Fresh Versus Frozen Stool for Fecal Microbiota Transplantation-Assessment by Multimethod Approach Combining Culturing, Flow Cytometry, and Next-Generation Sequencing.

The objective of this work was to compare the quality of FMT preparations made from fresh feces with those made from feces frozen at -30°C without any pre-processing or cryopreservation additives. The research hypothesis was that such preservation protocol (frozen whole stool, then thawed and processed) is equipotent to classical fresh FMT preparation. For that, three complementary methods were applied, including: (i) culturing in aerobic and anaerobic conditions, (ii) measuring viability by flow cytometry, and (iii) next-generation sequencing. Flow cytometry with cell staining showed that the applied freezing protocol causes significant changes in all of the observed bacterial fractions. Alive cell counts dropped four times, from around 70% to 15%, while the other two fractions, dead and unknown cell counts quadrupled and doubled, with the unknown fraction becoming the dominant one, with an average contribution of 57.47% per sample. It will be very interesting to uncover what this unknown fraction is (e.g., bacterial spores), as this may change our conclusions (if these are spores, the viability could be even higher after freezing). Freezing had a huge impact on the structure of cultivable bacterial communities. The biggest drop after freezing in the number of cultivable species was observed for Actinobacteria and Bacilli. In most cases, selected biodiversity indices were slightly lower for frozen samples. PCoA visualization built using weighted UniFrac index showed no donor-wise clusters, but a clear split between fresh and frozen samples. This split can be in part attributed to the changes in the relative abundance of Bacteroidales and Clostridiales orders. Our results clearly show that whole stool freezing without any cryoprotectants has a great impact on the cultivability and biodiversity of the bacterial community, and possibly also on the viability of bacterial cells.

Faecal Microbiota Transfer - a new concept for treating cytomegalovirus colitis in children with ulcerative colitis.

INTRODUCTION: Cytomegalovirus (CMV) infection in patients with inflammatory bowel disease (IBD) is reactivated by the use of immunosuppressive drugs. CMV infection may produce IBD flares refractory to standard therapy. OBJECTIVE: The aim of our study was to assess the efficacy and safety of faecal microbiota transplantation (FMT) for the treatment of CMV colitis in patients with ulcerative colitis (UC) flare. MATERIAL AND METHODS: A total of 8 children, with mild to severe UC, positive for CMV PCR in colonic biopsies, received 50-100 ml FMT by nasogastric tube on 5 consecutive days in each of 2 weeks. During the study, the subjects were treated with 5ASA and FMT. Immunosuppressant therapy was withdrawn, when CMV colitis was diagnosed by positive DNA PCR in colonic tissues. The clinical response was defined as a decrease of Paediatric UC Activity Index by ≥20 points. RESULTS: At the 6th week of the study, negative colonic CMV DNA PCR was measured after 10 infusions in 7/8 patients. For one boy, 20 infusions were administered to assess CMV elimination from colonic biopsies. A clinical response was observed in 3/8 patients, with clinical remission in 3/8 patients. Faecal calprotectin decreased significantly in 3 patients. CRP normalized in 2 patients after 6 weeks. No serious adverse effects were observed during and after infusions. CONCLUSIONS: FMT seems to be an effective and safe treatment option for CMV colitis in children with UC. This is the first study to demonstrate the application of FMT as a new therapeutic option for CMV colitis.

Eosinophilic gastroenteritis and graft-versus-host disease induced by transmission of Norovirus with fecal microbiota transplant.

Fecal microbiota transplantation (FMT) was performed to decolonize gastrointestinal tract from antibiotic-resistant bacteria before allogeneic hematopoietic cells transplantation (alloHCT). AlloHCT was complicated by norovirus gastroenteritis, acute graft-versus-host disease, and eosinophilic pancolitis. Norovirus was identified in samples from FMT material. Symptoms resolved after steroids course and second norovirus-free FMT from another donor.

Multimodal Approach to Assessment of Fecal Microbiota Donors based on Three Complementary Methods.

Methods of stool assessment are mostly focused on next-generation sequencing (NGS) or classical culturing, but only rarely both. We conducted a series of experiments using a multi-method approach to trace the stability of gut microbiota in various donors over time, to find the best method for the proper selection of fecal donors and to find "super-donor" indicators. Ten consecutive stools donated by each of three donors were used for the experiments (30 stools in total). The experiments assessed bacterial viability measured by flow cytometry, stool culturing on different media and in various conditions, and NGS (90 samples in total). There were no statistically significant differences between live and dead cell numbers; however, we found a group of cells classified as not-dead-not-alive, which may be possibly important in selection of "good" donors. Donor C, being a regular stool donor, was characterized by the largest number of cultivable species (64). Cultivable core microbiota (shared by all donors) was composed of only 16 species. ANCOM analysis of NGS data highlighted particular genera to be more abundant in one donor vs. the others. There was a correlation between the not-dead-not-alive group found in flow cytometry and Anaeroplasma found by NGS, and we could distinguish a regular stool donor from the others. In this work, we showed that combining various methods of microbiota assessment gives more information than each method separately.

A Two-Week Fecal Microbiota Transplantation Course in Pediatric Patients with Inflammatory Bowel Disease.

Dysbiosis plays a major role in the etiology of inflammatory bowel disease (IBD). Fecal microbiota transplantation (FMT) is a new promising option for IBD treatment. We aimed to assess the effectiveness of a two-week FMT course in children with IBD. Ten patients, 10-17 years of age with moderate to severe IBD received a course of eight doses of freshly prepared FMT via a naso-duodenal tube or gastroscopy. All of the patients had pancolitis. There were eight cases of ulcerative colitis (UC) and two of Crohn's disease (CD). Disease activity was evaluated using the Pediatric UC Activity Index (PUCAI) and Pediatric CD Activity Index (PCDAI) for UC and CD, respectively, CRP, and fecal calprotectin on the day before the first infusion and then on the day before the next course of FMT. Clinical response, defined as a decrease of 15 points in either index, was observed in 9/10 patients (seven UC and two CD). Clinical remission, defined as a PCDAI score ≤ 10 and PUCAI score < 10 measured at the same time point, was observed in 3/8 UC patients and 2/2 CD patients. Side effects observed were self-limiting and benign. We conclude that a short, intensive course of FMT has a beneficial effect on UC and CD colitis. FMT was well-tolerated and safe. Nonetheless, an optimal protocol of FMT administration is crucial for treatment efficacy.

Fecal Microbiota Transplantation in Patients With Blood Disorders Inhibits Gut Colonization With Antibiotic-Resistant Bacteria: Results of a Prospective, Single-Center Study.

Background: Patients with blood disorders colonized with antibiotic-resistant bacteria (ARB) are prone to systemic infections that are difficult to treat. Reintroduction of commensal bacteria in a murine model of enterococcal colonization of the gut can lead to eradication of enterococci. We hypothesized that fecal microbiota transplantation (FMT) could be used to eradicate ARB in humans. Methods: Participants colonized with ARB were treated with intraduodenal FMT according to a prospective protocol (NCT02461199). The primary endpoint was complete ARB decolonization at 1 month after FMT. Secondary endpoints included safety assessment and partial ARB decolonization. Microbiome sequencing was performed to investigate the influence of microbial composition of the transplanted material on the outcome of FMT. Results: Twenty-five FMTs were performed in 20 participants (including 40% who had neutropenia) who were colonized by a median of 2 (range, 1-4) strains of ARB. The primary endpoint was reached in 15/25 (60%) of the FMTs and more frequently in cases in which there was no periprocedural use of antibiotics (79% vs 36%, P < .05). Among participants, 15/20 (75%) experienced complete ARB decolonization. There were no severe adverse events, and partial ARB decolonization was observed in 20/25 (80%) of the FMTs. The microbiota composition analysis revealed higher abundance of Barnesiella spp., Bacteroides, and Butyricimonas and greater bacterial richness in the fecal material, resulting in eradication of Klebsiella pneumoniae compared with nonresponders. Conclusions: FMT in patients with blood disorders is safe and promotes eradication of ARB from the gastrointestinal tract. Clinical Trials Registration: NCT02461199.

Fecal Microbiota Transplantation Inhibits Multidrug-Resistant Gut Pathogens: Preliminary Report Performed in an Immunocompromised Host.

Colonization of the gastrointestinal tract with multidrug-resistant (MDR) bacteria is a consequence of gut dysbiosis. We describe the successful utilization of fecal microbiota transplantation to inhibit Klebsiella pneumoniae MBL(+) and Escherichia coli ESBL(+) gut colonization in the immunocompromised host as a novel tool in the battle against MDR microorganisms. ClinicalTrials.gov identifier NCT02461199.

Rotavirus vaccination in central Europe.

Each year, rotavirus (RV) infection is the leading cause of acute gastroenteritis requiring hospitalisation and of nosocomially transmitted diseases in children younger than 5 years across Central European Vaccination Awareness Group (CEVAG) countries; however, inadequate surveillance systems and lack of routine RV testing still exist in most CEVAG countries, making it difficult to accurately assess the present burden of acute RV gastroenteritis in the younger population. Furthermore, routine immunisation of infants with RV vaccines has not been implemented, and no official and uniform recommendations exist in most of the countries in these territories. The present study provides CEVAG country-specific estimates of the disease burden of RV gastroenteritis among the youngest population and presents evidence-based advice on the use of RV vaccines in the region, while providing a framework for vaccination at the national level.

Cost-effectiveness of polysaccharide pneumococcal vaccination in people aged 65 and above in Poland.

INTRODUCTION: Invasive pneumococcal disease is associated with substantial morbidity, mortality and cost implications, which could be reduced by vaccination. AIM: To assess the cost-effectiveness of a 23-valent pneumococcal vaccine in the elderly (65 and older) in Poland. METHODS: A Markov model with a 1-year cycle length was developed, allowing up to 10 cohorts to enter the model over the lifetime horizon (35 years). In the base case, costs and benefits were assessed using the public health care payer (NFZ) perspective. The analysis included routine vaccination of all elderly and high-risk (HR) elderly versus no vaccination. The analysis assumed that the government would reimburse 50% of the vaccine price. Costs and benefits were discounted 5%, with costs expressed in 2009 Polish Zloty (PLN). Extensive sensitivity analyses were carried out. RESULTS: PPV23 vaccination targeting all elderly and HR elderly in Poland would avoid 8,935 pneumococcal infections, 2,542 hospitalisations, 671 deaths and 5,886 infections, 1,673 hospitalisations and 441 deaths respectively. The incremental cost per QALY gained would be PLN 3,382 in all elderly and PLN2,148 in HR elderly. CONCLUSION: Vaccinating adults 65 and older regardless of risk status with a 23-valent pneumococcal vaccine, is cost-effective, resulting in clinical and economic benefits including a non-negligible reduction of ambulatory doctor visits, hospitalizations and, deaths in Poland.

Progress towards implementing the e-Bug Project in Poland.

The e-Bug Project provides useful educational tools that can be implemented in primary and secondary schools, and plays important role in reducing the burden of community-acquired infections. Consideration of the Polish education system and educational programmes in schools facilitated the selection of the best age groups for the implementation of e-Bug materials in Poland. The Polish version of the resources has been reviewed by a special group of experts and teachers from pilot schools. The pilot implementation is currently provided by demonstration lessons for educators and recorded as a demonstration material. The e-Bug teaching pack is a valuable resource that is useful for improving knowledge about microorganisms, hygiene and antimicrobial agents, and is aimed at pupils, teachers and parents.

[Paediatric Expert Group on the Immunization Programme. Activities focusing on effective immunization in Poland]. / Pediatryczny Zespól Ekspertów ds. Programu Szczepien Ochronnych. Dzialania na rzecz poprawy efektywnosci szczepien w Polsce.

INTRODUCTION: The Paediatric Expert Group on the Immunization Programme was established in January 2007. It is an independent advisory body to the Minister of Health. The Expert Group consists of paediatricians from various sub-specialities. Most of them are members of the Polish Society of Vaccinology (Table I). The Group started their activities informally in 2005. The first project concerned changes in immunization against tuberculosis and prophylaxis of measles, mumps and rubella. The project was fully implemented in 2006. The changes initiated three years ago, gradually implemented in the Immunization Programme are a result of wide cooperation with the Ministry of Health, Department of Health Policy Chief Sanitory Inspector, as well as the Institute of Tuberculosis and Pulmonary Diseases. The aim of the Paediatric Expert Group on the Immunization Programme is to present a unified policy in matters related to vaccination, leading to rapid changes in the prophylaxis of infective diseases which are still a threat to the life and health of children.

Clonal diversity and resistance mechanisms in tetracycline-nonsusceptible Streptococcus pneumoniae isolates in Poland.

The frequency of tetracycline resistance in Streptococcus pneumoniae isolates in Poland is one of the highest in Europe. The aim of this study was to analyze the clonal diversity and resistance determinants of tetracycline-nonsusceptible S. pneumoniae isolates identified in Poland and to investigate the effect of tetracycline resistance on their susceptibilities to tigecycline, doxycycline, and minocycline. We have analyzed 866 pneumococcal isolates collected from 1998 to 2003 from patients with respiratory tract diseases, and 242 of these (27.9%) were found to be resistant to tetracycline. All of the resistant isolates were characterized by testing of their susceptibilities to other antimicrobials, serotyping, pulsed-field gel electrophoresis (PFGE), and identification of tetracycline resistance genes and transposons. Selected isolates representing the main PFGE types were analyzed by multilocus sequence typing. Among the isolates investigated, 27 serotypes and 146 various PFGE patterns, grouped into 90 types, were discerned. The most common PFGE type, corresponding to serotype 19F and sequence type 423, was represented by 22.3% of all of the tetracycline-resistant isolates. The tet(M) gene was the sole resistance gene in the group of isolates studied, and in over 96% of the isolates, the Tn916 family of tet(M)-containing conjugative transposons was detected. Several isolates contained specific variants of the transposons, the Tn1545-like, Tn3872-like, or Tn2009-like element. The correlation between the MICs of tetracycline, doxycycline, and minocycline was revealed, whereas no cross-resistance to tetracycline and tigecycline was observed.

Phenotypic and molecular analysis of penicillin-nonsusceptible Streptococcus pneumoniae isolates in Poland.

beta-Lactams are the drugs of choice for the treatment of infections caused by the important bacterial pathogen Streptococcus pneumoniae. The recent growth of resistance of this organism to penicillin observed worldwide is of the highest concern. In this study, using 887 surveillance pneumococcal isolates recovered in Poland from 1998 to 2002, we observed the increase in penicillin nonsusceptibility from 8.7% to 20.3%. All of the 109 penicillin-nonsusceptible S. pneumoniae (PNSP) isolates identified, together with 22 archival PNSP isolates from 1995 to 1997, were subsequently analyzed by susceptibility testing, serotyping, profiling of pbp genes, pulsed-field gel electrophoresis, and multilocus sequence typing (MLST). Four predominant serotypes, serotypes 6B, 9V, 14, and 23F, characterized 85.5% of the isolates. MLST revealed the presence of 34 sequence types, 15 of which were novel types. Representatives of seven multiresistant international clones (Spain(23F)-1, Spain(6B)-2, Spain(9V)-3, Taiwan(23F)-15, Poland(23F)-16, Poland(6B)-20, and Sweden(15A)-25) or their closely related variants comprised the majority of the study isolates. The spread of Spain(9V)-3 and its related clone of serotype 14/ST143 has remarkably contributed to the recent increase in penicillin resistance in pneumococci in the country.

[Self-medication with antibiotics in Poland]. / Leczenie antybiotykami bez konsultacji lekarskiej w Polsce.

UNLABELLED: Antibiotic resistance, the important public health threat, depends on antibiotic overuse/misuse. Self-medication with antibiotics is of serious medical concern. The aim of the study, as a part of SAR project (Self-medication with antibiotic in Europe) was to survey the incidence of this phenomenon. MATERIAL AND METHODS: In Poland among 938 respondents of the questionnaire 27% adults were treated with antibiotics at least once a year, 13% of the therapies were undertaken without medical consultation. The most frequently prescribed antibiotic was amoxicillin, the most often self-ordered--fusafungine. RESULTS: The main self-medicated symptoms were sore throat and common cold. The duration of self-ordered therapy was less then 5 days. The sources of antibiotics used for self-medication were leftovers and those given by friends or family. CONCLUSION: The results of the study confirmed that antibiotic self-medication is relatively frequent problem in Poland and interventions are required in order to reduce the frequency of antibiotics misuse.

[Prevalence of antibodies against Anaplasma phagocytophilum, Babesia microti i Borrelia burgdorferi in adults in North-Eastern Poland]. / Wystepowanie przeciwcial przeciw Anaplasma phagocytophilum, Babesia microti i Borrelia burgdorferi u doroslych z pólnocno-wschodnich rejonów Polski.

THE AIM: of the study was to evaluate the seroprevalance of antibodies against Anaplasma phagocytophilum and Babesia Microti in healthy north-eastern Poland, adult population. MATERIAL AND METHODS: The study was conducted in a group of 142 healthy adults (mean age 19-22), bitten by ticks within last 2 years. The control group consisted of 50 adults from central Poland (nonendemic area). The antibody levels for A. phagocytophilum (IgG/Ap-Ab) and B. microti (IgM/Bm-Ab) were evaluated in two series of samples from the same persons (interval 5-6 months) by immunoenzymatic tests (Borrelia Biomedica, Austria), immunofluorescence test (Human Granulotic Ehrlichiosis IFA IgG and Babesia microti IFA IgG from MRL Diagnostics). RESULTS: Positive results for A. phagocytophilum were defined as titres > or =1:256 and for B. microti > or =1:64 and B. burgdorferi > or = 11 BBU/ml. Positive results for IgG B. burgdorferi during the first collection were revealed in 16% (n=24/142) of individuals from endemic area and in 4% (n=2/50) of the control group, which was statistically relevant (p<0,05). IgG A. phagocytophilum antibodies were present in 3,5% (n=5/142) of individuals from the endemic area, but for IgG B. microti antibodies (IgG/Bm-Ab) no positive results were found. No IgG antibodies against A. phagocytophilum and B.microti, were found in individuals from non-endemic area. During the second collection, in individuals from the endemic area, the antibodies against B. burgdorferi were found in 9,8% (n=14/142), IgG A. phagocytophilum antibodies (IgG/Ap-Ab) in 4,9% (n=7/142) and against B. microti (IgG/ Bm-Ab) in 1,4% (n=2/142). The antibodies against B. Burgdorferi were found in 2% (n=1/150) of the control group during the second collection, and no IgG against A. phagocytophilum and B. microti were found. CONCLUSION: [corrected] Evaluating the seroprevalance of the studied antibodies in both collections, a conclusion was drawn that there was no significant increase of antibodies levels directly after the highest exposition to tick bites. None of individuals showed 4-fold antibody level increase between the first and second collection. The seroconversion for IgG/Bm-Ab antibodies was present in 1,4% (n=2/142) of individuals, in those 2 cases a 2-fold antibodies level increase was observed. As far as IgG/Ap-Ab antibodies are concerned the seroconversion was observed in 2,1% (n-3/142), but only one case shown a 3-fold antibodies level increase. No seroconversion of B. burgdorferi antibodies were found in the second collection.

[Anthrax as a bioweapon]. / Waglik jako zagrozenie bioterrorystyczne.

The risk of biological and chemical terrorism is growing according to availability of modern biotechnologies and financial resources. The most potent biological weapon mentioned in the last decade is anthrax. The number of naturally acquired infections in humans is constantly reduced, however endemic areas are located in South and Middle Americas, South Europe, Asia and Africa. In any case of infection laboratory confirmation is indicated. Primary basic testing is available in all microbiology laboratories. In the current situation the medical and epidemiological infrastructure and microbiology diagnostics must be prepared for early and rapid coordinated action in order to detect and respond to biological and chemical attacks.

Characteristics of Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis isolated from the nasopharynges of asymptomatic children and molecular analysis of S. pneumoniae and H. influenzae strain replacement in the nasopharynx.

Nasopharyngeal carriage of Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis in 226 children in different settings (in a crèche [day care center], in an orphanage, and at home) during two seasons (winter and spring) was studied. The rates of carriage of S. pneumoniae and H. influenzae were markedly higher in the crèche and in the orphanage than in the home setting (e.g., 56.5, 63.3, and 25.9%, respectively, for S. pneumoniae in winter). Approximately 80% of the S. pneumoniae isolates identified in the crèche and in the orphanage belonged to the serotypes represented in the seven-valent pneumococcal vaccine, and 4.4% of the children were colonized by H. influenzae type b. Almost all H. influenzae isolates were fully susceptible to the antimicrobial agents tested, and only five (3.6%) produced beta-lactamase; in contrast, 100% of the M. catarrhalis isolates were beta-lactamase positive. Among S. pneumoniae isolates, 36.2% were nonsusceptible to penicillin (PNSP) and 11.8% were fully resistant to penicillin (PRP). All PNSP isolates were obtained from children at the crèche and at the orphanage but not among children brought up at home, and all PRP isolates showed a multiresistant phenotype. Colonization by PRP isolates correlated well with prior treatment with beta-lactams. For the majority of children colonized at both sampling times, strain replacement of S. pneumoniae and H. influenzae was observed; long-term colonization by a single strain was rare.

[Chlamydia pneumoniae infection in patients with advanced carotid atherosclerosis]. / Zakazenia Chlamydia pneumoniae u chorych z zaawansowana miadyca tetnic szyjnych.

Despite the growing number of scientific reports showing different markers of infection caused by Chlamydia pneumoniae in patients with advanced atherosclerosis, there is still no clear confirmation of a pathogenetic link between this infection and atherogenesis. The aim of the present study was to investigate the presence C. pneumoniae DNA in peripheral blood mononuclear cells and carotid endarterectomy samples obtained from patients with advanced atherosclerosis according to the presence specific antibodies against C. pneumoniae in serum. The levels of IgG, IgM and IgA antibodies to C. pneumoniae were determined by enzyme immunoassay (EIA) in sera of 36 patients with advanced atherosclerosis undergoing carotid endarterectomy. The presence of C. pneumoniae DNA in the carotid samples and in peripheral blood mononuclear cells was investigated by a nested polymerase chain reaction (PCR). We have not confirm the presence of C. pneumoniae DNA either in the carotid endarterectomy samples or in peripheral blood mononuclear cells, both in patients having the specific antibodies against C. pneumoniae and in seronegative patients. In the studied group of patients with advanced carotid atherosclerosis there was no correlation between the presence of specific antibodies against C. pneumoniae and the presence of C. pneumoniae DNA in endarterectomy samples and peripheral blood mononuclear cells.

[Modern possibilities of bioterrorism diagnosis]. / Wspólczesne mozliwosci diagnostyczne czynników bioterrorystycznych.

One of the most important components of early detecting and response to biological attack are microbiological diagnostic methods. In order to provide the effectiveness of diagnosis continuous training in detection of potentially dangerous bioterrorist agents is needed. Basic information about selected microbiological agents most dangerous for public health, belonging to A and B CDC categories are presented in the article. The most potent biological weapon mentioned in the last decade is anthrax. Other potential bioterrorist agents discussed in this article are Francisella tularensis, Yersinia pestis, Brucella species and Coxiella burnetii.