoculum was
added to each of the dilution tubes containing the
different concentrations of antimicrobial. Tubes were
vortexed for 30 seconds every hour for four hours.
A 1 mL aliquot was removed from each tube at 24
hours and serially diluted in Butterfield’s phosphate
buffer. Dilutions were plated on selective agars as
recommended for each type of bacteria. Plates were
incubated at 35°C for 48 hours prior to enumeration.
Clostridium, Lactobacillus and Campylobacter plates
were incubated under anaerobic conditions.
Determination of minimal inhibitory concentration
Isolates of 8 serovars of Salmonella were tested
(Heidelberg, Montevideo, Enteritidis, Typhimurium,
Worthington, Kentucky, Senftenberg and Infantis); all
isolates were obtained from the culture collection of
the Center for Food Safety of the University of Arkan-
sas. Overnight cultures were prepared by inoculating
10 ml of sterile LB broth (EMD Millipore, Billerica,
MA) with a single isolate of a serotype of Salmonella
and incubating at 37°C for 18 to 24 h. Minimal inhibi-
tory concentration (MIC) levels were determined in
96-well clear microtiter plates (NUNC, Rochester,
N.Y., U.S.A.) with lids. A stock solution of the antimi-
crobial was prepared at 1%. Prepared sterile LB broth
was aseptically pipetted (100 μl) into all wells of the
microtiter plate. A 100 μl aliquot of the antimicrobi-
al was pipetted into the first row of wells and serial
2-fold dilutions were performed to the end point of
0.25% of the antimicrobial, and 100 μl of excess solu-
tion was discarded from the last row to keep well vol-
umes equal. One row was used as a positive control
and contained 5 μl ml-1 of butyric acid; another row of
wells was used as a negative control and contained
bacteria and LB only. A 100 μL aliquot (containing
approximately 105 cfu) of a single Salmonella culture
was pipetted into each well. Microtiter plates were
incubated statically at 37°C for 18 hours and optical
density (OD) was read at 600 nm. The MIC was de-
fined as the first well that had an OD no greater than
the wells containing butyric acid. The experiment
was repeated in triplicate.
Efficacy of antimicrobial in animal feed
A culture of each serovar of Salmonella was pre-
pared by individually inoculating into 10 ml of sterile
TSB with a single serovar and incubating in a shaking
incubator at 37°C for 24 hours. One ml aliquots from
each of the 8 cultures were mixed to form a cocktail.
Cell density of the inoculum was adjusted to approxi-
mately 108 cfu ml-1.
Meat and bone meal (MBM) was used as the carri-
er matrix to inoculate the feed. The autoclaved MBM
was weighed out into 20 g aliquots and each aliquot
was mixed with 90 ml of 0.01% peptone water and
autoclaved. Four of the five samples were inoculated
with the cocktail and shaken well. All samples were
subsequently centrifuged (Beckman JR-21, Beckman
Coulter, Indianapolis, IN) for 15 minutes at 27,000 x g,
the excess peptone was poured off and the MBM was
placed into deep petri dishes, covered with a sterile
filter paper, and allowed to dry at ambient tempera-
ture for 48 hours in a biosafety cabinet.
68 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 5, Issue 2 - 2015
The inoculated MBM was scraped out from the
deep plates and placed in a stomacher bag and
stomached to a powder. An aliquot of 10 g of the
inoculum was placed with 990 g of ground dog food
(from a commercial source) in a lab scale mixer (Fig-
ure 1) and mixed for 2 minutes. The antimicrobial was
added to the inoculated feed using a nebulizer fit-
ted into the mixer and at a positive air pressure of
8 PSI. The antimicrobial was injected through a sep-
tum with a 19-gauge needle. The mixer was set to a
speed of 60 rpm and allowed to mix for 2 minutes.
Levels of antimicrobial were equivalent to 0, 5.0, 7.5
and 10 m kg-1 of feed. Each group was sampled on
days 0 (immediately after treatment), 1, 4, 7 and 14
after treatment.
For each sample of inoculated ground dog food
mixed with antimicrobial, 1 g was placed in 9 mL of
sterile 0.1 % peptone water (initial 1:10 dilution) and
further diluted to the appropriate end point by serial
dilution. An aliquot of 0.1 ml of each dilution was dis-
pensed onto duplicate xylose lysine desoxycholate
(XLD; BD Diagnostics) agar plates and spread with
a sterile spreader. Uninoculated MBM (UMBM) was
used as a negative Salmonella control, which was
cultured as described for samples. Plates were incu-
bated at 37°C for 24 hours and then enumerated for
the amount of Salmonella remaining. The entire ex-
periment was replicated three times.
RESULTS
Antimicrobial spectrum
The antimicrobial was observed to have efficacy
against both Gram-positive and Gram-negative bac-
teria (Table 1). The degree of efficacy was similar to
that obtained with formaldehyde and formic acid
under similar test conditions (Carrique-Mas et al.
2006). A 0.05% dilution (0.5 ml kg-1) of the antimicro-
bial gave 100% reduction of S. Typhimurium, E. coli,
S. aureus, S. agalactiae and C. jejuni after 24 hours
of exposure. C. perfringens and L. plantarum were
observed to be more resistant than the other organ-
isms, with C. perfringens reduced 100% at 0.1% (1
ml kg-1) and L. plantarum reduced 100% at 0.5% (5
ml kg-1).
Figure 1. Lab scale mixer used to mix antimicrobial with feed
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 5, Issue 2 - 2015 69
Minimal inhibitory concentration
Minimal inhibitory concentrations of Salmonella
serotypes varied between 1.5 μl ml-1 to 2.0 μl ml-1,
which is equivalent to 1.5 ml kg-1 and 2.0 ml kg-1 of
feed respectively.
Efficacy in pet food
The antimicrobial inhibited Salmonella survival in
the feed at all levels of application (Fig 2). Numbers
of Salmonella in the untreated control increased
from 8.1 log cfu g-1 at time 0 to 8.2 log cfu g-1 at 4
days. Numbers of Salmonella in the untreated con-
trol decreased from day 4 to day 14 with the final
number being 7.3 log cfu g-1. At time 0 all levels of
treatment had a lower count by almost 1 log cfu g-1
and within 24 hours all levels of antimicrobial were a
full 1 log cfu g-1 lower than the untreated control. At
the end of 14 days all levels were close to 2.0 log cfu
g-1 lower than the untreated feed. The UMBM was
negative for Salmonella growth.
DISCUSSION
Organic acids are often used as preservatives of
human foods (Brul and Coote, 1999) and have also
been used in poultry feed to control mold and bac-
teria (Paster et al., 1987). Treatment of poultry feed
with organic acids has been shown to have the po-
tential to reduce infection levels of Salmonella (Khan
and Katamay, 1969; Matlho et al., 1997). Any chemical
used to control Salmonella in feeds must also either
be metabolized by the animal or excreted without
absorption (Carrique-Mas et al., 2007). Hume et al.
(1993) found that organic acids used to treat poultry
feed were rapidly metabolized by the birds.
Researchers have suggested that small chain fatty
acids exhibit antimicrobial activity in the undissoci-
ated form because they are lipid permeable in this
form and can cross the microbial cell wall and dis-
sociate in the more alkaline interior of the microor-
ganism making the cytoplasm unstable for survival.
(Paster, 1979; Van Immerseel et al., 2006). Butyric
acid when used alone was been found to inhibit Sal-
Table 1. Results of efficacy testing of antimicrobial on various bacteria regularly found in pet food and animal feed. Initial inoculum was 5.0 log cfu/mL of bacterial culture. Exposure time was 24 hours
Treatment
Level
Percent reduction compared to the control
S. Ty-phimurium
E. coliS.
aureus
Cl. perfrin-gens
L.
planta-rum
S. agalac-tiae
C.
jejuni
0.005% 36 7 43 0 0.0 92.3 6.1
0.01% 59 29 39 4 0.0 98.3 59.2
0.05% 100 100 100 85 75 100 100
0.1% 100 100 100 100 99 100 100
0.5% 100 100 100 100 100 100 100
70 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 5, Issue 2 - 2015
monella (Khan and Katamay, 1969). Khan and Khata-
may (1969) found that butyric acid completely inhib-
ited the growth of Salmonella in media, and when it
was used to treat meat and bone meal artificially in-
oculated with Salmonella no viable organisms were
recovered even after a week. Nonanoic acid (also
known as pelargonic acid) is a naturally occurring
fatty acid with a faint odor compared to butyric acid
and is almost insoluble in water (EPA, 2004). Nona-
noic acid is found in a variety of fruits as well as in
dairy products, and is on the FDA generally recog-
nized as safe (GRAS) list as a synthetic food flavoring
agent, as an adjuvant, production aid and sanitizer
to be used on food contact surfaces. Very few have
studied the effects of nonanoic acid as an antimicro-
bial, but Khan and Khatamay (1969) found essentially
no activity against Salmonella artificially inoculated
into meat and bone meal.
Another volatile compound contained in the stud-
ied antimicrobial is trans-2-hexenal, which is present
in many edible plants such as apples, pears, grapes,
strawberries, kiwi, and tomatoes and has been an ef-
fective antimicrobial against Helicobacter pylori and
S. Cholerasuis (Kubo et al., 1999; 2001). Kim and Shin
(2004) found that trans-2-hexenal (247 mg/L) was
effective against Bacillus cereus, S. Typhimurium,
Vibrio parahemolyticus, Listeria monocytogenes,
Staphylococcus aureus and Escherichia coli 0157:H7.
Nakamura and Hatanaka (2002) demonstrated that
trans-2-hexenal was effective in controlling S. Ty-
phimurium at a level of 3 - 30 ug ml-1. The suggested
mode of action of trans-2 hexenal is the destruction
of electron transport systems and the perturbation
of membrane permeability (Gardini et al., 2001).
Previous research has shown that the reduction of
Salmonella in feed by treatment with organic acids
may require up to a week of contact to achieve re-
sults (Iba and Berchieri, 1995). Our results suggest a
Figure 2. Efficacy of antimicrobial against a cocktail of Salmonella inoculated into pet food. Antimicrobial was added at 0, 5.0 ml/kg, 7.5 ml/kg or 10 ml/kg of pet food. Error bars represent standard deviation from the mean.
4.5
5
5.5
6
6.5
7
7.5
8
8.5
9
9.5
0 1 4 7 14
Log
CFU
Sal
mo
nella
Day
0
5.0 mL/kg
7.5 mL/kg
10 mL/kg
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 5, Issue 2 - 2015 71
reduction of a high level of contamination with Sal-
monella within 24 hours of application by this com-
bination of organic acids with trans-2-hexenal. Addi-
tionally, the reduction was much greater after 4 days
of contact as compared to the control, where Salmo-
nella growth actually increased. Wales et al. (2013)
studied various feed treatment formulations contain-
ing organic acids and found reductions in Salmonel-
la of around 1 log unit after 7 days. They also found
that those formulations that ultimately had greater
reductions also reduced Salmonella numbers much
sooner, often within 24 hours of incorporation.
The tested antimicrobial was effective in feed at
all levels tested regardless of MIC determined in
vitro. All components are generally recognized as
safe (GRAS) in the US, and thus are approved for
use in animal feeds. This antimicrobial is a promising
new treatment to reduce Salmonella carriage in pet
foods.
ACKNOWLEDGEMENTS
This research was funded by a grant from Anitox
Corp., Lawrenceville, GA.
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www.afabjournal.comCopyright © 2015
Agriculture, Food and Analytical Bacteriology
ABSTRACT
Netting is a common characteristic in predominant cantaloupe (Cucumis melo L) varieties. Over the
past several years, Listeria and Salmonella outbreaks associated with cantaloupes have become a subject
of concern to consumers. It is hypothesized that unlike non-netted melons, the netted structure of the
cantaloupe rind could be a host for pathogens. Therefore, we investigated whether pathogen contamina-
tion in the field setting is closely associated with the netted rind. Twenty one netted cantaloupe genotypes
consisting of experimental F1 hybrids, inbred lines from the Texas A&M melon breeding program and com-
mercial cultivars were tested for the presence of Listeria spp. and Salmonella serotypes. Pathogen isolation
was performed using selective/differential media after pre-enrichment and selective enrichment. Use of
selective media resulted in the occurrence of 36.36% false positives for Listeria spp. and 16.25% false posi-
tives for Salmonella serovars. Isolates were confirmed using biochemical tests (Listeria API and API 20E) for
both pathogens and real time PCR for Listeria. Testing resulted in one of the triplicates in the cantaloupe
breeding line, ‘1405’ being positive for Listeria innocua. None of the genotypes were positive for Salmo-
nella serovars indicating that there was a low prevalence of the pathogens in the melon genotypes tested
in our study. The occurrence of false positives on selective/differential media highlights the importance
of developing sound selective protocols for the detection and isolation of pathogens from cantaloupes.
Understanding the natural prevalence of foodborne pathogens under growing conditions will help in de-
veloping field-based risk assessments for cantaloupes.
Keywords: cantaloupe lines, foodborne pathogens, field level assessment, rind netting, contamination, Salmonella. Listeria, false positives, PCR, chromogenic media
Correspondence: Sadhana Ravishankar, [email protected], Tel: +1 -520-626-1499
BRIEF COMMUNICATIONA Surveillance of Cantaloupe Genotypes
for the Prevalence of Listeria and Salmonella
G. Dev Kumar1, K. Crosby2, D. Leskovar2.3, H. Bang2, G.K. Jayaprakasha2, B. Patil2, and S. Ravishankar1
1School of Animal and Comparative Biomedical Sciences, University of Arizona, 1117, E. Lowell Street, Tucson, AZ 85721, USA
2 Vegetable and Fruit Improvement Center, Department of Horticultural Sciences, Texas A&M University, College Station, TX 77843, USA
3Texas A&M AgriLife Research and Extension Center, Texas A&M System, Uvalde, TX 78801, USA
Agric. Food Anal. Bacteriol. 5: 73-84, 2015
74 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 5, Issue 2 - 2015
INTRODUCTION
Fresh fruits and vegetables are profitable com-
modities for growers and distributors as consump-
tion trends are on the rise (Hanning et al., 2009).
Cantaloupes and honeydew melons are popular
among the United States (US) consumers because of
their sweetness and nutritional content (Ukuku and
Sapers, 2007). The high antioxidant value of canta-
loupes and the convenience of prepackaged ready
to eat (RTE) fruits have further contributed to the
popularity of this type of melon in the US (Lester and
Hodges, 2008). However, sales of cantaloupes are
often severely affected during and after an outbreak
implication, making it important to mitigate contam-
ination by pathogenic bacteria (Ribera et al., 2012).
Cantaloupes contaminated by foodborne patho-
gens such as Listeria monocytogenes and Salmonel-
la enterica have resulted in widespread diseases and
associated economic losses. In 2011, a multistate
outbreak caused by L. monocytogenes contaminat-
ed cantaloupes resulted in 146 cases, 30 fatalities and
one miscarriage (Laksanalamai et al., 2012). In 2012,
another cantaloupe outbreak caused by Salmonella
serotypes Typhimurium and Newport involving 24
states resulted in 261 cases, three deaths and 94 hos-
pitalizations (CDC, 2012). These outbreaks highlight
the susceptibility of cantaloupes to contamination
by foodborne pathogenic bacteria. Various factors
such as netting of the rind, presence of pathogens in
soil, water and manure, hydrophobicity and attach-
ment appendages of the pathogens, as well as bio-
film formation could result in colonization of enteric
pathogens on cantaloupe rinds (Ukuku and Sapers,
2007; Hanning et al., 2009). Rainfall, water runoff, un-
derground water, and surface water currents can all
aid in the dissemination of foodborne pathogens in
soils and sediments (Bech et al., 2010).
Sweet melons such as the netted cantaloupe
(Cucumis melo L. reticulatus) can get contaminated
during pre-harvest operations in the field or during
post-harvest processing (Ukuku and Sapers, 2007).
Aggregates of foodborne pathogens on cantaloupe
rinds could result in contamination of the fruit in the
field and consequently cross-contamination of other
fruits in packaging houses (Morris and Monier, 2003).
The factors responsible for the 2011 L. monocyto-
genes contaminated cantaloupe outbreak were at-
tributed to packing house design, ineffective equip-
ment sanitation and lack of pre-cooling of melons
before cold storage (Laksanalamai et al., 2012). Mel-
ons with a contaminated rind are a food safety risk,
as pathogenic bacteria can potentially be transferred
from the surface to the flesh by cutting tools (Lin and
Wei, 1997), especially in RTE pre-cut products.
A study by Gagliardi et al. (2003) indicated that
following post-harvest processing, there were higher
bacterial counts on cantaloupe rind compared to
those still in the field (Gagliardi et al., 2003). The ineffi-
ciency of washing is most likely due to the porous sur-
face characteristics of cantaloupe and the increased
roughness resulting from the microstructures present
in the netting which could favor bacterial attachment
(Webster and Craig, 1976; Chen et al., 2012). Averting
on-field contamination of melons by pathogenic bac-
teria could be preemptive, as it is known that melon
rinds retain bacteria even after washing and chemical
sanitizer treatments (Sapers et al., 2001).
Using cantaloupe genotypes that have lower re-
tention of foodborne pathogenic bacteria on their
rinds could help reduce the risk of fruit contamination
in the field. Determining genotypes of cantaloupes
that are less susceptible to bacterial attachment and
contamination could contribute to enhanced micro-
bial safety of cantaloupes. Hence, the objective of
this study was to survey the prevalence of Listeria
and Salmonella spp. amongst various genotypes of
cantaloupes harvested directly from the field.
MATERIALS AND METHODS
Cantaloupe genotypes
The test cantaloupes consisted of 14 experimen-
tal hybrids, three inbred lines and four commercial
cultivars. These cantaloupe genotypes had been
selected for high yield, disease resistance, and firm,
high quality fruit (Crosby et al., 2006). Seeds of all
genotypes were sown directly in a silty-clay soil at
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 5, Issue 2 - 2015 75
the Texas A&M AgriLife Research Center, Uvalde, TX
(long. 29º1’N, lat. 99º5’W, elevation 283 m) on March
30, 2012. Plants were grown with standard com-
mercial practices of sub-surface drip irrigation and
fertigation, on black plastic mulch at a spacing of 2
m between beds and 0.30 m between plants on the
bed. All fruits were allowed to reach half slip matu-
rity before harvesting. Slip is considered as the ab-
scission zone between the fruit and peduncle. Half
slip maturity is a standard commercial harvest pro-
cedure used by growers in Texas and other southern
regions of the US. Net characteristics ranged from
complete coverage, high off the epidermis (ropy) to
sparse coverage of short netting. Some genotypes
had a higher incidence of splitting in the net tracts
than others. The majority had some resistance to
Fusarium induced rind lesions, but some genotypes
did exhibit this damage when fruit contacted the
clay loam soil. Fruits were harvested between July
1 and July 7, 2012 and shipped to the Ravishankar
laboratory in the Department of Veterinary Science
and Microbiology (Currently School of Animal and
Comparative Biomedical Sciences) at the University
of Arizona within 2 days. No post-harvest methods
were performed on the cantaloupes before analysis.
Storage and inspection
Upon arrival to the laboratory, cantaloupes were
initially inspected for any visible damage or spoilage.
The longitudinal circumference from the stem scar
of each fruit was measured using a measuring tape.
Cantaloupe fruits were given alternate numerical
codes to prevent bias and maintain anonymity. Can-
taloupes were stored at 4°C for 24 h and were evalu-
ated for the presence of Listeria and Salmonella.
Surveillance of cantaloupes for Listeria spp.
Plugs of cantaloupe (20 mm length) were ob-
tained from the stem scar, the bottom of the fruit
and the sides of each fruit using a sterile cork borer
(20 mm diameter) in order to collect both rind and
flesh tissues. A total of 25 g of tissue was taken
from each cantaloupe for sampling. Isolation of Lis-
teria spp. was performed based on the procedure
adapted from the “FDA-Bacteriological Analytical
Manual (BAM) for the isolation of L. monocytogenes
from foods” (Hitchins and Jinneman, 2011). Briefly,
tissue samples were mixed with 225 ml of basal Buff-
ered Listeria Enrichment Broth (BLEB) (EMD Chemi-
cals Inc, Gibbstown, NJ) in a stomacher (Stomacher
Lab-Blender 400, Tekmar Co., Cincinnati, OH) for 2
min and incubated for 4 h at 30°C. Following this,
cycloheximide (Sigma-Aldrich, St. Louis, MO) was
added and the suspension was incubated at 30°C
for 48 h. After incubation, loopful of the suspensions
were streaked on to petri dishes containing modified
Oxford formulation (MOX; Becton, Dickinson and
Co, Sparks, MD) agar and Listeria CHROMagarTM
(CHROMagar, Paris, France) which were incubated at
37°C for 48 h. Typical black colonies formed on MOX
and blue colonies on CHROMagar irrespective of
halo formation were Gram stained and streaked for
isolation to account for Listeria spp. Those colonies
that were Gram positive were further confirmed us-
ing Listeria API strips (bioMerieux, Hazelwood, MO),
accessory tests (catalase, oxidase and hemolysis) ac-
cording to the manufacturer’s instructions, and real
time PCR (iQ Check Listeria spp. Kit, Bio-Rad labora-
tories, Hercules, CA)
Real time PCR confirmation of Listeria spp.
One isolated colony from a MOX plate was added
to 100 μl of lysis buffer (Bio-Rad Laboratories) and
incubated for 15 min at 95°C. To 45 μL of the PCR
amplification master mix, 5 μL of the lysed DNA
sample was added along with 5 μL fluorogenic oli-
gonucleotide molecular beacon probe solution (iQ
Check Listeria spp. Kit, Bio-Rad Laboratories). The
thermocycler (MiniOpticon™ real-time PCR detec-
tion system, Bio-Rad Laboratories) was programmed
as follows: 50°C for 2 min, 95°C for 5 min followed
by 95°C for 20 s, 55°C for 30 s, 72°C for 30 s for 50
cycles, and 72°C for 5 min. An increase in fluores-
cence from the amplification of the target sequence
resulting in a Ct value ≥10 was considered positive.
76 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 5, Issue 2 - 2015
Surveillance of cantaloupes for Salmo-nella serovars
A total of 25 g of tissue was taken from each can-
taloupe using techniques similar to those described
earlier for Listeria. Isolation of Salmonella spp. was
performed based on the procedure adapted from the
“FDA-BAM for the isolation of Salmonella spp. from
foods” (Andrews and Hammack, 2007). Briefly, tissue
samples were mixed with 225 ml sterile universal pre-
enrichment broth (UPB; Becton, Dickinson and Co.)
for 2 min using a stomacher (Stomacher Lab-Blender
400, Tekmar Co.). The suspension was incubated for
24 h at 37°C following which 100 μl was transferred
to 10 ml Rappaport-Vassiliadis (RV; EMD Chemicals
Inc.) medium and another 1 ml to 10 ml tetrathionate
(TT; EMD Chemicals Inc.) broth. The RV suspension
was vortexed and incubated at 42°C for 24 h in a wa-
ter bath. The TT broth suspension was vortexed and
incubated at 37°C for 24 h. Following incubation,
the suspensions from the broths were streaked on
to xylose lysine desoxycholate (XLD) agar (Becton,
Dickinson and Co.) and CHROMagarTM Salmonella
(CHROMagar) and incubated for 48 h. Typical colo-
nies were Gram stained and Gram negative isolates
were confirmed as Salmonella using API 20E strips
(bioMerieux), and by conducting biochemical tests
(catalase, oxidase) according to the manufacturer’s
recommendations.
Statistical Analysis
Geometric means and standard deviations were
calculated for the incidences of false positives on
selective plating media. A t-test was performed to
determine significant differences (p<0.05) between
false positive rates on different selective media. Sta-
tistical analysis was performed using Microsoft Excel
2007 (Microsoft Corp., Seattle, WA). Means and stan-
dard deviations were calculated for the longitudinal
circumference values of melons.
RESULTS
Sizes of cantaloupes based on their di-ameter
A total of 21 cantaloupe genotypes (3 fruits each
for most genotypes) were surveyed for the pres-
ence of Listeria and Salmonella spp. The cantaloupe
genotypes with the maximum average longitudinal
circumference were lines 18 and 20 with 60.53±4.06
and 62.23±4.58 cm, respectively (Table 1). Canta-
loupe breeding line 6 had the smallest melons with
an average longitudinal circumference of 46.57±1.50
cm. Cantaloupe genotypes 17 and Oro Duro were
also some of the smaller lines tested with an average
longitudinal circumference of 50.37±2.61 cm and
49.97±1.44 cm, respectively.
Surveillance of cantaloupes for Listeria spp.
None of the 21 cantaloupe genotypes were posi-
tive for the presence of L. monocytogenes. One
cantaloupe sample from the breeding line 1405 was
positive for the presence of L. innocua after enrich-
ment and plating on selective media (Table 1). This
sample was further confirmed through Listeria API
tests and real Time PCR. Real Time PCR analysis re-
sulted in one isolate from cantaloupe line 1405 hav-
ing an increase in the fluorescence curve indicating
amplification and a Ct>10, indicating a positive result
for Listeria spp. Sixteen of the 21 melon lines tested
demonstrated black and blue colored colonies on
MOX and Listeria CHROMagar, respectively. The
blue colonies were chosen to determine the pres-
ence of other Listeria spp. All these colonies were
Gram positive. However, the results of API Listeria
test and real-time PCR indicated that 15 of these iso-
lates were negative for L. monocytogenes.
Surveillance of cantaloupes for Salmo-nella
None of the 21 cantaloupe genotypes were posi-
tive for the presence of Salmonella serovars. Out of
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 5, Issue 2 - 2015 77
Table 1. Rind net characteristics and surveillance tests for the presence of Listeria spp. and Sal-monella on various cantaloupe genotypes using API Listeria for Listeria and API 20E for Salmo-nella.
C a n t a -loupe
g e n o -type
Rind net characteristics Pathogen surveil-lance testz
PhotographLongitudinal c i r c u m f e r -ence (cm)
Net
Coverage (%)
Splitting
/corkinessAPI Liste-ria strip
API 20E strip
Experimental Hybrid
1 50.37±2.62 100 Low Neg Neg
2 52.90±0.69 100 Low Neg Neg
3 54.60±1.30 100 Low Neg Neg
6 46.57±1.50 90 Low Neg Neg
78 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 5, Issue 2 - 2015
C a n t a -loupe
g e n o -type
Rind net characteristics Pathogen surveil-lance testz
PhotographLongitudinal c i r c u m f e r -ence (cm)
Net
Coverage (%)
Splitting
/corkinessAPI Liste-ria strip
API 20E strip
7* 58.40 100 Low Neg Neg
9 53.95±6.29 100 Low Neg Neg
10 59.70±5.86 100 Low Neg Neg
11 50.20±2.69 100 Low Neg Neg
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 5, Issue 2 - 2015 79
C a n t a -loupe
g e n o -type
Rind net characteristics Pathogen surveil-lance testz
PhotographLongitudinal c i r c u m f e r -ence (cm)
Net
Coverage (%)
Splitting
/corkinessAPI Liste-ria strip
API 20E strip
12* 53.30 100 Low Neg Neg
14 54.17±1.50 100 Low Neg Neg
15* 67.30 100 Low Neg Neg
17 50.37±2.61 100 Low Neg Neg
80 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 5, Issue 2 - 2015
C a n t a -loupe
g e n o -type
Rind net characteristics Pathogen surveil-lance testz
PhotographLongitudinal c i r c u m f e r -ence (cm)
Net
Coverage (%)
Splitting
/corkinessAPI Liste-ria strip
API 20E strip
18 60.53±4.06 100 Med Neg Neg
20 62.23±4.58 100 Med Neg Neg
Inbred
146 51.67±3.87 100 Low Neg Neg
F39 49.53±4.58 100 Low Neg Neg
1405 57.60±1.93 100 Med Pos+Neg Neg
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 5, Issue 2 - 2015 81
C a n t a -loupe
g e n o -type
Rind net characteristics Pathogen surveil-lance testz
PhotographLongitudinal c i r c u m f e r -ence (cm)
Net
Coverage (%)
Splitting
/corkinessAPI Liste-ria strip
API 20E strip
Commercial variety
Mission 53.33±2.55 100 Low Neg Neg
Oro Duro 49.97±1.44 100 Low Neg Neg
Sol Real 52.07±2.19 100 Low Neg Neg
Journey 54.60±5.86 90 Med Neg Neg
z Positive colonies isolated from selective media were tested for the presence of pathogen using an API Listeria strip for Listeria and an API 20E strip for Salmonella. Results were Negative (Neg) or Postive (Pos) for Listeria or Salmonella. One of the triplicates in 1405 showed positive for Listeria while two other replicates came out nega-tive.
* Only one sample was available in experimental hybrid 7, 12 and 15, because it is difficult to synchronize the maturity of all genotypes and open pollinated fruits in a field trial or some fruits may have aborted during fruit development.
82 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 5, Issue 2 - 2015
the 21 cantaloupe genotypes tested, 8 genotypes- 3,
9, 10, 18, 146, 1405, Mission and Oro Duro showed
typical black colonies on XLD agar indicative of H2S
production and typical mauve colonies on Salmo-
nella CHROMagar. All the isolates from the 8 lines
were Gram negative rods. However, when API 20E
tests were conducted, all 8 were negative for the
presence of Salmonella serovars (Table 1). A total of
10 and 8 melons (from the 8 genotypes tested) re-
sulted in false positives on XLD agar and Salmonella
CHROMagar, respectively. The same melons from
genotypes- 9, 18, 146, 1405, and Oro Duro resulted
in false positives on both selective media, while dif-
ferent melons from genotypes 3, 10 and Mission re-
sulted in false positives on XLD agar and Salmonella
CHROMagar.
DISCUSSION
A total of 21 cantaloupe genotypes were surveyed
for the presence of Salmonella and Listeria spp. Of
all the lines surveyed, line 1405 was positive for the
presence of L. innocua (Table 1). This is an inbred
line with a typical, complete net of medium height
and low incidence of splitting. The heavier net com-
pletely covers the rind (most Western shipper canta-
loupes) and could result in improved attachment of
microbiota.
L. innocua has been used as a surrogate for L.
monocytogenes (Buchholz et al., 2011), because of
similar growth and survival characteristics (McKinney
et al., 2009). L. monocytogenes might be capable
of surviving in similar or harsher environments than
L. innocua (Buchholz et al., 2011). The presence of
L. innocua on cantaloupe could indicate conditions
suitable for the possible survival and contamination
by L. monocytogenes.
L. monocytogenes is commonly found in the envi-
ronment and on plant material (Laksanalamai et al.,
2012) and can survive under adverse environmental
conditions (Tompkin, 2002). Johnston et al., (2005)
surveyed cantaloupes and other produce from the
southern regions of the US for the presence of patho-
gens (L. monocytogenes and Salmonella serovars)
and indicator organisms. Of the 398 produce items
sampled, none were positive for L. monocytogenes.
Of all the produce tested for Salmonella, three of the
90 cantaloupes were positive for Salmonella Monte-
video (Johnston et al., 2005). While lower numbers
of contaminated produce can occur in the field,
cross-contamination in the packing house may result
in higher volumes of product getting contaminated
and thereby causing outbreaks.
In our study, the cantaloupe genotypes tested
were not positive for Salmonella. The use of manure,
or presence of wild life, birds, or compost piles in the
field vicinity could serve as reservoirs of contamina-
tion. While the research farm at the Texas A&M AgriL-
ife Research Center did not contain these pathogen
reservoirs, farms could potentially be subjected to
pathogen introduction through environmental con-
tamination and animal or bird intrusion. Previous
studies have indicated that foodborne pathogens
can survive in soil and water for extended periods of
time and can be transferred to fruit tissue (Baloda et
al., 2001; Gupta et al., 2007; Barak and Liang, 2008).
Factors that affect the survival of the pathogen in soil
include soil type, nutrient availability, manure and
temperature (Andrews-Polymenis et al., 2010).
Selective isolation of pathogens from cantaloupes
resulted in false positive samples for both Listeria
spp. and Salmonella on selective media. In a study
to evaluate chromogenic agar media for the recov-
ery and detection of L. monocytogenes in foods, it
was observed that natural microbiota in foods are
capable of overgrowing pathogenic target microor-
ganisms (Michael, 2004). In our study, the sensitivity
of non-chromogenic plating media was not signifi-
cantly different (P>0.05) from chromogenic plating
media for distinguishing false positives, in case of
both pathogens.
Cantaloupes are rich in sugars and the rinds of
cantaloupes are capable of harboring high amounts
of microbiota because of the naturally present net-
ting (Ukuku and Sapers, 2007). The breakage or
rupture of the rinds could potentially result in cross-
contamination of the microorganisms from one fruit
to another, due to spilling of the juice, which is rich in
antioxidants and sugars (Lester and Hodges, 2008).
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 5, Issue 2 - 2015 83
While our study indicated that the various genotypes
did not result in significant differences in pathogen
attachment, mitigation strategies should be ex-
plored to reduce the risk of on-field contamination
of cantaloupes.
CONCLUSIONS
A survey of 21 cantaloupe genotypes resulted in
a single line of netted cantaloupe being positive
for Listeria spp., which was confirmed as L. innocua.
The presence of L. innocua on cantaloupe indicates
the existence of conditions wherein pathogenic L.
monocytogenes could survive. More research is
needed to understand the role of cantaloupe net-
ting on microbial attachment and persistence.
ACKNOWLEDGEMENTS
The authors would like to thank Libin Zhu and Jen-
nifer Todd of the Ravishankar lab for their technical
support.
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15
ARTICLES
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Hops (Humulus lupulus) ß-Acid as an Inhibitor of Caprine Rumen Hyper-Ammonia-Produc-ing Bacteria In VitroM. D. Flythe, G. E. Aiken1, G. L. Gellin, J. L. Klotz, B. M. Goff, K. M. Andries
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PEER REVIEW PROCESS
Authors will be requested to provide the names
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Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 5, Issue 2 - 2015 91
MANUSCRIPT CONTENT REQUIREMENTS
Preparing the Manuscript File
Manuscripts must be written in grammatically
correct English. AFAB offers a fee based language
service upon request ([email protected]).
Manuscripts should be typed double-spaced, with
lines and pages numbered consecutively. All docu-
ments must be submitted in Microsoft Word (.doc or
.docx, PC or Mac). All special characters (e.g., Greek,
math, symbols) should be inserted using the sym-
bols palette available in this font. Tables and figures
should be placed in separate sections at the end of
the manuscript (not placed in the text). Failure to fol-
low these instructions will cause delays of the pro-
cessing and review of the manuscript.
Title Page
At the very top of the title page, include a title of
not more than 100 characters. Format the title with
the first letter of each word capitalized. No abbre-
viations should be used. Under the title, the authors
names are listed. Use the author’s initials for both first
and middle names with a period (full-stop) between
initials (e.g., W. A. Afab). Underneath the authors, a
list affiliations must be listed. Please use numerical
superscripts after the author’s names to designate
affiliation. If an authors address has changed since
the research was completed, this new information
must be designated as “Current address:”. The cor-
responding author should be indicated with an aster-
isk e.g., * Corresponding author. The title page shall
include the name and full address of the correspond-
ing author. Telephone and e-mail address must also
be provided for the corresponding author, and email-addresses must be provided for all authors.
Editing
Author-derived abbreviations should be defined
at first use in the abstract and again in the body of
the manuscript. If abbreviations are extensive au-
thors may need to provide a list of abbreviations
at the beginning of the manuscript. In vivo, in vitro
and bacterial names must be italicized (obligatory).
Authors must avoid single sentence paragraphs and
merge such paragraphs appropriately. Authors must
not begin sentences with “Figure or Table shows…”
as these are inanimate objects and cannot “show”
anything. When number are reported in text or in ta-
bles, always put a zero in front of decimal numbers:
“0.10” instead of “.10”.
MANUSCRIPT SECTIONS
Abstract
The abstract provides an abridged version of the
manuscript. Please submit your abstract on a sepa-
rate page after the title page. The abstract should
provide a justification of your work, objectives, meth-
ods, results, discussion and implications of study or
review findings . Your abstract must consist of com-
plete sentences without references to other work or
footnotes and must not exceed 250 words. On the
same page as your abstract, please provide at least ten (10) keywords to be used for linking and index-
ing. Ideally, these keywords should include signifi-
cant words from the title.
Introduction
The introduction should clearly present the foun-
dation of the manuscript topic and what makes the
research or the review unique. The introduction
should validate why this topic is important based on
previously published literature, and the relevance of
the current research. Overall goals and project ob-
jectives must be clearly stated in the final sentence
of the last paragraphs of the introduction.
Materials and Methods
Information on equipment and chemicals used
must include the full company name, city, and state
(country if outside the United States or Province if
in Canada) [i.e., (Model 123, ACME Inc., Afab, AR)].
92 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 5, Issue 2 - 2015
Variability, Replication, and Statistical Analysis
To properly assess biological systems indepen-
dent replication of experiments and quantification
of variation among replicates is required by AFAB.
Reviewers and/or editors may request additional
statistical analysis depending on the nature of the
data and it will be the responsibility of the authors
to respond appropriately. Statistical methods com-
monly used in the bacteriology do not need to be
described in detail, but an adequate description
and/or appropriate references should be provided.
The statistical model and experimental unit must
be designated when appropriate. The experimen-
tal unit is the smallest unit to which an individual
treatment is imposed. For bacterial growth stud-
ies, the average of replicate tubes per single study
per treatment is the experimental unit; therefore,
individual studies must be replicated. Repeated
time analyses of the same sample usually do not
constitute independent experimental units. Mea-
surements on the same experimental unit over time
are also not independent and must not be consid-
ered as independent experimental units. For analy-
sis of time effects, assess as a rate of change over
time. Standard deviation refers to the variability
in the biological response being measured and is
presented as standard deviation or standard error
according to the definitions described in statistical
references or textbooks.
Results
Results represent the presentation of data in
words and all data should be described in same
fashion. No discussion of literature is included in
the results section.
Discussion
The discussion section involves comparing the
current data outcomes with previously published
work in this area without repeating the text in the
results section. Critical and in-depth dialogue is
encouraged.
Results and Discussion
Results and discussion can be under combined or
separate headings.
Conclusions
State conclusions (not a summary) briefly in one
paragraph.
Acknowledgments
Acknowledgments of individuals should include
institution, city, and state; city and country if not U.S.;
and City or Province if in Canada. Copies being re-
viewed shall have authors’ institutions omitted to re-
tain anonymity.
References
a) Citing References In Text
Authors of cited papers in the text are to be pre-
sented as follows: Adams and Harry (1992) or Smith
and Jones (1990, 1992). If more than two authors of
one article, the first author’s name is followed by the
abbreviation et al. in italics. If the sentence structure
requires that the authors’ names be included in pa-
rentheses, the proper format is (Adams and Harry,
1982; Harry, 1988a,b; Harry et al., 1993). Citations to a
group of references should be listed first alphabeti-
cally then chronologically. Work that has not been
submitted or accepted for publication shall be listed
in the text as: “G.C. Jay (institution, city, and state,
personal communication).” The author’s own un-
published work should be listed in the text as “(J.
Adams, unpublished data).” Personal communica-
tions and unsubmitted unpublished data must not
be included in the References section. Two or more
publications by the same authors in the same year
must be made distinct with lowercase letters after
the year (2010a,b). Likewise when multiple author ci-
tations designated by et al. in the text have the same
first author, then even if the other authors are differ-
ent these references in the text and the references
section must be identified by a letter. For example
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 5, Issue 2 - 2015 93
“(James et al., 2010a,b)” in text, refers to “James,
Smith, and Elliot. 2010a” and “James, West, and Ad-
ams. 2010b” in the reference section.
b) Citing References In Reference Section
In the References section, references are listed in
alphabetical order by authors’ last names, and then
chronologically. List only those references cited in the
text. Manuscripts submitted for publication, accepted
for publication or in press can be given in the refer-
ence section followed by the designation: “(submit-
ted)”, “(accepted)’, or “(In Press), respectively. If the
DOI number of unpublished references is available,
you must give the number. The year of publication fol-
lows the authors’ names. All authors’ names must be
included in the citation in the Reference section. Jour-
nals must be abbreviated. First and last page num-
bers must be provided. Sample references are given
below. Consult recent issues of AFAB for examples
not included in the following section.
Journal manuscript:
Examples:
Chase, G., and L. Erlandsen. 1976. Evidence for a
complex life cycle and endospore formation in the
attached, filamentous, segmented bacterium from
murine ileum. J. Bacteriol. 127:572-583.
Jiang, B., A.-M. Henstra, L. Paulo, M. Balk, W. van
Doesburg, and A. J. M. Stams. 2009. A typical
one-carbon metabolism of an acetogenic and
hydrogenogenic Moorella thermioacetica strain.
Arch. Microbiol. 191:123-131.
Book:
Examples:
Hungate, R. E. 1966. The rumen and its microbes
Academic Press, Inc., New York, NY. 533 p.
Book Chapter:
Examples:
O’Bryan, C. A., P. G. Crandall, and C. Bruhn. 2010.
Assessing consumer concerns and perceptions
of food safety risks and practices: Methodologies
and outcomes. In: S. C. Ricke and F. T. Jones. Eds.
Perspectives on Food Safety Issues of Food Animal
Derived Foods. Univ. Arkansas Press, Fayetteville,
AR. p 273-288.
Dissertation and thesis:
Maciorowski, K. G. 2000. Rapid detection of Salmo-
nella spp. and indicators of fecal contamination
in animal feed. Ph.D. Diss. Texas A&M University,
College Station, TX.
Donalson, L. M. 2005. The in vivo and in vitro effect
of a fructooligosacharide prebiotic combined with
alfalfa molt diets on egg production and Salmo-
nella in laying hens. M.S. thesis. Texas A&M Uni-
versity, College Station, TX.
Van Loo, E. 2009. Consumer perception of ready-to-
eat deli foods and organic meat. M.S. thesis. Uni-
versity of Arkansas, Fayetteville, AR. 202 p.
Web sites, patents:
Examples:
Davis, C. 2010. Salmonella. Medicinenet.com.
http://www.medicinenet.com/salmonella /article.
htm. Accessed July, 2010.
Afab, F. 2010, Development of a novel process. U.S.
Patent #_____
Author(s). Year. Article title. Journal title [abbreviated].
Volume number:inclusive pages.
Author(s) [or editor(s)]. Year. Title. Edition or volume (if
relevant). Publisher name, Place of publication. Number
of pages.
Author(s) of the chapter. Year. Title of the chapter. In:
author(s) or editor(s). Title of the book. Edition or vol-
ume, if relevant. Publisher name, Place of publication.
Inclusive pages of chapter.
Author. Date of degree. Title. Type of publication, such
as Ph.D. Diss or M.S. thesis. Institution, Place of institu-
tion. Total number of pages.
94 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 5, Issue 2 - 2015
Abstracts and Symposia Proceedings:
Fischer, J. R. 2007. Building a prosperous future in
which agriculture uses and produces energy effi-
ciently and effectively. NABC report 19, Agricultural
Biofuels: Tech., Sustainability, and Profitability. p.27
Musgrove, M. T., and M. E. Berrang. 2008. Presence
of aerobic microorganisms, Enterobacteriaceae and
Salmonella in the shell egg processing environment.
IAFP 95th Annual Meeting. p. 47 (Abstr. #T6-10)
Vianna, M. E., H. P. Horz, and G. Conrads. 2006. Op-
tions and risks by using diagnostic gene chips. Pro-
gram and abstracts book , The 8th Biennieal Con-
gress of the Anaerobe Society of the Americas. p.
86 (Abstr.)
Data Presentation in Tables and Figures
Figures and tables to be published in AFAB must
be constructed in such a fashion that they are able
to “stand alone” in the published manuscript. This
means that the reader should be able to look at
the figure or table independently of the rest of the
manuscript and be able to comprehend the experi-
mental approach sufficiently to interpret the data.
Consequently, all statistical analyses should be very
carefully presented along with variation estimates
and what constitutes an independent replication
and the number of replicates used to calculate the
averages presented in the table or figure.
Each table and figure must be on a separate
page in the submitted paper. In addition, you will
need to submit all data for charts, tables and
figures in native format when possible (e.g., Mi-
crosoft Excel, Powerpoint). Photographs should
be submitted as high-resolution (600 dpi) .jpg or
tif. files. All figures should be clearly presented with
well defined axis and units of measurement. Sym-
bols, lines, and bars must be made distinct as “stand
alone” black and white presentations. Stippling,
dashed lines etc. are encouraged for multiple com-
parison but shades of gray are discouraged. Color
images, micrographs, pictures are recommended
and there is no additional fee for their submission.
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