Studies into the Genetic Diversity and Complement Resistance Phenotype of Moraxella catarrhalis

The aim of this thesis was to help define the contribution of Moraxella catarrhalis genetic diversity on the ability of the bacterium to colonise and cause infection in the human host, as well as to investigate novel genes/mechanisms associated with isolates exhibiting the complement resistance phenotype. In this respect, we show that M. catarrhalis is a genetically diverse organism in hospitalised children, with type-switching not leading to an increase in disease burden. Further, vaccination with a Streptococcus pneumoniae vaccine (an organism that shares the same biological niche as M. catarrhalis) does not affect M. catarrhalis diversity. Finally, genetically diverse isolates were found within otitis media-prone children, as well as a low frequency of intra-genomic variation in two complement resistance associated genes. With respect to genes/mechanisms associated with the complement phenotype, a novel major outer membrane protein of M. catarrhalis (OMP J) associated (but not conferring) complement resistance was identified and characterised. This protein existed in two major forms, was immunogenic, but was found not to be a suitable vaccine candidate. In a further study, both complement resistant and complement sensitive isolates of M. catarrhalis were found to be only weak activators of the mannose-binding lectin (MBL) pathway of complement activation (a useful survival strategy). Finally, a novel plasmid of M. catarrhalis is described named pEMCJH03 (and only the second plasmid to be identified within this species). This plasmid may be useful as a cloning and expression vector for putative M. catarrhalis virulence genes (including the testing of complement resistance-associated genes)

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)

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

Pre-implementation guidelines for infectious disease point-of-care testing in medical institutions

Infectious disease point-of-care test (ID-POCT) devices are becoming widely available, and in this respect, international quality standards and guidelines are available for consultation once ID-POCT has been implemented into medical institutions. However, specific guidelines for consultation during the initial pre-implementation decision-making process are currently lacking. Further, there exist pre-implementation issues specific to ID-POCT. Here we present pre-implementation guidelines for consultation when considering the implementation of ID-POCT in medical institutions

Antibiotic misuse in respiratory tract infections in children and adultsa prospective, multicentre study (TAILORED Treatment)

Respiratory tract infections (RTI) are more commonly caused by viral pathogens in children than in adults. Surprisingly, little is known about antibiotic use in children as compared to adults with RTI. This prospective study aimed to determine antibiotic misuse in children and adults with RTI, using an expert panel reference standard, in order to prioritise the target age population for antibiotic stewardship interventions. We recruited children and adults who presented at the emergency department or were hospitalised with clinical presentation of RTI in The Netherlands and Israel. A panel of three experienced physicians adjudicated a reference standard diagnosis (i.e. bacterial or viral infection) for all the patients using all available clinical and laboratory information, including a 28-day follow-up assessment. The cohort included 284 children and 232 adults with RTI (median age, 1.3 years and 64.5 years, respectively). The proportion of viral infections was larger in children than in adults (209(74%) versus 89(38%), p < 0.001). In case of viral RTI, antibiotics were prescribed (i.e. overuse) less frequently in children than in adults (77/ 209 (37%) versus 74/89 (83%), p < 0.001). One (1%) child and three (2%) adults with bacterial infection were not treated with antibiotics (i.e. underuse); all were mild cases. This international, prospective study confirms major antibiotic overuse in patients with RTI. Viral infection is more common in children, but antibiotic overuse is more frequent in adults with viral RTI. Together, these findings support the need for effective interventions to decrease antibiotic overuse in RTI patients of all ages