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33ISOLATION AND CHARACTERIZATION OF PSEUDOMONAS AERUGINOSA BACTERIOPHAGES — POTENTIAL AGENTS FOR PHAGE THERAPY
Kornienko MA Kuptsov NS, Danilov DI, Gorodnichev RB, Malakhova MV, Bespiatykh DA, Veselovsky VA, Shitikov EA, Ilina EN Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency, Moscow, Russia
Pseudomonas aeruginosa — is one of the pathogens characterized by the critical number of multidrug-resistant (MDR) strains. Phage therapy is considered an alternative to antibiotics, especially in treatment of infections caused by MDR strains. The aim of this study was to isolate and characterize P. aeruginosa phages that could potentially be suitable for treating infectious diseases. To isolate the P. aeruginosa phages, enrichment cultures were used. The lytic activity spectrum was confirmed by spot testing on 40 P. aeruginosa strains. Whole-genome sequencing was performed using Illumina MiSeq instrument. Phylogenetic analysis was done using VICTOR tool. Isolated phages vB_PaeA-55-1w and vB_PaeM-198 from Autographiviridae and Myoviridae families, respectively, had a broad spectrum of lytic activity (about 50% each), including lysis of MDR strains. The genomes vB_PaeA-55-1w and vB_PaeM-198 comprise double-stranded DNA of 42.5 and 66.3 kbp in length, respectively. Open reading frames were annotated for both phages (52 for vB_PaeA-55-1w, and 95 for vB_PaeM-198), no integrases and toxins were detected. On a phylogenetic tree, vB_PaeA-55-1w phage was clustered with phages from the Phikmvvirus genus (Autographiviridae family), which are also used in phage therapy. vB_PaeM-198 phage was clustered with phages from the Pbunavirus genus (Myoviridae family). vB_PaeA-55-1w and vB_PaeM-198 phages could be considered as candidates for phage therapy and may be used to treat infections caused by MDR P. aeruginosa.
Keywords: Pseudomonas aeruginosa, virulent bacteriophages, phage therapy, Autographiviridae, Myoviridae, whole genome sequencing, phylogenetic analysis Funding: The study was supported by the State Assignment on the Development of a personalized approach to the therapy of infections using virulent bacteriophages (Code: Bacteriophage) (Russia).
Acknowledgments: the authors thank the Center for Precision Genome Editing and Genetic Technologies for Biomedicine, the Federal Research and Clinical Center of Physical-Chemical Medicine of the Federal Medical Biological Agency for their help with sequencing the genomes of bacteriophages. Author contribution: Kornienko MA — study plan, data collection and processing, article authoring; Kuptsov NS — data collection and processing, article authoring; Danilov DI, Gorodnichev RB, Malakhova MV, Veselovsky VA — data collection; Bespiatykh DA — data processing, Shitikov EA — research plan, data processing, article authoring; Ilina EN — research plan, article authoring.
Compliance with ethical standards: the experiment was carried out in compliance with the Sanitary and Epidemiological Rules SP 1.3.2322-08 "Safe work with microorganisms of III-IV pathogenicity (hazardousness) groups and pathogens of parasitic diseases"; Sanitary and Epidemiological Rules SP 1.3.2518-09 "Amendments and additions #1 to the sanitary and epidemiological rules" Safe work with microorganisms of III-IV pathogenicity (hazardousness) groups and pathogens of parasitic diseases"; Sanitary and Epidemiological Rules SanPiN 2.1.7.2790-10 "Sanitary and epidemiological requirements for medical waste management"; Federal Clinical Recommendations "Rational use of bacteriophages in medical and anti-epidemic practice." [><1 Correspondence should be addressed: Maria A. Kornienko
Malaya Pirogovskaya, 1a, Moscow, 119435; [email protected] Received: 19.07.2021 Accepted: 05.08.2021 Published online: 18.09.2021 DOI: 10.47183/mes.2021.027
ВЫДЕЛЕНИЕ И ХАРАКТЕРИСТИКА БАКТЕРИОФАГОВ PSEUDOMONAS AERUGINOSA — ПОТЕНЦИАЛЬНЫХ АГЕНТОВ ДЛЯ ФАГОВОЙ ТЕРАПИИ
М. А. Корниенко Н. С. Купцов, Д. И. Данилов, Р. Б. Городничев, М. В. Малахова, Д. А. Беспятых, В. А. Веселовский, Е. А. Шитиков, Е. Н. Ильина
Федеральный научно-клинический центр физико-химической медицины Федерального медико-биологического агентства, Москва, Россия
Одним из патогенов, характеризующихся критическим показателем доли штаммов с множественной лекарственной устойчивостью (МЛУ), является Pseudomonas aeruginosa. В качестве альтернативы антибиотикам при терапии инфекций, вызванных штаммами с МЛУ, рассматривают фаготерапию. Целью исследования было выделить и охарактеризовать бактериофаг P. aeruginosa, потенциально пригодный для терапии инфекционных заболеваний. Выделение проводили методом накопительных культур. Спектр литической активности устанавливали спот-тестированием на коллекции из 40 штаммов P. aeruginosa. Полногеномное секвенирование выполняли на платформе MiSeq (Illumina). Филогенетический анализ геномов проводили с помощью VICTOR. Выделенные бактериофаги vB_PaeA-55-1w и vB_PaeM-198, принадлежащие к семействам Autographiviridae и Myoviridae соответственно, обладали широким спектром литической активности (около 50% каждый), в том числе вызывали лизис штаммов с МЛУ Геномы vB_PaeA-55-1w и vB_PaeM-198 представлены двухцепочечной ДНК длиной 42,5 и 66,3 т.п.н. соответственно. В составе геномов аннотировано 52 (vB_PaeA-55-1w) и 95 (vB_PaeM-198) открытых рамок считывания, среди них гены интеграз и токсинов не обнаружены. На филогенетическом древе vB_PaeA-55-1w располагался в кластере совместно с бактериофагами рода Phikmvvirus семейства Autographiviridae, в том числе с используемыми в фаготерапии, а vB_ PaeM-198 входил в кластер, включающий бактериофаги рода Pbunavirus семейства Myoviridae. Бактериофаги vB_PaeA-55-1w и vB_PaeM-198 можно рассматривать в качестве кандидатов для применения в фаготерапии, в том числе и для лечения инфекций, вызванных штаммами P. aeruginosa с МЛУ. Ключевые слова: Pseudomonas aeruginosa, вирулентные бактериофаги, фаготерапия, Autographiviridae, Myoviridae, полногеномное секвенирование, филогенетический анализ
Финансирование: исследование выполнено за счет средств, предоставленных для выполнения государственного задания «Разработка персонализированного подхода терапии инфекционных процессов с применением вирулентных бактериофагов» (ШИФР: Бактериофаг).
Благодарности: авторы благодарят Центр высокоточного редактирования и генетических технологий для биомедицины ФГБУ ФНКЦ ФХМ ФМБА России за секвенирование геномов бактериофагов.
Вклад авторов: М. А. Корниенко — план исследований, набор и обработка данных, написание статьи; Н. С. Купцов — набор и обработка данных, написание статьи; Д. И. Данилов, Р Б. Городничев, М. В. Малахова, В. А. Веселовский — набор данных; Д. А. Беспятых — обработка данных, Е. А. Шитиков — план исследований, обработка данных, написание статьи; Е. Н. Ильина — план исследований, написание статьи. Соблюдение этических стандартов: экспериментальная работа выполнена с соблюдением норм Санитарно-эпидемиологических правил «Безопасность работы с микроорганизмами III-IV групп патогенности (опасности) и возбудителями паразитарных болезней» СП 1.3.2322-08; Санитарно-эпидемиологических правил СП 1.3.2518-09 — «Дополнения и изменения № 1 к санитарно-эпидемиологическим правилам «Безопасность работы с микроорганизмами III-IV групп патогенности (опасности) и возбудителями паразитарных болезней» СП 1.3.2322-08; Санитарно-эпидемиологических правил «Санитарно-эпидемиологические требования к обращению с медицинскими отходами» СанПиН 2.1.7.2790-10, а также Федеральных клинических рекомендаций «Рациональное применение бактериофагов в лечебной и противоэпидемической практике». [><3 Для корреспонденции: Мария Андреевна Корниенко
ул. Малая Пироговская, д. 1а, г. Москва, 119435; [email protected]
Статья получена: 19.07.2021 Статья принята к печати: 05.08.2021 Опубликована онлайн: 18.09.2021 DOI: 10.47183/mes.2021.027
According to the World Health Organization, antibiotic resistance is rising to dangerously high levels in all parts of the world [1]. Gram-negative bacteria, including Pseudomonas aeruginosa, occupy the first places in the global priority list of antibiotic-resistant bacteria posing the greatest threat to human health
[1]. The bacteria of this species are ubiquitous, their genetic plasticity and environmental adaptability are high. The wide variety of pathogenicity mechanisms often makes the infection general in cases of infestation with P. aeruginosa strains [2]. P. aeruginosa causes a wide range of diseases, from intoxication to extensive pyoinflammatory processes and septic shock
[2]. According to AMRmap portal, the share of P. aeruginosa isolates, which occupy the top lines in the list of most common nosocomial pathogens in Russia, was 16.83% among all such pathogens isolated in 2015-2020 [3]. Besides, about 30% of P. aeruginosa strains circulating in the population are multidrug-resistant (MDR), i.e., they are resistant to at least one antibiotic drug out of three or more antibiotic groups, and about 15% of the circulating P. aeruginosa strains show extreme drug resistance (XDR), i.e., they are resistant to at least one antibiotic from all groups of antibiotics, with the exception of 1-2 groups; these factors drive the related patient mortality up [4].
An urgent task currently is to develop alternative, non-antibiotic treatments for infectious diseases caused by MDR and XDR pathogens. One of the most promising alternatives are virulent bacteriophages, which are the basis of phage therapy [5]. The promise is in the capability of virulent bacteriophages to lyse both antibiotic-sensitive and antibiotic-resistant strains of bacteria. In addition, bacteriophages do not cause toxic and allergic side effects, have no contraindications [6] and can be prescribed to pregnant women in combination with other medications [7].
A number of reports and preclinical and clinical trials [8, 9] confirm the success of phage therapy against P. aeruginosa infections both in animals and humans. There are several commercial therapeutic drugs designed to counter infections caused by P. aeruginosa that are produced in Russia: Pseudomonas aeruginosa bacteriophage, Intesti-bacteriophage, Polyvalent purified pyobacteriophage (Microgen; Russia).
Despite the availability of bacteriophage preparations active against P. aeruginosa and the successful experience of their use, phage collections need to be constantly updated, as the specifics of modern phage therapy require. Since virulent bacteriophages have a rather narrow specificity and usually target only several strains, updating the collections means extending them with phages that cause lysis of current bacterial strains. Moreover, there are reported cases [10] when bacteria mutate and acquire resistance to bacteriophages [10]. Isolation of new bacteriophages and their inclusion in the composition of therapeutic drugs solves this problem.
In connection with the above, the purpose of this work is to isolate and characterize P. aeruginosa bacteriophages that can be used to treat infectious diseases.
METHODS Bacterial strains
The study used P. aeruginosa strains (n = 40) selected from the collection of bacterial strains of the laboratory of molecular genetics of microorganisms of the Federal Research and Clinical Center of Physical-chemical Medicine of Federal Medical Biological Agency of Russia. The strains of the collection were characterized by the profile of drug susceptibility (to ceftriaxone, gentamicin, ciprofloxacin, and meropenem), as well as by genotypes according to the results of multilocus sequence typing (MLST) [11]. The bacteria were cultivated for 18-24 h in the lysogeny broth (LB) nutrient medium (Oxoid; UK) at 37 °C.
Isolation of bacteriophages
The bacteriophages were isolated by enriching cultures from natural sources (sewage, water samples from various rivers) with P. aeruginosa strains PA55 and PA198 (Table 1). A sample of water (50 ml) was drawn through a Millipore filter with a polyvinylidene fluoride membrane, pore diameter of 0.45 pm (Merck Millipore; USA), and then two-fold LB broth was added to it. Subsequently, 300 ml of an overnight culture of the host strain was added and incubated on a shaker at 37 °C for 18 h. After cultivation, bacterial cells were centrifuged for 10 minutes at 3500 g to achieve precipitation. The supernatant was put through a Millipore filter with a polyethersulfone membrane and a pore diameter of 0.22 pm (Merck Millipore; USA). Individual bacteriophages were obtained by sequential (threefold) isolation from individual negative colonies. Further on, bacteriophages were grown in 50 ml of LB broth containing 300 pl of the bacterial strain overnight culture. The concentration of the bacteriophage in the phage lysate was estimated by the standard Grazia titration method [12].
Bacteriophage lytic capability range determination
Bacteriophage lytic capability range was determined by spot testing. Phagolysates with a titer of 3 x 106 PFU/ml (plaque-forming units per ml) were used in the tests with the aim to prevent non-specific lysis. An overnight culture (1010 CFU/ml) of the tested bacterial strain was sequentially diluted in LB broth to a cell concentration of 106 CFU/ml, then 0.1 ml thereof was mixed with 5 ml of semisolid LB agar (0.6% agar) and added to a Petri dish containing a thin layer of LB agar (1.5% agar). After solidification of the semisolid agar, a drop (5 pL) of the studied bacteriophage was applied to the dish's surface. Petri dishes were incubated at 37 °C for 18-24 h. Lytic capability was assessed visually: the bacterial strain was considered sensitive to the bacteriophage in case there appeared a transparent spot or separate negative colonies. In the absence of such a lysis spot or if it was opaque the bacterial strain was classified as resistant.
Table 1. Characteristics of P. aeruginosa bacteriophage host strains
Strain MLST sequence type Antibacterial susceptibility pattern
ceftriaxone gentamicin ciprofloxacin meropenem
РА198 ST508 R S S R
PA55 ST2690 R S S S
Table 2. Genetic characteristics of the collection strains and range of the hosts of vB_PaeA-55-1w and vB_PaeM-198 bacteriophages
MLST sequence typing Total number of strains Share of strains lysed by vB_PaeA-55-1w, % Share of strains lysed by vB_ PaeM-198, %
ST12 5 0 100
ST17 1 0 0
ST186 1 100 100
ST198 100 50
ST207 100 0
ST233 1 100 100
ST235 1 0 0
ST244 100 75
ST266 1 100 100
ST357 1 100 0
ST395 1 0 0
ST483 1 0 0
ST498 1 0 0
ST499 100 100
ST508 1 0 100
ST569 1 0 0
ST589 1 0 100
ST654 33,3 0
ST1094 1 100 100
ST1292 1 0 0
ST1527 1 0 0
ST2427 1 0 0
ST2690 1 100 100
Unique type 15-5-11-8-4-4-1* 2 0 50
Unique type 15-2-11- 3-3-38-3* 2 100 50
Unique type 17 -5-12-3-14-4-7* 1 0 0
Note: * — the allele numbers of genes included in the standard MLST pattern (arcC-aroE-glpF-gmk-pta-tpi-yq) [21] are indicated for each unique sequence type.
Isolation of bacteriophage DNA and whole genome sequencing
Total bacteriophage DNA was isolated by phenol-chloroform extraction method [13] with preliminary enzymatic treatment of phage lysates with RNase A, DNase I, and proteinase K (Thermo Fisher Scientific; USA) in accordance with the manufacturer's instructions.
The library was prepared with 250 ng of genomic DNA. Covaris S220 System (Covaris; United States) enabled DNA fragmentation to 400-500 bps. Quality of the fragmented samples was assessed with Agilent 2100 bioanalyzer (Agilent; USA) in accordance with the manufacturer's instructions. The NEBNext Ultra II DNA Library Prep Kit (New England Biolabs; USA) was used to prepare genomic libraries, and the NEBNext Multiplex Oligos kit for Illumina (96 index primers, New England Biolabs; USA) was used to index the libraries. Quantitative analysis of the libraries was performed with the help of Quant-iT DNA Assay Kit, High Sensitivity (Thermo Scientific; USA). Sequencing was done with the MiSeq system and MiSeq Reagent Nano Kit v2 (500 cycle) (Illumina; USA) in accordance with the manufacturer's recommendations.
Bioinformatic analysis of bacteriophage genomes
Prokka v1.14.6 software [14] was used to assemble the whole genome sequences of bacteriophages. Rapid Annotation
using Subsystem Technology (RAST) enabled bacteriophage genome annotation [15]. The functions of some open reading frames (ORF) have been predicted using BLASTP (https:// blast.ncbi.nlm.nih.gov/Blast.cgi) and HHpred (https://toolkit. tuebingen.mpg.de/#/tools/ hhpred). Transport RNAs (tRNAs) were searched for with ARAGORN software [16]. The obtained genomes were deposited into the GenBank database under numbers MZ553931 and MZ553930 for bacteriophages vB_ PaeA-55-1w and vB_PaeM-198, respectively.
The taxonomy of the studied bacteriophages was determined based on the homology of their genomic sequences with the sequences of bacteriophages registered in the GenBank database using the BLASTN service (https://blast.ncbi.nlm.nih.gov/Blast. cgi). Naming of the bacteriophages followed ICTV (International Committee on Taxonomy of Viruses) recommendations [17] and depended on their taxonomic position.
Phylogenetic analysis of the genomes was performed with the help of VICTOR online tool (Genome-BLAST Distance Phylogeny); the settings were as recommended for prokaryotic viruses [18]. The branches were processed with FASTME [19] according to the DO formula, visualization done with FigTree [20]. For bacteriophage vB_PaeA-55-1w, we used the following bacteriophage genomes (GenBank database numbers) for comparison: NC_054890, NC_047953, NC_047967, NC_048201, NC_047965, NC_026602, NC_027375, NC_047956, NC_047957, NC_031014, NC_016764, NC_030923, NC_028836, NC_013638, NC_004665,
А
B
I Pseudomonas aeruginosa Pseudomonas agarici
| Pseudomonas sp Pseudomonas syringae
Pseudomonas fluorescens Pseudomonas tolaasii
Pseudomonas p
Pseudomonas aeruginosa Pseudomonas fluorescens
Pseudomonas sp Pseudomonas syringae
Pseudomonas putida
Fig. 1. Phylogenetic analysis of the whole genome sequences of Pseudomonas spp bacteriophages A. Phylogenetic tree of the Autographiviridae family bacteriophages (n = 50). B. Phylogenetic tree of the Myoviridae family bacteriophages (n = 60). Bacteriophages vB_PaeA-55-1w and vB_PaeM-198 are marked in red
NC_047922, NC_009936, NC_022091, NC_047933, NC_047997, NC_024362, NC_023005, NC 048168,
NC_01 5264, NC_009935, NC_047955, NC_047952, NC_01 5208, NC_021 062, NC_047981, NC_048200,
NC_01 241 8, NC_01 0326, NC_047852, NC_027292, NC_005045, NC_0421 04, NC_0111 07, NC_047894,
NC_047827 NC_022746 NC_047954 MG250485 NC_047747 NC_028661 NC_0111 05 NC_047873
NC_041885, NC_047826. For bacteriophage vB_PaeM-198 the genomes were as follows: NC_048675, NC_048744 NC_048626, NC_048662, NC_048663, NC_048676 NC_048745, NC_011703, NC_026587, NC_026600 NC_04211 3, NC_01 991 8, NC_028745, NC_028971 NC_048109, NC_041870.1, NC_042080, NC_042079 NC_007623, NC_007810, NC_041968, NC_019450 NC_017674, NC_042054, NC_030934, NC_015272 NC_019935, NC_041865, NC_011165, NC_011166 NC_048699.1, NC_031073, NC_031073, MN871467 NC_041994, NC_041903, NC_041902.1, NC_042060 NC_048806, NC_022096, NC_015294, NC_022967.1 NC_022966, NC_022986, NC_019913, NC_011810 NC_041881, NC_030940, NC_003278, NC_004629 NC_03111 0, NC_041 880, NC_028882, NC_028939 NC_023601, NC_042081, NC_042081, NC_042081 NC_011756, NC_042092, NC_029065, NC_041904.
Modular structure of the genomes was determined based on the annotation and when establishing homology of the nucleotide sequences of individual ORFs with BLASTN (https:// blast.ncbi.nlm.nih.gov/Blast.cgi).
RESULTS
Isolation of bacteriophages and characterization of the lytic capability range
To isolate bacteriophages active against the P. aeruginosa species we selected strains PA55 and PA198 from the bacterial strains collection of the Federal Research and Clinical Center of Physical-Chemical Medicine (Table 1 ). These host strains allowed isolating two bacteriophages, which were later named vB_PaeA-55-1w and vB_PaeM-198. As for their lytic capability, vB_PaeA-55-1 w phage caused lysis of 1 9 strains of the collection (47.5%) and vB_PaeM-198 lysed 20 strains (50%) (Table 2). It should also be noted that out of 17 MDR strains vB_PaeA-55-1w lysed 8 (47%) and vB_PaeM-198 — 6 (35%).
Whole genome sequencing of the bacteriophages
Detailed characterization of the studied bacteriophages relied on the whole genome sequencing data and annotation thereof. Genomes of the bacteriophages were double-stranded DNA 42.5 kbp (vB_PaeA-55-1 w) and 66.3 kbp (vB_PaeM-198) long. Bacteriophage vB_PaeA-55-1w encoded 52 ORFs, while vB_PaeM-198 encoded 95 ORFs. None of the analyzed bacteriophages contained tRNA in the genome.
Taxonomic position of the bacteriophages and their closest relative were established by comparing the obtained genome-wide sequences with the genomes available in the Genbank database. Bacteriophage vB_PaeA-55-1w belonged to the
vB PaeA 55 1W
DNA putative
binding DNA
domain-containing primase protein
putative DnaG-like
Im
DNA
helicase
putative exonuclease
primase synthetase DNA
.L polymerase A 1
putative 3'-5' T3/T7-like putative putative putative putative capsid putative
DNA exonuclease RNA acetyltransferase structural head-tail scaffolding protein tail tail endonuclease
tail terminase
putative Class
fibre fibre fibre small putative " j protein protein protein subunit dna holin . protein III/ \
^ 1 1 11 maturase \
vB_PaeM_198-1
subunit scaffold
protein
putative
DNA helicase
_______
putative putative assembly polynucleotide DNA protein kinase polymerase
epsilon • subunit
10000
putative putative putative putative putative putative putative structural putative tail putative
DNA helicase DNA tail tail structural baseplate protein lytic fiber structural
helicase / ligase putative fiber fiber protein protein f tail protein
ail putative structural putative
ber structural protein structural
Dtein protein protein
putative putative putative structural structural minor protein
protein
I Hypothetical protein M Lysis M DNA metabolism M Morphogenesis and DNA packaging | Another function
Fig. 2. Main structural modules of the genomes of vB_PaeA-55-1w and vB_PaeM-198 P. aeruginosa bacteriophage
Phikmvvirus genus of the Autographiviridae family, and the closest genome corresponded to the Pseudomonas phage MYY9 (95% similarity, 97.59% alignment length, Genbank number — MW406975.1). Bacteriophage vB_PaeM-198 belonged to the Pbunavirus genus of the Myoviridae family and was highly similar to Pseudomonas phage phiKT28 (99% similarity, 96.34% alignment length, Genbank number — KP340287.1).
Phylogenetic analysis of Pseudomonas spp. bacteriophages
To position the studied bacteriophages within their respective families we relied on the reference genomes recommended by ICTV [17]. Phylogenetic analysis of the Autographiviridae family included 50 genomes of Pseudomonas spp. bacteriophages (Fig. 1A), that of the Myoviridae family — 60 genomes (Fig. 1B).
Two large clusters can be identified on the phylogenetic tree of Pseudomonas spp. bacteriophages belonging to the Autographiviridae family (Fig. 1A). The first cluster includes bacteriophages that have P. aeruginosa species as hosts, and Pseudomonas agarici, Pseudomonas putida and Pseudomonas fluorescens bacteriophages (one of each). It should be noted that phylogenetic analysis results match taxonomic classification of these viruses. Within the first cluster, P. aeruginosa bacteriophages are grouped separately and belong to the Phikmvvirus genus, including the investigated vB_PaeA-55-1w bacteriophage. The only exception is Pseudomonas phage LKA1, which has its own branch on the phylogenetic tree. The taxonomy has this bacteriophage belonging to another Stubburvirus genus. As for Pseudomonas agarici, Pseudomonas putida and Pseudomonas fluorescens in the first large cluster, each of them also occupies an individual branch of the phylogenetic tree and belongs to a separate taxon, namely, genera Kirikabuvirus, Tunggulvirus, and Kantovirus, respectively. The second large cluster on the phylogenetic tree is comprised of the bacteriophages that have Pseudomonas syringae, Pseudomonas tolaasii, Pseudomonas putida, Pseudomonas fluorescens as hosts, as well as Pseudomonas genus bacteria with unestablished
species identity. Phylogenetic subgroups of the second cluster are also consistent with genera of their bacteriophages, namely Pifdecavirus, Ghunavirus, Troedvirus, Pollyceevirus, Phutvirus, Napahaivirus, Pijolavirus, Pifdecavirus, Bifseptvirus, Uliginvirus. It should be noted that several genera within the family had hosts belonging to different species: Bifseptvirus — hosts of P. syringae and P. tolaasii; Ghunavirus — hosts of P. fluorescens, P. putida, P. syringae.
P. aeruginosa bacteriophages of the Myoviridae family were found in different parts of the phylogenetic tree and corresponded to seven genera: Baikalvirus, Citexvirus, Elvirus, Nankokuvirus, Pakpunavirus, Pbunavirus and Phikzvirus (Fig. 1B). The studied vB_PaeM-198 bacteriophage shares the position on the phylogenetic tree with 26 other Pbunavirus bacteriophages. P. fluorescens, P. putida, P. syringae bacteriophages of the Myoviridae families belong to 6 different genera: Chakrabartyvirus, Flaumdravirus, Noxifervirus, Otagovirus, Plaisancevirus and Tabernariusvirus.
Modular structure of the vB_PaeA-55-1w and vB_PaeM-198 bacteriophages
To describe the genomic organization of bacteriophages vB_PaeA-55-1 w and vB_PaeM-198, we analyzed functional modules of the genomes. It should be noted that the number of genes the functions of which were identified was higher for the vB_PaeA-55-1w bacteriophage (n = 30/52, 58%) than for vB_PaeM-198 (n = 24/95, 25%) (Fig. 2). The analysis also yielded localization of the modules of nucleic acid metabolism and morphogenesis and packaging. The location of the lysis module was established for the vB_PaeA-55-1w bacteriophage. Targeted search revealed no known bacterial toxins and various integrases in the vB_PaeA-55-1w and vB_ PaeM-198 genomes.
DISCUSSION
Due to antibiotic crisis associated with the spread of MDR and XDR bacteria, P. aeruginosa bacteriophages are used in therapeutic practice more and more often. Infections caused
by P. aeruginosa are most often treated with phages of the Autographiviridae and Myoviridae families [22, 23]. For this study, we isolated Autographiviridae (VB_PaeA-55-1w) and Myoviridae (vB_PaeM-198) bacteriophages from natural sources; these bacteriophages offer a wide range of lysing capability (47.5% and 50%, respectively), which is comparable to the P. aeruginosa bacteriophages of the corresponding families [24, 25]. It should be noted that the bacteriophages lysed various strains, which can make therapy more efficient with a phage cocktail that includes both of the studied bacteriophages.
It should also be emphasized that the bacteriophages caused lysis of the strains that belong to different sequence types, including ST235 (n = 1), ST244 (n = 4), and ST395 (n = 1). Isolates belonging to these sequence types are among the most widespread throughout the world; they are often associated with outbreaks of infectious diseases, and they have higher resistance to antibacterial drugs [26]. It seems interesting to further study the lytic capability of vB_PaeA-55-1w and vB_PaeM-198 on a collection of S. aureus strains of the high epidemic risk sequence types.
Current requirements for therapeutic drugs prescribe describing them in detail, and in the case of bacteriophages, it is also necessary to determine their genomic sequences [27] and thus confirm the virulent nature thereof through showing there are no integrase genes in their genomes. Temperate bacteriophages are not used for therapy because they can facilitate transfer of genes of bacterial toxins and determinants of antibiotic resistance in the bacterial population [27]. Besides, in order to assess therapeutic safety of a bacteriophage its genome is searched for genes of known toxins [27].
The studied bacteriophages (both families) were shown to have typical modular genome structure [24, 28], including
a nucleic acid metabolism module, a morphogenesis and packaging module. In addition, localization of the lysis module was established for vB_PaeA-55-1w (Autographiviridae). In case of the vB_PaeM-198 (family Myoviridae) bacteriophage, no genes highly similar to the known genes of lysines or cholines were found. Both studied bacteriophages were found to have no lysogeny module with integrase genes, which confirms their virulent nature, nor were they established to contain known genes of toxins, which makes them potentially usable in therapy.
Based on the results of phylogenetic analysis of Pseudomonas spp. bacteriophages belonging to the Autographiviridae and Myoviridae families, it was shown that P. aeruginosa bacteriophages, regardless of their family, form separate clusters on phylogenetic trees that correspond to genera Phikmvvirus and Stubburvirus (family Autographiviridae), as well as Elvirus, Nankokuvirus, Pakpunavirus, Pbunavirus, Phikzvirus, Phitrevirus (Myoviridae family). This fact indicates that P. aeruginosa bacteriophages are species-specific. On a separate note, bacteriophages previously described and used in phage therapy (phiKMV, PPA-ABTNL, MPK6, RLP), including ФЫИ-4 (Pbunaviruses) and PAK_P1 (Pakpunavirus), have shown their efficacy in animal models [29, 30], and they also cluster together with the studied vB_PaeA-55-1w and vB_PaeM-198 bacteriophages.
CONCLUSIONS
Based on the analysis performed, bacteriophages vB_PaeA-55-1w and vB_PaeM-198 can be recommended for use in phage therapy, including the protocols designed to combat infections caused by MDR strains of P. aeruginosa.
References
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28. Guo Y, Chen P, Lin Z, Wang T. Characterization of Two Pseudomonas aeruginosa Viruses vB_PaeM_SCUT-S1 and vB_PaeM_SCUT-S2. Viruses. 2019; 11 (4): 318. DOI: 10.3390/ v11040318.
29. Alemayehu D, Casey PG, McAuliffe O, Guinane CM, Martin JG, Shanahan F, et al. Bacteriophages ^MR299-2 and ^NH-4 can eliminate Pseudomonas aeruginosa in the murine lung and on cystic fibrosis lung airway cells. MBio. 2012; 3 (2): e00029-12. DOI: 10.1128/mBio.00029-12.
30. Debarbieux L, Leduc D, Maura D, Morello E, Criscuolo A, Grossi O, et al. Bacteriophages can treat and prevent Pseudomonas aeruginosa lung infections. J Infect Dis. 2010; 201 (7): 1096-104.
Литература
1. Tacconelli E, Carrara E, Savoldi A, Harbarth S, Mendelson M, Monnetet DL, et al. Discovery, research, and development of new antibiotics: the WHO priority list of antibiotic-resistant bacteria and tuberculosis. Lancet Infect Dis. 2018; 18 (3): 318-27.
2. Horcajada JP, Montero M, Oliver A, Sorlí L, Luque S, Gómez-Zorrilla S, et al. Epidemiology and treatment of multidrug-resistant and extensively drug-resistant Pseudomonas aeruginosa infections. Clin Microbiol Rev. 2019; 32 (4).
3. Kuzmenkov AY, Trushin IV, Vinogradova AG, Avramenko AA, Sukhorukova MV, Malhotra-Kumar S, et al. AMRmap: an interactive web platform for analysis of antimicrobial resistance surveillance data in Russia. Front Microbiol. 2021; 12: 620002.
4. Pena C, Cabot G, Gómez-Zorrilla S, Zamorano L, Ocampo-Sosa A, Murillas J, et al. Influence of virulence genotype and resistance profile in the mortality of Pseudomonas aeruginosa bloodstream infections. Clin Infect Dis. 2015; 60 (4): 539-48.
5. 2020 Antibacterial agents in clinical and preclinical development: an overview and analysis. Geneva: World Health Organization, 2021. Available from: https://www.who.int/publications/i/ item/9789240021303.
6. Gordillo Altamirano FL, Barr JJ. Phage therapy in the postantibiotic era. Clinical Microbiology Reviews. 2019; 32 (2): e00066-18. DOI: 10.1128/CMR.00066-18.
7. Акимкин В. Г., Дарбеева О. С., Колков В.Ф. Бактериофаги: исторические и современные аспекты их применения: опыт и перспективы. Клиническая практика. 2010; 1 (4): 48-54.
8. Chen F, Cheng X, Li J, Yuan X, Huang X, Lian M, et al. Novel lytic phages protect cells and mice against Pseudomonas aeruginosa infection. J Virol. 2021; 95 (8): e01832-20. DOI: 10.1128/ JVI.01832-20.
9. Jault P, Cheng X, Li J, Yuan X, Huang X, Lian M, et al. Efficacy and tolerability of a cocktail of bacteriophages to treat burn wounds infected by Pseudomonas aeruginosa (PhagoBurn): a randomised, controlled, double-blind phase 1/2 trial. Lancet Infect Dis. 2019; 19 (1): 35-45.
10. Oechslin F. Resistance development to bacteriophages occurring during bacteriophage therapy. Viruses. 2018; 10 (7): 351. DOI: 10.3390/v10070351.
11. Купцов Н. С. и др. Эффективность препаратов бактериофагов против патогенов группы ESKAPE. Вестник РГМУ. 2020; (3): 19-26.
12. Mazzocco A, et al. Enumeration of bacteriophages using the small drop plaque assay system. Methods Mol Biol. 2009; 501: 81-85.
13. Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Pr. 1989, 2200 p.
14. Seemann T. Prokka: Rapid prokaryotic genome annotation. Bioinformatics. Oxford University Press. 2014; 30 (14): 2068-9.
15. Aziz RK, et al. The RAST Server: Rapid annotations using
subsystems technology. BMC Genomics. BioMed Central. 2008; 9: 75.
16. Laslett D. ARAGORN, a program to detect tRNA genes and tmRNA genes in nucleotide sequences. Nucleic Acids Res. 2004; 32 (1): 11-16.
17. Lefkowitz EJ, et al. Virus taxonomy: The database of the International Committee on Taxonomy of Viruses (ICTV). Nucleic Acids Res. 2018: 46 (1): 708-17.
18. Meier-Kolthoff JP, Göker M. VICTOR: genome-based phylogeny and classification of prokaryotic viruses. Bioinformatics. 2017; 33 (21): 3396-404.
19. Lefort V, Desper R, Gascuel O. FastME 2.0: A comprehensive, accurate, and fast distance-based phylogeny inference program. Mol Biol. 2015; 32 (10): 2798-800.
20. FigTree. Available from: http://tree.bio.ed.ac.uk/software/figtree/. (Дата обращения: 16.07.2021).
21. Enright MC, et al. Multilocus sequence typing for characterization of methicillin-resistant and methicillin-susceptible clones of Staphylococcus aureus. J Clin Microbiol. 2000; 38 (3): 1008-15.
22. Alvi IA, et al. RLP, a bacteriophage of the family Podoviridae, rescues mice from bacteremia caused by multi-drug-resistant Pseudomonas aeruginosa. Arch Virol. 2020. 165 (6): 1289-97.
23. Farlow J, et al. Complete Genome Sequences of 10 Phages Lytic against Multidrug-Resistant Pseudomonas aeruginosa. Microbiol Resour. 2020. 9: 29.
24. Alvi IA, Asif M, Rehman S. A single dose of a virulent bacteriophage vB PaeP-SaPL, rescues bacteremic mice infected with multi drug resistant Pseudomonas aeruginosa. Virus Res. 2021; 292: 198250.
25. Adnan M, et al. Isolation and characterization of bacteriophage to control multidrug-resistant Pseudomonas aeruginosa planktonic cells and biofilm. Biologicals. 2020; 63: 89-96.
26. Treepong P, et al. Global emergence of the widespread Pseudomonas aeruginosa ST235 clone. Clin Microbiol Infect. 2018; 24 (3): 258-66.
27. Principi N, Silvestri E, Esposito S. Advantages and Limitations of Bacteriophages for the Treatment of Bacterial Infections. Front Pharmacol. 2019; 10: 513.
28. Guo Y, Chen P, Lin Z, Wang T. Characterization of Two Pseudomonas aeruginosa Viruses vB_PaeM_SCUT-S1 and vB_PaeM_SCUT-S2. Viruses. 2019; 11 (4): 318. DOI: 10.3390/ v11040318.
29. Alemayehu D, Casey PG, McAuliffe O, Guinane CM, Martin JG, Shanahan F, et al. Bacteriophages фMR299-2 and фNH-4 can eliminate Pseudomonas aeruginosa in the murine lung and on cystic fibrosis lung airway cells. MBio. 2012; 3 (2): e00029-12. DOI: 10.1128/mBio.00029-12.
30. Debarbieux L, Leduc D, Maura D, Morello E, Criscuolo A, Grossi O, et al. Bacteriophages can treat and prevent Pseudomonas aeruginosa lung infections. J Infect Dis. 2010; 201 (7): 1096-104.
