Traditional Typing Methods Research Articles

Differences of microRNA expression profiles between monozygotic twins’ blood samples

Monozygotic (MZ) twins are widely regarded as genetically identical, and traditional DNA typing methods are insufficient in identifying MZ twins. So the discrimination of MZ twins become a forensic problem. MicroRNAs (miRNAs) are a class of small, endogenous, non-protein-coding RNA molecules of approximately 22 nucleotides in length, and exist extensively in a variety of eukaryotic cells. MiRNAs regulate gene expression and play fundamental roles in multiple biological processes, including cell differentiation, proliferation and apoptosis as well as aging and disease processes. The goal of this study is to explore the differential expression of miRNAs within MZ twin pairs, and aimed to find new biomarkers for distinguishing MZ twins. Thus, the miRNA expression profiles of seven pairs of healthy MZ twins of different sex and age were analyzed by miRNA microarray. A total of 545 miRNAs were found to be differentially expressed in these MZ twin pairs, and 2, 5, 22, 53 and 132 differentially expressed miRNAs were shared across six, five, four, three and two pairs of MZ twins respectively. These findings had been confirmed by real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assays on select miRNAs, including miR-151a-3p, miR-3653-3p, miR-142-3p, miR-4325, miR-16-5p, let-7i-5p, miR-222-3p, miR-550b-3p, miR-4791 and miR-27a-3p. The results demonstrated that there are differences in the expression of miRNAs within MZ twin pairs, suggesting a role of miRNAs in identifying MZ twins.

Analysis on brucellosis epidemiological characteristics in Hainan province

Objective: To understand the epidemiological characteristics of brucellosis in Hainan province. Methods: Automatic microbial identification system of Vitek 2 compact was used for the preliminary identification of 16 brucellosis cases in Hainan province from 2012 to 2017, and further confirmation was performed with traditional biological typing methods. The epidemiological and clinical characteristics of the patients were analyzed in combination with the results of serological and etiological tests for raised livestock. Results: Vitek 2 compact detection results showed that 12 strains were Brucella (B.) melitensis and 4 strains were Ochrobactrum anthropi. Traditional biological typing methods showed that 11 strains were B. melitensis biovar 3 and 5 strains were B. suis biovar 3. Sixteen cases were found in Dongfang, Lingao, Haikou, Wanning, Ledong and Ding'an with 1 case in 2012, 2 cases in 2013, 4 cases in 2014, 1 case in 2015, 2 cases in 2016 and 6 cases in 2017 respectively. At the same time, 745 sheep serum samples from the epidemic area (Dongfang) were collected for Brucella serum antibody detection, in which 47 were positive (6.3%). And B. melitensis biovar 3 was isolated from samples collected from sick sheep in Dongfang. Conclusions: Vitek 2 compact is an simple and convenient method for Brucella identification, but it cannot replace traditional biological typing methods yet. The major epidemic strains of Brucella in Hainan were B. melitensis biovar 3 and B. suis biovar 3. The epidemic of brucellosis in Dongfang in 2017 indicated that brucellosis had spread from animal to human in Hainan, and it is very important to strengthen the prevention and control of brucellosis in Hainan.

Air-conditioner cooling towers as complex reservoirs and continuous source of Legionella pneumophila infection evidenced by a genomic analysis study in 2017, Switzerland.

IntroductionWater supply and air-conditioner cooling towers (ACCT) are potential sources of Legionella pneumophila infection in people. During outbreaks, traditional typing methods cannot sufficiently segregate L. pneumophila strains to reliably trace back transmissions to these artificial water systems. Moreover, because multiple L. pneumophila strains may be present within these systems, methods to adequately distinguish strains are needed. Whole genome sequencing (WGS) and core genome multilocus sequence typing (cgMLST), with their higher resolution are helpful in this respect. In summer 2017, the health administration of the city of Basel detected an increase of L. pneumophila infections compared with previous months, signalling an outbreak.AimWe aimed to identify L. pneumophila strains populating suspected environmental sources of the outbreak, and to assess the relations between these strains and clinical outbreak strains.MethodsAn epidemiological and WGS-based microbiological investigation was performed, involving isolates from the local water supply and two ACCTs (n = 60), clinical outbreak and non-outbreak related isolates from 2017 (n = 8) and historic isolates from 2003–2016 (n = 26).ResultsIn both ACCTs, multiple strains were found. Phylogenetic analysis of the ACCT isolates showed a diversity of a few hundred allelic differences in cgMLST. Furthermore, two isolates from one ACCT showed no allelic differences to three clinical isolates from 2017. Five clinical isolates collected in the Basel area in the last decade were also identical in cgMLST to recent isolates from the two ACCTs.ConclusionCurrent outbreak-related and historic isolates were linked to ACCTs, which form a complex environmental habitat where strains are conserved over years.

Epidemiological investigation of an Acinetobacter baumannii outbreak using core genome multilocus sequence typing.

Carbapenem-resistant Acinetobacter baumannii (CRAB) is a serious nosocomial pathogen that causes a variety of serious, often life-threatening, infections and outbreaks. This study aimed to investigate the molecular epidemiology of clinical CRAB isolates from an outbreak that occurred in the intensive care unit (ICU) of an Italian hospital. From December 2016 to April 2017, 13 CRAB isolates were collected from seven patients treated in the ICU at 'L. Spallanzani' Hospital (Rome, Italy). Typing was performed by repetitive extragenic palindromic PCR (rep-PCR) using a DiversiLab® system. Whole-genome sequencing (WGS) data were used for in silico analysis of traditional multilocus sequence typing (MLST) results, to identify resistance genes and for core genome MLST (cgMLST) analysis. Epidemiological data were obtained from hospital records. All isolates showed a carbapenem-resistant profile and carried the blaOXA-23 carbapenemase gene. Typing performed by rep-PCR and MLST showed that the isolates clustered into one group, whilst the cgMLST approach, which uses 2390 gene targets to characterise the gene-by-gene allelic profile, highlighted the presence of two cluster types. These results allowed us to identify two patients who were likely to be the source of two separate transmission chains. These results show that WGS by cgMLST is a valuable tool, better suited for prompt epidemiological investigations than traditional typing methods because of its higher discriminatory ability in determining clonal relatedness.

Staphylococcus aureus and Staphylococcus epidermidis Strain Diversity Underlying Pediatric Atopic Dermatitis

AL Byrd, Deming, SKB Cassidy; NISC Comparative Sequencing Program. Sci Transl Med. 2017;9(397):eaal4651 Atopic dermatitis (AD) is a complex and multifactorial disease. Authors of previous studies have used traditional typing methods on cultivated isolates or have sequenced a bacterial marker gene to study the skin microbial communities of patients with AD. A shotgun metagenomic sequencing analysis provides greater resolution, elucidating multiple levels of microbial community assembly. Eleven children with moderate to severe AD and 7 healthy children were recruited to the National …

Genomic epidemiology of Shigella in the United Kingdom shows transmission of pathogen sublineages and determinants of antimicrobial resistance

Shigella are globally important diarrhoeal pathogens that are endemic in low-to-middle income nations and also occur in high income nations, typically in travellers or community-based risk-groups. Shigella phylogenetics reveals population structures that are more reliable than those built with traditional typing methods, and has identified sublineages associated with specific geographical regions or patient groups. Genomic analyses reveal temporal increases in Shigella antimicrobial resistance (AMR) gene content, which is frequently encoded on mobile genetic elements. Here, we whole genome sequenced representative subsamples of S. flexneri 2a and S. sonnei (n = 366) from the United Kingdom from 2008 to 2014, and analysed these alongside publicly available data to make qualitative insights on the genomic epidemiology of shigellosis and its AMR within the broader global context. Combined phylogenetic, epidemiological and genomic anlayses revealed the presence of domestically-circulating sublineages in patient risk-groups and the importation of travel-related sublineages from both Africa and Asia, including ciprofloxacin-resistant sublineages of both species from Asia. Genomic analyses revealed common AMR determinants among travel-related and domestically-acquired isolates, and the evolution of mutations associated with reduced quinolone susceptibility in domestically-circulating sublineages. Collectively, this study provides unprecedented insights on the contribution and mobility of endemic and travel-imported sublineages and AMR determinants responsible for disease in a high-income nation.

Comparison of advanced whole genome sequence-based methods to distinguish strains of Salmonella enterica serovar Heidelberg involved in foodborne outbreaks in Québec

Salmonella enterica serovar Heidelberg (S. Heidelberg) is one of the top serovars causing human salmonellosis. This serovar ranks second and third among serovars that cause human infections in Québec and Canada, respectively, and has been associated with severe infections. Traditional typing methods such as PFGE do not display adequate discrimination required to resolve outbreak investigations due to the low level of genetic diversity of isolates belonging to this serovar. This study evaluates the ability of four whole genome sequence (WGS)-based typing methods to differentiate among 145 S. Heidelberg strains involved in four distinct outbreak events and sporadic cases of salmonellosis that occurred in Québec between 2007 and 2016. Isolates from all outbreaks were indistinguishable by PFGE. The core genome single nucleotide variant (SNV), core genome multilocus sequence typing (MLST) and whole genome MLST approaches were highly discriminatory and separated outbreak strains into four distinct phylogenetic clusters that were concordant with the epidemiological data. The clustered regularly interspaced short palindromic repeats (CRISPR) typing method was less discriminatory. However, CRISPR typing may be used as a secondary method to differentiate isolates of S. Heidelberg that are genetically similar but epidemiologically unrelated to outbreak events. WGS-based typing methods provide a highly discriminatory alternative to PFGE for the laboratory investigation of foodborne outbreaks.

Whole-genome sequencing reveals transmission of gonococcal antibiotic resistance among men who have sex with men: an observational study

ObjectivesDrug-resistant Neisseria gonorrhoeae are now a global public health threat. Direct transmission of antibiotic-resistant gonococci between individuals has been proposed as a driver for the increased transmission of resistance, but...

A Comprehensive Evaluation of the Genetic Relatedness of Listeria monocytogenes Serotype 4b Variant Strains.

Recently, we have identified a link between four listeriosis incidents/outbreaks to a variant of Listeria monocytogenes (Lm) serotype 4b strains, 4bV. Although 4bV strains have been reported from clinical specimens as well as from foods, listeriosis outbreaks occurring in 2014–2016 were the first reported outbreaks involving 4bV in the USA. Since traditional typing methods do not detect members of this group, we undertook a systematic and retrospective analysis of all Lm in the NCBI WGS Sequence Read Archive database to investigate the burden of 4bV strains among all listeriosis cases. This analysis identified the presence of isolates causing sporadic cases as well as those associated with the aforementioned outbreaks, as determined by WGS and traditional epidemiology. In total, approximately 350 Lm 4bV strains were identified from multiple parts of the USA as well as from Australia and Chile, dating back to 2001. The genomic relatedness of these strains was compared using the CFSAN SNP Pipeline and multi-virulence-locus sequence typing (MVLST). Using the CFSAN Pipeline tool, the 4bV strains were found to group into seven clusters that were separate from 4b strains. All seven clades appeared to contain isolates from both clinical and non-clinical sources. Conversely, the MVLST analysis revealed that practically all of the strains belonged to a single clade, suggesting that 4bV strains from disparate geographic regions and sources are under varied selective pressure, restricting diversity across these six virulence loci while allowing more variability across the genome as a whole. Further evaluation of these 4bV strains identified genes potentially acquired from a lineage II source external to the lmo0733–lmo0739 region, as well as highly conserved SNPs unique to the 4bV strains when compared to those from other lineages. Taken together, these data suggest that 4bV strains have undergone adaptive responses to selective pressures that may enhance survival in the environment while maintaining the pathogenic potential of serotype 4b strains.

Staphylococcus aureus and Staphylococcus epidermidis strain diversity underlying pediatric atopic dermatitis.

The heterogeneous course, severity, and treatment responses among patients with atopic dermatitis (AD; eczema) highlight the complexity of this multifactorial disease. Prior studies have used traditional typing methods on cultivated isolates or sequenced a bacterial marker gene to study the skin microbial communities of AD patients. Shotgun metagenomic sequence analysis provides much greater resolution, elucidating multiple levels of microbial community assembly ranging from kingdom to species and strain-level diversification. We analyzed microbial temporal dynamics from a cohort of pediatric AD patients sampled throughout the disease course. Species-level investigation of AD flares showed greater Staphylococcus aureus predominance in patients with more severe disease and Staphylococcus epidermidis predominance in patients with less severe disease. At the strain level, metagenomic sequencing analyses demonstrated clonal S. aureus strains in more severe patients and heterogeneous S. epidermidis strain communities in all patients. To investigate strain-level biological effects of S. aureus, we topically colonized mice with human strains isolated from AD patients and controls. This cutaneous colonization model demonstrated S. aureus strain-specific differences in eliciting skin inflammation and immune signatures characteristic of AD patients. Specifically, S. aureus isolates from AD patients with more severe flares induced epidermal thickening and expansion of cutaneous T helper 2 (TH2) and TH17 cells. Integrating high-resolution sequencing, culturing, and animal models demonstrated how functional differences of staphylococcal strains may contribute to the complexity of AD disease.

Comparison of classical multi-locus sequence typing software for next-generation sequencing data.

Multi-locus sequence typing (MLST) is a widely used method for categorizing bacteria. Increasingly, MLST is being performed using next-generation sequencing (NGS) data by reference laboratories and for clinical diagnostics. Many software applications have been developed to calculate sequence types from NGS data; however, there has been no comprehensive review to date on these methods. We have compared eight of these applications against real and simulated data, and present results on: (1) the accuracy of each method against traditional typing methods, (2) the performance on real outbreak datasets, (3) the impact of contamination and varying depth of coverage, and (4) the computational resource requirements.

Multi-provincial Salmonellosis Outbreak Related to Newly Hatched Chicks and Poults: A Genomics Perspective.

Background:A multi-provincial outbreak of Salmonella enterica serovar Enteritidis was linked to newly hatched chicks and poults from a single hatchery during the spring of 2015. In total, there were 61 human cases that were epidemiologically confirmed to be linked to the chicks and poults and the outbreak was deemed to have ended in the summer of 2015.Methods:PulseNet Canada, in coordination with the affected provinces, used genome sequencing of human and agricultural Salmonella Enteritidis isolates to aid in the epidemiological investigation, while also using traditional typing methods such as phagetyping and pulsed-field gel electrophoresis (PFGE).Results:All human outbreak cases, except one, were Phage Type (PT) 13a. Single nucleotide variant analysis (SNV) was able to provide a level of resolution commensurate with the results of the epidemiological investigation. SNV analysis was also able to separate PT13a outbreak-related isolates from isolates not linked to chicks or poults, while clustering some non-PT13a agricultural strains with the outbreak cluster.Conclusions:Based on conventional typing methods (phagetyping or PFGE), clinical and agricultural PT13a SE isolates would have been considered as part of a related cluster. In contrast, phagetyping would have led to the exclusion of several non- PT13a strains that clustered with the outbreak isolates using the genome sequence data. This study demonstrates the improved resolution of genome sequence analysis for coordinated surveillance and source attribution of both human and agricultural SE isolates.

CRISPR Diversity and Microevolution in Clostridium difficile.

Virulent strains of Clostridium difficile have become a global health problem associated with morbidity and mortality. Traditional typing methods do not provide ideal resolution to track outbreak strains, ascertain genetic diversity between isolates, or monitor the phylogeny of this species on a global basis. Here, we investigate the occurrence and diversity of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated genes (cas) in C. difficile to assess the potential of CRISPR-based phylogeny and high-resolution genotyping. A single Type-IB CRISPR-Cas system was identified in 217 analyzed genomes with cas gene clusters present at conserved chromosomal locations, suggesting vertical evolution of the system, assessing a total of 1,865 CRISPR arrays. The CRISPR arrays, markedly enriched (8.5 arrays/genome) compared with other species, occur both at conserved and variable locations across strains, and thus provide a basis for typing based on locus occurrence and spacer polymorphism. Clustering of strains by array composition correlated with sequence type (ST) analysis. Spacer content and polymorphism within conserved CRISPR arrays revealed phylogenetic relationship across clades and within ST. Spacer polymorphisms of conserved arrays were instrumental for differentiating closely related strains, e.g., ST1/RT027/B1 strains and pathogenicity locus encoding ST3/RT001 strains. CRISPR spacers showed sequence similarity to phage sequences, which is consistent with the native role of CRISPR-Cas as adaptive immune systems in bacteria. Overall, CRISPR-Cas sequences constitute a valuable basis for genotyping of C. difficile isolates, provide insights into the micro-evolutionary events that occur between closely related strains, and reflect the evolutionary trajectory of these genomes.

Phylogenetic structure of European Salmonella Enteritidis outbreak correlates with national and international egg distribution network.

Outbreaks of Salmonella Enteritidis have long been associated with contaminated poultry and eggs. In the summer of 2014 a large multi-national outbreak of Salmonella Enteritidis phage type 14b occurred with over 350 cases reported in the United Kingdom, Germany, Austria, France and Luxembourg. Egg supply network investigation and microbiological sampling identified the source to be a Bavarian egg producer. As part of the international investigation into the outbreak, over 400 isolates were sequenced including isolates from cases, implicated UK premises and eggs from the suspected source producer. We were able to show a clear statistical correlation between the topology of the UK egg distribution network and the phylogenetic network of outbreak isolates. This correlation can most plausibly be explained by different parts of the egg distribution network being supplied by eggs solely from independent premises of the Bavarian egg producer (Company X). Microbiological sampling from the source premises, traceback information and information on the interventions carried out at the egg production premises all supported this conclusion. The level of insight into the outbreak epidemiology provided by whole-genome sequencing (WGS) would not have been possible using traditional microbial typing methods.

Molecular Typing of Methicillin-sensitive and Methicillin-resistant <em>Staphylococcus aureus</em>: What Epidemiologists need to know from Plasmid Analysis to Whole Genome Sequencing.

Methicillin-resistant Staphylococcus aureus is an important nosocomial pathogen. The early identification of an outbreak, making use of a rapid, precise and simple methicillin-resistant Staphylococcus aureus typing technique, can lead to prompt and effective precautions that avoid further spread of the infection. Pulsed-field gel electrophoresis is considered the gold standard for methicillin-resistant Staphylococcus aureus typing and has been recently supported by multilocus sequence typing. SmaI-multiplex polymerase chain reaction typing is a novel polymerase chain reaction -based technique that combines the principles of pulsed-field gel electrophoresis, with simplicity of polymerase chain reaction and can be used in many routine clinical laboratories. Whole genome sequencing typing is a second generation sequencing technology and may ultimately replace the traditional molecular typing methods. This review aims to survey existing molecular typing techniques for Staphylococcus aureus and to discuss information for each method in order to aid researchers and clinical professionals in the selection and implementation of an optimal technique.

Nested Russian Doll-Like Genetic Mobility Drives Rapid Dissemination of the Carbapenem Resistance Gene blaKPC.

The recent widespread emergence of carbapenem resistance in Enterobacteriaceae is a major public health concern, as carbapenems are a therapy of last resort against this family of common bacterial pathogens. Resistance genes can mobilize via various mechanisms, including conjugation and transposition; however, the importance of this mobility in short-term evolution, such as within nosocomial outbreaks, is unknown. Using a combination of short- and long-read whole-genome sequencing of 281 blaKPC-positive Enterobacteriaceae isolates from a single hospital over 5 years, we demonstrate rapid dissemination of this carbapenem resistance gene to multiple species, strains, and plasmids. Mobility of blaKPC occurs at multiple nested genetic levels, with transmission of blaKPC strains between individuals, frequent transfer of blaKPC plasmids between strains/species, and frequent transposition of blaKPC transposon Tn4401 between plasmids. We also identify a common insertion site for Tn4401 within various Tn2-like elements, suggesting that homologous recombination between Tn2-like elements has enhanced the spread of Tn4401 between different plasmid vectors. Furthermore, while short-read sequencing has known limitations for plasmid assembly, various studies have attempted to overcome this by the use of reference-based methods. We also demonstrate that, as a consequence of the genetic mobility observed in this study, plasmid structures can be extremely dynamic, and therefore these reference-based methods, as well as traditional partial typing methods, can produce very misleading conclusions. Overall, our findings demonstrate that nonclonal resistance gene dissemination can be extremely rapid, presenting significant challenges for public health surveillance and achieving effective control of antibiotic resistance.

Advances in Blood Typing.

The clinical importance of blood group antigens relates to their ability to evoke immune antibodies that are capable of causing hemolysis. The most important antigens for safe transfusion are ABO and D (Rh), and typing for these antigens is routinely performed for patients awaiting transfusion, prenatal patients, and blood donors. Typing for other blood group antigens, typically of the Kell, Duffy, Kidd, and MNS blood groups, is sometimes necessary, for patients who have, or are likely to develop antibodies to these antigens. The most commonly used typing method is serological typing, based on hemagglutination reactions against specific antisera. This method is generally reliable and practical for routine use, but it has certain drawbacks. In recent years, molecular typing has emerged as an alternative or supplemental typing method. It is based on detecting the polymorphisms and mutations that control the expression of blood group antigens, and using this information to predict the probable antigen type. Molecular typing methods are useful when traditional serological typing methods cannot be used, as when a patient has been transfused and the sample is contaminated with red blood cells from the transfused blood component. Moreover, molecular typing methods can precisely identify clinically significant variant antigens that cannot be distinguished by serological typing; this capability has been exploited for the resolution of typing discrepancies and shows promise for the improved transfusion management of patients with sickle cell anemia. Despite its advantages, molecular typing has certain limitations, and it should be used in conjunction with serological methods.

Whole-genome sequencing for outbreak investigations of methicillin-resistant Staphylococcus aureus in the neonatal intensive care unit: time for routine practice?

BACKGROUND Infants in the neonatal intensive care unit (NICU) are at increased risk for methicillin-resistant Staphylococcus aureus (MRSA) acquisition. Outbreaks may be difficult to identify due in part to limitations in current molecular genotyping available in clinical practice. Comparison of genome-wide single nucleotide polymorphisms (SNPs) may identify epidemiologically distinct isolates among a population sample that appears homogenous when evaluated using conventional typing methods. OBJECTIVE To investigate a putative MRSA outbreak in a NICU utilizing whole-genome sequencing and phylogenetic analysis to identify recent transmission events. DESIGN Clinical and surveillance specimens collected during clinical care and outbreak investigation. PATIENTS A total of 17 neonates hospitalized in a 43-bed level III NICU in northeastern Florida from December 2010 to October 2011 were included in this study. METHODS We assessed epidemiological data in conjunction with 4 typing methods: antibiograms, PFGE, spa types, and phylogenetic analysis of genome-wide SNPs. RESULTS Among the 17 type USA300 isolates, 4 different spa types were identified using pulsed-field gel electrophoresis. Phylogenetic analysis identified 5 infants as belonging to 2 clusters of epidemiologically linked cases and excluded 10 unlinked cases from putative transmission events. The availability of these results during the initial investigation would have improved infection control interventions. CONCLUSION Whole-genome sequencing and phylogenetic analysis are invaluable tools for epidemic investigation; they identify transmission events and exclude cases mistakenly implicated by traditional typing methods. When routinely applied to surveillance and investigation in the clinical setting, this approach may provide actionable intelligence for measured, appropriate, and effective interventions.

Dissemination of cephalosporin resistance genes between Escherichia coli strains from farm animals and humans by specific plasmid lineages.

Third-generation cephalosporins are a class of β-lactam antibiotics that are often used for the treatment of human infections caused by Gram-negative bacteria, especially Escherichia coli. Worryingly, the incidence of human infections caused by third-generation cephalosporin-resistant E. coli is increasing worldwide. Recent studies have suggested that these E. coli strains, and their antibiotic resistance genes, can spread from food-producing animals, via the food-chain, to humans. However, these studies used traditional typing methods, which may not have provided sufficient resolution to reliably assess the relatedness of these strains. We therefore used whole-genome sequencing (WGS) to study the relatedness of cephalosporin-resistant E. coli from humans, chicken meat, poultry and pigs. One strain collection included pairs of human and poultry-associated strains that had previously been considered to be identical based on Multi-Locus Sequence Typing, plasmid typing and antibiotic resistance gene sequencing. The second collection included isolates from farmers and their pigs. WGS analysis revealed considerable heterogeneity between human and poultry-associated isolates. The most closely related pairs of strains from both sources carried 1263 Single-Nucleotide Polymorphisms (SNPs) per Mbp core genome. In contrast, epidemiologically linked strains from humans and pigs differed by only 1.8 SNPs per Mbp core genome. WGS-based plasmid reconstructions revealed three distinct plasmid lineages (IncI1- and IncK-type) that carried cephalosporin resistance genes of the Extended-Spectrum Beta-Lactamase (ESBL)- and AmpC-types. The plasmid backbones within each lineage were virtually identical and were shared by genetically unrelated human and animal isolates. Plasmid reconstructions from short-read sequencing data were validated by long-read DNA sequencing for two strains. Our findings failed to demonstrate evidence for recent clonal transmission of cephalosporin-resistant E. coli strains from poultry to humans, as has been suggested based on traditional, low-resolution typing methods. Instead, our data suggest that cephalosporin resistance genes are mainly disseminated in animals and humans via distinct plasmids.

METHODOLOGICAL APPROACHES TO MYCOBACTERIUM TUBERCULOSIS GENOTYPING FOR EVOLUTIONARY AND EPIDEMIOLOGICAL RESEARCH

Abstract. Current genome evolution of Mycobacterium tuberculosis is marked by virtual absence of the lateral gene transfer leading to the clonal population of this species consisting of separate genetic families. Standard typing method of M. tuberculosis (IS6110-RFLP, spoligo- and VNTR-typing) are based on variation of mobile and repetitive elements and provide sufficient strain discrimination for epidemiological purposes such as, estimation of recent transmission versus reactivation of latent tuberculosis, laboratory contamination, mixed infection. At the same time, rapid evolution of some markers may lead to emergence of identical profiles in the non-related strains (homoplasy) due to convergent evolution. Use of different independent markers may help solve this problem. Regularly updated databases are available for global and local analysis and are also important for standardised terminology and designation of the genotypes. Some of the M. tuberculosis genetic families continue to circulate in the limited areas while other families have become omnipresent due to their likely increased transmissibility and pathogeneicity (e.g., Beijing and LAM). The most frequently isolated Russian subvariant Beijing B0/W148 is marked by significantly higher population growth compared to the Russian Beijing population as a whole and hence may be defined as a successful clone in Russia. Recent years revealed higher than previously thought level of genome variation in M. tuberculosis even between related isolates. The whole-genome sequencing may become a useful typing method if its cost is reduced to be similar to that of the traditional typing methods. Accumulation of the data on old and new markers, development and use of new algorithms of their analysis will help to refine our knowledge about evolution of M. tuberculosis and its families, will provide better tools for epidemiological monitoring of the circulating strains on local and global scale.