Rapid and precise alignment of raw reads against redundant databases with KMA

Abstract Background As the cost of sequencing has declined, clinical diagnostics based on next generation sequencing (NGS) have become reality. Diagnostics based on sequencing will require rapid and precise mapping against redundant databases because some of the most important determinants, such as antimicrobial resistance and core genome multilocus sequence typing (MLST) alleles, are highly similar to one another. In order to facilitate this, a novel mapping method, KMA (k-mer alignment), was designed. KMA is able to map raw reads directly against redundant databases, it also scales well for large redundant databases. KMA uses k-mer seeding to speed up mapping and the Needleman-Wunsch algorithm to accurately align extensions from k-mer seeds. Multi-mapping reads are resolved using a novel sorting scheme (ConClave scheme), ensuring an accurate selection of templates. Results The functionality of KMA was compared with SRST2, MGmapper, BWA-MEM, Bowtie2, Minimap2 and Salmon, using both simulated data and a dataset of Escherichia coli mapped against resistance genes and core genome MLST alleles. KMA outperforms current methods with respect to both accuracy and speed, while using a comparable amount of memory. Conclusion With KMA, it was possible map raw reads directly against redundant databases with high accuracy, speed and memory efficiency

Validating Whole Genome Nanopore Sequencing, using Usutu Virus as an Example

Whole genome sequencing can be used to characterize and to trace viral outbreaks. Nanopore-based whole genome sequencing protocols have been described for several different viruses. These approaches utilize an overlapping amplicon-based approach which can be used to target a specific virus or group of genetically related viruses. In addition to confirmation of the virus presence, sequencing can be used for genomic epidemiology studies, to track viruses and unravel origins, reservoirs and modes of transmission. For such applications, it is crucial to understand possible effects of the error rate associated with the platform used. Routine application in clinical and public health settings require that this is documented with every important change in the protocol. Previously, a protocol for whole genome Usutu virus sequencing on the nanopore sequencing platform was validated (R9.4 flowcell) by direct comparison to Illumina sequencing. Here, we describe the method used to determine the required read coverage, using the comparison between the R1

Novel 16S rRNA methyltransferase RmtE3 in acinetobacter baumannii ST79.

Introduction. The 16S rRNA methyltransferase (16S RMTase) gene armA is the most common mechanism conferring high-level aminoglycoside resistance in Acinetobacter baumannii, although rmtA, rmtB, rmtC, rmtD and rmtE have also been reported.Hypothesis/Gap statement. The occurrence of 16S RMTase genes in A. baumannii in the UK and Republic of Ireland is currently unknown.Aim. To identify the occurrence of 16S RMTase genes in A. baumannii isolates from the UK and the Republic of Ireland between 2004 and 2015.Methodology. Five hundred and fifty pan-aminoglycoside-resistant A. baumannii isolates isolated from the UK and the Republic of Ireland between 2004 and 2015 were screened by PCR to detect known 16S RMTase genes, and then whole-genome sequencing was conducted to screen for novel 16S RMTase genes.Results. A total of 96.5 % (531/550) of isolates were positive for 16S RMTase genes, with all but 1 harbouring armA (99.8 %, 530/531). The remaining isolates harboured rmtE3, a new rmtE variant. Most (89.2 %, 473/530) armA-positive isolates belonged to international clone II (ST2), and the rmtE3-positive isolate belonged to ST79. rmtE3 shared a similar genetic environment to rmtE2 but lacked an ISCR20 element found upstream of rmtE2.Conclusion. This is the first report of rmtE in A. baumannii in Europe; the potential for transmission of rmtE3 to other bacterial species requires further research

Genetic Background of β-Lactamase Genes in Extraintestinal Pathogenic Escherichia coli ST131 in Indonesia Based on Whole-Genome Sequencing (WGS) Sequences

Extraintestinal pathogenic Escherichia coli (ExPEC) is a group of pathogens that can colonize the outside of the intestine, such as the kidney, urinary tract, or bloodstream. This study analyzed more about the genetic background of β-lactamase genes among Indonesian ExPEC ST131. Whole-genome sequencing (WGS) sequences from the National Center for Biotechnology Information (NCBI) of Indonesian ExPEC ST131 were taken, then analyzed. Circular genomic mapping and genomic comparison of surrounding genes of β-lactamase in these isolates were generated. blaOXA-1 and blaDHA-1 were analyzed deeply due to their presence in a relatively long contig, making them available for analysis. Indonesian ExPEC ST131 isolates had blaOXA-1 with an identical genetic background of E. coli originating from China and Austria with aac (6’)-1b-cr5 in the downstream and cab83 in the upstream. The blaOXA-1 was also detected in other species, including Klebsiella pneumoniae INF142 originating from Australia and Salmonella enterica S146 from China. While, blaDHA-1 in EC_0406 had an identical genetic background to E. coli 142 and other species such as Shigella sonei FC1428 from India and S. flexneri M2901 from Australia, with Globulin-encoded gene in the upstream and lysR in the downstream. Our findings demonstrate the global spread of β-lactamase genes detected in Indonesian ExPEC ST131. These genes were identical to those isolated from some countries around the world

Genomic palaeoparasitology traced the occurrence of Taenia asiatica in ancient Iran (Sassanid Empire, 2th cent. CE-6th cent. CE)

Palaeoparasitology investigates parasitological infections in animals and humans of past distance by examining biological remains. Palaeofaeces (or coprolites) are biological remains that provide valuable information on the disease, diet, and population movements in ancient times. Today, advances in detecting ancient DNA have cast light on dark corners that microscopy could never reach. The archaeological site of the Chehrabad salt mine of Achaemenid (550-330 BC) and Sassanid (third-seventh century AD) provides remains of various biotic and abiotic samples, including animal coprolites, for multidisciplinary studies. In the present work, we investigated coprolites for helminth eggs and larvae by microscopy and traced their biological agents' DNA by Next Generation Sequencing. Our results revealed various helminths, including Taenia asiatica, the species introduced in the 1990s. Implementing advanced modern molecular techniques like NGS gives a paramount view of pathogenic agents in space and time

Hypermutation as an Evolutionary Mechanism for Achromobacter xylosoxidans in Cystic Fibrosis Lung Infection

Achromobacter xylosoxidans can cause chronic infections in the lungs of patients with cystic fibrosis (CF) by adapting to the specific environment. The study of longitudinal isolates allows to investigate its within\u2010host evolution to unravel the adaptive mechanisms contributing to successful colonization. In this study, four clinical isolates longitudinally collected from two chronically infected patients underwent whole genome sequencing, de novo assembly and sequence analysis. Phenotypic assays were also performed. The isolates coming from one of the patients (patient A) presented a greater number of genetic variants, diverse integrative and conjugative elements, and different protease secretion. In the first of these isolates (strain A1), we also found a large deletion in the mutS gene, involved in DNA mismatch repair (MMR). In contrast, isolates from patient B showed a lower number of variants, only one integrative and mobilizable element, no phenotypic changes, and no mutations in the MMR system. These results suggest that in the two patients the establishment of a chronic infection was mediated by different adaptive mechanisms. While the strains isolated from patient B showed a longitudinal microevolution, strain A1 can be clearly classified as a hypermutator, confirming the occurrence and importance of this adaptive mechanism in A. xylosoxidans infection

Recurrent spontaneous Escherichia coli meningitis in an adult: a case report

Objectives The aim of this study was to characterize an unusual case of spontaneous, community-acquired Escherichia coli meningitis in an adult presenting to a general hospital in Kenya, where initial clinical recovery was followed by reinfection with an MDR, hospital-acquired strain. Patient and methods An adult presented to a hospital in Kenya with meningitis symptoms. E. coli was cultured from CSF. Treatment with ceftriaxone was successful; however, the patient relapsed a few days later. E. coli was cultured from CSF and blood during the reinfection episode, though the patient died during admission. We sequenced the isolates using Illumina MiSeq and performed antimicrobial susceptibility testing, fitness and virulence assays on the bacteria. Results The E. coli isolates from the two episodes were found to be distinct: the initial strain was ST88, serotype O8 H17 while the subsequent episode was caused by an ST167, serotype O101 H5 MDR strain. The ST88 strain was susceptible to all drugs except ampicillin and amoxicillin/clavulanate while the ST167 strain was MDR, including to all β-lactam drugs due to the presence of the carbapenemase gene blaNDM-5. The hospital-acquired ST167 strain was also resistant to newer drugs such as cefiderocol and eravacycline, which are currently not available locally, and had overall lower fitness and virulence in vitro compared with the initial infecting strain. Conclusions Though less fit and virulent in vitro, the MDR strain was fatal, suggesting that host factors, rather than bacterial virulence, may have been of greater importance in this patient’s outcome

Setting a baseline for global urban virome surveillance in sewage

The rapid development of megacities, and their growing connectedness across the world is becoming a distinct driver for emerging disease outbreaks. Early detection of unusual disease emergence and spread should therefore include such cities as part of risk-based surveillance. A catch-all metagenomic sequencing approach of urban sewage could potentially provide an unbiased insight into the dynamics of viral pathogens circulating in a community irrespective of access to care, a potential which already has been proven for the surveillance of poliovirus. Here, we present a detailed characterization of sewage viromes from a snapshot of 81 high density urban areas across the globe, including in-depth assessment of potential biases, as a proof of concept for catch-all viral pathogen surveillance. We show the ability to detect a wide range of viruses and geographical and seasonal differences for specific viral groups. Our findings offer a cross-sectional baseline for further research in viral surveillance from urban sewage samples and place previous studies in a global perspective

Genomic characterization of ESBL/AmpC-producing and high-risk clonal lineages of Escherichia coli and Klebsiella pneumoniae in imported dogs with shelter and stray background

Objectives: Extended spectrum ,B-lactamase (ESBL)-and ampicillinase C (AmpC)-carrying Enterobacteriaceae have been widely reported among companion animals. According to previous studies, dogs with a shelter or stray background might be at risk of carrying such bacteria. The aim of this study was to ex-plore, with whole-genome sequencing (WGS), the genomic characteristics of ESBL-producing Escherichia coli and Klebsiella pneumoniae isolated from imported dogs with a stray or shelter background. Methods: E. coli (n = 58) and K. pneumoniae (n = 2) isolates from imported dogs originating from seven countries were included. Phenotypic resistance was investigated by selective isolation and antibiotic sus-ceptibility testing. Whole-genome sequencing was used to study the genomic characteristics and the pres-ence of antimicrobial resistance genes (ARGs) and virulence determinants of the ESBL/AmpC-producing E. coli and K. pneumoniae isolates. Results: A high diversity of different ARGs (n = 56) and sequence types (STs) (n = 32), including high -risk clonal lineages ST410 (n = 3) and ST307 (n = 1), was identified in E. coli and K. pneumoniae isolates, respectively. Genes encoding resistance to ,beta-lactams accounted for the majority, with the most frequent being blaCTX-M-15. Moreover, 17 (29%) E. coli isolates qualified as presumptive extraintestinal pathogenic and/or uropathogenic E. coli. Conclusions: Our results highlight the multiplicity of genetic backgrounds disseminating ESBL/AmpC-genes in the studied dogs, calling for further investigation of possible drivers responsible for the dissem-ination of ARGs in animal shelters and amongst stray dogs. From a public health perspective, enhanced genomic surveillance of ESBL/AmpC-producing Enterobacteriaceae in dogs is needed in Finland. (C) 2022 The Authors. Published by Elsevier Ltd on behalf of International Society for Antimicrobial Chemotherapy.Peer reviewe

Evaluation of Fourier Transform Infrared Spectroscopy as a First-Line Typing Tool for the Identification of Extended-Spectrum β-Lactamase-Producing Klebsiella pneumoniae Outbreaks in the Hospital Setting

Early detection of pathogen cross-transmission events and environmental reservoirs is needed to control derived nosocomial outbreaks. Whole-genome sequencing (WGS) is considered the gold standard for outbreak confirmation, but, in most cases, it is time-consuming and has elevated costs. Consequently, the timely incorporation of WGS results to conventional epidemiology (CE) investigations for rapid outbreak detection is scarce. Fourier transform infrared spectroscopy (FTIR) is a rapid technique that establishes similarity among bacteria based on the comparison of infrared light absorption patterns of bacterial polysaccharides and has been used as a typing tool in recent studies. The aim of the present study was to evaluate the performance of the FTIR as a first-line typing tool for the identification of extended-spectrum β-lactamase-producing Klebsiella pneumoniae (ESBL-Kp) outbreaks in the hospital setting in comparison with CE investigations using WGS as the gold standard method. Sixty-three isolates of ESBL-Kp collected from 2018 to 2021 and classified according to CE were typed by both FTIR and WGS. Concordance was measured using the Adjusted Rand index (AR) and the Adjusted Wallace coefficient (AW) for both CE and FTIR clustering considering WGS as the reference method. Both AR and AW were significantly higher for FTIR clustering than CE clustering (0.475 vs. 0.134, p = 0.01, and 0.521 vs. 0.134, p = 0.009, respectively). Accordingly, FTIR inferred more true clustering relationships than CE (38/42 vs. 24/42, p = 0.001). However, a similar proportion of genomic singletons was detected by both FTIR and CE (13/21 vs. 12/21, p = 1). This study demonstrates the utility of the FTIR method as a quick, low-cost, first-line tool for the detection of ESBL-Kp outbreaks, while WGS analyses are being performed for outbreak confirmation and isolate characterization. Thus, clinical microbiology laboratories would benefit from integrating the FTIR method into CE investigations for infection control measures in the hospital setting.We thank the Germans Trias i Pujol Research Institute (IGTP) Translational Genomics Core Facility and staff for their contribution to this publication. We also thank CERCA Programme/Government of Catalonia for their support to the IGTP.S