WGS-based Typing Research Articles

Characterisation of Neisseria gonorrhoeae strain differences in patients with multisite infection

BackgroundNeisseria gonorrhoeae, the aetiological agent of gonorrhoea, is an increasing global health priority due to high levels of antimicrobial resistance (AMR). It is estimated that up to 42% of patients...

Three prolonged outbreaks of metallo-β-lactamase-producing Pseudomonas aeruginosa in an Upper Austrian hospital, 2017-2023.

The significance of our work lies in the successful resolution of three prolonged outbreaks of MBL-Pa infections in a hospital in Upper Austria. Through a comprehensive epidemiological investigation coupled with WGS-based typing of P. aeruginosa isolates, the study identified three distinct genomic clusters responsible for prolonged outbreaks involving 47 cases. The investigation pinpointed sinks in the ICU washroom as the likely source of infection for two of the clusters. The study demonstrates the effectiveness of control measures such as hand hygiene, gowning, patient isolation, screening, and disinfection in stopping further transmission and bringing the outbreaks to a close. This underscores the critical role of genomic surveillance and control measures, particularly in high-risk settings like ICUs, in reducing nosocomial transmission of MBL-Pa infections.

First Data on WGS-Based Typing and Antimicrobial Resistance of Human Salmonella Enteritidis Isolates in Greece.

Salmonella enterica subsp. enterica serotype Enteritidis (S. Enteritidis) is one of the major causes of foodborne infections and is responsible for many national and multi-country foodborne outbreaks worldwide. In Greece, human salmonellosis is a mandatory notifiable disease, with laboratory surveillance being on a voluntary basis. This study aims to provide the first insights into the genetic characteristics and antimicrobial resistance profiles of 47 S. Enteritidis human isolates using whole-genome sequencing (WGS) technology. The S. Enteritidis population was mainly resistant to fluoroquinolones due to gyrA point mutations, whereas one isolate presented a multi-resistant plasmid-mediated phenotype. ST11 was the most frequent sequence type, and phylogenetic analysis through the cgMLST and SNP methods revealed considerable genetic diversity. Regarding virulence factors, 8 out of the 24 known SPIs and C63PI were detected. Due to the observed variability between countries, it is of utmost importance to record the circulating S. Enteritidis strains' structure and genomic epidemiology at the national level. WGS is a valuable tool that is revolutionizing our approach to Salmonella by providing a deeper understanding of these pathogens and their impact on human health.

Genomic classification and antimicrobial resistance profiling of Streptococcus pneumoniae and Haemophilus influenzae isolates associated with paediatric otitis media and upper respiratory infection

Acute otitis media (AOM) is the most common childhood bacterial infectious disease requiring antimicrobial therapy. Most cases of AOM are caused by translocation of Streptococcus pneumoniae or Haemophilus influenzae from the nasopharynx to the middle ear during an upper respiratory tract infection (URI). Ongoing genomic surveillance of these pathogens is important for vaccine design and tracking of emerging variants, as well as for monitoring patterns of antibiotic resistance to inform treatment strategies and stewardship.In this work, we examined the ability of a genomics-based workflow to determine microbiological and clinically relevant information from cultured bacterial isolates obtained from patients with AOM or an URI. We performed whole genome sequencing (WGS) and analysis of 148 bacterial isolates cultured from the nasopharynx (N = 124, 94 AOM and 30 URI) and ear (N = 24, all AOM) of 101 children aged 6–35 months presenting with AOM or an URI. We then performed WGS-based sequence typing and antimicrobial resistance profiling of each strain and compared results to those obtained from traditional microbiological phenotyping.WGS of clinical isolates resulted in 71 S. pneumoniae genomes and 76 H. influenzae genomes. Multilocus sequencing typing (MSLT) identified 33 sequence types for S. pneumoniae and 19 predicted serotypes including the most frequent serotypes 35B and 3. Genome analysis predicted 30% of S. pneumoniae isolates to have complete or intermediate penicillin resistance. AMR predictions for S. pneumoniae isolates had strong agreement with clinical susceptibility testing results for beta-lactam and non beta-lactam antibiotics, with a mean sensitivity of 93% (86–100%) and a mean specificity of 98% (94–100%). MLST identified 29 H. influenzae sequence types. Genome analysis identified beta-lactamase genes in 30% of H. influenzae strains, which was 100% in agreement with clinical beta-lactamase testing. We also identified a divergent highly antibiotic-resistant strain of S. pneumoniae, and found its closest sequenced strains, also isolated from nasopharyngeal samples from over 15 years ago.Ultimately, our work provides the groundwork for clinical WGS-based workflows to aid in detection and analysis of H. influenzae and S. pneumoniae isolates.

Evaluation of IR Biotyper for carbapenem-resistant Pseudomonas aeruginosa typing and its application potential for the investigation of nosocomial infection.

Pseudomonas aeruginosa is one of the most common opportunistic pathogens causing severe nosocomial infections for its patterns of multidrug resistance, particularly for carbapenems. Timely epidemiological surveillance could greatly facilitate infection control of P. aeruginosa and many deadly pathogens alike. IR Biotyper (IRBT), is a novel real-time typing tool, based on a Fourier-transform infrared (FTIR) spectroscopy system. It is critical to comprehensively establish and evaluate the feasibility of IRBT in P. aeruginosa strain typing. In the current study, we first established standards and schemes for its routine laboratory application, and we found that Mueller-Hinton agar plates give better discriminatory power than blood agar plates. Data showed that the cut-off value of 0.15 with an additional 0.025 range was optimal. Secondly, 27 clinically isolated carbapenem-resistant P. aeruginosa (CRPA) strains collected from October 2010 to September 2011 were evaluated for typing effectiveness by comparing IRBT to the other commonly used typing methods, such as multi-locus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE) and whole-genome sequencing (WGS)-based typing. When using WGS-based typing as the reference method, the typing method of FTIR spectroscopy (AR = 0.757, SID = 0.749) could better cluster P. aeruginosa strains than MLST and in silico serotyping (AR = 0.544, SID = 0.470). Though PFGE showed the highest discriminatory power, low concordance was observed between PFGE and the other methods. Above all, this study demonstrates the utility of the IRBT as a quick, low-cost, real-time typing tool for the detection of CRPA strains.

Evaluation of Genomic Typing Methods in the Salmonella Reference Laboratory in Public Health, England, 2012–2020

We aim to provide an evidence-based evaluation of whole genome sequence (WGS) methods, employed at the Salmonella reference laboratory in England, in terms of its impact on public health and whether these methods remain a fit for purpose test under UKAS ISO 15189. The evaluation of the genomic methods were mapped against the value of detecting microbiological clusters to support the investigation of food-borne outbreaks of Salmonella in England between 2012-2020. The analysis of WGS with both SNP- and allelic-based methods provided an unprecedented level of strain discrimination and detection of additional clusters when comparing to all of the previous typing methods. The robustness of the routine genomic sequencing at the reference laboratory ensured confidence in the microbiological identifications, even in large outbreaks with complex international food distribution networks. There was evidence that the phylogeny derived from the WGS data can be used to inform the provenance of strains and support discrimination between domestic and non-domestic transmission events. Further insight on the evolutionary context of the emerging pathogenic strains was enabled with a deep dive of the phylogenetic data, including the detection of nested clusters. The public availability of the WGS data linked to the clinical, epidemiological and environmental context of the sequenced strains has improved the trace-back investigations during outbreaks. The global expansion in the use of WGS-based typing in reference laboratories has shown that the WGS methods are a fit for purpose test in public health as it has ensured the rapid implementation of interventions to protect public health, informed risk assessment and has facilitated the management of national and international food-borne outbreaks of Salmonella.

Сравнение инструментов биоинформатики для HLA-типирования на основе данных WGS

Сравнение инструментов биоинформатики для HLA-типирования на основе данных WGS

Characterization of a Pathogenic <em>Escherichia coli</em> Strain Derived from <em>Oreochromis</em> spp. Farms Using Whole-Genome Sequencing

Aquaculture is one of the fastest-growing food-producing sectors worldwide and tilapia (Oreochromis spp.) farming constitutes the major freshwater fish variety cultured. Because aquaculture practices are susceptible to microbial contamination derived from anthropogenic sources, extensive antibiotic usage is needed, leading to aquaculture systems becoming an important source of antibiotic-resistant and pathogenic bacteria of clinical relevance such as Escherichia coli (E. coli). Here, the antimicrobial resistance, virulence, and mobilome features of a pathogenic E. coli strain, recovered from inland farmed Oreochromis spp., were elucidated through whole-genome sequencing (WGS) and in silico analysis. Antimicrobial susceptibility testing (AST) and WGS were performed. Furthermore, phylogenetic group, serotype, multilocus sequence typing (MLST), acquired antimicrobial resistance, virulence, plasmid, and prophage content were determined using diverse available web tools. The E. coli isolate only exhibited intermediate susceptibility to ampicillin and was characterized as ONT:H21-B1-ST40 strain by WGS-based typing. Although only a single antimicrobial resistance-related gene was detected [mdf(A)], several virulence-associated genes (VAGs) from the atypical enteropathogenic E. coli (aEPEC) pathotype were identified. Additionally, the cargo of plasmid replicons from large plasmid groups and 18 prophage-associated regions were detected. In conclusion, the WGS characterization of an aEPEC isolate, recovered from a fish farm in Sinaloa, Mexico, allows insights into its pathogenic potential and the possible human health risk of consuming raw aquacultural products. It is necessary to exploit next-generation sequencing (NGS) techniques for studying environmental microorganisms and to adopt a one health framework to learn how health issues originate.

Bestandsaufnahme der verfügbaren und aktuell eingesetzten Typisierungsmethoden einschließlich genombasierter Verfahren von Zoonoseerregern am Beispiel von Salmonella enterica

ZusammenfassungHintergrundIn den vergangenen Jahren hat sich die Gesamtgenomsequenzierung („whole genome sequencing“; WGS) in Kombination mit bioinformatischen Analysen zum Stand der Technik bei der Bewertung des Pathogenitäts- und Resistenzpotenzials sowie der Verwandtschaftsgrade zwischen Bakterien entwickelt. Die WGS-Analyse stellt somit ein zentrales Instrument bei der Typisierung von Erregern und der Untersuchung von Krankheits- und Ausbruchsclustern im Rahmen der molekularen Epidemiologie dar. Ziel der Studie war die Generierung eines Überblicks der in Deutschland auf Landes- und Bundesebene verfügbaren Erregertypisiermethoden von Salmonellen und Shiga-Toxin-bildenden bzw. enterohämorrhagischen Escherichia coli (STEC/EHEC) und den angewandten geno- und phänotypischen Methoden sowie über die Verfügbarkeit der genombasierten Typisierung und entsprechenden Analyseverfahren.MethodenIm Zeitraum vom Februar bis Juni 2020 wurde eine elektronische Umfrage bei Laboratorien durchgeführt, die für den öffentlichen Gesundheitsschutz und gesundheitlichen Verbraucherschutz tätig sind.Ergebnisse und FazitDie Ergebnisse der Umfrage zeigten, dass viele der teilnehmenden Laboratorien über eine große Auswahl an phänotypischen und molekularbiologischen Methoden verfügen. Molekularbiologische Typisierungen werden am häufigsten für die Speziesidentifizierung von Salmonellen herangezogen. WGS-Verfahren sind vielfach schon bei Einrichtungen auf Bundes- und Landesebene etabliert oder befinden sich im Aufbau. Die Illumina-Sequenzierung ist dabei die am weitesten verbreitete Technologie. Die Umfrage bestätigt die Bedeutung von molekularbiologischen und genombasierten Typisierungstechnologien für die Laboratorien bei der Diagnostik von bakteriellen zoonotischen Erregern.

Whole genome sequencing reveals new links between spa t172/CC59 methicillin-resistant Staphylococcus aureus cases in low-endemicity region of Southwest Finland, 2007‒2016

Methicillin-resistant Staphylococcus aureus (MRSA) rates have remained relatively low in Finland. In Southwest Finland, however, annual MRSA incidence increased from 12 to 25/100,000 between 2007 and 2016 with spa t172 strain causing one fourth (237/983) of all cases. This provoked us to study the molecular epidemiology of t172-MRSA, aiming to better understand the transmission of this strain type. We combined epidemiological data and whole genome sequencing (WGS) of a set of 64 (27%, 64/237) t172-MRSA isolates covering 10years. Isolates represented sporadic and index cases of all identified healthcare-associated outbreaks (HAOs) and family clusters (FCs). Among the included 62 isolates, core-genome MLST analysis revealed eight genomic clusters comprising 24 (38.7%) isolates and 38 (61.3%) non-clustered isolates. Cluster 1 comprised ten and the remaining seven clusters two isolates each, respectively. Two epidemiologically distinct HAOs were linked in cluster 1. FCs were involved in all clusters. All strains were associated with epidemic clonal complex CC59. We were able to confirm the spread of several successful t172-MRSA subclones in regional healthcare and the community. WGS complemented routine surveillance by revealing undetected links between t172-MRSA cases. Targeted, WGS-based typing could enhance MRSA surveillance without the need for routine WGS diagnostics.

Spa Typing of Methicillin-Resistant Staphylococcus aureus Based on Whole-Genome Sequencing: the Impact of the Assembler.

Sequencing of the spa gene of methicillin-resistant Staphylococcus aureus (MRSA) is used for assigning spa types to e.g., detect transmission and control outbreaks. Traditionally, spa typing is performed by Sanger sequencing but has in recent years been replaced by whole-genome sequencing (WGS) in some laboratories. Spa typing by WGS involves de novo assembly of millions of short sequencing reads into larger contiguous sequences, from which the spa type is then determined. The choice of assembly program therefore potentially impacts the spa typing result. In this study, WGS of 1,754 MRSA isolates was followed by de novo assembly using the assembly programs SPAdes (with two different sets of parameters) and SKESA. The spa types were assigned and compared to the spa types obtained by Sanger sequencing, regarding the latter as the correct spa types. SPAdes with the two different settings resulted in assembly of the correct spa type for 84.8% and 97.6% of the isolates, respectively, while SKESA assembled the correct spa type in 98.6% of cases. The misassembled spa types were generally two spa repeats shorter than the correct spa type and mainly included spa types with repetition of the same repeats. WGS-based spa typing is thus very accurate compared to Sanger sequencing, when the best assembly program for this purpose is used. IMPORTANCE spa typing of methicillin-resistant Staphylococcus aureus (MRSA) is widely used by clinicians, infection control workers, and researchers both in local outbreak investigations and as an easy way to communicate and compare MRSA types between laboratories and countries. Traditionally, spa types are determined by Sanger sequencing, but in recent years a whole-genome sequencing (WGS)-based approach has become increasingly used. In this study, we compared spa typing by WGS using different methods for assembling the genome from short sequencing reads and compared to Sanger sequencing as the gold standard. We find substantial differences in correct assembly of spa types between the assembly methods. Our findings are therefore important for the quality of WGS based spa typing data being exchanged by clinical microbiology laboratories.

Role of Efflux in Antibiotic Resistance of Achromobacter xylosoxidans and Achromobacter insuavis Isolates From Patients With Cystic Fibrosis.

Achromobacter genus (including Achromobacter xylosoxidans, the most prevalent Achromobacter species in patients with cystic fibrosis) is poorly susceptible to most conventional antibiotics. Contribution of efflux by AxyABM, AxyXY-OprZ, and AxyEF-OprN and of target mutations were studied in clinical isolates of A. xylosoxidans and Achromobacter insuavis. Forty-one isolates longitudinally collected from 21 patients with CF were studied by whole-genome sequencing (WGS)-typing, determination of minimum inhibitory concentrations (MICs) of β-lactams, aminoglycosides, colistin, azithromycin, ciprofloxacin, chloramphenicol, and doxycycline, and expression (quantitative RT-PCR) and function (measure of the uptake of a fluorescent substrate) of efflux pumps. WGS-based typing resulted in 10 clusters comprising 2 or 3 isolates and 20 singletons. The efflux activity was high in strains with elevated MICs for amikacin or azithromycin. This work sheds a new light on the impact of efflux and target mutations in resistance of Achromobacter to several drugs.

Colonization of Dogs and Their Owners with Staphylococcus aureus and Staphylococcus pseudintermedius in Households, Veterinary Practices, and Healthcare Facilities.

There are uncertainties with respect to the transmission of methicillin-susceptible and methicillin-resistant Staphylococcus aureus (MSSA and MRSA) and Staphylococcus pseudintermedius between dogs and humans. In this study, we investigated concomitant nasal colonization of dogs and humans in three cohorts. Cohort I, households owning dogs: In 42 of 84 households, 66 humans (36.9%) and 10 dogs (8.9%) carried S. aureus. MRSA, attributed to sequence type (ST) 22 and ST130, were detected in two (1.1%) of the humans but in none of the dogs. Typing by means of spa-typing and whole-genome sequencing (WGS) indicated eight transmissions of S. aureus between humans and dogs in 8 of 42 (19.0%) households with human S. aureus carriers, whereas in 11 of 38 (29.0%) households with ≥two persons and S. aureus colonization of humans, 15 human-to-human transmissions were observed (p = 0.43). S. pseudintermedius was isolated from 42 dogs (37.5%), but from only one human (0.6%). In this case, WGS-based typing indicated strong relatedness of this isolate with a canine isolate from the same household. Cohort II, dogs and their owners visiting a veterinary practice: Among 17 humans and 17 dogs attending a veterinary practice, MSSA was detected in three humans and two dogs, and S. pseudintermedius in only six dogs. Cohort III, dogs used for animal-assisted interventions in human healthcare facilities and their owners: MSSA was obtained in 1 of 59 dogs (1.7%) and in 17 of 60 (28.3%) of the dog owners, while S. pseudintermedius was isolated from seven (12%) dogs and one (1.7%) human owner. We conclude that the risk of exchanging S. aureus/MRSA between humans and dogs is higher than that for S. pseudintermedius.

Genomic Profiling of Antibiotic-Resistant Escherichia coli Isolates from Surface Water of Agricultural Drainage in North-Western Mexico: Detection of the International High-Risk Lineages ST410 and ST617.

Aquatic environments are recognized as one of the main reservoirs for the emergence and dissemination of high-risk lineages of multidrug-resistant (MDR) bacteria of public health concern. However, the genomic characteristics of antibiotic-resistant Escherichia coli isolates from aquatic origins remain limited. Herein, we examined the antibiotic resistance and virulence genomic profiles of three E. coli recovered from surface water in northwest Mexico. Antimicrobial susceptibility testing, whole-genome sequencing (WGS), and in-depth in silico analysis were performed. Two E. coli exhibited MDR phenotypes. WGS-based typing revealed genetic diversity, and phylogenetic analysis corroborated a notable divergent relationship among the studied E. coli. One E. coli strain, harboring enterotoxigenic and extraintestinal pathogenic-associated virulence genes, was assigned to the ST4 lineage. MDR E. coli, belonging to the international high-risk clones ST410 and ST617, carried genes and mutations conferring resistance to aminoglycosides, β-lactams, quinolones, sulfonamides, tetracyclines, and trimethoprim. This study describes, for the first time, the detection and genomic profiling of high-risk lineages of E. coli ST410 and ST617 from surface water in Mexico. Additionally, our results underscore the role of surface water as a reservoir for critical pathogenic and MDR E. coli clones and the need for the surveillance and monitoring of aquatic environments via WGS from the One Health perspective.

Agrobacterium species bacteraemia, Switzerland, 2008 to 2019: a molecular epidemiological study

BackgroundAgrobacterium spp. are infrequent agents of bloodstream infections linked to healthcare-associated outbreaks. However, it is unclear if outbreaks also occur across larger geographic areas. Triggered by two local clusters from putative point sources, our aim was to detect potential additional clusters in Switzerland.MethodsWe performed a nationwide descriptive study of cases in Switzerland based on a prospective surveillance system (Swiss Centre for Antibiotic Resistance, anresis.ch), from 2008 to 2019. We identified patients with Agrobacterium spp. isolated from blood cultures and used a survey to collect clinical-epidemiological information and susceptibility testing results. We performed whole genome sequencing (WGS) of available clinical isolates and determined their relatedness by single nucleotide polymorphism (SNP) variant calling analysis.ResultsWe identified a total of 36 cases of Agrobacterium spp. from blood samples over 10 years. Beyond previously known local clusters, no new ones were identified. WGS-based typing was performed on 22 available isolates and showed no clonal relationships between newly identified isolates or to those from the known clusters, with all isolates outside these clusters being at least 50 SNPs apart.Conclusion and relevanceAgrobacterium spp. bacteraemia is infrequently detected and, given that it may be healthcare-associated and stem from a point source, occurrence of multiple episodes should entail an outbreak investigation. With the help of the national antimicrobial resistance surveillance system we identified multiple clinical cases of this rare pathogen but found no evidence by WGS that suggested a nation-wide outbreak.Graphical abstract

Comparison of Whole Genome Sequencing and Repetitive Element PCR for Multidrug-Resistant Pseudomonas aeruginosa Strain Typing

Hospital-acquired infections pose significant costly global challenges to patient care. Rapid and sensitive methods to identify potential outbreaks are integral to infection control measures. Whole-genome sequencing (WGS)-based bacterial strain typing provides higher discriminatory power over standard nucleotide banding pattern-based methods such as repetitive sequence-based PCR (rep-PCR). However, integration of WGS into clinical epidemiology is limited by the lack of consensus in methodology and data analysis/interpretation. In this study, WGS was performed on genomic DNA extracted from 22 multidrug-resistant Pseudomonas aeruginosa (MDR-PA) isolates using next-generation sequencing. Resulting high-quality reads were analyzed for phylogenetic relatedness using a whole-genome multilocus sequence typing (wgMLST)-based software program and single-nucleotide variant phylogenomics (SNVPhyl). WGS-based results were compared with conventional MLST and archived rep-PCR results. Rep-PCR identified three independent clonal clusters of MDR-PA. Only one clonal cluster identified by rep-PCR, an endemic strain within the pediatric cystic fibrosis population at Texas Children's Hospital, was concordantly identified using wgMLST and SNVPhyl. Results were highly consistent between the three sequence-based analyses (conventional MLST, wgMLST, and SNVPhyl), and these results remained consistent with the addition of 74 MDR-PA genomes. These WGS-based methods provided greater resolution for strain discrimination than rep-PCR or standard MLST classification, and the ease of use of wgMLST software renders it clinically viable for analysis, interpretation, and reporting of WGS-based strain typing.

Transition From PCR-Ribotyping to Whole Genome Sequencing Based Typing of Clostridioides difficile.

Clostridioides difficile causes nosocomial outbreaks which can lead to severe and even life-threatening colitis. Rapid molecular diagnostic tests allow the identification of toxin-producing, potentially hypervirulent strains, which is critical for patient management and infection control. PCR-ribotyping has been used for decades as the reference standard to investigate transmission in suspected outbreaks. However, the introduction of whole genome sequencing (WGS) for molecular epidemiology provides a realistic alternative to PCR-ribotyping. In this transition phase it is crucial to understand the strengths and weaknesses of the two technologies, and to assess their correlation. We aimed to investigate ribotype prediction from WGS data, and options for analysis at different levels of analytical granularity. Ribotypes cannot be directly determined from short read Illumina sequence data as the rRNA operons including the ribotype-defining ISR fragments collapse in genome assemblies, and comparison with traditional PCR-ribotyping results becomes impossible. Ribotype extraction from long read Oxford nanopore data also requires optimization. We have compared WGS-based typing with PCR-ribotyping in nearly 300 clinical and environmental isolates from Switzerland, and in addition from the Enterobase database (n=1778). Our results show that while multi-locus sequence type (MLST) often correlates with a specific ribotype, the agreement is not complete, and for some ribotypes the resolution is insufficient. Using core genome MLST (cgMLST) analysis, there is an improved resolution and ribotypes can often be predicted within clusters, using cutoffs of 30-50 allele differences. The exceptions are ribotypes within known ribotype complexes such as RT078/RT106, where the genome differences in cgMLST do not reflect the ribotype segregation. We show that different ribotype clusters display different degrees of diversity, which could be important for the definition of ribotype cluster specific cutoffs. WGS-based analysis offers the ultimate resolution to the SNP level, enabling exploration of patient-to-patient transmission. PCR-ribotyping does not sufficiently discriminate to prove nosocomial transmission with certainty. We discuss the associated challenges and opportunities in a switch to WGS from conventional ribotyping for C. difficile.

Investigating Major Recurring Campylobacter jejuni Lineages in Luxembourg Using Four Core or Whole Genome Sequencing Typing Schemes.

Campylobacter jejuni is the leading cause of bacterial gastroenteritis, which has motivated the monitoring of genetic profiles circulating in Luxembourg since 13 years. From our integrated surveillance using a genotyping strategy based on an extended MLST scheme including gyrA and porA markers, an unexpected endemic pattern was discovered in the temporal distribution of genotypes. We aimed to test the hypothesis of stable lineages occurrence by implementing whole genome sequencing (WGS) associated with comprehensive and internationally validated schemes. This pilot study assessed four WGS-based typing schemes to classify a panel of 108 strains previously identified as recurrent or sporadic profiles using this in-house typing system. The strain collection included four common lineages in human infection (N = 67) initially identified from recurrent combination of ST-gyrA-porA alleles also detected in non-human samples: veterinary (N = 19), food (N = 20), and environmental (N = 2) sources. An additional set of 19 strains belonging to sporadic profiles completed the tested panel. All the strains were processed by WGS by using Illumina technologies and by applying stringent criteria for filtering sequencing data; we ensure robustness in our genomic comparison. Four typing schemes were applied to classify the strains: (i) the cgMLST SeqSphere+ scheme of 637 loci, (ii) the cgMLST Oxford scheme of 1,343 loci, (iii) the cgMLST INNUENDO scheme of 678 loci, and (iv) the wgMLST INNUENDO scheme of 2,795 loci. A high concordance between the typing schemes was determined by comparing the calculated adjusted Wallace coefficients. After quality control and analyses with these four typing schemes, 60 strains were confirmed as members of the four recurrent lineages regardless of the method used (N = 32, 12, 7, and 9, respectively). Our results indicate that, regardless of the typing scheme used, epidemic or endemic signals were detected as reflected by lineage B (ST2254-gyrA9-porA1) in 2014 or lineage A (ST19-gyrA8-porA7), respectively. These findings support the clonal expansion of stable genomes in Campylobacter population exhibiting a multi-host profile and accounting for the majority of clinical strains isolated over a decade. Such recurring genotypes suggest persistence in reservoirs, sources or environment, emphasizing the need to investigate their survival strategy in greater depth.

Translatability of WGS typing results can simplify data exchange for surveillance and control of Listeria monocytogenes

Where classical epidemiology has proven to be inadequate for surveillance and control of foodborne pathogens, molecular epidemiology, using genomic typing methods, can add value. However, the analysis of whole genome sequencing (WGS) data varies widely and is not yet fully harmonised. We used genomic data on 494 Listeria monocytogenes isolates from ready-to-eat food products and food processing environments deposited in the strain collection of the German National Reference Laboratory to compare various procedures for WGS data analysis and to evaluate compatibility of results. Two different core genome multilocus sequence typing (cgMLST) schemes, different reference genomes in single nucleotide polymorphism (SNP) analysis and commercial as well as open-source software were compared. Correlation of allele distances from the different cgMLST approaches was high, ranging from 0.97 to 1, and unified thresholds yielded higher clustering concordance than scheme-specific thresholds. The number of detected SNP differences could be increased up to a factor of 3.9 using a specific reference genome compared with a general one. Additionally, specific reference genomes improved comparability of SNP analysis results obtained using different software tools. The use of a closed or a draft specific reference genome did not make a difference. The harmonisation of WGS data analysis will finally guarantee seamless data exchange, but, in the meantime, knowledge on threshold values that lead to comparable clustering of isolates by different methods may improve communication between laboratories. We therefore established a translation code between commonly applied cgMLST and SNP methods based on optimised clustering concordances. This code can work as a first filter to identify WGS-based typing matches resulting from different methods, which opens up a new perspective for data exchange and thereby accelerates time-critical analyses, such as in outbreak investigations.

Analytical Performance Validation of Next-Generation Sequencing Based Clinical Microbiology Assays Using a K-mer Analysis Workflow.

Next-generation sequencing (NGS) enables clinical microbiology assays such as molecular typing of bacterial isolates which is now routinely applied for infection control and epidemiology. Additionally, feasibility for NGS-based identification of antimicrobial resistance (AMR) markers as well as genetic prediction of antibiotic susceptibility testing results has been demonstrated. Various bioinformatics approaches enabling NGS-based clinical microbiology assays exist, but standardized, computationally efficient and scalable sample-to-results workflows including validated quality control parameters are still lacking. Bioinformatics analysis workflows based on k-mers have been shown to allow for fast and efficient analysis of large genomics data sets as obtained from microbial sequencing applications. We here demonstrate applicability of k-mer based clinical microbiology assays for whole-genome sequencing (WGS) including variant calling, taxonomic identification, bacterial typing as well as AMR marker detection. The wet-lab and dry-lab workflows were developed and validated in line with Clinical Laboratory Improvement Act (CLIA) guidelines for laboratory-developed tests (LDTs) on multi-drug resistant ESKAPE pathogens. The developed k-mer based workflow demonstrated ≥99.39% repeatability, ≥99.09% reproducibility and ≥99.76% accuracy for variant calling and applied assays as determined by intra-day and inter-day triplicate measurements. The limit of detection (LOD) across assays was found to be at 20× sequencing depth and 15× for AMR marker detection. Thorough benchmarking of the k-mer based workflow revealed analytical performance criteria are comparable to state-of-the-art alignment based workflows across clinical microbiology assays. Diagnostic sensitivity and specificity for multilocus sequence typing (MLST) and phylogenetic analysis were 100% for both approaches. For AMR marker detection, sensitivity and specificity were 95.29 and 99.78% for the k-mer based workflow as compared to 95.17 and 99.77% for the alignment-based approach. Summarizing, results illustrate that k-mer based analysis workflows enable a broad range of clinical microbiology assays, potentially not only for WGS-based typing and AMR gene detection but also genetic prediction of antibiotic susceptibility testing results.