Afab Volume 5 Issue 1

Volume 5 Issue 12015

ISSN: 2159-8967www.AFABjournal.com

2 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 5, Issue 1 - 2015

Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 5, Issue 1 - 2015 3

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 MuriasInstituto 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


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

ABOUT THIS PUBLICATION

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 pend-

ing manuscripts should be addressed Ellen Van Loo,

Managing Editor, Agriculture, Food & Analytical

Bacteriology: [email protected]

Information for Potential Editors: If you are interested

in becoming a part of our editorial board, please con-

tact Editor-in-Chief, Steven Ricke, Agriculture, Food &

Analytical Bacteriology: [email protected]

Advertising: If you are interested in advertising with

our journal, please contact us at advertising@afab-

journal.com for a media kit and current rates.

Reprint Permission: Correspondence regarding re-

prints should be addressed Ellen Van Loo, Managing

Editor, Agriculture, Food & Analytical Bacteriology

[email protected]

Ordering Print Copies: print editions of this journal

may be purchased and shipped internationally from

our website order form at www.afabjournal.com

Subscription Rates: Subscriptions are not available

at this time. To be advised when subscriptions plans

are made available, please join our newsletter at

www.afabjournal.com

Mailing Address: 2138 Revere Place . Fayetteville, AR . 72701 Website: www.AFABjournal.com

EDITORIAL STAFF


Salmonella Transfer to the Lymph Nodes and Synovial Fluid of Experimentally Orally Inocu-lated SwinP.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

6

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 ProcessB. T. Q. Hoa, T. Sajjaanantakul, V. Kitpreechavanich, W. Mahakarnchanakul

15

ARTICLES

Instructions for Authors41

Introduction to Authors

The publishers do not warrant the accuracy of the articles in this journal, nor any views or opinions by their authors.

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

29

TABLE OF CONTENTS


www.afabjournal.comCopyright © 2015

Agriculture, Food and Analytical Bacteriology

ABSTRACT

Salmonella is a foodborne pathogen that may be associated with the consumption of meat products.

Failure of current interventions to control Salmonella in the food supply of the U.S. has led researchers to

believe that atypical carcass reservoirs may be partially responsible for harboring this pathogen. In this two

phase study, pigs (n = 36/Phase 1; n = 38/Phase 2) were experimentally infected orally with Salmonella Ty-

phimurium to monitor the spread of the organism within the animal body. Fecal samples were collected 24,

48, and 72 h post-infection and tested for the presence of the Salmonella. After the pigs were euthanized,

Ileocecal, subiliac, popliteal, and mandibular lymph nodes were collected, and synovial fluid was collected

from the coxofemoral, shoulder, and stifle joints at the same post-infection timepoints to test for the ex-

perimentally inoculated bacteria. Fecal prevalence tended to be greater in Phase 1 (P = 0.06; 52.8 versus

31.6%). Ileocecal lymph node prevalence was 41.67% for Phase 1 and 37.00% for Phase 2. Both mandibular

and subiliac lymph node prevalence was determined to be 2.78% in Phase 1; however, no Salmonella were

detected in Phase 2. Examination of synovial fluid yielded a prevalence of 2.63% in all locations (from a

single pig) in Phase 2 but was not different from Phase 1 (P = 0.30) in which no samples were positive for

Salmonella. These results suggest that it is possible for orally contracted Salmonella to migrate to muscu-

loskeletal lymph nodes. Contamination in these areas may lead to cross-contamination of meat products.

Further research is needed to determine routes and migration patterns of Salmonella from the gastrointes-

tinal tract to peripheral tissues to further elucidate how these infections impact food safety.

Keywords: swine, Salmonella, lymph node, synovial

Correspondence: Jeff Carroll, [email protected]: +1 -806-746-5353 Ext. 120 Fax: +1-806-746-5028

Salmonella prevalence of lymph nodes and synovial fluid of orally inoculated swine

P.R. Broadway1, J.A. Carroll2, J.C. Brooks1, J.R. Donaldson3, N.C. Burdick Sanchez2, T.B. Schmidt4, T.R. Brown1, and T.R. Callaway5

1Department of Animal and Food Sciences, Texas Tech University, Lubbock, TX2Livestock Issues Research Unit, Agricultural Research Service, USDA, Lubbock, TX

3Department of Biological Sciences, Mississippi State University, Mississippi State, MS4Department of Animal Science, University of Nebraska Lincoln, Lincoln, NE

5Food and Feed Safety Research Unit, Southern Plains Agricultural Research Center, Agricultural Research Service, USDA, College Station, TX

Proprietary or brand names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by the USDA implies no approval of the product, or

exclusion of others that may be suitable.” USDA is an equal opportunity provider and employer

Agric. Food Anal. Bacteriol. 5: 6-14, 2015


INTRODUCTION

Salmonella was the most commonly reported

foodborne bacterial infection in 2011 (16.42 cases

per 100,000 people), thus failing to meet the objec-

tives to reduce the incidence of foodborne Salmo-

nella illness set forward by the 2010 national health

objective (6.8 cases per 100,000 people; Centers for

Disease Control and Prevention; CDC, 2012). The

“Healthy People 2010” report, a publication of the

United States Department of Health and Human

Services, indicated a failure to mitigate Salmonella

illness (Johnston, 2012). A broader understanding of

etiological and ecological characteristics and strat-

egies to reduce and control the prevalence of this

pathogen remains a priority in all meat producing

species.

Recent estimates suggest that among foodborne

illness in the U.S., 9 to 15% of all Salmonella infec-

tions, and 7.5% of Salmonella enterica serotypes

Enteritis and Typhimurium infections, are associated

with the consumption of pork or pork products (Hald

et al., 2004; Pires et al., 2010). The risk of Salmonella

infection from pork consumption is often considered

minimal when compared to Salmonella infections

stemming from the consumption of other food prod-

ucts (especially poultry). However, the commonality

in serotypes isolated from pigs and human infection

(USDA, 2013) combined with Salmonella’s ubiqui-

tous nature in swine production settings makes the

pathogen an area of focus for the pork industry.

With regard to Salmonella in the food supply,

lymph nodes have recently become a harbor of in-

terest. Lymph nodes in musculoskeletal tissues of

beef carcasses are often included in retail cuts and/

or ground beef, and have been targeted as an atypi-

cal reservoir of Salmonella (Arthur et al., 2008; Gragg

et al., 2013). Lymph nodes collected from the gas-

trointestinal tract and head of pigs at harvest have

also been reported to harbor Salmonella (Vieira-

Pinto et al., 2005); however, there has been minimal

research conducted to evaluate the occurrence of

Salmonella in peripheral lymph nodes of seemingly

healthy swine that are more likely to be introduced

into the food supply.

In addition to lymph nodes, synovial fluid may also

harbor Salmonella, thus serving as another potential

source of contamination within food products. While

investigations into Salmonella and other foodborne

pathogens in pork synovial fluid are limited, an in-

creased prevalence of Salmonella in human joints af-

ter trauma or invasive procedures has been reported,

and supports the hypothesis that joint synovial fluids

can harbor Salmonella (Fihman et al., 2007). Nairn

(1973) associated Salmonella and other pathogenic

bacteria with osteomyelitis and synovitis in commer-

cial turkeys. Additionally, 55 to 93% of commercial

swine suffer from hind foot lesions, abrasions, and/or

infections (Gentry et al., 2002; Mouttotou et al., 1999).

Thus, the prevalence of hind foot lesions/infections

combined with the ubiquitous nature of Salmonella

supports the theory of Salmonella manifestation in

bone joints of infected and/or sick animals, making

synovial fluid a possible contamination vector.

There is limited information within the literature

to elucidate the mechanisms of translocation of

Salmonella from the gastrointestinal tract into the

circulating lymph system and ultimate migration to

musculoskeletal lymph nodes in apparently healthy

animals. Thus, the overall objective of this study was

to determine if pigs that were orally inoculated with

Salmonella would harbor the pathogen in mesen-

teric and musculoskeletal lymph nodes, as well as

synovial fluid.

MATERIALS AND METHODS

All procedures in this study were reviewed and ap-

proved by the USDA-ARS, Livestock Issues Research

Unit’s Institutional Animal Care and Use Committee

(IACUC protocol 2010-10-JAC8).

Animals

Pigs, diet, and experimental design

This experiment was conducted in two phases

in which Yorkshire/Duroc crossbred pigs (n = 36 for

Phase 1; n = 38 for Phase 2; average 10 ± 1.4 kg BW)

were purchased from a commercial swine producer


and transported to the USDA Livestock Issues Re-

search Unit’s Swine Facility in Lubbock, TX. Pigs

were fed a non-medicated commercial diet com-

posed of (dry matter basis): ground corn 56.2%,

soybean meal 23.25%, rice bran 9%, fish meal 4%,

soyhulls 3.3%, Pork Flex 110 2.75%, tallow 1%, L-thre-

onine 0.2%, L-lysine 0.15%, and methionine 0.15%.

The diet was formulated according to Nutrient Re-

quirements of Swine recommendations and pigs

were allowed ad libitum access to water. Pigs were

individually penned and housed in an environmen-

tally controlled facility with an average air tempera-

ture of 28.2 ± 0.4°C. Fecal samples were collected

from each pig upon arrival and each subsequent day

(for 5 d) during the dietary/facility adaptation period

to verify that no growth occurred on novobiocin (20

µg/ml) and nalidixic acid (25 µg/ml) supplemented

Brilliant Green agar (BGANN). Prior to inoculation,

no colonies grew on any of the BGANN plates. Fecal

samples were also analyzed during the adaptation

period by enrichment for the presence of bacterio-

phages that could lyse the Salmonella Typhimurium

(Callaway et al., 2010), strain used in the present in-

oculation study.

In phase one, pigs were supplemented via the

diet with phosphate buffered saline (PBS) or PBS

with Enterobacter cloacae. In phase 2, pigs were fed

with and without the inclusion of yeast cell wall prod-

ucts. For both phases, at the end of the 5 d adapta-

tion period, each pig was inoculated with Salmonella

Typhimurium (2 x 1010 CFU/pig) via oral gavage (10

mL total volume per pig) at 0 h. The concentration of

Salmonella Typhimurium was utilized to ensure rela-

tively elevated concentrations of Salmonella in the

gastrointestinal tract to further enhance the possible

transfer of the pathogen into systemic lymph.

Bacterial cultures

Salmonella enterica serotype Typhimurium (ATCC

BAA-186) from the USDA Food and Feed Safety Re-

search Unit culture collection was repeatedly grown

(4 passages) by 10% (vol/vol) transfer in anoxic

(85% N2, 10% CO2, 5% H2 atmosphere) Tryptic soy

broth (TSB) medium at 37ºC to adapt the culture for

growth in the anaerobic intestinal tract. This strain

was made resistant to novobiocin and nalidixic acid

(20 and 25 µg/mL, respectively) by repeated transfer

and selection in the presence of sub-lethal concen-

trations of each antibiotic. This resistant phenotype

was stable through multiple unselected transfers in

batch culture and through repeated culture vessel

turnovers in continuous culture (data not shown).

Overnight cultures (1 L) contained populations of

Salmonella Typhimurium that were determined to be

4 x 109 CFU/ml by serial dilution and plating.

Gastrointestinal sample collection

Pigs (n = 12/d) in Phase 1 of the study were hu-

manely euthanized at 24, 48, and 72 h after inocula-

tion with Salmonella. For Phase 2, all pigs were euth-

anized 72 h post-inoculation in an effort to increase

the possibility of finding Salmonella in peripheral

lymph nodes and joints based on results from Phase

1. Ileocecal lymph nodes were aseptically collected

and enriched following maceration (Figure 1). Di-

gesta and epithelial tissues from the terminal rectum

were also aseptically collected upon necropsy.

Lymph node collection

Mandibular, subiliac, and popliteal lymph nodes

were collected from each animal following collec-

tion of gastrointestinal contents. Each lymph node

was collected aseptically from the right side of the

carcass. Samples were subjected to a surface disin-

fectant dip with 70% ethanol and were subsequently

macerated and placed in tetrathionate broth for

enrichment. Isolation of the inoculated Salmonella

strain was conducted as described below.

Synovial Fluid Collection

Synovial fluid was collected from each pig (n = 74)

at three anatomical locations (i.e., shoulder, coxo-

femoral, and stifle) on the right side of the carcass.

These joints were selected because each of these

anatomical locations represent an area of the carcass

that may be included in a retail cut or a joint that may


be exposed during fabrication that could lead to

possible cross contamination. Joints were exposed

using leverage on one side of the joint along with a

sterile scalpel cutting the skin on the opposite side of

the joint. Both the skin of the animal and the instru-

ments used were disinfected with 70% ethanol prior

to incision. Expressed synovial fluid was collected

with environmental sponges (EZ 10BPW; World Bio-

products, Woodinville, WA) that were placed in 10

mL buffered peptone water (BPW). Sponges were

stomached at 230 RPM for 2 min. (Stomacher 400

Circulator, Seward, Davie, FL), and the enrichment

was incubated at 37ºC prior to detection.

Salmonella Detection

To qualitatively confirm the presence of inocu-

lated Salmonella Typhimurium in lymph nodes, sy-

novial fluid, rectal contents, and epithelial samples,

macerated samples and sponges were incubated

overnight in tetrathionate broth at 39ºC and a 200 µL

aliquot was transferred to Rappaport-Vassiliadis R10

Broth which was subsequently incubated at 42ºC for

24 h. Following this secondary enrichment, samples

were streaked on BGANN plates. Plates that exhib-

ited colonies after 24 h incubation were classified as

positive for experimentally innoculated Salmonella

Typhimurium. Unless otherwise noted, all media

and agar were from Difco Laboratories (Sparks, MD).

Reagents and antibiotics were obtained from Sigma

Chemical Co., St. Louis, MO. Post-enrichment syno-

vial samples were subjected to real-time PCR analy-

sis (BAX ®; Dupont, Wilimington, DE; AOAC 100201)

to confirm the presence of Salmonella.

Statistical Analysis

The experimental unit in both phases was the indi-

vidual pig. Pigs were randomly assigned to a harvest

day for Phase 1. Data from pigs positive for Salmo-

nella were analyzed using Pearson Exact Chi Square

analysis of SAS (v. 9.3 SAS Inst. Inc., Cary, NC). In-

teractions between fecal and illeocecal lymph node

prevalence were analyzed using binomial logistic re-

gression in PROC GLIMMIX of SAS. For Phase 1, day

was considered a random variable. Significance was

determined at P < 0.05 for all data.

Figure 1. Graphical representation of lymph node and synovial sampling locations. a. Mandibular lymph node, b. Shoulder joint, c. Subiliac lymph node, d. Stifle Joint, e. Popliteal lymph node, f. Hip joint


RESULTS AND DISCUSSION

Animals

Pigs did not demonstrate any visual signs or

symptoms of disease during this short term infection

study. However, quantified immunological markers

were consistent with an infection. Additionally, a

small, yet significant, febrile response was observed

after the Salmonella challenge (data not shown).

Fecal

Fecal prevalence of the experimentally inocu-

lated Salmonella in Phase 1 was 52.8% (85%, 46%,

and 30% for 24, 48, and 72 h post-inoculation, re-

spectively) and 31.6% in Phase 2 of the study (Table

1). There was a tendency (P = 0.06) for Phase 1 fecal

prevalence to be greater than Phase 2 across all col-

lection timepoints. This tendency is understandable

due to Phase 2 sample collection only occurring at

72 h post-infection. Also, there was no fecal x day

interaction (P = 0.18). The presence of Salmonella

in the feces of pigs plays a major role in the cross-

contamination of pork carcasses and ultimately the

food supply. While the pigs in this study were in a

controlled environment, many factors can influence

infection and fecal shedding of Salmonella such as

transportation (Hurd et al., 2002), lairage (Hurd et al.,

2001), and co-mingling with other animals and new

environments (Hurd et al., 2001). Fecal prevalence in

pigs raised in commercial swine production opera-

tions has been reported to be between 1 and 33%

(Davies et al., 1998; Rodriguez et al., 2006; Barber,

2002; Foley et al., 2008). Gebreyes et al., (2004) re-

ported that swine herds with a greater prevalence

of fecal Salmonella had the greatest incidence of

carcass contamination at harvest, further solidifying

the correlation between fecal and carcass contami-

nation. Furthermore, Ojha and Kostrzynska (2007)

stated that pigs may shed 10 million cells/g in feces

during a Salmonella infection. Salmonella shedding

in feces may transfer to other pigs or lead to cross-

contamination via lairage, feed, or water.

Salmonella in Lymph Nodes

In Phase 1 of the study, a total of 41.8% of pigs

(n = 15) experimentally inoculated were positive for

Salmonella in ileocecal lymph nodes at necropsy.

The pigs were euthanized at three timepoints (24,

48, and 72 h post-inoculation; n = 13, 13, 10, respec-

tively) in Phase 1, and ileocecal lymph node preva-

lence of Salmonella at these time points was 46.15%,

46.15%, and 30.00%, respectively (Table 1). Salmo-

nella prevalence in ileocecal lymph nodes was less

than expected considering the concentration and

dosage of innocula introduced into the gastrointes-

tinal tract of the animal. Callaway and colleagues

Table 1. The prevalence of Salmonella detected in four different lymph nodes from pigs experi-mentally inoculated with Salmonella.

% Positive for Salmonella

Feces Illeocecal Popliteal Mandibular Subiliac

Phase 11 52.7 41.6 0.0 2.7 2.7

Phase 22 31.5 37.0 0.0 0.0 0.0

P-value3 0.0 0.6 1.0 0.3 0.3

1 n = 36 pigs2 n = 38 pigs3 SEM = 2.34


(2011) reported that in a trial in which pigs were inoc-

ulated with a similar dose of Salmonella, 77 to 83% of

ileocecal lymph nodes were positive for Salmonella.

In Phase 1 of the present study, all popliteal lymph

nodes collected tested negative for Salmonella, but

subiliac and mandibular lymph nodes were positive

for the experimentally infected Salmonella strain at

a prevalence of 2.78% (Table 1). Interestingly, all of

these positive peripheral lymph nodes were isolated

from pigs necropsied only at 48 h after inoculation.

A total of 36.80% of ileocecal lymph nodes col-

lected from pigs in Phase 2 (at 72 h post-inoculation)

of the study were positive for the inoculated Salmo-

nella strain; however, no peripheral lymph nodes col-

lected from Phase 2 pigs harbored Salmonella (Table

1). These results are consistent with data reported by

Gray et al., (1996) which indicated that lymph nodes

near the ileocolic junction were the lymph nodes with

the greatest Salmonella prevalence at harvest. While

there were numerical differences, there were no sta-

tistical differences in lymph node prevalence of Sal-

monella between the two phases of the present study

for ileocecal (P = 0.67), popliteal (P = 1.00), mandibu-

lar (P = 0.30), and subiliac (P = 0.30) lymph nodes.

Our results associated with the presence of Sal-

monella in subiliac and mandibular lymph nodes are

of great importance to food safety as these lymph

nodes are anatomically located in areas of the pork

carcass that are commonly included in trim and retail

cuts. These lymph nodes may also be exposed and

evaluated during post-mortem USDA inspection and

ultimate fabrication of the carcass. Exposure of in-

fected lymph tissues during fabrication could poten-

tially lead to cross contamination of equipment and/

or other carcasses. The presence of Salmonella in

the feces of live pigs has been previously examined

in conjunction with subiliac lymph nodes; however,

little association was present to suggest feces as a

predictor of lymph node contamination (Wang et al.,

2010). Studies conducted by Hurd et al. (2001, 2002)

reported less incidence of Salmonella in feces from

swine at the farm when compared to ileocecal lymph

nodes positive for Salmonella at harvest. Wood et

al. (1989) reported the prevalence of Salmonella

infected ileocecal lymph nodes to be between 30

and 50% less than the prevalence of Salmonella in

feces from the same animals. In the present study,

there was no interaction between fecal and ileocecal

lymph node prevalence (P = 0.15). These previously

reported data as well as data from the current study

help elucidate a possible disconnect between fecal

shedding of Salmonella from pigs and the incidence

of Salmonella harbored in the lymphatic system (Fol-

ey et al., 2008).

Berends et al. (1996) stated that the gastrointesti-

nal tract and lymph nodes may be major sources of

Salmonella carcass contamination and subsequent

transfer to human consumers. Positive correlations

have been reported between Salmonella in feces and

on carcasses (Berends et al., 1997) and between Sal-

monella in the intestinal tract and carcasses (Swan-

burg et al., 1999). A study conducted by Vieira-Pinto

et al, (2005) sampled carcasses that swabbed positive

for Salmonella, and the researchers reported that

18.8% of ileocecal lymph nodes from those carcasses

contained Salmonella. Additionally, pig mandibular

lymph nodes and tonsils have also been reported to

harbor Salmonella (12.9 and 9.9%, respectively; Viei-

ra-Pinto et al. 2005). Based on data from the current

study, we can infer that there is a relatively miniscule

possibility that Salmonella (contracted orally) will be

harbored in musculoskeletal lymph nodes at 72 h

post-inoculation. While the Salmonella prevalence

of these lymph nodes was very limited at 72 h post-

infection, further investigation should be conducted

to elucidate the timeframe of migration of oral Sal-

monella infections as well as other factors that may

impact pathogen migration in the lymphatic system.

While musculoskeletal lymph node prevalence was

not great, a single contaminated lymph node could

potentially contaminate thousands of pounds of

product when comminuted together with other trim.

Given that Salmonella can be internalized within the

lymph nodes of pigs, the bacterial cells are not as

susceptible to topical in-plant pathogen reduction

systems implemented by most packers and proces-

sors. For these reasons, some packers excise lymph

nodes as a routine part of the fabrication process in

an effort to reduce potential contamination of pork

trim.


Synovial

Synovial fluid prevalence was 0 and 2.63% for

Phase 1 and 2 respectively. There was no statisti-

cal difference (P = 0.30; Table 2) between Salmonella

positive synovial samples in Phase 1 and Phase 2

of the study. Of the synovial swabs collected (n =

222; 3/pig), only three swabs were positive for Sal-

monella, all of which were from the same pig. This

particular pig was noted to have a large abscess

on the abdomen at the time of harvest. While this

phenomenon was only observed in one animal, we

hypothesize that the immunocompromised state of

the pig during the experimental infection may have

played a role in the transmission of Salmonella into

the synovia. This hypothesis is supported by the

fact that no visible lesions were noted on this par-

ticular animal at any of the joint sampling locations.

While little is known about Salmonella in the joints of

swine, Varley and Wiseman (2001) suggest that im-

munosuppression due to porcine reproductive and

respiratory syndrome (PRRS) may predispose the

swine to synovial infections by Haemophilus para-

suis. More research is needed to determine if im-

munocompromised pigs translocate infections from

the gastrointestinal tract to other peripheral tissues.

Similar to lymph nodes, Salmonella in synovial fluid

is not susceptible to post-harvest topical pathogen

reduction interventions applied to the carcass. The

original hypothesis of this study stated that synovial

fluid may harbor Salmonella and be a possible vec-

tor for potential cross-contamination when exposed

during fabrication. However, based on the data from

the current study, we can infer that the possibility of

Salmonella cross-contamination via synovial fluid

from pigs that orally acquire this pathogen is rela-

tively low.

CONCLUSIONS

Overall, this study determined that experimental

oral inoculation of pigs with Salmonella may result

in Salmonella penetrating the lymphatic system and

reaching peripheral lymph nodes. While most of the

infection was localized to the illeocecal lymph nodes

and feces, the infection was also shown to reach pe-

ripheral lymph in some animals. While only being

observed in one animal, we hypothesize that oral in-

fection with Salmonella may be able to influence Sal-

monella prevalence in the joints of immunocompro-

mised animals. Elucidating the transmission routes

of Salmonella to peripheral tissues in the carcasses

of pigs that enter the food chain is vital to the estab-

lishment of interventions and control points to pre-

vent foodborne illness and cross contamination in

the pork production process. More research needs

to be conducted to determine how Salmonella infec-

tions with different routes of entry migrate through

the lymphatic system and how these infections im-

pact animal health and food safety.

Table 2. The prevalence of Salmonella detected in synovial fluid from three different joints from pigs experimentally inoculated with Salmonella.

% Positive for Salmonella

Shoulder Hip Stifle

Phase 11 0.0 0.0 0.0

Phase 22 2.6 2.6 2.6

P-value3 0.3 0.3 0.3

1 n = 36 pigs 2 n = 38 pigs 3 SEM = 1.32


REFERENCES

Arthur, T. M. et al. 2008. Prevalence and character-

ization of Salmonella in bovine lymph nodes po-

tentially destined for use in ground beef. J. Food

Prot. 71:1685-1688.

Barber, D.A., P.B. Bahnson, R. Isaacson, C.J. Jones,

and R.M. Weigel. 2002. Distribution of Salmonel-

la in swine production ecosystems. J. Food Prot.

65:1861-1868.

Berends, B.R., F. Van Knapen, J.M.A. Snijders, and

D.A. Mossel. 1997. Identification and quantifica-

tion of risk factors regarding Salmonella spp. on

pork carcasses. Int. J. Food Microbiol. 36:199-206.

Berends, B.R., H.A.P. Urlings, J.M.A. Snijders, and

F. Van Knapen. 1996. Identification and quanti-

fication of risk factors in animals management

and transport regarding Salmonella in pigs. Int. J.

Food Microbiol. 30:37-53.

Callaway, T. R., T.S. Edrington, A. Brabban, B. Kut-

ter, L. Karriker, C. Stahl, E. Wagstrom, R. Anderson,

T.L. Poole, K. Genovese, N. Krueger, R. Harvey, and

D.J. Nisbet. 2011. Evaluation of phage treatment

as a strategy to reduce Salmonella populations in

growing swine. Foodborne Pathog. Dis. 8:261-266.

Davies, P.R., F.G. Bovee, J.A. Funk, W.E. Morrow, F.T.

Jones, and J. Deen. 1998. Isolation of Salmonella

serotypes from feces of pigs raised in a multiple-

site production system. J. Am. Vet. Med. Assoc.

212:1925-1929.

Fihman, V., D. Hannouche, V. Bousson, T. Bardin, F.

Liote, L. Raskine, J. Riahi, M.J. Sanson-Le Pors, and

B. Bercot. 2007. Improved diagnosis specificity

in bone and joint infections using molecular tech-

niques. J. Infection. 55:510-517.

Foley, S.L., A.M. Lyne, and R. Nayak. 2008. Salmo-

nella challenges: prevalence in swine and poultry

and potential pathogenicity of such isolates. J.

Anim. Sci. 89:149-162.

Gentry, J.G., J.J. McGlone, J.R. Blanton Jr., and M.F.

Miller. 2002. Alternative housing systems for pigs:

influences on growth, composition, and pork qual-

ity. J. Anim. Sci. 80:1781-1790.

Gragg, S. E., G.H. Loneragan, M.M. Brashears, T.M.

Arthur, J.M. Bosilevac, N. Kalchayanand, R. Wang,

J.W. Schmidt, J.C. Brooks, S.D. Shackelford, T.L.

Wheeler, T.R. Brown, T.S. Edrington, and D.M.

Brichta-Harhay. 2013. Cross-sectional study ex-

amining Salmonella enterica carriage in subiliac

lymph nodes of cull and feedlot cattle at harvest.

Foodborne Pathog. Dis. 10:367-374.

Gray, J.T., T.J. Stabel, and P.J. Fedorka-Cray. 1996.

Effect of dose on the immune response and persis-

tence of Salmonella Cholerasuis infecton in swine.

Am. J. Vet. Res. 57:313-319.

Hald, T., D. Vose, H.C. Wegener, and T. Koupeev.

2004. A bayesian approach to quantify the contri-

bution of animal-food sources to human salmonel-

losis. Risk Anal. 24:255-269.

Hurd, H.S., J.D. McKean, I.V. Wesley, and L.A. Karrik-

er. 2001. The effect of lairage on Salmonella isola-

tion from market swine. J. Food Prot. 64:939-944.

Hurd, H.S., J.D. McKean, R.W. Griffith, I.V. Wesley,

and M.H. Rostango. 2002. Salmonella enterica in-

fections in market swine with and without transport

and holding. Appl. Environ. Microbiol. 68:2376-

2381.

Hurd, H.S., J.K. Gailer, J.D. McKean, M.H. Rostagno.

2001. Rapid infection in market-weight swine fol-

lowing exposure to a Salmonella Typhimurium-

contaminated environment. Abstract. Am. J. Vet.

Res. 62:1194-1197.

Johnston, T. 2012. “Pathogen of Interest”. Meating-

place. May 2012. Pages 57-61.

Mouttotou, N., F.M. Hatchell, and L.E. Green. 1999.

Prevalence and risk factors associated with adven-

titious bursitis in live growing and finishing pigs in

south-west England. Prev. Vet. Med. 39:39-52.

Ojha, S. and M Kostrzynska. 2007. Approaches for

reducing Salmonella in pork production. J. Food

Prot. 70:2676-2694.

Nairn, M.E. 1973. Bacterial osteomyelitis and syno-

vitis of the turkey. Avain Diseases. 17:504-517.

Pires, S.M. and T. Hald. 2010. Assessing the differ-

ences in public health impact of Salmonella sub-

types using a Bayesian microbial subtyping ap-

proach for source attribution. Foodborne Pathog.

Dis. 7:143-151.

Rodriguez, A., P. Panlgoli, H.A. Richards, J.R. Mount,

and F.A. Draughon. 2006. Prevalence of Salmo-


nella in diverse environmental farm samples. J.

Food Prot. 69:2576-2580.

Swanburg, M., H.A.P. Urlings, D.A. Keuzenkamp, and

J.M.A. Snijders. 1999. Tonsils of slaughtered pigs

as a marker sample for Salmonella positive pork.

In: Bahnson, P.B. (ed.). Proceedings of the 3rd In-

ternational Symposium on the Epidemiology and

Control of Salmonella in Pork, pp. 264-265. Wash-

ington DC, USA.

Varley, M.A. and J. Wiseman. 2001. The weaner pig:

nutrition and management. P. 243. CABI Publish-

ing, New York, NY.

Vieira-Pinto, M., P. Temudo, and C. Martins. 2005.

Occurrence of Salmonella in the ileum, ileocolic

lymph nodes, tonsils, mandibular lymph nodes

and carcasses of pigs slaughtered for consump-

tion. J. Vet. Med. 52:476-481.

Vieira-Pinto, M., P. Temudo, and C. Martins. 2005.

Occurrence of Salmonella in the ileum, ileocolic

lymph nodes, tonsils, mandibular lymph nodes

and carcasses of pigs slaughtered for consump-

tion. J. Vet. Med. 52:476-481.

Wang, B., I. V. Wesley, J. D. McKean, and A. M.

O’connor. 2010. Sub-iliac lymph nodes at slaughter

lack ability to predict Salmonella enterica for swine

farms. Foodborne Path. Dis. 7:795-800.

Wood, R.L., A. Pospischil, and R. Rose. 1989. Distri-

bution of persistent Salmonella typhimurium infec-

tion in internal organs of swine. Am. J. Vet. Res.

50:1015-1021.




ABSTRACT

Control and elimination of biofilm formation in the food processing environment is vital for food safety.

This study was designed to investigate the efficacy of hydrogen peroxide (H2O2) pre-treatment combined

with the regular daily cleaning procedure used in a shrimp plant to control biofilm formation. Single and

mixed species biofilms of Listeria, Salmonella and Pseudomonas were used as the test model. Single bio-

films on stainless steel (SS) coupons were formed under nutrient stress and harvested at 3 and 7 days to as-

sess four cleaning procedures. Using 2% alkaline detergent for 10 minutes followed by two types of quater-

nary ammonium compounds (QACs) - based sanitizers completely eliminated single biofilms of Listeria and

Salmonella. When alkaline was replaced to acidic type, microbial reduction achieved 5 log colony forming

units (CFU)/cm2 (or more). For the mixed species biofilm study, biofilms were formed under the simulated

seafood processing plant conditions for 7 days, on SS, Teflon and rubber coupons. After pre-treated mixed

species biofilms with H2O2 at 1% and 2% for 5 and 10 minutes followed by the regular cleaning procedure,

2% of H2O2 for 10 minutes reduced microorganisms by 6 log CFU/cm2. Mixed biofilm on SS was easier to

remove compared to the other surfaces. Overall these results suggest that the application of H2O2 prior to

the regular cleaning process in food processing facilities may help to reduce and control biofilm formation,

particularly biofilms composed of mixed species.

Keywords: Biofilm, Listeria, Salmonella, Pseudomonas, hydrogen peroxide, cleaning, sanitizer,

stainless steel, Teflon, rubber

INTRODUCTION

Microorganism contamination on food products

has been increasing in the food processing environ-

ment even though routine cleaning and sanitizing

using various detergents and chemicals is employed

Correspondence: Warapa Mahakarnchanakul,[email protected]: +66-2562-5036 Fax: +66 2 562 5021

(Bridier et al., 2015; Food & Water-Watch, 2007;

Norhana et al., 2010). Contaminated food causes

harm to consumer’s heath and economic issues as

well as losses related to the brand name of the re-

spective food producer (Bohme et al., 2013). Biofilm

formation is a serious concern due to inappropriate

cleaning process (Brooks and Flint, 2008; Chmielews-

ki and Frank, 2003; Giaouris et al., 2014; Gibson et

al., 1999). Moreover, not only spoilage bacteria

Efficacy of Sanitizers on Listeria, Salmonella, and Pseudomonas Single and Mixed Biofilms in a Seafood Processing Environment

B. T. Q. Hoa1, W. Mahakarnchanakul1, T. Sajjaanantakul1, V. Kitpreechavanich2

1Department of Food Science and Technology, Faculty of Agro-Industry, Kasetsart University, Bangkok 10900, Thailand2Department of Microbiology, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand



(e.g., Pseudomonas, Klebsiella) but also most of the

foodborne pathogens (e.g., Listeria monocytogenes,

Salmonella enterica serovar Typhimurium, E. coli H7:

O157) are able to adhere to biofilms on most materi-

als and under almost all of the environmental con-

ditions in food production plants (Beauchamp et al.,

2012; Bridier et al., 2015; Giaouris et al., 2012; Joseph

et al., 2001; Marchand et al., 2012; Ryder et al., 2007).

In addition, viable microorganisms in biofilm are tol-

erant to sanitizers because some microorganisms

have specific mechanisms to resist sanitizing agents.

A few examples include Pseudomonas spp., Listeria

spp., Salmonella spp., Escherichia coli, etc. (Bridier

et al., 2015; Chmielewski and Frank, 2003; Dourou et

al., 2011; Duong, 2012; Ibusquiza et al, 2011; Myszka

and Czaczyk, 2011). Furthermore, extracellular poly-

meric substances (EPS), which are produced by mi-

croorganisms to protect themselves against other

species or under stress growth conditions, act as glue

which help microorganisms to effectively adhere on

the surfaces of equipment, leading to the failure of

the removal of the biofilm during the cleaning pro-

cess (Bridier et al., 2015; Giaouris et al., 2012; 2013).

Therefore, finding the appropriate method for re-

moving biofilm is the interest of both food scientists

and food manufacturers. Numerous studies on eval-

uation of the effectiveness of sanitizer on single bio-

films of both pathogenic and spoilage bacteria have

been reported (Beauchamp et al., 2012; Belessi et al.,

2011; Choi et al., 2012; Elmali et al., 2012; Ölmez and

Temur, 2010; Oz et al., 2012). However, research on

the efficacy of sanitizers to remove the mixed species

biofilms at a mature stage remain limited, especially

mixed species biofilm formation under simulated

food processing ecosystem conditions. In addition,

most research only focuses on the effect of sanitizers

to eliminate organisms in biofilms (Aase et al., 2000;

Fatemi and Frank, 1999; Joseph et al., 2001; Norwood

and Gilmour, 2000). There is still a lack of studies that

fully applies the simulation of likely cleaning process

at the seafood processing facility, using both clean-

ing with detergent and disinfectant with sanitizers.

Furthermore, employing a method to degrade the

EPS in a biofilm before cleaning procedure has not

been fully investigated (Giaouris et al., 2014; Xavier

et al., 2005). Therefore, the objectives of this study

were focused on the assessment of cleaning process

at food processing plants through single mature bio-

film formation under nutrient stress followed by a

proposed modified cleaning method that combines

EPS degradation by H2O2 and a cleaning process to

assess the effectiveness of removing a mixed species

biofilms under simulated food processing ecosystem

conditions.


Sanitizers and detergents

Sanitizers and detergents were supplied by a local

seafood processing facility in Thailand where these

chemicals are regularly used in cleaning process.

These included an alkaline foam cleaner (Superp

foam), acidic foam cleaner (Dilac Z – descaler), QACs

(Spectrum- broad spectrum liquid disinfectant), PAA,

oxidizing disinfectant- peracetic acid (Zal Perax II)

and QACs- based (Quatdet clear -broad disinfectant,

fogging). These chemicals were products of Diversey

Hygiene (Thailand) Co., Ltd. Hydrogen peroxide

(grade AR) was a product of QReC, New Zealand.

Bacteria cultures and stock preparation

Three types of bacteria (Listeria monocytogenes

101; Salmonella enterica serovar Aberdeen and

Pseudomonas aeruginosa) were obtained from

the Department of Food Science and Technology,

Agro-Industry Faculty, Kasetsart University, Thailand.

These bacteria (Listeria monocytogenes 101, Salmo-

nella enterica serovar Aberdeen and Pseudomonas

aeruginosa) were commonly present on foodborne

pathogenic and spoilage bacteria, especially in sea-

food processing (Gram and Huss, 1996; Gram et al.,

1987; Koonse et al., 2005; Norhana et al., 2010). All

bacterial species were cultured (37°C, 24 h) and sub-

cultured (37°C, 18 h) individually in 10 mL tryptic soy

broth (TSB; Difco). Cells of the individual cultures

were then harvested by centrifugation (10000 ×g at

4 °C for 10 min) followed by washing twice in 1 mL


phosphate buffered saline (PBS, pH 7.4) (Bae et al.,

2012; Stewart and Costerton, 2001). Washed cell pel-

lets of each species were resuspended in 1 mL TSB.

To prepare the single species stock, the resuspension

of each species was mixed with sterilized glycerol

36% by a ratio of 1: 1 (v/v). Finally, stock cultures were

stored in a refrigerator at -20°C.

Similar with the method to apply for preparing

single species stock, the mixed species stock, which

was used for the mixed species experiment, was pre-

pared with 80% of volume cell suspension of Pseu-

domonas spp., 10% of volume of Listeria spp., and

10% of volume of Salmonella spp. Following this, the

mixed species were added to glycerol and kept in a

refrigerator at -20°C.

Biofilm formation

Single species biofilm formation

Stocks of three species Listeria spp., Salmonella

spp., and Pseudomonas spp. were individually cul-

tured (37°C, 24 h) and sub-cultured (37°C, 18 h) again

to prepare single species biofilms for this experi-

ment. Sterilized SS coupons (304, finish # 2B) with

dimensions of 5 × 2 × 0.08 cm were transferred to

50 mL conical centrifuge tubes containing 40 mL of

TSB (1% or 10%; Difco); which were inoculated with a

0.1mL suspension of sub-cultured bacterial cells; the

bacterial population in each sample was approxi-

mately 2 × 107 CFU/mL. Following this, samples were

incubated at 16°C for 3 days or 7 days. At sampling

time (3 days or 7 days) single species biofilms were

collected to conduct the experiments.

Mixed species biofilm formation under a simu-

lated food processing ecosystem (SFP)

The SS coupons (304, finish # 2B); teflon® coupons

and rubber coupons representing common materi-

als that have been employed in food processing sys-

tems were used in this study. A modified CDC biofilm

reactor (Centers for Disease Control, CDC; BioSur-

face Technologies Corp., Bozeman, MT, USA) (modi-

fied from Hadi et al., 2010) was used for conducting

this experiment. It included two main components,

namely, a bioreactor and a frame holding coupons.

Forty eight coupons were set up under the same

conditions. First, coupons were soaked overnight

in commercial detergent solution, degreased with

70% ethanol, thoroughly rinsed with tap water and

distilled water (Hoa et al., 2015), and coupons were

subsequently vertically placed sequentially at coupon

gaps on a frame of the biofilm reactor (bioreactor).

These CDC bioreactors were autoclaved at 121° C for

15 min prior to use (Pan, 2005; Stewart and Coster-

ton, 2001). Next, four liters of broth (1% TSB, 16°C)

and 1 mL of mixed species stock were added into the

bioreactor. The bacterial population in the bioreactor

was approximately 4.4 × 106 CFU/mL. Following this,

the bioreactor was placed in an incubator for 7 days,

at 16°C under simulated food processing ecosystem

with daily released and refreshed 4 liters of broth.

Bacteria in the biofilm were subjected to broth for 8

hours/day, followed by starvation for nearly 16 hours

/day. Finally, mixed species biofilms were harvested

for conducting the experiment.

Cleaning process and modified cleaning process by pretreatment with hydrogen peroxide

Cleaning process

The cleaning process used at the seafood com-

pany was simulated for this experiment. The regime

for the cleaning process consisted of rinsing, clean-

ing with detergent, rinsing, disinfecting, rinsing, re-

disinfecting and rinsing. The four cleaning processes

were respectively, (Fig. 1), D1S1- regular daily clean-

ing; D1S2- weekly cleaning; D2S1 and D2S2 - bi-

monthly cleaning. To perform the cleaning process,

biofilm coupons were placed in a cleaning holding

coupon frame which had been designed for assess-

ing cleaning performance. The cleaning procedure

was conducted as indicated by the flow chart shown

in Fig. 1. The spraying method was applied for all

steps of each treatment. Distilled water, detergents,

sanitizers and hydrogen peroxide were contained in

the same type of bottles (distilled water bottle 16 oz

# 500 mL). Spraying distance was approximately 10

cm from the top of bottle to coupons. Spraying time

was approximately 30 seconds per treatment (4 cou-


pons and both sides of coupons). Spraying volume

was approximate 60 mL per treatment. At the end

of each step, samples were covered and placed in

a laminar flow biological safety cabinet for specified

exposure times. The concentration of detergents,

concentration of sanitizers and exposure time were

determined based on the flow chart of each clean-

ing process. Detergents and sanitizers were diluted

and placed in the refrigerator for an hour prior to the

cleaning process. After the cleaning process, biofilm

coupons were collected in duplicate and placed into

sterilized petri dishes for evaluating the efficacy of

each cleaning process.

Modified cleaning process by pre-treatment

with H2O2

Similarly, a modified cleaning process was applied

the pre-treatment with H2O2 before cleaning with a

detergent-based stage. The pre-treatment with H2O2

was performed with two levels of concentrations (1%

and 2%) and two levels of exposure time (5 and 10

minutes). Following this, biofilm coupon continuous

performances were evaluated over a regular daily

cleaning process (D1S1). For D1S1 samples (no pre-

treatment with H2O2), coupons were rinsed with 10

mL distilled water, followed by continuous perfor-

mance evaluations over a regular daily cleaning pro-

cess (D1S1).

Determination of number of surviving bacteria

Surviving bacteria on inoculated coupons with

single species or mixed species bacteria were de-

tached carefully using two cotton swabs. For con-

trol samples (Biofilm, no cleaning process), coupons

were rinsed twice with 10 mL distilled water in order

to remove the loosely attached cells. Following this,

Figure 1. Flow chart of four cleaning processes (D1S1) daily; (D1S2) weekly; (D2S1 & D2S2) bi-monthly


all samples were swabbed thoroughly and the swab

heads were broken off into a glass tube containing

10mL sterilized saline peptone water (SPW) (0.85%

of salt and 0.1% of bacteriological peptone, Difco;

Bagge-Ravn et al., 2003; Gibson et al., 1999). Next,

suspensions were left for 30 minutes at 16°C to re-

cover cells after treatment with the sanitizing agent.

The bacteria on the swabs were re-suspended by

vortexing for 1 min at high speed (Vortex Genie 2 G

– 560E, speed 8). The re-suspension fluid was serially

diluted in SPW and spread in duplicate on Tryptic

soy agar (TSA) (Difco). The TSA plates were incu-

bated at 37°C for 1 day and bacterial viability was

quantified (Bae et al., 2012; Nguyen and Yuk, 2013).

Data analysis

Results of experiments were converted into log

values and averages were reported. Mean values

and standard deviations were determined for results

of two biological independent tests in duplicates.

To calculate means and standard deviations, a

value of 0.69 was assigned to sample outcomes that

were below the lower limit of detection (<10 CFU/

mL) of the spread method. This value was equal to

100 CFU/1 coupon (# 20 cm2), or 5 CFU/cm2. SPSS

version 18 was used for statistical analyses. Statistical

significance was set at P-value less than 0.05.

RESULTS AND DISCUSSION

Assessment of efficacy of four cleaning processes by removing the differences of mature single species biofilms

Effect of growth conditions on biofilm population

Listeria spp., Salmonella spp., and Pseudomonas

spp. have been demonstrated to be significant haz-

ards in food production environments, especially in

seafood processing ecosystems (Ababouch et al.,

2005; Bridier et al., 2015; Giaouris et al., 2014; Van

Houdt and Michiels, 2010). In addition, L. monocy-

togenes, Salmonella spp. and Pseudomonas spp.

have been widely studied and shown to have con-

siderable ability to form single and mixed species

biofilms (Bridier et al., 2015; Dourou et al., 2011;

Giaouris et al., 2013; Slama et al., 2012). However,

the ability to eliminate bacteria in biofilms depends

on the characteristics of the biofilms and the clean-

ing process. In this experiment, biofilms which were

formed on SS with two conditions of nutrient stress

(TSB 10% or TSB 1%) and two different ages of bio-

films (3 days or 7 days) and three species bacteria

at 16 °C. These results are demonstrated in Fig. 2.

Four cleaning procedures which have been used in

the seafood plant were applied to examine the ef-

ficacy of each cleaning process.

Populations of Listeria, Pseudomonas and Salmo-

nella biofilms are presented as log CFU/cm2 values.

Figure 2 presents the population of single species

biofilms of L. monocytogenes, Salmonella spp., and

Pseudomonas spp. ranging from 3.5 log CFU/cm2 to

7.7 log CFU/cm2. In general, it was observed that

the population of cells in biofilm depended on the

type of cultures and nutrient levels (TSB 10% and

TSB 1%). There was no significant difference in the

population of cells for 3 days biofilms or 7 days bio-

films. Similar results were obtained from a study

of Giaouris et al. (2013) for Pseudomonas putida at

18°C. Their study showed that during a 10 day sam-

pling interval, Pseudomonas putida generated two

biofilm-formed cycles, one reached on day 4 and

another at day 8. The cell population was approxi-

mately 7 log CFU/cm2. The results of this experiment

indicated that the population of Pseudomonas cells

was significantly higher than the population of Sal-

monella cells, but there was no significant difference

with Listeria population in TSB 10%. This result may

be explained due to the temperature of this experi-

ment. In this experiment, the temperature of biofilm

formation was set up at 16°C to simulate the work-

ing temperature of the seafood processing plant.

Therefore, this temperature may not have influenced

the growth of Listeria or Pseudomonas since they are

psychrotrophic microorganisms, whereas Salmonella

is a mesophilic bacteria. Moreover, due to the pres-

ence of a complex enzymatic system, Pseudomonas

spp. can metabolize various materials around them

to serve as their nutrient sources (Franzetti and Scar-


pellini, 2007). Hence, under nutrient stress in TSB

1%, the population of Pseudomonas biofilm reached

approximately 5.5 log CFU/cm2 whereas the Listeria

biofilms reached only 3.5 and 4.5 log CFU/cm2 for 3

days and 7 days biofilms, respectively.

Efficacy of four cleaning processes on different

single biofilms

We applied cleaning procedures (Fig. 1), that

were used in the seafood factory in daily, weekly and

bimonthly. This procedure included: 1. rinsing with

fresh water, 2. cleaning with 2% alkaline or acidic

detergent for 10 min, 3. rinsing with fresh water to

remove detergent, 4. sanitizing with QACs 1 or PAA

0.2% for 10 min, 5. repeating the rinsing process with

fresh water to remove the sanitizers, 6. re-sanitizing

by fogging with Quatdet clear - a QACs 2 based 1%

for 10 min. Finally, equipment was rinsed with fresh

water again and allowed to dry before production.

Among the four cleaning processes, treatment

D1S1 was the least effective and treatment D2S2

was the most effectiveness for eliminating bacteria in

biofilms (Table 1 and Table 2). In terms of efficacy to

remove biofilm of various single species, the results

show that the Pseudomonas biofilms had the highest

ability to survive after the cleaning process followed

by Salmonella and Listeria. All the cleaning process-

es were effective at removing biofilms of Salmonella

and Listeria. According to Somers and Wong (2004)

a combination of cleaning and sanitizing was more

effective than sanitizer alone in terms of eliminating

Listeria monocytogenes biofilms. Moreover, QACs,

which was used in four cleaning processes as sani-

tizer and re-sanitizer, was more effective against Sal-

monella spp. (Gram–negative) (Sinde and Carballo,

2000) and Listeria spp. (Gram-positive) (Buffet-Ba-

taillon et al., 2012); even though they differ in their

cell wall characterstics. Therefore the Pseudomonas

Figure 2. Population of viable cells in single biofilms of Listeria, Pseudomonas and Salmonella for 3 days or 7 days in TSB 1% and 10%, at 16°C. Results were expressed as mean values ± standard deviation from two independent tests in duplicate. Among different nutrient conditions, incuba-tion times and type of cultures (a, b, c); the mean values with the same letters are not significantly different (p>0.05).

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

9.0

Log

(CFU

/cm

2 )

cd

a

abcab

cdabc

bcbcd

d

bcd bc

cd

Listeria Pseudomonas Salmonella

TSB 1% - 3 days TSB 10% - 3 days TSB 1% - 7 days TSB 10% - 7 days


biofilm would be considered a main concern. The

results in Table 1 and Table 2 showed that treatment

D1S1 and D1S2 removed only 3 to 4 log CFU/cm2

Pseudomonas; whereas treatment D2S1 or D2S2 re-

moved 5 to 6 log CFU/cm2 Pseudomonas. The main

difference between the four treatments was the

types of detergents; treatment D1S1 and D1S2 us-

ing alkaline detergent, (D1- superp foam, 2%) and

treatment D2S1 and D2S2 using acidic detergent

(D2 - Dilac Z, 2%).

The characteristics of different bacteria, which

contribute to the formation of their corresponding

biofilms, affects their survival under the cleaning

and disinfecting process was examined in the cur-

rent study. Our results were similar to the results

obtained by Gibson et al. (1999). The investigators

showed that Pseudomonas aeruginosa was more re-

sistant to the detergent products, greater than 3 log

CFU/cm2 still remained on SS coupons after clean-

ing; their research also reported that the maximum

removal cells was a 4 log CFU/cm2 reduction (Gib-

son et al., 1999). The reason Pseudomonas biofilms

were more resistant than Listeria and Salmonella

biofilms may be because of the difference in colo-

nization mechanisms (Gibson et al., 1999). Pseudo-

monas aeruginosa attached to surfaces produces

a variety of extracellular polymeric substance (EPS)

such as cellulose, alginate, Pel and PsI exopolysac-

Table 1. Efficacy of 4 cleaning processes on different single biofilms of Pseudomonas, Listeria, Salmonella for 3 and 7 days in TSB 1% and TSB 10%

Organism T ime (days)

TSB (%)

log (CFU/cm2)

Treatment

Biofilm D1S1 D1S2 D2S1 D2S2

Pseudomonas 3 1 5.50 ± 0.11 2.63 ± 0.01ab 0.79 ± 1.11ce ND ND

Listeria 3 1 3.51 ± 0.04 0.70 ± 0.00c ND 0.70 ± 0.00c ND

Salmonella 3 1 5.49 ± 0.06 NDd ND ND ND

Pseudomonas 3 10 7.65 ± 0.54 4.13 ± 1.19a 2.87± 0.66ab 1.55 ± 1.20bc ND

Listeria 3 10 6.96 ± 0.38 0.70 ± 0.00c ND 0.70 ± 0.00c ND

Salmonella 3 10 4.33 ± 1.38 ND ND ND ND

Pseudomonas 7 1 5.36 ± 0.19 1.74 ± 0.13bc 0.59 ± 0.83ce 0.35 ± 0.49ce 0.35 ± 0.49ce

Listeria 7 1 4.55 ± 0.23 ND ND ND ND


Pseudomonas 7 10 5.84 ± 0.73 2.71 ± 0.75ab 0.50 ± 0.71ce 1.61 ± 0.81bc 0.70 ± 0.00c

Listeria 7 10 5.88 ± 0.98 ND ND ND ND


a,b,c significant decrease within 4 cleaning processes (D1S1, D1S2, D2S1 and D2S2) in both rows and columnsd ND, not detectable, less than 0.69 log CFU/cm2 e one sample detectable

Results were expressed as mean values ± standard deviation from two independent tests in duplicate. The mean values with the same letters are not significantly different (p>0.05).


charides which help them to strongly adhere and

form strong biofilms on surfaces (Giaouris et al.,

2013; Gibson et al., 1999). Moreover, EPS also forms

a matrix around the cells, which can protect the cells

from adverse conditions (Gibson et al., 1999). In ad-

dition, the larger population of Pseudomonas meant

thicker biofilm compared to those of Listeria or Sal-

monella biofilms, causing less diffusion of sanitizers

in Pseudomonas biofilms. Therefore, Pseudomonas

spp. may survive better than the others under the

same treatment (Giaouris et al., 2013). The results

revealed that under simultaneous growth conditions

(3 days - TSB 1% or 7 days - TSB 10%) and having

a similar cellular populations (approximately 5.5 log

CFU/cm2), there was significant differences in terms

of viable cells of Pseudomonas and Salmonella bio-

films; Pseudomonas spp. remained at approximately

2.7 log CFU/cm2 while no Salmonella spp. colonies

were recovered. Similar results were demonstrated

by Giaouris et al. (2013). Gram-negative P. putida

showed higher tolerance to benzalkonium chloride

(BC- QACs -based) compared with the Gram posi-

tive L. monocytogenes.

When comparing the effectiveness of biofilm re-

moval based on detergent pH, an acidic detergent

(Dilac Z, pH 1.6; treatment D2) was more effective

than an alkaline product (Superp foam, pH 12.2;

treatment D1) in terms of effect on cell viability. Sim-

Table 2. Percentage reduction of 4 cleaning processes on different single biofilms of Pseu-domonas, Listeria, Salmonella for 3 and 7 days in TSB 1% and TSB 10%

OrganismTime (days)

TSB (%)

Percentage reduction*

Treatment

D1S1 D1S2 D2S1 D2S2

Pseudomonas 3 1 99.8685 99.9943 100.0000 100.0000

Listeria 3 1 99.8435 100.0000 99.8435 100.0000

Salmonella 3 1 100.0000 100.0000 100.0000 100.0000

Pseudomonas 3 10 99.9235 99.9980 99.9998 100.0000

Listeria 3 10 99.9999 100.0000 99.9999 100.0000

Salmonella 3 10 100.0000 100.0000 100.0000 100.0000

Pseudomonas 7 1 99.9764 99.9968 99.9989 99.9989

Listeria 7 1 100.0000 100.0000 100.0000 100.0000

Salmonella 7 1 100.0000 100.0000 100.0000 100.0000

Pseudomonas 7 10 99.9232 99.9996 99.9935 99.9996

Listeria 7 10 100.0000 100.0000 100.0000 100.0000

Salmonella 7 10 100.0000 100.0000 100.0000 100.0000

*Each value is a mean of duplicate replication of two independent tests.


ilarly, Gibson et al. (1999) found that the acidic de-

tergent reduced the viable population of attached

Staphylococcus aureus but not P. aeruginosa. Our re-

sults may be explained by source of Pseudomonas,

Pseudomonas genus which can be found as an bun-

dance species in fish and seafood ecosystem grew at

pH 6 to 9; they did not growth at pH 4 (Shivaji et al.,

1989) thus leading to acidic detergent being more

effective in cleaning than the alkaline detergent.

Indeed, PAA may have a stronger effect to antimi-

crobial activity compared with QACs, because PAA

had both oxidizing and low pH functions in killing

bacteria whereas QACs had only one antimicro-

bial mechanism, namely, interaction with cell mem-

branes, disruption of membranes integrity and leak-

age of cellular content (Buffet-Bataillon et al., 2012;

Giaouris et al., 2013).

Assessment cleaning and sanitizing pro-cedure based on the efficacy to remove mixed species biofilms

Although combining detergent and sanitizer in

the cleaning process showed more effectiveness to

eliminate biofilms (Somers and Wong, 2004), Pseu-

domonas biofilms were still present on SS coupons

after the cleaning process, particularly the D1S1

method. Hence, it can be concluded that the regu-

lar daily cleaning process D1S1 is an improper clean-

ing procedure to remove Pseudomonas biofilms.

However, the cleaning process D1S1 was still applied

daily because alkaline detergents have a higher po-

tential to remove organic material and prevent the

corosion of equipment in the food procesing envi-

ronment. In addition, the ability to eliminate micro-

Figure 3. Efficacy of combination of H2O2 pre-treatment and regular daily cleaning process (D1S1) on mixed species biofilms on SS, Teflon and rubber. Results were expressed as mean values ± standard deviation from two independent tests in duplicate. Among different biofilm and clean-ing processes (A, B, C, D, E) and among different materials at the same cleaning process (a, b, c); the mean values with the same letters are not significantly different (p>0.05).

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

Biofilm

Log

(CFU

/cm

2 )

Stainless steellTeflonRubber

aa a

b

b ab

c

a

aaaaaaa

a

b b

Pre-treatment H2O2 + D1S1

1%, 5 min

E

1%, 10 min 2%, 5 min 2%, 10 min

A B C D D

(Before cleaning)

D1S1Biofilm


organisms in biofilms depends on many factors such

as the microbial population of the biofilm, quorum

sensing, and material where biofilms form and in-

habit. From previous experiments, it was demon-

strated that mixed species biofilms may be formed

by numerous different species. Moreover, mixed

species biofilms are usually more stable than single

species biofilm (Giaouris et al., 2014). Consequently,

there was a need to evaluate the efficacy of clean-

ing process on mixed species biofilms of Listeria,

Salmonella and Pseudomonas under simulated food

processing conditions in the current experiment. Ac-

cording to the result from Gram et al. (1987), Guðb-

jörnsdóttir et al.(2005) and our previous experiment

(data unpublished), the microflora in seafood plants

was shown to be 80% Gram negative microorgan-

isms; Pseudomonas spp. was shown to be the pre-

dominant species. Therefore, the mixed species

stock which included 80% of volume cell suspen-

sion of Pseudomonas spp., 10% of volume of Liste-

ria spp., and 10% of volume of Salmonella spp. was

used for this experiment. The efficacy of the cleaning

process was determined by the surviving bacteria at-

tached on the surface of coupons after performing

the cleaning process. The results were reported as a

mean with duplicate replication of two independent

experiments.

The modified cleaning process was applied to re-

move mixed species of 7 day biofilms on different

substratum such as SS, Teflon and rubber. Biofilm

formation on these materials ranged from 7.2 to 7.7

log CFU/cm2. There were no significant difference

in the population of cells adhering on SS, Teflon and

rubber for 7 days biofilms. D1S1 sample consisted of

no treatment with H2O2 using only the cleaning pro-

cess of D1S1. Overall, the surviving bacterial cells of

all samples decreased when H2O2 concentration and

exposure time were increased (Fig. 3). Without pre-

treatment with H2O2, viable cells on SS, Teflon and

rubber were 3.3, 4.3 and 5.2 log CFU/cm2, respec-

tively. The low efficacy for removing mixed species

biofilms may be attributed to the large population of

microbial cells in the biofilms, and interactions be-

tween cells and surfaces (Giaouris et al., 2013, 2014;

Lagha et al., 2014; Norwood and Gilmour, 1999).

Moreover, limiting the diffusion of sanitizers within

the biofilm occurred because these strong biofilms

formed under nutrient limitation (they were subject-

ed to TSB 1%, 8 h per day) and 7 days.

Indeed, Giaouris et al. (2014) reported that one of

the four mechanisms leading to increase resistance

of organisms with sanitizers is a physical barrier

formed by the EPS matrix. Therefore, disruption EPS

matrix will increase the effectiveness of the cleaning

procedure by increasing the penetration of sanitizer

into biofilms (Simões et al., 2010; Xavier et al., 2005).

There are several methods which could be used

to destabilize the EPS matrix of biofilms including

biological methods, physical methods and chemi-

cal methods; among them H2O2 (chemical method)

was selected because of its advantages, namely,

low-cost, ease of use, minimal impact on the envi-

ronment, broad spectrum capabilities and high po-

tential to degrade the EPS matrix (Back et al., 2014;

Gao et al., 2014; Imamura et al., 2010). According to

Imamura et al. (2010) H2O2 molecules degenerate

and produce °OH radicals by accepting an electron

from the metal surface. Due to an extremely high

oxidation potential, °OH radicals are generated on

the metal surface at high concentrations (Imamura

et al., 2002). As a consequence, organic substances,

which include in EPS, are instantaneously oxidized.

The oxidized substances become soluble fragments

which can easily be removed from surfaces by a reg-

ular cleaning procedure (Imamura et al., 2002, 2010).

The effectiveness of pretreatment with H2O2 is

associated with H2O2 concentration and exposure

time. Average survival of microbial cells in different

substratum ranged from 3.1 to 1.3 log CFU/cm2 fol-

lowed by H2O2 1% for 5 minutes and H2O2 2% for

10 minutes, respectively. The results showed that

there was more than a 6 log CFU/cm2 reduction in

terms of pre-treatment with H2O2 2% for 10 minutes.

A similar result was obtained by DeQueiroz and Day

(2007). In their study, when sodium hypochlorite was

combined with H2O2, cell numbers were reduced

by 5 log to 6 log of P. aeruginosa biofilms after 1

min exposure while sodium hypochlorite reduced

viable numbers by 3 log to 4 log under an equiva-

lent concentration. In addition, P. aeruginosa biofilm


formation on SS and aluminum surfaces were also

removed (DeQueiroz and Day, 2007). The results of

this experiment was supported by Choi et al. (2012)

where pathogen biofilms on the SS surfaces were

reduced when treated with aerosolized hydrogen

peroxide-based sanitizer. However, there was no sig-

nificant difference between pre-treatment with H2O2

1% for 10 minutes and H2O2 2% for 5 minutes. It was

suggested that the treatment of 1% of H2O2 for 10

minutes or 2% of H2O2 for 5 minutes should result in

a low concentration or short exposure time for °OH

radicals to decompose the EPS matrix of the highly

mixed microbial species generated in these biofilms.

Attachment or detachment of microorganisms on

surfaces depends on both characteristics of bacte-

ria and material surfaces (Sinde and Carballo, 2000).

Among the three materials SS, Teflon and rubber,

there were no significant difference in the number of

adhesive cells but there was a significant decrease in

the number of viable cells during the cleaning pro-

cess. The number of viable cells on rubber remained

high in both the control and the pre-treatment with

H2O2. In fact, when applying the pre-treatment H2O2

2% for 10 minutes to the biofilm formation on the

rubber coupon, there was greater than a 5 log CFU/

cm2 reduction, however there remained 2 log CFU/

cm2 of viable cells on the surfaces of rubber cou-

pons. While applying the same treatment condition,

biofilms on SS and Teflon were removed, reaching a

6 log CFU/cm2 reduction. Higher efficacy in clean-

ing process to SS and Teflon could be explained

because a high concentration of °OH radicals was

produced on the metal surface (Imamura et al., 2010)

and the more hydrophobic characteristics of the Tef-

lon surface.

CONCLUSIONS

This study demonstrated the efficacy of combi-

nation of H2O2 pre-treatment with the regular daily

cleaning procedure used in a shrimp plant to control

biofilm formation. For a single biofilm, the popula-

tion of bacteria in biofilms depended on bacteria

characteristics and nutrient availability. Pseudomo-

nas biofilms demonstrated higher adaptability for all

growth conditions; as indicated by the higher num-

ber of cells in biofilms. Four existing cleaning pro-

cesses were effective against Listeria and Salmonella

biofilms, with Pseudomonas biofilms being the ex-

ception. For mixed-species biofilm, when combined

with pretreatment of H2O2, the cleaning process was

more effective when compared to using the existing

cleaning process alone; there was a 4 log CFU/cm2

reduction for the control method compared to a 6

log CFU/cm2 reduction for the method that com-

bined pre-treatment H2O2 2% for 10 minutes and a

cleaning process for cleaning mixed species biofilms

on SS coupons. SS is ideally suited for food indus-

try, especially in terms of eliminating and prevent-

ing biofilm formation. Therefore, the combination

of H2O2 pre-treatment and cleaning process can be

used as an alternative method to remove mixed spe-

cies biofilms in food processing equipment.

ACKNOWLEDGEMENTS

The authors are grateful for financial support from:

the Faculty of Agro-Industry, Kasetsart University,

Thailand (through the Scholarships for International

Graduate Students 2012) and Agri-Biotech and Fish-

eries project, Vietnamese Government grant for this

study. The authors would like to thank Professor Dr.

Steven C. Ricke, Editor-in-Chief and Professor Dr.

Md. Latiful Bari, University of Dhaka, Bangladesh for

their valuable comments and suggestions for this

manuscript.

REFERENCES

Aase, B., G. Sundheim, S. Langsrud, & L. M. Rørvik.

2000. Occurrence of and a possible mechanism for

resistance to a quaternary ammonium compound

in Listeria monocytogenes. International Journal of


Ababouch, L., G. Gandini & J. Ryder. 2005. Causes

of detentions and rejections in international food

trade. FAO fisheries technical paper 473, Rome:


Food and Agricultural Organization of the United

Nations.

Back, K.-H., J.-W. Ha & D.-H. Kang. 2014. Effect of

hydrogen peroxide vapor treatment for inacti-

vating Salmonella Typhimurium, Escherichia coli

O157:H7 and Listeria monocytogenes on organic

fresh lettuce. Food Control 44:78-85.

Bae, Y.-M., S.-Y. Baek & S.-Y. Lee. 2012. Resistance

of pathogenic bacteria on the surface of stainless

steel depending on attachment form and efficacy

of chemical sanitizers. Int. J. Food Microbiol.153:

465-473.

Bagge-Ravn, D., Y. Ng, M. Hjelm, J. N. Christian-

sen, C. Johansen & L. Gram. 2003. The microbial

ecology of processing equipment in different fish

industries—analysis of the microflora during pro-

cessing and following cleaning and disinfection.

Int. J. Food Microbiol. 87:239–250.

Beauchamp, C. S., D. Dourou, I. Geornaras, Y. Yoon,

J. Scanga, K. E. Belk, , G. C. Smith, G.-J. E. Nychas

& J. N. Sofos. 2012. Transfer, attachment, and for-

mation of biofilms by Escherichia coli O157:H7 on

meat-contact surface materials. Food Microbiol. &

Safety 77: M343-347.

Belessi, C.-E. A., A. S. Gounadaki, A. N. Psomas, & P.

N. Skandamis. 2011. Efficiency of different sanita-

tion methods on Listeria monocytogenes biofilms

formed under various environmental conditions.

Int. J. Food Microbiol. 145: S46-52.

Bohme, K., I. C. Fernandez-No, M. Pazos, J. M. Gal-

lardo, J. Barros-Velazquez, , B. Canas & P. Calo-

Mata. 2013. Identification and classification of

seafood-borne pathogenic and spoilage bacteria:

16S rRNA sequencing versus MALDI-TOF MS fin-

gerprinting. Electrophoresis 34: 877-887.

Bridier, A., P. Sanchez-Vizuete, M. Guilbaud, J. C.

Piard, M. Naïtali & R. Briandet. 2015. Biofilm-as-

sociated persistence of food-borne pathogens.


Brooks, J. D. and S. H. Flint. 2008. Biofilms in the

food industry: problems and potential solutions.

Int. J. Food Science & Technol. 43: 2163-2176.

Buffet-Bataillon, S., P. Tattevin, M. Bonnaure-Mallet

& A. Jolivet-Gougeon. 2012. Emergence of resis-

tance to antibacterial agents: the role of quater-

nary ammonium compounds--a critical review. Int.

J. Antimicrob. Agents. 39: 381-389.

Chmielewski, R. A. N. and J. F. Frank. 2003. Biofilm

formation and control in food processing facilities.

Compr. Rev. Food Sci. Food Safety 2: 22-32.

Choi, N.-Y., S.-Y. Baek, J.-H. Yoon, M.-R. Choi, D.-H.

Kang and S.-Y. Lee. 2012. Efficacy of aerosolized

hydrogen peroxide-based sanitizer on the reduc-

tion of pathogenic bacteria on a stainless steel sur-

face. Food Control 27: 57-63.

DeQueiroz, G. A. and D. F. Day. 200). Antimicrobial

activity and effectiveness of a combination of sodi-

um hypochlorite and hydrogen peroxide in killing

and removing Pseudomonas aeruginosa biofilms

from surfaces. J. Appl. Microbiol. 103: 794-802.

Dourou, D., C. S. Beauchamp, Y. Yoon, I. Geornaras,

K. E. Belk, G. C. Smith, G.-J. E. Nychas and J. N.

Sofos. 2011. Attachment and biofilm formation

by Escherichia coli O157:H7 at different tempera-

tures, on various food-contact surfaces encoun-

tered in beef processing. Int. J. Food Microbiol.

149:262-268.

Duong, N. N. H. 2012. Formation of Salmonella

Typhimurium biofilm under various growth con-

ditions and its sensitivity to industrial sanitizers.

Master thesis. National University of Singapore,

National University of Singapore.

Elmali, M., H. Yaman, K. K. Tekinsen, S. Öner & E.

Çekin. 2012. Inhibitory effects of different decon-

tamination agents on the levels of Listeria monocy-

togenes in the experimentally inoculated raw beef

samples in the laboratory conditions. Kafkas Univ

Vet Fak Derg. 18:763-768.

Fatemi, P. and J. F. Frank. 1999. Inactivation of Lis-

teria monocytogenes and Pseudomonas biofilms

by peracid sanitizers. J. of Food Prot. 62: 761-765.

Food & Water-Watch. 2007. Import Alert: Govern-

ment Fails Consumers, Falls Short on Seafood In-

spections. In (pp. 28). Food & Water Watch: www.

foodandwaterwatch.org.

Franzetti, L. and M. Scarpellini. 2007. Characterisa-

tion of Pseudomonas spp. isolated from foods.

Ann. Microbiol. 57: 39-47.

Gao, L., K. M. Giglio, J. L. Nelson, H. Sondermann

and A. J. Travis. 2014. Ferromagnetic nanoparticles


with peroxidase-like activity enhance the cleavage

of biological macromolecules for biofilm elimina-

tion. Nanoscale 6:2588-2593.

Giaouris, E., E. Heir, M. Hebraud, N. Chorianopou-

los, S. Langsrud, T. Moretro, O. Habimana, M. Des-

vaux, S. Renier & G. J. Nychas. 2014. Attachment

and biofilm formation by foodborne bacteria in

meat processing environments: causes, implica-

tions, role of bacterial interactions and control by

alternative novel methods. Meat Sci. 97: 298-309.

Giaouris, E., N. Chorianopoulos, A. Doulgeraki & G.-

J. Nychas. 2013. Co-culture with Listeria monocy-

togenes within a dual-species biofilm community

strongly increases resistance of Pseudomonas pu-

tida to benzalkonium chloride. PloS One 8: 1-14.

Giaouris, E., N. Chorianopoulos, P. Skandamis & G.-

J. Nychas. 2012. Attachment and biofilm formation

by Salmonella in food processing environments.

In: B. S. M. Mahmoud (Ed.), Salmonella - A Dan-

gerous Foodborne Pathogen (pp. 450). Croatia:

InTech. www.intechopen.com.

Gibson, H., J. H. Taylor, K. E. Hall & J. T. Holah. 1999.

Effectiveness of cleaning techniques used in the

food industry in terms of the removal of bacterial

biofilms. Appl. Microbiol. 87: 41–48.

Gram, L. & H. H. Huss. 1996. Microbiological spoil-

age of fish and fish products. Int. J. Food Micro-

biol. 33: 121-137.

Gram, L., G. Trolle & H. H. Huss. 1987. Detection of

specific spoilage bacteria from fish stored at low

(0 °C) and high (20 °C) temperatures. Int. J. Food

Microbiol. 4: 65-72.

Guðbjörnsdóttir, B., H. Einarsson & G. Thorkelsson.

2005. Microbial adhesion to processing lines for

fish fillets and cooked shrimp: Influence of stainless

steel surface finish and presence of Gram-negative

bacteria on the attachment of Listeria monocyto-

genes. Food Technol. and Biotechnol. 43: 55–61.

Hadi, R., K. Vickery, A. Deva and T. Charlton. 2010.

Biofilm removal by medical device cleaners: com-

parison of two bioreactor detection assays. J. Hos-

pital Infection 74:160-167.

Hoa, B. T. Q., W. Mahakarnchanakul,T. Sajjaanantakul

& V. Kitpreechavanich. 2015. Adhesive microflora

on stainless steel coupons in seafood processing

plant. Food Nutr. Sci. 3:28-32.

Ibusquiza, P. S., J. J. R. Herrera & M. L. Cabo. 2011.

Resistance to benzalkonium chloride, peracetic

acid and nisin during formation of mature biofilms

by Listeria monocytogenes. Food Microbiol. 28:

418-425.

Imamura, K., M. Oshita, M. Iwai, T. Kuroda, I. Wata-

nabe, T. Sakiyama &, K. Nakanishi. 2010. Influences

of properties of protein and adsorption surface

on removal kinetics of protein adsorbed on metal

surface by H2O2-electrolysis treatment. J. Colloid

Interface Sci. 345: 474-480.

Imamura, K., Y. Tada, H. Tanaka, T. Sakiyama & K.

Nakanishi. 2002. Removal of proteinaceous soils

using hydroxyl radicals generated by the electroly-

sis of hydrogen peroxide. J. Colloid Interface Sci.

250: 409-414.

Joseph, B., S. K. Otta, I. Karunasagar & I. Karuna-

sagar. 2001. Biofilm formation by Salmonella spp.

on food contact surfaces and their sensitivity to

sanitizers. Int. J. Food Microbiol. 64: 367-372.

Koonse, B., W. Burkhardt, S. Chirtel & G. P. Hoskin.

2005. Salmonella and the sanitary quality of aqua-

cultured shrimp. J. of Food Prot. 68: 2527-2532.

Lagha, R., M.-N. Bellon-Fontaine, M. Renault, R. Bri-

andet, J.-M. Herry, B. Mrabet, , A. Bakhrouf & M.

M. Chehimi. 2014. Impact of long-term starvation

on adhesion to and biofilm formation on stainless

steel 316 L and gold surfaces of Salmonella en-

terica serovar Typhimurium. Ann. Microbiol. April:

1-11.

Marchand, S., J. D. Block, V. D. Jonghe, A. Coorevits,

M. Heyndrickx & L. Herman. 2012. Biofilm forma-

tion in milk production and processing environ-

ments; Influence on milk quality and safety. Comp.

Rev. Food Sci. Food Safety 11:133-147.

Myszka, K. and K. Czaczyk. 2011. Bacterial biofilms

on food contact surfaces - A Review. Pol. J. Food

Nutr. Sci. 61:173-180.

Nguyen, H. D. N. and H.-G. Yuk. 2013. Changes in

resistance of Salmonella Typhimurium biofilms

formed under various conditions to industrial

sanitizers. Food Control 29:236-240.

Norhana, M. N. W., S. E. Poole, H. C. Deeth and G. A.

Dykes. 2010. Prevalence, persistence and control


of Salmonella and Listeria in shrimp and shrimp

products: A review. Food Control 21:343-361.

Norwood, D. E. and A. Gilmour. 1999. Adherence of

Listeria monocytogenes strains to stainless steel

coupons. J. Appl Microbiol. 86: 576–582.

Norwood, D. E. and A. Gilmour. 2000. The growth

and resistance to sodium hypochlorite of Listeria

monocytogenes in a steady-state multispecies

biofilm. Appl. Microbiol. 88: 512–520.

Ölmez, H. and S. D. Temur. 2010. Effects of different

sanitizing treatments on biofilms and attachment

of Escherichia coli and Listeria monocytogenes

on green leaf lettuce. LWT - Food Sci. Technol.

43:964-970.

Oz, Y., I. Dag & N. Kiraz. 2012. Efficacy of disinfec-

tants on Candida biofilms at different concentra-

tions and contact times. Brit. Microbiol. Research

2: 40-52.

Pan, Y. 2005. Behavior of Listeria monocytogenes

biofilms in a simulated food processing (SFP) eco-

system. Master Thesis. North Carolina State Uni-

versity, North Carolina State University.

Ryder, C., M. Byrd and D. J. Wozniak. 2007. Role

of polysaccharides in Pseudomonas aeruginosa

biofilm development. Curr. Opinion Microbiol.

10:644-648.

Shivaji, S., N. S. Rao, L. Saisree, V. Sheth, G. S. Reddy

and P. M. Bhargava. 1989. Isolation and identifica-

tion of Pseudomonas spp. from Schirmacher Oasis,

Antarctica. Appl. Environ. Microbiol. 55:767-770.

Simões, M., L. C. Simões and M. J. Vieira. 2010. A re-

view of current and emergent biofilm control strat-

egies. LWT - Food Sci. Technol. 43:573–583.

Sinde, E. and J. Carballo. 2000. Attachment of Sal-

monella spp. and Listeria monocytogenes to stain-

less steel, rubber and polytetrafluor- ethylene: the

influence of free energy and the effect of commer-

cial sanitizers. Food Microbiol. 17:439-447.

Slama, R. B., K. Bekir, H. Miladi, A. Noumi and A.

Bakhrouf. 2012. Adhesive ability and biofilm met-

abolic activity of Listeria monocytogenes strains

before and after cold stress. Afric. J. Biotechnol.

11:12475-12482.

Somers, E. B. and A. C. L. Wong. 2004. Efficacy of

two cleaning and sanitizing combinations on Lis-

teria monocytogenes biofilms formed at low tem-

perature on a variety of materials in the presence

of ready-to-eat meat residue. J. Food Prot. 67:

2218-2229.

Stewart, P. S. and W. J. Costerton. 2001. Antibi-

otic resistance of bacteria in biofilms. The Lancet

358:135-138.

Van Houdt, R. and C. W. Michiels. 2010. Biofilm for-

mation and the food industry, a focus on the bac-

terial outer surface. J. Appl. Microbiol. 109:1117-

1131.

Xavier, J. B., C. Picioreanu, S. A. Rani, M. C. van

Loosdrecht and P. S. Stewart. 2005. Biofilm-control

strategies based on enzymic disruption of the ex-

tracellular polymeric substance matrix--a model-

ling study. Microbiol. 151:3817-3832.




ABSTRACT

Antimicrobial plant secondary metabolites increase rumen efficiency and decrease waste products (i.e.

ammonia, methane) in some cases. A promising source of bioactive secondary metabolites is the hops

plant (Humulus lupulus L.), which produces ß-acid, a suite of structurally similar, potent antibacterial com-

pounds. The efficacy of hops has been shown in bovines. Additionally, the ß-acid mechanism of antimicro-

bial action was determined on rumen bacteria of bovine origin. The objective of the current study was to

determine the effect of hops ß-acid on amino acid degradation and ammonia production by goat rumen

bacteria. The growth of two rumen hyper-ammonia-producing bacteria of caprine origin (Peptostreptococ-

cus spp. BG1 and BG2) was inhibited by ß-acid (≥ 45 ppm and ≥ 4.5 ppm, respectively) when either amino

acids or peptides were the growth substrate. Uncultivated, mixed rumen microorganisms harvested from

goats produced approximately 35 mM ammonia from amino acids and 50 mM ammonia from peptides

during 24 h incubation. The addition of ß-acid reduced the final ammonia concentration, and there was a

dose-response relationship between the ß-acid concentration and the ammonia concentration. Peptide

catabolism was more sensitive to ß-acid inhibition than free amino acid catabolism to ß-acid inhibition

because as little as 3 ppm resulted in less ammonia production than the control (P < 0.05). These results

demonstrate that hops ß-acid is effective against caprine HAB and suggest that hops could be useful in

controlling wasteful metabolic processes in the caprine rumen.

Keywords: antibiotic alternative, ammonia, beta-acid, bypass protein, feed efficiency, hops, goat, hyper ammonia producing bacteria, inhibition, ionophore, lupulone, phytochemical, phytoprotectant, plant secondary metabolite, rumen

Correspondence: Michael D. Flythe, [email protected]: +1 -859-421-5699 Fax: +1-859-257-3334

Hops (Humulus lupulus) ß-Acid as an Inhibitor of Caprine Rumen Hyper-Ammonia-Producing Bacteria In Vitro

M. D. Flythe1, 2, G. E. Aiken1, 3, G. L. Gellin1, J. , L. Klotz1, 2, B. M. Goff3, K. M. Andries4

1 Forage-Animal Production Research Unit, Agricultural Research Service, United States Department of Agriculture 2 Department of Animal & Food Sciences, University of Kentucky

3 Department of Plant & Soil Sciences, University of Kentucky4 College of Agriculture, Food Science & Sustainable Systems, Kentucky State University

“Proprietary or brand names are necessary to report factually on available data; however, the USDA neither guaran-

tees nor warrants the standard of the product, and the use of the name by the USDA implies no approval of the product,

nor exclusion of others that may be suitable.”



INTRODUCTION

A symbiotic relationship with the rumen microflo-

ra is the adaptation that gives ruminants metabolic

access to fibrous plant tissues (Russell and Rychlik,

2001). This dense community of microorganisms in-

cludes bacteria, archaea, fungi and protists, which

collectively degrade fiber and convert the resulting

sugars to fermentation acids and gases. The fermen-

tation acids can be transported across the rumen ep-

ithelium and utilized by the ruminant host, and the

gases, which are essential for reducing equivalent

cycling in the microorganisms, are eructated (Russell

and Rychlik, 2001).

In addition to carbohydrates, plant proteins, pep-

tides and amino acids are also catabolized. Rumen

proteolysis and deamination (diagrammed in Figure

1) are not inherently harmful to ruminants. In fact,

some cellulolytic bacteria, such as Ruminococcus

albus, require the aromatic- and branched-acids

produced by deamination of particular amino acids

(Caldwell and Bryant, 1966). Bacteria can assimilate

some of the ammonia allowing the ruminant host

to utilize the resulting microbial protein (Satter and

Slyter, 1974). The ruminant converts excess ammo-

nia to urea, some of which can be recycled into the

rumen, and goats are exceptionally good at this

process (Kohn et al., 2005). Ruminants have lower

nitrogen clearance rates than non-ruminants and

goats tend to be lower than other ruminants (Kohn

et al., 2005). The urea transport rate through the ru-

men epithelum and kidney increases as nitrogen in

the diet decreases (Muscher et al., 2010; Starke et

al., 2014). When urea is not recycled it is lost in the

urine. The loss of feed amino-nitrogen in the urine

is an economic loss to the rancher or dairyman, and

a source of environmental pollution (Tedeschi et al.,

2003). Additionally, amino acid fermentation results

in the production of indole and related compounds,

which can cause flavor notes in milk that are undesir-

able to some consumers (Attwood et al., 2006).

Proteolysis is carried out by a variety of rumen mi-

croorganisms, but most of the ammonia production

from the resulting peptides and amino acids is at-

tributable to ciliate protozoa (Abou Akkada and El-

Shazly, 1964) and to a guild of bacteria termed Hyper

Ammonia-producing Bacteria, HAP or HAB (Russell

et al., 1988). The contribution of each type of micro-

organism to rumen ammonia depends on host, diet

and other factors. However, ammonia production

in defaunated ruminants supports the hypothesis

that bacteria are sufficient for amino acid degrada-

tion (Abou Akkada and El-Shazly, 1964). Fortunately,

both HAB and ciliates are susceptible to ionophores,

which are included in many ruminant diets (Chen and

Russell, 1989; Dennis et al., 1986). Ionophores (e.g.

monensin, lasalocid) inhibit ammonia production,

increase feed efficiency, promote growth and de-

crease amino-nitrogen release into the environment

(Tedeschi et al., 2003; Callaway et al., 2008).

In spite of the production and environmental ad-

vantages and limited clinical utility of ionophores,

the fact that they are antibiotics has led to some

resistance to their use in industrialized nations (Rus-

sell and Houlihan, 2003). For more than a decade,

researchers have looked for phytochemical alterna-

tives to control ammonia production and other as-

pects of rumen fermentation (Wallace, 2004). Some

phytochemicals, like those derived from the hops

plant (Humulus lupulus) even have an ionophore-like

mechanism of action on HAB (Flythe 2009). Antimi-

crobial phytochemicals have the added advantage

of production from local botanical sources. For ex-

ample, hops might be an appropriate choice in

northern Europe or northern North America where

this plant is cultivated. Spearmint might be a better

local source of phytochemicals in the Middle East,

and spearmint essential oil has been shown to have

antimicrobial activity on HAB from Mehraban sheep

(Taghavi-Nezhad et al., 2013).

The use of regionally available antimicrobial phy-

tochemicals in ruminant production raises the ques-

tion of which animals and microorganisms should be

used to test these compounds. Most rumen micro-

biology research has been conducted on dairy cows

in North America and Western Europe, but goats

are the predominant domestic ruminants in many

parts of the world (FAO, 2011). Goat production

increased more than other species, except poultry,

in both developed and developing countries (FAO,


2011). The goat industry in the US increased by 23%

between 1997 and 2007, this increase was 116% for

the 12 Southeastern states (USDA 2004 and 2009;

USDA-APHIS 2005), which is where the current work

was conducted. The size of the industry steadied be-

tween 2002 and 2012 goat numbers increased by just

over 1% in these same states (USDA 2004 and 2014).

The purpose of the study was to test the effect of a

model antimicrobial phytochemical on: 1) the growth

of caprine HAB, and 2) ammonia production by un-

cultivated rumen microorganisms from goats. Hops

ß-acid is a suite of structurally similar compounds

(lupulone, colupulone, adlupulone) that are potently

inhibitory to Gram-positive bacteria. It was selected

as the model antimicrobial phytochemical because

inhibition and the mechanism of action have already

been determined on bovine HAB (Flythe 2009).


Animals and diet

All animal husbandry and procedures were ap-

proved by the University of Kentucky care and use

committee. Rumen fistulated Kiko goat wethers

(n=4; 2 y, 40-50 kg) were used as rumen digesta do-

nors. They were maintained on pasture in a herd of

Kiko goats at the University of Kentucky’s Research

Farm, Lexington, Kentucky, USA. The botanical com-

position of the pasture is shown in Table 1. The herd

was supplemented with 1.0 kg head-1 d-1 orchard

grass hay (Dactylis glomerata; 18% crude protein),

and 0.25 kg head-1d-1 soya-based supplement (16%

crude protein; Southern States Cooperative, Rich-

mond, Virginia, USA). Water and a mineral mixture

(Southern States Cooperative, Richmond, Virginia,

USA) were provided free choice.

Figure 1. Simplified schematic of amino-nitrogen cycling the rumen. Processes are labeled: 1- Proteolysis by proteolytic microorganisms, 2- “By-pass” protein that is not deconstructed in the rumen, 3- Deamination by HAB and other microorganisms, 4- Assimilation of ammonia and amino acids by microorganisms for anabolic purposes.


Bacterial strains and media composition

The isolation and characterization of the HAB cul-

tures (Peptostreptococcus spp. BG1 and BG2) used

in these experiments were previously reported (Fly-

the and Andries, 2009). The HAB medium contained

(per liter): 240 mg K2HPO4, 240 mg KH2PO4, 480 mg

NaCl, 480 mg Na2SO4, 64 mg CaCl2·2H2O, 100 mg

MgSO4·7H2O, 600 mg cysteine hydrochloride, min-

eral and vitamin solutions as previously described

(Russell et al., 1988). The initial pH was adjusted to

6.5 with 20% NaOH. The liquid was autoclaved to re-

move O2 and cooled under O2 -free CO2. The buffer

(4.0 g Na2CO3) was added before dispensing anaero-

bically and autoclaving again for sterility. Casamino

acids and Trypticase (Fisher BioReagents, Fair Lawn,

New Jersey, USA) were prepared separately using

anaerobic technique, as described above. They were

aseptically added when indicated (15 mg ml-1 final

concentration).

Pure culture growth experiments

The HAB medium was amended with Casamino

acids or Trypticase (15 mg ml-1). Overnight Pepto-

streptococcus spp. BG1 and BG2 cultures were used

as the inocula (10% v/v). “Beta-Bio” hops extract was

added to the media, vigorously mixed and serially

diluted with to achieve the concentration indicated.

The propylene glycol-based extract contained 45%

ß-acid and no detectable ß-acid, as reported by the

manufacturer (S.S. Steiner, Inc., Yakima, Washington,

USA). The concentrations were verified by HPLC

(Dionex; Sunny Vale, California). The Phenomineex

250×4.6 mm Luna 5u C18 column (Torrance, Califor-

nia) was 30˚C. The mobile phase was 80% methanol,

20% water, pH 2.5. Eluting compounds were de-

tected by UV absorbance (314 nm). The standard

curve was generated using hops standards obtained

from the American Society of Brewing Chemists (St.

Paul, Minnesota). The limits of detection were 3.3

ppm and 4.1 ppm for colupulone and adlupulone,

respectively. The extract was not autoclaved, but

was added to uninoculated media and incubated

as a control. Controls for the effect of the propylene

glycol carrier were also performed. The incubations

were conducted in a shaking incubator (150 rpm,

39˚C). Growth was determined by optical density at

24 and 48 h (600 nm).

Table 1. The botanical composition of the pasture

Pasture Component Percent

Forage

Tall Fescue Lolium arundinaceum (Schreb.) Darbysh. 44.4

Orchardgrass Dactylis glomerata L. 14.9

White Clover Trifolium repens L. 14.5

Kentucky Bluegrass Poa pratensis L. 8.9

Red Clover Trifolium pratense L. 3.3

Bermudagrass Cynodon dactylon (L.) Pers. 1.4

Weeds* 12.6

* Weed species include: Rumex crispus L., Taraxacum officinale F.H. Wigg., Plantago coronopus L.,

Solanum carolinense L., Convolvulus arvensis L., Digitaria sp., Ambrosia artemisiifolia L.


Ammonia production by uncultivated rumen microorganisms

The rumen digesta (approx. 0.5 kg) was collected

from each goat individually (n=4) and transported

back to the laboratory in an insulated, airtight con-

tainer within 30 min. Rumen contents were removed

and filtered through four layers of muslin cheese-

cloth into centrifuge bottles. The fluid was subject-

ed to low-speed centrifugation (100 x g, 10 min) to

remove feed particles. The supernatants were trans-

ferred to new bottles and subjected to high-speed

centrifugation (25,600 x g, 10 min) to harvest the mi-

croorganisms. The pellets were resuspended in HAB

medium without a growth substrate, and low-speed

centrifugation was repeated to wash the cells. The

pellets were resuspended in HAB medium, pooled

into a glass vessel (500 ml), and sparged with O2-free

CO2. Microscopic examination revealed that the cell

suspension (approximately 15.0 OD, pH 6.7) con-

tained no visible plant fiber and few protozoa. The

cell suspension was dispensed into serum bottles

that contained CO2. Growth substrates (Trypticase

or casamino acids, 15 mg ml-1) and hops extract were

added as indicted. The bottles were incubated in a

shaking water bath (39˚C 150 rpm). Samples were

clarified by centrifugation and frozen (-20°C) until

ammonia analysis. Ammonia concentrations were

determined with a colorimetric method (Chaney and

Marbach, 1962).

Replication and statistics

The pure culture growth experiments were repeat-

ed three times with identical results. The in vitro am-

monia production experiments were repeated four

times using cell suspensions from a different goat

each time. The data were subjected to an analysis

of variance with Tukey’s test post hoc. Results were

considered significant when P < 0.05.

Figure 2. Effect of hops extract on ammonia production by uncultivated microorganisms from the goat rumen. Ammonia concentrations from peptides (squares) or amino acids (circles) after 24 h incubation (39 ° C) are shown. Error bars indicate standard error of the mean. Asterisks indicate treatments that are different than the 0 ppm control (P < 0.05).


RESULTS

Two caprine HAB pure cultures (Peptostreptococ-

cus spp. BG1 and BG2) grew when either free amino

acids (Casamino acids) or peptides (Trypticase) were

the sole growth substrate, and produced ammonia

at rates as great as 600 nmol mg cell protein-1min-1

(data not shown). Hops extract inhibited growth of

the two HAB cultures when the ß-acid concentrations

were ≥ 45 ppm and ≥ 4.5 ppm, respectively. The in-

hibitory concentration was not changed if amino ac-

ids or peptides were used as the growth substrate.

Cell suspensions of uncultivated rumen microor-

ganisms from goats produced approximately 35 mM

ammonia from free amino acids (Casamino acids)

and 50 mM ammonia from peptides (Trypticase) in

24 h (Fig. 2). The addition of hops extract decreased

the total ammonia produced during the incubations.

Ammonia from amino acids was not significantly less

than the control unless the ß-acid concentration was

30 ppm or greater. When the ß-acid concentration

was 30 ppm, ammonia production from amino acids

was 30% less than the control (P < 0.05). When pep-

tides were the growth substrate as little as 3 ppm

ß-acid caused a decrease in ammonia production

(P < 0.05). Ammonia production from peptides was

decreased by 50% relative to the control when the ß-

acid concentration was 12 ppm or greater (P < 0.05).

DISCUSSION

Several other experiments have been performed

to assess the usefulness of hops as a feed additive,

and all of these employed cows or microorganisms

of bovine origin. Previous in vitro experiments indi-

cated that hops ß-acid did not inhibit bovine ruminal

protozoa (Schmidt et al., 2006). However, hops ß-ac-

id did inhibit Streptococcus bovis and decreased the

acetate: propionate ratio during in vitro experiments

(Flythe and Aiken, 2010). S. bovis is proteolytic (Rus-

sell et al., 1981) thus, the results of the latter study

is consistent with recent work by Lavrenčič and col-

leagues (2013), who determined that two varieties

of hops decreased proteolysis by bovine rumen mi-

croorganisms. Wang and colleagues (2010) showed

the in vivo efficacy of hops in a feeding trial. In that

experiment, the average daily gain of steers was im-

proved by the inclusion of hops cones in the diet.

The results of the current study indicate that hops,

specifically the ß-acid component, can decrease am-

monia production by microorganisms from the goat

rumen. Inhibition of caprine HAB in pure culture

demonstrated that the decrease in ammonia could

be accomplished by antimicrobial of the ß-acid on

HAB. These results are consistent with previous

work that showed antimicrobial action on three bo-

vine HAB species (Flythe, 2009).

ACKNOWLEDGEMENTS

This work was funded by the United States Depart-

ment of Agriculture, Agricultural Research Service.

S.S. Steiner, Inc. (Yakima, WA) donated the hops ex-

tract used in this study. The authors acknowledge

the technical assistance of Adam Barnes and Tracy

Hamilton.

REFERENCES

Abou Akkada, A. R., and K. El-Shazly. 1964. Effect

of absence of ciliate protozoa from the rumen on

microbial activity and growth of lambs. Appl. Mi-

crobiol. 12: 384-390.

Attwood, G., D. Li, D. Pacheco, and M. Tavendale.

2006. Production of indolic compounds by rumen

bacteria isolated from grazing ruminants. J. Appl.

Microbiol. 100: 1261-1271.

Caldwell, D. R., and M. P. Bryant. 1966. Medium

without rumen fluid for non-selective enumera-

tion and isolation of rumen bacteria. Appl. Micro-

biol. 14: 794-801.

Callaway, T. R., T. S. Edrington, J. L. Rychlik, K. J.

Genovese, T. L. Poole, Y. S. Jung, K. M. Bischoff,

R. C. Anderson, and D. J. Nisbet. 2003. Iono-

phores: Their use as ruminant growth promotants

and impact on food safety. Curr. Issues Intest. Mi-

crobiol. 4: 43-51.


Chaney, A. L., and E. P. Marbach. 1962. Modified

reagents for determination of urea and ammonia.

Clin. Chem. 8: 130-132.

Chen, G. J., and J. B. Russell. 1989. More monensin-

sensitive, ammonia-producing bacteria from the

rumen. Appl. Environ. Microbiol. 55: 1052-1057.

Dennis, S. M., T. G. Nagaraja, and A. D. Dayton.

1986. Effect of lasalocid, monensin and thiopeptin

on rumen protozoa. Res. Veter. Sci. 41: 251-256.

Flythe, M. 2009. The antimicrobial effects of hops

(Humulus lupulus l.) on ruminal hyper ammonia-

producing bacteria. Letters Appl. Microbiol. 118:

242-248.

Flythe, M., and K. Andries. 2009. The effects of mo-

nensin on amino acid catabolizing bacteria iso-

lated from_the Boer goat rumen. Small Ruminant

Res. 81:178–181.

Flythe, M. D., and G. E. Aiken. 2010. Effects of hops

(Humulus lupulus l.) extract on volatile fatty acid

production by rumen bacteria. J. Appl. Microbiol.

109: 1169-1176.

Russell, J.B., Bottje, W.G., and M.A Cotta. 1981.

Degradation of protein by mixed cultures of ru-

men bacteria: identification of Streptococcus bo-

vis as an actively proteolytic rumen bacterium. J.

Anim. Sci. 53: 242-252.

Russell, J. B., and A. J. Houlihan. 2003. Ionophore

resistance of ruminal bacteria and its potential

impact on human health. FEMS Microbiol. Rev.

27: 65-74.

Kohn, R. A., M. M. Dinneen; and E. Russek-Cohen.

2005. Using blood urea nitriogen to predict nitro-

gen excretion and efficiency of nitrogen utiliza-

tion in cattle, sheep, goats, horses, pigs, and rats.

J. Anim. Sci. 83:879-889.

Lavrenčič, A., A. Levart, I. J. Košir, and A. Čerenak.

2013. Influence of two hop (Humulus lupulus L.)

varieties on in vitro dry matter and crude protein

degradability and digestibility in ruminants. J. Sci.

Food Agricult. 94: 1248-1252.

Muscher, A. S., B. Schröder, G. Breves, and K. Hu-

ber. 2010. Dietary nitrogen reduction enhances

urea transport across goat rumen epithetium. J.

Animal Sci. 88:3390-3398.

Russell, J. B., H. J. Strobel, and G. J. Chen. 1988.

Enrichment and isolation of a ruminal bacterium

with a very high specific activity of ammonia pro-

duction. Appl. Environ. Microbiol. 54: 872-877.

Russell, J., and J. Rychlik. 2001. Factors that alter ru-

men microbial ecology. Sci. 11: 1119-1122.

Schmidt, M. A., M. L. Nelson, J. J. Michal, and H.

H. Westberg. 2006. Effects of hop acids. II. Beta

acids on ruminal methane emission, protozoal

population, fermentation, and coM concentration

in cannulated finishing steers. J. Anim. Sci. Suppl.

84: 240.

Satter, L. D., and L. L. Slyter. 1974. Effect of am-

monia concentration on rumen microbial protein

production in vitro. Brit. J. Nutr. 32: 199-208.

Starke, S., A. S. Muscher, N. Hirschhausen, E. Pfef-

fer, G. Breves, and K. Huber. 2012. Expression of

urea transporters is affected by dietary nitrogen

restriction in goat kidney. J. Anim. Sci. 90:3889-

3897.

Taghavi-Nezhad, M., D. Alipour, M. D. Flythe, P.

Zamani, and G. Khodakaramian. 2013. The effect

of essential oils of Zataria multiflora and Mentha

spicata on the in vitro rumen fermentation, and

growth and deaminative activity of amino acid-

fermenting bacteria isolated from Mehraban

sheep. Anim. Prod. Sci. 54: 299-307.

Tedeschi, L. O., D. G. Fox, and T. P. Tylutki. 2003.

Potential environmental benefits of ionophores

in ruminant diets. J. Environ. Qual. 32: 1591-1602.

USDA-APHIS. 2005. The goat industry structure,

concentration, demand and growth. Electronic Re-

port. http://www.aphis.usda.gov/animal_health/

emergingissues/downloads/goatreport090805.

pdf

USDA. 2004. 2002 Census of Agriculture. AC-

02-A-51. http://www.agcensus.usda.gov/Publica-

tions/2002/USVolume104.pdf

USDA. 2009. 2007 Census of Agriculture . AC-


tions/2007/Full_Report/Volume_1,_Chapter_1_

US/usv1.pdf

USDA. 2014. 2012 Census of Agriculture. AC-


tions/2012/Full_Report/Volume_1,_Chapter_1_

US/usv1.pdf


Wallace, R. 2004. Antimicrobial properties of plant

secondary metabolites. Proceedings of the Nutri-

tion Society 63: 621-629.

Wang, Y., A. V. Chaves, F. L. Rigby, M. L. He, and

T. A. McAllister. 2010. Effects of hops on ruminal

fermentation, growth, carcass traits and shedding

of Escherichia coli of feedlot cattle. Livestock Sci.

129: 135-140.



VOLUME 4 ISSUE 1

Preventing Post-Processing Contamination in a Food Nugget Processing Line When Lan-guage Barriers ExistJ. A. Neal, C. A. O’Bryan and P. G. Crandall

20

A Personal Hygiene Behavioral Change Study at a Midwestern Cheese Production PlantJ. A. Neal, C. A. O’Bryan and P. G. Crandall

13

Behavioral Change Study at a Western Soup Production Plant

C. A. O’Bryan, J. A. Neal, and P. G. Crandall

27

Salmonella in Cantaloupes: You Make Me Sick!B. A. Almanza

35

The Hurricane Sandy DilemmaB. A. Almanza

43

Introduction Special IssueP. G. Crandall

8

Case Studies



Intellect-u-ale: A Smart Approach to Quality Assurance in a Micro-BreweryA. J. Corsi, M. Goodman, and J. A. Neal

50


Antibiotic Use in Livestock ProductionBroadway, P. R., J. A. Carroll, and T. R. Callaway

76

Effects of Co-nutrients in Foods and Bioremediation in the Environment on Methylmercury

P. G. Crandall, C. A. O’Bryan

86

Alternative antimicrobial supplements that positively impact animal health and food safety Broadway, P. R., J. A. Carroll, and T. R. Callaway

109

Human Health Benefits of Isoflavones from Soybeansk. Kushwaha, C. A. O’Bryan, D. Babu, P. G. Crandall, P. Chen, and S.-O. Lee

122

REVIEW

Contribution of Chemical and Physical Factors to Zoonotic Pathogen Inactivation during Chicken Manure CompostingM.C. Erickson, J. Liao, X. Jiang, and M.P. Doyle

96

ARTICLES




VOLUME 4 ISSUE 2


Batch Culture Characterization of Acetogenesis in Ruminal Contents: Influence of Aceto-

gen Inocula Concentration and Addition of 2-Bromoethanesulfonic AcidP. Boccazzi and J. A. Patterson

177

Survival of Salmonella enterica and Listeria monocytogenes in manure-based compost mix-tures at sublethal temperatures M.C. Erickson, C. Smith, X. Jiang, I.D. Flitcroft, and M.P. Doyle

224

The Effect of Phytochemical Tannins-Containing Diet on Rumen Fermentation Characteris-tics and Microbial Diversity Dynamics in Goats Using 16S rDNA Amplicon PyrosequencingB. R. Min, C. Wright, P. Ho, J.-S. Eun, N. Gurung, and R. Shange

195

Characterization of the Novel Enterobacter cloacae Strain JD6301 and a Genetically Modified Variant Capable of Producing Extracellular LipidsJ. R. Donaldson, S. Shields-Menard, J. M. Barnard, E. Revellame, J. I. Hall, A. Lawrence, J. G. Wilson, A. Lipzen, J. Martin, W. Schackwitz, T. Woyke, N. Shapiro, K. S. Biddle, W. E. Holmes, R. Hernandez, and W. T. French

212

ARTICLES

The Prevalence of E. coli O157:H7 in the Production of Organic Herbs and a Case Study of Organic Lemongrass Intended for Use in Blended TeaS. Zaman, Md. K. Alam, S. S. Ahmed, Md. N. Uddin, and Md. L. Bari

164




VOLUME 4 ISSUE 3



MANUSCRIPT SUBMISSION

Authors must submit their papers electronically

([email protected]). According to instruc-

tions provided online at our site: www.afabjournal.

com. Authors who are unable to submit electroni-

cally should contact the editorial office for assistance

by email at [email protected].

INSTRUCTIONS TO AUTHORS

• Aerobic microbiology

• Aerobiology

• Anaerobic microbiology

• Analytical microbiology

• Animal microbiology

• Antibiotics

• Antimicrobials

• Bacteriophage

• Bioremediation

• Biotechnology

• Detection

• Environmental microbiology

• Feed microbiology

• Fermentation

• Food bacteriology

• Food control

• Food microbiology

• Food quality

• Food Safety

• Foodborne pathogens

• Gastrointestinal microbiology

• Microbial education

• Microbial genetics

• Microbial physiology

• Modeling and microbial kinetics

• Natural products

• Phytoceuticals

• Quantitative microbiology

• Plant microbiology

• Plant pathogens

• Prebiotics

• Probiotics

• Rumen microbiology

• Rapid methods

• Toxins

• Veterinary microbiology

• Waste microbiology

• Water microbiology

CONTENT OF MANUSCRIPT

We invite you to consider submitting your re-

search and review manuscripts to AFAB. The jour-

nal serves as a peer reviewed scientific forum for to

the latest advancements in bacteriology research

on Agricultural and Food Systems which includes

the following fields:


With an open access publication model of this

journal, all interested readers around the world can

freely access articles online. AFAB publishes origi-

nal papers including, but not limited to the types

of manuscripts described in the following sections.

Papers that have been, or are scheduled to be, pub-

lished elsewhere should not be submitted and will

not be reviewed. Opinions or views expressed in pa-

pers published by AFAB are those of the author(s)

and do not necessarily represent the opinion of the

AFAB or the editorial board.

MANUSCRIPT TYPES

Full-Length Research Manuscripts

AFAB accepts full-length research articles con-

taining four (4) figures and/or tables or more. AFAB

emphasizes the importance of sound scientific ex-

perimentation on any of the topics listed in the focus

areas followed by clear concise writing that describes

the research in its entirety. The results of experi-

ments published in AFAB must be replicated, with

appropriate statistical assessment of experimental

variation and assignment of significant difference.

Major headings to include are: Abstract, Introduc-tion, Materials and Methods, Results, Discussion (or Results and Discussion), Conclusion, Acknowl-edgements (optional), Appendix for abbreviations (optional), and References.

Manuscripts clearly lacking in language will be re-

turned to author without review, with a suggestion

that English editing be sought before the paper is

reconsidered. AFAB offers a fee based language

service upon request. Please contact [email protected] for more information about our fees

and services.

Rapid Communications

Under normal circumstances, AFAB aims for re-

ceipt-to-decision times of approximately one month or less. Accepted papers will have priority for publi-

cation in the next available issue of AFAB. However,

if an author chooses or requires a much more rapid

peer review, the journal editorial office has the capa-

bility to shorten the review timing to one week or less.

Any type of manuscript whether it be a full length

manuscript, brief communication or review paper can

be submitted as a rapid communication. There will be

additional costs for processing and page charges will

be double the normal rate. Authors who choose this

option must select Rapid Communications as the pa-

per type when submitting the paper and the editors

will judge whether a rapid review is possible and let

the author know immediately.

Brief Communications

Brief communications are short research notes giv-

ing the results of complete experiments but are con-

sidered less comprehensive than full-length articles

with three (3) figures and/or tables or less. Manuscripts

should be prepared with the same subheadings as full

length research papers. The running head above the

title of the paper is “Brief Communications.”

Unsolicited Review Papers

Review papers are welcome on any topic listed in

the focus section and have no page limits. Reviews

are assessed the same pages charges as all other

manuscripts. All AFAB guidelines for style and form

apply. Major headings to include are: Abstract, In-troduction, Main discussion topics and appropri-ate subheadings, Conclusions, Acknowledgements (optional) and References. Review papers shorter

than 20 pages of double spaced text and references

will be considered mini-reviews with the subhead-

ing above the title on the first page. The running

head above the title of the paper is either “Review”

or “Mini-review”.

Solicited Review Papers

Solicited reviews will have no page limits. The

editor-in-chief will send invitations to the authors

and then review these contributions when they are

submitted. Nominations or suggestions for potential

timely reviews are welcomed by the editors or edito-


rial board members and should be sent to submit@

afabjournal.com. There will be no page charges for

solicited review papers but the solicitation must origi-

nate from the editor-in-chief or one of the editors. Re-

quests from authors will automatically be classified as

unsolicited review papers. The running head above

the title of the paper will be “Invited Review.”

Conference and Special Issues Reviews

AFAB welcomes opportunities to publish papers

from symposia, scientific conference, and/or meet-

ings in their entirety. Conference organizers need

simply to contact AFAB at [email protected]

and a rapid decision is guaranteed. If in agreement,

the conference organizers must guarantee delivery

of a set number of peer reviewed manuscripts within

a specified time and submitted in the same format

as that described for unsolicited review papers. Con-

ference papers must be prepared in accordance with

the guidelines for review articles and are subject to

peer review. The conference chair must decide

whether or not they wish to serve as Special Issue

Editor and conduct the editorial review process. If

the conference chair/organizer chooses to serve as

special issue editor, this will involve review of the pa-

pers and, if necessary, returning them to the authors

for revision. The conference organizer then submits

the revised manuscripts to the journal editorial of-

fice for further editorial examination. Final revisions

by the author and recommendations for acceptance

or rejection by the chair must be completed by a

mutually agreed upon date between the editor and

the conference organizer. Manuscripts not meeting

this deadline will not be included in the published

symposium proceedings but if submitted later can

still be considered as unsolicited review papers. Al-

though offprints and costs of pages are the same

as for all other papers, the symposium chair may be

asked to guarantee an agreed upon number of hard

copies to be purchased by conference attendees. If

the decision is not to publish the symposium as a

special issue, the individual authors retain the right

to submit their papers for consideration for the jour-

nal as ordinary unsolicited review manuscripts.

Book Reviews

AFAB publishes reviews of books considered to

be of interest to the readers. The editor-in-chief ordi-

narily solicits reviews. Book reviews shall be prepared

in accordance to the style and form requirements of

the journal, and they are subject to editorial revision.

No page charges will be assessed solicited reviews

while unsolicited book reviews will be assigned the

regular page charge rate.

Opinions and Current Viewpoints

The purpose of this section will be to discuss, cri-

tique, or expand on scientific points made in articles

recently published in AFAB. Drafts must be received

within 6 months of an article’s publication. Opinions

and current perspectives do not have page limits.

They shall have a title followed by the body of the

text and references. Author name(s) and affiliation(s)

shall be placed between the end of the text and list

of references. If this document pertains to a par-

ticular manuscript then the author(s) of the original

paper(s) will be provided a copy of the letter and of-

fered the opportunity to submit for consideration a

reply within 30 days. Responses will have the same

page restrictions and format as the original opinion

and current viewpoint, and the titles shall end with

“Opinions.” They will be published together. Letters

and replies shall follow appropriate AFAB format

and may be edited by the editor-in-chief and a tech-

nical editor. If multiple letters on the same topic are

received, a representative set of opinions concern-

ing a specific article will be published. A disclaimer

will be added by the editorial staff that the opinion

expressed in this viewpoint is the authors alone and

does not necessarily represent the opinion of AFAB

or the editorial board.

COPYRIGHT AGREEMENT

The copyright form is published in AFAB as space

permits and is available online (www.afabjournal.com).


AFAB grants to the author the right of re-publication

in any book of which he or she is the author or edi-

tor, subject only to giving proper credit to the original

journal publication of the article by AFAB. AFAB re-

tains the copyright to all materials accepted for pub-

lication in the journal. If an author desires to reprint

a table or figure published from a non-AFAB source,

written evidence of copyright permission from an au-

thority representing that source must be obtained by

the author and forwarded to the AFAB editorial office.

PEER REVIEW PROCESS

Authors will be requested to provide the names

and complete addresses including emails of five (5) potential reviewers who have expertise in the research

area and no conflict of interest with any of the authors.

Except for manuscripts designated as Rapid Commu-

nication each reviewer has two (2) weeks to review

the manuscript, and submit comments electronically

to the editorial office. Authors have three (3) weeks

to complete the revision, which shall be returned to

the editorial office within six (6) weeks after which the

authors risk having their manuscript removed from

AFAB files if they fail to ask the editorial office for

an extension by email. Deleted manuscripts will be

reconsidered, but they will have to be processed as

new manuscripts with an additional processing fee as-

sessed upon submission. Once reviewed, the author

will be notified of the outcome and advised accord-

ingly. Editors handle all initial correspondence with

authors during the review process. The editor-in chief

will notify the author of the final decision to accept or

reject. Rejected manuscripts can be resubmitted only

with an invitation from the editor or editor-in chief. Re-

vised versions of previously rejected manuscripts are

treated as new submissions.

PRODUCTION OF PROOFS

Accepted manuscripts are forwarded to the edito-

rial office for technical editing and layout. The manu-

script is then formatted, figures are reproduced, and

author proofs are prepared as PDFs. Author proofs

of all manuscripts will be provided to the correspond-

ing author. Author proofs should be read carefully and

checked against the typed manuscript, because the

responsibility for proofreading is with the author(s).

Corrections must be returned by e-mail. Changes

sent by e-mail to the technical editor must indicate

page, column, and line numbers for each correction

to be made on the proof. Corrections can also be

marked using “track changes” in Microsoft Word or

using e-annotation tools for electronic proof correc-

tion in Adobe Acrobat to indicate necessary chang-

es. Author alterations to proofs exceeding 5% of the

original proof content will be charged to the author. All

correspondence of proofs must be agreed to by the

editorial office and the author within 48 hours or proof

will be published as is and AFAB will assume no re-

sponsibility for errors that result in the final publication.

PUBLICATION CHARGES

AFAB has two publication charge options: conven-

tional page charges and rapid communication. The

current charge for conventional publication is $25 per printed page in the journal. There is no additional

charge for the publication of pages containing color

images, micrographs or pictures. For authors who

wish to have their papers processed as a rapid com-

munication, authors will pay the rapid communication

fee when proofs are returned to the editorial office

in addition to twice the conventional page charges.

Charges for rapid communications are $1000 per manuscript for guaranteed peer review within one

week and $100 per journal page.

HARD COPY OFFPRINTS

If you are wishing to obtain a physical hard copy of

the AFAB journal, offprints are available in any quan-

tity at an additional charge: $100/page for black-white

and $150/page for color prints. You may order your

offprints at any time after publication on our website.

Scientific conference organizers may be expected to

agree to a set number of offprints as a part of their

agreement with AFAB.


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)].


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


“(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.


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.

AFAB Online Edition is Now Available!

www.AFABjournal.com

• Free Access

• Print PDFs

• Flip Through Issues

• Search Article Archives

• Order Reprints

• Submit a Paper

Online Publication: www.AFABjournal.com

Documents

Afab Volume 5 Issue 1