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Isolation, identification and characterization of lytic, wide host range

bacteriophages from waste effluents against Salmonella enterica serovars

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DOI: 10.1016/j.foodcont.2013.09.064

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Food Control 38 (2014) 67e74

Contents lists available at ScienceDirect

Food Control
journal homepage: www.elsevier.com/locate/foodcont

Isolation, identification and characterization of lytic, wide host range

bacteriophages from waste effluents against Salmonella enterica
serovars
Mastura Akhtar, Stelios Viazis, Francisco Diez-Gonzalez*
Department of Food Science and Nutrition, University of Minnesota, St. Paul, MN 55108, USA

a r t i c l e i n f o a b s t r a c t

Article history: The use of bacteriophages is considered as a viable alternative to chemical antimicrobials against
Received 25 July 2013 foodborne pathogens. The objective of this study was to develop a collection of lytic bacteriophages
Received in revised form which will be able to infect different pathogenic Salmonella enterica serovars. Phages were isolated from
26 September 2013
animal feces and sewage samples, purified, characterized morphologically and by DNA fingerprinting,
Accepted 28 September 2013
and host ranges were determined. Spot test and efficiency of plaquing (EOP) data indicated that two
phages, SEA1 and SEA2 had the broadest host range against Salmonella among all isolated phages. SEA2
Keywords:
was highly efficient to infect S. Typhimurium DT104 (0.5e1 EOP value). Only phage SSA1 was able to
Phages
Salmonella
infect S. Montevideo. Transmission electron microscopy (TEM) revealed the phages in the collection were
Antimicrobial mostly (4 out of 6) Siphoviridae, while SEA1 and SEA2 were Myoviridae T4-like phages. SEA1 and SEA2
Anti-Salmonella phages had the largest genome sizes in the collection, 190 and 170 kb, respectively. Pulsed field gel electro-
Biocontrol phoresis (PFGE) analysis demonstrated distinct digestion profiles with EcoRI for phages SSA1, STD3, STE3
Food safety and STF1. However, SEA1 and SEA2 shared a similar restriction enzyme (RE) digestion pattern with same
morphotype, but distinct profiles in lysing Salmonella strains. These anti-Salmonella phages were highly
host specific with few exceptions of lytic phages that were able to infect a wide variety of Salmonella.
These phages have potential for use in applications controlling Salmonella on different matrices.
Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction an important public health risk that needs great attention and a
pursuit for effective antimicrobial alternatives. In this context,
Salmonella infections are related to a wide diversity of food bacteriophages have potential as an alternative to antibiotics or
products and their source of contamination ranges from human other conventional chemical control methods against bacterial
through food processing facilities (Miao & Miller, 1999; Tauxe, pathogens. Bacteriophages are viruses capable of lysing bacteria,
Doyle, Kuchenmüller, Schlundt, & Stein, 2010). The estimated and specific lytic phages can kill pathogenic bacteria in their own
annual cost due to foodborne Salmonella infections is $2.4 billion in habitat. Phages are ubiquitous in nature and can often be found in a
the United States ((USDA-ERS), 2001). According to the most recent variety of environments related to their host such as soil, sewage,
report, non-typhoidal Salmonella has been identified as the leading water, manure, animal and produce farms, as well as different food
cause of foodborne illnesses in USA with estimates of 1 million processing plant effluents.
cases and 378 deaths per year (Scallan et al., 2011). Outbreaks have The application of bacteriophages as a food safety intervention
been associated with multidrug resistant serovars such as S. has been recently investigated and a few commercial preparations
Typhimurium DT104 that often cause serious problems because of have been approved and marketed. Bacteriophages are often used
the limited treatment options (CDC, 2001). Though preventative in high concentrations to inactivate foodborne pathogens, such as
measures are available, the recurring outbreaks of Salmonella pose Escherichia coli O157:H7, Salmonella, Listeria, and Campylobacter in
different foods (Carlton, Noordman, Biswas, De Meester, & Loessner,
2005; Greer, 2005; O’Flynn, Coffey, Fitzgerald, & Ross, 2006;
O’Flynn, Ross, Fitzgerald, & Coffey, 2004). Also, in production fa-
* Corresponding author. Department of Food Science and Nutrition, University of
Minnesota, 225A, 1334 Eckles Ave., St. Paul, MN 55108, USA. Tel.: þ1 612 624 9756;
cilities, phages also have been used to control specific bacteria at
fax: þ1 612 625 5272. pre-harvest and post-harvest stages of food production and storage
E-mail address: [email protected] (F. Diez-Gonzalez). (Greer, 2005). The Food and Drug Administration (FDA) has

0956-7135/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodcont.2013.09.064
 Author's personal copy

68 M. Akhtar et al. / Food Control 38 (2014) 67e74

approved Listeria specific phages for use in foods (FDA, 2006) Lake City, Wisconsin; and 3) raw sewage sludge, wastewater
indicating the promising use of bacteriophages in food applications. treatment plant, City of St. Paul. Samples were collected by several
Salmonella specific phages have been isolated and a few lytic wide- visits throughout the spring and summer seasons.
activity range phages have been identified. Bielke et al. found only
two potent bacteriophages from the isolated pool were able to lyse 2.3. Enrichment, isolation, purification and preparation of
7 out of 10 Salmonella isolates (Bielke, Higgins, Donoghue, bacteriophages
Donoghue, & Hargis, 2007). Flynn et al. described that two lytic
phages, st104a and st104b were screened from 100 fecal samples All waste samples were collected and centrifuged at 10,000  g
and evaluated as effective therapy to reduce Salmonella enterica for 10 min to remove solid particles and then filtered with 0.45 mm
serovar Typhimurium DT104 in vitro (O’Flynn et al., 2006). How- pore syringe filters (Nalgene, Thermo Fisher Scientific Inc. USA). For
ever, despite the existing inadequate library of lytic Salmonella enrichment, isolation and purification of bacteriophages, modified
phages, literature search indicated that there is no currently methods were used from previously published reports (Klieve,
available phage treatment to control the diverse group of Salmo- 2005; Mullan, 2002; Twest & Kropinski, 2009). Salmonella strains
nella serovars. were grown overnight at 37  C in TSB to obtain pure bacterial
In this study, waste effluents from cattle and swine manure pit cultures. 0.1 mL of overnight pure cultures of Salmonella strains
and raw sewage from the wastewater treatment plant were were inoculated into 10 mL TSB and incubated at 37  C shaker for
screened to isolate a collection of broad spectrum lytic Salmonella 3e4 h to grow exponential phase cultures. Filtered sample super-
phages. The selected potent lytic phages were identified morpho- natant (4.5 mL) was then mixed with 0.5 mL exponential phase
logically and further characterized to evaluate their lysis efficacy for bacterial cultures and 0.5 mL 10 concentrated TSB, and incubated
infecting a wide range of S. enterica serovars. This library of lytic at 37  C for 24e48 h. After incubation, samples were centrifuged at
phages may be prospective to use with other isolated lytic phages 10,000  g for 10 min, supernatants were filtered with a 0.45 mm
for further food-related applications against Salmonella. filter syringe, and used as enriched phage (EP) samples.
Initially, spot testing was used to isolate the phages. The host
2. Materials and methods bacterial lawn was made by using a tryptone top agar (TTA) (con-
taining per liter: bacteriological agar, 4; tryptone, 10 g; yeast
2.1. Bacterial strains and culture conditions extract, 5 g; NaCl, 7.5 g; Glucose, 10 g; 12 mmol MgSO4; 12 mmol
CaCl2) containing host bacterial suspensions that were overlaid on
A total of 34 different of S. enterica strains belonging to eight top of TSA agar plates. When the agar overlays were solidified,
serovars were used to serve as host strains and to screen phages several EP samples were spotted (1:10, phage:host) onto the lawns
from isolation sources (Table 1). All bacterial strains were streaked in a row and plates were incubated at 37  C for 18e24 h. After in-
from the frozen culture stock onto tryptic soy agar (TSA; Neogen cubation, all plates were examined for positive and clear spot for-
Corp., Lansing, MI). Prior to experiments, each strain was grown by mation. The lysed agar spot was cut and dissolved into phosphate
picking an isolated colony from TSA plates and inoculating into buffer saline (PBS; Neogen, Inc.), centrifuged and filtered again to
tryptic soy broth (TSB; Neogen Corp.) and incubated at 37  C to collect the supernatants. The supernatants were used as spot
obtain fresh overnight cultures. filtrate.
Plaque assays were conducted to isolate and purify individual
2.2. Sample collection phages from spot filtrates. For plaque assay, a series of 10-fold di-
lutions were made of spot filtrates. Exponential phase host cultures
Several waste effluent samples were collected from 1) cattle and were mixed with dilutions of spot filtrates as host: spot filtrate (2:1)
swine manure pit from the dairy cattle and swine barns of the and incubated at 37  C for 15 min for propagation. These phages
University of Minnesota; 2) city sewage treatment plant in Rice and bacterial suspensions were mixed with the TTA agar and
poured onto TSA. Plates were incubated at 37  C for 24 h for plaque
formation. To purify the phages, isolated plaques were picked by
Table 1 using a pipette or a wire loop and suspended with PBS. The sus-
List of bacterial strains that were used for this study. pensions were centrifuged and the supernatants were filter steril-
Salmonella Strain ID number Number Source ized and used as a single phage culture. This purification process of
serovars of strains of strains an individual plaque through plaque assay, centrifugation and
S. Agona Agona FDA 1 FDA resuspension was repeated at least 3 times to ensure a pure phage
S. Typhimurium UK-1, 3019907 2 FSML stock.
ATCC 14028, ATCC 700408 2 ATCC The high titer phage stocks were prepared by inoculating 1 mL
I598, I503, I527, I534, I535, 11 VBS
of overnight host bacterial cultures with 100 ml of purified phage
I649, I526, I758, I536, I740, I600
E2009005811 1 MDH stock (105e106 PFU mL1) into TSB (with 5 mol l1 CaCl2) and
S. Enteritidis 2009595, 95657613, I823 3 FSML incubated at 37  C for 18e20 h. When lysis of liquid TSB cultures
S. Heidelberg FSIS10 1 FSML was visually observed, a few drops of CHCl3 were added for com-
S. Montevideo 95573473 1 MDH plete lysis of the bacterial cells. The amplified, purified phages were
S. Newport AMO7076, AMO7073, AMO5104, 5 CDC
AMO5313, B4442
centrifuged at 10,000  g for 10 min to pellet debris and super-
Newport FDA 1 FDA natants were filtered to remove bacterial contaminants. The filtered
2006036 1 FSML supernatants were stored as high titer (108e1010 PFU mL1) phage
S. Saintpaul E2008001236, E2008001177, 5 MDH stocks at 4  C and used for the different analysis throughout the
E2003002913, E2009010674,
study.
E2008001358
S. Tennessee E200700502 1 MDH
2.4. Determination of host range
Abbreviation: FDA, Food and Drug Administration; FSML, Food Safety Microbiology
Laboratory, University of Minnesota; ATCC, American Type Culture Collection; VBS,
Department of Veterinary and Biomedical Sciences, University of Minnesota; MDH, Spot testing was conducted to measure the ability of individual
Minnesota Department of Health; CDC, Centers for Disease Control and Prevention. phages to infect different serovars of Salmonella. First, bacterial
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M. Akhtar et al. / Food Control 38 (2014) 67e74 69

lawns were made with exponential phase cultures on TSA plates as England Biolabs, Ipswich, MA, USA) following the manufacturer’s
described above and purified phages were spotted as 1:10 (phag- protocol. All digested plugs were run on a PFGE gel in a CHEF-DR II
e:host) on each plate. All plates were incubated at 37  C for 24 h. PFGE (BioRad, Inc) for 18 h and imaged on an ethidium bromide-
After incubation, all clear spots were identified as positive and lytic stained agarose gel (1%, wt/vol) by a BioRad gel imaging system.
spots on each strain of bacterial lawns and then analyzed to report The RE digested DNA images were analyzed to identify the distinct
the host range of isolated phages. Phages were selected based on DNA features among purified phages.
the spot test for further analysis by efficiency of plaquing (EOP)
using a modified version of previously published protocols (O’Flynn 2.7. Data analysis
et al., 2004; Viazis, Akhtar, Feirtag, Brabban, & Diez-Gonzalez,
2011). EOP was conducted on selected phages to determine their All reported results were calculated by averaging two inde-
ability of plaque formation on a diverse set of Salmonella serovars. pendent experimental data in which duplicate samples with
When a phage formed a plaque on the lawn of specific Salmonella duplicate counts were tested at each time interval. Mean and
strain of interest, the efficiency of plaquing (EOP) was measured by standard deviations were determined using Microsoft Excel
using the Salmonella host strain as the reference strain. The EOP (Version 14.0; Microsoft, Corp; Redmond, WA). Two factor Analysis
was determined by expressing the phage titer of the susceptible of variances (ANOVA) of EOP values among the phages were
strain relative to the phage titer of the reference strain. EOP of calculated in respect of infecting different serovars of Salmonella.
bacteriophages was identified on the tested strains from eight First, all tested Salmonella strains were grouped by serovars. Mean
different serovars, those are Typhimurium (N ¼ 15), Saintpaul EOP were measured for different Salmonella serovars for each
(N ¼ 1), Tennessee (N ¼ 1), Enteritidis (N ¼ 3), Montevideo (N ¼ 1), phage, and ANOVA was performed by using Microsoft Excel.
Newport (N ¼ 4), Agona (N ¼ 1), Heidelberg (N ¼ 1) (Table 4).
3. Results
2.5. Morphology
3.1. Collection and isolation of phages
Stock cultures of purified phages were used to image the
morphology by transmission electron microscopy (TEM). Phages As many as 52 waste samples were tested for the presence of
were diluted into Phosphate buffer saline (PBS) to avoid the gelatin bacteriophages against Salmonella. A total of 59 phages were iso-
microstructures and negatively stained with either ammonium lated from natural waste. Twelve phages were isolated from dairy
molybdate or 0.5% phosphotungstic acid or 3% uranyl acetate cattle manure and nine phages from swine manure using S.
(Ackermann, 2009b). The fixed phages on copper grid were Typhimurium UK-1 and S. Typhimurium ATCC 14028 as host
pictured and analyzed by a Philips CM12 transmission electron strains. A third group of phages (N ¼ 38) were obtained from raw
microscope (TEM) with 60 kV, at the Imaging Center, College of sewage samples of a wastewater treatment facility. Eleven strains of
Biological Sciences, and by a JEOL 1200 EXII TEM, with accelerating four different Salmonella serovars were used as host for phage
voltage from 40 kV to 120 kV, at the Characterization Facility, Col- isolation from sewage samples. Thirty phages were isolated using S.
lege of Science and Engineering, University of Minnesota. Typhimurium, five phages with S. Enteritidis, one with S. Newport,
and two with S. Saintpaul (Table 2). We could not isolate any phage
2.6. Phage genome size determination with the Tennessee serovar.

The genome sizes of the phages were measured by pulsed field 3.2. Host range of phages
gel electrophoresis (PFGE). Phage DNA plugs were made by mixing
purified phage (1:1) with 1.4% (w/v) molten PFGE grade agarose 3.2.1. Spot testing
(Ultra Pure DNA Grade Agarose: BioRad, Hercules, CA). DNA plugs All 59 isolated phages were capable of lysing their host strains
were treated with proteinase K (0.1 mg mL1) and lysis buffer to throughout the purification process. When these phages were
extract the phage DNA and stored in 0.5  Tris EDTA (TE) at 4  C for screened by spot testing against a total of thirty strains of six Sal-
further analysis. Phage DNA was tested by PFGE to ensure the purity monella serovars including Typhimurium, Enteritidis, Newport,
of isolated phage. Restriction enzyme digestion was used for Heidelberg, Saintpaul and Tennessee (Table 3), less than 25% of
further characterization of the isolated phages. These sets of DNA isolated phages (13 out of 59) formed clear plaques and were
plugs were made with higher DNA concentration than the genome capable to lyse at least two serovars (Table 3) while the rest of them
size to obtain the clear multiple bands of digested DNA. Phage DNA were highly specific in infecting only their host serovars and pla-
plugs were then digested with restriction enzyme (RE) EcoRI (New ques were not well defined. Among phages against Typhimurium,

Table 2
Collection and isolation of bacteriophages against different Salmonella serovars from natural sources.

Host bacterial strain List of isolated phages Number

of phages

S. Typhimurium UK-1 STA1, STA2, STA3, STA9/STA4, STA5, STA6, STA7, STA8, STA10/STA11, STA12, STA13, STA14 14
S. Typhimurium ATCC 14028 STB1, STB2, STB3, STB4, STB5, STB9, STB10, STB11/STB6, STB7, STB8/STB13, STB14, STB15, STB16, STB17, STB18, STB19 18
S. Typhimurium DT104 STC1, STC2, STC3, STC4, STC5, STC6, STC7, STC8, STC9, STC10, STC11, STC12, STC13 13
S. Typhimurium I600 STD1, STD2, STD3 3
S. Typhimurium I536 STE1 1
S. Typhimurium I527 STF1 1
S. Typhimurium 758 STG1 1
S. Enteritidis 2009595 SEA1, SEA2, SEA3 3
S. Enteritidis 95657613 SEB1, SEB2 2
S. Newport AMO7076 SNA1 1
S. Saintpaul E2008001236 SSA1, SSA2 2
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70 M. Akhtar et al. / Food Control 38 (2014) 67e74

Table 3
Sensitivity of Salmonella serovars to lysis by selected isolated bacteriophages determined by spot testing.

Phage % Of positive spots against Salmonella serovars

Typhimurium (N ¼ 14) Enteritidis (N ¼ 3) Newport (N ¼ 6) Heidelberg (N ¼ 1) Saintpaul (N ¼ 5) Tennessee (N ¼ 1)

STA1 92.9 0 0 100 20 0

STA3 92.9 33.3 16.7 100 0 0
STA9 92.9 0 0 100 0 0
STA10 64.3 66.7 33.3 100 80 0
STA11 85.7 100 0 100 80 0
SEA1 64.3 100 83.3 0 100 100
SEA2 64.3 100 66.7 100 100 100
SSA1 78.6 100 50 100 80 100
SSA2 78.6 100 50 100 100 100
STD3 78.6 100 50 100 100 100
STE1 78.6 100 50 100 80 100
STF1 35.7 33.3 16.7 0 100 0

STD3 and STE1 were capable of lysing at least three other serovars 3.3. Characterization of phages
in addition to the host. Two S. Enteritidis phages, SEA1 and SEA2
were isolated from two different samples using one strain, S. 3.3.1. Morphology
Enteritidis 2009595. These two phages displayed similar host range TEM was conducted after negative staining to classify the
activity, but SEA1 did not lyse the Heidelberg strain. The S. Saint- selected phages by morphological characteristics. All of the phages
paul phages, SSA1 and SSA2 had broad host range and lysed 50e that were identified by TEM (Fig. 1) were tailed phages and
100% strains of all six serovars. SEA1 and SEA2 infected lower appeared to fall into the order Caudovirales. The isolated phages
number of Typhimurium strains but produced larger clearing spots belonged to two different families, Myoviridae and Siphoviridae
than SSA1 and SSA2. SEA2, SSA1, SSA2, STD3 and STE1 were the (Ackermann, 2009a). SEA1 and SEA2 resembled the typical
phages that formed visibly clear spots on the majority of bacterial morphological features of T4 Myoviridae phages with icosahedral
lawns (from 50 to 100%) of all six tested serovars. head and contractile tail with fixation structures. The head and tail
length were approx. 111 and 100 nm, respectively, for these Myo-
3.2.2. EOP viridae phages. The Saintpaul-specific phage, SSA1 belonged to
In the process of identifying the most effective phages, the Siphoviridae with a non-contractile tail. TEM pictures of Typhi-
selected phages were further analyzed to determine the host range murium phages, STA9, STA1 and STA2 indicated that they were
and lytic capacity by EOP. The EOP assay was conducted on several Siphoviridae, non-contractile, long tailed phages.
representative phages which had lysed 50% or more tested strains.
Results are presented for nine phages (SEA1, SEA2, SSA1, SSA2, STE1, 3.3.2. Genome size
STA3, STA9, STA10 and STD3) which were the most efficient among We determined the genome size for a variety for isolated phages
all the isolated 59 phages in generating plaques against tested strains adopting the narrow and broad host range. According to the TEM
(Tables 4 and 5). SEA1 and SEA2 were found to have the broadest pictures (Fig. 1), phages were tailed Myoviridae or Siphoviridae
spectrum of lytic ability. SEA1 was found to be the most efficient phages. These typical phages have been described previously con-
with a wide range of lysis ability among all the isolated phages taining double stranded DNA (Ackermann, 2009a). The most vari-
(according to ANOVA, p < 0.05). SEA2 showed medium to high ef- able genome sizes were observed among the screened
ficiency (EOP ¼ 0.2e1) to lyse all Newport, Enteritidis, Tennessee, Typhimurium phages with a range from 35 to 190 kb. The Enter-
Saintpaul strains and the majority of tested Typhimurium strains. itidis phages had comparatively larger genomes and had minimal
SEA1 was medium to highly efficient in producing plaques from size differences among each other, SEA1 and SEA2 sizes were
most Newport, Enteritidis, Saintpaul, Tennessee and few Typhimu- approx. 190 and 170 kb, respectively. The Saintpaul-specific phage,
rium strains. Similar to the spot test, SSA1 and SSA2 also lyzed a wide SSA1 genome size was estimated to be 125 kb.
variety of hosts in EOP analysis. SSA1 had medium to highly effi-
ciency (0.2e1) to infect majority of Typhimurium strains, but EOP 3.3.3. Restriction enzyme digestion
values were low (0.001e0.2) or negative for other serovars. SSA2 had Isolated DNAs of selected phages were digested with restriction
the greatest efficiency (EOP ¼ 0.5e1) infecting Typhimurium UK-1, endonuclease EcoRI. The compared digestion patterns were quite
among the phages that had been isolated with that strain. different for the digested phage DNA. EcoRI digestion of SSA1
Only SSA2 phage had the capacity to infect the serovar Mon- produced17 visible bands, 22 fragments are obtained from STE2,
tevideo strain with very high efficiency (EOP ¼ 120.58). STA3, STA9 and phage STD3 DNA were cut into 3 clear fragments. SEA1, SEA2
and STA10 were highly infection ability with EOP values from 0.5 to and STE1 were not digested at all.
1 against most S. Typhimurium strains. These Typhimurium phages
had very low EOP values when tested against other serovars. 4. Discussion
Interestingly, STA3 and STA9 had high efficiency values (EOP ¼ 0.5e
1) while infecting the Heidelberg strain. STE1 was the only phage This study isolated and partially characterized virulent and
isolated with S. Typhimurium strain I536, and also the only phage broad-spectrum Salmonella-specific bacteriophages from natural
with higher EOP values than 0.2 against three additional strains of sources with the ultimate goal of developing a collection of phages
the same serovar. However, it was the only Typhimurium phage to biocontrol Salmonella. It has long been recognized that the
that had medium to low EOP values against Tennessee, Newport presence of bacteriophages is tightly associated with their natural
and Enteritidis strains. The variances of EOP values among the hosts. Because Salmonella is a natural inhabitant of the gastroin-
phages in respect of infecting different Salmonella serovars were testinal tract of animals (Guttman, Raya, & Kutter, 2005) and
statistically different (P  0.05) with ANOVA. abundant in animal feces, various waste effluents typically provide
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M. Akhtar et al. / Food Control 38 (2014) 67e74 71

Table 4
Efficiency of plaque formation (EOP) by selected bacteriophages against Salmonella serovars.

Bacterial strains SEA1 SEA2 SSA1 SSA2 STE1 STA3 STA9 STA10 STD3

S. Typhimurium I598 þþ þþþ þþþ þ  þþþ þþþ þþþ 

S. Typhimurium I527 þþþ þþþ þþþ þ  þþþ þþþ þþþ 
S. Typhimurium I534 þþþ þþþ þþ þþ  þþþ þþþ þþþ 
S. Typhimurium I503   þ þ  þþþ þþþ þþþ þ
S. Typhimurium I535 þþþ þþþ þ þ  þþþ þþþ þþþ 
S. Typhimurium I649   þþ þþ  þþþ þþþ þþþ þ
S. Typhimurium I526 þ þþþ þþþ þ  þþþ þþþ þþþ 
S. Typhimurium E2009005811 þ þþ þ   þþþ þþ þþþ 
S. Typhimurium 14028 þþþ þþþ þ  þþ þþþ þþþ þþþ 
S. Typhimurium I758   þþ þ þþþ þþ þþ þþ þ
S. Typhimurium I536  þþ þþþ þ Host þþþ þþþ þþþ þþþ
S. Typhimurium I740 þþþ þþþ þþ þþ þþþ þþ þþ þþ þ
S. Typhimurium DT104  þþþ    þþþ þþþ þ þ
S. Typhimurium UK-1  þþ þ þþþ  Host Host Host 
S. Typhimurium I600  þþ þþþ þ  þþþ þþþ þþþ Host
S. Saintpaul E2008001236 þþ þþ Host Host     þ
S. Tennessee E200700502 þþþ þþþ þ  þþ    
S. Enteritidis 95657613 þ þþþ þ þþþ þ    
S. Enteritidis 2009595 Host Host þ þ     
S. Enteritidis I823 þþ þþ   þ    
S. Montevideo 95573473    þþþ     
S. Newport AMO7076 þþþ þþþ þ      
S. Newport AMO7073 þþþ þþþ  þ     
S. Newport B4442CDC  þþ  þ þþ    
S. Newport AMO5313 þþþ þþþ þ þ þþ    
S. Agona FDA  þþ  þ     
S. Heidelberg FSIS10 þ   þþ  þþþ þþþ þþ 

þþþ, EOP 0.5 to 1.0; þþ, EOP 0.2 to 0.5; þ, EOP 0.001 to 0.2; , (<0.001) bacterial strain was not susceptible to phage attack.

the best source for phage isolation. In this study, swine manure, to different environment and exploitation of different vectors. In
cattle manure and sewage samples were collected at various time fact, phages present in animal feces would need their bacterial host
points to isolate the lytic bacteriophages against a selected group of as a vector to better survive and disseminate from one GI tract to
Salmonella serovars. Though the same set of Salmonella host strains another. On the other hand, phages in water sewage can reach a
were used for screening all effluent samples (Table 2) for isolation wide variety of bacterial hosts (e.g., different Salmonella serovars).
purposes, sewage samples were found to have the most diverse Lysogenic bacterium plays a role in dissemination of phage genes
phages. Wide host range phages against Typhimurium, Enteritidis, which is also in the evolutionary interest of the bacteriophage
Saintpaul and Newport serovars with clear plaques were predom- (Boyd & Brüssow, 2002; Desiere, Mcshan, Van Sinderen, Ferretti, &
inantly isolated from sewage effluents. Brüssow, 2001). All of our fecal samples were collected from a
In earlier reports, pooled fecal samples have been continuously single swine and dairy barn, which may also limit the presence of
investigated as a rich source for collecting lytic Salmonella phages wide variety of Salmonella serovars in comparison to the samples
(Dhillon, Dhillon, Chau, LI, & Tsang, 1976; O’Flynn et al., 2006). from a community wastewater treatment plant. Studies with E. coli
However, Ibrahim et al. evaluated three different sources, fecal O157:H7 phages also provided similar conclusions; the virulent
samples, intestinal contents of turkey poults, and carrier cultures of phage, SP15 was isolated from activated sludge at wastewater
Salmonella and E. coli to determine the suitability of different treatment plant and showed to infect a diverse range of E. coli
habitats for lytic Salmonella phage isolation. In their study, fecal strains whereas PP17 was found in pig feces lysing only O157:H7
samples were the least diverse source and carrying only S. Typhi- strains (Yoichi et al., 2004).
murium phages. However, carrier cultures were identified to be the Several methods have been adopted previously to characterize
most diverse source with the isolation of S. Typhimurium, S. isolated phages (Mclaughlin, Balaa, Sims, & King, 2006; O’Flynn
Copenhagen and S. Heidelberg phages. The authors also reported et al., 2004). Our study was run through a specific screening
that phages from fecal samples did not show wide host range and scheme effluent samples were first filtered, centrifuged and
were able to infect 20% of the tested strains compared to 57% lysis enriched for phage isolation. To ensure the phage isolation from
activity of carrier culture phages (Ibrahim, 1969). Feces from swine low concentration population from effluent samples, initial filtering
commercial farms were investigated by Callaway et al. and indi- was done to have a rich phage flora as well as removing debris and
cated that phages are ubiquitous in pig manure but the population other background bacterial flora. This method also stated earlier to
of lytic phages against S. Typhimurium might be very low (Callaway isolate lytic and diverse Salmonella phages from wastewater sam-
et al., 2010). On the other hand, studies with wastewater samples ples (Bielke et al., 2007). In comparison, chloroform was also used
were found to be more successful attempts for phage isolation. to clean the background flora by other researchers (Callaway et al.,
Bacteriophages from sewage were able to infect broad host range 2010) prior to enrichment. Studies were also described the isolation
and not highly serovar specific (Carey-Smith, Billington, Cornelius, method of two lytic O157:H7 phages by processing the different
Hudson, & Heinemann, 2006) or genus-restricted (Bielke et al., fecal samples for direct enrichment without any pre filtering step
2007). In our study, manure samples carried mostly temperate (O’Flynn et al., 2004) might be indicative of having less success of
phages with strict host specificity which corresponds with previous isolating phages from a lower concentrated population. Interest-
findings. In contrast, lytic and wide host range phages were pre- ingly, in a previous research of Salmonella phage isolation, E. coli B
dominantly isolated from sewage samples of community waste- host strain was used in parallel to enrich phages followed by
water plant. We speculate that this difference mirror the adaptation isolation with direct spot testing against S. Typhimurium. The
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72 M. Akhtar et al. / Food Control 38 (2014) 67e74

Table 5 exhibited the most extensive lysis over a wide range of Salmonella
Determination of phage genome size by pulsed field gel electrophoresis (PFGE). compared to other screened phages. SSA1 was efficient to infect
Bacteriophage Whole genome size (wkb) several Typhimurium strains other than host Saintpaul. In
SEA1, STA11 190
contrast, SSA2 lysed serovars Enteritidis and Montevideo strains
SEA2, STF1 170 as well as Typhimurium. Spot test results indicated that Typhi-
STA1, STA2, STB3, STB4, STE1 145 murium phages were moderately specific in infecting different
SSA1 125 Salmonella serovars, however, most of them appeared to be
STA4, STA3, STA3, STA4, STB5, STB6, STB7 45
strictly host specific in EOP tests. Interestingly, STA3 and STA9
STD3 35
were able to lyse Heidelberg which was a rare phenomenon for
other isolated phages.
Similar to this study, previously isolated virulent phages, st104a,
authors suggested that E. coli B was used as a very sensitive strain to st104b and Felix 01 were also reported to be infective over a wider
aid the phage isolation from a lower phage concentrated environ- range of serovar hosts (O’Flynn et al., 2006). However, supporting to
ment (Callaway et al., 2010). In our approach, host strains of eight our findings, several research groups also observed the high degree
different Salmonella serovars were used as a mechanism to increase of specificity among Salmonella phages. In one report, 34 Salmonella
the chances of finding lytic phages that can infect a variety of phages were isolated from sewage samples and host range was
serovars. In this study, lytic phages were initially isolated based on determined by spot testing against 53 different serovars. Host range
the capacity of forming clear spots and plaques on host lawns. comparison data revealed a high specificity of most of those phages
While purifying, plaque uniformity was carefully observed because were only effective to lyse the host strains, but only 5 phages had a
uniform plaques are considered (Ibrahim, 1969) to be critical to for wider range and none of them were able to infect all tested strains
obtaining a single phage strain isolation. A large portion of isolated (Heringa, Kim, Jiang, Doyle, & Erickson, 2010). In accordance with
phages was not selected for further characterization due to their our findings, Callaway et al. reported the lytic phages against S.
inability to make clear plaques with host serovars. Typhimurium showed a high degree of specificity (Callaway et al.,
The host range of the phages was determined by spot testing 2010). In contrast, Ibrahim (1969) observed a variation in host
and EOP for a wide variety of Salmonella serovars. Spot testing range of isolated E. coli phages that were capable of lysing Salmo-
was used as the first criteria to select wide host range phages nella as well. One study (Mclaughlin & King, 2008) showed an
which were eventually selected for EOP analysis to determine the interesting phenomenon that phages isolated from swine lagoons
efficiency of infecting capacity relative to host strains (O’Flynn were able to infect the ATCC reference strains but not effective
et al., 2004; Viazis et al., 2011; Viscardi et al., 2008). Based on against the lagoon strains of Salmonella. The authors of the latter
the spot test (Table 3) and EOP results (Table 4), SEA1 and SEA2 study hypothesized that long term co-culture of host and phages

Fig. 1. Transmission electron micrographs of negatively stained bacteriophages that caused lysis to Salmonella serovars.
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M. Akhtar et al. / Food Control 38 (2014) 67e74 73

can be a selection factor for developing a phage-resistant bacterial This study presents an alternate option to biocontrol of the
population. undesirable foodborne pathogen, Salmonella. Any effective imple-
Our results indicated that the majority of the phages were mentation of these phages must have a clear understanding with
specific for the host serovars, somewhat specific to the strain and high quality supporting data and effective trials to meet the regu-
few were able to cross-infect other serovars. The distinct profiles of latory standards. Though the results have demonstrated the lysis
Salmonella susceptibility may be explained by the non-specific efficiency of these phages, most importantly, the ability to infect a
binding receptors on the bacterial host or different resistant wide range of Salmonella hosts; these isolated phages need to be
mechanisms during phage infection (Duckworth, Glenn, & further explored as an effective antimicrobial to control the target
Mccorquodale, 1981). Most dsDNA phage DNAs are known to be pathogens on different matrices. It is fundamental to characterize
cased into an icosahedral shell of protein attached to a tail and understand the microbial ecosystem in molecular level prior to
(Calender & Inman, 2005). The tail-end contains fiber proteins utilize these bacteriophages as biological control approach to
which help them to recognize the receptor molecules on the bac- improve food safety.
terial cell wall and also restrain them to bind onto non-specific
bacterial cells (Kutter & Sulakvelidze, 2005; Singh et al., 2010). Acknowledgment
Also, bacteria could resist the phage infections through superin-
fection exclusion (Sie) systems. Sie systems are predicted to be the We thank Mike Magee and Mike Larose, Rice Lake Wastewater
membrane associated proteins which prevent the phage DNA to Treatment facilities, Rice Lake, WI for their cooperation and help to
enter into bacterial hosts. Though the molecular mechanisms are provide sewage samples for this study. We would like to thank Dr.
not clearly described yet, but the prophages of some Enter- Andrew Brabban and Dr. Elizabeth Kutter of Evergreen State Col-
obacteriaceae species were identified to have the genes that encode lege, WA for all of their suggestions help with developing the
the Sim and SieA proteins. The virulent coliphage, T4 possess two methods. This project was funded by the USDA’s Integrated Organic
different Sie systems which offers resistance against other T-even Program under award No. 2007-51300-03796.
like phages by blocking the phage DNA injection in Gram negative
bacteria. Interestingly, S. enterica subsp. Enterica serovar Typhi-
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