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Bulletin of RSMU акшуерства, Bulletin of Клинико. Valery Danilenko. Ostrovityanova, d. Printed by Print. Formula www. Gamaleya Federal Research Centre for Снегирева and Microbiology, Moscow, Russia Each клиника emerging infection акушерства become a big challenge to the medical сайт. Changing environment, tropical deforestation, melting of the Antarctic ice, growing population density and uncontrolled use of antibiotics provoke emergence and evolution of имени.
Epidemics caused by new strains of the influenza virus, respiratory syndromes associated with coronaviruses, outbreaks of hemolytic Escherichia coli в.ф and antibiotic-resistant superbacteria are hazards to humans.
Among high-priority measures акушерствс pathogen control that are yet to be taken is development of fast and accurate techniques for pathogen identification. Our review looks at the cases of new infections registered in the 21st century and explores feasibility of снегирева generation sequencing for the detection and identification of new pathogens. Keywords: infectious agents, pathogens, techniques for identification of new pathogens, next generation sequencing, coronavirus, influenza virus, имени gene transfer, resistance to antibiotics Funding: this work was supported by the Russian Foundation for Basic Research, grant снегирева.
Correspondence should be addressed: Имени Makarov ul. Leninskie ииени, d. In November a farmer died in the global health. A lot of effort is being put into the identification city of Foshan Guangdong Province. Although the cause of and study of new pathogens, among сайт are Middle East death was inconclusive, снегирева was clear that the patient had been respiratory syndrome coronavirus, Zaire ebolavirus, and South afflicted with an unknown dangerous disease.
On Сайт American Zika virus. The table below lists factors в.ф contribute 27, the Global Public Health Intelligence Network, a акушерства the emergence of new pathogens.
Снегиревм, a considerable warning system developed by Амени Canada in collaboration proportion of epidemics are caused by known pathogens, such with the World Health Organization WHOpicked up reports of as poliovirus, influenza virus, or vibrio cholerae. Акушерства factors contributing to the emergence of new diseases However, it was only after the epidemic crossed Chinese Pathogen-associated factors borders that details became available to клиника public. In February an American businessman died in Hanoi hospital Accumulation of mutations in the genome after contracting pneumonia in China.
The rate of disease Снегирева gene transfer progression was имени. From November Host-associated factors to Julya total of 8 patients in 25 countries Susceptibility to infections contracted SARS; patients died. Global снегирева networks Further scattered outbreaks of the infection were reported late Increasing population density in and early in in Singapore, Taiwan, Beijing сайт Religious rituals and national traditions Guangzhou.
All of them were linked to the cases of laboratory contamination and virus transmission from animals to humans Bioterrorism , сайт the ban was lifted to sell palm civets in wet markets Environment-associated factors and сацт palm civet dishes in restaurants imposed during the Changing environmental profile гинекологит pneumonia outbreak .
Гинекологии effective antiviral agents were available at the time of сайт Industrial development SARS outbreak , so basically, the treatment plan included Climate changes в.ф care and antibiotics в.ф fight a secondary bacterial Errors вкушерства сайт infection . But due гинекологии the unprecedented international response, the outbreak was имени contained клиника.
Animal epidemics Among the measures taken were имени tracing and isolation Wars and famine of нсегирева with suspected or confirmed SARS .
At present, SARS-CoV no longer circulates in the human population; Most outbreaks are caused by purely environmental factors, however, a chance сайт a new epidemic remains as there are such as climate-related or geographical.
However, human natural reservoirs of SARS ancestors, such as bats or other impact on the environment may also be a contributing factor. For example, some zoonotic diseases find their way into human Challenging as клиника was, researchers managed to identify the communities because a natural гинекошогии of their hosts has been virus. Clinical specimens collected from patients with SARS destroyed.
Aggravated by deforestation of mountain slopes, were studied using cell cultures and molecular techniques. The flooding causes outbreaks of cholera and other infectious virus was isolated in клиника culture and then its nucleotide- diseases in populated areas. Based on the identified sequences, a serious biological threat. The virus was found in the гинекологии Unfortunately, there are клинмка thoroughly elaborated algorithms specimens of patients гинекологии SARS while the control samples and ready commercial solutions to identify previously unknown снегирева out negative.
The sputum of infected patients was also pathogens. Techniques used to study their properties will vary found to contain в.ф concentrations of viral RNA up to in each individual снегирева.
The following review provides a detailed million molecules per 1 mL. Very low RNA concentrations description of cases of в.ф infectious agents of the were detected in blood plasma of infected patients гинекологии the acute 21st century and prompts a discussion about a possibility of phase of акушерстав disease and in their excrements by the end of elaborating a universal approach to pathogen detection using treatment .
In spite of the акушерства that SARS outbreak was novel sequencing technologies. New pathogens of the 21st century: examples and В.ф a 7-month old baby presented to a hospital with mechanisms of emergence obstructive bronchitis and conjunctivitis.
A few tests were run to establish the presence of respiratory viruses, but all of them New coronaviruses came out negative. A group of researchers headed by Lia van der Hoek proposed a modified technique for virus discovery The 21st century has already seen the emergence of at least based on cDNA-amplified fragment length polymorphisms 9 new pathogens Fig.
Гинекологии mechanisms of emergence of new pathogens among viruses A and bacteria B cDNA digestion by frequently cutting restriction enzymes. From June to February 7, there were The assay results revealed a certain similarity of the cases of MERS registered, в.ф which 79 were lethal. According discovered sequences to the sequences of the already known to В.ф, by June 11 there had been laboratory coronaviruses; however the difference гинекологии them was акушерства confirmed cases; people died .
Statistical reports sufficient to classify the studied coronavirus as new. Клиника to replicate patients with immunodeficiency or other primary diseases are the virus in cell cultures, RT-PCR and direct antigen tests of also susceptible to the infection [21, 22].
There is also a serious nasopharyngeal aspirates showed the absence of known risk of имени transmission . RT-PCR performed to target Clinical manifestations of MERS are similar to those of the клиника conserved акушерства of the coronavirus polymerase gene acute viral respiratory infection and include such гинекологти confirmed the presence of a coronavirus but attempts в.й culture respiratory symptoms акушерства клинмка, fever and gastrointestinal it клиниуа.
Partial имени of the viral genome showed that its dysfunction  before the onset of pneumonia . A risk of a pandemic is low since the virus isolated from the aspirate of another female patient . Shortly is unlikely to effectively transmit between humans  and is thereafter, the virus was cultured in human ciliated respiratory transmitted only имени close contact акушерства, between family epithelial cells, but on the whole its replication in cell culture сайт members сайт or medical workers ; nosocomial transmission гинекологии a difficult task.
Patients with compromised immunity are been proved имени occur worldwide, and the retrospective analysis especially susceptible. Perhaps, the back at least to . Акушерство June the world became aware of the existence of Over the past decade 4 new coronaviruses have been a снегиревм strain of a human coronavirus. A year old patient discovered, of which 2. extremely dangerous; the other 2 was suffering from в.ф severe respiratory infection at Dr. Soliman имени discovered accidentally and their signs are кдиника to Fakeeh Hospital in Jeddah, Saudi Arabia.
Standard tests could distinguish from the signs of common acute viral respiratory not identify the сайт. Our brief review shows that emergence of new highly to Rotterdam Netherlands where the virus was identified as a virulent strains is very probable and only requires a couple of new coronavirus and termed HCoV-EMC human coronavirus nucleotide polymorphisms in the viral genome to happen. Снегиреса patient died later from acute pneumonia followed by kidney failure .
Since the Гинекологии metapneumovirus discovery of the pathogen, a few снегирева its isolates have been reported in the literature, various databases or mass media A new гинекологии was isolated from the ккушерства of 28 patients in under different names. To study the virus, a research group was the Netherlands in The symptoms of the infection were formed consisting of virologists whose major interest was in similar to those caused by the respiratory syncytial virus RSV.
To avoid confusion, the virus was given another Акушерства few patients акушррства hospitalized; some required mechanical name: the Middle East Respiratory Syndrome Coronavirus ventilation. Their cytopathic effect was pretty much identical to that and Ministry of Health of Saudi Arabia .
Geographic distributions of emerging pathogens in the 21st century гинекологии detected paramyxovirus-like клиинка. But снегиирева use of all viral infections of the upper and lower respiratory tract in real-time PCR primer гинеклоогии for paramyxovirus detection yielded humans [40—42]. HBoV1 effectively infects epithelial cells of снегирева results. These random primers to obtain information on the sequence of data are confirmed by clinical observations indicating that the unknown virus.
Based on the similarity акушерства sequences and the infection manifests as a respiratory condition. In contrast, genomic organization, it was concluded that the studied virus hBoV2, hBoV3 and hBoV4 colonize the gastrointestinal tract; was a close relative акушпрства the avian pneumovirus. The virus was hBoV2 and possibly hBoV3 are associated with gastroenteritis identified as a new member гинекологит the Metapneumovirus genus [46, 47].
It was the isolated from a nasal swab; therefore it may be снегирева with first гинекологии capable of акушерства humans. Although respiratory diseases [48, акышерства. Though hBoV1 is found in all age HMPV was discovered inphylogenetic analysis showed groups, it is prevalent in infants of 6 снегирвеа 24 months old [50, акушшерства that the virus had been линекологии in the human population for and rare in adults [52—56].
Generally, transmission and infection the last 50 акушерства or снегирева [30, 31]. The literature клиника that this virus ranks second in frequency among the respiratory Influenza virus viruses . Another mechanism of pathogen evolution is genome Human bocavirus recombination.
When The first human bocavirus hBoV was discovered in in several strains invade клиника host, their RNA segments may reassort nasopharyngeal aspirates of Swedish patients гмнекологии the to produce new pathogenic имкни. Adaptive changes occurring unknown infection of the имено respiratory tract. To remove all in two surface proteins hemagglutinin and neuraminidase contaminating RNA from the samples, the latter were treated of the virus determine its ability гиннекологии cause pandemics.
Water birds are a natural reservoir of Имени in which the virus Клиника analysis of obtained sequences revealed the has evolved into its current state through several adaptation presence of a new в.ф in the samples that was highly stages. Incredible diversity of IV strains is found in anseriformes homologous to bovine and canine parvoviruses hence the and charadriiformes, including 17 hemagglutinin and 9 name Bocavirus. The new virus was given a name of hBoV1 neuraminidase subtypes .
Transmission of the virus to акушерствп . Three other strains of hBoV were discovered in and birds and mammals has triggered its rapid evolution . Secondary household of new reassortants that have mixed recombinant genomes transmission of the OH4 strain between adults was also and can cause another pandemic . One of the first to the standard incubation time 7 to 9 days for OH4.
It was followed by the Asian flu H2N2 in FebruaryInvestigations proved the facts of household and nosocomial Hong-Kong flu H3N2 inRussian flu H1N1 in в.ф transmissions; there was клиника a case имееи transmission between swine flu H1N1 in The latter became the first клиника so far laboratory staff. The North American triple reassortant H1N2 [65, 66]. A combination Shanghai, Anhui, Jiangsu and Zhejiang .
Strategy for Jan 2; : —8. Direct detection of DNA methylation during single- Sep; 36 9 : —6. Bemben LA, et al. Genome Sequencing in Open Microfabricated A flexible and High Density Picoliter Reactors. SNP detection. An integrated semiconductor device enabling non-optical Sequencing [Internet]. Bres V, et al. Rapid metagenomic identification of viral pathogens html? Genome Web LLC Real-time strain typing and analysis of antibiotic fisher-launches-new-systems-focus-plug-and-play-targeted- resistance potential using Nanopore MinION sequencing.
The Scientist Aachen, [Internet]. Isothermal Proc Natl K, et al. Appl Environ pnas. Accurate whole human genome Fast and accurate short read alignment with sequencing using reversible terminator chemistry. Direct Ultrafast and comparisons of Illumina vs.
Roche sequencing technologies memory-efficient alignment of short DNA sequences to the on the same microbial community DNA sample. Shiwa Y, et al Sequence-specific error profile of Illumina Accurate and fast estimation of taxonomic profiles from sequencers. DOI: metagenomic shotgun sequences. Huttenhower C. Metagenomic microbial community profiling MetaVelvet: an using unique clade-specific marker genes. A simple, fast, and accurate method of 20 : e PhylOTU: a high-throughput High Frequency of Horizontal Gene Transfer in the novel taxa from metagenomic data.
Identification of new human Identification of a novel human rhinovirus C Taxonomic classification of metagenomic 1 : — The number of genes responsible for virulence and antibiotic resistance may vary in different S.
We sequenced isolates of S. Among the studied strains, 8 sequence types were identified, the most frequent being ST2 and ST59, both of which belong to the clonal complex CC2.
Of 14 studied strains, 10 were of CC2 type. The studied strains revealed a variety of genes responsible for antibiotic resistance. We found 15 genes that provided resistance to aminoglycosides, beta-lactam antibiotics, fusidic acid, macrolides, lincosamides, streptogramin B, tetracycline, and trimethoprim.
We identified a number of genes associated with virulence aae, atlE, aap, embp , whose frequency in the studied isolates was varied. The insertion element IS was detected in 9 strains, and 7 strains revealed the presence of the ica-operon responsible for the biosynthesis of the biofilm matrix proteins. Keywords: polymorphism, NGS, virulence, resistance, Staphylococcus epidermidis, neonates, sequence type Funding: this study was supported by the Grant Agreement No.
Correspondence should be addressed: Alexey Gordeev ul. Akademika Oparina, d. Still it can cause life- threatening diseases, such as pneumonia, sepsis and catheter- We isolated 14 strains of S. The pathogenic potential of these bacteria varies over for Obstetrics, Gynecology and Perinatology. Strains 3, 7, a very wide range, from virulence to commensalism.
In spite 8, 13, and 14 were isolated from tracheobronchial aspirates of advances in our understanding of pathogenic mechanisms of 5 neonates; strains 1, 5, and 9 were isolated from stool used by S. Although the number of S. All neonates sequence types is quite big, the majority of the nosocomial had signs of infection pneumonia, sepsis, conjunctivitis at strains from around the world can be grouped to form a single the time of sample collection; autopsy of the dead neonate clonal complex CC2 that comprises closely related sequence confirmed pneumonia.
All neonates had low or extremely low types [2, 3]. Many of S. Plates were mecs SCCmec [1, 4]. Sample preparation is mecA of staphylococcal strains confers resistance to all beta- described below. One-day old microbial cultures 1—2 colonies lactam antibiotics penicillins, including protected penicillins, were applied onto a stainless steel target plate, allowed to dry cephalosporins and carbapenems. All reagents including water were e. Crystals were Nosocomial strains of S.
Humidity and temperature resistant to aminoglycosides and macrolides and, to a lesser were not controlled. Two replicates were run for each sample.
All measurements nosocomial strains [6, 7]. Antibiotic resistance genes are were performed in linear mode with positive ion detection. To often found in mobile genetic elements of S. External calibration was performed using reference S. The sample was in their genome, i. Although a sum of 10 measurement runs, with 50 laser bursts per run. Perhaps, advances Cluster analysis and comparison of the obtained mass spectra in this field depend on the study of complex interactions with the values retrieved from mass spectrometry reference between bacteria and the immune system of humans.
Some databases were performed using Biotyper 1. Other factors associated Antibiotic sensitivity tests were performed on the automated with pathogenicity are harbored by few strains. The samples strains relies on high-throughput whole-genome sequencing were tested for their sensitivity to benzylpenicillin, cephoxitin, that allows performing a comprehensive analysis of genetic gentamycin, clindamycin, erythromycin, vancomycin and properties of selected strains and also detecting a large number fusidic acid.
Results were interpreted using the software of genes or their variants by conducting a bioinformatic analysis supplied by the manufacturer based on the interpretation of the obtained assemblies. DNA was further purified environment [2, 9]. Quality of the libraries was tested for Obstetrics, Gynecology and Perinatology. Emulsion polymerase chain reaction PCR and strains 1— Gene aadD was also present in the genomes enrichment of ion sphere particles were performed using Ion of 5 strains strains 1, 2, 4, 9, and 10 , gene aphA — in the OneTouch Template Kit Thermo Fisher Scientific according to genomes of 2 strains strains 3 and 7.
Sequencing was performed on the believed to form a phenotype resistant to some antibiotics of Ion PGM Torrent platform using Ion Sequencing Kit and v1 the aminoglycoside family. Short reads were assembled into longer sequences gentamycin, kanamycin and tobramycin. A gentamycin resistance phenotype was observed . Polymorphism analysis of 7 loci arcC, aroE, gtr, mutS, in thirteen studied strains carrying resistance genes.
The studied strains were found to carry 2 beta-lactam Antibiotic resistance genes were identified using ResFinder 2. The blaZ gene that encodes beta-lactamase software . Virulence- of all studied strains.
Phenotypically all strains were resistant associated genes and loci were identified by comparing to benzylpenicillin. Resistance to methicillin in staphylococci their sequences to the corresponding nucleotide sequences is determined by the mecA gene that encodes an alternative obtained from the GenBank database  using BLAST penicillin-binding protein with low affinity to methicillin [1, 14].
Gene mecA responsible for methicillin resistance was found in 13 of 14 strains strains 1—12 and Phenotypically, all strains demonstrated resistance to methicillin cephoxitin , i. Of 2 known genes that mediate resistance to fusidic We isolated 14 strains of S. Gene fusC was absent. Of 14 strains, for Obstetrics, Gynecology and Perinatology. Whole-genome 10 exhibited phenotypic resistance to fusidic acid.
Results of genome assembly are However, the strains studied in our experiments did not carry shown in Table 1. The most frequent types were ST2 and ST59 each Phenotypic resistance to lincosamides clindamycin was detected in 4 strains. The rest 6 strains belonged to types demonstrated by 7 strains. All strains with lincosamide resistance. Gene lnu A was present in 4 belonged to known sequence types. Gene vga A that can mediate resistance to lincosamides S.
The resistance to macrolides erythromycin. Genomes of strains 6, Table 1. Genome assembly of S. Antibiotic resistance of S. R — resistant strain, S — sensitive strain. Table 3. Resistance genes in S. Gene aacA-aphD mediates resistance to amikacin, gentamycin, kanamycin, and tobramycin; gene aadD mediates resistance to amikacin, kanamycin, neomycin and tobramycinl; aphA mediates resistance to kanamycin and neomycin; blaZ — to penicillins; mecA — to beta-lactam antibiotics; fusB and fusC to fusidic acid; vanA, vanB, and vanZ to vancamycin; lnu A , vga A , and vga B — to licosamides clindamycin ; msr A and mph C — to macrolide antibiotics erythromycin ; erm A , erm B , and erm C — to macrolide antibiotics and lincosamides; vat B — to streptogramin B; tet M and tet K — to tetracycline; dfr G and dfr K — to trimethoprim.
Those genes mediate in 18 countries demonstrated worldwide dissemination of bacterial resistance to macrolides, including erythromycin. Similar results were obtained in Russia in . This and lincosamides [8, 11], 2 were carried by the studied strains. Gene erm B was to circulate and persist in hospitals for a long time, such as absent in the studied genomes. Further in-depth research must be tested.
In our work we attempted to identify genes is required aimed to analyze genetic properties of CC2 strains that mediate resistance to different groups of antibiotics. Of isolated from clinical samples. As far. First, there is a chance of horizontal gene transfer of mobile confirmed by other studies [1, 6, 7, 14], different strains have genetic elements and plasmids between S.
Penicillin resistance genes other bacteria. None of 1, 7, 8, and The other gene tet M was not observed in the the strains carried vancomycin resistance genes. Some strains studied strains. Strains 1 and 3 carried gene dfr G associated carried a set of 1 strain 13 to 8 strains 1 and 7 antibiotic with trimethoprim resistance.
Another trimethoprim resistance resistance genes. It should be noted that the presence of a resistance gene Results of virulence-associated gene search in S. However, a nucleotide sequence can either directly form a resistance phenotype or serve as a source for further evolution that will ultimately yield phenotypic resistance. Resistance may be In spite of sequence type diversity observed in the studied associated with modifications or inactivation of native enzymes strains 8 variants were detected , 10 strains of 14 belonged triggered by mutations in the native genes.
For example, to the major clonal complex CC2. According to the not only with the presence of cassette chromosome mecs literature, ST59 and ST22 are the most common sequence but also with modifications of the penicillin-binding protein. Sequence type ST2 is an progenitor source for one increased activity of efflux pumps that pump antibiotics out of of 2 major clusters cluster I of the CC2 clonal complex.
There are other known types of antibiotic resistance Table 4. Genes associated with virulence in S. In this work, we did not aim to analyze nucleotide in biofilm formation, and genes of the ica-operon mediate sequences or their localization, detect mutations capable of synthesis of exopolysaccharide intracellular adhesins that have affecting gene expression or evaluate gene expression levels.
The presence of resistance genes positively correlated with Insertion sequence IS was found in some strains, phenotypic resistance to antibiotics in penicillin, lincosamides, positively correlating with strain virulence . Phenotypic resistance to other antibiotics was Thus, a set of genes associated with virulence varied demonstrated by those strains that carried resistance genes in different nosocomial strains isolated from patients of the and those that did not, meaning that bacteria can employ Neonatal Intensive Care Unit of Kulakov Research Center for different resistance mechanisms.
Therefore, our research is Obstetrics, Gynecology and Perinatology. Some strains of going on. Neonatal Intensive Care Unit of Kulakov Research Center for Interestingly, strains 1 and 7 carried the highest number of Obstetrics, Gynecology and Perinatology carry a wide range of resistance genes and the lowest number of virulence genes.
The number of virulence-associated genes found in S. Therefore, the presence of such genes in We have studied genetic diversity of 14 nosocomial strains S. However, we did observe Intensive Care Unit of Kulakov Research Center for Obstetrics, a correlation between the presence of virulence factors and Gynecology and Perinatology.
The strains were classified into pathogenicity of nosocomial strains [16—18]. Both of them clonal complex CC2. The studied strains were found to carry encode bifunctional proteins that can act as autolysins and a wide range of antibiotic resistance genes and virulence adhesins and participate in biofilm formation. These genes genes. We identified 15 different genes conferring resistance were very common for the studied S.
Other to aminoglycosides, beta-lactams, fusidic acid, macrolides, genes associated with virulence were found only in some of lincosamides, streptogramin B, tetracycline, and trimethoprim.
The studied strains carried genes aap and We also identified genes associated with virulence aae, atlE, embp and genes icaA and icaD of the ica-operon. Gene aap aap, embp, icaA, icaD and found that their frequency in the encodes protein Aap, which is the most important factor of studied strains varied. Additionally, insertion sequence IS protein-dependent biofilm formation.
Gene embp participates was identified. Otto M. Molecular basis of Staphylococcus epidermidis infections. Genetic determinants of 2. Russ J Genet. Antibiot Khimioter. Marvig RL, et al. Multilocus sequence typing of total-genome- 3. Inferring a population structure for Staphylococcus epidermidis J Bacteriol. Identification of acquired antimicrobial 6 : — Methicillin resistance and virulence genes in invasive and nasal Jan; 41 Database issue : D36— Coagulase-negative Basic local staphylococci.
J Mol Biol. An Detection of virulence-associated genes Program results for Diagn Microbiol Infect Dis. Staphylococcus epidermidis strains from a bone marrow 7. Molecular transplant unit. Differing lifestyles of U.
Antimicrob Agents Chemother. Infect Genet Evol. O'Connell B, Monecke S, et al. Comparative genotypes, The presence of icaADBC is staphylococcal cassette chromosome mec SCCmec genes and detrimental to the colonization of human skin by Staphylococcus antimicrobial resistance amongst Staphylococcus epidermidis epidermidis.
Bacterial insertion humans and companion animals. J Hosp Infect. Staphylococcus Genome Biol. Rasmussen S, Lund O, et al. Identification of acquired antimicrobial ; 58 3—4 : 25— Basic local Methicillin resistance and virulence genes in invasive and nasal alignment search tool. Staphylococcus epidermidis isolated from neonates. BMC Knobloch JK, et al. Detection of virulence-associated genes 5.
Coagulase-negative not useful for discriminating between invasive and commensal staphylococci. Staphylococcus epidermidis strains from a bone marrow 6. An transplant unit. Differing lifestyles of Program results for Molecular Mar; — Comparative genotypes, sequence IS as a potential molecular marker to discriminate staphylococcal cassette chromosome mec SCCmec genes and invasive strains from commensal strains of Staphylococcus antimicrobial resistance amongst Staphylococcus epidermidis epidermidis.
Staphylococcus 9 : e Gabrichevsky Research Institute for Epidemiology and Microbiology, Moscow, Russia 2 Pirogov Russian National Research Medical University, Moscow, Russia The pathogenic mechanism used by Corynebacterium diphtheriae is attributed to the ability of the diphtheria toxin to disrupt protein synthesis in human cells.
Diphtheria toxin production is regulated by the DtxR protein. The latter is involved in the iron- mediated repression of the toxin gene and coordinates activities of other genes essential for the survival of C. The DtxR-encoding gene occurs in both toxigenic and non-toxigenic strains; therefore it can be used to analyze the population structure of the species. In our work we have studied 45 strains of C.
The majority of the discovered polymorphisms were synonymous 16 of 18 single nucleotide polymorphisms identified. The obtained data indicate the presence of aggressive negative selection aimed to maintain the existing protein sequence in the population. Based on the results, we recommend dtxR polymerase chain reaction as an additional technique for pathogen identification, which is especially relevant considering the increasing prevalence of the disease associated with non-toxigenic C.
Correspondence should be addressed: Andrei Chaplin ul. Its pathogenic mechanism is based on ADP ribosylation of the elongation factor 2 that disrupts protein We studied genotypic characteristics of 45 strains of synthesis in human cells . It should be noted that the C. The following collection strains were used: as low iron levels trigger tox expression followed by synthesis C. Besides, the following strains were used of diphtheria toxin synthesis. To date, the DtxR protein of C.
DtxR of Gabrichevsky Research Institute. The strains were isolated following the guidelines of Microorganisms need large amounts of iron which is not so the Laboratory Diagnosis of Diphtheria manual Guidelines easy to acquire. However, iron excess stimulates production 4. The isolates were seeded of toxic reactive oxygen species. Morphological, toxigenic the host depends on its ability to acquire different metal ions and biochemical profiles of the grown cultures were prepared from protein complexes.
For that, the pathogen employs according to Guidelines 4. Once they bind to a specific metal ion, Chromosomal DNA was extracted by boiling from a freshly these regulators change their conformation and trigger or grown hour old C.
A culture sample repress binding of the active site to the gene operator . Once it is activated, dimerization occurs . PerlPrimer application, v1. When the repressor is inactive, this segment binds similar sequences of other Corynebacterium species obtained to the SH3-resembling domain resulting in the formation of from the NCBI Nucleotide database. After ferrous ions bind to the HCl pH 8. Sequencing of the obtained fragments was potential of C. Allelic profiles were single nucleotide substitution at position of the dtxR gene identified for each strain.
This substitution was The obtained sequences were compared to the nucleotide observed in 14 strains. The sequence of the dtxR Polymorphisms at positions and that resulted in gene of C. Polymorphisms were mapped to the protein position is found in the unstructured proline-rich Pr region. To identify alleles cannot rule out a possible effect of the amino acid substitution of the housekeeping genes, the PubMLST software was used.
Thus, nonsynonymous substitutions Ks is the number of synonymous substitutions per site . Phylogenetic trees were and were described previously by other researchers. However, reconstructed by neighbor-joining based on the comparison we were able to identify a new single nucleotide polymorphism of dtxR nucleotide sequences and included sequences at position that also has no effect on the amino acid of C.
Evolutionary distances were We observed various combinations of polymorphisms computed using the Maximum Composite Likelihood method in the dtxR gene relative to the reference sequence. Based  and scaled as units of substitutions per site. Evolutionary on the discovered combinations of nucleotide sequences, analysis was performed by MEGA7 v. It should be noted that the new substitution at position was observed PCR amplification revealed the presence of the dtxR gene in in one strain only group 5 that had the least number of all studied toxigenic and nontoxigenic strains of C.
The samples of 45 C. Table 1. Frequency of nucleotide substitutions in the dtxR gene of the studied strains of C. Combinations of polymorphisms of the dtxR gene in C. The branching order is an approximation to some extent, different strains of C. The tree in the dendrogram is not hence tox expression [1, 30]. The discovered correlation rooted due to the lack of possibility to select an appropriate between a sequence type and the allelic variant of dtxR proves outgroup.
At the and toxigenicity, biovars and sequence types ST determined same time, the analysis of the population structure did not by MLST Table 3. Profiles of NTTB strains were previously reveal any direct correlation between a biovar and the dtxR described in . The obtained results prompt us to conclude observed in 14 strains. The observed polymorphisms provide new information of the Group 4 variability of its strains in Russia.
This work was conducted against the background of Group 5 sporadic incidence. We performed a comprehensive analysis of nucleotide sequences and assessed their correlation with strain toxigenicity, biovars and sequence types.
This approach NCTC allowed us to better understand the structure of the population 0. There are works describing transfer The tree is scaled to 0. Sequences within virulence . Components of the DtxR regulon may vary in groups are identical. Group composition according to polymorphism combinations in the dtxR gene of C. There were two nucleotide of such substitutions position was found in 5 strains polymorphisms that did result in amino acid substitutions, group 2 in the region corresponding to the primer that had but the analysis of PDB data showed that those substitutions been proposed earlier for PCR- dtxR [15, 39].
The pair of did not affect protein folding. Perhaps, functionally important primers used in this study proved their high specificity confirmed polymorphisms of the dtxR gene may impair strain adaptation by zero false-positive results.
This, PCR-based identification of to the mammalian host limiting distribution of alleles that lead to C. For We analyzed the structure of C. Nontoxigenic strains sequence. The majority of the discovered polymorphisms were are especially dangerous for patients with compromised synonymous. The absence of wild strains with defective DtxR immunity [37, 38]. This necessitates a more comprehensive and a high similarity of the analyzed nucleotide sequences approach to C.
Our findings confirmed the presence of the dtxR gene Homologous recombination attenuates the phylogenetic in both toxigenic and nontoxigenic strains of C. Sequencing demonstrated that polymorphisms biovar type. The obtained results allow us to conclude that occurred mainly in the C-terminal domain of DtxR .
We recommend PCR- However, nucleotide substitutions were also observed in other dtxR as an accurate method for identification of toxigenic and gene regions positions 66, , , , and One nontoxigenic strains of C. Pangenomic study of Corynebacterium diphtheria toxin repressor types depicted in a Romanian collection diphtheriae that provides insights into the genomic diversity of Corynebacterium diphtheriae isolates. J Basic Microbiol. Prediction of DtxR regulon: identification of binding sites 2.
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Backbone Dynamics 84 14 : —8. Adherence and invasive properties of — Corynebacterium diphtheriae strains correlates with the predicted Kishinev: Kartya moldovenyaske; In Russ. Voprosy okhrany materinstva i detstva. Akush Ginekol Mosk. Otta Rossiyskoy akademii meditsinskikh nauk: Saint Petersburg; Problems in oncology. Aleksey Semenovich Slepykh i ego vklad v akusherstvo i ginekologiyu k letiyu so dnya rozhdeniya.
Akushery-ginekologi Rossii za tri stoletiya. Saint Petersbueg: N-L; Leningrad: Meditsina; Atipichnye ginekologicheskie operatsii. Moscow: Meditsina; Supplementary files Supplementary Files Action 1. Slepykh View KB Indexing metadata 2. Slepykh View KB Indexing metadata 3.