Single Nucleotide Polymorphism Differences Research Articles

Comparison of IncK-blaCMY-2 Plasmids in Extended-Spectrum Cephalosporin-Resistant Escherichia coli Isolated from Poultry and Humans in Denmark, Finland, and Germany

Escherichia coli carrying IncK-blaCMY-2 plasmids mediating resistance to extended-spectrum cephalosporins (ESC) has been frequently described in food-producing animals and in humans. This study aimed to characterize IncK-blaCMY-2-positive ESC-resistant E. coli isolates from poultry production systems in Denmark, Finland, and Germany, as well as from Danish human blood infections, and further compare their plasmids. Whole-genome sequencing (Illumina) of all isolates (n = 46) confirmed the presence of the blaCMY-2 gene. Minimum inhibitory concentration (MIC) testing revealed a resistant phenotype to cefotaxime as well as resistance to ≥3 antibiotic classes. Conjugative transfer of the blaCMY-2 gene confirmed the resistance being on mobile plasmids. Pangenome analysis showed only one-third of the genes being in the core with the remainder being in the large accessory gene pool. Single nucleotide polymorphism (SNP) analysis on sequence type (ST) 429 and 1286 isolates showed between 0–60 and 13–90 SNP differences, respectively, indicating vertical transmission of closely related clones in the poultry production, including among Danish, Finnish, and German ST429 isolates. A comparison of 22 ST429 isolates from this study with 80 ST429 isolates in Enterobase revealed the widespread geographical occurrence of related isolates associated with poultry production. Long-read sequencing of a representative subset of isolates (n = 28) allowed further characterization and comparison of the IncK-blaCMY-2 plasmids with publicly available plasmid sequences. This analysis revealed the presence of highly similar plasmids in ESC-resistant E. coli from Denmark, Finland, and Germany pointing to the existence of common sources. Moreover, the analysis presented evidence of global plasmid transmission and evolution. Lastly, our results indicate that IncK-blaCMY-2 plasmids and their carriers had been circulating in the Danish production chain with an associated risk of spreading to humans, as exemplified by the similarity of the clinical ST429 isolate to poultry isolates. Its persistence may be driven by co-selection since most IncK-blaCMY-2 plasmids harbor resistance factors to drugs used in veterinary medicine.

Genomic Characterization of a Carbapenem-Resistant Raoultella planticola Strain Co-Harboring blaIMP-4 and blaSHV-12 Genes.

Raoultella planticola is an emerging bacterial pathogen responsible for causing infections in both humans and animals. Unfortunately, sporadic reports of carbapenem-resistant R. planticola (CRRP) have been documented worldwide. Here we first reported the complete genome sequence of a CRRP isolate RP_3045 co-carrying blaIMP-4 and blaSHV-12, recovered from a patient in China, and its genetic relatedness to 82 R. planticola strains deposited in the NCBI GenBank database, sourced from humans, animals, and the environment. Whole-genome sequencing was performed using the Illumina NovaSeq 6000 and Oxford Nanopore MinION platforms. Phylogenetic analysis was also performed and visualized using a single nucleotide polymorphism (SNP)-based strategy. The complete genome of R. planticola strain RP_3045 was determined to be 6,312,961 bp in length, comprising five contigs that included one chromosome and four plasmids. RP_3045 was found to be multidrug-resistant and harbored several antimicrobial resistance genes, including both blaIMP-4 and blaSHV-12 genes located on a single plasmid. The most closely related strain was hkcpe63, recovered from humans in Hong Kong, China, in 2014, with 506 SNP differences. R. planticola strains were distributed globally and exhibited strong associations among isolates obtained from different sectors. This study provides evidence for the potential of R. planticola to disseminate carbapenem resistance across different sectors, highlighting the critical need for active and continuous surveillance of CRRP.

Genomic characterization of Listeria monocytogenes and Listeria innocua isolated from milk and dairy samples in Ethiopia

Listeriosis caused by Listeria monocytogenes often poses a significant threat to vulnerable populations. Dairy products have been implicated in outbreaks of listeriosis worldwide. In Ethiopia, studies have identified Listeria spp. and L. monocytogenes in various dairy products, but the genetic diversity and phylogenetic relationships of these bacteria remain largely unknown in the low- and middle-income countries. Therefore, we conducted whole-genome sequencing on 15 L. monocytogenes and 55 L. innocua isolates obtained from different levels of the dairy supply chains across three regions in Ethiopia. Genomes were assembled and used for MLST genotyping and single nucleotide polymorphism (SNP) analysis to infer phylogenetic relationships. We identified a total of 3 L. monocytogenes (i.e., 2, 145, and 18) and 12 L. innocua (i.e., 1489, 1619, 603, 537, 1010, 3186, 492, 3007, 1087, 474, 1008, and 637) MLST sequence types among the studied isolates. Some of these sequence types showed region-specific occurrence, while others were broadly distributed across regions. Through high-quality SNP analysis, we found that among 13 L. monocytogenes identified as ST 2, 11 of them were highly similar with low genetic variation, differing by only 1 to 10 SNPs, suggesting potential selection in the dairy food supply chain. The L. innocua isolates also exhibited low intra-ST genetic variation with only 0–10 SNP differences, except for the ST 1619, which displayed a greater diversity.

Insidious transmission of Mycobacterium tuberculosis in Ordos, China: a molecular epidemiology study.

In this study, we conducted this population-based study to evaluate the genetic diversity and clustering rate of Mycobacterium tuberculosis (MTB) strains using the whole-genome sequencing (WGS), to better understand its transmission in Ordos. All patients with culture-positive TB notified in Ordos from January 2021 to December 2022 were recruited. WGS was performed to analyze single-nucleotide polymorphism (SNP) and to identify genotypic drug susceptibilities of MTB isolates. Overall, a total of 186 patients were included in the present study, of whom 35 (18.8%) had no symptoms suggestive of active TB. Lineage 2 was the predominant MTB sublineage, accounting for 186 of isolates tested. When the pairwise SNP difference ≤ 12 was used as the cutoff for WGS-based clusters, we identified 17 genotypic clusters, and 38 isolates belonged to these 17 clusters, resulting in a clustering rate of 20.4%. The Beijing genotype was an independent factor associating with genomic-clustering (adjusted OR 4.219, 95% CI 0.962-18.502). The overall sensitivity on WGS-based resistance prediction was 85.7% for rifampicin, 73.1% for isoniazid, 60.0% for Ethambutol, 72.7% for streptomycin, and 72.7% for fluoroquinolones. To conclude, the present study demonstrates the extensive recent transmission of Beijing genotype strains in the community of Ordos. The failure to provide a comprehensive pattern of transmission indicated the missed diagnosis of active TB within the community. A substantial proportion of subclinical TB cases are recognized in the bacteria-positive cases, emphasizing that we must interrupt transmission by finding people with active TB before they infect others.

Exploring programmatic indicators of tuberculosis control that incorporate routine Mycobacterium tuberculosis sequencing in low incidence settings: a comprehensive (2017–2021) patient cohort analysis

Routine whole genome sequencing of Mycobacterium tuberculosis has been implemented with increasing frequency. However, its value for tuberculosis (TB) control programs beyond individual case management and enhanced drug resistance detection has not yet been explored. We analysed routine sequencing data of culture-confirmed TB cases notified between 1st January 2017 and 31st December 2021 in New South Wales (NSW), Australia. Genomic surveillance included evidence of local TB transmission, defined by single nucleotide polymorphism (SNP) clustering over a variable (0-25) SNP threshold, and drug resistance conferring mutations. M.tuberculosis sequences from 1831 patients were examined, representing 64.8% of all notified TB cases and 96.2% of culture-confirmed cases. Applying a traditional 5-SNP cluster threshold identified 62 transmission clusters with 183 clustered cases; 101/183 (55.2%) had 0 SNP differences. Cluster assessment over a 5-year period, using a 5-SNP threshold, provided a comprehensive overview of likely recent transmission within NSW, Australia, as an indicator of local TB control. Genotypic drug susceptibility testing (DST) was highly concordant with phenotypic DST and provided a 6.8% increase in antimycobacterial resistance detection. Importantly, it detected mutations missed by routine molecular tests. Lineage 2 strains were more likely to be drug resistant (p<0.0001) and locally transmitted if drug resistant (p<0.0001). Performing routine prospective WGS in a low incidence country like Australia, provides genomically informed programmatic indicators of local TB control. A rolling 5-year cluster assessment reflects epidemic containment and progress towards 'zero TB transmission'. Genomic DST also provides valuable information for clinical care and drug resistance surveillance. NHMRC Centre for Research Excellence in Tuberculosis (www.tbcre.org.au) and NSW Health Prevention Research Support Program.

Whole genome sequencing analysis demonstrates therapy-induced echinocandin resistance in Candida auris isolates.

Candida auris is an emerging, multidrug-resistant yeast, causing outbreaks in healthcare facilities. Echinocandins are the antifungal drugs of choice to treat candidiasis, as they cause few side effects and resistance is rarely found. Previously, immunocompromised patients from Kuwait with C. auris colonisation or infection were treated with echinocandins, and within days to months, resistance was reported in urine isolates. To determine whether the development of echinocandin resistance was due to independent introductions of resistant strains or resulted from intra-patient resistance development, whole genome sequencing (WGS) single-nucleotide polymorphism (SNP) analysis was performed on susceptible (n = 26) and echinocandin-resistant (n = 6) isolates from seven patients. WGS SNP analysis identified three distinct clusters differing 17-127 SNPs from two patients, and the remaining isolates from five patients, respectively. Sequential isolates within patients had a maximum of 11 SNP differences over a time period of 1-10 months. The majority of isolates with reduced susceptibility displayed unique FKS1 substitutions including a novel FKS1M690V substitution, and nearly all were genetically related, ranging from only three to six SNP differences compared to susceptible isolates from the same patient. Resistant isolates from three patients shared the common FKS1S639F substitution; however, WGS analysis did not suggest a common source. These findings strongly indicate that echinocandin resistance is induced during antifungal treatment. Future studies should determine whether such echinocandin-resistant strains are capable of long-term colonisation, cause subsequent breakthrough candidiasis, have a propensity to cross-infect other patients, or remain viable for longer time periods in the hospital environment.

A Novel Insight into the Identification of Potential SNP Markers for the Genomic Characterization of Buffalo Breeds in Pakistan.

Domestic buffaloes (Bubalus bubalis), known as water buffaloes, play a key role as versatile multipurpose agricultural animals in the Asiatic region. Pakistan, with the second-largest buffalo population in the world, holds a rich domestication history of buffaloes. The overall trends in buffalo production demand the genomic characterization of Pakistani buffalo breeds. To this end, the resequencing data of Pakistani breeds, along with buffalo breeds from 13 other countries, were retrieved from our previous study. This dataset, which contained 34,671,886 single-nucleotide polymorphisms (SNPs), was analyzed through a pipeline that was developed to compare possible allele differences among breeds at each SNP position. In contrast, other available tools only check for positional SNP differences for breed-specific markers. In total, 1918, 1549, 404, and 341 breed-specific markers were identified to characterize the Nili, Nili-Ravi, Azakheli, and Kundi breeds of Pakistani buffalo, respectively. Sufficient evidence in the form of phenotypic data, principal component analysis, admixture analysis, and linkage analysis showed that the Nili breed has maintained its distinct breed status despite sharing a close evolutionary relationship with the Nili-Ravi breed of buffalo. In this era of genome science, the conservation of these breeds and the further validation of the given selection markers in larger populations is a pressing need.

Implementation of routine genomic surveillance provided insights into a locally acquired outbreak caused by a rare clade of Salmonella enterica serovar Enteritidis in Queensland, Australia.

Salmonellosis is a significant public health problem globally. In Australia, Salmonella enterica serovar Enteritidis is one of the main causes of salmonellosis. This study reports how the implementation of routine genetic surveillance of isolates from human S. Enteritidis cases enabled identification of the likely source of an outbreak that occurred in a remote town in Far North Queensland, Australia. This study included patient, food and water samples collected during an outbreak investigation. S. Enteritidis of the novel sequence type 5438 was isolated from all seven patient samples and one bore water sample but not any of the food samples. Both whole-genome single nucleotide polymorphism (SNP) and core-genome multilocus sequence typing analysis revealed that S. Enteritidis isolated from outbreak-related patient samples and the bore water isolates clustered together with fewer than five SNP differences and ten allelic differences. This genetic relatedness informed the outbreak response team around public health interventions and no further cases were identified post-treatment of the bore water. This disease cluster was identified through the routine sequencing of S. Enteritidis performed by the state public health laboratory in an actionable time frame. Additionally, genomic surveillance captured a case with unknown epidemiological links to the affected community, ruled out a simultaneous outbreak in an adjacent state as the source and provided evidence for the likely source preventing further transmission. Therefore, this report provides compelling support for the implementation of whole-genome sequencing based genotyping methods in public health microbiology laboratories for better outbreak detection and management.

Whole-genome sequencing of carbapenem-resistant Enterobacterales isolates and evaluation of hospital-acquired infections

Background: Multidrug-resistant organisms (MDROs) are increasingly implicated in nosocomial outbreaks worldwide. We evaluated whole-genome sequencing (WGS) as an adjunctive epidemiological tool to identify infection clusters and MDRO transmission in the healthcare setting. Methods: Clinical isolates of carbapenem-resistant Enterobacterales (CRE) from July 1, 2021, to June 30, 2022, underwent Illumina WGS. Assembled genomes were taxonomically classified with GTDB-Tk software and were typed using multilocus sequence typing (MLST). Average nucleotide identity (ANI) was calculated between genomes. Numbers of differences among core single-nucleotide polymorphisms (SNPs) were calculated for pairs within taxonomic groups, and the data were evaluated in the context of patient dates and locations of care obtained from the electronic medical record. Results: In total, 39 CRE isolates underwent WGS (Fig. 1). Klebsiella pneumoniae represented the largest number of isolates (n = 18). Using MLST, 2 distinct groups of K. pneumoniae were identified (ST307 and ST258) with 5 and 4 isolates, respectively (Fig. 2). Within ST307, SNP differences ranged between 8 and 115. 3 isolates (CRE8, CRE10, and CRE12) were collected within 4 weeks of each other and had ≤26 pairwise SNP differences. Notably, CRE8 and CRE10 were located on the same unit at the same time and used the same MRI scanner on the same day. CRE35 had &gt;95 SNP differences and was admitted 8 months prior to others in ST307 but had surgery in the same OR as CRE8. Within ST258, pairwise comparison of samples revealed 139–588 SNP differences. CRE21, CRE31, and CRE33 had SNP differences of ≤150. These patients were in the same hospital room (CRE33 and CRE21) and unit (CRE31 and CRE33), but they did not overlap temporally. CRE37 had &gt;580 SNP differences, with no overlap in hospitalization dates or locations with other patients. Conclusions: Two closely related K. pneumoniae isolate populations were identified using WGS. Strong temporal and spatial commonalities were identified among isolates with few SNP differences. Isolate pairs with intermediate SNP differences shared spatial commonalities, suggesting possible indirect transmission between patients. No common exposures were identified for pairs with large numbers of SNP differences. WGS is an evolving tool to detect outbreak clonal populations of MDRO not identified through traditional epidemiologic techniques. WGS can provide insight into transmission patterns and the role of environmental contamination in propagating these nosocomial infections. More studies are needed to define the role and clinical significance of isolates with intermediate SNP differences in transmission of these pathogens between hosts and the healthcare environment.Disclosures: None

Whole-genome sequencing cluster analysis reveals complex healthcare-associated COVID-19 dynamics

Background: Identifying and interrupting transmission of severe acute respiratory syndrome coronavirus 2 and resulting disease (COVID-19) in acute-care settings can be challenging due to incubation period, asymptomatic infection, and prevalent community disease. To elucidate routes of infection and interrupt COVID-19 outbreaks with uncertain epidemiological chains of transmission, UPMC utilized reactive whole-genome sequencing (WGS) of viral specimens. Methods: UPMC infection prevention teams identified healthcare-associated COVID-19 clusters with uncertain transmission pathways among patients and/or healthcare personnel (HCP) in acute-care hospitals. Nasopharyngeal samples preserved in viral transport media were obtained for genetic analyses. Nucleic acids were extracted and WGS libraries were prepared by targeted enrichment or multiplex PCR methodologies. Resulting sequencing reads were aligned to the Wuhan-1 reference genome, followed by identification of single-nucleotide polymorphisms (SNPs) among the genomes and construction of a phylogenetic tree. Specimens were considered genetically similar if there were ≤2 SNP differences between viral genomes within a cluster. Results: Between May 2020 until August 2022, infection prevention teams requested WGS for 17 healthcare-associated clusters of COVID-19 involving 182 individuals across 8 UPMC facilities (median outbreak size, 9 individuals; range, 2–26). Of the 182 individuals, 36 lacked clinical specimens and 30 did not pass WGS quality-control criteria of ≥95% of the reference genome with a minimum of 10× coverage. Of the 116 sequenced genomes, 94 (81%) had virus genetically similar to ≥1 other specimen, including 87 (83.6%) of 104 patient viruses and 7 (58.3%) of 12 HCP viruses, comprising 22 clusters (Fig. 1). The remaining 22 (20.6%) specimens were genetically unrelated. In total, 16 (94.1%) of the 17 epidemiologically identified clusters had 2 or more individuals with a genetically similar virus. Also, 7 (41.1%) of these clusters had genetically similar viral genomes for every individual within each cluster. Also, 9 (52.9%) clusters contained both genetically related and unrelated specimens: 5 of these had more complex genomic profiles (including 4 clusters containing 2 distinct subclusters of ≥2 genetically related viruses) and 1 cluster contained 3 subclusters of ≥2 genetically related viruses. In the outbreak with 3 clusters, 3 SNPs separated specimens from 2 temporally proximal clusters, suggesting possible propagation between clusters (cluster B-3 in Fig. 1). Conclusions: WGS can complement traditional epidemiological investigations of healthcare-associated COVID-19 outbreaks, revealing complex transmission dynamics. Future investigations will characterize the impact of WGS on determining specific transmission pathways in acute-care facilities.Disclosures: None

Molecular epidemiological characteristics and genetic evolutionary relationships of methicillin-resistant Staphylococcus aureus of different avian origins in Qingdao, China, using whole-genome sequencing.

To understand the prevalence of avian methicillin-resistant Staphylococcus aureus (MRSA) and the current status of drug resistance in Qingdao, a comprehensive molecular epidemiological investigation and analysis of evolutionary relationships of MRSA isolates from broiler and layer chickens and waterfowl was conducted. One hundred and two avian MRSA strains were identified by multi-locus sequence typing, staphylococcal protein A (spa) and staphylococcal cassette chromosome mec (SCCmec) typing, and whole-genome sequencing. The sequence type (ST) 9-t899-SCCmec IVb type represented the highest proportion of avian-derived MRSA strains (71.57%), with ST398 type strains occasionally observed in broilers and waterfowl. The poultry-derived MRSA strains were all resistant to eight or more antimicrobials. Avian-derived MRSA strains carried 20 resistance genes, 109 virulence genes and 10 plasmids. Strains carrying the cfr oxazolidinone resistance gene were occasionally seen in broiler- and layer-derived MRSA. Single nucleotide polymorphism (SNP) core genome evolution and locus difference analysis showed that the closest strains were all of ST9-t899 type (to which also affiliated the highest number of strains) and this type occurred on all three kinds of poultry farm, but the SNP difference loci between strains of the same type ranged from 0 to 1472. The dominant type of MRSA from different poultry sources in Qingdao is ST9-t899-SCCmec IVb, which is commonly resistant to a variety of antimicrobial drugs and carries a variety of resistance genes and a large number of virulence genes. Sequence type 9-t899 type is widely spread among the three kinds of poultry investigated, but there are differences in affiliations.

Whole-Genome Analysis of Bordetella pertussis MT27 Isolates from School-Associated Outbreaks: Single-Nucleotide Polymorphism Diversity and Threshold of the Outbreak Strains.

Bordetella pertussis, the causative agent of whooping cough, can cause pertussis outbreaks in humans, especially in school-aged children. Here, we performed whole-genome sequencing of 51 B. pertussis isolates (epidemic strain MT27) collected from patients infected during 6 school-associated outbreaks lasting less than 4 months. We compared their genetic diversity with that of 28 sporadic isolates (non-outbreak MT27 isolates) based on single-nucleotide polymorphisms (SNPs). Our temporal SNP diversity analysis revealed a mean SNP accumulation rate (time-weighted average) of 0.21 SNPs/genome/year during the outbreaks. The outbreak isolates showed a mean of 0.74 SNP differences (median, 0; range, 0 to 5) between 238 isolate pairs, whereas the sporadic isolates had a mean of 16.12 SNP differences (median, 17; range 0 to 36) between 378 isolate pairs. A low SNP diversity was observed in the outbreak isolates. Receiver operating characteristic analysis demonstrated that the optimal cutoff value to distinguish between the outbreak and sporadic isolates was 3 SNPs (Youden's index of 0.90 with a true-positive rate of 0.97 and a false-positive rate of 0.07). Based on these results, we propose an epidemiological threshold of ≤3 SNPs per genome as a reliable marker of B. pertussis strain identity during pertussis outbreaks that span less than 4 months. IMPORTANCE Bordetella pertussis is a highly infectious bacterium that easily causes pertussis outbreaks in humans, especially in school-aged children. In detection and investigation of outbreaks, excluding non-outbreak isolates is important for understanding the bacterial transmission routes. Currently, whole-genome sequencing is widely used for outbreak investigations, and the genetic relatedness of outbreak isolates is assessed based on differences in the number of single-nucleotide polymorphisms (SNPs) in the genomes of different isolates. The optimal SNP threshold defining strain identity has been proposed for many bacterial pathogens, but not for B. pertussis. In this study, we performed whole-genome sequencing of 51 B. pertussis outbreak isolates and identified a genetic threshold of ≤3 SNPs per genome as a marker defining the strain identity during pertussis outbreaks. This study provides a useful marker for identifying and analyzing pertussis outbreaks and can serve as a basis for future epidemiological studies on pertussis.

Surveillance of carbapenem-resistant organisms using next-generation sequencing.

The genomic data generated from next-generation sequencing (NGS) provides nucleotide-level resolution of bacterial genomes which is critical for disease surveillance and the implementation of prevention strategies to interrupt the spread of antimicrobial resistance (AMR) bacteria. Infection with AMR bacteria, including Gram-negative Carbapenem-Resistant Organisms (CRO), may be acute and recurrent-once they have colonized a patient, they are notoriously difficult to eradicate. Through phylogenetic tools that assess the single nucleotide polymorphisms (SNPs) within a pathogen genome dataset, public health scientists can estimate the genetic identity between isolates. This information is used as an epidemiologic proxy of a putative outbreak. Pathogens with minimal to no differences in SNPs are likely to be the same strain attributable to a common source or transmission between cases. These genomic comparisons enhance public health response by prompting targeted intervention and infection control measures. This methodology overview demonstrates the utility of phenotypic and molecular assays, antimicrobial susceptibility testing (AST), NGS, publicly available genomics databases, and open-source bioinformatics pipelines for a tiered workflow to detect resistance genes and potential clusters of illness. These methods, when used in combination, facilitate a genomic surveillance workflow for detecting potential AMR bacterial outbreaks to inform epidemiologic investigations. Use of this workflow helps to target and focus epidemiologic resources to the cases with the highest likelihood of being related.

Whole-genome sequencing of Listeria innocua recovered from retail milk and dairy products in Egypt.

The similarity of the Listeria innocua genome with Listeria monocytogenes and their presence in the same niche may facilitate gene transfer between them. A better understanding of the mechanisms responsible for bacterial virulence requires an in-depth knowledge of the genetic characteristics of these bacteria. In this context, draft whole genome sequences were completed on five L. innocua isolated from milk and dairy products in Egypt. The assembled sequences were screened for antimicrobial resistance and virulence genes, plasmid replicons and multilocus sequence types (MLST); phylogenetic analysis of the sequenced isolates was also performed. The sequencing results revealed the presence of only one antimicrobial resistance gene, fosX, in the L. innocua isolates. However, the five isolates carried 13 virulence genes involved in adhesion, invasion, surface protein anchoring, peptidoglycan degradation, intracellular survival, and heat stress; all five lacked the Listeria Pathogenicity Island 1 (LIPI-1) genes. MLST assigned these five isolates into the same sequence type (ST), ST-1085; however, single nucleotide polymorphism (SNP)-based phylogenetic analysis revealed 422-1,091 SNP differences between our isolates and global lineages of L. innocua. The five isolates possessed an ATP-dependent protease (clpL) gene, which mediates heat resistance, on a rep25 type plasmids. Blast analysis of clpL-carrying plasmid contigs showed approximately 99% sequence similarity to the corresponding parts of plasmids of L. monocytogenes strains 2015TE24968 and N1-011A previously isolated from Italy and the United States, respectively. Although this plasmid has been linked to L. monocytogenes that was responsible for a serious outbreak, this is the first report of L. innocua containing clpL-carrying plasmids. Various genetic mechanisms of virulence transfer among Listeria species and other genera could raise the possibility of the evolution of virulent strains of L. innocua. Such strains could challenge processing and preservation protocols and pose health risks from dairy products. Ongoing genomic research is necessary to identify these alarming genetic changes and develop preventive and control measures.

Assessment of NLRP3 Gene Polymorphisms with Periodontitis as Compared with Healthy Periodontium in Iraqi Arabs Patients.

The nod-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome regulates the maturation and release of the cytokines as well as the activation of caspase in response to danger signals derived from pathogenic infection, tissue damage, andmetabolic changes that have a role in the pathogenesis of different diseases as periodontitis. Yet, the susceptibility to this illness could be determined by population-based genetic differences. The aim of this study was to determine whether periodontitis in Arab populations from Iraq is correlated with NLRP3 gene polymorphisms and measure clinical periodontal parameters and investigate their association with genetic polymorphisms of the NLRP3. The study sample consisted of 94 participants ranging from 30 to 55 years old, both males and females who fulfilled the study's criteria. The selected participants were divided into two groups: the periodontitis group (62 subjects) and the healthy control group (32 subjects). The examination of clinical periodontal parameters of all participants was carried out, followed by a collection of venous blood for NLRP3 genetic analysis using the polymerase chain reaction-sequencing technique. The genetic analysis of NLRP3 genotypes at four single nucleotide polymorphisms (SNPs) (rs10925024, rs4612666, rs34777555, and rs10754557), by Hardy-Weinberg equilibrium, identified nonsignificant differences in studied groups. The C-T genotype among periodontitis was significantly different from controls, while the C-C genotype among control was significantly different from periodontitis at NLRP3 rs10925024. Overall, there were 35 SNPs in the periodontitis group and 10 SNPs in the control group for rs10925024 with significant differences versus nonsignificant differences of the other SNPs between the studied groups. Clinical attachment loss and NLRP3 rs10925024 additionally demonstrated a significant positive correlation in the periodontitis subjects. The findings suggested that polymorphisms of the NLRP3 gene may have a role and increasing the genetic susceptibility to periodontal disease in Arabs Iraqi patients.

Presence and Persistence of Listeria monocytogenes in the Danish Ready-to-Eat Food Production Environment

Listeria monocytogenes is an ubiquitously occurring foodborne bacterial pathogen known to contaminate foods during the production processes. To assess the presence and persistence of L. monocytogenes in Danish ready-to-eat (RTE) food production companies in response to a Listeria awareness campaign, the production environment of selected companies were sampled in 2016 and in 2020. Whole genome sequencing (WGS) was performed to characterize the isolates (n = 50, plus 35 isolates obtained from the routine surveillance during 2016–2020), including investigation of the presence of virulence, persistence and resistance genes. The number of companies that tested positive by culture was 17/39 (43.6%) in 2016 and 11/34 (32.4%) in 2020, indicating a limited effect of the campaign. WGS analyses of the 85 isolates showed that the most common sequence types (STs) were ST8 and ST121. The single nucleotide polymorphism (SNP) analysis showed that isolates coming from the same company and belonging to the same ST exhibited &lt;10 SNP differences regardless of the sampling year and whether the samples came from the environment or products, indicating the persistence of the specific STs. Several prevalent STs were found in clinical cases concurrently, including genetically similar isolates. This highlights the issue of persistent L. monocytogenes in the food production environment and the need for improved risk communication and mitigation strategies.

Haplotypes of Dirofilaria repens from Poland and selected countries of Central, North-Eastern Europe and the Middle East: An evaluation on the relation between the genetic diversity and the geographic distribution of the fast-spreading parasite

Subcutaneous dirofilariosis is a fast-spreading infection of dogs, and occasionally of other carnivores and humans. Several factors contribute to its spread, including climate change, which facilitates development and survival of Dirofilaria repens in the mosquito vector. Movement/relocation of infected definitive hosts (dogs) from endemic regions to non-endemic regions is another possible cause of local emergence and the presence of a wide variety of wild reservoirs of the parasite may also contribute to its spread. The main aim of this study was to evaluate the genetic diversity of D. repens from different regions of Europe and to evaluate the spread of identified haplotypes and their geographic origin. A total of 95 D. repens isolates were obtained from Central and Eastern Europe (Poland, Belarus, Ukraine, Austria, Romania), NE Europe (Lithuania, Latvia, Estonia), Italy and Israel. All but two positive samples were obtained from the blood of dogs while one positive sample was obtained from an adult worm from a human case from the Lublin area in SE Poland and one sample was obtained from Anopheles plumbeus mosquito from Austria. Genetic diversity in D. repens isolates was evaluated by PCR amplification and sequencing of three genetic markers, including two mitochondrial genes (mtDNA): the cytochrome c oxidase subunit I (COI) and dehydrogenase subunit I (NADH). Additionally, the genomic marker, internal transcribed spacer 1 (ITS-1) was amplified and sequenced. Haplotypes were differentiated based on sequence alignments by identifying Single Nucleotide Polymorphism (SNPs) using DnaSP and Mega X. PopArt was used to construct a haplotype network including all identified haplotypes. Both mtDNA sequences (COI and NADH) were combined together for phylogenetic and network analyses. Altogether 18 haplotypes (DR1-DR18) were identified in combined mtDNA markers among 95 analysed samples. Haplotype DR1 was the most common encompassing 66 isolates: 42 isolates from Poland (41 from dogs and one from a human), 13 from Lithuania, 4 from Latvia, 2 from Ukraine and 5 from Romania. All other haplotypes grouped around haplotype DR1 separated by 1–5 SNPs, forming a star-like shape. Haplotype DR2 was the second most common haplotype, formed by six isolates from Romania. Interestingly, haplotype DR3 was represented only by four isolates from Israel. The remaining 15 haplotypes were represented by 1–4 isolates of different origins. Our study showed that only minor genetic diversity was found in D. repens since all isolates appear to have clustered in or branched out from haplotype DR1 with 1–5 SNP differences. The genetic diversity appears to be governed by geographic origin since isolates from neighbouring populations (countries) appear to share unique haplotypes while other populations that are geographically distant from individual haplotypes.

1374. Utilizing Genomic Epidemiology to Explore SARS CoV-2 Transmission Patterns and Support Outbreak Investigations in Long Term Care Facilities, Washington State, April-October 2021

Abstract Background In Washington State, COVID-19 cases in long-term care facilities (LTCF) have accounted for less than 3% of all cases, yet 30% of all COVID-19 deaths. Understanding transmission patterns and outbreak epidemiology informs outbreak management. From April to October 2021, two large LTCFs experienced COVID-19 outbreaks. Whole genome sequencing and phylogenetic analysis were leveraged to explore transmission patterns and complement outbreak epidemiology. Methods Epidemiologic data was exported from the Washington Disease Reporting System. Sequences, retrieved from GISAID, were aligned to the Wuhan-1 reference genome using Nextalign version 1.11.0. Pairwise single nucleotide polymorphism (SNP) distance matrices were calculated using SNP-Dists version 0.8.2. Phylogenetic trees for each outbreak were generated using IQ-Tree multicore version 2.2.0-beta COVID-edition using the GTR+F+G4 nucleotide substitution model with 1000 bootstrap replicates. MicrobeTrace was used to visualize the phylogeny, SNP heatmap, and identify clusters among sequences. Results Weekly, LTCF A tested 162 residents and 800 staff, and LTCF B tested 60 residents and 144 staff. Of all cases in LTCF A (n= 119), 23% (n =27) were residents and 77% (n = 92) were staff, compared to 78% (n =28) residents and 22% (n =7) staff among total LTCF B cases (n=36). In LTCF A, 34% (n=40) of the cases had high-quality sequences available. Seven clusters of two or more genetically related sequences and thirteen genetically unrelated sequences were identified. Five of the clusters involved resident and staff cases, linked by unit. Two clusters and remaining unrelated sequences were among staff. In LTCF B, 40% (n=14) of the cases had high-quality sequences available. One cluster of genetically related sequences was identified, all from residents of two floors. The SNP differences between sequences from LTCF A ranged from 0 to 70, whereas SNP differences between LTCF B sequences ranged from 0 to 6. Conclusion Phylogenetic analysis of the two outbreaks confirms differences in disease transmission patterns. Multiple independent introductions of SARS-CoV-2 were identified in LTCF A, compared to a single introduction in LTCF B. Genomic epidemiology is a valuable resource for outbreak investigation and management. Disclosures All Authors: No reported disclosures.

CD33 rs12459419 SNP Regulated the Total CD33 Expression Level

Background: Gemutuzumab ozogamicin (GO) is a molecularly targeted drug which combines a humanized anti-CD33 antibody (p67.6) with calicheamicin. GO has differed therapeutic efficacy in cases of acute myeloid leukemia (AML). Single nucleotide polymorphisms (SNPs) in the CD33 gene have been investigated as one of the reasons for this difference. SNPs (CC, CT, and TT types) in rs12459419 have been reported to be associated with clinical outcomes in pediatric AML patients treated with GO-containing chemotherapy. On the other hand, there are reports of poor association in adult AML patients, and the significance of rs12459419 SNPs in remains to be clarified. CD33 is composed of two extracellular domains, IgV and IgC. p67.6, anti-CD33 antibodies, recognize IgV, while HIM3-4 antibodies recognize IgC. rs12459419 SNP is located in the splicing acceptor region of exon 2 of the CD33 gene, when the SNP is T, transcripts that skip exon 2 which encodes the IgV domain, are more likely to occur. The increasing expression of this exon skipping product may be accompanied by a decreasing amount of wild-type CD33 (CD33FL) and an increasing amount of IgV domain-deficient CD33 (CD33ΔE2). GO only recognizes CD33FL, but not CD33ΔE2. We used CRISPR/Cas9 genome editing system to generate cell lines with specifically modified CD33 SNPs to elucidate the expression of CD33FL and CD33ΔE2 by CD33 SNPs and its effect on sensitivity to GO in a uniform genetic background. Methods and Results: First, we analyzed CD33 SNPs in 23 AML patients treated with GO (CC type: 13; CT type; 9: TT type: 1). The outcome of 17 patients, excluding a history of hematopoietic stem cell transplantation before treated with GO, was evaluated. Three-year overall survival rate for the type of CC vs. CT was 70.7% vs. 83.3% (p value = 0.609), and 3-year disease-free survival rate for the type of CC vs. CT was 80.8% vs. 83.3% (p value = 0.895). There was no significant difference in CC and CT. Next, using p67.6 recognizes only CD33FL and HIM3-4 recognizes both CD33FL and CD33ΔE2, we evaluated the expression ratio of CD33FL and CD33ΔE2 in relation with CD33 SNP. We assumed that the expression levels of MFI(median fluorescence intensity) were roughly similar in HIM3-4, but the MFI evaluated by both antibodies tended to be higher in the CT and CC types than in the TT type (MFI for p67.6 of CC, CT and TT was 2675, 1752 and 245, respectively; MFI for HIM3-4 of CC, CT and TT was 699, 629 and 53, respectively). The patient samples differ in many genetic backgrounds besides CD33 SNPs, making accurate evaluation difficult. Genome editing was performed by co-transfection of guide RNAs targeting CD33 SNPs, Cas9 protein and with single-stranded oligonucleotides to the target leukemia cell line. Single cell cloning was performed by limiting dilution to generate single nucleotide altered cells for the CD33 SNP. OCI-AML3 wild-type (TT type) and the obtained CD33 SNP-modified clones (CC type, 4 clones and CT type, 4 clones) were evaluated by flow cytometry with p67.6 and HIM3-4.The results showed that the MFI of both antibodies was higher in the CT and CC types than in the TT, the wild type (MFI for p67.6 of CC, CT and TT was 10760, 13686 and 4801, respectively; MFI for HIM3-4 of CC, CT and TT was 3115, 4067 and 887, respectively). These results showed similar trends to the clinical samples implying that differences in CD33 SNPs affect the amount of total CD33 expression on the cell surface itself. Conclusion: The CD33 rs12459419 SNP regulated the expression level of total CD33 in clinical samples and gene-edited cell lines. There was no obvious difference in the expression levels of total CD33 and CD33FL at the cell surface between the CC and CT types, but it was higher than that of the TT type. The difference in CD33 expression may influence the therapeutic efficacy of GO.

Large-Scale Evaluation of a Rapid Fully Automated Analysis Platform to Detect and Refute Outbreaks Based on MRSA Genome Comparisons.

Genomic epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) could transform outbreak investigations, but its clinical introduction is hampered by the lack of automated data analysis tools to rapidly and accurately define transmission based on sequence relatedness. We aimed to evaluate a fully automated bioinformatics system for MRSA genome analysis versus a bespoke researcher-led manual informatics pipeline. We analyzed 781 MRSA genomes from 777 consecutive patients identified over a 9-month period in a clinical microbiology laboratory in the United Kingdom. Outputs were bacterial species identification, detection of mec genes, assignment to sequence types (STs), identification of pairwise relatedness using a definition of ≤25 single nucleotide polymorphisms (SNPs) apart, and use of genetic relatedness to identify clusters. There was full concordance between the two analysis methods for species identification, detection of mec genes, and ST assignment. A total of 3,311 isolate pairs ≤25 SNPs apart were identified by at least one method. These had a median (range) SNP difference between the two methods of 1.2 SNPs (0 to 22 SNPs), with most isolate pairs (87%) varying by ≤2 SNPs. This similarity increased when the research pipeline was modified to use a clonal-complex-specific reference (median 0 SNP difference, 91% varying by ≤2 SNPs). Both pipelines clustered 338 isolates/334 patients into 66 unique clusters based on genetic relatedness. We conclude that the automated transmission detection tool worked at least as well as a researcher-led manual analysis and indicates how such tools could support the rapid use of MRSA genomic epidemiology in infection control practice. IMPORTANCE It has been clearly established that genome sequencing of MRSA improves the accuracy of health care-associated outbreak investigations, including the confirmation and exclusion of outbreaks and identification of patients involved. This could lead to more targeted infection control actions but its use in clinical practice is prevented by several barriers, one of which is the availability of genome analysis tools that do not depend on specialist knowledge to analyze or interpret the results. We evaluated a prototype of a fully automated bioinformatics system for MRSA genome analysis versus a bespoke researcher-led manual informatics pipeline, using genomes from 777 patients over a period of 9 months. The performance was at least equivalent to the researcher-led manual genomic analysis. This indicates the feasibility of automated analysis and represents one more step toward the routine use of pathogen sequencing in infection prevention and control practice.