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8. Hunt A. E., Smith R. M. Mechanics and control of the flat versus normal foot during the stance phase of walking. Clinical Biomechanics, 2004, vol. 19, no. 4, pp. 391-397.
9. Iaquinto J. M., Wayne J. S. Effects of Surgical Correction for the Treatment of Adult Acquired Flatfoot Deformity: A Computational Investigation. Journal of Orthopaedic Research, 2011, vol. 29, no. 7, pp. 1047-1054.
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УДК 578.5:579.61 14.03.00 - Медико-биологические науки
© E.O. Rubalskii, A.V. Aleshkin, S.S. Afanasiev, 03.02.00 - Общая биология
V.A. Aleshkin, Kh.M. Galimzyanov, A.R. Umerova,
O.V. Rubalsky, O.N. Ershova, E.E. Rubalskaya, I.A. Kiseleva,
S.S. Bochkareva, E.R. Zul'karneev, A.Kh. Akhmineeva,
M.O. Rubalsky, I.O. Lunina, V.V. Uskov,
M.M. Karnaukh, O.Yu. Borisova, N.T. Gadua, A.D. Teply,
S. Rümke, Ch. Salmoukas, Ch. Kühn, A. Haverich, 2017
INTEGRATIVE APPROACH FOR CONTROL OF TEMPERATE BACTERIOPHAGES
IN PHAGE-BASED PRODUCTS
Rubalskii Evgenii O., Cand. Sci. (Biol.), Research Associate, Laboratory of Applied Immunochemis-try, G.N. Gabrichevsky Moscow Research Institute for Epidemiology and Microbiology, 10 Admiral Makarov St., Moscow, 125212, Russia; Research Fellow, Department of Cardiothoracic, Transplantation and Vascular Surgery, Hannover Medical School, 1 Carl-Neuberg-Straße, Hannover, 30625, Germany, tel.: +7-961-798-37-53, e-mail: e.o.rubalsky@gmail.com.
Aleshkin Andrey V., Dr. Sci. (Biol.), Head of Laboratory of Clinical Microbiology and Biotechnology of Bacteriophages, G.N. Gabrichevsky Moscow Research Institute for Epidemiology and Microbiology, 10 Admiral Makarov St., Moscow, 125212, Russia, tel.: +7 (495) 452-18-16, e-mail: ava@gabri.ru.
Afanasiev Stanislav S., Dr. Sci. (Med.), Professor, Honored Scientist, Deputy Director, G.N. Gabrichevsky Moscow Research Institute for Epidemiology and Microbiology, 10 Admiral Makarov St., Moscow, 125212, Russia, tel.: +7-903-667-20-68, e-mail: afanasievss409.4@bk.ru.
Aleshkin Vladimir A., Dr. Sci. (Biol.), Professor, Honored Scientist, Director, G.N. Gabrichevsky Moscow Research Institute for Epidemiology and Microbiology, 10 Admiral Makarov St., Moscow, 125212, Russia, tel.: +7 (495) 452-18-16, e-mail: info@gabrich.ru.
Galimzyanov Khalil M., Dr. Sci. (Med.), Professor, Head of Department of Infectious Diseases, Astrakhan State Medical University, 121 Bakinskaya St., Astrakhan, 414000, Russia, tel.: +7 (8512) 52-41-43, e-mail: agma@astranet.ru.
Umerova Adelya R., Dr. Sci. (Med.), Head of Department of Clinical Pharmacology, Astrakhan State Medical University, 121 Bakinskaya St., Astrakhan, 414000, Russia, tel.: + 7 (8512) 25-33-91, e-mail: klinfarm_agma@mail. ru.
Rubalsky Oleg V., Dr. Sci. (Med.), Professor, Head of Department of Microbiology and Virology, Astrakhan State Medical University, 121 Bakinskaya St., Astrakhan, 414000, Russia, tel.: +7 (8512) 52-35-99, e-mail: rubalsky.innovation@gmail.com.
Ershova Ol'ga N., Dr. Sci. (Med.), Associate Professor, Deputy Chief Physician for Epidemiology, N.N. Burdenko Neurosurgery Research Institute, 16 4th Tverskaya Yamskaya St., Moscow, 125047, Russia, tel.: +7 (499) 972-85-70, e-mail: OErshova@nsi.ru.
Rubalskaya Elena E., Head of Laboratory of Clinical Laboratory Diagnostics, Research Institute of Regional Infectious Pathology, Astrakhan State Medical University, 121 Bakinskaya St., Astrakhan, 414000, Russia, tel: +7-908-616-90-66, e-mail: e.e.rubalskaya@gmail.com.
Kiseleva Irina A., Research Associate, BPhage LLC, Butyrskiy Val St., 10, Moscow, 1250476, Russia, Research Associate, Laboratory of Clinical Microbiology and Biotechnology of Bacteriophages, G.N. Gabrichevsky Moscow Research Institute for Epidemiology and Microbiology, 10 Admiral Makarov St., Moscow, 125212, Russia, tel.: +7-916-397-22-38, e-mail: irina6804@mail.ru.
Bochkareva Svetlana S., Cand. Sci. (Biol.), Senior Research Associate, Laboratory of Immunobi-ological Preparations, G.N. Gabrichevsky Moscow Research Institute for Epidemiology and Microbiology, 10 Admiral Makarov St., Moscow, 125212, Russia, tel.: +7-495-452-38-03, e-mail: cip1989@gmail.com.
Zul'karneev Eldar R., Junior Research Associate, Laboratory of Clinical Microbiology and Biotechnology of Bacteriophages, G.N. Gabrichevsky Moscow Research Institute for Epidemiology and Microbiology, 10 Admiral Makarov St., Moscow, 125212, Russia, tel.: +7 (495) 452-18-16, е-mail: elzz89@mail.ru.
Akhmineeva Aziza Kh., Dr. Sci. (Med.), Associate Professor, Astrakhan State Medical University, 121 Bakinskaya St., Astrakhan, 414000, Russia, tel.: +7 (8512) 52-36-55, e-mail: asmafordec@mail. ru.
Rubalsky Maxim O., Post-graduate Student, Department of Infectious Diseases, Astrakhan State Medical University, 121 Bakinskaya St., Astrakhan, 414000, Russia, tel.: +7 (8512) 52-35-99, e-mail: m.o.rubalsky@gmail.com.
Lunina Iraida O., Head of Technology and Innovation Support Center, Astrakhan State Medical University, 121 Bakinskaya St., Astrakhan, 414000, Russia, tel.: +7-927-88-97, e-mail: iraida3000.91@mail.ru.
Uskov Vladislav V., Cand. Sci. (Econ.), Specialist in Innovations of Technology and Innovation Support Center, Astrakhan State Medical University, 121 Bakinskaya St., Astrakhan, 414000; Russia, Associate Professor, Department of Legal Support of Economic Security, St. Petersburg State University of Architecture and Construction, 4 2-ya Krasnoarmeyskaya St., St.-Petersburg, 190005, Russia, tel: +7-927-577-11-55, e-mail: wtovl@yandex.ru.
Karnaukh Mariya M., Post-graduate student, G.N. Gabrichevsky Moscow Research Institute for Epidemiology and Microbiology, 10 Admiral Makarov St., Moscow, 125212, Russia, tel.: +7-917-196-16-06, e-mail: mascha.karnaukh@yandex.ru.
Borisova Olga Yu., Dr. Sci. (Med.), Head of Laboratory for Diagnostics of Diphtheria and Pertussis Infections, G.N. Gabrichevsky Moscow Research Institute for Epidemiology and Microbiology, 10 Admiral Makarov St., Moscow, 125212, Russia, tel.: +7 (495) 452-18-16, e-mail: olgborisova@mail.ru.
Gadua Natiya T., Cand. Sci. (Med.), Senior Research Associate, Laboratory for Diagnostics of Diphtheria and Pertussis Infections, G.N. Gabrichevsky Moscow Research Institute for Epidemiology and Microbiology, 10 Admiral Makarov St., Moscow, 125212, Russia, tel.: +7 (495) 452-18-16, е-mail: 8nati8@mail.ru.
Teply Aleksandr D., post-graduate student, Astrakhan State Medical University, 121 Bakinskaya St., Astrakhan, 414000, Russia, tel.: (8512) 52-41-43, e-mail: tkleon@mail.ru.
Rümke Stefan, MD, Resident, Department of Cardiothoracic, Transplantation and Vascular Surgery, Hannover Medical School, 1 Carl-Neuberg-Straße, Hannover, 30625, Germany, tel.: +49-511-532-2151, e-mail: Ruemke.Stefan@mh-hannover.de.
Salmoukas Christina, Dr. med., MD, Resident, Department of Cardiothoracic, Transplantation and Vascular Surgery, Hannover Medical School, 1 Carl-Neuberg-Straße, Hannover, 30625, Germany, tel.: +49-511-532-2151, e-mail: Salmoukas.Christina@mh-hannover.de.
Kühn Christian, Dr. Med., PD, Cardiothoracic and Transplant Surgeon, Consultant, Head of ECMO Surgery Division, Department of Cardiothoracic, Transplantation and Vascular Surgery, Hannover Medical School, 1 Carl-Neuberg-Straße, Hannover, 30625, Germany, tel.: +49-511-532-3448, e-mail: Kuehn.Christian@mh-hannover. de.
Haverich Axel, Dr. Med., Professor, Director of Department of Cardiothoracic, Transplantation and Vascular Surgery, Hannover Medical School, 1 Carl-Neuberg-Straße, Hannover, 30625, Germany, tel.: +49-511-532-6580, e-mail: Haverich.Axel @mh-hannover.de.
Modern requirements for bacteriophages for therapeutic and preventive products recommend using only lytic (virulent) phages and avoiding temperate phages or other phage-like elements of genetic exchange. The presence of at least one temperate bacteriophage strain in the cocktail may lead to negative consequences, such as spread of antibiotic resistance, transfer of bacterial factors of pathogenicity, formation of bacteria resistant to phages. The sources of temperate phages in preparations may be both incorrectly characterized candidate strains of bacteriophages and lysogenic host bacteria cultures. Classical microbiological methods of temperate bacteriophages induction (heating, ultraviolet irradiation, mitomycin C) often give false negative results and therefore cannot provide a reliable approach of production control of phage-based preparations. As a basis of the developed methodology, we chose the sequencing of viral metagenomes including a database of known integrases.
Key words: lytic bacteriophages, temperate bacteriophages, phage therapy, phage-based products, viral me-tagenomics, integrase, bioinformatics.
Introduction. Quality and safety requirements for bacteriophages for therapeutic and preventive products recommends to use only lytic (virulent) phages and avoid of temperate phages or other phage-like elements of genetic exchange. The presence of temperate bacteriophage strains in phage-based products may lead to negative consequences due to horizontal gene transfer, such as spread of antibiotic resistance, transfer of bacterial factors of pathogenicity, formation of bacteria resistant to phages [4, 5, 15].
Phage-based products designed against pathogenic bacteria as a rule consist of bacteriophage strains members of the order Caudovirales. Possibility of temperate life cycle of these dsDNA viruses depends on the presence of integrase genes in their genomes which allow recombinate with hosts genomes. The phage integrases belong to tyrosine or serine recombinase family and able to provide site-specific recombination. The sources of temperate phages in preparations may be both newly isolated strains of bacteriophages and lysogenic host bacteria cultures on which phages are propagated. Procedures for detection of temperate bacteriophages include methods of phage induction (heating, ultraviolet irradiation, mitomycin C) and molecular genetic screening (DNA sequencing and further bioinformatic analysis of phages and hosts genomes) [10, 15, 17].
In our experience the classical microbiological methods of temperate bacteriophages induction from their hosts often give false negative results and therefore cannot provide a reliable approach of production control of phage-based preparations. Fortunately there has already been developed some bioinformatics software for detection of prophage motifs in bacterial genomes such as Prophinder [11], PHAST, PHASTER [3], and others. Despite the usability of these bioinformatics approaches it is still a nontrivial objective to distinguish inducible prophages and defective ones.
Reliable bioinformatics prediction of bacteriophage lifecycle is not a routine procedure as well. For sure metagenome and virome analysis servers such as MG-RAST [13], Metavir [16] and iVirus [7] are very useful in taxonomy prediction of viruses. However it can be rather sophisticated because of high mosaicism in phage genomes. Moreover using these services is relatively time consuming due to a very deep metage-nomic analysis. This is also a disadvantage in terms of developing new and updating the existing bacterio-phage preparations.
Prediction of phage lifestyle with a certain level of reliability is also possible since such software as PHACTS is developed. The alhorythm of this set of tools is based on comparison of annotated protein sequences of phages. However some mistakes occur because some phages that carry integrases may have strictly lytic livestyle. Nevertheless it does not stop horizontal gene transfer which makes these phages unacceptable for therapeutic and prevention purposes [12]. That's why presence of any bacteriophage with an integrase gene in the phage-based product is unacceptable.
Thus, there are two main challenges in terms of control of temperate bacteriophages in phage-based products:
1. time and money consuming detection of active prophages in host bacteria;
2. incorrect or time consuming characterization of candidate strains of bacteriophages.
Solving these problems is a priority either for huge bacteriophage prodicung companies which need constantly update a pool of relevant host bacteria strains and circulating isolates, or for services of personalized phage therapy which require isolating and charachterizing new bacteriophages pretty often.
The aim of this work was to develop an integrative molecular-genetic, bioinformatic and biotechno-logical approach for detection and elimination of temperate bacteriophages from phage-based products.
Materials and methods. Phage cocktail KPV 05196 which contained previousely not characterized bacteriophages active against Klebsiella pneumoniae was investigated as an example of the effective control of temperate phages absence. Model K. pneumoniae isolates number 246 and 509 were used to illustrate a case with and without temperate phage respectively. Clean bacteriophage strains were obtaind through at least 3 consequtive times picking up of single plaques from the Gratia method Petri dishes [1].
Metaviromic sequencing. Preliminary the phage cocktail was passed through 0,22 ^m PES syringe filter (Millipore, USA) followed by treatment with DNase I (New England Biolabs, USA). Then total phage DNA was extracted with a microspin K-Sorb Kit (Syntol LLC, Russia) according with a standard protocol modified for the elution step with nuclease-free water. Extracted DNA was sonicated with the Bioruptor UCD-200 device (Diagenode Inc., USA) with further preparation of shotgun libraries with the NEBNext Fast DNA Library Prep Set for Ion Torrent (New England Biolabs, USA) and standard barcodes from the Ion Xpress Barcode Adapters 1-16 Kit (Thermo Fisher Scientific, USA). Clonal amplification performed with Ion PI Template OT2 200 Kit v3 followed by sequencing on the Ion Proton machine with Ion PI Sequencing 200 Kit v3 and Ion PI Chip Kit v2 (Thermo Fisher Scientific, USA).
Bioinformatic analysis. Obtained reads were filtered with Trimmomatic v.0.33 [8] followed by de-novo assembly with Newbler software (Roche Diagnostics). Resulting contigs with minimal length of 100 bp were analyzed on MG-RAST server and in parallel with a database of sequences of known integrases complied from the GenBank and runned on the Blast2GO software [9]. The constantly updated and structured database is available under the following link: https://github.com/rubalsky/MetaPhOr
Visualization of the MG-RAST results we performed with the Krona tools [14]. Phylogenetic analysis of the found integrase was done in the Geneious sotware (Biomatters, New Zeland).
PCR confirmation of the interase gene. DNA isolation performed for the phage cocktail and for the K. pneumoniae cultures as before for the metagenomics sequencing included DNase I treatment. PCR for detection and distinguishing of source of the most common temperate phages performed according with Balding C. et al [6]. Specific PCR was developed to the found sequence of the P2virus integrase. Following primers were designed for it (Table) and PCR was performed using standard recombinant Taq-polymerase chemistry (Thermo Fisher Scientific, USA) on the Tercyc multi-block amplifier (DNA-Technology LLC, Russia).
Table
Oligonucleotide primers for detection of the found integrase
Primer name Oligonucleotide sequence, 5'-3' Size of the amplicon, bp Annealing temperature, °C MgCl concentration, mM
KPV int fw3 ata gcc act tcg gta tcg gc 620 59 1.8
KPV int rv3 ata caa aaa ccg aga gcg cg
Results and discussion. Metagenomic analysis on the MG-RAST server showed the presence of phages in the cocktail from all the three families of the Caudovirales order: Myoviridae, Siphoviridae and Podoviridae (figure 1). Different temperate phages were found in relatively low amounts: P22virus (from the family Podoviridae), Lambdavirus (from the family Siphoviridae), P2virus (from the family Myoviridae, figure 2). Confrimation of their presence was done by previously devepoled PCR assays [6]. We detected all of the temperate phages above except the P2-viruses. Nevertheless running the compiled database of integrases (E-value cutoff 1.0E-3) uniquely showed the presence of the integrase, which had more than 85% of homology with known integrases of P2virus genus (figure 3). That's why we developed our own PCR to find a source of this temperate phage.
Results of the PCR tests demonstrated that the source of the temperate P2-like phage was a K. pneumoniae 246 culture (figure 4a). Purification of the lytic phage strains growed previously on the K. pneumoniae 246 was done through passaging of their single plaques on the prophage-free K. pneumoniae 509 isolate. Results of the PCR presented on the figure 4b show the absence of the P2-like integrase.
Figure 1. Visualization of the MG-RAST results of the level of Caudovirales order
Figure 2. Visualization of the MG-RAST results of the level of Myoviridae family
0.4
gij 17313217:33616-34735 , PseudoiriDiiBS pliage pliiCTX, integrase
tfi|DQ42fflD4.1|:332S7-342S5 , Bacteriophage plii-MliaAl-PHLlOl, integrase
gij 145 70S0 74:35 709-36791, Ralstoiiia pliage phiRSAl, integrase
gi| 1116 76329:9314-10(539 , BuikhoBeriii prophage plii5223 7, integrase
gij 1342SS 739:7912-9093 , Bmkliolderia pliage phiE2D2a integrase
gij9(53032 7:24462-25475 , Enteiobacteria pliage P2, integiase
gjb| A Y135 739.1|:22712-23692 , Bacteriophage WFlii, integiase
g¡b|KC61S326.11:1S 7-116 7 , Enteiobacteria pliage P2, integiase
gij30065 704:23342-24322 , Yeisinia pliage L-413C, integLase
^b|U32222.1 |B 1U32222:21423-22433 , Bacteiiopliage 136, integrase
gij 169936017:32527-33552 , Enteiobacteria pliage Fels-2, integrase
qneiy, KPV cocktail 05196, integrase
^b|KT630647.11:20 799-21343 , Salmonella pliage SENS, integrase ^b|KT630644.11:20 79S-21347 , Salmonella pliage SEN1, integrase gij4105 7352:21799-22343 , Entenobacteiia pliage PsP3, integiase
Figure 3. Phylogenetic analysis of aminoacid sequences of integrases belonging to phages of P2virus genus; in the
frame highlighted the found integrase from the cocktail
b)
Figure 4. Results of PCR detection of the P2virus integrase: a) before themperate phage control; b) after passaging on the prophage-free klebsiella: 100 bp - DNA ladder;
PC - positive control (DNA KPV_05196 cocktail); KP246 - K. pneumoniae 246 culture DNA;
KP509 - K. pneumoniae 509 culture DNA; NC - negative control (control of extraction);
MC - negative control (reaction mix)
Different efficacy of the PCR shows higher P2-like prophage concentration in bacteriophage cocktail in comparison with a culture. This phenomenon allows suggesting that the bacteriophage propagation process itself makes a signal (probably induced SOS-like response) for the prophage induction. Consequently the importance of temperate phage absence control grows for the previous stages of development and manufacturing processes.
Further metaviromic confirmation of any other induced prophage probably coming from the substituted bacterial cultures might be required. However this test can be performed beforehand in a batch mode
for a number of different isolates. These isolates can be further better characterized and used as constant host strains for large bacteriophage production.
Reliability of temperate phage induction may vary for different bacterial species and strains as well as depend on prophages, their defectiveness and quantity within certain bacterial genomes. The developed approach may be combined with the classical in vitro induction methods to increase probability of temperate bacteriophage detection. Under these conditions the approach may be suitable for all stages of development and manufacture of phage-based products.
It's interesting that run of the compiled database of integrases showed 2648 positive results among total 6019 sequences of all complete genomes of bacteriophages available on the GenBank. Corresponding BLAST hits (maximally 20 per sequence) with certain levels of similarity are presented on figure 5. Probably further optimization of the developing of integrases database is necessary as well as the E-value cutoff level has to be justified.
9000 8000 7000 % 6000 £ 5000 < 4000 CO 3000 2000
l0000 ...........Illlllllllllllllllllllllllll.l.....ll.lllHlllllllll
0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100
Sequence similarity, %
Figure 5. Sequence similarity distribution of the detected integrases whithin public available bacteriophage
genomic sequences
Conclusions. Thus it is possible to formulate methodological approach for production control of bacteriophage preparations that allows identify and eliminate temperate bacteriophages (figure 6).
Bacteriophage-based product / candidate cocktail
Confirmation of temperate phage absence
Excluding of temperate phage and it's source
/ N
Metaviromic
sequencing
V J
f
Bioinformatic
analysis
V J
Design and conduction of specific PCR
Figure 6. Integrative approach for production control of temperate phages in preparations
The developed approach can be used with different procedures of preparation of phage stocks and final products. For example it is constantly used within previously disclosed pipeline for the development
of phage-based products [2].
Further update and structurization of the presented database lead to a powerfull tool for rapid and reliable charachterization of bacteriophages and their hosts. This tool might be improved by the development of specialized software based on it for selection of the appropriate microorganisms.
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