Utilization of Benchtop Next Generation Sequencing Platforms Ion Torrent PGM and MiSeq in Noninvasive Prenatal Testing for Chromosome 21 Trisomy and Testing of Impact of In Silico and Physical Size Selection on Its Analytical Performance

OBJECTIVES: The aims of this study were to test the utility of benchtop NGS platforms for NIPT for trisomy 21 using previously published z score calculation methods and to optimize the sample preparation and data analysis with use of in silico and physical size selection methods. METHODS: Samples from 130 pregnant women were analyzed by whole genome sequencing on benchtop NGS systems Ion Torrent PGM and MiSeq. The targeted yield of 3 million raw reads on each platform was used for z score calculation. The impact of in silico and physical size selection on analytical performance of the test was studied. RESULTS: Using a z score value of 3 as the cut-off, 98.11% - 100% (104-106/106) specificity and 100% (24/24) sensitivity and 99.06% - 100% (105-106/106) specificity and 100% (24/24) sensitivity were observed for Ion Torrent PGM and MiSeq, respectively. After in silico based size selection both platforms reached 100% specificity and sensitivity. Following the physical size selection z scores of tested trisomic samples increased significantly-p = 0.0141 and p = 0.025 for Ion Torrent PGM and MiSeq, respectively. CONCLUSIONS: Noninvasive prenatal testing for chromosome 21 trisomy with the utilization of benchtop NGS systems led to results equivalent to previously published studies performed on high-to-ultrahigh throughput NGS systems. The in silico size selection led to higher specificity of the test. Physical size selection performed on isolated DNA led to significant increase in z scores. The observed results could represent a basis for increasing of cost effectiveness of the test and thus help with its penetration worldwide

The Influence of Food Intake Specificity in Children with Autism on Gut Microbiota

Autism spectrum disorder (ASD) is a complex of neurodevelopmental conditions with increasing incidence. The microbiota of children with ASD is distinct from neurotypical children, their food habits are also different, and it is known that nutrient intake influences microbiota in a specific way. Thus, this study investigates the food habits of children with ASD and their association with the gut microbiota. Children with ASD had their dietary energy intakes similar to controls, but they more often demonstrated food selectivity, which seemed to result in deficiency of micronutrients such as vitamins K, B6, C, iron, cooper, docosahexaenoic and docosapentanoic acid. Using high-throughput sequencing, a DNA library of intestinal microbiota was performed. Core microbiota was similar in children with and without ASD, but Dichelobacter, Nitriliruptor and Constrictibacter were found to be putative markers of ASD. The changes in gut microbiota that we observed in connection to food selectivity, intake of fats and omega-3 in particular, fermented milk products and animal/plant protein consumption had similar character, independent of diagnosis. However, high fibre intake was connected with a decreased α-diversity only in children with ASD. High carbohydrate and fibre intake influenced β-diversity, changing the abundance of Bacteroides and other genera, many of them members of the Clostidiaceae. Modulating food habits of ASD children can influence their gut microbiota composition

Table_2_What do we know about the microbiome of I. ricinus?.pdf

I. ricinus is an obligate hematophagous parasitic arthropod that is responsible for the transmission of a wide range of zoonotic pathogens including spirochetes of the genus Borrelia, Rickettsia spp., C. burnetii, Anaplasma phagocytophilum and Francisella tularensis, which are part the tick´s microbiome. Most of the studies focus on “pathogens” and only very few elucidate the role of “non-pathogenic” symbiotic microorganisms in I. ricinus. While most of the members of the microbiome are leading an intracellular lifestyle, they are able to complement tick´s nutrition and stress response having a great impact on tick´s survival and transmission of pathogens. The composition of the tick´s microbiome is not consistent and can be tied to the environment, tick species, developmental stage, or specific organ or tissue. Ovarian tissue harbors a stable microbiome consisting mainly but not exclusively of endosymbiotic bacteria, while the microbiome of the digestive system is rather unstable, and together with salivary glands, is mostly comprised of pathogens. The most prevalent endosymbionts found in ticks are Rickettsia spp., Ricketsiella spp., Coxiella-like and Francisella-like endosymbionts, Spiroplasma spp. and Candidatus Midichloria spp. Since microorganisms can modify ticks’ behavior, such as mobility, feeding or saliva production, which results in increased survival rates, we aimed to elucidate the potential, tight relationship, and interaction between bacteria of the I. ricinus microbiome. Here we show that endosymbionts including Coxiella-like spp., can provide I. ricinus with different types of vitamin B (B2, B6, B7, B9) essential for eukaryotic organisms. Furthermore, we hypothesize that survival of Wolbachia spp., or the bacterial pathogen A. phagocytophilum can be supported by the tick itself since coinfection with symbiotic Spiroplasma ixodetis provides I. ricinus with complete metabolic pathway of folate biosynthesis necessary for DNA synthesis and cell division. Manipulation of tick´s endosymbiotic microbiome could present a perspective way of I. ricinus control and regulation of spread of emerging bacterial pathogens.</p

Table_1_What do we know about the microbiome of I. ricinus?.pdf

I. ricinus is an obligate hematophagous parasitic arthropod that is responsible for the transmission of a wide range of zoonotic pathogens including spirochetes of the genus Borrelia, Rickettsia spp., C. burnetii, Anaplasma phagocytophilum and Francisella tularensis, which are part the tick´s microbiome. Most of the studies focus on “pathogens” and only very few elucidate the role of “non-pathogenic” symbiotic microorganisms in I. ricinus. While most of the members of the microbiome are leading an intracellular lifestyle, they are able to complement tick´s nutrition and stress response having a great impact on tick´s survival and transmission of pathogens. The composition of the tick´s microbiome is not consistent and can be tied to the environment, tick species, developmental stage, or specific organ or tissue. Ovarian tissue harbors a stable microbiome consisting mainly but not exclusively of endosymbiotic bacteria, while the microbiome of the digestive system is rather unstable, and together with salivary glands, is mostly comprised of pathogens. The most prevalent endosymbionts found in ticks are Rickettsia spp., Ricketsiella spp., Coxiella-like and Francisella-like endosymbionts, Spiroplasma spp. and Candidatus Midichloria spp. Since microorganisms can modify ticks’ behavior, such as mobility, feeding or saliva production, which results in increased survival rates, we aimed to elucidate the potential, tight relationship, and interaction between bacteria of the I. ricinus microbiome. Here we show that endosymbionts including Coxiella-like spp., can provide I. ricinus with different types of vitamin B (B2, B6, B7, B9) essential for eukaryotic organisms. Furthermore, we hypothesize that survival of Wolbachia spp., or the bacterial pathogen A. phagocytophilum can be supported by the tick itself since coinfection with symbiotic Spiroplasma ixodetis provides I. ricinus with complete metabolic pathway of folate biosynthesis necessary for DNA synthesis and cell division. Manipulation of tick´s endosymbiotic microbiome could present a perspective way of I. ricinus control and regulation of spread of emerging bacterial pathogens.</p

Table_3_What do we know about the microbiome of I. ricinus?.pdf

I. ricinus is an obligate hematophagous parasitic arthropod that is responsible for the transmission of a wide range of zoonotic pathogens including spirochetes of the genus Borrelia, Rickettsia spp., C. burnetii, Anaplasma phagocytophilum and Francisella tularensis, which are part the tick´s microbiome. Most of the studies focus on “pathogens” and only very few elucidate the role of “non-pathogenic” symbiotic microorganisms in I. ricinus. While most of the members of the microbiome are leading an intracellular lifestyle, they are able to complement tick´s nutrition and stress response having a great impact on tick´s survival and transmission of pathogens. The composition of the tick´s microbiome is not consistent and can be tied to the environment, tick species, developmental stage, or specific organ or tissue. Ovarian tissue harbors a stable microbiome consisting mainly but not exclusively of endosymbiotic bacteria, while the microbiome of the digestive system is rather unstable, and together with salivary glands, is mostly comprised of pathogens. The most prevalent endosymbionts found in ticks are Rickettsia spp., Ricketsiella spp., Coxiella-like and Francisella-like endosymbionts, Spiroplasma spp. and Candidatus Midichloria spp. Since microorganisms can modify ticks’ behavior, such as mobility, feeding or saliva production, which results in increased survival rates, we aimed to elucidate the potential, tight relationship, and interaction between bacteria of the I. ricinus microbiome. Here we show that endosymbionts including Coxiella-like spp., can provide I. ricinus with different types of vitamin B (B2, B6, B7, B9) essential for eukaryotic organisms. Furthermore, we hypothesize that survival of Wolbachia spp., or the bacterial pathogen A. phagocytophilum can be supported by the tick itself since coinfection with symbiotic Spiroplasma ixodetis provides I. ricinus with complete metabolic pathway of folate biosynthesis necessary for DNA synthesis and cell division. Manipulation of tick´s endosymbiotic microbiome could present a perspective way of I. ricinus control and regulation of spread of emerging bacterial pathogens.</p

Characterization of Clinical and Carrier Streptococcus agalactiae and Prophage Contribution to the Strain Variability

Streptococcus agalactiae (group B Streptococcus, GBS) represents a leading cause of invasive bacterial infections in newborns and is also responsible for diseases in older and immunocompromised adults. Prophages represent an important factor contributing to the genome plasticity and evolution of new strains. In the present study, prophage content was analyzed in human GBS isolates. Thirty-seven prophages were identified in genomes of 20 representative sequenced strains. On the basis of the sequence comparison, we divided the prophages into eight groups named A&ndash;H. This division also corresponded to the clustering of phage integrase, even though several different integration sites were observed in some relative prophages. Next, PCR method was used for detection of the prophages in 123 GBS strains from adult hospitalized patients and from pregnancy screening. At least one prophage was present in 105 isolates (85%). The highest prevalence was observed for prophage group A (71%) and satellite prophage group B (62%). Other groups were detected infrequently (1&ndash;6%). Prophage distribution did not differ between clinical and screening strains, but it was unevenly distributed in MLST (multi locus sequence typing) sequence types. High content of full-length and satellite prophages detected in present study implies that prophages could be beneficial for the host bacterium and could contribute to evolution of more adapted strains

Gut diversity and the resistome as biomarkers of febrile neutropenia outcome in paediatric oncology patients undergoing hematopoietic stem cell transplantation

Abstract The gut microbiota of paediatric oncology patients undergoing a conditioning regimen before hematopoietic stem cell transplantation is recently considered to play role in febrile neutropenia. Disruption of commensal microbiota and evolution of opportune pathogens community carrying a plethora of antibiotic-resistance genes play crucial role. However, the impact, predictive role and association of patient´s gut resistome in the course of the therapy is still to be elucidated. We analysed gut microbiota composition and resistome of 18 paediatric oncology patients undergoing hematopoietic stem cell transplantation, including 12 patients developing febrile neutropenia, hospitalized at The Bone Marrow Transplantation Unit of the National Institute of Children´s disease in Slovak Republic and healthy individuals (n = 14). Gut microbiome of stool samples obtained in 3 time points, before hematopoietic stem cell transplantation (n = 16), one week after hematopoietic stem cell transplantation (n = 16) and four weeks after hematopoietic stem cell transplantation (n = 14) was investigated using shotgun metagenome sequencing and bioinformatical analysis. We identified significant decrease in alpha-diversity and nine antibiotic-resistance genes msr(C), dfrG, erm(T), VanHAX, erm(B), aac(6)-aph(2), aph(3)-III, ant(6)-Ia and aac(6)-Ii, one week after hematopoietic stem cell transplantation associated with febrile neutropenia. Multidrug-resistant opportune pathogens of ESKAPE, Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae and Escherichia coli found in the gut carried the significant subset of patient’s resistome. Over 50% of patients treated with trimethoprim/sulfamethoxazole, piperacillin/tazobactam and amikacin carried antibiotic-resistance genes to applied treatment. The alpha diversity and the resistome of gut microbiota one week after hematopoietic stem cell transplantation is relevant predictor of febrile neutropenia outcome after hematopoietic stem cell transplantation. Furthermore, the interindividual diversity of multi-drug resistant opportunistic pathogens with variable portfolios of antibiotic-resistance genes indicates necessity of preventive, personalized approach

Z score values of samples calculated by three different previously published methods.

<p><b>□</b> –trisomic samples, <b>○</b> –euploid samples, horizontal line—mean z score value of trisomic samples. Dotted lines represent the standard limit for identification of a trisomic sample (z score = 3). A—Ion Torrent PGM analyzed samples, B—MiSeq analyzed samples.</p