Strains In Faecal Samples Research Articles

Highly accurate and sensitive absolute quantification of bacterial strains in human fecal samples

BackgroundNext-generation sequencing (NGS) approaches have revolutionized gut microbiome research and can provide strain-level resolution, but these techniques have limitations in that they are only semi-quantitative, suffer from high detection limits, and generate data that is compositional. The present study aimed to systematically compare quantitative PCR (qPCR) and droplet digital PCR (ddPCR) for the absolute quantification of Limosilactobacillus reuteri strains in human fecal samples and to develop an optimized protocol for the absolute quantification of bacterial strains in fecal samples.ResultsUsing strain-specific PCR primers for L. reuteri 17938, ddPCR showed slightly better reproducibility, but qPCR was almost as reproducible and showed comparable sensitivity (limit of detection [LOD] around 104 cells/g feces) and linearity (R2 > 0.98) when kit-based DNA isolation methods were used. qPCR further had a wider dynamic range and is cheaper and faster. Based on these findings, we conclude that qPCR has advantages over ddPCR for the absolute quantification of bacterial strains in fecal samples. We provide an optimized and easy-to-follow step-by-step protocol for the design of strain-specific qPCR assays, starting from primer design from genome sequences to the calibration of the PCR system. Validation of this protocol to design PCR assays for two L. reuteri strains, PB-W1 and DSM 20016 T, resulted in a highly accurate qPCR with a detection limit in spiked fecal samples of around 103 cells/g feces. Applying our strain-specific qPCR assays to fecal samples collected from human subjects who received live L. reuteri PB-W1 or DSM 20016 T during a human trial demonstrated a highly accurate quantification and sensitive detection of these two strains, with a much lower LOD and a broader dynamic range compared to NGS approaches (16S rRNA gene sequencing and whole metagenome sequencing).ConclusionsBased on our analyses, we consider qPCR with kit-based DNA extraction approaches the best approach to accurately quantify gut bacteria at the strain level in fecal samples. The provided step-by-step protocol will allow scientists to design highly sensitive strain-specific PCR systems for the accurate quantification of bacterial strains of not only L. reuteri but also other bacterial taxa in a broad range of applications and sample types.-oGjUmj5e8MXZDxyDjzcm-Video

Time-Dependent Shifts in Intestinal Bacteriome, Klebsiella Colonization and Incidence of Antibiotic-Resistance Genes after Allogeneic Hematopoietic Stem Cell Transplantation.

Dose-intensive cytostatic therapy and antibiotic treatment in allogeneic hematopoietic stem cell transplantation (allo-HSCT) cause severe abnormalities in a composition of gut microbiota as well as the emergence of antibiotic resistance. The data on the longitudinal recovery of major bacterial phyla and the expansion of genes associated with antibiotic resistance are limited. We collected regular stool samples during the first year after allo-HSCT from 12 adult patients with oncohematological disorders after allo-HSCT and performed 16SrRNA sequencing, multiplex PCR, conventional bacteriology and CHROMagar testing. We observed a decline in Shannon microbiota diversity index as early as day 0 of allo-HSCT (p = 0.034) before any administration of antibiotics, which persisted up to 1 year after transplantation, when the Shannon index returned to pre-transplant levels (p = 0.91). The study confirmed the previously shown decline in Bacillota (Firmicutes) genera and the expansion of E. coli/Shigella, Klebsiella and Enterococci. The recovery of Firmicutes was slower than that of other phyla and occurred only a year post-transplant. A positive correlation was observed between the expansion of E. coli/Shigella genera and blaKPC, blaCTX-M-1 and blaTEM (p < 0.001), Klebsiella spp. and blaOXA-48-like, blaNDM, blaCTX-M-1, blaTEM, and blaSHV (p < 0.001), Pseudomonas spp. and blaNDM (p = 0.002), Enterococcus spp. and blaOXA-48-like, blaNDM, blaCTX-M-1, blaSHV (p < 0.01). The correlation was observed between the expansion of Enterobacterales and and carbapenemase-positive CHROMagar samples (p < 0.001). Samples positive for carbapenem-resitant bacteria were at their maximum levels on day +30, and were gradually diminishing one year after allo-HSCT. From day +30 to +60, all isolated K. pneumoniae strains in fecal samples proved to be resistant to the main antibiotic groups (carbapenems, aminoglycosides, fluoroquinolones, third-generation cephalosporins). One year after HSCT, we documented the spontaneous decolonization of K. pneumoniae. The sensitivity of molecular biology techniques in the search for total and antibiotic-resistant Klebsiella seems to be superior to common bacteriological cultures. Future studies should be focused on searching for novel approaches to the efficient reconstitution and/or maintenance of strictly anaerobic microbiota in oncological patients.

Dissemination and characterization of Stenotrophomonas maltophilia isolates from Dairy Cows in Northeast China.

This work investigated the genetic relationship among Stenotrophomonas maltophilia strains in fecal samples from dairy cows in northeast China and identified the dominant β-lactamase genotype. One hundred and six samples were collected from two randomly selected cow farms in northeast China, and the isolates were identified with MALDI-TOF/MS. Whole-genome sequencing was conducted using Illumina HiSeq 4000-PE150 platform (Illumina, Inc., USA). The antimicrobial resistance genes were detected using CGE services. The phylogenetic analysis of S. maltophilia strains was performed by Roary and MEGA X. In total, 24 S. maltophilia isolates were isolated. The results of resistome analysis showed all S. maltophilia strains carrying bla L1 gene, which was the only β-lactamase genotype. In addition, the aminoglycoside resistance genes aac(6')-Iz and aph(3')-IIc were found. The phylogenetic tree indicated the clonal diversity of S. maltophilia in these two regions and the clonal relatedness of the strains from these regions. This study first investigated the dissemination and characterization of S. maltophilia isolates from dairy cows in northeast China and provided evidence of the potential transmission between two provinces. Furthermore, it indicated bla L1 was the most prevalent genotype of β-lactamase in these regions.

How is gut microbiome of patients with familial adenomatous polyposis different from healthy people?

The gut microbiome has been increasingly suggested as an underlying cause of various human diseases. In this study, we hypothesized that the gut microbiomes of patients with familial adenomatous polyposis (FAP) are different from those of healthy people and attempted to identify the associations between gut microbiome characteristics and FAP. We collected fecal samples from patients with FAP and healthy volunteers and evaluated the diversity, composition, and distribution of the gut microbiome between the 2 groups via 16S rRNA-based taxonomic profiling of the fecal samples. Fecal samples were collected from 10 patients with FAP (4 men and 6 women, mean age 39.2 ± 13.8 years) and 10 healthy volunteers (4 men and 6 women, mean age 40.9 ± 9.8 years). The microbial richness in patients with FAP was significantly lower than that in healthy people. Regarding microbial composition, the Firmicutes/Bacteroidetes ratio in patients with FAP was higher than that in healthy people, especially in those with a lower proportion of Bacteroidetes and a higher proportion of Proteobacteria. We also found 7 specific abundant strains in fecal samples of patients with FAP. Patients with FAP had different Firmicutes/Bacteroidetes ratios and Proteobacteria abundance compared to healthy people and showed the presence of specific bacteria. These findings suggest a promising role of the gut microbiome in patients with FAP, although further studies are needed.

DETECTION OF CARBAPENEMASE PRODUCING ENTEROBACTERALES AMONG THE CLINICAL ISOLATES OF DIARRHOEAGENIC ESCHERICHIA COLI CAUSING ACUTE GASTROENTERITIS IN CHILDREN BELOW 2 YEARS IN TERTIARY CARE HOSPITAL OF NORTH EAST INDIA

Introduction: Enterobacterales that test resistant to at least one of the carbapenem antibiotics (ertapenem, meropenem, doripenem, or imipenem) are called Carbapenem resistant Enterobacterales (CRE) and if they produce a carbapenemase (an enzyme that can make them resistant to carbapenem antibiotics) they are called Carpenemase producing Enterobacterales (CPE). Children with CRE strains in fecal samples are considered as a high risk group by World Health Organization (WHO), which can spread CRE by intimate contact and travel. This cross-sectional study was conducted in the Department of Microbiology, RIMS, Imphal, Manipur, India fromMethods: Jan 2020 to Feb 2022. A total of 157 children under 2 years of age whose stool culture was positive for diarrhoeagenic Escherichia coli were included in the study. The modied carbapenem inactivation method (mCIM) has been done for detection of carbapenemase producers and the addition of EDTA in eCIM to further differentiate between serine and metallo-β-lactamase producers. Out of 157Result and Discussion: Diarrhoegenic E.coli (DEC) ,Carbapenem resistance was seen in 9 isolates i.e 5.7 %. Out of these 9 isolates, 3 were MBL producers tested by the phenotypic test mCIM and eCIM. All the three MBL producers carried bla NDM-1 gene. mCIM/eCIM assay is designed to simultaneously detect and distinguish the different types of carbapenemases. Carbapenemase genes are often located on plasmids that can be exchanged between Enterobacteriaceae and other Gram-negative bacteria. Carbapenem-resistant K. pneumoniae are currently more frequent and more likely to cause healthcareassociated outbreaks, carbapenem-resistant E. coli pose a greater risk for spread in the community. Conclusion: Screening for carbapenemase producer using mCIM and eCIM essay is important along with infection control measure such as active surveillance through rectal screening for CRE carriage on hospital admission, contact precautions, hand hygiene, patient isolation, environmental sanitation, case notication/agging, antibiotic restriction.

Validation and improvement of a multiplex PCR method to detect murine Helicobacter species in feces samples of mice.

Murine Helicobacter species have gained increasing awareness in mouse facilities over the last years. Infections with Helicobacter species may have an impact effect on the health of mice and might pose a zoonotic risk to researchers. To minimize the interference with experiments and hence contribute to the 3Rs, a reliable method of monitoring Helicobacter infections in animal facilities needs to be available. The aim of this study was to improve and validate the detection of the most common murine Helicobacter species. A multiplex PCR assay was developed for identification of Helicobacter hepaticus, H.bilis, H.muridarum, H.rodentium, and H.typhlonius that could simultaneously detect these five strains in fecal samples. To ensure the quality of the results, the method was validated based on recommendations for in-house developed tests. The method established was highly sensitive and specific. All five strains were detectable with a detection limit of 102 bacteria. Eight different mouse facilities were tested with the validated assay, and the following prevalence were found: H.rodentium 57%, H.hepaticus 46%, H.typhlonius 17%, H.bilis 12%, and H.muridarum 0%. The multiplex PCR is a reliable, economic, and time-saving diagnostic tool for routine health monitoring. Further prevalence studies are needed to confirm the high prevalence and hence importance of H.rodentium, as until now this agent is not yet listed in FELASA recommendations.

Abundance of Multiple-Antibiotic-Resistant Salmonella Strains in Fecal Samples of Rhinoceros unicornis of the Kaziranga National Park, India

Aims: This study was undertaken to assess the abundance of multiple-antibiotic-resistant (MAR) Salmonella strains in fecal samples of Rhinoceros unicornis of the Kaziranga National Park (KNP), India.&#x0D; Study Design: Antibiotic-resistance profile of the Salmonella isolates from fecal samples of rhinoceros was carried out by replica plating on Muller Hinton Agar (MHA) plates containing antibiotics. The presence of class 1 integrons in metallo-β-lactamase (MBL) producing Salmonella isolates was determined by multiplex PCR.&#x0D; Place and Duration of Study: The study was carried out on rhinos of KNP situated in the Golaghat district of Assam, India in April 2015.&#x0D; Methodology: Fresh rhino fecal samples (designated as 1R, 2R, 3R, 4R and 5R) were dilution plated onto MacConkey agar. Purified bacterial colonies were then streaked separately on bismuth sulphite (BS) agar plates. All black colonies which are characteric growth of Salmonella were selected and used to make master plates on Luria Agar. To determine the antibiotic-resistance profile of the isolates, master-plates of purified single colonies of Salmonella spp. were replicate-printed on plates containing antibiotics from the β-lactam, aminoglycoside, or quinolone groups. To detect the presence of an integron, a conserved segment polymerase chain reaction was used.&#x0D; Results: 97.6% of the Salmonella isolates tested were resistant to &gt;1 antibiotics (multidrug resistant or MAR). A total of 100 isolates from two fecal samples, 4R and 5R, were found to be imipenem resistant; 52 of them tested positive for the presence of MBLs. Five of the twenty MBL producing strains contained class 1 integrons.&#x0D; Conclusion: Because Salmonella is usually spread by drinking contaminated water, it is likely that water bodies in KNP are contaminated with MAR Salmonella strains. In case of Salmonellosis outbreak among Indian one-horned rhinos, our antibiogram will assist the veterinarians to choose the appropriate regimen of antibiotics for the rhinos in the KNP.

Quantitative Detection of Bifidobacterium longum Strains in Feces Using Strain-Specific Primers.

We adopted a bioinformatics-based technique to identify strain-specific markers, which were then used to quantify the abundances of three distinct B. longum sup. longum strains in fecal samples of humans and mice. A pangenome analysis of 205 B. longum sup. longum genomes revealed the accumulation of considerable strain-specific genes within this species; specifically, 28.7% of the total identified genes were strain-specific. We identified 32, 14, and 49 genes specific to B. longum sup. longum RG4-1, B. longum sup. longum M1-20-R01-3, and B. longum sup. longum FGSZY6M4, respectively. After performing an in silico validation of these strain-specific markers using a nucleotide BLAST against both the B. longum sup. longum genome database and an NR/NT database, RG4-1_01874 (1331 bp), M1-20-R01-3_00324 (1745 bp), and FGSZY6M4_01477 (1691 bp) were chosen as target genes for strain-specific quantification. The specificities of the qPCR primers were validated against 47 non-target microorganisms and fecal baseline microbiota to ensure that they produced no PCR amplification products. The performance of the qPCR primer-based analysis was further assessed using fecal samples. After oral administration, the target B. longum strains appeared to efficiently colonize both the human and mouse guts, with average population levels of >108 CFU/g feces. The bioinformatics pipeline proposed here can be applied to the quantification of various bacterial species.

Phylogenetic analysis of partial transmembrane protein gene of canine coronaviruses detected in Turkey

Canine coronaviruses (CCoVs), a member of the family Coronaviridae, are the causative agents of acute gastroenteritis and are genetically divided into two groups, CCoV type I and CCoV type II. The aim of this study was to detect and characterize CCoV strains in fecal samples from six dogs exhibited gastrointestinal system symptoms. To determine the presence of the CCoV RNA, samples were tested by the reverse transcription-polymerase chain reaction (RT-PCR) assay targeting the partial M gene and then sequenced. Among six samples tested, two were found positive for CCoV RNA. Phylogenetic analysis was performed by Maximum-Likelihood (ML) method and revealed that one of the obtained field sequences was classified into CCoV-I genotype; and the other positive sample grouped in CCoV-II genotype. Both genogroups demonstrated broad genetic diversity. Phylogenetic analysis of amino acid sequences shows that our CCoV field strains was closely related to Italy and Brazil strains and placed on different genogroup clades in the CCoV cluster. Sequence comparison of the partial M gene revealed nucleotide identity of 71–100% and 68–100% similarity among the 25 coronavirus strains. TR/Ccv2 (MK636864) and TR/Ccv6 (MK636865) obtained in this study demonstrated 78.5-97.5% and 71–99% nucleotide identity with other CCoV strains around the world respectively. The results of the study demonstrate, CCoV strains from different genogroups are circulating in Turkey and this is a report on the phylogenetic analysis of a CCoV in Turkey, which there is limited information.

Lab Protocol Paper: Use of a High-throughput, Multiplex Reverse-transcription Quantitative Polymerase Chain Reaction Assay for Detection of Sabin Oral Polio Vaccine in Fecal Samples.

BackgroundGlobal polio eradication efforts rely in part on molecular methods of detecting polioviruses, both wild and vaccine strains, from human and environmental samples. Previous assays used for detection of Sabin oral polio vaccine (OPV) in fecal samples have been labor and time intensive and vary in their sensitivity and specificity.MethodsWe developed a high-throughput, multiplex reverse-transcription quantitative polymerase chain reaction assay able to detect all 3 OPV strains in fecal samples. The assay used a KingFisher Duo Prime system for viral RNA isolation and extraction. Positive samples were retested and Sanger sequenced for verification of Sabin serotype identity.ResultsThe 95% lower limit of detection was determined to be 3 copies per reaction for Sabin 1 and 3 and 4 copies per reaction for Sabin 2, with no cross-reactivity between the 3 serotypes and their primers. A total of 554 samples (3.6%) were positive, with 304 positive samples (54.9%) containing >1 serotype. Of the positive samples, 476 (85.9%) contained enough RNA to be sequenced, and of these all sequences were Sabin serotypes. The previous assay we used could process 48 samples in a 10-hour period, whereas the new assay processed >100 samples in 6 hours.ConclusionsThe new high-throughput, multiplex reverse-transcription quantitative polymerase chain reaction assay allowed for sensitive and specific detection of OPV serotypes while greatly decreasing sample handling and processing time. We were able to sequence 72.4% of the 210 positive samples in the cycle threshold range of 35–37.

Frequency of CagA-positive Helicobacter pylori strains in 160 patients subjected to endoscopy

Helicobacter pylori is the most significant pathogen associated with gastric diseases, including gastric cancer. Infected patients with strains that are CagA-positive generally have worse outcomes than those infected with CagA-negative strains. Patients infected with CagA-positive strains have a higher risk for developing gastric cancer. To determine the prevalence of CagA-positive H. pylori strains in fecal samples of patients from the Coquimbo Region of Chile, using a non-invasive, nested-qPCR method. We evaluated 160 patients with gastrointestinal symptoms subjected to an upper gastrointestinal endoscopy. DNA was extracted from fecal samples and tested for the presence of H. pylori using nested-qPCR for the ureC gene, and subsequently compared with the results of histology-Giemsa stain from the patients' endoscopic biopsies. When H. pylori was found, the presence of CagA-positive strains was determined via nested-qPCR. The histology-Giemsa stain was positive for H. pylori infection in 123 patients (76.9%), while the analysis of fecal samples detected H. pylori in 129 patients (80.6%). The sensitivity and specificity of nested-qPCR to detect the bacterium was 96.7 and 73.0% respectively. Among patients with the infection, 25% had CagA-positive strains. In this sample of patients, there is a low prevalence of CagA-positive H. pylori strains.

Potential of Lactobacillus plantarum IBB3036 and Lactobacillus salivarius IBB3154 to persistence in chicken after in ovo delivery.

The aim of this study was to characterize and compare selected Lactobacillus strains originating from different environments (cow milk and hen feces) with respect to their applicative potential to colonize gastrointestinal track of chickens before hatching from an egg. In vitro phenotypic characterization of lactobacilli strains included the investigation of the important prerequisites for persistence in gastrointestinal tract, such as a capability to survive in the presence of bile salts and at low pH, enzymatic and sugar metabolic profiles, adhesion abilities, and resistance to osmolytes, temperature, and antibiotics. Regarding the resistance of lactobacilli to most of the various stress factors tested, the milk isolate Lactobacillus plantarum IBB3036 showed better abilities than the chicken feces isolate Lactobacillus salivarius IBB3154. However, regarding the acidification tolerance and adherence ability, L. salivarius IBB3154 revealed better characteristics. Use of these two selected lactobacilli isolates together with proper prebiotics resulted in the preparation of two S1 and S2 bioformulations, which were injected in ovo into hen Cobb500 FF fertilized eggs. Furthermore, in vivo tests assessing the persistence of L. plantarum IBB3036 and L. salivarius IBB3154 in the chicken gastrointestinal tract was monitored by PCR‐based classical and quantitative techniques and revealed the presence of both strains in fecal samples collected 3 days after hatching. Subsequently, the number of L. salivarius IBB3154 increased significantly in the chicken intestine, whereas the presence of L. plantarum IBB3036 was gradually decreased.

Novel encapsulation improves recovery of probiotic strains in fecal samples of human volunteers

Probiotic supplements can contribute to maintaining health and ameliorating various disease symptoms. Probiotics can be delivered in many forms with crucial differences in their survival during gastrointestinal (GI) passage. Previously, a novel encapsulation, Probiotic Pearls™ Acidophilus, Integrative Therapeutics, LLC, USA (Pearls), was shown to increase survival in vitro after exposure to gastric conditions. Here, we compare fecal recovery in human volunteers consuming Pearls or a conventional hard-shelled gelatin capsule. We performed a randomized double-blinded, two-armed trial, with six healthy subjects in each 12-day study arm. In fecal samples collected at baseline, twice during the intervention period, and after washout, we compared colony counts between the two encapsulation methods. The identity of the colonies was confirmed by colony morphology, strain-specific PCR, and 16S rRNA gene sequencing. We further performed a comprehensive 16S rRNA gene sequencing-based analysis to identify differential effects on overall microbiota composition. We detected an average log increase in bifidobacteria of 0.152cfu/g with gelatin and 0.651cfu/g with Pearls capsules (p>0.05). Total lactobacilli counts increased in both groups with no difference between the groups. However, the supplemented Lactobacillus acidophilus NCFM decreased to baseline levels within 7days after end of supplementation with gelatin capsules while 3.11 log cfu/g higher counts compared to baseline (p=0.05) remained for Pearls. Targeted qPCR largely confirmed the trends observed by viable plate counts. Protecting the probiotic strains by Pearls encapsulation results in higher recovery rates of the supplemented lactobacilli and bifidobacteria in fecal samples and increased persistence, suggesting an improved survival and viability that might increase efficacy towards achieving desired health benefits.

Differentiation of RotaTeq® vaccine strains from wild-type strains using NSP3 gene in reverse transcription polymerase chain reaction assay

RotaTeq® is a live attenuated human-bovine reassortant vaccine against rotaviruses that is used worldwide. However, shedding of the virus used in RotaTeq® has been detected in the feces of children following vaccination by the oral route, possibly affecting community immunity. Therefore, a simple and efficient method to discriminate between virulent and RotaTeq® vaccine strains is required. In this study, a novel one-step multiplex reverse-transcription polymerase chain reaction (RT-PCR) assay targeting the NSP3 gene was developed to detect RotaTeq® vaccine strains in fecal samples. RotaTeq® vaccine viruses were successfully distinguished from known wild-type rotavirus genotypes. In addition, the developed assay was able to detect rotaviruses in clinical stool samples obtained from South Korea during the 2011–2013 rotavirus seasons. Of the 1106 stool specimens from children with acute gastroenteritis that were screened, 286 rotaviruses were genotyped. RotaTeq® vaccine strains were identified in 39 samples (13.6%). The novel RT-PCR assay that was developed could be used to detect and discriminate between RotaTeq® vaccine strains that are shed in fecal matter, and to estimate the quantification of virus that has been shed after vaccination.

Enterotoxigenic and non-enterotoxigenic Bacteroides fragilis from fecal microbiota of children.

Enterotoxigenic Bacteroides fragilis (ETBF) is an important part of the human and animal intestinal microbiota and is commonly associated with diarrhea. ETBF strains produce an enterotoxin encoded by the bft gene located in the B. fragilis pathogenicity island (BfPAI). Non-enterotoxigenic B. fragilis (NTBF) strains lack the BfPAI and usually show two different genetic patterns, II and III, based on the absence or presence of a BfPAI-flanking region, respectively. The incidence of ETBF and NTBF strains in fecal samples isolated from children without acute diarrhea or any other intestinal disorders was determined. All 84 fecal samples evaluated were B. fragilis-positive by PCR, four of them harbored the bft gene, 27 contained the NTBF pattern III DNA sequence, and 52 were considered to be NTBF pattern II samples. One sample was positive for both ETBF and NTBF pattern III DNA sequences. All 19 B. fragilis strains isolated by the culture method were bft-negative, 9 belonged to pattern III and 10 to pattern II. We present an updated overview of the ETBF and NTBF incidence in the fecal microbiota of children from Sao Paulo City, Brazil.

Long Term Development of Gut Microbiota Composition in Atopic Children: Impact of Probiotics.

IntroductionImbalance of the human gut microbiota in early childhood is suggested as a risk factor for immune-mediated disorders such as allergies. With the objective to modulate the intestinal microbiota, probiotic supplementation during infancy has been used for prevention of allergic diseases in infants, with variable success. However, not much is known about the long-term consequences of neonatal use of probiotics on the microbiota composition. The aim of this study was to assess the composition and microbial diversity in stool samples of infants at high-risk for atopic disease, from birth onwards to six years of age, who were treated with probiotics or placebo during the first year of life.MethodsIn a double-blind, randomized, placebo-controlled trial, a probiotic mixture consisting of B. bifidum W23, B. lactis W52 and Lc. Lactis W58 (Ecologic® Panda) was administered to pregnant women during the last 6 weeks of pregnancy and to their offspring during the first year of life. During follow-up, faecal samples were collected from 99 children over a 6-year period with the following time points: first week, second week, first month, three months, first year, eighteen months, two years and six years. Bacterial profiling was performed by IS-pro. Differences in bacterial abundance and diversity were assessed by conventional statistics.ResultsThe presence of the supplemented probiotic strains in faecal samples was confirmed, and the probiotic strains had a higher abundance and prevalence in the probiotic group during supplementation. Only minor and short term differences in composition of microbiota were found between the probiotic and placebo group and between children with or without atopy. The diversity of Bacteroidetes was significantly higher after two weeks in the placebo group, and at the age of two years atopic children had a significantly higher Proteobacteria diversity (p < 0.05). Gut microbiota development continued between two and six years, whereby microbiota composition at phylum level evolved more and more towards an adult-like configuration.ConclusionPerinatal supplementation with Ecologic® Panda, to children at high-risk for atopic disease, had minor effects on gut microbiota composition during the supplementation period. No long lasting differences were identified. Regardless of intervention or atopic disease status, children had a shared microbiota development over time determined by age that continued to develop between two and six years.

Experimental Induction of &amp;lt;i&amp;gt;Escherichia coli&amp;lt;/i&amp;gt; Diarrhoea in Weaned Piglets

Escherichia coli diarrhoea is a multifactorial condition which usually occurs during the post-weaning and is responsible for economic losses in pig production. One approach, to evaluate if substitute of antibiotic in vivo is effective in controlling postweaning diarrhea in the gastrointestinal tract ecosystem, is to use an appropriate disease model. However, there are still many criticisms related to the incidence and the severity of the diarrhoea in the experimental conditions. The aim of the study was to set up an Escherichia coli challenge model in order to induce a significant percentage of diarrhoea in weaned piglets for the evaluation of innovative compounds in vivo. A total of 35 piglets, weaned at 33±2 days were randomized into 3 groups: control (CG), infected 1 (IG1) and infected 2 (IG2). One day after arrival piglets of IG1 and IG2 were orally inoculated with 3.7 × 108 CFU of Escherichia coli O149. All piglets were fed a high protein ration for 3 days. Daily health status and faeces were recorded by a point scale individually. Challenge strains in faecal samples were evaluated by polymerase chain reaction, serotyping and biochemical identification. Diarrhoea was observed in 96.67% (58.6% severe; 41.4% mild) of all infected piglets and occurred on average 1.3 days after the challenge. The CG group presented one piglet with a transient mild diarrhoea. The E. coli challenge significantly affected the consistency and color of faeces (P E. coli O149, mainly hemolytic (88%), was isolated in 56% of faecal samples and the 70% of piglets with severe diarrhoea shed E. coli O149 in the faeces. Zootechnical parameters did not show significant differences. The experimental conditions described in this study allowed to effectively induce diarrhoea in weaned piglets. In conclusion a multifactorial approach (infectious, nutritional and management) is necessary to reproduce in vivo diarrhea in piglets.

Detection of Clostridium difficile in animals: comparison of real-time PCR assays with the culture method

Clostridium difficile has emerged as a pathogen or commensal in food animals. There is overlap between isolates from animals, retail meats and humans, suggesting that animals may be a C. difficile reservoir. For direct detection of variant C. difficile strains in faecal samples of symptomatic and asymptomatic animals, we developed and validated a new TaqMan real-time PCR (TMrtPCR) assay targeting the tcdA, tcdB and cdtB genes. We compared it with the enrichment culture method and with two real-time PCR (rtPCR) assays, BrtPCR and PCRFast, targeting tcdB and tcdA/tcdB, respectively. All ten tested C. difficile toxinotypes, except one (XIa) with PCRFast and two (X, XIa) with BrtPCR, were detected with the test assays. A total of 340 (100 %) samples were cultured and amplified with TMrtPCR. Results correlated in 75.3 % samples. Forty (11.8 %) samples were culture positive/TMrtPCR negative, possibly because of the low numbers of bacteria in the samples or because of DNA extraction failure. Forty (11.8 %) samples were TMrtPCR positive/culture negative. Among 79 samples included in the rtPCR assays/culture comparison, 50.6 % were in complete concordance. The results showed that TMrtPCR performed better than BrtPCR and PCRFast, and 67 % of the culture-positive samples were TMrtPCR positive in comparison to 40 % of the samples positive in BrtPCR and 7 % of the samples positive in PCRFast, respectively. Another advantage of TMrtPCR over BrtPCR and PCRFast is its ability to detect a binary toxin gene. Therefore, the TMrtPCR results can provide the first information about the toxin type present in the sample. According to the results of our study, TMrtPCR could be a preferred screening method for the rapid detection of C. difficile in animal faecal samples, although an enrichment culture has to be performed for the specimens with negative or inconclusive rtPCR results.

Occurrence and antimicrobial drug susceptibility patterns of commensal and diarrheagenic Escherichia coli in fecal microbiota from children with and without acute diarrhea

Acute diarrhea is a public health problem and an important cause of morbidity and mortality, especially in developing countries. The etiology is varied, and the diarrheagenic Escherichia coli pathotypes are among the most important. Our objectives were to determine the occurrence of commensal and diarrheagenic E. coli strains in fecal samples from children under five years old and their drug susceptibility patterns. E. coli were isolated from 141 fresh fecal samples; 84 were obtained from clinically injured donors with acute diarrhea (AD) and 57 from clinically healthy donors without diarrhea (WD). Presumptive phenotypic species identification was carried out and confirmed by amplification of specific 16S ribosomal RNA encoding DNA. Multiplex PCR was performed to characterize the diarrheagenic E. coli strains. Drug susceptibility patterns were determined by the disc-diffusion method. In total, 220 strains were recovered from the fecal specimens (61.8% from AD and 38.2% from WD). Diarrheagenic E. coli was identified at a rate of 36.8% (n=50) in diarrheic feces and 29.8% (n=25) in non-diarrheic feces. Enteroaggregative E. coli was the most frequently identified pathotype in the AD group (16.2%) and the only pathotype identified in the WD group (30.9%). Enteropathogenic E. coli was the second most isolated pathotype (10.3%), followed by Shiga toxin-producing E. coli (7.4%) and enterotoxigenic E. coli (2.9%). No enteroinvasive E. coli strains were recovered. The isolates showed high resistance rates against ampicillin, tetracycline, and sulfamethoxazole-trimethoprim. The most effective drugs were ceftazidime, ceftriaxone, imipenem and piperacillin-tazobactam, for which no resistance was observed. Differentiation between the diarrheagenic E. coli pathotypes is of great importance since they are involved in acute diarrheal diseases and may require specific antimicrobial chemotherapy. The high antimicrobial resistance observed in our study raises a broad discussion on the indiscriminate or improper use of antimicrobials, besides the risks of self-medication.

Molecular typing of Escherichia coli strains associated with threatened sea ducks and near‐shore marine habitats of south‐west Alaska

In Alaska, sea ducks winter in coastal habitats at remote, non-industrialized areas, as well as in proximity to human communities and industrial activity. We evaluated prevalence and characteristics of Escherichia coli strains in faecal samples of Steller's eiders (Polysticta stelleri; n = 122) and harlequin ducks (Histrionicus histrionicus; n = 21) at an industrialized site and Steller's eiders (n = 48) at a reference site, and compared these strains with those isolated from water samples from near-shore habitats of ducks. The overall prevalence of E. coli was 16% and 67% in Steller's eiders and harlequin ducks, respectively, at the industrialized study site, and 2% in Steller's eiders at the reference site. Based on O and H antigen subtyping and genetic characterization by enterobacterial repetitive intergenic consensus polymerase chain reaction and pulsed-field gel electrophoresis, we found evidence of avian pathogenic E. coli (APEC) strains associated with both species and detected E. coli strains carrying virulence genes associated with mammals in harlequin ducks. Steller's eiders that carried APEC had lower serum total protein and albumin concentrations, providing further evidence of pathogenicity. The genetic profile of two E. coli strains from water matched an isolate from a Steller's eider providing evidence of transmission between near-shore habitats and birds.