Core Genome Single Nucleotide Variant Research Articles

Rapid identification and subsequent contextualization of an outbreak of methicillin-resistant Staphylococcus aureus in a neonatal intensive care unit using nanopore sequencing.

Outbreaks of methicillin-resistant Staphylococcus aureus (MRSA) are well described in the neonatal intensive care unit (NICU) setting. Genomics has revolutionized the investigation of such outbreaks; however, to date, this has largely been completed retrospectively and has typically relied on short-read platforms. In 2022, our laboratory established a prospective genomic surveillance system using Oxford Nanopore Technologies sequencing for rapid outbreak detection. Herein, using this system, we describe the detection and control of an outbreak of sequence-type (ST)97 MRSA in our NICU. The outbreak was identified 13 days after the first MRSA-positive culture and at a point where there were only two known cases. Ward screening rapidly defined the extent of the outbreak, with six other infants found to be colonized. There was minimal transmission once the outbreak had been detected and appropriate infection control measures had been instituted; only two further ST97 cases were detected, along with three unrelated non-ST97 MRSA cases. To contextualize the outbreak, core-genome single-nucleotide variants were identified for phylogenetic analysis after de novo assembly of nanopore data. Comparisons with global (n=45) and national surveillance (n=35) ST97 genomes revealed the stepwise evolution of methicillin resistance within this ST97 subset. A distinct cluster comprising nine of the ten ST97-IVa genomes from the NICU was identified, with strains from 2020 to 2022 national surveillance serving as outgroups to this cluster. One ST97-IVa genome presumed to be part of the outbreak formed an outgroup and was retrospectively excluded. A second phylogeny was created using Illumina sequencing, which considerably reduced the branch lengths of the NICU isolates on the phylogenetic tree. However, the overall tree topology and conclusions were unchanged, with the exception of the NICU outbreak cluster, where differences in branch lengths were observed. This analysis demonstrated the ability of a nanopore-only prospective genomic surveillance system to rapidly identify and contextualize an outbreak of MRSA in a NICU.

Genome stability during serial subculturing in hyperepidemic multidrug-resistant Klebsiella pneumoniae and Escherichia coli

Core-genome single nucleotide variant (cgSNV) analysis represents a powerful tool for epidemiological investigations of multidrug-resistant (MDR) bacteria. However, cgSNV thresholds to confirm whether isolates are the same clone are not formally defined. We implemented hybrid whole-genome sequencing to study the genomic changes of four MDR isolates belonging to hyperepidemic sequence types (STs) during 20 propagation steps (T20) on MacConkey and CHROMID(R) ESBL plates. The following strains were analyzed: Klebsiella pneumoniae AE-2247421 (OXA-48/NDM-1-producing, ST101), K. pneumoniae MCL-2017-2 (CTX-M-15-producing, ST307), Escherichia coli Ec-042 (OXA-181-producing, ST410), and E. coli Ec-050 (NDM-5-producing, ST167). The genome assembly at T5 and T20 was compared to that at time point zero (T0) and to two reference genomes. At T20, AE-2247421 lost the IncL blaOXA-48-carrying plasmid when grown on CHROMID(R) ESBL plates, while a large fragment encompassing blaNDM-1 was lost from its IncC plasmid when grown on both plates. In contrast, no structural changes were noted for the other three strains. Regarding the cgSNVs, the following results were obtained at T5 and T20 (ranges considering the different agar plates and reference genomes): AE-2247421 (1-8 and 2-12 cgSNVs), MCL-2017-2 (both 1-2 cgSNVs), Ec-042 (both 0 cgSNVs), and Ec-050 (0-6 and 0-9 cgSNVs). We showed that structural changes and accumulation of cgSNVs can occur in few propagation steps under laboratory conditions. These changes might also arise in the clinical context in a short time, especially under antibiotics treatment. This phenomenon should be carefully considered because it might affect the final interpretation of epidemiological genomic analyses.

Assessment of the Transmission Dynamics of Clostridioides difficile in a Farm Environment Reveals the Presence of a New Toxigenic Strain Connected to Swine Production.

The recent increase in community-acquired Clostridioides difficile infections discloses the shift in this bacterium epidemiology. This study aimed at establishing a transmission network involving One Health components, as well as assessing the zoonotic potential and genomic features of dominant clones. Samples were collected from different compartments of animal, human and environmental origin, from an animal production unit. C. difficile isolates were characterized for toxigenic profile by multiplex-PCR, while genetic diversity was evaluated by PCR-ribotyping and whole genome-based analysis. The overall C. difficile prevalence was 37.2% (70/188), and included samples from environmental (58.3%, 35/60) and animal (31.5%, 35/111) compartments; human samples (n = 17) taken from healthy workers were negative. A predominant clone from RT033 was found in almost 90% of the positive samples, including samples from all compartments connected to the pig production unit, with core-genome single nucleotide variant (SNV)-based Analysis supporting a clonal transmission between them (mean distance of 0.1 ± 0.1 core-SNVs). The isolates from this clone (herein designated PT RT033) were positive for all C. difficile toxin genes (tcdA, tcdB, cdtA/cdtB). The phyloGenetic positioning of this clone was clearly distinct from the classical RT033 cluster, suggesting a different evolutionary route. This new clone shares genomic features with several RTs from the clade 5 Sequence Type (ST) 11, including a complete pathogenicity locus (PaLoc) that is more similar to the one found in toxigenic strains and contrasting to the less virulent classical RT033 (tcdA-, tcdB-, cdtA + /cdtB +). The presence of a tcdA gene truncated into two ORFs, not previously described, requires further evaluation concerning toxin functionality. We hypothesize that the unique combination of genetic elements found in the PT RT033 clone may contribute to host tropism and environmental dissemination and maintenance. This study constitutes the first report of a toxigenic RT033 clone and adds to the overall knowledge on Clade 5 sequence type 11, considered the C. difficile evolutionary lineage with the highest zoonotic potential. The presence of this clone in all compartments associated with the pig production unit suggests a transmission chain involving these animals and contributes to unveil the role played by animal and environmental reservoirs in this pathogen epidemiology.

Evaluation of whole-genome sequencing-based subtyping methods for the surveillance of Shigella spp. and the confounding effect of mobile genetic elements in long-term outbreaks.

Many public health laboratories across the world have implemented whole-genome sequencing (WGS) for the surveillance and outbreak detection of foodborne pathogens. PulseNet-affiliated laboratories have determined that most single-strain foodborne outbreaks are contained within 0–10 multi-locus sequence typing (MLST)-based allele differences and/or core genome single-nucleotide variants (SNVs). In addition to being a food- and travel-associated outbreak pathogen, most Shigella spp. cases occur through continuous person-to-person transmission, predominantly involving men who have sex with men (MSM), leading to long-term and recurrent outbreaks. Continuous transmission patterns coupled to genetic evolution under antibiotic treatment pressure require an assessment of existing WGS-based subtyping methods and interpretation criteria for cluster inclusion/exclusion. An evaluation of 4 WGS-based subtyping methods [SNVPhyl, coreMLST, core genome MLST (cgMLST) and whole-genome MLST (wgMLST)] was performed on 9 foodborne-, travel- and MSM-related retrospective outbreaks from a collection of 91 Shigella flexneri and 232 Shigella sonnei isolates to determine the methods’ epidemiological concordance, discriminatory power, robustness and ability to generate stable interpretation criteria. The discriminatory powers were ranked as follows: coreMLST<SNVPhyl<cgMLST<wgMLST (range: 0.970–1.000). The genetic differences observed for non-MSM-related Shigella spp. outbreaks respect the standard 0–10 allele/SNV guideline; however, mobile genetic element (MGE)-encoded loci caused inflated genetic variation and discrepant phylogenies for prolonged MSM-related S. sonnei outbreaks via wgMLST. The S. sonnei correlation coefficients of wgMLST were also the lowest at 0.680, 0.703 and 0.712 for SNVPhyl, coreMLST and cgMLST, respectively. Plasmid maintenance, mobilization and conjugation-associated genes were found to be the main source of genetic distance inflation in addition to prophage-related genes. Duplicated alleles arising from the repeated nature of IS elements were also responsible for many false cg/wgMLST differences. The coreMLST approach was shown to be the most robust, followed by SNVPhyl and wgMLST for inter-laboratory comparability. Our results highlight the need for validating species-specific subtyping methods based on microbial genome plasticity and outbreak dynamics in addition to the importance of filtering confounding MGEs for cluster detection.

Source Tracking Based on Core Genome SNV and CRISPR Typing of Salmonella enterica Serovar Heidelberg Isolates Involved in Foodborne Outbreaks in Québec, 2012.

Whole-genome sequencing (WGS) is the method of choice for bacterial subtyping and it is rapidly replacing the more traditional methods such as pulsed-field gel electrophoresis (PFGE). Here we used the high-resolution core genome single nucleotide variant (cgSNV) typing method to characterize clinical and food from Salmonella enterica serovar Heidelberg isolates in the context of source attribution. Additionally, clustered regularly interspaced short palindromic repeats (CRISPR) analysis was included to further support this method. Our results revealed that cgSNV was highly discriminatory and separated the outbreak isolates into distinct clusters (0–4 SNVs). CRISPR analysis was also able to distinguish outbreak strains from epidemiologically unrelated isolates. Specifically, our data clearly demonstrated the strength of these two methods to determine the probable source(s) of a 2012 epidemiologically characterized outbreak of S. Heidelberg. Using molecular cut-off of 0–10 SNVs, the cgSNV analysis of 246 clinical and food isolates of S. Heidelberg collected in Québec, in the same year of the outbreak event, revealed that retail and abattoir chicken isolates likely represent an important source of human infection to S. Heidelberg. Interestingly, the isolates genetically related by cgSNV also harbored the same CRISPR as outbreak isolates and clusters. This indicates that CRISPR profiles can be useful as a complementary approach to determine source attribution in foodborne outbreaks. Use of the genomic analysis also allowed to identify a large number of cases that were missed by PFGE, indicating that most outbreaks are probably underestimated. Although epidemiological information must still support WGS-based results, cgSNV method is a highly discriminatory method for the resolution of outbreak events and the attribution of these events to their respective sources. CRISPR typing can serve as a complimentary tool to this analysis during source tracking.

Molecular epidemiology of Clostridium difficile isolated from piglets

Information on the epidemiology of C. difficile infection (CDI) in South-East Asian countries is limited, as is data on possible animal reservoirs of C. difficile in the region. We investigated the prevalence and molecular epidemiology of C. difficile in piglets and the piggery environment in Thailand and Malaysia. Piglet rectal swabs (n = 224) and piggery environmental specimens (n = 23) were collected between 2015 and 2016 from 11 farms located in Thailand and Malaysia. All specimens were tested for the presence of C. difficile with toxigenic culture. PCR assays were performed on isolates to determine the ribotype (RT), and the presence of toxin genes. Whole genome sequencing was used on a subset of isolates to determine the evolutionary relatedness of RT038 (the most prevalent RT identified) common to pigs and humans from Thailand and Indonesia. C. difficile was recovered from 35% (58/165) and 92% (54/59) of the piglets, and 89% (8/9) and 93% (13/14) of the environmental specimens from Thailand and Malaysia, respectively. All strains from Thailand, and 30 strains from Malaysia (23 piglet and 7 environmental isolates) were non-toxigenic. To our knowledge, this is the first and only report with a complete lack of toxigenic C. difficile among piglets, a feature which could have a protective effect on the host. The most common strain belonged to RT038 (ST48), accounting for 88% (51/58) of piglet and 78% (7/9) of environmental isolates from Thailand, and all 30 isolates tested from Malaysia. Piglet RT038 isolates from Thailand and Malaysia differed by only 18 core-genome single nucleotide variants (cgSNVs) and both were, on average, 30 cgSNVs different from the human strains from Thailand and Indonesia, indicating a common ancestor in the last two decades.

Clostridioides difficile Whole-Genome Sequencing Reveals Limited Within-Host Genetic Diversity in a Pediatric Cohort.

Whole-genome sequencing (WGS) is a highly sensitive method for identifying genetic relatedness and transmission of Clostridioides difficile strains. Previous studies suggest that as few as 3 core genome single-nucleotide variants (SNVs) discriminate between genetically distinct isolates. Because a single C. difficile colony is selected from culture for WGS, significant within-host genetic diversity could preclude identification of transmission events. To evaluate the likelihood of missed transmission events using WGS of single colonies from culture, we examined within-host genetic diversity among C. difficile isolates collected from children. We performed WGS using an Illumina MiSeq instrument on 8 C. difficile colonies randomly selected from each culture performed on stool collected from 10 children (8 children diagnosed with C. difficile infection and 2 children with asymptomatic carriage); 77/80 (96%) isolate sequences were successfully assembled. Among 8/10 (80%) children, all isolates were the same sequence type (ST). The other 2 children each had mixed infection with two STs, although one ST predominated. Among 9/10 (90%) children, isotypic isolates differed by ≤2 SNVs; an isotypic isolate in the remaining child differed by 3 to SNVs relative to the other isolates from that child. Overall, among the 77 isolates collected from 10 stool cultures, 74/77 (96%) were clonal (i.e., same ST and ≤2 core genome SNVs) to other isolates in stool culture. In summary, we identified rare C. difficile within-host genetic diversity in children, suggesting that WGS of a single colony from stool is likely to appropriately characterize isolate clonality and putative transmission events in the majority of cases.

A series of three cases of severe Clostridium difficile infection in Australia associated with a binary toxin producing clade 2 ribotype 251 strain

Three patients with severe Clostridium difficile infection (CDI) caused by an unusual strain of C.difficile, PCR ribotype (RT) 251, were identified in New South Wales, Australia. All cases presented with severe diarrhoea, two had multiple recurrences and one died following a colectomy. C.difficile RT251 strains were isolated by toxigenic culture. Genetic characterisation was performed using techniques including toxin gene profiling, PCR ribotyping, whole genome sequencing (WGS), in-silico multi-locus-sequence-typing (MLST) and core-genome single nucleotide variant (SNV) analyses. Antimicrobial susceptibility was determined using an agar incorporation method. Invitro toxin production was confirmed by Vero cell cytotoxicity assay and pathogenicity was assessed in a murine model of CDI. All RT251 isolates contained toxin A (tcdA), toxin B (tcdB) and binary toxin (cdtA and cdtB) genes. Core-genome analyses revealed the RT251 strains were clonal, with 0-5 SNVs between isolates. WGS and MLST clustered RT251 in the same evolutionary clade (clade 2) as RT027. Despite comparatively lower levels of invitro toxin production, in the murine model RT251 infection resembled RT027 infection. Mice showed marked weight loss, severe disease within 48 h post-infection and death. All isolates were susceptible to metronidazole and vancomycin. Our observations suggest C.difficile RT251 causes severe disease and emphasise the importance of ongoing surveillance for new and emerging strains of C.difficile with enhanced virulence.

Impact of the choice of reference genome on the ability of the core genome SNV methodology to distinguish strains of Salmonella enterica serovar Heidelberg.

Salmonella enterica serovar Heidelberg (S. Heidelberg) is one of the top serovars causing human salmonellosis. The core genome single nucleotide variant pipeline (cgSNV) is one of several whole genome based sequence typing methods used for the laboratory investigation of foodborne pathogens. SNV detection using this method requires a reference genome. The purpose of this study was to investigate the impact of the choice of the reference genome on the cgSNV-informed phylogenetic clustering and inferred isolate relationships. We found that using a draft or closed genome of S. Heidelberg as reference did not impact the ability of the cgSNV methodology to differentiate among 145 S. Heidelberg isolates involved in foodborne outbreaks. We also found that using a distantly related genome such as S. Dublin as choice of reference led to a loss in resolution since some sporadic isolates were found to cluster together with outbreak isolates. In addition, the genetic distances between outbreak isolates as well as between outbreak and sporadic isolates were overall reduced when S. Dublin was used as the reference genome as opposed to S. Heidelberg.

Clostridium difficile Whole Genome Sequencing Reveals Limited Transmission Among Symptomatic Children: A Single-Center Analysis.

Although pediatric Clostridium difficile infections (CDIs) are increasing, C. difficile transmission patterns among children are poorly understood. We performed whole genome sequencing (WGS) on C. difficile isolates collected from children diagnosed with CDI between December 2012 and December 2013 at a single academic medical center. Genome sequences of isolates from CDIs diagnosed ≥8 weeks after study initiation were compared to all study isolate genome sequences. Among patients with isogenic isolates (≤2-3 core genome single nucleotide variants [SNVs] identified by pairwise SNV analyses), common inpatient and/or outpatient healthcare exposures were investigated. Among 131 CDIs in 107 children, WGS identified 104 genetically distinct isolates. Of 84 incident CDIs occurring ≥8 weeks after study initiation, only 10 (11.9%) were caused by a strain isogenic to another cohort CDI isolate (putative transmission events). Proportions of each CDI class putatively associated with transmission were hospital-onset healthcare facility-associated (HCFA), 2/16 (12.5%); community-onset HCFA, 1/17 (5.9%); indeterminate, 1/11 (9.1%); community-associated (CA), 5/40 (12.5%); and recurrent, 1/21 (4.8%). Transmission events among CA and HCFA CDIs were similarly infrequent (5/40 [12.5%] vs 3/33 [9.1%]; P = .64). Shared healthcare facility exposures were only identified among 7/10 putative transmission events. Potential community transmission (same postal code) was not identified. WGS identified a highly diverse group of C. difficile isolates among children with CDI, including those with HCFA CDI. Clostridium difficile transmission among symptomatic children was very uncommon. Among putatively transmitted cases, investigation of shared healthcare exposures often did not identify a potential transmission source.

Comparison of advanced whole genome sequence-based methods to distinguish strains of Salmonella enterica serovar Heidelberg involved in foodborne outbreaks in Québec

Salmonella enterica serovar Heidelberg (S. Heidelberg) is one of the top serovars causing human salmonellosis. This serovar ranks second and third among serovars that cause human infections in Québec and Canada, respectively, and has been associated with severe infections. Traditional typing methods such as PFGE do not display adequate discrimination required to resolve outbreak investigations due to the low level of genetic diversity of isolates belonging to this serovar. This study evaluates the ability of four whole genome sequence (WGS)-based typing methods to differentiate among 145 S. Heidelberg strains involved in four distinct outbreak events and sporadic cases of salmonellosis that occurred in Québec between 2007 and 2016. Isolates from all outbreaks were indistinguishable by PFGE. The core genome single nucleotide variant (SNV), core genome multilocus sequence typing (MLST) and whole genome MLST approaches were highly discriminatory and separated outbreak strains into four distinct phylogenetic clusters that were concordant with the epidemiological data. The clustered regularly interspaced short palindromic repeats (CRISPR) typing method was less discriminatory. However, CRISPR typing may be used as a secondary method to differentiate isolates of S. Heidelberg that are genetically similar but epidemiologically unrelated to outbreak events. WGS-based typing methods provide a highly discriminatory alternative to PFGE for the laboratory investigation of foodborne outbreaks.

Genome Analysis of Clostridium difficile PCR Ribotype 014 Lineage in Australian Pigs and Humans Reveals a Diverse Genetic Repertoire and Signatures of Long-Range Interspecies Transmission.

Clostridium difficile PCR ribotype (RT) 014 is well-established in both human and porcine populations in Australia, raising the possibility that C. difficile infection (CDI) may have a zoonotic or foodborne etiology. Here, whole genome sequencing and high-resolution core genome phylogenetics were performed on a contemporaneous collection of 40 Australian RT014 isolates of human and porcine origin. Phylogenies based on MLST (7 loci, STs 2, 13, and 49) and core orthologous genes (1260 loci) showed clustering of human and porcine strains indicative of very recent shared ancestry. Core genome single nucleotide variant (SNV) analysis found 42% of human strains showed a clonal relationship (separated by ≤2 SNVs in their core genome) with one or more porcine strains, consistent with recent inter-host transmission. Clones were spread over a vast geographic area with 50% of the human cases occurring without recent healthcare exposure. These findings suggest a persistent community reservoir with long-range dissemination, potentially due to agricultural recycling of piggery effluent. We also provide the first pan-genome analysis for this lineage, characterizing its resistome, prophage content, and in silico virulence potential. The RT014 is defined by a large “open” pan-genome (7587 genes) comprising a core genome of 2296 genes (30.3% of the total gene repertoire) and an accessory genome of 5291 genes. Antimicrobial resistance genotypes and phenotypes varied across host populations and ST lineages and were characterized by resistance to tetracycline [tetM, tetA(P), tetB(P) and tetW], clindamycin/erythromycin (ermB), and aminoglycosides (aph3-III-Sat4A-ant6-Ia). Resistance was mediated by clinically important mobile genetic elements, most notably Tn6194 (harboring ermB) and a novel variant of Tn5397 (harboring tetM). Numerous clinically important prophages (Siphoviridae and Myoviridae) were identified as well as an uncommon accessory gene regulator locus (agr3). Conservation in the pathogenicity locus and S-layer correlated with ST affiliation, further extending the concept of clonal C. difficile lineages. This study provides novel insights on the genetic variability and strain relatedness of C. difficile RT014, a lineage of emerging One Health importance. Ongoing molecular and genomic surveillance of strains in humans, animals, food, and the environment is imperative to identify opportunities to reduce the overall CDI burden.