Original article [618584]

Original article
www.germs.ro • GERMS 4 (4) • December 2014 • page 92
Center, Tbilisi, Republic of Georgia ; 6MD, PhD Student: [anonimizat], Carol Davila University
of Medicine and Ph armacy ; National Institute for Infectious
Diseases “Prof. Dr. Matei Bal ș”; 7PhD, Microbiology
Department, Faculty of Biology, University of Bucharest; Ștefan S. Nicolau Institute of Virology , Romanian
Academy, Bucharest, Romania ;
8MD, PhD, Professor,
Department of Microbiolog y II, Carol Davila University of
Medicine and Pharm acy, Bucharest, Romania ; 9PhD,
Professor, Microbiology Department, Faculty of Biology, University of Bucharest; Research Institute of the University of Bucharest, Romania ;
10MD, PhD, Professor,
Department of Infectious Diseases, Carol Davila Univ ersity
of Medicine and Pharmacy ; National Institute for Infectious
Diseases “Prof. Dr. Matei Bal ș”, Bucharest, Romania.
Experimental approach for bacteriophage susceptibility testing of
planktonic and sessile bacterial populations – Study protocol
Alina Cristina Negu ț1,*, Oana S ăndulescu2, Marcela Popa3, Anca Streinu -Cercel4, Zemphira Alavidze5, Ioana Berciu6,
Coralia Bleot u7, Mircea Ioan Popa8, Mariana Carmen Chifiriuc9, Adrian Streinu -Cercel10

Abstract
Introduction Antimicrobial resistance is a growing threat for all clinical branches. This phenomenon
poses important challenges in controlling infectious diseases. However, m ultidrug resistance is not the
only issue, as bacteria that are otherwise susceptible to common antibiotics express other patterns for
evading antibiotherapy , for example they can aggregate within a self -produced matrix to form biofilm.
Methods We inte nd to perform a prospective laboratory study of the germs isolated from different
samples collected from patients admitted with infectious pathology in reference hospitals in Romania.
We will perform antibiotic resistance testing as well as phage testing, both on solid and liquid growth
medium, for Staphylococcus spp., Enterococcus spp., and Pseudomonas spp. We intend to collect data
for 150 patients with different infections with these identified pathogens .
Phage susceptibility testing will be performed us ing 5 types of strain -specific bacteriophage mixtures:
PYO, INTESTI, STAPHYLOCOCCAL (Eliava BioPreparations, Tbilisi, Georgia), PHAGYO,
PHAGESTI (JSC "Biochimpharm", Tbilisi, Georgia). For phage -susceptible strains, w e will evaluate
biofilm form ation in th e presence of phages, as well as phage effect on already formed biofilm.
Expected results Through this study, we intend to provide the first set of results on bacteriophage-
susceptibility of bacteria isolated from patients with hard to treat infections, fr om reference hospitals in
Romania. By evaluating a large number of bacterial strains we aim to predict and project biofilm
kinetics, while adding binary phage dilutions at key timepoints during biofilm formation .
Keywords Bacteriophages, planktonic bacteri a, biofilm, lysis, spectrophotometry
Acknowledgments POSDRU/159/1.5/S/141531; Carol Davila University of Medicine and
Pharmacy, Young Researchers Grant no. 28341/2013.

Introduction 1
In 2011, the World Health Organization
(WHO) chose antimicrobial res istance as the
central theme for the World Health Day to acknowledge this growing threat for all clinical

Received: 06 November 2014; revised 27 November 2014;
accepted: 28 November 2014
1MD, PhD Student, Carol Davila Univ ersity of Medicine
and Pharmacy ; National Institute for Infectious Diseases
“Prof. Dr. Matei Bal ș”, Bucharest, Romania ; 2MD, Assistant
Lecturer, Department of Infectious Diseases, Carol Davila
University of Medicine and Pharmacy, National Institute for Infectious Diseases “Prof. Dr. Matei Bal
ș”, Bucharest,
Romania; 3PhD Student, Microbiology Departme nt, Faculty
of Biology, University of Bucharest; Research Institute of the University of Bucharest, Romania ;
4MD, PhD, Lecturer,
Department of Infectious Diseases, Carol Davila University of Medicine and Pharmacy, National Institute for Infectious Diseases “Prof. Dr. Matei Bal
ș”, Bucharest, Romania; 5PhD,
Lead Scientist, “ Eliava Biopreparation” ; Phage Therapy branches. This phenomenon poses important
challenges in controlling infectious diseases, the
outcomes being realized with high costs.
Antimicrobial re sistance has also been associated
with an increase in morbidity and mortality. Each

Bacteriophage susceptibility of planktonic and sessile bacteria – Neguț et al .• Original article

www.germs.ro • GERMS 4(4) • December 2014 • page 93 *Corre sponding author: Alina Cristina Negu ț, MD, PhD
Student, Carol Davila University of Medicine and
Pharmacy, Bucharest, Romania; National Institute for Infectious Diseases “Prof. Dr. Matei Bal
ș”, No. 1 Dr.
Calistrat Grozovici s treet, Bucharest, 021105, Romania .
negut.alina@gmail.com
Article downloaded from www.germs.ro
Published December 2014
© GERMS 201 4
ISSN 2248 – 2997
ISSN – L = 2248 – 2997 year, i n t h e E u r o p e a n U n i o n a l m o s t 2 5 , 000
patients die because of infections caused by
multidrug resistant germs and the costs are
estimated at about 1.5 billion euros per year.1
The problem deepens w i t h t h e l a c k o f n e w
antimicrobial agents entering practice in the past years.
2
However, multidrug resistance is not the only
issue that infectious diseases practitioners are
faced with. Bacteria that are otherwise susceptible
to common antibiotics express different patterns
for evading antibiotherapy, for example they can
aggregate within a self -produced matrix to form
biofilms, and become a metabolically -integrated
bacterial population3 displaying diverse metabolic
and resistan ce patterns within a multi -layered
distribution.
Apart from research for identifying and
synthetizing new antimicrobials, other modalities
of treatment have also become priorities of
modern medicine and biotechnology.
Bacteriophages are viruses that can i nfect
bacterial cells. Given the fact that phages have strain or species specificity, they associate less
interference with the human microbiome than regular antimicrobials. As bacteriophages are able to reproduce within bacterial cells, an important
prope rty is their exponential growth at the site of
infection.
4
Phage treatment was used in the pre -antibiotic
era, as a way of fighting fire with fire, but it was
temporarily abandon ed when the first antibiotics
were discovered , because of the uncertainty
regarding their exact mechanisms of action.5
A w i d e r a n g e o f a r t i c l e s w e r e p u b l i s h e d
between 1960 and 1975 by Romanian researchers from the Cantacuzino Institute, concerning the
use of bacteriophages for the
treatment of
infections with Enterobacteriaceae or S.
aureus.6-8 Even during the antibiotic era, phage therapy
development continued in count ries of the
former Soviet Union but starting with 2009 ,
European countries such as France and Poland ,
and the USA have also initiated experimental
therapy with phages.
In the past 15 years, articles and books about
the effect of bacteriophages on biofilm have been
published international ly, but no specific
information is currently available for Romania.9-11
With this background, bacteriophages have a
great unexplored po tential, in both preclinical
and clinical research.

Aims of the study
We aim to optimize an in vitro method for
assessing the effects of phages on strains of
Staphylococcus spp., Enterococcus spp. and
Pseudomonas aeruginosa, in planktonic and
metabolical ly-integrated state s. We will also
compare the antimicrobial sensitivity profiles
with the bacteriophage susceptibility profiles.
Methods
Study design
We intend to perform a prospective
laboratory study o n bacterial strains isolated
from different sample s collected from 150
patients admitted with clinically -significant
infections in reference hospitals from Bucharest.
We will perform antibiotic resistance testing as
well as phage susceptibility testing, both on solid
and liquid growth medium, for Staphylo coccus
spp., Enterococcus spp., and Pseudomonas spp.
strains.
After the informed consent is signed , the
samples will be collected and processed
according to the hospital protocol. Data will be
collected in a centralized database and e ach
patient will be assigned a study code, for
confidentiality and anonymization. We will collect information on age, medical history,
diagnosis at admission and discharge , admission
duration, complete blood count, inflammatory syndrome tests, microbiology samples collected, information about the germ (identification and
antibiogram), treatment received, clinical
evolution, follow -up, relapse of infection, etc.

Bacteriophage susceptibility of planktonic and sessile bacteria – Neguț et al .• Original article

www.germs.ro • GERMS 4(4) • December 2014 • page 94

Figure 1. Adding 100 μL broth per well
Figure 2. Adding the phages and performing
the binary dilutions
All the strains isolated from the enrolled
p a t i e n t s w i l l b e s t o r e d i n s o f t a g a r . After strain
identification, the pr ocedure for phage
susceptibi lity testing will be performed.
The study uses 5 bacteriophage mixture s:
PYO, INTESTI, STAPHYLOCOCCAL (Eliava
BioPreparations, Tbilisi, Georgia), PHAGYO,
PHAGESTI (JSC "Biochimpharm", Tbilisi, G e o r g i a ) . P Y O a n d P H A G Y O c o n t a i n p hages
specific for Staphylococcus spp., Streptococcus spp.,
E. coli , Pseudomonas aeruginosa , and Proteus spp.
INTESTI and PHAGESTI contain phages for
Staphylococcus spp., Enterococcus spp., Shigella
spp., Salmonella spp., Proteus spp., E. coli ,
Pseudomonas aeruginosa . STAPHYLOCOCCAL
contains phages for Staphylococcus spp.

Laboratory procedures
T h e s t o r e d b a c t e r i a w i l l b e c u l t u r e d o n
Columbia agar plus 5% sheep blood medium (BIOMÉRIEUX, France) for 24 hou rs in normal
atmosphere at 35±2°C.
A bacterial inocu lum standardized at 0.5
M c F a r l a n d w i l l b e p r e p a r e d a n d p l a t e d i n horizontal lines on Mueller -Hinton solid agar
(BIOMÉRIEUX, France) and then 20 μL of each
p h a g e m i x t u r e w i l l b e p l a c e d o n e a c h b a c t e r i a l line. The p l a t e s w i l l b e i n c u b a t e d a t 3 5 ± 2°C in
normal atmosphere for 24 hours.
The results will be read the following day,
quantifying the aspect of lysis: Positive result: confluent lysis, semi -confluent lysis, ++ (>50
plaques), + (20 -50 lysis plaques), ± (<20) or
Negative result: absence of lysis.
The strains with susceptibility for phages will
be tested with binary phage dilutions. The
bacterial capacity for adherence and biofilm formation wil l be evaluated th rough the
microtiter technique. The 96 -well plates will be
filled with 100 μL of li quid broth per well (Figure
1).
On the first line (A) 100 μ L of phage mixture
will be added. To obtain binary dilutions, from the A wells, after mixing, 100 μL will be added in
the B wells, and the procedure will be continued
from A to F and the la st 100 μ L extracted from F
will be thrown out, to ensure equal quantities
remain in all wells (Figure 2).
Figure 3. Adding the inoculum

Bacteriophage susceptibility of planktonic and sessile bacteria – Neguț et al .• Original article

www.germs.ro • GERMS 4(4) • December 2014 • page 95 As described, t he bacterial suspension (20 μL
of 0.5 McFarland inoculum) will be added in all
wells except for the H row. In conclusion we will
obtai n the negative, sterility control (sterile broth)
i n r o w H , p h a g e s i n b i n a r y d i l u t i o n a n d t h e
microbial culture in rows A to F. In the G row,
100 μL of broth and 20 μ L of bacterial inoculum
will be added, as culture positive control, while
the H row contai ned the negative control (Figure
3).
After 24 hours of incubation, the results will
be evaluated qualitatively based on the
macroscopic aspect of the liquid medium from
the wells . Quantification of bacterial growth will
be performed through plating 10 μL from each
well, in triplicate, on solid Mueller -Hinton agar
followed by 24 hours incubation at 35± 2°C in
normal atmosphere. The number o f colonies will
be evaluated.
The liquid left in the wells will be thrown out
a n d t h e p l a t e w i l l b e w a s h e d t h r e e t i m e s w ith
s t e r i l e w a t e r a n d f i x e d w i t h 1 3 0 μ L cold
m e t h a n o l f o r 1 0 m i n u t e s . T h e p l a t e s w i l l b e stained with 1% crystal violet. The colored
biofilm will be resuspended with 200 μL of 33%
acetic acid and the absorbance of the colored
suspension will be read through 492 nm
spectrophotometry using Huma Reader HS
(HUMAN Diagnostics Worldwide, Germany) . A
blank well containing 200 μL of 33% acetic acid
will be considered the control.
Through the technique described above,
biofilm formation in the presence of phages will
be evaluated.
The strains that formed biofilm may be
selected and used in the next step of the experiment, for evaluating the effect of phages on already formed biofilm.
Each bacterial strain capable of forming
biofilm will be grown in liquid broth in 2 wells ( n o t e d a , b ) f o r 2 4 h o u r s – the timespan
necessary to ensure biofilm formation . After 24
hours any planktonic bacteria left will be washed
out; in the “a” w ell broth will be added, an d in t h e “ b ” w e l l b r o t h a n d b a c t e r i o p h a g e s w i l l b e
added. These will then be incubated for 24
hours. T he next day, the “a” and “b” wells will be
w a s h e d a n d f i x e d . I n a n o t h e r p l a t e , each strain
will be incubated in liquid broth for 24 hours; after that the liquid will be washed out and the
wells fixed with methanol. Subsequently, all the
wells from both pla t e s w i l l b e s t a i n e d w i t h 1 %
crystal violet and the colored biofilm will be
resuspended with 200 μL 33% acetic acid and
the same spectrophotometry technique as
d e s c r i b e d a b o v e w i l l b e u s e d f o r r e a d i n g r e s u l t s .
A blank well containing 200 μL o f 3 3 % a c e t i c
acid will be considered the control value.
The value s from the wells with the same strain
will be compared, evaluating the effect of phages
on preformed biofilm .

Expected results
Given the major burden associated with
biofilm formation in patients with implanted foreign bodies and recurrent catheter or
prosthesis infection, the efficiency of phages
should be evaluated not only on planktonic
bacteria , but also on biofilm formation. The
described method will allow us to evaluat e a large
number of bacterial strains to predict and project
the kinetics of biofilm formation, while adding binary phage dilut ions. The effect of phages on
preformed biofilm could also be assessed.
Through this study, we intend to provide the
first set of results regarding susceptibility to
bacteriophages of bacterial strains isolated from
patients with hard to treat infections, from
reference hospitals in Romania. To further
describe the microbiological characteristics of these particular strains, we also aim to asses s the
correlation between phage susceptibility and
antimicrobial sensitivity profiles.
The results derived from th is experimental
approach will fill a gap in the current knowledge regarding the treatment of patients with problematic recurrent infections with bacterial
strains with potentially diminished suscept ibility
to current antibiotics.
By creating a database for each bacterial
species studied, we will collect data on the patient’s profile and through corroborating this
information with the laboratory results described
f o r t h i s s t u d y , w e w i l l b e a b l e t o b e t t e r
understand the physiologic and pathologic conditions f or developing chronic infections or
hard to treat infections associated with biofilm formation.

Bacteriophage susceptibility of planktonic and sessile bacteria – Neguț et al .• Original article

www.germs.ro • GERMS 4(4) • December 2014 • page 96 We intend to project and develop further
studies to assess the potential synergy for co –
administration of antibiotics and bacteriophages
on both planktonic and sessile bacterial
populations.
We intend to use the results derived from this
study to generate an algorithm for predicting the clinical outcome and monitoring patients with problematic infections, and to describe the
potential role of bacteriophages in clinical
practice, alongside antimicrobial therapy, either as synergic therapy providing an extra mechanism
of bactericidal action, or as adjuvant therapy
potentially disrupting the extracellular polymeric matrix of biofilm, and leaving the bacteria exposed to the direct action of conventional
antimicrobials.
Conclusion
To our knowledge, this is the first such study
to be performed in Romania, regarding the assessment of susceptibility to bacteriophages of planktonic and aggregated bacterial populations. We consider that this study has the potential to
redesign our current understanding of antimicrobial therapy options and to describe
the possible role of bacteriophages in clinical
practice for recurrent, hard to treat infections.

Acknowledgment s
This paper is partly supported by the Sectorial
Operational Programme Human Resources Development
(SOPHRD), financed by the European Social Fund and the Romanian Government under the contract number POSDRU 141531.
This paper is partly supported by the Carol Davila
University of Medicine and Pharmacy, Young Researchers
Grant, no . 28341/2013.
Authors’ contributions statement: All authors had
equal contributions.
Conflicts of interest: All authors – none to declare

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Please cite this article as:
Neguț AC, S ăndulescu O, Popa M, Streinu -Cercel A, Alavidze Z, Berciu I, Bleotu C, Popa MI, Chifiriuc
MC, Streinu -Cercel A . Experimental approach for bacteriophage susceptibility testing of planktonic and
sessile bacterial populations – Study protocol. GERMS. 2014; 4(4):92-96. doi: 10.11599/germs.2014. 1062