318 © IWA Publishing 2016 Water Science & Technology | 74.

2 | 2016

Total coliphages removal by activated sludge process and

their morphological diversity by transmission electron
microscopy
Sihem Jebri, Fatma Hmaied, Mariem Yahya, Aouatef Ben Ammar
and Moktar Hamdi

ABSTRACT
Sihem Jebri
This study was conducted to isolate phages in treated sewage collected from wastewater treatment
Fatma Hmaied (corresponding author)
plant, and explore their morphological diversity by transmission electron microscopy (TEM). Fates of Mariem Yahya
Unité de Microbiologie et de Biologie Moléculaire,
total bacteriophages and their reduction by biological treatment were also assayed. Phages were CNSTN, Technopôle de Sidi Thabet,
2020 Sidi Thabet,
isolated using the plaque assay then negatively stained and observed by electron microscope. Tunisia
E-mail: hmaiedfatma@yahoo.com
Electron micrographs showed different types of phages with different shapes and sizes. The majority
Aouatef Ben Ammar
of viruses found in treated sewage ranged from 30 to 100 nm in capsid diameter. Many of them were
Faculté de médecine de Tunis,
tailed, belonging to Siphoviridae, Myoviridae and Podoviridae families. Non-tailed phage particles Unité de Microscopie électronique, Bab Saâdoun,
1007 Tunis,
were also found at a low rate, presumably belonging to Leviviridae or Microviridae families. This Tunisia

study shows the diversity and the abundance of bacteriophages in wastewater after biological Moktar Hamdi
Tunis Carthage University,
treatment. Their persistence in wastewater reused in agriculture should raise concerns about their Institut National Sciences Appliquées de Tunis,
Laboratoire Ecologie Technologie Microbienne,
potential role in controlling bacterial populations in the environment. They should be also included in
BP 676,
water treatment quality controlling guidelines as fecal and viral indicators. 1080 Tunis,
Tunisia
Key words | bacteriophages, diversity, removal, transmission electron microscopy, treated sewage

INTRODUCTION

Bacteriophages can be found in all environments populated
by bacterial hosts, such as soil, water and animal guts. These
viruses that infect prokaryotes were first described in the
early 1900s (Twort ; d’Herelle ). Studies of phage
model systems revolutionized biology and established the
field of molecular biology (Cairns et al. ). Estimated to
be the most widely distributed and diverse entities in the bio-
sphere (Mc Grath & van Sinderen ), bacterial viruses
are extremely common in the environment. There are 1010
phage particles per liter of surface seawater (Bergh et al.
). Phages are also major conduits of genetic exchange,
transducing an estimated 1025 to 1028 bp of bacterial DNA
per year in the world’s oceans ( Jiang & Paul ; Paul
). Despite their great numbers and ubiquitous presence
(1031 in the biosphere), very little information is known
about phage biodiversity, biogeography, or phylogeny. Infor-
mation is limited, in part, because the current International
Committee on the Taxonomy of Viruses (ICTV) taxonomi-
cal system is based on culturing phages and measuring
physical parameters of the free virion. Many difficulties
are encountered for this purpose: the classification is
based on host genus-specificity of phages; however, poly-
valent phages exist especially in tectiviruses or
archeabacteria phages. In addition to phages of established
families, a number of archaeal phages have recently been
described; they still await classification by the ICTV (Acker-
mann ). The ICTV taxonomic system requires
visualization of the phage particles by electron microscopy
to determine capsid morphology. In fact, the taxonomic
system is regularly approved and updated (Murphy et al.
). Historically, phages have been characterized by
their host range and the physical characteristics of the free
virion, including capsid size, shape, resistance to organic
solvents, and structure, as well as genome size and type.
Their unique characteristics bring several advantages to
their use as pathogen surrogates. Hence, they have been suc-
cessfully used in a variety of environmental applications: as
fecal indicators, as process indicators, in microbial source

doi: 10.2166/wst.2016.178
 319 S. Jebri et al. | Phages in sewage: removal and morphological diversity
tracking, as comprehensive pathogenic virus indices and as
viral models and tracers (Stetler ; Payment et al. ;
Havelaar ; Durán et al. ; Davies-Colley et al. ;
Persson et al. ; Abbaszadegan et al. ). Bacterio-
phages infecting enteric bacteria seem good candidates for
enteric virus indicators sharing morphological similarities.
Their structure consists of a nucleic acid molecule sur-
rounded by capsid. Many phages also contain additional
structures such as tails and spikes. These features imply
that in terms of composition, structure, morphology and
capsid size, phages share many properties with human
viruses ( Jofre ). Somatic coliphages (Kott et al. ;
IAWPRC Study Group ), F-specific RNA bacteriophages
(Havelaar et al. ; IAWPRC Study Group ) and bac-
teriophages infecting Bacteroides fragilis (Tartera & Jofre
; IAWPRC Study Group ) have so far been advo-
cated as potential model microorganisms for various
aspects of water quality control, especially in reclaimed
water and treated sewage issued from wastewater treatment
plants. The aim of this study was to isolate total coliphages
from treated sewage collected from an urban wastewater
treatment plant using the plaque assay and to explore
their morphological diversity by transmission electron
microscopy (TEM) after negative staining.
METHODS
Enumeration of coliphages
Five samples of raw sewage and five samples of sewage trea-
ted by activated sludge process were collected from an
urban wastewater treatment plant in Northern Tunisia.
Total coliphages were enumerated on host strain CB390
(Guzmán et al. ) with the media recommended in
ISO10705-1 standard (TYGB, TYGA and ssTYGA) (ISO
). Reference phages ΦX174 and MS2 were used as posi-
tive controls (for somatic and F-specific phages,
respectively). Results are expressed in plaque-forming units
(PFU)/100 mL.
Phage isolation and detection
100 mL of the treated sewage samples after secondary treat-
ment were centrifuged for 20 min at 3,000 × g, then
decontaminated by filtration through 0.22 μm nitrocellulose
filters. 1 mL of an overnight Escherichia coli CB390 culture
was inoculated with 1 mL of the filtrate in 20 mL of TYGB
W
medium and incubated overnight at 37 C. This step is
Water Science & Technology | 74.2 | 2016
repeated at least three times for phage propagation. After
incubation, the culture is centrifuged and filtrated again.
50 μL of filtrates were spotted in TYGA plates. Clear plaques
W
appeared after 24 h of incubation at 37 C.
Phage propagation and purification
Thirty clear plaques were picked up randomly and placed in
W
5 mL of TYGB medium. After incubation for 24 h at 4 C,
0.5 mL of an overnight E. coli CB390 culture was added
and incubated for 8 h until lysis of bacterial cells. Phage par-
ticles were concentrated by polyethylene glycol. 15% of
glycerol was added, and the isolated phages were kept at
W
80 C.
Electron microscopy
TEM was performed to investigate isolated phage mor-
phology. 20 μL of the isolated phage solution was
deposited on a carbon copper coated grid; after adsorption,
a drop of phosphotungstate 2% (w/v) was added for negative
staining as described by Ackermann & Heldal (). Phage
morphology was examined with a TEM-1010 transmission
electron microscope (JEOL, Japan) operated at 80 kV and
at a magnification of 80,000 and 120,000.
RESULTS AND DISCUSSION
Currently, somatic and F-specific coliphages are considered
to be one of the best indicators of the presence of enteric
viruses, and the diversity and concentrations obtained in
this study continue to support their use. In fact, all samples
contained high amounts of total coliphages (somatic and
F-specific phages). Treated samples are considered ‘clean’
according to the existing Tunisian guidelines regarding
fecal coliforms in effluents issued from wastewater treat-
ment plants (Norme Tunisienne NT. . ).
However, fecal coliform numbers do not serve as adequate
indicators of enteric virus contamination. The water treat-
ment process is not efficient regarding the removal of
somatic and F-specific coliphages used as indicators of the
presence and behavior of animal viruses in terms of trans-
port and survival characteristics. The concentration of
total coliphages decreases by 1 Log after treatment. The
number of total coliphages in raw sewage ranged from
4.95 to 6.36 log10 PFU/100 mL. Their concentration aver-
aged between 3.57 log10 PFU/100 mL and 5.04 log10
PFU/100 mL in treated sewage (Figure 1).
 320 S. Jebri et al. | Phages in sewage: removal and morphological diversity
Figure 1 | Box plot of Log10 PFU/100 mL of total coliphages in raw sewage (influent) and
treated sewage (effluent).
These results are in compliance with data published
elsewhere (Contreras-Coll et al. ; Mandilara et al.
; Blanch et al. ; Gentry-Shields et al. ). High
concentration of total coliphages in treated sewage raises
concerns about the efficacy of biological treatment
Figure 2 | Electron micrographs showing tailed phage particles belonging to the Syphoviridae family.
Water Science & Technology | 74.2 | 2016
(activated sludge) in the elimination of viruses from
sewage and the reuse of treated sewage in agriculture. In
fact, somatic coliphages and F-specific phages were advo-
cated as viral indicators since they share the same
morphological characteristics with enteric viruses. The
removal of bacteriophages resembles the removal of viruses
(Jebri et al. ). As a result, they have become invaluable
tools in environmental research and are often successfully
used in a variety of applications.
A great variety of human origin viruses may be found in
sewage from domestic sources; some are present in very
large numbers. In our study, electron microscopy of purified
phages revealed different types of viral particles: the largest
community of phages was tailed, belonging to the Syphoviri-
dae family. Their shape is characteristic, with an icosahedral
head and a long, flexible non-contractile tail. Head diameter
was ranging from 30 to 100 nm. These phages are estimated
to represent 53% of randomly tested plaques (Figure 2).
Figure 3 shows phages belonging to the Myoviridae
family with a contractile tail ending with fibers and an icosa-
hedral head (70 to 90 nm diameter). These phages represent
27% of tested phages.
 321 S. Jebri et al. | Phages in sewage: removal and morphological diversity Water Science & Technology | 74.2 | 2016

Figure 3 | Electron micrographs showing tailed phage particles belonging to the Myoviridae family; white arrow indicates clear fibers at the end of the tail.

Figure 4 | Electron micrographs showing phage particles belonging to the Podoviridae family.
 322 S. Jebri et al. | Phages in sewage: removal and morphological diversity
Thirteen percent of tested plaques revealed short-tailed
phages with icosahedral head with a diameter ranging
between 60 and 70 nm (Figure 4).
This minor entity was assigned to the Podoviridae
family. Only 7% of tested plaque contained phage particles
without tails, presumably belonging to the Leviviridae or
Microviridae family (Figure 5).
The prevalence of each type of phages investigated by
TEM is not unusual, since comparable abundance has
been found in literature (Ackermann , ). Due to
their diversity and abundance, phages belonging to the
three families may play an important role in wastewater’s
ecological system. Coliphages are excreted at all times by a
certain percentage of all humans and other warm-blooded
animals (Grabow et al. ); this fact explains the high
titers found in influents. Their resistance to biological treat-
ment by activated sludge explains their persistence in
effluents. Furthermore, the population of bacteriophages is
highly dynamic; there are about 1,023 phage infections per
second worldwide (Hatfull & Hendrix ). In fact,
Figure 5 | Electron micrographs showing non-tailed phage particles.
Water Science & Technology | 74.2 | 2016
bacteriophages in wastewater are now being recognized as
important vectors for genomic reshuffling and are having a
significant impact on the understanding of genetic mechan-
isms for phage evolution, genetic diversity, and the genetic
structure of phage populations in the environment. A
better characterization of the diversity of bacteriophages
used as viral and fecal indicators of enteric viruses’ presence
and survival in treated sewage will help to contribute to the
management and evaluation of microbiological risk for agri-
cultural use and consumption of contaminated food or
water.
CONCLUSIONS
This study showed that total coliphages persist after a bio-
logical secondary treatment with activated sludge in
wastewater treatment plant residues. Three families of
tailed phages were identified according to their morphologi-
cal characteristics after TEM. The abundance of these
phages after biological treatment should raise concerns
about enteric viruses’ survival of the treatment process.
The diversity of these phages implies that they may play
an important role in the environmental ecological system
due to their ability to kill specific bacteria.
ACKNOWLEDGEMENTS
This work was supported by the National Center of Science
and Nuclear Technology.
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