Novel micelle PCR-based method for accurate, sensitive and quantitative microbiota profiling

In the last decade, many researchers have embraced 16S rRNA gene sequencing techniques, which has led to a wealth of publications and documented differences in the composition of microbial communities derived from many different ecosystems. However, comparison between different microbiota studies is currently very difficult due to the lack of a standardized 16S rRNA gene sequencing protocol. Here we report on a novel approach employing micelle PCR (micPCR) in combination with an internal calibrator that allows for standardization of microbiota profiles via their absolute abundances. The addition of an internal calibrator allows the researcher to express the resulting operational taxonomic units (OTUs) as a measure of 16S rRNA gene copies by correcting the number of sequences of each individual OTU in a sample for efficiency differences in the NGS process. Additionally, accurate quantification of OTUs obtained from negative extraction control samples allows for the subtraction of contaminating bacterial DNA derived from the laboratory environment or chemicals/reagents used. Using equimolar synthetic microbial community samples and low biomass clinical samples, we demonstrate that the calibrated micPCR/NGS methodology possess a much higher precision and a lower limit of detection compared with traditional PCR/NGS, resulting in more accurate microbiota profiles suitable for multi-study comparison

Micelle PCR reduces chimera formation in 16S rRNA profiling of complex microbial DNA mixtures

16S rRNA gene profiling has revolutionized the field of microbial ecology. Many researchers in various fields have embraced this technology to investigate bacterial compositions of samples derived from many different ecosystems. However, it is important to acknowledge the current limitations and drawbacks of 16S rRNA gene profiling. Although sample handling, DNA extraction methods and the choice of universal 16S rRNA gene PCR primers are well known factors that could seriously affect the final results of microbiota profiling studies, inevitable amplification artifacts, such as chimera formation and PCR competition, are seldom appreciated. Here we report on a novel micelle based amplification strategy, which overcomes these limitations via the clonal amplification of targeted DNA molecules. Our results show that micelle PCR drastically reduces chimera formation by a factor of 38 (1.5% vs. 56.9%) compared with traditional PCR, resulting in improved microbial diversity estimates. In addition, compartmentalization during micelle PCR prevents PCR competition due to unequal amplification rates of different 16S template molecules, generating robust and accurate 16S microbiota profiles required for comparative studies (e.g. longitudinal surveys)

Development and evaluation of a culture-free microbiota profiling platform (MYcrobiota) for clinical diagnostics

Microbiota profiling has the potential to greatly impact on routine clinical diagnostics by detecting DNA derived from live, fastidious, and dead bacterial cells present within clinical samples. Such results could potentially be used to benefit patients by influencing antibiotic prescribing practices or to generate new classical-based diagnostic methods, e.g., culture or PCR. However, technical flaws in 16S rRNA gene next-generation sequencing (NGS) protocols, together with the requirement for access to bioinformatics, currently hinder the introduction of microbiota analysis into clinical diagnostics. Here, we report on the development and evaluation of an “end-to-end” microbiota profiling platform (MYcrobiota), which combines our previously validated micelle PCR/NGS (micPCR/NGS) methodology with an easy-to-use, dedicated bioinf

Characterization of the nasopharyngeal and middle ear microbiota in gastroesophageal reflux-prone versus gastroesophageal reflux non-prone children

Otitis media (OM) is one of the most common pediatric infections worldwide, but the complex microbiology associated with OM is poorly understood. Previous studies have shown an association between OM and gastroesophageal reflux (GER) in children. Therefore, in order to bridge the gap in our current understanding of the interaction between GER and OM, we investigated the nasopharyngeal and middle ear microbiota of children suffering from GER-associated OM and OM only, using culture-independent 16S rRNA gene sequencing. Middle ear fluid, nasopharyngeal swabs, and clinical data were collected as part of a prospective pilot study conducted at the Department of Otorhinolaryngology of the Erasmus MC-Sophia Children’s Hospital, Rotterdam, the Netherlands. A total of 30 children up to 12 years of age who suffered from recurrent acute otitis media (AOM) (5), chronic otitis media with effusion (OME) (23), or both (2), and who were listed for tympanostomy tube placement, were included in the study. Nine children were included in the GER-associated OM cohort and 21 in the OM-only cohort. We found no obvious effect of GER on the nasopharyngeal and middle ear microbiota between the two groups of children. However, our results highlight the need to assess the true role of Alloiococcus spp. and Turicella spp. in children presenting with a high prevalence of recurrent AOM and chronic OME

Comparison of mycoplasma pneumoniae genome sequences from strains isolated from symptomatic and asymptomatic patients

Mycoplasma pneumoniae is a common cause of respiratory tract infections (RTIs) in children. We recently demonstrated that this bacterium can be carried asymptomatically in the respiratory tract of children. To identify potential genetic differences between M. pneumoniae strains that are carried asymptomatically and those that cause symptomatic infections, we performed whole-genome sequence analysis of 20 M. pneumoniae strains. The analyzed strains included 3 reference strains, 3 strains isolated from asymptomatic children, 13 strains isolated from clinically well-defined patients suffering from an upper (n = 4) or lower (n = 9) RTI, and one strain isolated from a follow-up patient who recently recovered from an RTI. The obtained sequences were each compared to the sequences of the reference strains. To find differences between strains isolated from asymptomatic and symptomatic individuals, a variant comparison was performed between the different groups of strains. Irrespective of the group (asymptomatic vs. symptomatic) from which the strains originated, subtype 1 and subtype 2 strains formed separate clusters. We could not identify a specific genotype associated with M. pneumoniae virulence. However, we found marked genetic differences between clinical isolates and the reference strains, which indicated that the latter strains may

Monitoring of microbial dynamics in a drinking water distribution system using the culture-free, user-friendly, MYcrobiota platform

Drinking water utilities currently rely on a range of microbiological detection techniques to evaluate the quality of their drinking water (DW). However, microbiota profiling using culture-free 16S rRNA gene next-generation sequencing (NGS) provides an opportunity for improved monitoring of the microbial ecology and quality of DW. Here, we evaluated the utility of a previously validated microbiota profiling platform (MYcrobiota) to investigate the microbial dynamics of a full-scale, non-chlorinated DW distribution system (DWDS). In contrast to conventional methods, we observed spatial and temporal bacterial genus changes (expressed as operational taxonomic units - OTUs) within the DWDS. Further, a small subset of bacterial OTUs dominated with abundances that shifted across the length of the DWDS, and were particularly affected by a post-disinfection step. We also found seasonal variation in OTUs within the DWDS and that many OTUs could not be identified, even though MYcrobiota is specifically designed to reduce potential PCR sequencing artefacts. This suggests that our current knowledge about the microbial ecology of DW communities is limited. Our findings demonstrate that the user-friendly MYcrobiota platform facilitates culture-free, standardized microbial dynamics monitoring and has the capacity to facilitate the introduction of microbiota profiling into the management of drinking water quality

Lactic acid production from lime-treated wheat straw by Bacillus coagulans: neutralization of acid by fed-batch addition of alkaline substrate

Conventional processes for lignocellulose-to-organic acid conversion requires pretreatment, enzymatic hydrolysis, and microbial fermentation. In this study, lime-treated wheat straw was hydrolyzed and fermented simultaneously to lactic acid by an enzyme preparation and Bacillus coagulans DSM 2314. Decrease in pH because of lactic acid formation was partially adjusted by automatic addition of the alkaline substrate. After 55 h of incubation, the polymeric glucan, xylan, and arabinan present in the lime-treated straw were hydrolyzed for 55%, 75%, and 80%, respectively. Lactic acid (40.7 g/l) indicated a fermentation efficiency of 81% and a chiral l(+)-lactic acid purity of 97.2%. In total, 711 g lactic acid was produced out of 2,706 g lime-treated straw, representing 43% of the overall theoretical maximum yield. Approximately half of the lactic acid produced was neutralized by fed-batch feeding of lime-treated straw, whereas the remaining half was neutralized during the batch phase with a Ca(OH)2 suspension. Of the lime added during the pretreatment of straw, 61% was used for the neutralization of lactic acid. This is the first demonstration of a process having a combined alkaline pretreatment of lignocellulosic biomass and pH control in fermentation resulting in a significant saving of lime consumption and avoiding the necessity to recycle lime

Advancing interdisciplinary research in head and neck cancer through a multicenter longitudinal prospective cohort study: The NETherlands QUality of life and BIomedical Cohort (NET-QUBIC) data warehouse and biobank

Background: Worldwide, over 500,000 people are diagnosed with head and neck cancer each year, a disease with major impact on life expectancy and quality of life. The purpose of the Netherlands Quality of life and Biomedical Cohort study (NET-QUBIC) is to advance interdisciplinary research that aims to optimize diagnosis, treatment, and supportive care for head and neck cancer patients and their informal caregivers. Methods: Using an extensive assessment protocol (electronic clinical record form, patient reported outcome measures and fieldwork (interviews and physical tests)), clinical data and data on quality of life, demographic and personal factors, psychosocial (depression, anxiety, fatigue, pain, sleep, mental adjustment to cancer, posttraumatic stress), physical (speech, swallowing, oral function, malnutrition, physical fitness, neurocognitive function, sexual function), lifestyle (physical activity, nutrition, smoking, alcohol, drugs), and social factors (social function, social support, work, health care use, and costs) are collected and stored in the data warehouse. A longitudinal biobank is built with tumor tissue, blood and blood components, saliva samples, and oral rinses. An infrastructure for fieldwork and laboratory protocols is esta

Patient-Reported Outcomes and Function after Surgical Repair of the Ulnar Collateral Ligament of the Thumb

Purpose: The purpose of this study was to report prospectively collected patient-reported outcomes of patients who underwent open thumb ulnar collateral ligament (UCL) repair and to find risk factors associated with poor patient-reported outcomes. Methods: Patients undergoing open surgical repair for a complete thumb UCL rupture were included between December 2011 and February 2021. Michigan Hand Outcomes Questionnaire (MHQ) total scores at baseline were compared to MHQ total scores at three and 12 months after surgery. Associations between the 12-month MHQ total score and several variables (i.e., sex, injury to surgery time, K-wire immobilization) were analyzed. Results: Seventy-six patients were included. From baseline to three and 12 months after surgery, patients improved significantly with a mean MHQ total score of 65 (standard deviation [SD] 15) to 78 (SD 14) and 87 (SD 12), respectively. We did not find any differences in outcomes between patients who underwent surgery in the acute (&lt;3 weeks) setting compared to a delayed setting (&lt;6 months). Conclusions: We found that patient-reported outcomes improve significantly at three and 12 months after open surgical repair of the thumb UCL compared to baseline. We did not find an association between injury to surgery time and lower MHQ total scores. This suggests that acute repair for full-thickness UCL tears might not always be necessary. Type of study/level of evidence: Therapeutic II.</p