AFAB Volume 5 Issue 2 by cielo shabatura

ISSN: 2159-8967 www.AFABjournal.com

Volume 5 Issue 2 2015

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EDITORIAL BOARD Sooyoun Ahn

Hae-Yeong Kim

University of Florida, USA

Kyung Hee University, South Korea

Walid Q. Alali

Woo-Kyun Kim

University of Georgia, USA

Kenneth M. Bischoff

M.B. Kirkham

NCAUR, USDA-ARS, USA

Kansas State University, USA

Debabrata Biswas

Todd Kostman

University of Maryland, USA

University of Wisconsin, Oshkosh, USA

Claudia S. Dunkley

Y. M. Kwon

University of Georgia, USA

University of Arkansas, USA

Michael Flythe

Maria Luz Sanz Murias

USDA, Agricultural Research Service

Instituto de Quimica Organic General, Spain

Lawrence Goodridge

Byeng R. Min

McGill University, Canada

Tuskegee University in Tuskegee, AL

Leluo Guan

Melanie R. Mormile

University of Alberta, Canada

Missouri University of Science and Tech., USA

Joshua Gurtler

Rama Nannapaneni

ERRC, USDA-ARS, USA

Mississippi State University, USA

Yong D. Hang

Jack A. Neal, Jr.

Cornell University, USA

University of Houston, USA

Armitra Jackson-Davis

Benedict Okeke

Alabama A&M University, USA

Auburn University at Montgomery, USA

Divya Jaroni

John Patterson

Oklahoma State University, USA

Purdue University, USA

Weihong Jiang

Toni Poole

Shanghai Institute for Biol. Sciences, P.R. China

FFSRU, USDA-ARS, USA

Michael Johnson

Marcos Rostagno

University of Arkansas, USA

LBRU, USDA-ARS, USA

Timothy Kelly

Roni Shapira

East Carolina University, USA

Hebrew University of Jerusalem, Israel

William R. Kenealy

Kalidas Shetty

Mascoma Corporation, USA

North Dakota State University, USA Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 5, Issue 2 - 2015

EDITORIAL STAFF EDITOR-IN-CHIEF Steven C. Ricke University of Arkansas, USA

EDITORS Todd R. Callaway FFSRU, USADA-ARS, USA Philip G. Crandall University of Arkansas, USA Janet Donaldson Mississippi State University, USA

MANAGING and LAYOUT EDITOR Ellen J. Van Loo Ghent, Belgium

TECHNICAL EDITOR Jessica C. Shabatura Fayetteville, USA

ONLINE EDITION EDITOR C.S. Shabatura Fayetteville, USA

Ok-Kyung Koo Korea Food Research Institute, South Korea

ABOUT THIS PUBLICATION Mailing Address: 2138 Revere Place . Fayetteville, AR . 72701 Agriculture, Food & Analytical Bacteriology (ISSN 2159-8967) is published quarterly. Instructions for Authors may be obtained at the back of this issue, or online via our website at www.afabjournal.com Manuscripts: All correspondence regarding pending manuscripts should be addressed Ellen Van Loo, Managing Editor, Agriculture, Food & Analytical Bacteriology: ellen@afabjournal.com Information for Potential Editors: If you are interested in becoming a part of our editorial board, please contact Editor-in-Chief, Steven Ricke, Agriculture, Food & Analytical Bacteriology: editor@afabjournal.com

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Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 5, Issue 2 - 2015

TABLE OF CONTENTS BRIEF COMMUNICATIONS 73

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

ARTICLES 56

The Mutating Gastrointestinal Flora, Multidrug Resistant Enterococcus faecium A. Limayem

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

Introduction to Authors 87

Instructions for Authors

The publishers do not warrant the accuracy of the articles in this journal, nor any views or opinions by their authors. Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 5, Issue 2 - 2015

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

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. Moreover, 87% of drug resistant microorganisms including Enterococcus faecium were detected in ground turkey 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 animals’ 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 including 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

Agric. Food Anal. Bacteriol. 5: 56-64, 2015

INTRODUCTION Increase in multi-drug resistance jeopardizes human and animal health in the U.S. and worldwide (Centers for Disease Control (CDC), 2013). Vancomycin resistant strains of Enterococcus including priCorrespondence: Alya Limayem, alimayem@usf.edu Tel: +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 Enterococcus 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

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further lead to endocarditis and death in immunocompromised populations (Lebreton et al., 2013). Enterococci can be carried on the hands of health care workers, transferred from one patient to another 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-

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 agriculture use. Virginiamycin has been used in agricultural system for growth promotion and bacterial control of farm animals (Barton et al., 2003; Claycamp and Hooberman, 2004). As such, without a similar streptogramin ban, MEF strains would disseminate in food animal products (Barton et al., 2003). As such, antibiotic resistance would disseminate in farming animals and subsequently pass through the food chain

nary tract producing cystisis, prostatitis, and epididymitis (Gilmore et al., 2002). This study demonstrated that Enterococci are also found in intra-abdominal, pelvic, and soft tissue infections and can cause nosocomial bacteremia. However, endocarditis is considered the most serious enterococcal infection, as it causes inflammation of the heart valves. In many cases of endocarditis, antibiotic treatment fails and surgery 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 hematologic 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-

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 mutate and generate intrinsic and extrinsic mechanisms of resistance to a broad continuum of antibiotics. (Tremblay et al., 2011). Moreover, MEF has a tremendous capability to gain drug resistance via conjugation mechanisms or gene transfer from other microorganisms (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 knowledge on the ecology, epidemiology and antibiotic resistance of some E. faecium strains from food animals to clinical settings. Beneficial attributes of some E. faecium strains are also discussed. Further studies on the risk from resistant E. faecium strains are

ically climbed in the last 27 years to achieve an unprecedented 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

suggested to ensure greater preventive actions and public health safety.

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ECOLOGY AND SPECIFIC CHARACTERISTICS E. faecium has a spherical shape and is a Grampositive 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 environments 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-

tas et al., 2011; Garcia-Migura et al., 2005; GarciaMigura 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 contamination of water supplies from farmland, its isolation is less prevalent from livestock than from human feces (Franz et al., 1999).

BENEFICIAL ATTRIBUTES

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 pathogenicity (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 substantial 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 vaginal 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 equipment such as medical and agricultural devices (klein,

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 diarrhea, enhance the immune system, and lower the cholesterol level (Franz et al., 2011), other strains are used for their food safety attributes in limiting zoonotic pathogens from food animals through bacteriocin production (Franz et al., 2011). As evidenced by De Kwaadsteniet et al. (2005), E. faecium P21 isolated from sausage produces both enterocins A and B. Enterocins proved to be active against a substantial range of Gram-positive bacteria including primarily 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 (Leroy et al., 2003). Nonvirulent strains of E. faecium have been suggested as a possible probiotic against microbes possessing antimicrobial resistances (Franz

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-

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-

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vert into pathogenic strains would hinder the beneficial attributes of E. faecium. This is increasingly more problematic due to the considerable ability of E. faecium to mutate and acquire virulent genes in multiple types of environment (Arias and Murray, 2012).

EPIDEMIOLOGY The previously named Streptococcus faecium started to evolve as a nosocomial vancomycin resistant 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 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 pathogens 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. faecium (Freitas et al., 2011): (1) the commensal/community-associated (CA) strains and (2) the hospitalassociated (HA) strains, Clonal Complex 17 (CC17). The CC17 strains gave rise to the worldwide noso-

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 inoculation 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 immunocompromised 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 further 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 epidemic began in the late 1980s and was partially associated with the extensive use of avoparcin in farming animals (Arias and Murray, 2012). Wegener et al (1999) have reported that glycopeptide avoparcin contained high levels of vanA encoding resistance

comial VRE as the most prevalent source of enterococcus 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. faeciumâ&#x20AC;&#x2122;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.

to glycopeptide agent found on the Tn1546 transposon (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).

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 reviewed by Arias and Murray (2012), the main antibi-

ANTIBIOTIC RESISTANCE The high intrinsic capability and phenotypic elasticity of the MEF strain enable it to acquire exogenous genes from the environment, mutate continuously, and transfer resistant genes to other

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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 suppress microbial contaminant load at slaughter (Donabedian 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

While numerous molecular investigations are conducted on Enterococcus strains, (Aslam et al., 2012; Cha et al., 2012; Garnier et al., 2004; Getachew et al., 2013) including their genetic relatedness (Freitas 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. Furthermore, 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

mechanism against streptogramin type A includes O-acetyltransferase. Acetyltransferase of virginiamycin agent is encoded by the gene vat. Among the most prevalent vat genes (Simjee et al., 2006) encoding 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 streptogramins via ABC porters (Sletvold et al., 2008) encoded 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).

the source to hospitals within a comprehensive modeling approach. Further investigations including a complete characterization of the MEF strain revealing rapid detection and quantification within a comprehensive risk model will enable the development of effective mitigation 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.

CONCLUSIONS-FUTURE DIRECTIONS

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 Sanitary Foundation Laboratory for their support in providing the poultry samples and the clinical isolates from Michigan State.

Extensive research studies have elucidated the antibiotic resistance profile of VRE in human populations and hospitals (Acharya et al., 2007; Harisberger 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

Acharya, A., A. Khanal, R. Kanungo, and T. Mohapa-

the resistance profile of Enterococcus genera in food animals (Persoons et al., 2010; Pesavento et al., 2014; Simjee et al., 2007) primarily in poultry (Fracalanzza et al., 2007; Ghidán et al., 2008a ; Ghidán et al., 2008b; Schwaiger et al., 2010; Usui et al., 2014).

tra. 2007. Characterization and susceptibility patterns of clinically important Enterococcus species in eastern Nepal. Nepal Med. Coll. J. 9:250-254. Arias, C.A. and B.E. Murray. 2012. The rise of the Enterococcus: beyond vancomycin resistance. Nat.

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Zoonoses Public Health. 57:171-180. Simjee, S., Y. Zhang, P.F. McDermott, S.M. Donabedian, M.J. Zervos, and J. Meng. 2006. Heterogeneity of vat(E)-carrying plasmids in Enterococcus faecium recovered from human and animal sources. Int. J. Antimicrob. Agents. 28:200-205. Simjee, S., P.F. McDermott, D.G. White, C. Hofacre, R.D. Berghaus, P.J. Carter, L. Stewart, T. Liu, M. Maier, and J.J. Maurer. 2007. Antimicrobial susceptibility and distribution of antimicrobial-resistance genes among Enterococcus and coagulase-negative Staphylococcus isolates recovered from poultry litter. Avian Dis. 51:884-892. Sletvold, H., P.J. Johnsen, I. Hamre, G.S. Simonsen,

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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 72704 2 Current address: Food Safety Research Group, Korea Food Research Institute, Seongnam-si, Gyeonggi-do, Republic of Korea.

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 determined 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 antimicrobial 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; antimicrobial; food safety; foodborne illness; companion animals Agric. Food Anal. Bacteriol. 5: 65-72, 2015

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, sricke@uark.edu Tel: +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 ingredients 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-

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tures which acts as a kill step for pathogens. However, high temperatures also destroy some of the nutrients 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-

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 isolated from unopened bags of dry dog food and fecal specimens from dogs that had eaten the food and

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 determined 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 serotype 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 Salmonella 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 program and was found to be due to 2 serotypes of Salmonella, Montevideo and Give. Dogs in 4 kennels were exposed and 63.8% of the dogs had positive

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 microbiological 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 microbiological integrity of pet foods must cover the entire production 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, nonanoic acid and trans-2-hexenal.

fecal samples, although only 9 dogs exhibited clinical 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 66

MATERIALS AND METHODS Determination of antimicrobial spectrum Lyophilized cultures of test organisms (Salmonella Typhimurium, Escherichia coli, Staphylococcus aureus, Clostridium perfringens, Lactobacillus plantarum, Streptococcus agalactiae and Campylobacter jejuni)

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were obtained from the American Type Culture Collection (ATCC; Manassas, VA). Cultures were resuscitated according to ATCC recommended methods and transferred to Standard Methods Agar (SMA; BD Diagnostics, Franklin Lakes, NJ). Agar plates were incubated for 24 hours at 35°C. After incubation, bacterial colonies were transferred 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.

MA) with a single isolate of a serotype of Salmonella and incubating at 37°C for 18 to 24 h. Minimal inhibitory 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 antimicrobial 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 antimicrobial 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 solution was discarded from the last row to keep well volumes equal. One row was used as a positive control and contained 5 µl ml-1 of butyric acid; another row of

One mL of CO-60 surfactant and 1 mL of the antimicrobial (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 diluted 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.

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 defined 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

Isolates of 8 serovars of Salmonella were tested (Heidelberg, Montevideo, Enteritidis, Typhimurium,

A culture of each serovar of Salmonella was prepared 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 approximately 108 cfu ml-1. Meat and bone meal (MBM) was used as the carrier 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

Worthington, Kentucky, Senftenberg and Infantis); all isolates were obtained from the culture collection of the Center for Food Safety of the University of Arkansas. Overnight cultures were prepared by inoculating 10 ml of sterile LB broth (EMD Millipore, Billerica,

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 temperature for 48 hours in a biosafety cabinet.

Determination of minimal inhibitory concentration

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Figure 1. Lab scale mixer used to mix antimicrobial with feed

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 (Figure 1) and mixed for 2 minutes. The antimicrobial was added to the inoculated feed using a nebulizer fitted into the mixer and at a positive air pressure of 8 PSI. The antimicrobial was injected through a septum 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

cultured as described for samples. Plates were incubated at 37°C for 24 hours and then enumerated for the amount of Salmonella remaining. The entire experiment was replicated three times.

dilution. An aliquot of 0.1 ml of each dilution was dispensed 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

observed to be more resistant than the other organisms, with C. perfringens reduced 100% at 0.1% (1 ml kg-1) and L. plantarum reduced 100% at 0.5% (5 ml kg-1).

RESULTS Antimicrobial spectrum The antimicrobial was observed to have efficacy against both Gram-positive and Gram-negative bacteria (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 antimicrobial 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

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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.

S. TyE. coli phimurium

aureus

Cl. perfringens

L. plantarum

S. agalactiae

C. jejuni

0.005%

0.0

92.3

6.1

0.01%

0.0

98.3

59.2

0.05%

100

0.1%

100

0.5%

100

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.

DISCUSSION

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 control 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

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 bacteria (Paster et al., 1987). Treatment of poultry feed with organic acids has been shown to have the potential 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 undissociated form because they are lipid permeable in this

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.

form and can cross the microbial cell wall and dissociate in the more alkaline interior of the microorganism making the cytoplasm unstable for survival. (Paster, 1979; Van Immerseel et al., 2006). Butyric acid when used alone was been found to inhibit Sal-

Efficacy in pet food

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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.

9.5

Log CFU Salmonella

9 8.5 8 7.5 0

5.0 mL/kg

6.5

7.5 mL/kg 10 mL/kg

6 5.5 5 4.5 0

Day

monella (Khan and Katamay, 1969). Khan and Khatamay (1969) found that butyric acid completely inhibited the growth of Salmonella in media, and when it was used to treat meat and bone meal artificially inoculated 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). Nonanoic acid is found in a variety of fruits as well as in dairy products, and is on the FDA generally recognized 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 antimicrobial, but Khan and Khatamay (1969) found essentially no activity against Salmonella artificially inoculated into meat and bone meal. Another volatile compound contained in the stud70

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 effective 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. Typhimurium 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 results (Iba and Berchieri, 1995). Our results suggest a

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reduction of a high level of contamination with Salmonella within 24 hours of application by this combination of organic acids with trans-2-hexenal. Additionally, the reduction was much greater after 4 days of contact as compared to the control, where Salmonella growth actually increased. Wales et al. (2013) studied various feed treatment formulations containing organic acids and found reductions in Salmonella 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

shared concern. Br. J. Nutr. 106: 878-884. Carrique-Mas, J.J., S. Bedford, and R.H. Davies. 2007. Organic acid and formaldehyde treatment of animal feeds to control Salmonella: Efficacy and masking during culture. J. Appl. Microbiol. 103: 88–96. CDC. 2006. Human salmonellosis associated with animal-derived pet treats—United States and Canada, 2005. MMWR 55: 702–705. Clark, C., J. Cunningham, R. Ahmed, D. Woodward, K. Fonseca, S. Isaacs, A. Ellis, C. Anand, K. Ziebell, A. Muckle, P. Sockett, and F. Rodgers. 2001 Characterization of Salmonella associated with pig ear dog treats in Canada. J. Clin. Microbiol. 39: 3962–3968.

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.

AVMA. 2014. U. S. pet ownership statistics. Available at: https://www.avma.org/KB/Resources/Statistics/ Pages/Market-research-statistics-US-pet-ownership.aspx Accessed 04 November 2014. Behravesh, C.B., A. Ferraro, M. Deasy, V. Dato, M. Moll, C. Sandt, N.K. Rea, R. Rickert, C. Marriott, K. Warren, V. Urdaneta, E. Salehi, E. Villamil, T. Ayers, R.M. Hoekstra, J.L. Austin, S. Ostroff, and I.T. Williams. 2010. Human Salmonella infections linked to contaminated dry dog and cat food, 2006–2008. Pediatrics 126: 477–483.

EPA. 2009. Pesticides: Regulating Pesticides. Pelargonic Acid (217500). U.S. Environmental Protection Agency. Retrieved 14 January 2015, from http://www.epa.gov/opp00001/chem_search/reg_ actions/registration/fs_PC-217500_01-Apr-00.pdf. Fernández-Rubio, C., C. Ordóñez, J. Abad-González, A. Garcia-Gallego, M.P. Honrubia, J..J. Mallo, and R. Balaña-Fouce. 2009. Butyric acid-based feed additives help protect broiler chickens from Salmonella Enteritidis infection. Poultry Sci. 88: 943-948. Gardini, F., R. Lanciotti, and M.E. Guerzoni. 2001. Effect of irans-2-hexenal on the growth of Aspergillus flavus in relation to its concentration, temperature and water activity. Letts. Appl. Microbiol. 33: 50-55. Hume, M.E., D.E. Corrier, G.W. Ivie, and J.R. Deloach. 1993. Metabolism of [14C] propionic acid in broiler chicks. Poultry Sci. 72: 786–793. Iba, A.M. and A. Berchieri. 1995. Studies on the use of a formic acid-propionic acid mixture (Bio-add) to control experimental Salmonella infection in broiler chickens. Avian Pathol. 24: 303–311. Imanishi, M., D.S. Rotstein, R. Reimschuessel, C.A. Schwensohn, D.H. Woody, Jr, S.W. Davis, A.D. Hunt, K.D. Arends, M. Achen, J. Cui, Y. Zhang, L.F. Denny, Q.N. Phan, L.A. Joseph, C.C.Tuite, J.R. Tataryn, and C.B. Behravesh. 2014. Outbreak of Sal-

Brul, S. and P. Coote. 1999. Preservative agents in foods: Mode of action and microbial resistance mechanisms. Int. J. Food Microbiol. 50: 1–17. Buchanan, R.L., R.C. Baker, A.J. Charlton, J.E. Riviere, and R. Standaert. 2011. Pet food safety: a

monella enterica serotype Infantis infection in humans linked to dry dog food in the United States and Canada, 2012. J. Am. Vet. Med. Assoc. 244: 545-553. Khan, M. and M. Katamay. 1969. Antagonistic effect

ACKNOWLEDGEMENTS This research was funded by a grant from Anitox Corp., Lawrenceville, GA.

REFERENCES

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of fatty acids against Salmonella in meat and bone meal. J. Appl. Microbiol. 17, 402–404. Kim, Y. S. and D. H. Shin. 2004. Volatile constituents from the leaves of Callicarpa japonica Thunb. and their antibacterial activities. J. Agric. Food Chem. 52: 781-787. Kubo, J., J. R. Lee, and I. Kubo. 1999. Anti-Helicobacter pylori agents from the cashew apple. J. Agric. Food Chem. 47: 533-537. Kubo, I. and K. Fujita. 2001. Naturally occurring antiSalmonella agents. J. Agric. Food Chem. 49: 57505754. Li, X., L.A. Bethune, Y. Jia, R.A. Lovell, T.A. Proescholdt, S.A. Benz, T.C. Schell, G. Kaplan, and

comparative study of the efficacy of calcium propionate, agrosil and adofeed as mold inhibitors in poultry feed. Poultry Science 66: 858–860. Schotte, U., D. Borchers, C. Wulff, and L. Geue. 2007. Salmonella Montevideo outbreak in military kennel dogs caused by contaminated commercial feed, which was only recognized through monitoring. Vet. Microbiol. 119: 316-323. Thompson, A. 2008. Ingredients: where pet food starts. Top Companion Anim. Med. 23: 127–132. Van Immerseel, F., J.B. Russell, M.D. Flythe, I. Gantois, L. Timbermont, F. Pasmans, F. Haesebrouck, and R. Ducatelle. 2006. The use of organic acids to combat Salmonella in poultry: a mechanistic ex-

D.G. McChesney. 2012. Surveillance of Salmonella prevalence in animal feeds and characterization of the Salmonella isolates by serotyping and antimicrobial susceptibility. Foodborne Pathog. Dis. 9: 692–698. Martinez-Mayorga, K., T.L. Peppard, F. Lopez-Vallejo, A.B. Yongye, and J.L. Medina-Franco. 2013. Systematic mining of generally recognized as safe (GRAS) flavor chemicals for bioactive compounds. J. Agric. Food Chem. 61: 7507-7514. Matlho, G., S. Himathongkham, H. Riemann, and P. Kass. 1997. Destruction of Salmonella Enteritidis in poultry feed by combination of heat and propionic acid. Avian Dis. 41: 58–61. Nakamura, S. and A. Hatanaka. 2002. Green-leaf-derived C6-aroma compounds with potent antibacterial action that act on both gram-negative and gram-positive bacteria. J. Agric. Food Chem. 50: 7639-7644. Nemser, S.M., T. Doran, M. Grabenstein, T. McConnell, T. McGrath, R. Pamboukian, A.C. Smith, M. Achen, G. Danzeisen, S. Kim, Y. Liu, S. Robeson, G. Rosario, K. McWilliams Wilson, and R. Reimschuessel. 2014. Investigation of Listeria, Salmonella, and toxigenic Escherichia coli in various pet foods. Foodborne Pathog. Dis. 11: 706-709.

planation of the efficacy. Avian Pathol. 35: 182-188. Wales, A., I. McLaren, A. Rabie, R.J. Gosling, F. Martelli, R. Sayers, and R. Davies. 2013. Assessment of the anti-Salmonella activity of commercial formulations of organic acid products. Avian Pathol. 42: 268-275. White, D.G., A. Datta, P. McDermott, S. Friedman, S. Qaiyumi, S. Ayers, L. English, S. McDermott, D.D. Wagner and S. Zhao. 2003. Antimicrobial susceptibility and genetic relatedness of Salmonella serovars isolated from animal-derived dog treats in the USA. J. Antimicrob. Chemother. 52: 860-863.

Paster, N. 1979. A commercial study of the efficiency of propionic acid and acid and calcium propionate as fungistats in poultry feed, Poultry Sci. 58: 572-576. Paster, N., E. Pinthus, and D. Reichman. 1987. A 72

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BRIEF COMMUNICATION A 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 School 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 3 Texas A&M AgriLife Research and Extension Center, Texas A&M System, Uvalde, TX 78801, USA 1

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 contamination 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 commercial 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 positives 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 Salmonella 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 developing field-based risk assessments for cantaloupes. Keywords: cantaloupe lines, foodborne pathogens, field level assessment, rind netting, contamination, Salmonella. Listeria, false positives, PCR, chromogenic media Agric. Food Anal. Bacteriol. 5: 73-84, 2015

Correspondence: Sadhana Ravishankar, sadhravi@email.arizona. edu, Tel: +1 -520-626-1499

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INTRODUCTION Fresh fruits and vegetables are profitable commodities for growers and distributors as consumption 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 cantaloupes 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

fruits in packaging houses (Morris and Monier, 2003). The factors responsible for the 2011 L. monocytogenes contaminated cantaloupe outbreak were attributed to packing house design, ineffective equipment sanitation and lack of pre-cooling of melons before cold storage (Laksanalamai et al., 2012). Melons 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-

implication, making it important to mitigate contamination by pathogenic bacteria (Ribera et al., 2012). Cantaloupes contaminated by foodborne pathogens such as Listeria monocytogenes and Salmonella enterica have resulted in widespread diseases and associated economic losses. In 2011, a multistate outbreak caused by L. monocytogenes contaminated 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 hospitalizations (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 attachment appendages of the pathogens, as well as biofilm formation could result in colonization of enteric pathogens on cantaloupe rinds (Ukuku and Sapers, 2007; Hanning et al., 2009). Rainfall, water runoff, underground 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

ciency of washing is most likely due to the porous surface 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 bacteria 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 retention 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 microbial 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.

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

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

MATERIALS AND METHODS Cantaloupe genotypes The test cantaloupes consisted of 14 experimen-

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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 commercial 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 maturity before harvesting. Slip is considered as the abscission zone between the fruit and peduncle. Half slip maturity is a standard commercial harvest procedure 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

from each cantaloupe for sampling. Isolation of Listeria 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 Buffered Listeria Enrichment Broth (BLEB) (EMD Chemicals 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

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.

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 using Listeria API strips (bioMerieux, Hazelwood, MO), accessory tests (catalase, oxidase and hemolysis) according to the manufacturer’s instructions, and real time PCR (iQ Check Listeria spp. Kit, Bio-Rad laboratories, Hercules, CA)

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. Cantaloupes were stored at 4°C for 24 h and were evaluated for the presence of Listeria and Salmonella.

Surveillance of cantaloupes for Listeria spp. Plugs of cantaloupe (20 mm length) were obtained 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

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 oligonucleotide molecular beacon probe solution (iQ Check Listeria spp. Kit, Bio-Rad Laboratories). The thermocycler (MiniOpticon™ real-time PCR detection 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 fluorescence from the amplification of the target sequence resulting in a Ct value ≥10 was considered positive.

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Surveillance of cantaloupes for Salmonella serovars A total of 25 g of tissue was taken from each cantaloupe 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 preenrichment 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 water 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 colonies 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. Statistical analysis was performed using Microsoft Excel 2007 (Microsoft Corp., Seattle, WA). Means and standard deviations were calculated for the longitudinal circumference values of melons.

RESULTS Sizes of cantaloupes based on their diameter A total of 21 cantaloupe genotypes (3 fruits each for most genotypes) were surveyed for the presence 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). Cantaloupe 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 positive for the presence of L. monocytogenes. One cantaloupe sample from the breeding line 1405 was positive for the presence of L. innocua after enrichment and plating on selective media (Table 1). This sample was further confirmed through Listeria API tests and real Time PCR. Real Time PCR analysis resulted in one isolate from cantaloupe line 1405 having 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 presence 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 isolates were negative for L. monocytogenes.

Surveillance of cantaloupes for Salmonella None of the 21 cantaloupe genotypes were positive for the presence of Salmonella serovars. Out of

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Table 1. Rind net characteristics and surveillance tests for the presence of Listeria spp. and Salmonella on various cantaloupe genotypes using API Listeria for Listeria and API 20E for Salmonella.

Cantaloupe

Rind net characteristics

Pathogen surveillance testz

Longitudinal Net Splitting API Liste- API 20E g e n o - c i rc u m f e rCoverage /corkiness ria strip strip type ence (cm) (%)

Photograph

Experimental Hybrid

50.37±2.62

100

Low

Neg

52.90±0.69

100

Low

Neg

54.60±1.30

100

Low

Neg

46.57±1.50

Low

Neg

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Cantaloupe

Rind net characteristics

Pathogen surveillance testz

Longitudinal Net Splitting API Liste- API 20E g e n o - c i rc u m f e rCoverage /corkiness ria strip strip type ence (cm) (%)

58.40

100

Low

Neg

53.95±6.29

100

Low

Neg

59.70±5.86

100

Low

Neg

50.20±2.69

100

Low

Neg

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Photograph

Cantaloupe

Rind net characteristics

Pathogen surveillance testz

Longitudinal Net Splitting API Liste- API 20E g e n o - c i rc u m f e rCoverage /corkiness ria strip strip type ence (cm) (%)

12*

53.30

100

Low

Neg

54.17±1.50

100

Low

Neg

15*

67.30

100

Low

Neg

50.37±2.61

100

Low

Neg

Photograph

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Rind net characteristics

Cantaloupe

Pathogen surveillance testz

Longitudinal Net Splitting API Liste- API 20E g e n o - c i rc u m f e rCoverage /corkiness ria strip strip type ence (cm) (%)

60.53±4.06

100

Med

Neg

62.23±4.58

100

Med

Neg

Inbred

146

51.67±3.87

100

Low

Neg

F39

49.53±4.58

100

Low

Neg

1405

57.60±1.93

100

Med

Pos+Neg

Neg

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Photograph

Cantaloupe

Rind net characteristics

Pathogen surveillance testz

Longitudinal Net Splitting API Liste- API 20E g e n o - c i rc u m f e rCoverage /corkiness ria strip strip type ence (cm) (%)

Photograph

Commercial variety

Mission

53.33±2.55

100

Low

Neg

Oro Duro

49.97±1.44

100

Low

Neg

Sol Real

52.07±2.19

100

Low

Neg

Journey

54.60±5.86

Med

Neg

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 negative. z

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. *

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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 Salmonella 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) resulted 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 different melons from genotypes 3, 10 and Mission re-

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 Montevideo (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 contamination. While the research farm at the Texas A&M AgriL-

sulted in false positives on XLD agar and Salmonella CHROMagar.

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 completely covers the rind (most Western shipper cantaloupes) 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 environment and on plant material (Laksanalamai et al.,

ife Research Center did not contain these pathogen reservoirs, farms could potentially be subjected to pathogen introduction through environmental contamination 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 recovery and detection of L. monocytogenes in foods, it was observed that natural microbiota in foods are capable of overgrowing pathogenic target microorganisms (Michael, 2004). In our study, the sensitivity of non-chromogenic plating media was not significantly 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-

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 pathogens (L. monocytogenes and Salmonella serovars)

ting (Ukuku and Sapers, 2007). The breakage or rupture of the rinds could potentially result in crosscontamination 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).

DISCUSSION

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While our study indicated that the various genotypes did not result in significant differences in pathogen attachment, mitigation strategies should be explored 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 netting on microbial attachment and persistence.

ca serovar Typhimurium in loamy and sandy soil monoliths with applied liquid manure. Appl. Environ. Microbiol. 76: 710-714. Buchholz, U., H. Bernard, D. Werber, M.M. Böhmer, C. Remschmidt, H. Wilking, Y. Deleré, M. an der Heiden, C. Adlhoch, J. Dreesman, J. Ehlers, S. Ethelberg, M. Faber, C. Frank, G. Fricke, M. Greiner, M. Höhle, S. Ivarsson, U. Jark, M. Kirchner, J. Koch, G. Krause, P. Luber, B. Rosner, K. Stark, and M. Kühne. 2011. German outbreak of Escherichia coli O104:H4 associated with sprouts. N. Engl. J. Med. e 365: 1763-1770. Centers for Disease Control and Prevention (CDC). 2012. Multistate outbreak of Salmonella Typhimuri-

Andrews-Polymenis, H.L., A.J. Bäumler, B.A. McCormick, and F.C. Fang. 2010. Taming the elephant: Salmonella biology, pathogenesis, and prevention. Infect. Immun. 78: 2356-2369. Baloda, S.B., L. Christensen and S. Trajcevska. 2001. Persistence of a Salmonella enterica serovar Typhimurium DT12 clone in a piggery and in agricultural soil amended with Salmonella-contaminated slurry. Appl. Environ. Microbiol. 67: 2859-2862. Barak, J.D. and A.S. Liang. 2008. Role of soil, crop debris, and a plant pathogen in Salmonella en-

um and Salmonella Newport infections linked to cantaloupe (Final Update). Available at: http:/ www.cdc.gov/salmonella/typhimurium-cantaloupe-08-12/. Chen, W., T.Z. Jin, J.B. Gurtler, D.J. Geveke, and X. Fan. 2012. Inactivation of Salmonella on whole cantaloupe by application of an antimicrobial coating containing chitosan and allyl isothiocyanate. Int. J. Food Microbiol. 155: 165-170. Crosby, K., G. Lester, and D. Leskovar. 2006. Genetic variation for beneficial phytochemical levels in melons (Cucumis melo L.). In: Cucurbitaceae 2006. September 17-21, 2006. Asheville, NC, USA, pp. 70-77. Gagliardi, J.V., P.D. Millner, G. Lester, and D. Ingram, 2003. On-farm and postharvest processing sources of bacterial contamination to melon rinds. J. Food Prot. 66: 82-87. Gupta, S., K. Nalluswami, C. Snider, M. Perch, M. Balasegaram, D. Burmeister, J. Lockett, C. Sandt, R. Hoekstra, and S. Montgomery. 2007. Outbreak of Salmonella Braenderup infections associated with Roma tomatoes, northeastern United States, 2004: a useful method for subtyping exposures in field investigations. Epidemiol. Infect. 135: 1165-1173. Hanning, I.B., J.D. Nutt, and S.C. Ricke. 2009. Sal-

terica contamination of tomato plants. PLoS ONE 3: e1657. Bech, T.B., K. Johnsen, A. Dalsgaard, M. Laegdsmand, O.H. Jacobsen, and C.S. Jacobsen. 2010. Transport and distribution of Salmonella enteri-

monellosis outbreaks in the United States due to fresh produce: sources and potential intervention measures. Foodborne Pathog. Dis. 6: 635-648. Hitchins, A.D. and K. Jinneman. 2011. BAM: Detection and Enumeration of Listeria monocytogenes.

ACKNOWLEDGEMENTS

The authors would like to thank Libin Zhu and Jennifer Todd of the Ravishankar lab for their technical support.

REFERENCES

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Bacterial analytical manual online [Online] US Food and Drug Administration, Washington, DC. Available at : http://www.fda.gov/food/foodscienceresearch/laboratorymethods/ucm071400.htm. Johnston, L.M., L.-A. Jaykus, D. Moll, M.C. Martinez, J. Anciso, B. Mora, and C.L. Moe. 2005. A field study of the microbiological quality of fresh produce. J. Food Prot. 68: 1840-1847. Laksanalamai, P., L.A. Joseph, B.J. Silk, L.S. Burall, C.L. Tarr, P. Gerner-Smidt, and A.R. Datta. 2012. Genomic characterization of Listeria monocytogenes strains involved in a multistate listeriosis outbreak associated with cantaloupe in US. PLoS ONE 7: e42448.

togenes in the food-processing environment. J. Food Prot. 65: 709-725. Ukuku, D.O. and G.M. Sapers, 2007. Effect of time before storage and storage temperature on survival of Salmonella inoculated on fresh-cut melons. Food Microbiol. 24: 288-295. Webster, B. and M. Craig. 1976. Net morphogenesis and characteristics of the surface of muskmelon fruit. J. Amer. Soc. Hort. Sci. 101: 412-415.

Lester, G.E. and D.M. Hodges. 2008. Antioxidants associated with fruit senescence and human health: Novel orange-fleshed non-netted honey dew melon genotype comparisons following different seasonal productions and cold storage durations. Postharvest Biol. Tech. 48: 347-354. Lin, C.-M. and C.-I. Wei. 997. Transfer of Salmonella Montevideo onto the interior surfaces of tomatoes by cutting. J. Food Prot. 60: 858-862. McKinney, J.M., R.C. Williams, G.D. Boardman, J.D. Eifert, and S.S. Sumner, 2009. Effect of acid stress, antibiotic resistance, and heat shock on the resistance of Listeria monocytogenes to UV light when suspended in distilled water and fresh brine. J. Food Prot. 72: 1634-1640. Michael, H., 2004. Evolution of pathogenicity islands of Salmonella enterica. Int J. Med. Microbiol. 294: 95-102. Morris, C.E. and J.M. Monier. 2003. The ecological significance of biofilm formation by plant-associated bacteria. Annu. Rev. Phytopathol. 41: 429-453. Ribera, L.A., M.A. Palma, M. Paggi, R. Knutson, J.G. Masabni, and J. Anciso. 2012. Economic analysis of food safety compliance costs and foodborne illness outbreaks in the United States. HortTechnol. 22: 150-156. Sapers, G.M., R.L. Miller, V. Pilizota, and A.M. Mattrazzo. 2001. Antimicrobial treatments for minimally processed cantaloupe melon. J. Food Sci. 66: 345-349. Tompkin, R.B. 2002. Control of Listeria monocy84

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VOLUME 5 ISSUE 1 ARTICLES 6

Salmonella Transfer to the Lymph Nodes and Synovial Fluid of Experimentally Orally Inoculated Swin P.R. Broadway, J.A. Carroll, J.C. Brooks, J.R. Donaldson, N.C. Burdick Sanchez, T.B. Schmidt, T.R. Brown, and T.R. Callaway

Efficacy of Elimination of Listeria spp., Salmonella spp. and Pseudomonas spp. in Single and Mixed Species Biofilms by Combination of Hydrogen Peroxide Pre-treatment and Cleaning Process B. T. Q. Hoa, T. Sajjaanantakul, V. Kitpreechavanich, W. Mahakarnchanakul

Hops (Humulus lupulus) ß-Acid as an Inhibitor of Caprine Rumen Hyper-Ammonia-Producing Bacteria In Vitro M. D. Flythe, G. E. Aiken1, G. L. Gellin, J. L. Klotz, B. M. Goff, K. M. Andries

Introduction to Authors 41

Instructions for Authors

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Aerobic microbiology Aerobiology Anaerobic microbiology Analytical microbiology Animal microbiology Antibiotics Antimicrobials Bacteriophage Bioremediation Biotechnology Detection Environmental microbiology Feed microbiology Fermentation Food bacteriology Food control

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Inclusive pages of chapter.

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:

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Maciorowski, K. G. 2000. Rapid detection of Salmonella spp. and indicators of fecal contamination in animal feed. Ph.D. Diss. Texas A&M University, College Station, TX.

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.

Donalson, L. M. 2005. The in vivo and in vitro effect of a fructooligosacharide prebiotic combined with alfalfa molt diets on egg production and Salmonella in laying hens. M.S. thesis. Texas A&M University, College Station, TX.

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.

Van Loo, E. 2009. Consumer perception of ready-toeat deli foods and organic meat. M.S. thesis. University of Arkansas, Fayetteville, AR. 202 p.

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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 #_____

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Abstracts and Symposia Proceedings: Fischer, J. R. 2007. Building a prosperous future in which agriculture uses and produces energy efficiently 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. Options and risks by using diagnostic gene chips. Program and abstracts book , The 8th Biennieal Congress of the Anaerobe Society of the Americas. p. 86 (Abstr.)

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