Overview of microbial NGS for clinical and public health microbiology

Next-generation sequencing (NGS) has revolutionised the way we perform clinical and public health microbiology. This technology combined with the bioinformatics research field has led to a deeper understanding of the mechanisms involved in the pathogenesis of clinical infections, antimicrobial-resistant determination and dissemination of microorganisms inside the hospital, in the community, in animals and the environment. New approaches, like machine-learning methods, will even further potentiate the understanding of the information that is generated through NGS. In this book chapter, we aim to show the possibilities for NGS, including whole-genome sequencing (WGS) and metagenomics, in the fields of clinical and public health microbiology and speculate on its future importance.</p

Self-collected gargle fluids and nasopharyngeal swabs as a strategy for molecular diagnostics of respiratory viruses

Diagnosis of respiratory viruses traditionally relies on deep oropharynx or nasopharynx swabs collected by healthcare workers (HCW). However, outpatients must make an appointment, and the procedure can cause discomfort in patients. Self-collecting has the potential as a strategy to improve participants’ willingness to participate in diagnostics, surveillance, or studies. We compared self-collected gargle fluids and nasopharyngeal swabs as a strategy for molecular diagnostics of respiratory viruses and compared the average cycle threshold (Ct)-values with those of samples collected by HCW. The study was conducted among technicians of the Laboratory of Clinical Microbiology and Infectious Diseases, Zwolle, the Netherlands, and their family members, between April 2019 and March 2020. It included a questionnaire regarding the severity and date of first symptoms and an assessment of the sampling experience. The primary outcome was the mean Ct of positive PCRs. Similar mean Ct values were obtained using self- or HCW-collected swabs. In addition, gargle fluids and self-swabbed specimens had comparable detection rates of respiratory viruses. Notably, most participants preferred gargling over self-swabbing. Interestingly, but not surprisingly, the time between the onset of symptoms and sampling was shorter in PCR-positive compared to PCR-negative participants. Though this study was abrogated by the SARS-CoV-2 pandemic, the results indicate that both self-swabs and gargle fluids are acceptable for diagnosing common respiratory viruses in the outpatient population, including influenza virus, rhinovirus, adenovirus, SARS-CoV-2 and endemic human coronaviruses. Gargling could be considered an alternative sampling strategy and may enhance willingness to participate in screenings or diagnostics for respiratory viruses

Antibiotic Resistance and Molecular Characterization of Cronobacter sakazakii Strains Isolated from Powdered Infant Formula Milk

Background: Cronobacter sakazakii is a new emerging foodborne bacterial pathogen associated with severe lethal diseases such as meningitis, necrotizing enterocolitis, and septicemia in infants and neonates. Powdered infant formula milk (PIFM) has been recognized as one of the main transmission vehicles and contaminated sources of this pathogen. This study aimed to investigate the prevalence rate, genotypic and phenotypic antibiotic resistance profile, and clonal relatedness of C. sakazakii strains isolated from 364 PIFM samples collected from Tehran city, Iran. Methods: Culture-based methods, Kirby–Bauer disk diffusion antibiotic resistance testing, conventional Polymerase Chain Reaction (PCR), and Enterobacterial Repetitive Intergenic Consensus PCR (ERIC-PCR) assays were used in this study to detect and characterize the C. sakazakii isolates. Results: We isolated 25 C. sakazakii strains from PIFM samples (6.86%). The isolates were highly resistant to amoxicillin-clavulanic acid, amoxicillin, ampicillin, cefoxitin, cefepime, erythromycin, ceftriaxone, ciprofloxacin, and chloramphenicol and susceptible to gentamicin, tetracycline, norfloxacin, and azithromycin antibiotics. The blaCTX-M-1 gene was detected in 96% of the isolates. The isolates were categorized into eight distinct clonal types using the ERIC-PCR method, showing a high genetic diversity among the isolates. However, there was a significant correlation between the genotypic and phenotypic antibiotic resistance properties of the isolates. Conclusions: Novel microbial surveillance systems for detecting multi-drug-resistant C. sakazakii are required to control the contamination of this foodborne pathogen in infant foods

Future potential of metagenomics in clinical laboratories

INTRODUCTION: Rapid and sensitive diagnostic strategies are necessary for patient care and public health. Most of the current conventional microbiological assays detect only a restricted panel of pathogens at a time or require a microbe to be successfully cultured from a sample. Clinical metagenomics next-generation sequencing (mNGS) has the potential to unbiasedly detect all pathogens in a sample, increasing the sensitivity for detection and enabling the discovery of unknown infectious agents. AREAS COVERED: High expectations have been built around mNGS; however, this technique is far from widely available. This review highlights the advances and currently available options in terms of costs, turnaround time, sensitivity, specificity, validation, and reproducibility of mNGS as a diagnostic tool in clinical microbiology laboratories. EXPERT OPINION: The need for a novel diagnostic tool to increase the sensitivity of microbial diagnostics is clear. mNGS has the potential to revolutionise clinical microbiology. However, its role as a diagnostic tool has yet to be widely established, which is crucial for successfully implementing the technique. A clear definition of diagnostic algorithms that include mNGS is vital to show clinical utility. Similarly to real-time PCR, mNGS will one day become a vital tool in any testing algorithm

Complete Genome Sequences of Two Methicillin-Resistant Staphylococcus haemolyticus Isolates of Multilocus Sequence Type 25, First Detected by Shotgun Metagenomics

The emergence of nosocomial infections by multidrug-resistantStaphylococcus haemolyticusisolates has been reported in several European countries. Here, we report the first two complete genome sequences ofS. haemolyticussequence type 25 (ST25) isolates 83131A and 83131B. Both isolates were isolated from the same clinical sample and were first identified through shotgun metagenomics

New Topoisomerase Inhibitors:Evaluating the Potency of Gepotidacin and Zoliflodacin in Fluoroquinolone-Resistant Escherichia coli upon tolC Inactivation and Differentiating Their Efflux Pump Substrate Nature

Inactivating tolC in multidrug-resistant Escherichia coli with differing sequence types and quinolone resistance-determining mutations reveals remarkably potentiated activity of the first-in-class topoisomerase inhibitors gepotidacin and zoliflodacin. Differences between both structurally unrelated compounds in comparison to fluoroquinolones regarding the selectivity of E. coli RND (resistance-nodulation-cell division)-type transporters, efflux inhibitors, and AcrB porter domain mutations were demonstrated. The findings should reinforce efforts to develop efflux-bypassing drugs and provide AcrB targets with critical relevance for this purpose

Complete Genome Sequences of Two Methicillin-Resistant Staphylococcus haemolyticus Isolates of Multilocus Sequence Type 25, First Detected by Shotgun Metagenomics

The emergence of nosocomial infections by multidrug-resistantStaphylococcus haemolyticusisolates has been reported in several European countries. Here, we report the first two complete genome sequences ofS. haemolyticussequence type 25 (ST25) isolates 83131A and 83131B. Both isolates were isolated from the same clinical sample and were first identified through shotgun metagenomics

Composition of C-S-H in pastes with increasing levels of silica fume addition

New results show that the microstructure development of cement–silica fume blends is very different from plain cement. Portlandite (CH) tends to precipitate as platelets and even around clinker grains as “CH rims” and is consumed by pozzolanic reaction with silica fume. The Ca/Si ratio in the inner product (IP) C–S–H decreases as CH is consumed to reach Ca/Si ≈ 1.40–1.50 at the point when CH has disappeared, and then drops down to 1.00 in absence of CH. At later ages, the IP C–S–H is often composed of two distinct regions. The outermost (formed first) consists of originally high Ca/Si C–S–H, which Ca/Si slowly decreases. The second (formed later) forms only once CH is no longer present and has a lower Ca/Si. Between 10 and 38 °C, the main effect of increasing the temperature is to accelerate the reaction of cement and increase the reactivity of silica fume. The changes in Ca and Si in the pore solution of similar systems suggest that the composition of the solution and the solids reciprocally influence each other