Antimicrobial susceptibility testing (AST) technologies help to accelerate the initiation of targeted antimicrobial therapy for patients with infections and could potentially extend the lifespan of current narrow-spectrum antimicrobials. Although conceptually new and rapid AST technologies have been described, including new phenotyping methods, digital imaging and genomic approaches, there is no single major, or broadly accepted, technological breakthrough that leads the field of rapid AST platform development. This might be owing to several barriers that prevent the timely development and implementation of novel and rapid AST platforms in health-care settings. In this Consensus Statement, we explore such barriers, which include the utility of new methods, the complex process of validating new technology against reference methods beyond the proof-of-concept phase, the legal and regulatory landscapes, costs, the uptake of new tools, reagent stability , optimization of target product profiles, difficulties conducting clinical trials and issues relating to quality and quality control, and present possible solutions.

Recent advancements in next-generation sequencing (NGS) have provided the foundation for modern studies into the composition of microbial communities. The use of these NGS methods allows for the detection and identification of (‘difficult-to-culture’) microorganisms using a culture-independent strategy. In the field of routine clinical diagnostics however, the application of NGS is currently limited to microbial strain typing for epidemiological purposes only, even though the implementation of NGS for microbial community analysis may yield clinically important information. This lack of NGS implementation is due to many different factors, including issues relating to NGS method standardization and result reproducibility. In this review article, the authors provide a general introduction to the most widely used NGS methods currently available (i.e., targeted amplicon sequencing and shotgun metagenomics) and the strengths and weaknesses of each method is discussed. The focus of the publication then shifts toward 16S rRNA gene NGS methods, which are currently the most cost-effective and widely used NGS methods for research purposes, and are therefore more likely to be successfully implemented into routine clinical diagnostics in the short term. In this respect, the experimental pitfalls and biases created at each step of the 16S rRNA gene NGS workflow are explained, as well as their potential solutions. Finally, a novel diagnostic microbiota profiling platform (‘MYcrobiota’) is introduced, which was developed by the authors by taking into consideration the pitfalls, biases, and solutions explained in this article. The development of the MYcrobiota, and future NGS methodologies, will help pave the way toward the successful implementation of NGS methodologies into routine clinical diagnostics.

The gut microbiota has been shown to play diverse roles in human health and disease although the underlying mechanisms have not yet been fully elucidated. Large cohort studies can provide further understanding into inter-individual differences, with more precise characterization of the pathways by which the gut microbiota influences human physiology and disease processes. Here, we aimed to profile the stool microbiome of children and adults from two population-based cohort studies, comprising 2,111 children in the age-range of 9 to 12 years (the Generation R Study) and 1,427 adult individuals in the range of 46 to 88 years of age (the Rotterdam Study). For the two cohorts, 16S rRNA gene profile datasets derived from the Dutch population were generated. The comparison of the two cohorts showed that children had significantly lower gut microbiome diversity. Furthermore, we observed higher relative abundances of genus Bacteroides in children and higher relative abundances of genus Blautia in adults. Predicted functional metagenome analysis showed an overrepresentation of the glycan degradation pathways, riboflavin (vitamin B2), pyridoxine (vitamin B6) and folate (vitamin B9) biosynthesis pathways in children. In contrast, the gut microbiome of adults showed higher abundances of carbohydrate metabolism pathways, beta-lactam resistance, thiamine (vitamin B1) and pantothenic (vitamin B5) biosynthesis pathways. A predominance of catabolic pathways in children (valine, leucine and isoleucine degradation) as compared to biosynthetic pathways in adults (valine, leucine and isoleucine biosynthesis) suggests a functional microbiome switch to the latter in adult individuals. Overall, we identified compositional and functional differences in gut microbiome between children and adults in a population-based setting. These microbiome profiles can serve as reference for future studies on specific human disease susceptibility in childhood, adulthood and specific diseased populations.

Moraxella catarrhalis is an emerging human-restricted respiratory tract pathogen that is a common cause of childhood otitis media and exacerbations of chronic obstructive pulmonary disease in adults. Here, we report the first completely assembled and annotated genome sequence of an isolate of M. catarrhalis, strain RH4, which originally was isolated from blood of an infected patient. The RH4 genome consists of 1,863,286 nucleotides that form 1,886 protein-encoding genes. Comparison of the RH4 genome to the ATCC 43617 contigs demonstrated that the gene content of both strains is highly conserved. In silico phylogenetic analyses based on both 16S rRNA and multilocus sequence typing revealed that RH4 belongs to the seroresistant lineage. We were able to identify almost the entire repertoire of known M. catarrhalis virulence factors and mapped the members of the biosynthetic pathways for lipooligosaccharide, peptidoglycan, and type IV pili. Reconstruction of the central metabolic pathways suggested that RH4 relies on fatty acid and acetate metabolism, as the genes encoding the enzymes required for the glyoxylate pathway, the tricarboxylic acid cycle, the gluconeogenic pathway, the nonoxidative branch of the pentose phosphate pathway, the beta-oxidation pathway of fatty acids, and acetate metabolism were present. Moreover, pathways important for survival under challenging in vivo conditions, such as the iron-acquisition pathways, nitrogen metabolism, and oxidative stress responses, were identified. Finally, we showed by microarray expression profiling that ϳ88% of the predicted coding sequences are transcribed under in vitro conditions. Overall, these results provide a foundation for future research into the mechanisms of M. catarrhalis pathogenesis and vaccine development.Moraxella catarrhalis is an emerging human-restricted unencapsulated Gram-negative mucosal pathogen. Long considered to be a commensal of the upper respiratory tract, this bacterium has now firmly been established to be an etiological cause of otitis media (OM) and exacerbations of chronic obstructive pulmonary disease (COPD). It is the third most common cause of childhood OM after Haemophilus influenzae and Streptococcus pneumoniae (37, 64), and it is responsible for up to 20% of the cases (64, 65). Further, M. catarrhalis is the second most common cause of exacerbations of COPD after H. influenzae; it is responsible for 10 to 15% of the exacerbations and annually causes 2 to 4 million episodes in the United States (47, 60). Antibiotics are widely used for treatment of OM, but the high prevalence of this disease and the increasing incidence of antibiotic-resistant strains mean that multivalent vaccines, preferably vaccines with protective antigens for all three causative bacterial agents, must be developed (46).M. catarrhalis is able to colonize the mucosal surfaces of the middle ear in OM patients and the lower respiratory tract in COPD patients (31,60). Successful colonization of the human host is a complex process which requires the expression ...

Moraxella catarrhalis is generally associated with upper respiratory tract infections in children and lower respiratory tract infections in adults. However, little is known regarding the population biology of isolates infecting these two age groups. To address this, a population-screening strategy was employed to investigate 195 worldwide M. catarrhalis isolates cultured from children (,5 years of age) and adults (.20 years of age) presenting with respiratory disease in the years [2001][2002]. Parameters compared included: genotype analysis; autoagglutination/biofilmforming ability; serum resistance; uspA1, uspA2, uspA2H, hag and mcaP incidence; copB/LOS/ ompCD/16S rRNA types; and UspA1/Hag expression. A significant difference in biofilm formation (P50.002), but not in autoagglutination or serum resistance, was observed, as well as significant differences in the incidence of uspA2-and uspA2H-positive isolates, and the distribution of lipooligosaccharide (LOS) types (P,0.0001 and P50.01, respectively). Further, a significant decrease in the incidence of Hag expression (for isolates possessing the hag gene) was observed in adult isolates (P50.001). Both uspA2H and LOS type B were associated with 16S rRNA type 1 isolates only, and two surrogate markers (copB and ompCD PCR RFLP types) for the two major M. catarrhalis 16S rRNA genetic lineages were identified. In conclusion, there are significant differences in phenotype and gene incidence between M. catarrhalis isolates from children and adults presenting with respiratory disease, possibly as a result of immune evasion in the adult age group. Our results should also be useful in the choice of effective vaccine candidates against M. catarrhalis.

S.A. SIMPKINS, A.B. CHAN, J. HAYS, B. PÖPPING & N. COOK.2000.Possession of mRNA is indicative of cell viability. RTPCR is not appropriate for mRNA detection as it cannot unambiguously detect mRNA in a DNA background. The alternative amplification technique, NASBA, avoids the disadvantages of RTPCR. We have devised a method for detection of viable Salmonella enterica. This involves NASBA amplification of mRNA transcribed from the dnaK gene. Amplification of mRNA extracted from viable and heat‐killed cells from the same population produced consistent and highly significant (P > 0·01) differences between the respective signals. The signal obtained from viable cells was completely eradicated by RNase treatment, while PCR amplification of treated and untreated samples was unaffected, indicating that NASBA was unaffected by background DNA.

Typhoid fever is caused by Salmonella enterica serovar Typhi, a major public health concern in developing countries. Recently, there has been an upsurge in the occurrence of bacterial isolates that are resistant to ciprofloxacin, and the emergence of broad spectrum β-lactamases in typhoidal salmonellae constitutes a new challenge for the clinician. A total of 337 blood culture isolates of S. Typhi, isolated from Pondicherry, India, between January 2005 and December 2009, were investigated using phenotypic, molecular and serological methods. Of the 337 isolates, 74 (22%) were found to be multidrug resistant (MDR) and 264 (78%) nalidixic acid resistant (NAR). Isolates with reduced susceptibility to ciprofloxacin possessed single mutations in the gyrA gene. A high rate of resistance (8%) was found to ciprofloxacin. All isolates with a ciprofloxacin MIC ≥ 4 mg/L possessed both double mutations in the QRDR of the gyrA gene and a single mutation in the parC gene. Active efflux pump mechanisms were also found to be involved in ciprofloxacin resistance. Finally, a large number of PFGE patterns (non-clonal genotypes) were observed among the S. Typhi isolates. In conclusion, a high rate of ciprofloxacin resistance was observed in comparison to other endemic areas in blood culture isolates of S. Typhi from Pondicherry, India, with steadily increasing NAR but decreasing MDR isolations over the study period. This is most likely to be due to an increased use of ciprofloxacin as a first-line drug of choice over more traditional antimicrobial agents for the treatment of typhoid fever.

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