Are there new biomarkers of the gastroduodenal microbiota useful in the diagnosis of coeliac disease in children? A pilot study.

The changing of microbiome could precede the development of coeliac disease (CeD). We compared the bacterial profile of microbiota of tissues collected simultaneously from the stomach and duodenum in newly diagnosed patients with CeD. Biopsies were collected from 60 children and adolescents aged 2-18 years: (1) 40 patients with CeD; (2) 20 children as control group. The evaluation of the bacterial microbiota was carried out by sequencing the V3-V4 regions of the 16S rRNA subunit, using next-generation sequencing (NGS). The composition of bacterial microbiota was correlated with clinical and blood parameters. The beta diversity analysis revealed a significant dissimilarity in the gastric samples between the CeD and control group (Bray-Curtis index, P = 0.008, and weighted UniFrac distance, P = 0.024). At L2 (phylum level), Campylobacterota was only present in the stomach of the CeD group. A comparison of the abundance of bacteria between the stomach and duodenum showed significant differences in 10 OTUs (operational taxonomic units) in the control and 9 OTUs in the CeD group at L6 (genus) and in 8 OTUs and in 6 OTUs, respectively, at L7 (species). A significant correlation was observed between the genus Novosphingobium in stomach of CeD group and possession of the DQ2.5 and DQ 8 allele, and in the duodenum - between the DQ 8 allele and the species Blautia wexlerae. Significant differences in selected, little-known genera of bacteria suggest their potential role as new biomarkers in the development of CeD. To fully understand the mechanism of CeD development in genetically predisposed individuals, it is necessary to take into account not only the abundance of a given genus or species of bacteria, but also the anatomical location of its occurrence.

Determinants of the microbiome spatial variability in chronic rhinosinusitis.

BACKGROUND: The sinus microbiome in patients with chronic rhinosinusitis (CRS) is considered homogenous across the sinonasal cavity. The middle nasal meatus is the recommended sampling site for 16S rRNA sequencing. However, individuals with unusually high between-site variability between the middle meatus and the sinuses were identified in previous studies. This study aimed to identify which factors determine increased microbial heterogeneity between sampling sites in the sinuses. METHODOLOGY: In this cross-sectional study samples for 16S rRNA sequencing were obtained from the middle meatus, the maxillary and the frontal sinus in 50 patients with CRS. The microbiome diversity between sampling sites was analysed in relation to the size of the sinus ostia and clinical metadata. RESULTS: In approximately 15% of study participants, the differences between sampling sites within one patient were greater than between the patient and other individuals. Contrary to a popular hypothesis, obstruction of the sinus ostium resulted in decreased dissimilarity between the sinus and the middle meatus. The dissimilarity between the sampling sites was patient-specific: greater between-sinus differences were associated with greater meatus-sinus differences, regardless of the drainage pathway patency. Decreased spatial variability was observed in patients with nasal polyps and extensive mucosal changes in the sinuses. CONCLUSIONS: Sampling from the middle meatus is not universally representative of the sinus microbiome. The differences between sites cannot be predicted from the patency of communication pathways between them.

Alterations in intestinal Archaea composition in pediatric patients with Crohn's disease based on next-generation sequencing - a pilot study.

Intestinal dysbiosis can lead to the induction of systemic immune-mediated inflammatory diseases, such as Crohn's disease Although archaea are part of the commensal microbiota, they are still one of the least studied microorganisms. The aim of our study was the standardization of the optimal conditions and primers for sequencing of the gut archaeome using Next Generation Sequencing, and evaluation of the differences between the composition of archaea in patients and healthy volunteers, as well as analysis of the changes that occur in the archaeome of patients depending on disease activity. Newly diagnosed patients were characterized by similar archeal profiles at every taxonomic level as in healthy individuals (the dominance of Methanobacteria at the class level, and Methanobrevibacter at the genus level). In turn, in patients previously diagnosed with Crohn's disease (both in active and remission phase), an increased prevalence of Thermoplasmata, Thermoprotei, Halobacteria (at the class level), and Halococcus, Methanospaera or Picrophilus (at the genus level) were observed. Furthermore, we have found a significant correlation between the patient's parameters and the individual class or species of Archaea. Our study confirms changes in archaeal composition in pediatric patients with Crohn's disease, however, only in long-standing disease. At the beginning of the disease, the archeal profile is similar to that of healthy people. However, in the chronic form of the disease, significant differences in the composition of archaeome begin to appear. It seems that some archaea may be a good indicator of the chronicity and activity of Crohn's disease.

Comprehensive detection and identification of bacterial DNA in the blood of patients with sepsis and healthy volunteers using next-generation sequencing method - the observation of DNAemia.

Blood is considered to be a sterile microenvironment, in which bacteria appear only periodically. Previously used methods allowed only for the detection of either viable bacteria with low sensitivity or selected species of bacteria. The Next-Generation Sequencing method (NGS) enables the identification of all bacteria in the sample with their taxonomic classification. We used NGS for the analysis of blood samples from healthy volunteers (n = 23) and patients with sepsis (n = 62) to check whether any bacterial DNA exists in the blood of healthy people and to identify bacterial taxonomic profile in the blood of septic patients. The presence of bacterial DNA was found both in septic and healthy subjects; however, bacterial diversity was significantly different (P = 0.002) between the studied groups. Among healthy volunteers, a significant predominance of anaerobic bacteria (76.2 %), of which most were bacteria of the order Bifidobacteriales (73.0 %), was observed. In sepsis, the majority of detected taxa belonged to aerobic or microaerophilic microorganisms (75.1 %). The most striking difference was seen in the case of Actinobacteria phyla, the abundance of which was decreased in sepsis (P < 0.001) and Proteobacteria phyla which was decreased in the healthy volunteers (P < 0.001). Our research shows that bacterial DNA can be detected in the blood of healthy people and that its taxonomic composition is different from the one seen in septic patients. Detection of bacterial DNA in the blood of healthy people may suggest that bacteria continuously translocate into the blood, but not always cause sepsis; this observation can be called DNAemia.

Control of an outbreak of Acinetobacter baumannii infections using vaporized hydrogen peroxide.

BACKGROUND: Multidrug-resistant Acinetobacter baumannii (MRAB) is a serious nosocomial pathogen characterized by its survival on inanimate surfaces for long periods, making control of outbreaks difficult. AIM: To analyse two hospital outbreaks caused by MRAB, determine their epidemiology, carbapenem-resistance mechanisms and assess the effectiveness of surface disinfection by vaporized hydrogen peroxide (VHP). METHODS: MRAB strains were isolated from patients in two intensive care units (ICUs). Antimicrobial susceptibility testing was performed by E-test. Polymerase chain reaction (PCR) was used to detect the presence of the most common A. baumannii carbapenemases. Epidemiological typing was performed by rep-PCR (DiversiLab) and pulsed-field gel electrophoresis. VHP was used to decontaminate the affected ICUs. FINDINGS: MRAB was isolated from 28 patients between January 2009 and September 2010. All isolates were resistant to ciprofloxacin and gentamicin. Twenty-one were also resistant to carbapenems. Carbapenem resistance was associated primarily with the acquired OXA-23-like enzyme. Genotyping revealed three clones; the predominant clone corresponded to the international clone (IC) 2. Typing of the isolates pointed to a multifocal outbreak without a single source of infection, with horizontal spread of the dominating clone among ICU patients. A combination of rigorous infection control measures including strict isolation, education of staff, hand hygiene and surface decontamination using VHP halted the outbreak. CONCLUSION: The results of this study confirm the importance of rigorous infection prevention and control measures, combined with VHP decontamination in controlling an outbreak of MRAB.

Molecular characterization of capsular polysaccharides and surface protein genes in relation to genetic similarity of group B streptococci isolated from Polish pregnant women.

Serotyping, subtyping and genotyping are important tools for epidemiological studies of group B streptococci (GBS). We investigated the genotype distribution of 353 GBS isolates originating from vaginal or rectal carriage to identify capsular serotypes and subtypes based on the surface protein genes of the alpha-like protein (Alp) family. GBS were recovered from 30% of 1176 pregnant women during the period 2007-2009, with a predominance of capsular genotypes III (35%), Ia (20%), V (17%), II (15%), Ib (8%) and IV (5%). The most common Alp gene was epsilon (26%), followed by rib (22%), alp2 (21%), bca (17%) and alp3 (14%). Several protein genes were significantly associated (G(2)=249·635, P<0·0001) with particular serotypes: epsilon with Ia, Ib, IV; bca with Ib, II; rib with II, III; alp3 with V; alp2 with III. High genetic diversity within GBS strains was observed using DNA macrorestriction. Serotypes Ib, II and III demonstrated the greatest genetic heterogeneity and serotype V the lowest heterogeneity (relative frequency coefficient ≥0·03 vs. -0·46, respectively). Macrolide-resistant strains with serotype V and alp3 gene, showed higher uniformity in genetic profile. The distribution of serotypes and surface proteins of GBS strains are necessary data to inform the design and formulation of new GBS vaccines for use in Poland and other countries.

A role of hydrogen peroxide producing commensal bacteria present in colon of adolescents with inflammatory bowel disease in perpetuation of the inflammatory process.

Bacteria in the gut play a central role in the initiation and progress of inflammatory bowel disease (IBD). This study was prepared to elucidate the role in the inflammatory process of the bacterial species which are able to produce hydrogen peroxide, present in samples taken from colon lesions in adolescents with inflammatory bowel disease. Fifty eight adolescents were enrolled into the study from January 2004 to October 2006 in Cracow, Poland. Biopsies and stool samples were collected. Bacteriological examinations and measurements of hydrogen peroxide production by enterococci, streptococci and lactobacilli were performed. For the first time it has been shown here that HP producing bacteria may contribute to increased amounts of hydrogen peroxide in the inflamed mucosa of Crohn's disease and ulcerative colitis patients. Moreover, we have been able to demonstrate an increase of total populations of aerobic bacteria but not anaerobes in the studied samples of mucosa of adolescents with inflammatory bowel disease which is an indirect evidence of higher oxygen tension present in inflamed tissues in IBD. We have also been able to demonstrate the direct relationship between presence of blood in stools of IBD adolescents and increased populations of Enterobacteriaceae but not streptococci in samples of colon mucosa. It is, therefore, possible that different products of Enterobacteriaceae and especially their lipopolysaccharides may also contribute to perpetuation of the chronic colon inflammation.