ORIGINAL RESEARCH ARTICLE
published: 06 June 2013
doi: 10.3389/fcimb.2013.00020
CELLULAR AND INFECTION MICROBIOLOGY
Phage biocontrol of enteropathogenic and shiga
toxin-producing Escherichia coli in meat products
David Tomat 1*, Leonel Migliore 1 , Virginia Aquili 1 , Andrea Quiberoni 2 and Claudia Balagué 1
1
2
Área de Bacteriología, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, Argentina
Facultad de Ingeniería Química, Instituto de Lactología Industrial (UNL - CONICET), Santa Fe, Argentina
Edited by:
Nora L. Padola, Universidad Nacional
del Centro de la Provincia de
Buenos Aires, Argentina
Reviewed by:
Mohamed H. Abdulla, Cochin
University of Science and
Technology, India
Adriana Bentancor, Universidad de
Buenos Aires, Argentina
*Correspondence:
David Tomat, Área de Bacteriología,
Facultad de Ciencias Bioquímicas y
Farmacéuticas, Universidad
Nacional de Rosario, Suipacha 531,
S2002LRK Rosario, Santa Fe,
Argentina
e-mail: dtomat@fbioyf.unr.edu.ar
Ten bacteriophages were isolated from faeces and their lytic effects assayed on 103
pathogenic and non-pathogenic Enterobacteriaceae. Two phages (DT1 and DT6) were
selected based on their host ranges, and their lytic effects on pathogenic E. coli strains
inoculated on pieces of beef were determined. We evaluated the reductions of viable
cells of Escherichia coli O157:H7 and non-O157 Shiga toxigenic E. coli strains on meat
after exposure to DT6 at 5 and 24◦ C for 3, 6, and 24 h and the effect of both phages
against an enteropathogenic E. coli strain. Significant viable cell reductions, compared to
controls without phages, at both temperatures were observed, with the greatest decrease
taking place within the first hours of the assays. Reductions were also influenced by
phage concentration, being the highest concentrations, 1.7 × 1010 plaque forming units
per milliliter (PFU/mL) for DT1 and 1.4 × 1010 PFU/mL for DT6, the most effective. When
enteropathogenic E. coli and Shiga toxigenic E. coli (O157:H7) strains were tested,
we obtained viable cell reductions of 0.67 log (p = 0.01) and 0.77 log (p = 0.01) after
3 h incubation and 0.80 log (p = 0.01) and 1.15 log (p = 0.001) after 6 h. In contrast,
all nonpathogenic E. coli strains as well as other enterobacteria tested were resistant.
In addition, phage cocktail was evaluated on two strains and further reductions were
observed. However, E. coli bacteriophage insensitive mutants (BIMs) emerged in meat
assays. BIMs isolated from meat along with those isolated by using the secondary
culture method were tested to evaluate resistance phenotype stability and reversion. They
presented low emergence frequencies (6.5 × 10−7 –1.8 × 10−6 ) and variable stability and
reversion. Results indicate that isolated phages were stable on storage, negative for all the
virulence factors assayed, presented lytic activity for different E. coli virotypes and could
be useful in reducing Shiga toxigenic E. coli and enteropathogenic E. coli viable cells in
meat products.
Keywords: Escherichia coli, bacteriophage, phage biocontrol, bacteriophage insensitive mutant, phage cocktail
INTRODUCTION
Shiga toxin-producing Escherichia coli (STEC) are human
pathogens that can cause diarrhea, as well as severe clinical manifestations including hemorrhagic enterocolitis, hemolytic uremic
syndrome (HUS), and thrombotic thrombocytopenic purpura
(Su and Brandt, 1995; Griffin et al., 2002; Yoon and Hovde,
2008). STEC produce several virulence factors which contribute
to their pathogenicity. Shiga toxins (Stx), AB type toxins that
inhibit protein synthesis in target cells, are the most characterized
virulence factors (Thorpe et al., 2002). Shiga toxins produced in
the intestines by STEC are able to enter the systemic circulation
causing severe damage to distal organs. The degree of damage
is related to the amount of toxin produced during the infection
(Ritchie et al., 2003). STEC synthesize two main types of Shiga
toxins encoded by stx1 and stx2 genes. Moreover, the enterocyte
attaching-and-effacing lesion gene (eaeA), which is also present in
enteropathogenic strains (EPEC), can contribute to the virulence
of STEC. The gene codes for the intimin protein, which allows
bacteria to attach themselves to the intestinal epithelium (Frankel
et al., 1998).
Frontiers in Cellular and Infection Microbiology
Foodborne disease-producing Enterobacteriaceae, such as
Shigella spp., Salmonella spp., EPEC and STEC, are important etiologic agents of infantile gastroenteritis in Argentina (Binsztein
et al., 1999; Rivas et al., 2008). In developing countries, EPEC are
the cause of outbreaks of infantile diarrhea with high mortality in
children under two years of age. In Argentina, HUS is endemic,
with approximately 400 new cases being reported annually by
National Health Surveillance System (Rivas et al., 2006), and more
than 7000 cases being reported since 1965 (NCASP, 1995). In
2005, the annual incidence of HUS is 13.9 cases/100,000 children
under five years of age (Rivas et al., 2006). Recent epidemiological
studies showed that there is a sustained global increase in the isolation of non-O157 STEC strains from humans (Tozzi et al., 2003;
Brooks et al., 2005; Bettelheim, 2007) and animals (Jenkins et al.,
2003; Fernandez et al., 2009), particularly STEC of serogroups
O26, O103, and O111 (Ogura et al., 2007).
The therapeutic potential of bacteriophages has been explored
since they were discovered by Felix d’Herelle (Summers, 1999).
Some of the attributes that make bacteriophages interesting as
tools for biological control are: (i) their ability to infect and lyse
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Tomat et al.
Phage biocontrol of Escherichia coli
specific bacterial target cells and their inability to infect eukaryotic cells; (ii) phages generally do not cross bacterial species or
genus barriers, and therefore do not affect desirable microorganisms commonly present in foods, the gastrointestinal tract or the
normal bacterial microbiota (Carlton et al., 2005); (iii) phages
need a bacterial host in which to multiply and therefore will persist only as long as the sensitive host is present (Clark and March,
2006). The potential of bacteriophages to control food pathogens
is reflected in recent studies involving various pathogens including Campylobacter jejuni (Atterbury et al., 2003; Bigwood et al.,
2008), E. coli O157:H7 (O’Flynn et al., 2004; Abuladze et al., 2008)
and Listeria monocytogenes (Leverentz et al., 2003; Guenther et al.,
2009; Holck and Berg, 2009). Several strategies are currently being
applied to preserve perishable refrigerated foods and extend their
shelf-life. However, physical processes and chemical compounds
(preservatives) used for this purpose may alter meat organoleptic
properties. Although bacteriophages represent a novel approach,
there are no reports of their industrial use to improve safety, even
if this “new, ecological, and specific” technology may be cheaper
than “older” technologies, since phages can be isolated from the
environment and are self-replicating entities. On the other hand,
their inclusion into a meat product can be seen as a less aggressive
approach.
The aim of this work was to isolate phages with specific lytic
capacity for E. coli strains in order to determine phage host range
and analyze their potential as biocontrol agents for STEC and
EPEC strains in beef products.
MATERIALS AND METHODS
BACTERIOPHAGE ISOLATION AND PREPARATION OF STOCKS
E. coli DH5α was used to isolate bacteriophages from fifty
stool samples of patients with diarrhea treated at the Centenary
Hospital, Rosario. This strain was grown up to an optical
absorbance of 1 (A600 = 1) in 10 mL of Hershey broth (8 g/L
Bacto nutrient broth, 5 g/L Bacto peptone, 5 g/L NaCl, and 1 g/L
glucose) (Difco, Detroit, MI, USA) supplemented with MgSO4
(5 mM) (Cicarelli, San Lorenzo, Santa Fe, Argentina). A portion of faeces (5 g) was added and the culture was incubated
for a further 12 h at 37◦ C. Next, chloroform (0.5 mL, Cicarelli)
was added and the preparation was mixed and centrifuged at
15,000× g for 10 min. The supernatant was then filtered through
a 0.45 μm pore size (Gamafil S.A., Buenos Aires, Argentina)
(Kudva et al., 1999). Bacteriophage isolation and purification
were performed by the double-layer plaque technique (Balagué
et al., 2006). Briefly, aliquots of filtrates (10 and 100 μL) were
mixed with 100 μL of recipient strain culture (A600 = 1), three
mL of molten soft agar at 45◦ C (Hershey broth supplemented
with 5 mM MgSO4 and 0.7% agar) were added to each suspension and the mixture was poured onto pre-solidified Hershey agar
plates and incubated overnight at 37◦ C. To isolate and purify
phages, well-defined single plaques on the soft agar were picked
and placed in 5 mL of Hershey medium supplemented with 5 mM
MgSO4 . Tubes were kept at 4◦ C for 2 h and then inoculated with
100 μL of recipient strain culture (A600 = 1). Inoculated tubes
were incubated at 37◦ C with intermittent shaking until complete lysis. Next, chloroform (0.1 mL) was added and cultures
were centrifuged at 4000× g for 10 min. Phage stocks were stored
Frontiers in Cellular and Infection Microbiology
at 4◦ C and enumerated by the double-layer plaque technique
(Jamalludeen et al., 2007). These steps were repeated three times.
Stability of phage stocks was evaluated after two months of storage
at 4◦ C.
BACTERIOPHAGE AND BACTERIA CHARACTERIZATION
Phage electron micrographs were obtained by the procedure of
Bolondi et al. (1995). Phage suspensions were concentrated by
centrifugation (1 h, 70,000 × g, 5◦ C) and subsequently stained
with phosphotungstic acid (2% w/v) (Biopack, Buenos Aires,
Argentina). Electron micrographs were obtained using a JEOL
1200 EX II electron microscope (INTA Castelar, Buenos Aires,
Argentina) operating at 85 kV. Phage morphologies and dimensions (head diameter, tail length, and diameter) were recorded.
Phages and strains of E. coli were tested for the presence of
toxin-encoding genes (stx1, Shiga toxin 1; stx2, Shiga toxin 2;
eaeA, attaching-and effacing; LT1, thermolabile toxin and ST1,
thermostable toxin) of diarrheogenic E. coli by the polymerase
chain reaction (PCR) using primers detailed in Table 1 (Pass et al.,
2000). PCR conditions were as follows: initial denaturing step at
95◦ C for 2 min, followed by 25 cycles of 95◦ C for 30 s, annealing at 63◦ C for 30 s and elongation at 72◦ C for 30 s, followed by
a final step at 72◦ C for 5 min to achieve complete product elongation. E. coli ATCC43889 (stx2 and eaeA), ATCC43890 (stx1),
and ATCC43895 (stx1, stx2, and eaeA, and also harboring the
stx2 phage, 933W) were used as positive controls, while enterotoxigenic E. coli ATCC35401 was used for LT1 and ST1 genes.
E. coli HB101 and ATCC98222 were utilized as negative controls.
Amplified products were resolved by electrophoresis using 3%
agarose gels in TBE buffer (89 mM Tris borate, 2 mM EDTA, pH
8.0) (Promega, Madison, WI, USA) at 100 V for 3 h. Gels were
stained with ethidium bromide (0.5 μg/mL) (Sigma, St. Louis,
MO, USA) and PCR products were visualized under UV light.
BACTERIOPHAGE SPECIFICITY
The host range of each phage was determined by the double layer agar technique using 44 strains isolated from stool
Table 1 | Sequences of primers used in this study.
Gene
Primera
Product size
(bp) expected
stx1
fp: 5 -ACGTTACAGCGTGTTGCRGGGATC-3
121
bp: 5 -TTGCCACAGACTGCGTCAGTRAGG-3
fp: 5 -TGTGGCTGGGTTCGTTTATACGGC-3
stx2
102
bp: 5 -TCCGTTGTCATGGAAACCGTTGTC-3
eaeA
fp: 5 -TGAGCGGCTGGCATGATGCATAC-3
241
bp: 5 -TCGATCCCCATCGTCACCAGAGG-3
fp: 5 -TGGATTCATCATGCACCACAAGG-3
LT1
360
bp: 5 -CCATTTCTCTTTTGCCTGCCATC-3
fp: 5 -TTTCCCCTCTTTTAGTCAGTCAACTG-3
ST1
160
bp: 5 -GGCAGGACTACAACAAAGTTCACAG-3
a fp,
forward primer; bp, backward primer. stxl and stx2: Shiga toxin1 and 2
encoding genes; eaeA: intimin encoding gene; LTl and STl: thermolabile and
thermostable toxins encoding genes.
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June 2013 | Volume 3 | Article 20 | 2
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Phage biocontrol of Escherichia coli
samples, and urine cultures (uropathogenic E. coli, UPEC).
Stool and urine samples were streaked in Cystine Lactose
Electrolyte Deficient (CLED) agar plates. Simmons citrate agar
test was performed on growing lactose positive colonies. After
incubation for 24 h at 35◦ C, only lactose positive and citrate negative colonies were further identified using API system
(Biomerieux, Bs. As., Argentina). Sixteen E. coli strain from
food (Balagué et al., 2006), one uropathogenic E. coli strain
(E. coli T149) which expresses fimbriae P and α-hemolysin
(Balagué et al., 2004) and five ATCC E. coli strains were
also tested (ATCC 43890; 43889; 43895; 35401 and 98222).
Previously characterized (API system) isolates from stool samples were also tested: Shigella flexneri, S. sonnei, Proteus mirabilis,
Citrobacter freundii, Klebsiella pneumoniae, Salmonella enteritidis,
Salmonella Typhi and Salmonella Typhimurium. Strains tested
against stock phages are listed in Table 2. Bacteriophage sensitivity was assayed by placing 10 μL of phage suspension on
the solidified soft-agar layer inoculated with 100 μL of each
bacterial culture, incubated for 24 h at 37◦ C, and the presence of lysis zones or plaques was examined (Goodridge et al.,
2003).
MEAT ASSAYS
Beef from cow hindquarter purchased from retail was aseptically cut into pieces (1 cm2 of surface and 0.4 cm thick), placed
in petri dishes and pre-equilibrated to 5 or 24◦ C. The required
pH was obtained by washing with sodium chloride-magnesium
sulfate (SM) buffer (0.05 M TRIS, 0.1 M NaCl, 0.008 M MgSO4 ,
0.01% w/v gelatin, pH = 7.5) prior to inoculation with bacteria
and phage. Host strains employed in this study, namely nonO157 STEC (ARG4827; serogroup O18; harboring stx1 and stx2
genes) (Balagué et al., 2006), O157:H7 STEC (464; harboring
stx1 and eaeA genes) and an EPEC (EPEC920; which harbors
Table 2 | Strains tested against stock phages.
Source
Strains (amount)
Strains characteristics/
description
Food
Escherichia coli (10)
8 non-O157 STEC and
2 O157:H7 STEC
Stool sample
Escherichia coli (9)
4 O157:H7 STEC and
5 EPEC
Non-pathogenic
Escherichia coli (18)
Shigella spp.
Salmonella spp.
Proteus mirabilis
Other enterobacteria
Citrobacter freundii
Klebsiella pneumoniae (17)
Urine culture
Escherichia coli (17)
UPEC
ATCC
Escherichia coli (5)
35401; 43889; 43890;
43895 and 98222
eaeA gene), were grown in Hershey medium supplemented with
MgSO4 (5 mM) for 12 h at 37◦ C. Bacterial strains and specific
bacteriophages added to the meat samples are detailed in Table 3.
Twenty μL of each diluted bacterial suspension (ranging from
5.9 × 105 to 3.9 × 107 CFU/mL) were pipetted onto the surface
of each meat piece and allowed to attach for 10 min at room
temperature. Another 20 μL of each bacteriophage were then
pipetted on the meat, at low multiplicity of infection (MOI),
1.7 × 109 PFU/mL for DT1 and 1.4 × 109 PFU/mL for DT6, or
high MOI, 1.7 × 1010 PFU/mL for DT1 and 1.4 × 1010 PFU/mL
for DT6. Pieces of meat were also added with SM buffer (pH
7.5), instead of phage suspension, as controls. At 3, 6, and 24 h,
meat pieces were transferred to a sterile bag, 5 mL SM buffer were
added and samples processed for 2 min in a Stomacher (Seward,
London, UK). A 1 mL portion of the stomacher fluid was transferred to a sterile tube and cells were pelleted by centrifugation
at 3000× g for 10 min. The supernatant was removed and cells
were resuspended in 1 mL SM buffer. Finally, a 0.1 mL sample
was removed, serially diluted (102 –104 -fold) in SM buffer and
0.1 mL volumes of each dilution were plated on Hershey agar
for viable cell enumeration (Bigwood et al., 2008). Phage cocktail (DT1 and DT6 in equal proportions) was assayed on E. coli
DH5α (indicator strain used for phage isolation) and in O157:H7
STEC (464) using the methodology employed for each individual phage described above. Three replicates were performed
for each treatment and one meat piece processed for replicate.
Uninoculated controls were tested to determine the presence of
naturally occurring bacteriophages. Plaques (PFU/mL) were evaluated by the double layer agar technique (Jamalludeen et al.,
2007).
BACTERIOPHAGE INSENSITIVE MUTANTS (BIMs) ISOLATION
Bacteriophage insensitive mutants (BIMs) were isolated by the
secondary culture method described by Guglielmotti et al. (2007)
with some modifications. E. coli sensitive strains (one EPEC, three
O157:H7 STEC and one non-O157 STEC) (A600 = 0.2 − 0.3)
were infected with a phage suspension at different infection ratios
(multiplicity of infection, MOI of ≈ 10 and 1), incubated in
Hershey broth at 37◦ C for 24 h and observed visually until complete lysis. An uninfected culture of each E. coli strain was used as
a control. Cultures exhibiting complete and delayed lysis were the
best candidates to isolate BIMs. After lysis, further incubation for
48 h at 37◦ C was required for secondary growth. Each tube with
secondary growth was spread on Hershey agar plates for colony
isolation.
BIMs were isolated from meat as described in meat assays
methodology described above modified with an extended incubation time (48 h) at 37◦ C. For both of the aforementioned
methodologies, after incubation of agar plates, eight different
colonies were randomly isolated (on agar plates) and cultured
overnight in Hershey broth at 37◦ C. These isolates were purified by three consecutive streakings on Hershey agar plates. The
growing colonies were isolated as presumptive BIMs.
EPEC, enteropathogenic E. coli; O157:H7 STEC, O157:H7 Shigatoxigenic E. coli;
BIMs CONFIRMATION
non-O157 STEC, Shigatoxigenic non-O157 E. coli; UPEC, urophatogenic E. coli;
Presumptive BIMs were confirmed by a liquid culture sensitivity test (Guglielmotti et al., 2007). Briefly, a log-phase culture
ATCC, american type culture collection.
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Table 3 | E. coli viable cell logarithmic reductions after phage treatment of contaminated meat products.
Phage stock/sensitive strain
DT1/EPEC (920)
DT6/EPEC (920)
DT6/non-O157 STEC (ARG4827)
DT6/O157:H7 STEC (464)
Cocktail/DH5α
Cocktail/O157:H7 STEC (464)
Log reduction in E. coli viable cellsa
after the incubation time (h)b
Assay conditions
T (◦ C)
MOI
3
6
24
5
4.4 × 102
NS
**0.80 ± 0.14
NS
24
4.8 × 102
**0.30 ± 0.05
NS
NS
5
4.4 × 101
NS
**0.49 ± 0.09
NS
24
4.8 × 101
NS
NS
**0.46 ± 0.08
5
5.2 × 102
**0.67 ± 0.12
**0.59 ± 0.11
*0.46 ± 0.15
24
6.5 × 103
*0.32 ± 0.09
NS
NS
5
5.2 × 101
NS
*0.30 ± 0.08
NS
24
6.5 × 102
NS
NS
NS
5
2.4 × 104
*0.33 ± 0.09
*0.47 ± 0.12
*0.56 ± 0.17
24
4.0 × 102
*0.43 ± 0.13
NS
NS
5
2.4 × 103
NS
*0.37 ± 0.09
*0.50 ± 0.16
24
4.0 × 101
*0.35 ± 0.11
NS
NS
5
2.3 × 103
*0.59 ± 0.16
**0.86 ± 0.15
*0.38 ± 0.10
24
5.8 × 103
**0.77 ± 0.14
***1.15 ± 0.12
NS
5
2.3 × 102
*0.38 ± 0.09
*0.62 ± 0.18
NS
24
5.8 × 102
NS
**0.74 ± 0.13
NS
5
2.25 × 104
*0.91 ± 0.19
**2.16 ± 0.20
**2.23 ± 0.21
24
1.75 × 104
*0.66 ± 0.15
NS
NS
5
1.56 × 105
NS
NS
NS
24
3.33 × 105
**1.43 ± 0.24
**2.58 ± 0.21
**2.20 ± 0.22
MOI, multiplicity of infection (PFU/CFU); NS, not significant. Mean values of treated and control samples not significantly different using the scheffé method
(*significant at p = 0.05; **significant at p = 0.01; ***significant at p = 0.001).
a Log
reduction in E. coli viable cells with respect to phage-free control.
b Mean
of three data points ± standard deviations.
(A600 = 0.2 − 0.3) of each presumptive BIM in Hershey broth
was infected with the phage suspension at various MOI (≈ 10
and 1). Uninfected cultures of each E. coli strain were used
as controls. BIMs cultures were incubated in Hershey broth
at 37◦ C until growth of control strains was evident. Infected
cultures that did not lyse at the first attempt were subcultured again. Each second subculture was prepared by transferring 2–3% of the final volume from the first culture to
another test tube with 1 mL of fresh broth. When no bacterial lysis was evident, the resulting culture was stored at 4◦ C
and subcultured under the same conditions. Presumptive BIMs
that survived the third subculture were considered to be confirmed BIMs. Sensitivity of each parent strain (sensitive) was
always determined in parallel to ensure lytic activity of phage
suspensions.
mixture was supplemented with MgSO4 (5 mM), plated by the
double-layer agar technique and incubated overnight at 37◦ C.
BIM frequency was estimated as the ratio of the number of confirmed BIM to the initial bacterial number. All the experiments
were performed in duplicate. Selected BIMs were propagated
through 50 generations at 37◦ C and then checked by a plaque
assay to evaluate reversion to phage sensitivity (O’Flynn et al.,
2004).
Phage resistance stability was assayed by seven sequential subcultures of 2% portions of BIM cultures (Hershey broth) with
independent addition of phage at each subculture (Guglielmotti
et al., 2007). The loss of phage resistance was determined by
comparing lysis of BIM culture with the control (mutant subculture without phage addition). The subculture where lysis first
occurred was recorded.
DETERMINATION OF BACTERIOPHAGE-INSENSITIVE MUTANT
FREQUENCY, REVERSION, AND STABILITY
STATISTICAL ANALYSIS
The emergence frequency of BIMs was evaluated by mixing
the appropriate volume of an overnight culture of each strain
(EPEC920 and O157:H7 STEC 464) and phage suspension (DT1
and DT6) to obtain a MOI of 100. The bacterium–phage
Frontiers in Cellular and Infection Microbiology
Means and standard deviations for data sets were calculated.
Differences between means for control (untreated) and treated
samples were compared by the Scheffé method and Origin 6.0
for graphics. Differences were considered statistically significant
when p-values were
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