Accessory Genome Research Articles

Geography, Antimicrobial Resistance, and Genomics of Salmonella enterica (Serotypes Newport and Anatum) from Meat in Mexico (2021–2023)

Salmonella enterica non-typhoidal is a major contributor to diarrheal diseases, with over 2600 serovars identified across diverse environments. In Mexico, serovars Newport and Anatum have shown a marked increase, especially in foodborne disease, posing a public health problem. We conducted a cross-sectional study from 2021 to 2023 using active epidemiological surveillance to assess contamination in ground beef and pork at butcher shops nationwide. It involved isolation, phenotypic antimicrobial resistance, comparative genomics, spatial distribution, antimicrobial-resistance genes, and pangenome analysis. A total of 402 non-typhoidal S. enterica strains were isolated, including 59 Newport and 50 Anatum. After curating for redundancy, 45 Newport and 32 Anatum strains remained. We found that 75% of Newport strains exhibited multidrug resistance (MDR), compared to 25% of Anatum strains. Salmonella Newport also showed a broader distribution and stronger antibiotic-resistance capacity, particularly due to genes such as mphA and ramA. Our pangenome analysis showed a predominance of cell maintenance and survival-process genes in the accessory genome of both serotypes. Considering unique genes, Salmonella Anatum and Newport showed a notorious abundance of genes with functions related to replication, recombination, and repair. The substantial rise of Anatum and Newport strains in meat samples for human consumption presents an epidemiological alert, highlighting the critical need for stringent surveillance programs to mitigate human and ecosystem health risks.

Niche-specific evolution and gene exchange of Salmonella in retail pork and chicken

Salmonella exhibits extensive genetic diversity, facilitated by horizontal gene transfer occurring within and between species, playing a pivotal role in this diversification. Nevertheless, most studies focus on clinical and farm animal isolates, and research on the pangenome dynamics of Salmonella isolates from retail stage of the animal food supply chain is limited. Here, we investigated the genomes of 950 Salmonella isolates recovered from retail chicken and pork meats in seven provinces and one municipality of China in 2018. We observed a strong correlation between Salmonella sublineage diversity and the accessory genome with meat type, revealing reduced diversity associated with increased resistance. Importantly, genes associated with antibiotic, biocide, and heavy metal resistance were unevenly distributed in Salmonella from retail chicken and pork. Pork Salmonella isolates showed a higher prevalence of copper and silver resistance genes, while chicken Salmonella isolates displayed a significant predominance of genetic determinants associated with cephalosporin and ciprofloxacin resistance. Moreover, co-occurrence patterns of resistance determinants and their interaction with mobile genetic elements also correlated with meat type. In summary, our findings shed light on how Salmonella achieves their ecological niche success driven by evolution and gene changes in the retail stage of the animal food supply chain.

Elucidating metabolic pathways through genomic analysis in highly heavy metal-resistant Halobacterium salinarum strains

The annotated and predicted genomes of five archaeal strains (AS1, AS2, AS8, AS11 and AS19), isolated from Sfax solar saltern sediments (Tunisia) and affiliated with Halobacterium salinarum, were performed by RAST webserver (Rapid Annotation using Subsystem Technology) and NCBI prokaryotic genome annotation pipeline (PGAP). The results showed the ability of strains to use a reduced semi-phosphorylative Entner-Doudoroff pathway for glucose degradation and an Embden-Meyerhof one for gluconeogenesis. They could use glucose, fructose, glycerol, and acetate as sole source of carbon and energy. ATP synthase, various cytochromes and aerobic respiration proteins were encoded. All strains showed fermentation capability through the arginine deiminase pathway and facultative anaerobic respiration using electron acceptors (Dimethyl sulfoxide and trimethylamine N-oxide). Several biosynthesis pathways for many amino acids were identified. Comparative and pangenome analyses between the strains and the well-studied halophilic archaea Halobacterium NRC-1 highlighted a notable dissimilarity. Besides, the strains shared a core genome of 1973 genes and an accessory genome of 767 genes. 129, 94, 67, 15 and 29 unique genes were detected in the AS1, AS2, AS8, AS11 and AS19 genomes, respectively. Most of these unique genes code for hypothetical proteins. The strains displayed plant-growth promoting characteristics under heavy metal stress (Ammonium assimilation, phosphate solubilization, chemotaxis, cell motility and production of indole acetic acid, siderophore and phenazine). Therefore, they could be used as a biofertilizer to promote plant growth. The genomes encoded numerous biotechnologically relevant genes responsible for vitamin biosynthesis, including cobalamin, folate, biotin, pantothenate, riboflavin, thiamine, menaquinone, nicotinate, and nicotinamide. The carotenogenetic pathway of the studied strains was also predicted. Consequently, the findings of this study contribute to a better understanding of the halophilic archaea metabolism providing valuable insights into their ecophysiology as well as relevant biotechnological applications.

The pangenome analysis of the environmental source Salmonella enterica highlights a diverse accessory genome and a distinct serotype clustering.

Salmonella remains the leading cause of foodborne infections globally. Environmental reservoirs, particularly aquatic bodies, serve as conduits for the fecal-oral transmission of this pathogen. While the gastrointestinal tract is traditionally considered the primary habitat of Salmonella, mounting evidence suggests the bacterium's capacity for survival in external environments. The application of advanced technological platforms, such as next-generation sequencing (NGS), facilitates a comprehensive analysis of Salmonella's genomic features. This study aims to characterize the genomic composition of Salmonella isolates from river water, contributing to a potential paradigm shift and advancing public health protection. A total of 25 river water samples were collected and processed, followed by microbiological isolation of Salmonella strains, which were then sequenced. Genomic characterization revealed adaptive mechanisms, including gene duplication. Furthermore, an open pangenome, predisposed to incorporating foreign genetic material, was identified. Notably, antibiotic resistance genes were found to be part of the core genome, challenging previous reports that placed them in the accessory genome.

Dissecting the genetic features and evolution of Staphylococcus aureus sequence type 88: a global perspective.

Staphylococcus aureus sequence type (ST) 88, encompassing both methicillin-resistant S. aureus (MRSA) and methicillin-sensitive S. aureus (MSSA) phenotypes, is globally prevalent and commonly associated with skin and soft tissue infections. Despite its widespread occurrence, comprehensive genomic studies on this clone remain scarce. In this study, we performed detailed genomic analyses on 130 ST88 isolates derived from severe bloodstream infections alongside 275 publicly available ST88 sequences. Our phylogenetic analysis identified four distinct clades, with evidence suggesting independent evolution and significant clonal expansion of ST88 in China, particularly within clade I, which appeared to have emerged circa 1964. We documented notable interregional, international, and even intercontinental transmission of ST88 isolates. Variability in the distribution of SCCmec and spa types was observed across clades. Our in silico analyses indicated distinct patterns in the distribution of resistance genes, virulence genes, and mobile genetic elements among the clades, with clade I notably harboring the highest prevalence of the intact sraP gene and an independently acquired novel prophage, φST88-1. Conversely, clade IV exhibited deletions within the sasC gene, with certain sub-clades lacking the sdrDE and fnbB genes, underscoring the superior adhesive capabilities of clade I. In vitro experiments confirmed enhanced biofilm formation in clade I isolates, although the levels of hemolysis and cytotoxicity were similar across clades. Pan-genome-wide association study revealed that core SNPs, rather than the accessory genome, are the primary contributors to the diversification of the ST88 clades. These findings enrich our understanding of the genetic foundations underpinning the transmission dynamics and phenotypic diversity of ST88 clones globally.IMPORTANCEUnderstanding the evolution and transmission of Staphylococcus aureus ST88 clones is critically important due to their spread within food, hospital, and community environments, leading to significant health issues. Despite its prevalence, detailed genomic insights into ST88, particularly regarding its diversity and evolutionary dynamics, have been lacking. Our comprehensive genomic analysis of 130 ST88 isolates from severe bloodstream infections, alongside 275 sequences from public databases, significantly advances our understanding of this pathogen. We identified four distinct evolutionary clades, demonstrating the independent evolution and substantial clonal expansion of ST88 in China, as well as its ability to spread across regions and continents. The diversity among the isolates was evident in their unique profiles of SCCmec elements, antibiotic resistance genes, virulence genes, and mobile genetic elements. Our findings underscore the critical role of core genomic variations over accessory elements in driving the diversification of ST88. This enhanced understanding provides new insights that could inform more effective control strategies, crucial for developing interventions to combat the global spread of this formidable pathogen.

Genomic characterization of a bla KPC-2-producing IncM2 plasmid harboring transposon ΔTn6296 in Klebsiella michiganensis.

Klebsiella michiganensis is an emerging hospital-acquired bacterial pathogen, particularly strains harboring plasmid-mediated carbapenemase genes. Here, we recovered and characterized a multidrug-resistant strain, bla KPC-2-producing Klebsiella michiganensis LS81, which was isolated from the abdominal drainage fluid of a clinical patient in China, and further characterized the co-harboring plasmid. K. michiganensis LS81 tested positive for the bla KPC-2 genes by PCR sequencing, with bla KPC-2 located on a plasmid as confirmed by S1 nuclease pulsed-field gel electrophoresis combined with Southern blotting. In the transconjugants, the bla KPC-2 genes were successfully transferred to the recipient strain E. coli EC600. Whole-genome sequencing and bioinformatics analysis confirmed that this strain belongs to sequence type 196 (ST196), with a complete genome comprising a 5,926,662bp circular chromosome and an 81,451bp IncM2 plasmid encoding bla KPC-2 (designated pLS81-KPC). The IncM2 plasmid carried multiple β-lactamase genes such as bla TEM-1B, bla CTX-M-3, and bla KPC-2 inserted in truncated Tn6296 with the distinctive core structure ISKpn27-bla KPC-2-ISKpn6. A comparison with 46 K. michiganensis genomes available in the NCBI database revealed that the closest phylogenetic relative of K. michiganensis LS81 is a clinical isolate from a wound swab in the United Kingdom. Ultimately, the pan-genomic analysis unveiled a substantial accessory genome within the strain, alongside significant genomic plasticity within the K. michiganensis species, emphasizing the necessity for continuous surveillance of this pathogen in clinical environments.

New Insights Into the Molecular Characteristics of Binary Toxin-Producing Clostridioides difficile Strains in China: A Comparative Genomics Study

Abstract Binary toxin (CDT)-producing Clostridioides difficile is associated with severe clinical symptoms and high mortality rates. However, the fundamental genomic factors that underpin the differentiated prevalence of CDT-producing C. difficile strains in China remain to be elucidated. To provide a comprehensive view of the genetic characteristics, this study comprehensively analyzed whole-genome sequences of CDT-producing C. difficile strains from China. The number of unique accessory genes in sequence type (ST)-5 strains, which are predominant in China, increased steadily with the increasing number of genomes sampled, while the accumulation curve for ST-1 strains was shallower. This suggests that ST-5 may have an open pangenome, whereas ST-1 may have a relatively conservative and stable pangenome. The pathogenicity locus (PaLoc) variants of Clades 1, 2 and 5 were found to share a common genetic organization, with the CDT locus (CdtLoc) showing lower variability than PaLoc. PaLoc in Clade 3 exhibits an insertion of transposon Tn6218, which is clade-specific. The flexibility of the C. difficile accessory genome has facilitated the development of divergent lineages characterized by the presence of evolutionarily advantageous genetic traits. This genetic diversification has led to enhanced antimicrobial resistance, increased virulence and improved metabolic capacities, which collectively augment the ability of C. difficile to survive and proliferate.

Evolution of the pheV-tRNA integrated genomic island in Escherichia coli.

Escherichia coli exhibit extensive genetic diversity at the genome level, particularly within their accessory genome. The tRNA integrated genomic islands (GIs), a part of the E. coli accessory genome, play an important role in pathogenicity. However, studies examining the evolution of GIs have been challenging due to their large size, considerable gene content variation and fragmented assembly in draft genomes. Here we examined the evolution of the GI integrated at pheV-tRNA (GI-pheV), with a primary focus on uropathogenic E. coli (UPEC) and the globally disseminated multidrug resistant ST131 clone. We show the gene content of GI-pheV is highly diverse and arranged in a modular configuration, with the P4 integrase encoding gene intP4 the only conserved gene. Despite this diversity, the GI-pheV gene content displayed conserved features among strains from the same pathotype. In ST131, GI-pheV corresponding to the reference strain EC958 (EC958_GI-pheV) was found in ~90% of strains. Phylogenetic analyses suggested that GI-pheV in ST131 has evolved together with the core genome, with the loss/gain of specific modules (or the entire GI) linked to strain specific events. Overall, we show GI-pheV exhibits a dynamic evolutionary pathway, in which modules and genes have evolved through multiple events including insertions, deletions and recombination.

Diverse and specialized metabolic capabilities of microbes in oligotrophic built environments

BackgroundBuilt environments (BEs) are typically considered to be oligotrophic and harsh environments for microbial communities under normal, non-damp conditions. However, the metabolic functions of microbial inhabitants in BEs remain poorly understood. This study aimed to shed light on the functional capabilities of microbes in BEs by analyzing 860 representative metagenome-assembled genomes (rMAGs) reconstructed from 738 samples collected from BEs across the city of Hong Kong and from the skin surfaces of human occupants. The study specifically focused on the metabolic functions of rMAGs that are either phylogenetically novel or prevalent in BEs.ResultsThe diversity and composition of BE microbiomes were primarily shaped by the sample type, with Micrococcus luteus and Cutibacterium acnes being prevalent. The metabolic functions of rMAGs varied significantly based on taxonomy, even at the strain level. A novel strain affiliated with the Candidatus class Xenobia in the Candidatus phylum Eremiobacterota and two novel strains affiliated with the superphylum Patescibacteria exhibited unique functions compared with their close relatives, potentially aiding their survival in BEs and on human skins. The novel strains in the class Xenobia possessed genes for transporting nitrate and nitrite as nitrogen sources and nitrosative stress mitigation induced by nitric oxide during denitrification. The two novel Patescibacteria strains both possessed a broad array of genes for amino acid and trace element transport, while one of them carried genes for carotenoid and ubiquinone biosynthesis. The globally prevalent M. luteus in BEs displayed a large and open pangenome, with high infraspecific genomic diversity contributed by 11 conspecific strains recovered from BEs in a single geographic region. The versatile metabolic functions encoded in the large accessory genomes of M. luteus may contribute to its global ubiquity and specialization in BEs.ConclusionsThis study illustrates that the microbial inhabitants of BEs possess metabolic potentials that enable them to tolerate and counter different biotic and abiotic conditions. Additionally, these microbes can efficiently utilize various limited residual resources from occupant activities, potentially enhancing their survival and persistence within BEs. A better understanding of the metabolic functions of BE microbes will ultimately facilitate the development of strategies to create a healthy indoor microbiome.Bh9ATT9ssZEtNaJHv1wg7GVideo

Prediction of post-PCV13 pneumococcal evolution using invasive disease data enhanced by inverse-invasiveness weighting.

After introducing pneumococcal conjugate vaccines (PCVs), serotype replacement occurred in Streptococcus pneumoniae. Predicting which pneumococcal strains will become common in carriage after vaccination can enhance vaccine design, public health interventions, and understanding of pneumococcal evolution. Invasive pneumococcal isolates were collected during 1998-2018 by the Active Bacterial Core surveillance (ABCs). Carriage data from Massachusetts (MA) and Southwest United States were used to calculate weights. Using pre-vaccine data, serotype-specific inverse-invasiveness weights were defined as the ratio of the proportion of the serotype in carriage to the proportion in invasive data. Genomic data were processed under bioinformatic pipelines to define genetically similar sequence clusters (i.e., strains), and accessory genes (COGs) present in 5-95% of isolates. Weights were applied to adjust observed strain proportions and COG frequencies. The negative frequency-dependent selection (NFDS) model predicted strain proportions by calculating the post-vaccine strain composition in the weighted invasive disease population that would best match pre-vaccine COG frequencies. Inverse-invasiveness weighting increased the correlation of COG frequencies between invasive and carriage data in linear or logit scale for pre-vaccine, post-PCV7, and post-PCV13; and between different epochs in the invasive data. Weighting the invasive data significantly improved the NFDS model's accuracy in predicting strain proportions in the carriage population in the post-PCV13 epoch, with the adjusted R2 increasing from 0.254 before weighting to 0.545 after weighting. The weighting system adjusted invasive disease data to better represent the pneumococcal carriage population, allowing the NFDS mechanism to predict strain proportions in carriage in the post-PCV13 epoch. Our methods enrich the value of genomic sequences from invasive disease surveillance.IMPORTANCEStreptococcus pneumoniae, a common colonizer in the human nasopharynx, can cause invasive diseases including pneumonia, bacteremia, and meningitis mostly in children under 5 years or older adults. The PCV7 was introduced in 2000 in the United States within the pediatric population to prevent disease and reduce deaths, followed by PCV13 in 2010, PCV15 in 2022, and PCV20 in 2023. After the removal of vaccine serotypes, the prevalence of carriage remained stable as the vacated pediatric ecological niche was filled with certain non-vaccine serotypes. Predicting which pneumococcal clones, and which serotypes, will be most successful in colonization after vaccination can enhance vaccine design and public health interventions, while also improving our understanding of pneumococcal evolution. While carriage data, which are collected from the pneumococcal population that is competing to colonize and transmit, are most directly relevant to evolutionary studies, invasive disease data are often more plentiful. Previously, evolutionary models based on negative frequency-dependent selection (NFDS) on the accessory genome were shown to predict which non-vaccine strains and serotypes were most successful in colonization following the introduction of PCV7. Here, we show that an inverse-invasiveness weighting system applied to invasive disease surveillance data allows the NFDS model to predict strain proportions in the projected carriage population in the post-PCV13/pre-PCV15 and pre-PCV20 epoch. The significance of our research lies in using a sample of invasive disease surveillance data to extend the use of NFDS as an evolutionary mechanism to predict post-PCV13 population dynamics. This has shown that we can correct for biased sampling that arises from differences in virulence and can enrich the value of genomic data from disease surveillance and advance our understanding of how NFDS impacts carriage population dynamics after both PCV7 and PCV13 vaccination.

Desiccation tolerance and reduced antibiotic resistance: Key drivers in ST239-III to ST22-IV MRSA clonal replacement at a Malaysian teaching hospital

Molecular surveillance of methicillin-resistant Staphylococcus aureus (MRSA) isolated from Hospital Canselor Tuanku Muhriz (HCTM), a Malaysian teaching hospital revealed clonal replacement events of SCCmec type III-SCCmercury to SCCmec type IV strains before the year 2017; however, the reasons behind this phenomenon are still unclear. This study aimed to identify factors associated with the clonal replacement using genomic sequencing and phenotypic investigations (antibiogram profiling, growth rate and desiccation tolerance determination, survival in vancomycin sub-minimum inhibitory concentration (MIC) determination) of representative HCTM MRSA strains isolated in four-year intervals from 2005 – 2017 (n = 16). HCTM Antimicrobial Stewardship (AMS) and Infection Prevention and Control (IPC) policies were also reviewed. Phylogenetic analyses revealed the presence of 3 major MRSA lineages: ST239-III, ST22-IV and ST6-IV; MRSAs with the same STs shared similar core and accessory genomes. Majority of the ST239-III strains isolated in earlier years of the surveillance (2005, 2009 and 2013) were resistant to many antibiotics and harboured multiple AMR and virulence genes compared to ST22-IV and ST6-IV strains (isolated in 2013 and 2017). Interestingly, ST22-IV and ST6-IV MRSAs grew significantly faster and were more resistant to desiccation than ST239-III (p < 0.05), even though the later clone survived better post-vancomycin exposure. Intriguingly, ST22-IV was outcompeted by ST239-III in broth co-cultures; though it survived better when desiccated together with ST239-III. Higher desiccation tolerance and fewer carriage of AMR genes by ST22-IV, together with reduction of antibiotic selection pressure in HCTM (due to AMS and IPC policies) during 2005 – 2017 may have provided the clone a competitive edge in replacing the previously dominant ST239-III in HCTM. This study highlights the importance of MRSA surveillance for a clearer picture of circulating clones and clonal changes. To our knowledge, this is the first genomic epidemiology study of MRSA in Malaysia, which will serve as baseline genomic data for future surveillance.

Pangenome – its Aspect and Prospect

Pangenome is a very new discipline showing a collection of unique and variable genomes of any species in one model. This discipline is a combination of three subjects of Biology like Computer Science and Applied Mathematics. All genes from all species or strains are called Pangenomes. Pangenome was first discovered during the preparation of protein-based vaccine of the G group of Streptococcus sp. which is the main cause of neonatal life-threatening disease by a team of scientists led by Tettelin. Conventional method of Vaccine preparation was not successful due to the existence of five different serotypes in this organism. Then Tettelin with groups of scientists tried to use the genome sequence information for preparing protein- based vaccine in G group of Streptococcus. During their studies, two gap free genomes were found in this G group of bacteria. After sequencing studies of many strains, they found several regions of diversity (genomic diversity) among different isolates of same strain. After sequencing a large number of genomes, they realized that genes belong to isolates present in the species but not present in genomes. Then the Pangenome concept came to their mind and genes were classified into Core genome, Accessory or Dispensable genome and Species-specific genomes. Bacteria genomes have a distinct number of genes but there are differences in the genomes of different strains of a single species that is mainly due to horizontal gene transfer of genes from other bacteria. The Pangenome concept and its discovery was discussed. Plant genomes are dynamic with whole genome duplication, tandem duplication, transposable element duplication, chromosomal rearrangements or structural variations like deletion, inversion, translocation and recombination between species. Several methods have already been tried for the improvement of crop plants like Marker Assisted Selection (MAS), QTL mapping, Single Nucleotide Polymorphisms (SNP), Copy number variations and Genome Wide Association Studies (GWAS) by comparing with Reference Genome Assemblies representing the genome sequence of the genome of an individual or organism. But the Reference genome is not able to locate all genetic variations of a species. To overcome this limitation the concept of Pangenomes may help in the crop improvement which has been discussed. Pangenomic studies have already been done in many crop plants like Rice, Wheat, Maize, Brassica, Soybean etc. Some of all these aspects have been discussed.

Pangenomic landscapes shape performances of a synthetic genetic circuit across Stutzerimonas species.

Engineering identical genetic circuits into different species typically results in large differences in performance due to the unique cellular environmental context of each host, a phenomenon known as the "chassis-effect" or "context-dependency". A better understanding of how genomic and physiological contexts underpin the chassis-effect will improve biodesign strategies across diverse microorganisms. Here, we combined a pangenomic-based gene expression analysis with quantitative measurements of performance from an engineered genetic inverter device to uncover how genome structure and function relate to the observed chassis-effect across six closely related Stutzerimonas hosts. Our results reveal that genome architecture underpins divergent responses between our chosen non-model bacterial hosts to the engineered device. Specifically, differential expression of the core genome, gene clusters shared between all hosts, was found to be the main source of significant concordance to the observed differential genetic device performance, whereas specialty genes from respective accessory genomes were not significant. A data-driven investigation revealed that genes involved in denitrification and components of trans-membrane transporter proteins were among the most differentially expressed gene clusters between hosts in response to the genetic device. Our results show that the chassis-effect can be traced along differences among the most conserved genome-encoded functions and that these differences create a unique biodesign space among closely related species.IMPORTANCEContemporary synthetic biology endeavors often default to a handful of model organisms to host their engineered systems. Model organisms such as Escherichia coli serve as attractive hosts due to their tractability but do not necessarily provide the ideal environment to optimize performance. As more novel microbes are domesticated for use as biotechnology platforms, synthetic biologists are urged to explore the chassis-design space to optimize their systems and deliver on the promises of synthetic biology. The consequences of the chassis-effect will therefore only become more relevant as the field of biodesign grows. In our work, we demonstrate that the performance of a genetic device is highly dependent on the host environment it operates within, promoting the notion that the chassis can be considered a design variable to tune circuit function. Importantly, our results unveil that the chassis-effect can be traced along similarities in genome architecture, specifically the shared core genome. Our study advocates for the exploration of the chassis-design space and is a step forward to empowering synthetic biologists with knowledge for more efficient exploration of the chassis-design space to enable the next generation of broad-host-range synthetic biology.

Pangenome analysis of Clostridium scindens : a collection of diverse bile acid and steroid metabolizing commensal gut bacterial strains.

Clostridium scindens is a commensal gut bacterium capable of forming the secondary bile acids deoxycholic acid and lithocholic acid from the primary bile acids cholic acid and chenodeoxycholic acid, respectively, as well as converting glucocorticoids to androgens. Historically, only two strains, C. scindens ATCC 35704 and C. scindens VPI 12708, have been characterized in vitro and in vivo to any significant extent. The formation of secondary bile acids is important in maintaining normal gastrointestinal function, in regulating the structure of the gut microbiome, in the etiology of such diseases such as cancers of the GI tract, and in the prevention of Clostridium difficile infection. We therefore wanted to determine the pangenome of 34 cultured strains of C. scindens and a set of 200 metagenome-assembled genomes (MAGs) to understand the variability among strains. The results indicate that the 34 strains of C. scindens have an open pangenome with 12,720 orthologous gene groups, and a core genome with 1,630 gene families, in addition to 7,051 and 4,039 gene families in the accessory and unique (i.e., strain-exclusive) genomes, respectively. The core genome contains 39% of the proteins with predicted metabolic function, and, in the unique genome, the function of storage and processing of information prevails, with 34% of the proteins being in that category. The pangenome profile including the MAGs also proved to be open. The presence of bile acid inducible ( bai ) and steroid-17,20-desmolase ( des ) genes was identified among groups of strains. The analysis reveals that C. scindens strains are distributed into two clades, indicating the possible onset of C. scindens separation into two species, confirmed by gene content, phylogenomic, and average nucleotide identity (ANI) analyses. This study provides insight into the structure and function of the C. scindens pangenome, offering a genetic foundation of significance for many aspects of research on the intestinal microbiota and bile acid metabolism.

Genomic Analyses of Methicillin-Susceptible and Methicillin-Resistant Staphylococcus pseudintermedius Strains Involved in Canine Infections: A Comprehensive Genotypic Characterization.

Staphylococcus pseudintermedius is frequently associated with several bacterial infections in dogs, highlighting a One Health concern due to the zoonotic potential. Given the clinical significance of this pathogen, we performed comprehensive genomic analyses of 28 S. pseudintermedius strains isolated from canine infections throughout whole-genome sequencing using Illumina HiSeq, and compared the genetic features between S. pseudintermedius methicillin-resistant (MRSP) and methicillin-susceptible (MSSP) strains. Our analyses determined that MRSP genomes are larger than MSSP strains, with significant changes in antimicrobial resistance genes and virulent markers, suggesting differences in the pathogenicity of MRSP and MSSP strains. In addition, the pangenome analysis of S. pseudintermedius from canine and human origins identified core and accessory genomes with 1847 and 3037 genes, respectively, which indicates that most of the S. pseudintermedius genome is highly variable. Furthermore, phylogenomic analysis clearly separated MRSP from MSSP strains, despite their infection sites, showing phylogenetic differences according to methicillin susceptibility. Altogether our findings underscore the importance of studying the evolutionary dynamics of S. pseudintermedius, which is crucial for the development of effective prevention and control strategies of resistant S. pseudintermedius infections.

Genomic epidemiology and longitudinal sampling of ward wastewater environments and patients reveals complexity of the transmission dynamics of bla KPC-carbapenemase-producing Enterobacterales in a hospital setting.

Healthcare-associated wastewater and asymptomatic patient reservoirs colonized by carbapenemase-producing Enterobacterales (CPE) contribute to nosocomial CPE dissemination, but the characteristics and dynamics of this remain unclear. We systematically sampled wastewater sites (n = 4488 samples; 349 sites) and patients (n = 1247) across six wards over 6-12 months to understand blaKPC-associated CPE (KPC-E) diversity within these reservoirs and transmission in a healthcare setting. Up to five KPC-E-positive isolates per sample were sequenced (Illumina). Recombination-adjusted phylogenies were used to define genetically related strains; assembly and mapping-based approaches were used to characterize antimicrobial resistance genes, insertion sequences (ISs) and Tn4401 types/target site sequences. The accessory genome was evaluated in some of the largest clusters, and those crossing reservoirs. Wastewater site KPC-E-positivity was substantial [101/349 sites (28.9%); 228/5601 (4.1%) patients cultured]. Thirteen KPC-E species and 109 strains were identified using genomics, and 24% of wastewater and 26% of patient KPC-E-positive samples harboured one or more strains. Most diversity was explained by the individual niche, suggesting localized factors are important in selection and spread. Tn4401 + flanking target site sequence diversity was greater in wastewater sites (P < 0.001), which might favour Tn4401-associated transposition/evolution. Shower/bath- and sluice/mop-associated sites were more likely to be KPC-E-positive (adjusted OR = 2.69; 95% CI: 1.44-5.01; P = 0.0019; and adjusted OR = 2.60; 95% CI: 1.04-6.52; P = 0.0410, respectively). Different strains had different blaKPC dissemination dynamics. We identified substantial and diverse KPC-E colonization of wastewater sites and patients in this hospital setting. Reservoir and niche-specific factors (e.g. microbial interactions, selection pressures), and different strains and mobile genetic elements likely affect transmission dynamics. This should be considered in surveillance and control strategies.

CC180 clade dynamics does not universally explain Streptococcus pneumoniae serotype 3 persistence post-vaccine: a global comparative population genomics study.

Background Clonal complex 180 (CC180) is currently the major clone of serotype 3 Streptococcus pneumoniae (Spn). The 13-valent pneumococcal conjugate vaccine (PCV13) does not have significant efficacy against serotype 3 despite polysaccharide inclusion in the vaccine. It was hypothesized that PCV13 may effectively control Clade I of CC180 but that Clades III and IV are resistant, provoking a population shift that enables serotype 3 persistence. This has been observed in the United States, England, and Wales but not Spain. We tested this hypothesis further utilizing a dataset from Portugal. Methods We whole-genome sequenced (WGS) 501 serotype 3 strains from Portugal isolated from patients with pneumococcal infections between 1999-2020. The draft genomes underwent phylogenetic analyses, pangenome profiling, and a genome-wide association study (GWAS). We also completed antibiotic susceptibility testing and compiled over 2,600 serotype 3 multilocus sequence type 180 (MLST180) WGSs to perform global comparative genomics. Findings CC180 Clades I, II, III, IV, and VI distributions were similar when comparing non-invasive pneumonia isolates and invasive disease isolates (Fisher's exact test, P=0.29), and adult and pediatric cases (Fisher's exact test, P=0.074). The serotype 3 CCs shifted post-PCV13 (Fisher's exact test, P&lt;0.0001) and Clade I became dominant. Clade I is largely antibiotic-sensitive and carries the phiOXC141 prophage but the pangenome is heterogenous. Strains from Portugal and Spain, where Clade I remains dominant post-PCV13, have larger pangenomes and are associated with the presence of two genes encoding hypothetical proteins. Interpretation Clade I became dominant in Portugal post-PCV13, despite the burden of the prophage and antibiotic sensitivity. The accessory genome content may mitigate these fitness costs. Regional differences in Clade I prevalence and pangenome heterogeneity suggest that clade dynamics is not a generalizable approach to understanding serotype 3 vaccine escape. Funding National Institute of Child Health and Human Development, Pfizer, and Merck Sharp &amp; Dohme.

Comparison of gene-by-gene and genome-wide short nucleotide sequence-based approaches to define the global population structure of Streptococcus pneumoniae.

Defining the population structure of a pathogen is a key part of epidemiology, as genomically related isolates are likely to share key clinical features such as antimicrobial resistance profiles and invasiveness. Multiple different methods are currently used to cluster together closely related genomes, potentially leading to inconsistency between studies. Here, we use a global dataset of 26 306 Streptococcus pneumoniae genomes to compare four clustering methods: gene-by-gene seven-locus MLST, core genome MLST (cgMLST)-based hierarchical clustering (HierCC) assignments, life identification number (LIN) barcoding and k-mer-based PopPUNK clustering (known as GPSCs in this species). We compare the clustering results with phylogenetic and pan-genome analyses to assess their relationship with genome diversity and evolution, as we would expect a good clustering method to form a single monophyletic cluster that has high within-cluster similarity of genomic content. We show that the four methods are generally able to accurately reflect the population structure based on these metrics and that the methods were broadly consistent with each other. We investigated further to study the discrepancies in clusters. The greatest concordance was seen between LIN barcoding and HierCC (adjusted mutual information score=0.950), which was expected given that both methods utilize cgMLST, but have different methods for defining an individual cluster and different core genome schema. However, the existence of differences between the two methods shows that the selection of a core genome schema can introduce inconsistencies between studies. GPSC and HierCC assignments were also highly concordant (AMI=0.946), showing that k-mer-based methods which use the whole genome and do not require the careful selection of a core genome schema are just as effective at representing the population structure. Additionally, where there were differences in clustering between these methods, this could be explained by differences in the accessory genome that were not identified in cgMLST. We conclude that for S. pneumoniae, standardized and stable nomenclature is important as the number of genomes available expands. Furthermore, the research community should transition away from seven-locus MLST, whilst cgMLST, GPSC and LIN assignments should be used more widely. However, to allow for easy comparison between studies and to make previous literature relevant, the reporting of multiple clustering names should be standardized within the research.

Analysis of the Borreliaceae Pangenome Reveals a Distinct Genomic Architecture Conserved Across Phylogenetic Scales.

The family Borreliaceae contains arthropod-borne spirochetes that cause two widespread human diseases, Lyme disease and relapsing fever. Lyme disease is a subacute, progressive illness with variable stage and tissue manifestations. Relapsing fever is an acute febrile illness with prominent bacteremia that may recur and disseminate, particularly to the nervous system. Clinical heterogeneity is a hallmark of both diseases. While human clinical manifestations are influenced by a wide variety of factors, including immune status and host genetic susceptibility, there is evidence that Borreliaceae microbial factors influence the clinical manifestations of human disease caused by this family of spirochetes. Despite these associations, the spirochete genes that influence the severity and manifestations of human disease are, for the most part, unknown. Recent work has identified lineage-specific expansions of lipoproteome-rich accessory genome elements in virulent clones of Borrelia burgdorferi. Using publicly available genome assemblies, it is shown that all Borreliaceae lineages for which sufficient sequence data are available harbor a similar pattern of strongly structured, lineage-specific expansions in their accessory genomes, particularly among lipoproteins, and that this pattern holds across phylogenetic scales including genera, species, and genotypes. The relationships among pangenome elements suggest that infrequent episodes of marked genomic change followed by clonal expansion in geographically and enzootically structured populations may account for the unique lineage structure of Borreliaceae. This analysis informs future genotype-phenotype studies among Borreliaceae and lays a foundation for studies of individual gene function guided by phylogenetic patterns of conservation, diversification, gain, and/or loss.

Comparative Genomic Analysis of Campylobacter rectus and Closely Related Species.

Campylobacter rectus is a gram-negative, anaerobic bacterium strongly associated with periodontitis. It also causes various extraoral infections and is linked to adverse pregnancy outcomes in humans and murine models. C. rectus and related oral Campylobacters have been termed "emerging Campylobacter species" because infections by these organisms are likely underreported. Previously, no comparative methods have been used to analyze more than single C. rectus strains and until recently, very few C. rectus genomes have been publicly available. More sequenced genomes and comparative analyses are needed to study the genomic features and pathogenicity of this species. We sequenced eight new C. rectus strains and used comparative methods to identify regions of interest. An emphasis was put on the type III flagellar secretion system (T3SS), type IV secretion system (T4SS), and type VI secretion system (T6SS) because these protein complexes are important for pathogenesis in other Campylobacter species. RAST, BV-BRC, and other bioinformatics tools were used to assemble, annotate, and compare these regions in the genomes. The pan-genome of C. rectus consists of 2670 genes with core and accessory genomes of 1429 and 1241 genes, respectively. All isolates analyzed in this study have T3SS and T6SS hallmark proteins, while five of the isolates are missing a T4SS system. Twenty-one prophage clusters were identified across the panel of isolates, including four that appear intact. Overall, significant genomic islands were found, suggesting regions in the genomes that underwent horizontal gene transfer. Additionally, the high frequency of CRISPR arrays and other repetitive elements has led to genome rearrangements across the strains, including in areas adjacent to secretion system gene clusters. This study describes the substantial diversity present among C. rectus isolates and highlights tools/assays that have been developed to permit functional genomic studies. Additionally, we have expanded the studies on C. showae T4SS since we have two new C. showae genomes to report. We also demonstrate that unlike C. rectus , C showae does not demonstrate evidence of intact T6SS except for the strain CAM. The only strain of sequenced C. massilensis has neither T4SS or T6SS.