A core-genome multilocus sequence typing scheme for the detection of genetically related Streptococcus pyogenes clusters

The recently observed increase in invasive Streptococcus pyogenes infections causes concern in Europe. However, conventional molecular typing methods lack discriminatory power to aid investigations of outbreaks caused by S. pyogenes. Therefore, there is an urgent need for high-resolution molecular typing methods to assess genetic relatedness between S. pyogenes isolates. In the current study, we aimed to develop a novel high-resolution core-genome multilocus sequence typing (cgMLST) scheme for S. pyogenes and compared its discriminatory power to conventional molecular typing methods. The cgMLST scheme was designed with the commercial Ridom SeqSphere+ software package. To define a cluster threshold, the scheme was evaluated using publicly available data from nine defined S. pyogenes outbreaks in the United Kingdom. The cgMLST scheme was then applied to 23 isolates from a suspected S. pyogenes outbreak and 117 S. pyogenes surveillance isolates both from the Netherlands. MLST and emm-typing results were used for comparison to cgMLST results. The allelic differences between isolates from defined outbreaks ranged between 6 and 31 for isolates with the same emm-type, resulting in a proposed cluster threshold of </p

Viral metagenomic sequencing in a cohort of international travellers returning with febrile illness

Background: Diagnosis of infections in returning international travellers can be challenging because of the broad spectrum of potential infectious etiologies potentially involved. Viral metagenomic next-generation sequencing (mNGS) has the potential to detect any virus present in a patient sample and is increasingly being used for difficult to diagnose cases. The aim of this study was to analyze the performance of mNGS for viral pathogen detection in the clinical setting of international travellers returning with febrile illness. Methods: Thirty-eight serum samples from international travellers returning with febrile illness and presenting at the outpatient clinic of the Leiden University Medical Center in the Netherlands in the time period 2015-2016 were selected retrospectively. Samples were processed for viral metagenomic sequencing using a probe panel capturing all known vertebrate viruses. Bioinformatic analysis was performed using Genome Detective software for metagenomic virus detection. Metagenomic virus findings were compared with viral pathogen detection using conventional methods. Results: In 8 out of the 38 patients (21%), a pathogenic virus was detected by mNGS. All viral pathogens detected by conventional assays were also detected by mNGS: dengue virus (n=4 patients), Epstein-Barr virus (n=2), hepatitis B virus (n=1). In addition, mNGS resulted in additional pathogenic findings in 2 patients (5%): dengue virus (n=1), and hepatitis C virus (n=1). Non-pathogenic viruses detected were: GB virus C (n=1) and torque teno viruses (n=3). High genome coverage and depth using capture probes enabled typing of the dengue viruses detected. Conclusions: Viral metagenomics has the potential to assist the detection of viral pathogens and co-infections in one step in international travellers with a febrile syndrome. Furthermore, viral enrichment by probes resulted in high genome coverage and depth which enabled dengue virus typing.Molecular basis of virus replication, viral pathogenesis and antiviral strategie

Distinct evolution of colistin resistance associated with experimental resistance evolution models in Klebsiella pneumoniae

Pharmacolog

A comparison of five Illumina, Ion Torrent, and nanopore sequencing technology-based approaches for whole genome sequencing of SARS-CoV-2

Rapid identification of the rise and spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern remains critical for monitoring of the efficacy of diagnostics, therapeutics, vaccines, and control strategies. A wide range of SARS-CoV-2 next-generation sequencing (NGS) methods have been developed over the last years, but cross-sequence technology benchmarking studies have been scarce. In the current study, 26 clinical samples were sequenced using five protocols: AmpliSeq SARS-CoV-2 (Illumina), EasySeq RC-PCR SARS-CoV-2 (Illumina/NimaGen), Ion AmpliSeq SARS-CoV-2 (Thermo Fisher), custom primer sets (Oxford Nanopore Technologies (ONT)), and capture probe-based viral metagenomics (Roche/Illumina). Studied parameters included genome coverage, depth of coverage, amplicon distribution, and variant calling. The median SARS-CoV-2 genome coverage of samples with cycle threshold (Ct) values of 30 and lower ranged from 81.6 to 99.8% for, respectively, the ONT protocol and Illumina AmpliSeq protocol. Correlation of coverage with PCR Ct values varied per protocol. Amplicon distribution signatures differed across the methods, with peak differences of up to 4 log(10) at disbalanced positions in samples with high viral loads (Ct values <= 23). Phylogenetic analyses of consensus sequences showed clustering independent of the workflow used. The proportion of SARS-CoV-2 reads in relation to background sequences, as a (cost-)efficiency metric, was the highest for the EasySeq protocol. The hands-on time was the lowest when using EasySeq and ONT protocols, with the latter additionally having the shortest sequence runtime. In conclusion, the studied protocols differed on a variety of the studied metrics. This study provides data that assist laboratories when selecting protocols for their specific setting.Medical Microbiolog

BacTag - a pipeline for fast and accurate gene and allele typing in bacterial sequencing data based on database preprocessing

Molecular Technology and Informatics for Personalised Medicine and Healt

Characterization of fluoroquinolone and cephalosporin resistance mechanisms in Enterobacteriaceae isolated in a Dutch teaching hospital reveals the presence of an Escherichia coli ST131 clone with a specific mutation in parE

Molecular basis of bacterial pathogenesis, virulence factors and antibiotic resistanc

Exploring the Contribution of Efflux on the Resistance to Fluoroquinolones in Clinical Isolates of Escherichia coli

Molecular basis of bacterial pathogenesis, virulence factors and antibiotic resistanc

Borderline oxacillin-resistant Staphylococcus aureus carriage among healthcare workers at neonatal intensive care unit and paediatric ward

Background: During a meticillin-resistant Staphylococcus aureus contact tracing and screening investigation, two borderline oxacillin-resistant Staphylococcus aureus (BORSA)positive screening cultures were encountered among neonatal intensive care unit (NICU) healthcare workers (HCWs). This finding led to further investigations. Aim: To assess the likelihood of an outbreak with direct transmission among HCWs. Methods: An infection control team was initiated after the discovery. The team initiated additional infection control measures and evaluated new findings. All NICUs and paediatric ward HCWs were screened for BORSA carriage, and a prospective BORSA seven-week monitoring period for patients was observed. To assess the likelihood of an outbreak with direct transmission among HCWs, the BORSA isolates were analysed using augmented fragment length polymorphism and whole-genome sequencing (WGS). Findings: Positive HCWs were prohibited from clinical work while awaiting the results from the screening programme. In all, 127 NICU and 77 general paediatric ward HCWs were screened for BORSA carriage; five HCWs were BORSA positive. Seventy-two patients were screened during the seven-week period yielding a total of 138 cultures, ranging from one to nine cultures per patient. No spread from HCWs to patients occurred, and the BORSA screening programme was discontinued. WGS analysis with core genome multi-locus sequence typing of all five BORSA strains showed relatedness between two NICU strains. Conclusion: During a seven-week period, no transmission from BORSA-positive HCWs to neonates was observed in either screening or clinical cultures. More vigilance and experience is needed to design adequate evidence-based interventions in the future for this vulnerable population. (c) 2020 Published by Elsevier Ltd on behalf of The Healthcare Infection Society.Molecular basis of bacterial pathogenesis, virulence factors and antibiotic resistanc

Ceftazidime-avibactam resistance and restoration of carbapenem susceptibility in KPC-producing Klebsiella pneumoniae infections: A case series

Objectives: Since the introduction of the 13-lactam/13-lactamase inhibitor ceftazidime-avibactam (CZA), rapid evolution of resistance has been reported in different KPC-producing Klebsiella pneumoniae isolates. In this multicenter retrospective study, we describe the emergence of CZA resistance and evaluate the mutations that might be responsible for the restoration of carbapenem susceptibility. Methods: During a study period of 18 months, KPC-producing K. pneumoniae isolates of five hospitalized patients were collected with phenotypic development of CZA resistance. Results: In vitro restoration of carbapenem susceptibility during treatment was observed in 3 isolates. Whole genome sequencing of these isolates showed a D179Y mutation in the KPC gene of 2 variants and a KPC-2 with a A242-GT-243 deletion (KPC-14). Two KPC-3 variants showed CZA resistance with sustained carbapenemase activity without genomic adaptations in the KPC gene. Conclusions: This study confirms the emergence of CZA resistance in KPC K. pneumoniae. The role of carbapenems in treating patients with these variants is unclear and combination therapies warrant further investigation.(c) 2021 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.Molecular basis of bacterial pathogenesis, virulence factors and antibiotic resistanc

Taxonomic classification and abundance estimation using 16S and WGS-A comparison using controlled reference samples

The assessment of microbiome biodiversity is the most common application of metagenomics. While 16S sequencing remains standard procedure for taxonomic profiling of metagenomic data, a growing number of studies have clearly demonstrated biases associated with this method. By using Whole Genome Shotgun sequencing (WGS) metagenomics, most of the known restrictions associated with 16S data are alleviated. However, due to the computationally intensive data analyses and higher sequencing costs, WGS based metagenomics remains a less popular option. Selecting the experiment type that provides a comprehensive, yet manageable amount of information is a challenge encountered in many metagenomics studies. In this work, we created a series of artificial bacterial mixes, each with a different distribution of skin-associated microbial species. These mixes were used to estimate the resolution of two different metagenomic experiments - 16S and WGS - and to evaluate several different bioinformatics approaches for taxonomic read classification. In all test cases, WGS approaches provide much more accurate results, in terms of taxa prediction and abundance estimation, in comparison to those of 16S. Furthermore, we demonstrate that a 16S dataset, analysed using different state of the art techniques and reference databases, can produce widely different results. In light of the fact that most forensic metagenomic analysis are still performed using 16S data, our results are especially important.Molecular Epidemiolog