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ВНУТРИВИДОВОЕ
ГЕНЕТИЧЕСКОЕ
РАЗНООБРАЗИЕ
НОЗОКОМИАЛЬНЫХ
ОППОРТУНИСТИЧЕСКИХ
ПАТОГЕНОВ: КЛИНИЧЕСКОЕ
ЗНАЧЕНИЕ (МИНИОБЗОР)
1Тараскина А.Е. (зав. лаб.)*, 2 Ли Фан (ректор научного центра),1Скорбунова О.В. (лаборант-исследователь), 1,3Пчелин И.М. (н.с., аспирант), 3Степанов А.С. (аспирант), 3Рябинин И.А. (аспирант), 1Шульгина М.В. (зам. директора института),1,3Васильева Н.В. (директор института, зав. кафедрой)
Северо-Западный государственный медицинский университет им. И.И. Мечникова: 1 НИИ медицинской микологии им. П.Н. Кашкина и 2 кафедра медицинской микробиологии, Санкт-Петербург, Россия; 3 Научный центр здоровья им. Норманна Бетюна, Джилинский Университет, Китай
©Коллектив авторов, 2016
Нозокомиальные инфекции являются актуальной проблемой современного здравоохранения в силу широкого распространения и негативных последствий для здоровья пациентов, персонала и экономики государства. Особую роль в нозокомиальных инфекциях отводят оппортунистическим микроорганизмам. В настоящий момент установлено, что возбудители внутрибольничных инфекций, в отличие от природных популяций микроорганизмов тех же видов, обладают сравнительно невысокой степенью генетического разнообразия. Эпидемические клоны возбудителей внутрибольнич-ных инфекций формируются как результат их внутривидового генетического разнообразия. Эпидемические клоны условно-патогенных микроорганизмов представляют собой наиболее приспособленные к ведению паразитического образа жизни варианты возбудителей, а процесс их формирования отражает эволюционные процессы, приводящие к приобретению факультативными паразитами патогенных свойств.
В обзоре приведены примеры внутривидового генетического разнообразия различных видов оппортунистических микроорганизмов, клиническое значение определенных клонов, обсуждается значимость внутривидового типирования в клинической практике. Внутривидовое типирование клинических изолятов позволит вывести эпидемиологические исследования на новый уровень: изучить эпидемиологические и эпизоотические связи источников возбудителя и очагов заболевания и выявить типы (варианты) возбудителя, ассоциированные с наличием определенных факторов вирулентности и резистентности к антимикробным препаратам.
Ключевые слова: генетическое внутривидовое разнообразие, Escherichia coli, Candida spp., нозокомиальные инфекции, оппортунистические микроорганизмы, Staphylococcus aureus, Streptococcus pyogenes
INTRASPECIFIC GENETIC DIVERSITY OF NOSOCOMIAL OPPORTUNISTIC PATHOGENS: CLINICAL SIGNIFICANCE (MINIREVIEW)
1Taraskina A.E. (head of the laboratory), 2 Li Fan (chancellor of science center),1Skorbunova O.V.
* Контактное лицо: Тараскина Анастасия Евгеньевна,
e-mail: Anastasiya.Taraskina@szgmu.ru
(assistant researcher), 1,3Pchelin I.M. (scientific collaborator, postgraduate student), 3Stepanov A.S. (postgraduate student), 3Ryabinin I.A. (postgraduate student), 1Shulgina M.V. (deputy director of the Institute), 1,3Vasilyeva N.V. (director of the institute, head of the chair)
1 Kashkin Research Institute of Medical Mycology, Northwestern State Medical University named after I.I. Mechnikov, St. Petersburg, Russia; 2 Norman Bethune Health Science Center Jilin University, China; 3 North-Western State Medical University named after I.I. Mechnikov (chair of medical microbiology), St. Petersburg, Russia
©Collective of authors, 2016
Nosocomial infections are a current problem of the modern healthcare due to its widespread and negative consequences on health of the patients, staff and state economy. Opportunistic pathogens play a special role in nosocomial infections. Currently it has been established that causative agents of nosocomial infections have a relatively low degree of genetic diversity compared with natural populations of microorganisms of the same species. Epidemic clones of nosocomial pathogens are formed as a result of the intraspecific diversity. Epidemic clones of opportunistic pathogens are the most fitted to parasitic lifestyle types of pathogens. Their evolutionary formation reflects processes acquisition of pathogenic properties by facultative parasites. Clinical significance of pathogenic clones of opportunistic pathogens and their role in epidemics of nosocomial infections are under intensive investigations in many laboratories all over the world now.
This review presents examples of intraspecific genetic diversity of different types of opportunistic microorganisms, and the data the clinical significance of specific clones. The importance of typing in diagnostic assays is discussed. This may bring epidemiological and epizootic investigations of sources of infections and infectious foci to the new level. Secondly it will extend our knowledge of pathogenic clones acquiring factors of virulence and resistance to antimicrobial drugs.
Key words: Candida spp., Escherichia coli, genetic intraspecific diversity, nosocomial infections, opportunistic pathogens, Staphylococcus aureus, Streptococcus pyogenes
INTRODUCTION
A nosocomial infection - also called «hospital-ac-quired» can be defined as: «An infection occurring in a patient in a hospital or other health care facility in whom the infection was not present or incubating at the time of administration. This includes infections acquired in the hospital but appearing after discharge, and also occupational infections among staff of the facility» (Prevention of hospital-acquire infection: a practical guide-WHO/2002). Nosocomial infections occur worldwide and affect both developed and resource-poor countries. They are important contributors to morbidity and mortality. They will become even more important as a public health problem with increasing economic and human impact because of: increasing numbers and crowding of people, more frequent impaired immunity (age, illness, treatments), new microorganisms, increasing microbial resistance to antimicrobial drugs.
Many different pathogens may cause nosocomial infections. The infecting organisms vary among different patient populations, health care settings, facilities and countries. The most common nosocomial pathogens are both pathogenic and commensal microorganisms. The mechanism of a commensal microorganism to pathogenic is still unknown. However impaired host defence mechanisms are considered to be fundamental. Systemic infections caused by commensals are becoming increasingly common in modern hospitals and frequently occur in patients with inherited and acquired immunodeficiencies, particularly in risk groups of patients, such as neutropenic patients, HIV-positive patients, and patients under intensive care.
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Many opportunistic fungi and some parasites (e.g. Giardia lamblia) may cause infections during extended antibiotic treatment and severe immunosuppression (Candida spp., Aspergillus spp., Cryptococcus neoformans). These are major cause of systemic infections in immunocompromised patients. Environmental contamination by airborne organisms such as Aspergillus spp. which originate in dust and soil is also a concern, as it may contaminate hospital building during construction.
In fact, opportunistic pathogens rarely produce disease in nondebilitated patients. Therefore, environmental and clinical isolates of opportunistic pathogens such as P. aeruginosa and Stenotrophomonas maltophilia are genetically indistinguishable. Analysis of two collections of P. ae-ruginosa isolates from environmental and clinical settings has demonstrated that they were functionally equivalent in traits relevant for both their pathogenic (host-oriented) and biodegradative (environment-oriented) abilities [1].
Intraspecific genetic diversity of microorganisms is a material for evolution and determines the success of a particular type in inhabiting of new ecological niches with unusual conditions of existence. Microorganisms that inhabit the macroorganism need to resist unfavorable influence of chemical and physical factors of the environment and also stress factors of immune response of the macroorganism or antimicrobial agents. Under the influence of these factors some groups of genetically similar microorganisms may gain selective advantageous and are able to survive in these conditions. Therefore these groups of microorganisms are able to develop mechanisms protecting them from antimicrobial substances and factors of immune system of the host [Martínez J.L., Baquero F. //Clin. Microb. Rev. -2002. - Vol. 15, №4].
Groups of genetically similar microorganisms originating from a common predecessor are called clones. Further analysis has demonstrated that the degree of clonality varies depending on the species analyzed. In that way, pathogenic species such as E. coli and Salmonella spp. are highly clonal [Martínez J.L., Baquero F. //Clin. Microb. Rev. -2002. - Vol. 15, №4].
Intraspecific differences in pathogenicity of microorganisms and their resistance to antimicrobial drugs are long known. Individual clones are often characterized by unique sets of virulence genes and/or resistance to antimicrobial agents or alleles of such genes.
This highlights the need for efficient methods for in-traspecific typing and delineation of strains. The earliest methods used in the typing of microorganisms were based on phenotypic characteristics including serotyping, biotyping, morphotyping, resistance to various chemicals and toxins and antimicrobial susceptibility profiles. However, phenotypic techniques have a very low degree of discrimination and reproducibility, which obviously constitutes a limitation for reliable epidemiological analysis. The advent of the molecular DNA-based techniques revolutionized the knowledge in the biology and epidemiology of many opportunistic pathogens. Development of sequencing technology facilitated the introduction of gene chip technology widely, followed with changes in research pattern in microbiology. For instance, the genome bar code on the micro-bial genome structure achieved visual annotation, which provided a powerful opportunity to the classification and identification of pathogenic bacteria as well as the prediction of horizontal transfer fragments (such as pathogenic-
ity islands), and it provided a powerful tool for the study of gene function and pathogenic mechanism of pathogenic bacteria [2]. Since then, various molecular approaches have been described that targeted various levels of polymorphism within a species.
Intraspecific typing can resolve two clinically important problems:
1. Uncovering epidemiological linkages of infection sources and disease areas. Linkages of this kind can be traced, for example, between units of a hospital, relying on exact knowledge of hospital personnel and patient translocation routes.
2. Revealing strain types, harbouring certain virulence and antimicrobial drug resistance factors. Direct detection of these factors is important for a patient management.
Examples of intraspecific diversity of different opportunistic pathogens species
The strains of the Enterobacteriaceae family are distinguished by antigenic characteristics and classified as se-rotypes. Specific serotypes are associated with severity of infection process. For many Enterobacteriaceae species particular serotypes are associated with certain sets of patho-genicity factors. Recently, the molecular genetic basis of serotypes differences was revealed [3, 4].
^-hemolytic streptococci (Streptococcus pyogenes) is responsible for a variety of infectious diseases and immu-nological complications, which differ in localization and severity. Traditionally this group of bacteria was thought to be the commensals, causing non-threatening disease of life, such as pharyngitis and impetigo. However, group A Streptococcus (GAS) is one of the most clinicaly relevant species of Streptococcus. It has been associated with human life-threatening infections, such as necrotising fasciitis and toxic shock syndrome. Moreover, immunological complications such as acute rheumatic fever, rheumatic heart disease and post-streptococcal acute glomerulonephritis are significant streptococcal disease burdens, especially in the developing world.
Most frequently invasive diseases were confirmed by several streptococcal isolates with M1serotype. This serotype is associated with isoform of protein M - a cell surface protein that inhibits phagocytosis. M proteinis considered to be the most important epidemiological marker of GAS infections. Serological methods were not able to type a significant number of the GAS isolates. emm gene (encoding M protein) sequencing based on 5' region is becoming a universal method for characterization of GAS infection[5]. Today more than 200 types of this gene are revealed. Beta-lactams are used for treatment of GAS infections, because these bacteria retain susceptible to penicillin G. Macrolides, lincosamides and fluoroquinolones are recommended for individuals with allergic reaction on penicillins. However, GAS resistance to these later antimicrobials has been described worldwide. While fluoroquinolone resistance in GAS emerges due to point mutations in gyr A, gyr B and parC genes, macrolide resistance is a result of active efflux of the drug or modification of the target site. Mutations conferring resistance to macrolides are located in mefA/E, ermA and ermB genes [6].
Knowledge of antimicrobial resistance rates and emm types prevalence in a given population are essential for development of effective strategies for prevention and treatment of GAS infections.
Gram-positive Staphylococcus aureus is a typical representative of normal biota in approximately 30% of the human population, but is capable to cause severe infections in immunocompromised patients in hospitals and in healthy humans in the community. S. aureus species includes diverse types of strains that differ in their sensitivity to various phages (phage groups) and in their pathogenicity. Molecular mechanisms, ensuring the success of various phage groups the disease development were described. S. aureus strains of phage group I produced toxic shock syndrome toxin 1 (TSST-1) encoded by tstH gene which is localized in mobile genetic elements. TSST-1 is exotoxin associated with the majority of cases of toxic shock syndrome (TSS) [Deresiewicz et al., 1996]. In 1996 90% TSS cases were caused by of S. aureus strains producing TSST-1. TSST-1 is regarded as a major virulence factor in these outbreaks. All strains isolated from the vagina in women with TSS associated with menstruation, belonged to a single clone characterized by increased ability to adhere to the vaginal mucosa and the cervical canal, and designated as the ET-41.
Methicillin-resistant Staphylococcus aureus (MRSA) clones are causing a global public health concern. MRSA clones diversify occur through point mutations, recombination, or the acquisition/deletion of mobile genetic elements, giving rise to extensive genomic and phenotypic diversity. First MRSA clones had genetic and phenotypic properties similar to methicillin-susceptible Staphylococcus aureus (MSSA) clones that were epidemic in Europe in the early 1960s. First MRSA bacteria emerged as result of acquisition of mecA gene by successful MSSA clones from an unknown heterologous source. mecA gene encodes a penicillin-binding protein conferring resistance to methicillin. Mobile genetic element caring mecA, is called the staphy-lococcal cassette chromosome mec (SCCmec), and has five forms (I, II, III, IV, V). The forms appeared as a result of independent events of the horizontal transfer of mecA . Five major lineages of MRSA (CC5, CC8, CC22, CC45 and CC30) with corresponding SCCmec forms circulate worldwide and cause most of nosocomial infections (up to 68%) in the world [7, 8].
Clonal origins of their genetic resistance determinants can eventually be traced even for highly recombinogenic bacterial species. For example, analysis ofpbp genes coding penicillin-binding protein synthesis demonstrated their high uniformity in different penicillin-susceptible isolates of N. meningitis, N. gonorrhoeae, and S. pneumonia, whereas those from penicillin-resistant strains had mosaic structure resulting from the horizontal transfer DNA blocks from other commensal species with natural penicillinresistance. Analysis of beta-lactam-resistant populations demonstrated that these novel pbp genes have occasionally been selected, as single gene «clones», and disseminates and evolves further among the bacterial population [Martínez J.L., Baquero F. //Clin. Microb. Rev. -2002. - Vol. 15, №4].
Presently, Candida spp. are the main causative agents of opportunistic mycoses in different regions of the World. Several species of the genus Candida are representatives of human normobiota. In persons with impaired immune system, however, these fungi may induce a pathogenic process.
The incidence of systemic candidiasis has been steadily increasing and it also has been associated with high rates of mortality (over 30%) even among appropriately treated
patients. Candida is now the fourth pathogen among microorganisms responsible for nosocomial bloodstream infections [9].
Epidemiological investigations of infectious outbreaks in hospitals are targeted to reveal the source and the route of an infection dissemination to effectively eradicate it. Although most Candida infections appear to originate from an endogenous source, nosocomial transmission is not uncommon and may occur either by cross-infection or by exposure to a common infecting source. Indeed, the same healthy individual can harbor the same strain in different body locations, or carry unrelated strains at the same or different body sites. Strains can replace each other in recurrent infections, or undergo microevolution (minor changes in genotype over a relatively small number of cell generations) and substrain shuffling (changes in subpopulations within individuals over time), be transferred from one individual to another. Specific strains may predominate in particular geographical areas and some strains may be endemic in some hospitals and can undergo microevolution in the hospital setting [10].
The most frequent isolates are Candida albicans. Standard genome of C. albicans is diploid and contains eight pairs of chromosome homologs. C. albicans has a predominantly clonal mode of reproduction. It was the first medically significant pathogenic fungus which completes genome sequence was published. It was genome of SC5314 clinical isolate. It has high degree of heterozygosity and more than 55700 single-nucleotide polymorphisms (SNPs) in 32-Mb diploid genome. Despite its diploid genome and clonal reproduction, C. albicans has high rate of genetic diversity, provided by recombination, chromosomal polymorphisms, gene replacement and cryptic mating. They ensure the plasticity of the genome [11].
There are five principal phylogenetic clades in C. albi-cans, revealed by Southern hybridization and multilocus sequence typing (MLST) techniques. These clades are designated as I, II, III, С.А. and Е. They differ by geographic distribution and phenotypic characteristics. Clades C.A. and E are prevalent in South Africa and Europe, respectively, while clade I strains occur worldwideJn vitro studies of clade I strains revealed that they are naturally resistant to flucytosine and terbinafine. Furthermore, each clade has a specific VNTR pattern. Clades are not associated with virulence factors and/or infection type [12].
Incidence of non-albicans Candida species has been increasing during last decades. These species have also been associated with higher mortality. C. glabrata and C. krusei have lower sensitivity to fluconazole differently from C. al-bicans. Acquired resistance to amphotericin B in C. lusita-niae has also been reported [9]. C. parapsilosis is a predominant species isolated from patients with systemic infections. It causes up to 45% of all candidemia cases. This yeast causes several infections in human, including life-threatening bloodstream infections. They are associated with catheters, premature birth, and contaminated prosthetic devices. Unlike other Candida species, C. parapsilosis was found on the hands of health care workers, who install and maintain medical devices, suggesting its potential route of transmission. Physiologically uniform C. parapsilosis isolates were reported genetically heterogeneous. Several reports suggested that C. parapsilosis includes three distinct groups (I, II and III). Group I isolates retain the name of the specie, C. parapsilosis (sensu stricto). For fungi belonging to groups
ПРОБЛЕМЫ МЕДИЦИНСКОЙ МИКОЛОГИИ. 2016. Т.18. №4
II and III new names are proposed: C. orthopsilosis and C. metapsilosis, respectively. C. parapsilosis persists in hospital environment, thereby enhancing the chance of nosocomial infections. Therefore, among the three species fungi show important substain differences in the degree of virulence (adhesive properties and ability to form biofilms) and antifungal susceptibility [13-15].
CONCLUSION
Examples of intraspecific diversity in different bacteria and fungi demonstrate clinical importance of intraspecific characteristics of pathogenic isolates. Fine characteristics of pathogen may have an input to the disease course prognosis and to prescribing of the rational patients' management regime. It may also provide more details for epidemiologic surveys and investigations of nosocomial infections outbreaks. This supports the importance of detailed identification of pathogenic isolates, expanding researches to intraspecific level, performing molecular genetic analysis of isolates and characterizing its genotype. Selection of a typing method depends on the nature and specific targets of each molecular study. Efficiency of each typing method should be evaluated in terms of its discriminating ability, reproducibility and ease of use
and interpretation. Discriminating power of the method depends on the ability of the method to detect genomes of one and the same strain or highly related strains in different isolates. A high resolution methods are preferable to monitor the microevolution of epidemic clones due the ability to segregate unrelated isolates.
Internal analysis of the reproducibility (Interassay reproducibility) is estimated by repeated testing of the same strain. Analysis should give identical results. In newly presented or developed genomes characteristics databases utilized by international researchers community standardization and reproducibility of methods used by different laboratories are vitally important. Ease of use and efficiency depend on the cost of specialized equipment and reagents, facilities for technical implementation in routine use, complexity of technical procedures and the ease results interpreting. In addition, the data must be suitable for computer analysis and storage, which is required for databases.
The study was supported by the Russian Foundation for Basic Research (grant № 16-54-53109 a «Intraspecies typing of pathogenic microorganisms (genotyping and proteomic features)».
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Поступила в редакцию журнала 30.11.2016
Рецензент: А.Е. Гончаров
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