cgMLST Profiles Research Articles

Unraveling the impact of genome assembly on bacterial typing: a one health perspective

BackgroundIn the context of pathogen surveillance, it is crucial to ensure interoperability and harmonized data. Several surveillance systems are designed to compare bacteria and identify outbreak clusters based on core genome MultiLocus Sequence Typing (cgMLST). Among the different approaches available to generate bacterial cgMLST, our research used an assembly-based approach (chewBBACA tool).MethodsSimulations of short-read sequencing were conducted for 5 genomes of 27 pathogens of interest in animal, plant, and human health to evaluate the repeatability and reproducibility of cgMLST. Various quality parameters, such as read quality and depth of sequencing were applied, and several read simulations and genome assemblies were repeated using three tools: SPAdes, Unicycler and Shovill. In vitro sequencing were also used to evaluate assembly impact on cgMLST results, for six bacterial species: Bacillus thuringiensis, Listeria monocytogenes, Salmonella enterica, Staphylococcus aureus, Vibrio parahaemolyticus and Xylella fastidiosa.ResultsThe results highlighted variability in cgMLST, which not only related to the assembly tools, but also induced by the intrinsic composition of the genomes themselves. This variability observed in simulated sequencing was further validated with real data for six of the bacterial pathogens studied.ConclusionThis highlights that the intrinsic genome composition affects assembly and resulting cgMLST profiles, and that variability in bioinformatics tools can induce a bias in cgMLST profiles. In conclusion, we propose that the completeness of cgMLST schemes should be considered when clustering strains.

Oxford Nanopore's 2024 sequencing technology for Listeria monocytogenes outbreak detection and source attribution: progress and clone-specific challenges.

Whole genome sequencing is an essential cornerstone of pathogen surveillance and outbreak detection. Established sequencing technologies are currently being challenged by Oxford Nanopore Technologies (ONT), which offers an accessible and cost-effective alternative enabling gap-free assemblies of chromosomes and plasmids. Limited accuracy has hindered its use for investigating pathogen transmission, but recent technology updates have brought significant improvements. To evaluate its readiness for outbreak detection, we selected 78 Listeria monocytogenes isolates from diverse lineages or known epidemiological clusters for sequencing with ONT's V14 Rapid Barcoding Kit and R10.4.1 flow cells. The most accurate of several tested workflows generated assemblies with a median of one error (SNP or indel) per assembly. For 66 isolates, the cgMLST profiles from ONT-only assemblies were identical to those generated from Illumina data. Eight assemblies were of lower quality, with more than 20 erroneous sites each, primarily caused by methylations at the GAAGAC motif (5'-GAAG6mAC-3'/5'-GT4mCTTC-3'). This led to inaccurate clustering, failing to group isolates from a persistence-associated clone that carried the responsible restriction-modification system. Out of 50 methylation motifs detected among the 78 isolates, only the GAAGAC motif was linked to substantially increased error rates. Our study shows that most L. monocytogenes genomes assembled from ONT-only data are suitable for high-resolution genotyping, but further improvements of chemistries or basecallers are required for reliable routine use in outbreak and food safety investigations.

Co-Occurrence of Two Plasmids Encoding Transferable blaNDM-1 and tet(Y) Genes in Carbapenem-Resistant Acinetobacter bereziniae.

Acinetobacter bereziniae has emerged as a significant human pathogen, acquiring multiple antibiotic resistance genes, including carbapenemases. This study focuses on characterizing the plasmids harboring the blaNDM-1 and tet(Y) genes in two carbapenem-resistant A. bereziniae isolates (UCO-553 and UCO-554) obtained in Chile during the COVID-19 pandemic. Methods: Antibiotic susceptibility testing was conducted on UCO-553 and UCO-554. Both isolates underwent whole-genome sequencing to ascertain their sequence type (ST), core genome multilocus sequence-typing (cgMLST) profile, antibiotic resistance genes, plasmids, and mobile genetic elements. Conjugation experiments were performed for both isolates. Results: Both isolates exhibited broad resistance, including resistance to carbapenems, third-generation cephalosporins, fluoroquinolones, tetracycline, cotrimoxazole, and aminoglycosides. Both isolates belong to sequence type STPAS1761, with a difference of 17 out of 2984 alleles. Each isolate carried a 47,274 bp plasmid with blaNDM-1 and aph(3')-VI genes and two highly similar plasmids: a 35,184 bp plasmid with tet(Y), sul2, aph(6)-Id, and aph(3″)-Ib genes, and a 6078 bp plasmid containing the ant(2″)-Ia gene. Quinolone-resistance mutations were identified in the gyrA and parC genes of both isolates. Importantly, blaNDM-1 was located within a Tn125 transposon, and tet(Y) was embedded in a Tn5393 transposon. Conjugation experiments successfully transferred blaNDM-1 and tet(Y) into the A. baumannii ATCC 19606 strain, indicating the potential for horizontal gene transfer. Conclusions: This study highlights the critical role of plasmids in disseminating resistance genes in A. bereziniae and underscores the need for the continued genomic surveillance of this emerging pathogen. The findings emphasize the importance of monitoring A. bereziniae for its potential to cause difficult-to-treat infections and its capacity to spread resistance determinants against clinically significant antibiotics.

Corynebacterium pseudotuberculosis: Whole genome sequencing reveals unforeseen and relevant genetic diversity in this pathogen.

Corynebacterium pseudotuberculosis (CPS) is an important bacterial animal pathogen. CPS causes chronic, debilitating and currently incurable infectious diseases affecting a wide range of livestock and wild herbivores including camelids worldwide. Belonging to the Corynebacterium diphtheriae complex, this pathogen can also infect humans. The classical characterization of CPS is typically based on the testing of nitrate reductase activity, separating the two biovars Equi and Ovis. However, more refined resolutions are required to unravel routes of infection. This was realized in our study by generating and analyzing whole genome sequencing (WGS) data. Using newly created core genome multilocus sequence typing (cgMLST) profiles we were the first to discover isolates grouping in a cluster adjacent to clusters formed by CPS biovar Equi isolates. This novel cluster includes CPS isolates from alpacas, llamas, camels and dromedaries, which are characterized by a lack of nitrate reductase activity as encountered in biovar Ovis. This is of special interest for molecular epidemiology. Nevertheless, these isolates bear the genes of the nitrate locus, which are characteristic of biovar Equi isolates. However, sequence analysis of the genes narG and narH of the nitrate locus revealed indels leading to frameshifts and inactivity of the enzymes involved in nitrate reduction. Interestingly, one CPS isolate originating from another lama with an insertion in the MFS transporter (narT) is adjacent to a cluster formed by ovine CPS isolates biovar Equi. Based on this knowledge, the combination of biochemical and PCR based molecular biological nitrate reductase detection can be used for a fast and uncomplicated classification of isolates in routine diagnostics in order to check the origin of camelid CPS isolates. Further analysis revealed that partial sequencing of the ABC transporter substrate binding protein (CP258_RS07935) is a powerful tool to assign the biovars and the novel genomovar.

Genetic Diversity in Salmonella enterica in Outbreaks of Foodborne and Zoonotic Origin in the USA in 2006-2017.

Whole genome sequencing is replacing traditional laboratory surveillance methods as the primary tool to track and characterize clusters and outbreaks of the foodborne and zoonotic pathogen Salmonella enterica (S. enterica). In this study, 438 S. enterica isolates representing 35 serovars and 13 broad vehicle categories from one hundred epidemiologically confirmed outbreaks were evaluated for genetic variation to develop epidemiologically relevant interpretation guidelines for Salmonella disease cluster detection. The Illumina sequences were analyzed by core genome multi-locus sequence typing (cgMLST) and screened for antimicrobial resistance (AR) determinants and plasmids. Ninety-three of the one hundred outbreaks exhibited a close allele range (less than 10 allele differences with a subset closer than 5). The remaining seven outbreaks showed increased variation, of which three were considered polyclonal. A total of 16 and 28 outbreaks, respectively, showed variations in the AR and plasmid profiles. The serovars Newport and I 4,[5],12:i:-, as well as the zoonotic and poultry product vehicles, were overrepresented among the outbreaks, showing increased variation. A close allele range in cgMLST profiles can be considered a reliable proxy for epidemiological relatedness for the vast majority of S. enterica outbreak investigations. Variations associated with mobile elements happen relatively frequently during outbreaks and could be reflective of changing selective pressures.

Genomic Epidemiology Dataset for the Important Nosocomial Pathogenic Bacterium Acinetobacter baumannii

The infections caused by various bacterial pathogens both in clinical and community settings represent a significant threat to public healthcare worldwide. The growing resistance to antimicrobial drugs acquired by bacterial species causing healthcare-associated infections has already become a life-threatening danger noticed by the World Health Organization. Several groups or lineages of bacterial isolates, usually called ‘the clones of high risk’, often drive the spread of resistance within particular species. Thus, it is vitally important to reveal and track the spread of such clones and the mechanisms by which they acquire antibiotic resistance and enhance their survival skills. Currently, the analysis of whole-genome sequences for bacterial isolates of interest is increasingly used for these purposes, including epidemiological surveillance and the development of spread prevention measures. However, the availability and uniformity of the data derived from genomic sequences often represent a bottleneck for such investigations. With this dataset, we present the results of a genomic epidemiology analysis of 17,546 genomes of a dangerous bacterial pathogen, Acinetobacter baumannii. Important typing information, including multilocus sequence typing (MLST)-based sequence types (STs), intrinsic blaOXA-51-like gene variants, capsular (KL) and oligosaccharide (OCL) types, CRISPR-Cas systems, and cgMLST profiles are presented, as well as the assignment of particular isolates to nine known international clones of high risk. The presence of antimicrobial resistance genes within the genomes is also reported. These data will be useful for researchers in the field of A. baumannii genomic epidemiology, resistance analysis, and prevention measure development.

International Clones of High Risk of Acinetobacter Baumannii-Definitions, History, Properties and Perspectives.

Acinetobacter baumannii is a Gram-negative coccobacillus with exceptional survival skills in an unfavorable environment and the ability to rapidly acquire antibiotic resistance, making it one of the most successful hospital pathogens worldwide, representing a serious threat to public health. The global dissemination of A. baumannii is driven by several lineages named 'international clones of high risk' (ICs), two of which were first revealed in the 1970s. Epidemiological surveillance is a crucial tool for controlling the spread of this pathogen, which currently increasingly involves whole genome sequencing. However, the assignment of a particular A. baumannii isolate to some IC based on its genomic sequence is not always straightforward and requires some computational skills from researchers, while the definitions found in the literature are sometimes controversial. In this review, we will focus on A. baumannii typing tools suitable for IC determination, provide data to easily determine IC assignment based on MLST sequence type (ST) and intrinsic blaOXA-51-like gene variants, discuss the history and current spread data of nine known ICs, IC1-IC9, and investigate the representation of ICs in public databases. MLST and cgMLST profiles, as well as OXA-51-like presence data are provided for all isolates available in GenBank. The possible emergence of a novel A. baumannii international clone, IC10, will be discussed.

Core Genome Multilocus Sequence Typing Scheme for Improved Characterization and Epidemiological Surveillance of Pathogenic Brucella.

ABSTRACTBrucellosis poses a significant burden to human and animal health worldwide. Robust and harmonized molecular epidemiological approaches and population studies that include routine disease screening are needed to efficiently track the origin and spread of Brucella strains. Core genome multilocus sequence typing (cgMLST) is a powerful genotyping system commonly used to delineate pathogen transmission routes for disease surveillance and control. Except for Brucella melitensis, cgMLST schemes for Brucella species are currently not established. Here, we describe a novel cgMLST scheme that covers multiple Brucella species. We first determined the phylogenetic breadth of the genus using 612 Brucella genomes. We selected 1,764 genes that were particularly well conserved and typeable in at least 98% of these genomes. We tested the new scheme on 600 genomes and found high agreement with the whole-genome-based single nucleotide polymorphism (SNP) analysis. Next, we applied the scheme to reanalyze the genome of Brucella strains from epidemiologically linked outbreaks. We demonstrated the applicability of the new scheme for high-resolution typing required in outbreak investigations as previously reported with whole-genome SNP methods. We also used the novel scheme to define the global population structure of the genus using 1,322 Brucella genomes. Finally, we demonstrated the possibility of tracing distribution of Brucella strains by performing cluster analysis of cgMLST profiles and found nearly identical cgMLST profiles in different countries. Our results show that sequencing depth of more than 40-fold is optimal for allele calling with this scheme. In summary, this study describes a novel Brucella-wide cgMLST scheme that is applicable in Brucella molecular epidemiology and helps in accurately tracking and thus controlling the sources of infection. The scheme is publicly accessible and should represent a valuable resource for laboratories with limited computational resources and bioinformatics expertise.

Demographic Features of Invasive Meningococcal Disease in Taiwan, 1993 to 2020, and Genetic Characteristics of Neisseria meningitidis Isolates, 2003 to 2020.

ABSTRACTWe present the demographic features of invasive meningococcal disease (IMD) in Taiwan between 1993 and 2020 and the genetic characteristics of Neisseria meningitidis isolates recovered from 2003 to 2020. IMD was rare in Taiwan between 1993 and 2020, with an annual incidence ranging from 0.009 to 0.204 per 100,000 people. The case fatality rate (CFR) declined from 18.1% for patients in 1993 to 2002 to 9.8% in 2003 to 2020. Infants less than 12 months were most susceptible to the disease. N. meningitidis serogroup B (NmB) was most predominant, responsible for 81.2% (134/165) of the IMD cases in 2003 to 2020. The majority of the isolates recovered from 2003 to 2020 belonged to 4 worldwide-spread hyperinvasive clonal complexes (cc), cc4821 (30.3%), cc32 (19.4%), cc41/44 (12.7%), cc23 (7.3%), and also a newly assigned clonal complex, cc3439 (10.3%). Core genome multilocus sequence typing (cgMLST) profile comparisons revealed that the cc4821 isolates with a T-to-I substitution at position 91 in gyrA were closely related to those originating from China. Of the 165 isolates, 20.0% and 53.3% were predicted to be covered by the Bexsero and Trumenba vaccines, respectively, whereas, 77.0% and 46.7% remained indeterminate. In conclusion, N. meningitidis isolates recovered in Taiwan between 2003 and 2020 were mostly highly diverse. Most IMD cases appeared sporadically and were caused by localized strains, although some patients were infected by recently introduced strains. cgMLST is a powerful tool for the rapid comparison of genetic relatedness among a large number of isolates. cgMLST profiling, based on 1,241 core genes, and strain tracking can be performed on the website of cgMLST@Taiwan (http://rdvd.cdc.gov.tw/cgMLST/).IMPORTANCE N. meningitidis can cause life-threatening invasive meningococcal disease (IMD), including meningitis and sepsis, resulting in a high CFR and long-term sequelae in survivors. Here, we report the demographic features of IMD in Taiwan over a 28-year period (1993 to 2020) and the genetic characteristics of N. meningitidis isolates recovered from patients with IMD over an 18-year period (2003 to 2020). We conducted a whole-genome sequence analysis to characterize the genetic features of the isolates and developed a cgMLST scheme for epidemiological investigation and strain tracking. The findings can be beneficial in understanding the epidemiology of IMD in Taiwan, the genetic characteristics of the bacterial strains, and the distribution of vaccine antigens for vaccine development and implementation.

Guidelines for reporting Whole Genome Sequencing‐based typing data through the EFSA One Health WGS System

In 2019, the European Food Safety Authority (EFSA) and the European Centre for Disease Prevention and Control (ECDC) received a mandate from the European Commission (EC) to implement and manage two interoperable systems for the collection and analysis of Whole Genome Sequence (WGS) data of Salmonella enterica, Listeria monocytogenes and Escherichia coli, including Shiga toxin-producing E. coli (STEC) from humans and from food, feed, animals and the related environment, to contribute to the epidemiological investigations of food-borne outbreaks and to the identification of emerging health threats. As response to this EC mandate, EFSA has developed the EFSA One Health WGS System that interoperates with the ECDC Molecular Typing system exchanging core genome Multi Locus Sequence Typing (cgMLST) profiles and minimum metadata. Interested European Union (EU) Member States (MS) and European Economic Area (EEA) countries can submit their typing data from food, feed and animal samples to the EFSA One Health WGS system either through an user-friendly interface or programmatically. This technical report describes the EFSA One Health WGS system, including his architecture, the users and their relative data visibility, as well as data ownership and intellectual property. This report provides also detailed information on the types and formats of data that can be submitted to the EFSA One Health WGS system. Moreover, these guidelines describe the individual steps of the reporting procedure in order to support the EU MSs and EEA countries in data submission and data management.

Genomic Characterization of Clinical Acinetobacter baumannii Isolates Obtained from COVID-19 Patients in Russia.

The coronavirus disease 2019 (COVID-19) pandemic has already affected all realms of public healthcare and, in particular, has led to increasing use of various antibiotics to treat possible bacterial coinfections even in cases for which such infections were not confirmed clinically. This could lead to an increase in the fraction and severity of multidrug-resistant bacterial isolates in healthcare facilities, especially in intensive care units (ICU). However, detailed epidemiological investigations, possibly including whole genome sequencing (WGS), are required to confirm the increase in antibiotic resistance and changes, if any, in the population and clonal structures of bacterial pathogens. In this study, we performed a comprehensive genomic and phenotypic characterization of selected multidrug-resistant A. baumannii isolates obtained from the patients of a dedicated COVID-19 ICU in Moscow, Russia. Hybrid short- and long-read sequencing allowed us to obtain complete profiles of genomic antimicrobial resistance and virulence determinants, as well as to reveal the plasmid structure. We demonstrated the genomic similarity in terms of cgMLST profiles of the isolates studied with a clone previously identified in the same facility. We believe that the data provided will contribute to better understanding the changes imposed by the COVID-19 pandemic on the population structure and the antimicrobial resistance of bacterial pathogens in healthcare facilities.

Campylobacter coli isolated in Brazil typed by core genome Multilocus Sequence Typing shows high genomic diversity in a global context.

The current study aimed to determine the phylogenetic relationships among Campylobacter coli strains isolated in Brazil and to compare them with international Campylobacter isolates available in some public databases. A total of 63C. coli strains isolated in Brazil were studied. The MLST analysis showed 18 different STs including three STs not yet described in the PubMLST database. The cgMLST allocated the Brazilian strains studied into five main clusters and each cluster comprised groups of strains with nearly identical cgMLST profiles and with significant genetic distance observed among the distinct clusters. The comparison of the Brazilian strains with 3401 isolates from different countries showed a wide distribution of these strains isolated in this country. The results showed a high similarity among some strains studied and a wide distribution of the Brazilian strains when compared to isolates from different countries, which is an interesting data set since it showed a high genetic diversity of these strains from Brazil in a global context. This study contributed for a better genomic characterization of C. coli strains isolated in Brazil and provided important information about the diversity of this clinically-relevant pathogen.

Investigation of MALDI-TOF Mass Spectrometry for Assessing the Molecular Diversity of Campylobacter jejuni and Comparison with MLST and cgMLST: A Luxembourg One-Health Study.

There is a need for active molecular surveillance of human and veterinary Campylobacter infections. However, sequencing of all isolates is associated with high costs and a considerable workload. Thus, there is a need for a straightforward complementary tool to prioritize isolates to sequence. In this study, we proposed to investigate the ability of MALDI-TOF MS to pre-screen C. jejuni genetic diversity in comparison to MLST and cgMLST. A panel of 126 isolates, with 10 clonal complexes (CC), 21 sequence types (ST) and 42 different complex types (CT) determined by the SeqSphere+ cgMLST, were analysed by a MALDI Biotyper, resulting into one average spectra per isolate. Concordance and discriminating ability were evaluated based on protein profiles and different cut-offs. A random forest algorithm was trained to predict STs. With a 94% similarity cut-off, an AWC of 1.000, 0.933 and 0.851 was obtained for MLSTCC, MLSTST and cgMLST profile, respectively. The random forest classifier showed a sensitivity and specificity up to 97.5% to predict four different STs. Protein profiles allowed to predict C. jejuni CCs, STs and CTs at 100%, 93% and 85%, respectively. Machine learning and MALDI-TOF MS could be a fast and inexpensive complementary tool to give an early signal of recurrent C. jejuni on a routine basis.

CgMLST@Taiwan: A web service platform for Vibrio cholerae cgMLST profiling and global strain tracking.

Cholera, a rapidly dehydrating diarrheal disease caused by toxigenic Vibrio cholerae, is a leading cause of morbidity and mortality in some regions of the world. Core genome multilocus sequence typing (cgMLST) is a promising approach in generating genetic fingerprints from whole-genome sequencing (WGS) data for strain comparison among laboratories. We constructed a V. cholerae core gene allele database using an in-house developed computational pipeline, a database with cgMLST profiles converted from genomic sequences from the National Center for Biotechnology Information, and built a REST-based web accessible via the Internet. We built a web service platform-cgMLST@Taiwan and installed a V. cholerae allele database, a cgMLST profile database, and computational tools for generating V. cholerae cgMLST profiles (based on 3,017 core genes), performing rapid global strain tracking, and clustering analysis of cgMLST profiles. This web-based platform provides services to researchers, public health microbiologists, and physicians who use WGS data for the investigation of cholera outbreaks and tracking of V. cholerae strain transmission across countries and geographic regions. The cgMLST@Taiwan is accessible at http://rdvd.cdc.gov.tw/cgMLST.

Multidrug Resistant Klebsiella pneumoniae ST101 Clone Survival Chain From Inpatients to Hospital Effluent After Chlorine Treatment.

In this paper we describe the transmission of a multi-drug resistant Klebsiella pneumoniae ST101 clone from hospital to wastewater and its persistence after chlorine treatment. Water samples from influents and effluents of the sewage tank of an infectious diseases hospital and clinical strains collected from the intra-hospital infections, during a period of 10 days prior to wastewater sampling were analyzed. Antibiotic resistant K. pneumoniae strains from wastewaters were recovered on selective media. Based on antibiotic susceptibility profiles and PCR analyses of antibiotic resistance (AR) genetic background, as well as whole-genome sequencing (Illumina MiSeq) and subsequent bioinformatic analyses, 11 ST101 K. pneumoniae strains isolated from hospital wastewater influent, wastewater effluent and clinical sector were identified as clonally related. The SNP and core genome analyses pointed out that five strains were found to be closely related (with ≤18 SNPs and identical cgMLST profile). The strains belonging to this clone harbored multiple acquired AR genes [blaCTX–M–15, blaOXA–48, blaOXA–1, blaSHV–106, blaTEM–150, aac(3)-IIa, aac(6′)-Ib-cr, oqxA10, oqxB17, fosA, catB3, dfrA14, tet(D)] and chromosomal mutations involved in AR (ΔmgrB, ΔompK35, amino acid substitutions in GyrA Ser83Tyr, Asp87Asn, ParC Ser80Tyr). Twenty-nine virulence genes involved in iron acquisition, biofilm and pili formation, adherence, and the type six secretion system – T6SS-III were identified. Our study proves the transmission of MDR K. pneumoniae from hospital to the hospital effluent and its persistence after the chlorine treatment, raising the risk of surface water contamination and further dissemination to different components of the trophic chain, including humans.

831. Analysis of a Worldwide Collection of Klebsiella pneumoniae CC258 with Reference to Carbapenemase Production Using the 1928 Core Genome (cg) Multilocus Sequence Type (MLST) Reveals Endemicity and Global Dissemination of Clones

Background K. pneumoniae (KPN) has emerged as a major hospital associated pathogen in the recent years. Clonal complex (CC) 258 constitutes an international epidemic clone responsible for spread of extended spectrum beta-lactamases (ESBL) and carbapenemases. We evaluated the core genome (cg) MLST, resistance (R) gene and plasmid profiles of a worldwide collection of KPN using the 1928 bioinformatic cloud platform.MethodsA total of 155 KPN clinical isolates (CC258, n=129; non-CC258, n=26) collected from 19 countries during 2018 of SENTRY Program were analyzed. Isolates included carbapenem resistant (CR; n=120) and non-CR (n=35). Whole genome sequencing FASTQ files were uploaded to the 1928 pipeline for analysis.ResultsMost CC258 isolates belonged to ST258, ST11 and ST512 (Table), and separated from unrelated ST by allelic distance (ad) > 2000. cgMLST grouped together isolates from the same STs, and those from similar geographies had greater homology, except isolates from US showed greater heterogenicity (ad 620). Applying an ad cutoff of < 100, ST258 isolates grouped in 4 clades with a predominance of either KPC-2 or -3. An ad cutoff of < 200 identified 7 clades within ST11 that were related by geography and R genes. Among CC258 isolates KPC was the major carbapenemase (58.1%) and associated with Tn4401 (a or b in 84% KPC); NDM-1 was detected (8.5%) only in ST11 and ST395. KPC-2 was more prevalent in Latin America and the isolates were closely related (ad 104) among ST258 compared to ST11 (ad 355). KPC-3 CC258 isolates showed ad of 164 and were from Italy, Russia, Greece, and US. CTX-M-14 was prevalent in ST258 while CTX-M-15 was common in other STs, except ST512 which carried no CTX-M despite clustering within ST258.Conclusion1928 generated cgMLST showed good correlation with MLST in classifying all KPN isolates. ST258 isolates showed tight clustering, no NDM genes and distribution in the Americas while ST11 showed global dissemination and diversity of carbapenemases.Table 1 DisclosuresFredrik Dyrkell, n/a, 1928 Diagnostics (Employee) Dimitrios Arnellos, n/a, 1928 Diagnostics (Employee) Mariana Castanheira, PhD, 1928 Diagnostics (Research Grant or Support)A. Menarini Industrie Farmaceutiche Riunite S.R.L. (Research Grant or Support)Allergan (Research Grant or Support)Allergan (Research Grant or Support)Amplyx Pharmaceuticals (Research Grant or Support)Cidara Therapeutics (Research Grant or Support)Cidara Therapeutics (Research Grant or Support)Cipla Ltd. (Research Grant or Support)Cipla Ltd. (Research Grant or Support)Fox Chase Chemical Diversity Center (Research Grant or Support)GlaxoSmithKline (Research Grant or Support)Melinta Therapeutics, Inc. (Research Grant or Support)Melinta Therapeutics, Inc. (Research Grant or Support)Melinta Therapeutics, Inc. (Research Grant or Support)Merck (Research Grant or Support)Merck (Research Grant or Support)Merck & Co, Inc. (Research Grant or Support)Merck & Co, Inc. (Research Grant or Support)Paratek Pharma, LLC (Research Grant or Support)Pfizer (Research Grant or Support)Qpex Biopharma (Research Grant or Support)

Optical maps of plasmids as a proxy for clonal spread of MDR bacteria: a case study of an outbreak in a rural Ethiopian hospital.

ObjectivesMDR bacteria have become a prevailing health threat worldwide. We here aimed to use optical DNA mapping (ODM) as a rapid method to trace nosocomial spread of bacterial clones and gene elements. We believe that this method has the potential to be a tool of pivotal importance for MDR control.MethodsTwenty-four Escherichia coli samples of ST410 from three different wards were collected at an Ethiopian hospital and their plasmids were analysed by ODM. Plasmids were specifically digested with Cas9 targeting the antibiotic resistance genes, stained by competitive binding and confined in nanochannels for imaging. The resulting intensity profiles (barcodes) for each plasmid were compared to identify potential clonal spread of resistant bacteria.ResultsODM demonstrated that a large fraction of the patients carried bacteria with a plasmid of the same origin, carrying the ESBL gene blaCTX-M-15, suggesting clonal spread. The results correlate perfectly with core genome (cg)MLST data, where bacteria with the same plasmid also had very similar cgMLST profiles.ConclusionsODM is a rapid discriminatory method for identifying plasmids and antibiotic resistance genes. Long-range deletions/insertions, which are challenging for short-read next-generation sequencing, can be easily identified and used to trace bacterial clonal spread. We propose that plasmid typing can be a useful tool to identify clonal spread of MDR bacteria. Furthermore, the simplicity of the method enables possible future application in low- and middle-income countries.

The core genome multi-locus sequence typing of Mycoplasma anserisalpingitidis

BackgroundMycoplasma anserisalpingitidis is a waterfowl pathogen that mainly infects geese, can cause significant economic losses and is present worldwide. With the advance of whole genome sequencing technologies, new methods are available for the researchers; one emerging methodology is the core genome Multi-Locus Sequence Typing (cgMLST). The core genome contains a high percentage of the coding DNA sequence (CDS) set of the studied strains. The cgMLST schemas are powerful genotyping tools allowing for the investigation of potential epidemics, and precise and reliable classification of the strains. Although whole genome sequences of M. anserisalpingitidis strains are available, to date, no cgMLST schema has been published for this species.ResultsIn this study, Illumina short reads of 81 M. anserisalpingitidis strains were used, including samples from Hungary, Poland, Sweden, and China. Draft genomes were assembled with the SPAdes software and analysed with the online available chewBBACA program. User made modifications in the program enabled analysis of mycoplasmas and provided similar results as the conventional SeqSphere+ software. The threshold of the presence of CDS in the strains was set to 93% due to the quality of the draft genomes, resulting in the most accurate and robust schema. Three hundred thirty-one CDSs constituted our cgMLST schema (representing 42,77% of the whole CDS set of M. anserisalpingitidis ATCC BAA-2147), and a Neighbor joining tree was created using the allelic profiles. The correlation was observed between the strains’ cgMLST profile and geographical origin; however, strains from the same integration but different locations also showed close relationship. Strains isolated from different tissue samples of the same animal revealed highly similar cgMLST profiles.ConclusionsThe Neighbor joining tree from the cgMLST schema closely resembled the real-life spatial and temporal relationships of the strains. The incongruences between background data and the cgMLST profile in the strains from the same integration can be because of the higher probability of contacts between the flocks. This schema can help with the epidemiological investigation and can be used as a basis for further studies.

Core/Whole Genome Multilocus Sequence Typing and Core Genome SNP-Based Typing of OXA-48-Producing Klebsiella pneumoniae Clinical Isolates From Spain.

Whole-genome sequencing (WGS)-based typing methods have emerged as promising and highly discriminative epidemiological tools. In this study, we combined gene-by-gene allele calling and core genome single nucleotide polymorphism (cgSNP) approaches to investigate the genetic relatedness of a well-characterized collection of OXA-48-producing Klebsiella pneumoniae isolates. We included isolates from the predominant sequence type ST405 (n = 31) OXA-48-producing K. pneumoniae clone and isolates from ST101 (n = 3), ST14 (n = 1), ST17 (n = 1), and ST1233 (n = 1), obtained from eight Catalan hospitals. Core-genome multilocus sequence typing (cgMLST) schemes from Institut Pasteur’s BIGSdb-Kp (634 genes) and SeqSphere+ (2,365 genes), and a SeqSphere+ whole-genome MLST (wgMLST) scheme (4,891 genes) were used. Allele differences or allelic mismatches and the genetic distance, as the proportion of allele differences, were used to interpret the results from a gene-by-gene approach, whereas the number of SNPs was used for the cgSNP analysis. We observed between 0–10 and 0–14 allele differences among the predominant ST405 using cgMLST and wgMLST from SeqSphere+, respectively, and <2 allelic mismatches when using Institut Pasteur’s BIGSdb-Kp cgMLST scheme. For ST101, we observed 14 and 54 allele differences when using cgMLST and wgMLST SeqSphere+, respectively, and 2–5 allelic mismatches for BIGSdb-Kp cgMLST. A low genetic distance (<0.0035, a previously established threshold for epidemiological link) was generally in concordance with a low number of allele differences (<8) when using the SeqSphere+ cgMLST scheme. The cgSNP analysis showed 6–29 SNPs in isolates with identical allelic SeqSphere+ cgMLST profiles and 16–61 cgSNPs among ST405 isolates. Furthermore, comparison of WGS-based typing results with previously obtained MLST and pulsed-field gel electrophoresis (PFGE) data showed some differences, demonstrating the different molecular principles underlying these techniques. In conclusion, the use of the different WGS-based typing methods that were used to elucidate the genetic relatedness of clonal OXA-48-producing K. pneumoniae all led to the same conclusions. Furthermore, threshold parameters in WGS-based typing methods should be applied with caution and should be used in combination with clinical epidemiological data and population and species characteristics.

239. Epidemiologic Analysis of a Worldwide Collection of Escherichia coli ST131 Using the 1928D Core Genome (cg) Multilocus Sequence Type (MLST) Reveals Country Specific and Globally Disseminated Clades

BackgroundIncreasing antimicrobial resistance (R) among Escherichia coli (EC) isolates can be associated with the expansion of the pandemic sequence type (ST) 131 that harbors virulence factors and causes more severe infections when compared with other antimicrobial-R EC. We evaluated the core genome MLST (cgMLST) profiles and R genes using the bioinformatics tool 1928D to evaluate the epidemiology of a global ST131 EC collection and unrelated STs.MethodsA total of 259 EC clinical isolates belonging to ST131 (n = 206), ST131-single loci variant (SLV; n = 25), and 28 non-ST131 isolates collected from 27 countries during 2016–2018 were selected. Whole-genome sequencing FASTQ files were uploaded to the 1928D pipeline to generate MLST, cgMLST and R gene prediction. cgMLST assignment was based on comparing >2,500 genes.ResultsAmong 231 ST131 and SLV EC isolates, 7 clades were identified (3 major [178 isolates]; Table) applying cgMLST allele distance (ad) of ≤50 as a cutoff. A total of 21 isolates were not assigned to clades (>50 ad from ST131 and SLV). Based on >95% concordance, 11 alleles differentiated clades II and III from clade I, while 6 alleles separated clades I and III from clade II. Isolates in clades I to IV were ciprofloxacin R (MIC, ≥4 mg/L); clades I and III predominantly carried blaCTX-M-15 (39/43 and 61/66), blaOXA-1 (35/43 and 43/66), and aac(6’)-Ib-cr (39/43, 45/66) while clade II carried blaCTX-M-14-like and rarely aac(6’)Ib-cr (3/69). The most ad between the 7 ST131 clades was 216, while unrelated STs showed variable ad among isolates within that ST. Isolates bellowing to ST1193 were closely related genetically (ad of 30), but other STs had more variability among isolates (ST167, ad 552; ST38, ad 150; and ST69, ad 179).Conclusion1928D is a robust platform for epidemiological analysis of isolates, providing additional granularity when compared with MLST. Clades II and III were closely related, but carried different blaCTX-M genes, while clades I and III were not as closely related, but both carried blaCTX-M-15, blaOXA-1, and aac(6’)Ib-cr. These findings suggest that these clades might have acquired R genes at different points in their genetic evolution. A threshold of ≤50 (cgMLST distance) was useful for classifying isolates into clades. Disclosures All authors: No reported disclosures.