Genomic analysis of a cAmpC (CMY-41)-producing Citrobacter freundii ST64 isolated from patient.

Antibiotic resistance in Citrobacter freundii is a public health concern. This study evaluated the closed genome of a C. freundii isolated from the stool of a hospitalized patient initially related to a Salmonella outbreak. Confirmation of the isolate was determined by whole-genome sequencing. Nanopore sequencing was performed using a MinION with a Flongle flow cell. Assembly using SPAdes and Unicycler yielded a closed genome annotated by National Center for Biotechnology Information Prokaryotic Genome Annotation Pipeline. Genomic analyses employed MLST 2.0, ResFinder4.1, PlasmidFinder2.1, and VFanalyzer. Phylogenetic comparison utilized the Center for Food Safety and Applied Nutrition (CFSAN)-single nucleotide polymorphism pipeline and Genetic Algorithm for Rapid Likelihood Inference. Antimicrobial susceptibility was tested by broth microdilution following Clinical and Laboratory Standards Institute criteria. Multi-locus sequence type in silico analysis assigned the C. freundii as sequence type 64 and the blaCMY-41 gene was detected in resistome investigation. The susceptibility to antibiotics, determined using Sensititre® plates, revealed resistance to aztreonam, colistin, cefoxitin, amoxicillin/clavulanic acid, sulfisoxazole, ampicillin, and streptomycin. The genetic relatedness of the C. freundii CFSAN077772 with publicly available C. freundii genomes revealed a close relationship to a C. freundii SRR1186659, isolated in 2009 from human stool in Tanzania. In addition, C. freundii CFSAN077772 is nested in the same cluster with C. freundii clinical strains isolated in Denmark, Mexico, Myanmar, and Canada, suggesting a successful intercontinental spread.

Closed genome sequence of Clostridium botulinum type B1 strain isolated from an infant botulism case in the United States.

We present the closed genome sequence of the Clostridium botulinum BT-22100019 strain isolated from the stool specimen of an infant diagnosed with botulism. With 4.33-Mb genome size and 28.0% G + C content, the bont/B1 gene encoded for botulinum neurotoxin serotype B was found on a 262 kb plasmid arranged in a ha+ orfx - cluster.

Closed genomes of four multidrug resistance Salmonella enterica serotype Infantis isolated in Costa Rica.

Here, we report the complete genome of four S. enterica Infantis isolated in Costa Rica from human, poultry rinse, and raw chicken meat from 2017 to 2019. All genomes belonged to ST32 and carried a 310-kb plasmid with many antimicrobial resistance genes including the bla CTX-M65 gene.

Identification and Characterization of ten Escherichia coli Strains Encoding Novel Shiga Toxin 2 Subtypes, Stx2n as Well as Stx2j, Stx2m, and Stx2o, in the United States.

The sharing of genome sequences in online data repositories allows for large scale analyses of specific genes or gene families. This can result in the detection of novel gene subtypes as well as the development of improved detection methods. Here, we used publicly available WGS data to detect a novel Stx subtype, Stx2n in two clinical E. coli strains isolated in the USA. During this process, additional Stx2 subtypes were detected; six Stx2j, one Stx2m strain, and one Stx2o, were all analyzed for variability from the originally described subtypes. Complete genome sequences were assembled from short- or long-read sequencing and analyzed for serotype, and ST types. The WGS data from Stx2n- and Stx2o-producing STEC strains were further analyzed for virulence genes pro-phage analysis and phage insertion sites. Nucleotide and amino acid maximum parsimony trees showed expected clustering of the previously described subtypes and a clear separation of the novel Stx2n subtype. WGS data were used to design OMNI PCR primers for the detection of all known stx1 (283 bp amplicon), stx2 (400 bp amplicon), intimin encoded by eae (221 bp amplicon), and stx2f (438 bp amplicon) subtypes. These primers were tested in three different laboratories, using standard reference strains. An analysis of the complete genome sequence showed variability in serogroup, virulence genes, and ST type, and Stx2 pro-phages showed variability in size, gene composition, and phage insertion sites. The strains with Stx2j, Stx2m, Stx2n, and Stx2o showed toxicity to Vero cells. Stx2j carrying strain, 2012C-4221, was induced when grown with sub-inhibitory concentrations of ciprofloxacin, and toxicity was detected. Taken together, these data highlight the need to reinforce genomic surveillance to identify the emergence of potential new Stx2 or Stx1 variants. The importance of this surveillance has a paramount impact on public health. Per our description in this study, we suggest that 2017C-4317 be designated as the Stx2n type-strain.

Precision metagenomics sequencing for food safety: hybrid assembly of Shiga toxin-producing Escherichia coli in enriched agricultural water.

Culture-independent metagenomic sequencing of enriched agricultural water could expedite the detection and virulotyping of Shiga toxin-producing Escherichia coli (STEC). We previously determined the limits of a complete, closed metagenome-assembled genome (MAG) assembly and of a complete, fragmented MAG assembly for O157:H7 in enriched agricultural water using long reads (Oxford Nanopore Technologies, Oxford), which were 107 and 105 CFU/ml, respectively. However, the nanopore assemblies did not have enough accuracy to be used in Single Nucleotide Polymorphism (SNP) phylogenies and cannot be used for the precise identification of an outbreak STEC strain. The present study aimed to determine the limits of detection and assembly for STECs in enriched agricultural water by Illumina MiSeq sequencing technology alone, followed by establishing the limit of hybrid assembly with nanopore long-read sequencing using three different hybrid assemblers (SPAdes, Unicycler, and OPERA-MS). We also aimed to generate a genome with enough accuracy to be used in a SNP phylogeny. The classification of MiSeq and nanopore sequencing identified the same highly abundant species. Using the totality of the MiSeq output and a precision metagenomics approach in which the E. coli reads are binned before assembly, the limit of detection and assembly of STECs by MiSeq were determined to be 105 and 107 CFU/ml, respectively. While a complete, closed MAG could not be generated at any concentration, a complete, fragmented MAG was produced using the SPAdes assembler with an STEC concentration of at least 107 CFU/ml. At this concentration, hybrid assembled contigs aligned to the nanopore-assembled genome could be accurately placed in a neighbor-joining tree. The MiSeq limit of detection and assembly was less sensitive than nanopore sequencing, which was likely due to factors including the small starting material (50 vs. 1 µg) and the dilution of the library loaded on the cartridge. This pilot study demonstrates that MiSeq sequencing requires higher coverage in precision metagenomic samples; however, with sufficient concentration, STECs can be characterized and phylogeny can be accurately determined.

Whole-genome sequencing reveals changes in genomic diversity and distinctive repertoires of T3SS and T6SS effector candidates in Chilean clinical Campylobacter strains.

Campylobacter is the leading cause of bacterial gastroenteritis worldwide and an emerging and neglected pathogen in South America. This zoonotic pathogen colonizes the gastrointestinal tract of a wide range of mammals and birds, with poultry as the most important reservoir for human infections. Apart from its high morbidity rates, the emergence of resistant strains is of global concern. The aims of this work were to determine genetic diversity, presence of antimicrobial resistance determinants and virulence potential of Campylobacter spp. isolated from patients with acute gastrointestinal disease at 'Clinica Alemana', Santiago de Chile. The study considered the isolation of Campylobacter spp., from stool samples during a 20-month period (January 2020 to September 2021). We sequenced (NextSeq, Illumina) and performed an in-depth analysis of the genome sequences of 88 Campylobacter jejuni and 2 Campylobacter coli strains isolated from clinical samples in Chile. We identified a high genetic diversity among C. jejuni strains and the emergence of prevalent clonal complexes, which were not identified in our previous reports. While ~40% of strains harbored a mutation in the gyrA gene associated with fluoroquinolone resistance, no macrolide-resistance determinants were detected. Interestingly, gene clusters encoding virulence factors such as the T6SS or genes associated with long-term sequelae such as Guillain-Barré syndrome showed lineage-relatedness. In addition, our analysis revealed a high degree of variability regarding the presence of fT3SS and T6SS effector proteins in comparison to type strains 81-176, F38011, and NCTC 11168 and 488. Our study provides important insights into the molecular epidemiology of this emerging foodborne pathogen. In addition, the differences observed regarding the repertoire of fT3SS and T6SS effector proteins could have an impact on the pathogenic potential and transmissibility of these Latin American isolates, posing another challenge in characterizing the infection dynamics of this emergent and neglected bacterial pathogen.

The composition of environmental microbiota in three tree fruit packing facilities changed over seasons and contained taxa indicative of L. monocytogenes contamination.

BACKGROUND: Listeria monocytogenes can survive in cold and wet environments, such as tree fruit packing facilities and it has been implicated in outbreaks and recalls of tree fruit products. However, little is known about microbiota that co-occurs with L. monocytogenes and its stability over seasons in tree fruit packing environments. In this 2-year longitudinal study, we aimed to characterize spatial and seasonal changes in microbiota composition and identify taxa indicative of L. monocytogenes contamination in wet processing areas of three tree fruit packing facilities (F1, F2, F3). METHODS: A total of 189 samples were collected during two apple packing seasons from floors under the washing, drying, and waxing areas. The presence of L. monocytogenes was determined using a standard culturing method, and environmental microbiota was characterized using amplicon sequencing. PERMANOVA was used to compare microbiota composition among facilities over two seasons, and abundance-occupancy analysis was used to identify shared and temporal core microbiota. Differential abundance analysis and random forest were applied to detect taxa indicative of L. monocytogenes contamination. Lastly, three L. monocytogenes-positive samples were sequenced using shotgun metagenomics with Nanopore MinION, as a proof-of-concept for direct detection of L. monocytogenes' DNA in environmental samples. RESULTS: The occurrence of L. monocytogenes significantly increased from 28% in year 1 to 46% in year 2 in F1, and from 41% in year 1 to 92% in year 2 in F3, while all samples collected from F2 were L. monocytogenes-positive in both years. Samples collected from three facilities had a significantly different microbiota composition in both years, but the composition of each facility changed over years. A subset of bacterial taxa including Pseudomonas, Stenotrophomonas, and Microbacterium, and fungal taxa, including Yarrowia, Kurtzmaniella, Cystobasidium, Paraphoma, and Cutaneotrichosporon, were identified as potential indicators of L. monocytogenes within the monitored environments. Lastly, the DNA of L. monocytogenes was detected through direct Nanopore sequencing of metagenomic DNA extracted from environmental samples. CONCLUSIONS: This study demonstrated that a cross-sectional sampling strategy may not accurately reflect the representative microbiota of food processing facilities. Our findings also suggest that specific microorganisms are indicative of L. monocytogenes, warranting further investigation of their role in the survival and persistence of L. monocytogenes. Video Abstract.

Phylogenetic analyses of Salmonella detected along the broiler production chain in Trinidad and Tobago.

This study was conducted to determine the phylogenies of Salmonella strains isolated from cross-sectional studies conducted at hatcheries, broiler farms, processing plants, and retail outlets (broiler production chain) in Trinidad and Tobago over 4 yr (2016-2019). Whole-genome sequencing (WGS) was used to characterize Salmonella isolates. Core genome phylogenies of 8 serovars of public health significance were analyzed for similarities in origin and relatedness. In addition, Salmonella strains isolated from human salmonellosis cases in Trinidad were analyzed for their relatedness to the isolates detected along the broiler production chain. The common source of these isolates of diverse serovars within farms, within processing plants, between processing plants and retail outlets, and among farm-processing plant-retail outlet continuum was well-supported (bootstrap value >70%) by the core genome phylogenies for the respective serovars. Also, genome analyses revealed clustering of Salmonella serovars of regional (intra-Caribbean) and international (extra-Caribbean) origin. Similarly, strains of S. Enteritidis and S. Infantis isolated from human clinical salmonellosis in 2019 from Trinidad and Tobago clustered with our processing plant isolates recovered in 2018. This study is the first phylogenetic analysis of Salmonella isolates using WGS from the broiler industry in the Caribbean region. The use of WGS confirmed the genetic relatedness and transmission of Salmonella serovars contaminating chickens in broiler processing, and retailing in the country, with zoonotic and food safety implications for humans.

Pathogenome comparison and global phylogeny of Escherichia coli ST1485 strains.

Escherichia coli ST1485 strains belong to the clinically important phylogroup F and have disseminated worldwide in humans, animals, and the environment. Here, we elucidated the pathogenome of a global collection of E. coli ST1485 isolates from diverse sources retrieved from public databases and a high-quality sequenced complete genome of colistin-resistant E. coli strain CFSAN061771 isolated from raw milk cheese which designated as a reference strain. CFSAN061771 belongs to O83:H42-ST1485 pathotype and carries a conjugative ColV plasmid, pCFSAN061771_01, combining extraintestinal virulence genes (ompt, sitA, iroN, etsC, traT, cvaC, hylF, iss, tsh, mchf, iucC, iutA) with a multidrug resistance island (blaTEM-1, aph(6)-Id, aph(3″)-Ib, sul2, dfrA14). Comparative genomic analysis revealed a high frequency of pCFSAN061771_01-like plasmids in E. coli ST1485. A notable evolutionary genetic event in E. coli ST1485 strains is the acquisition of a pCFSAN061771_02-like plasmid, which confers resistance to several antimicrobials, tellurium, and quaternary ammonium compounds. The identical virulence and antibiotic resistance profiles identified in some human and animal strains are worrisome. This is the first study to emphasize the significance of E. coli ST1485 as a global high-risk virulent and multidrug-resistant clone with zoonotic potential.

Revealing Genomic Insights of the Unexplored Porcine Pathogen Actinobacillus pleuropneumoniae Using Whole Genome Sequencing.

Actinobacillus pleuropneumoniae (APP) is the causative agent of pleuropneumonia in pigs, one of the most relevant bacterial respiratory diseases in the swine industry. To date, 19 serotypes have been described based on capsular polysaccharide typing with significant virulence dissimilarities. In this study, 16 APP isolates from Spanish origin were selected to perform antimicrobial susceptibility tests and comparative genomic analysis using whole genome sequencing (WGS). To obtain a more comprehensive worldwide molecular epidemiologic analyses, all APP whole genome assemblies available at the National Center for Biotechnology Information (NCBI) at the time of the study were also included. An in-house in silico PCR approach enabled the correct serotyping of unserotyped or incorrectly serotyped isolates and allowed for the discrimination between serotypes 9 and 11. A pangenome analysis identified the presence or absence of gene clusters to be serotype specific, as well as virulence profile analyses targeting the apx operons. Antimicrobial resistance genes were correlated to the presence of specific plasmids. Altogether, this study provides new insights into the genetic variability within APP serotypes, correlates phenotypic tests with bioinformatic analyses and manifests the benefits of populated databases for a better assessment of diversity and variability of relatively unknown pathogens. Overall, genomic comparative analysis enhances the understanding of transmission and epidemiological patterns of this species and suggests vertical transmission of the pathogen, including the resistance genes, within the Spanish integrated systems. IMPORTANCE Pleuropneumonia is one of the most relevant respiratory infections in the swine industry. Despite Actinobacillus pleuropneumoniae (APP) being one of the most important pathogens in the pig production, this is the first comparative study including all available whole genome sequencing data from NCBI. Moreover, this study also includes 16 APP isolates of Spanish origin with known epidemiological relationships through vertical integrated systems. Genomic comparisons provided a deeper understanding of molecular and epidemiological knowledge between different APP serotypes. Furthermore, determination of resistance and toxin profiles allowed correlation with the presence of mobile genetic elements and specific serotype, respectively.

Genomic Comparison of Eight Closed Genomes of Multidrug-Resistant Salmonella enterica Strains Isolated From Broiler Farms and Processing Plants in Trinidad and Tobago.

Salmonella enterica is an important foodborne pathogen worldwide. We used long and short-read sequencing to close genomes of eight multidrug-resistant (MDR) S. enterica strains, belonging to serovars Infantis (2), Albany, Oranienburg, I 4,[5],12:i:-, Javiana, Schwarzengrund, and Kentucky from broiler chicken farms and processing plants in Trinidad and Tobago. They also belonged to seven different sequence types (STs- 32, 292, 1510, 19, 24, 152, and 96). Among the strains, seven had demonstrated multi-drug resistance with the presence of at least three AMR genes, whereas three isolates contained the quinolone resistance gene qnr B19 in plasmids (CFSAN103840, CFSAN103854, and CFSAN103872). The extended-spectrum ß-lactamase genes bla CTX-M-65 (CFSAN103796) and bla TEM-1 (CFSAN103852) were detected in this study. The genomes closed in this study will be useful for future source tracking and outbreak investigations in Trinidad and Tobago and worldwide.

Molecular Characterization of Salmonella Detected along the Broiler Production Chain in Trinidad and Tobago.

This cross-sectional study determined the serovars, antimicrobial resistance genes, and virulence factors of Salmonella isolated from hatcheries, broiler farms, processing plants, and retail outlets in Trinidad and Tobago. Salmonella in silico serotyping detected 23 different serovars where Kentucky 20.5% (30/146), Javiana 19.2% (28/146), Infantis 13.7% (20/146), and Albany 8.9% (13/146) were the predominant serovars. There was a 76.0% (111/146) agreement between serotyping results using traditional conventional methods and whole-genome sequencing (WGS) in in silico analysis. In silico identification of antimicrobial resistance genes conferring resistance to aminoglycosides, cephalosporins, peptides, sulfonamides, and antiseptics were detected. Multidrug resistance (MDR) was detected in 6.8% (10/146) of the isolates of which 100% originated from broiler farms. Overall, virulence factors associated with secretion systems and fimbrial adherence determinants accounted for 69.3% (3091/4463), and 29.2% (1302/4463) counts, respectively. Ten of 20 isolates of serovar Infantis (50.0%) showed MDR and contained the blaCTX-M-65 gene. This is the first molecular characterization of Salmonella isolates detected along the entire broiler production continuum in the Caribbean region using WGS. The availability of these genomes will help future source tracking during epidemiological investigations associated with Salmonella foodborne outbreaks in the region and worldwide.

In vivo commensal control of Clostridioides difficile virulence.

Leveraging systems biology approaches, we illustrate how metabolically distinct species of Clostridia protect against or worsen Clostridioides difficile infection in mice by modulating the pathogen's colonization, growth, and virulence to impact host survival. Gnotobiotic mice colonized with the amino acid fermenter Paraclostridium bifermentans survive infection with reduced disease severity, while mice colonized with the butyrate-producer, Clostridium sardiniense, succumb more rapidly. Systematic in vivo analyses revealed how each commensal alters the gut-nutrient environment to modulate the pathogen's metabolism, gene regulatory networks, and toxin production. Oral administration of P. bifermentans rescues conventional, clindamycin-treated mice from lethal C. difficile infection in a manner similar to that of monocolonized animals, thereby supporting the therapeutic potential of this commensal species. Our findings lay the foundation for mechanistically informed therapies to counter C. difficile disease using systems biology approaches to define host-commensal-pathogen interactions in vivo.

Diversity of Non-O157 Shiga Toxin-Producing Escherichia coli Isolated from Cattle from Central and Southern Chile.

Cattle are the main reservoir of Shiga toxin-producing Escherichia coli (STEC), one of the world's most important foodborne pathogens. The pathogen causes severe human diseases and outbreaks. This study aimed to identify and characterize non-O157 STEC isolated from cattle feces from central and southern Chile. We analyzed 446 cattle fecal samples and isolated non-O157 STEC from 12.6% (56/446); a total of 93 different isolates were recovered. Most isolates displayed ß-glucuronidase activity (96.8%; 90/93) and fermented sorbitol (86.0%; 80/93), whereas only 39.8% (37/93) were resistant to tellurite. A subgroup of 30 representative non-O157 STEC isolates was selected for whole-genome sequencing and bioinformatics analysis. In silico analysis showed that they grouped into 16 different sequence types and 17 serotypes; the serotypes most frequently identified were O116:H21 and O168:H8 (13% each). A single isolate of serotype O26:H11 was recovered. One isolate was resistant to tetracycline and carried resistance genes tet(A) and tet(R); no other isolate displayed antimicrobial resistance or carried antimicrobial resistance genes. The intimin gene (eae) was identified in 13.3% (4/30) of the genomes and 90% (27/30) carried the stx2 gene. A phylogenetic reconstruction demonstrated that the isolates clustered based on serotypes, independent of geographical origin. These results indicate that cattle in Chile carry a wide diversity of STEC potentially pathogenic for humans based on the presence of critical virulence genes.

AMRFinderPlus and the Reference Gene Catalog facilitate examination of the genomic links among antimicrobial resistance, stress response, and virulence.

Antimicrobial resistance (AMR) is a significant public health threat. With the rise of affordable whole genome sequencing, in silico approaches to assessing AMR gene content can be used to detect known resistance mechanisms and potentially identify novel mechanisms. To enable accurate assessment of AMR gene content, as part of a multi-agency collaboration, NCBI developed a comprehensive AMR gene database, the Bacterial Antimicrobial Resistance Reference Gene Database and the AMR gene detection tool AMRFinder. Here, we describe the expansion of the Reference Gene Database, now called the Reference Gene Catalog, to include putative acid, biocide, metal, stress resistance genes, in addition to virulence genes and species-specific point mutations. Genes and point mutations are classified by broad functions, as well as more detailed functions. As we have expanded both the functional repertoire of identified genes and functionality, NCBI released a new version of AMRFinder, known as AMRFinderPlus. This new tool allows users the option to utilize only the core set of AMR elements, or include stress response and virulence genes, too. AMRFinderPlus can detect acquired genes and point mutations in both protein and nucleotide sequence. In addition, the evidence used to identify the gene has been expanded to include whether nucleotide or protein sequence was used, its location in the contig, and presence of an internal stop codon. These database improvements and functional expansions will enable increased precision in identifying AMR genes, linking AMR genotypes and phenotypes, and determining possible relationships between AMR, virulence, and stress response.

Closed Genome Sequence of a Salmonella enterica Serotype Senftenberg Strain Carrying the mcr-9 Gene Isolated from Broken Chicken Eggshells in Trinidad and Tobago.

Salmonella enterica is a highly important foodborne pathogen worldwide. We report the complete genome sequence of a sequence type 14 Salmonella enterica serotype Senftenberg strain carrying the mcr-9 gene in a plasmid isolated from broken chicken eggshells in Trinidad and Tobago, obtained by using a combination of long- and short-read sequencing.

Evaluating the accuracy of Listeria monocytogenes assemblies from quasimetagenomic samples using long and short reads.

BACKGROUND: Whole genome sequencing of cultured pathogens is the state of the art public health response for the bioinformatic source tracking of illness outbreaks. Quasimetagenomics can substantially reduce the amount of culturing needed before a high quality genome can be recovered. Highly accurate short read data is analyzed for single nucleotide polymorphisms and multi-locus sequence types to differentiate strains but cannot span many genomic repeats, resulting in highly fragmented assemblies. Long reads can span repeats, resulting in much more contiguous assemblies, but have lower accuracy than short reads. RESULTS: We evaluated the accuracy of Listeria monocytogenes assemblies from enrichments (quasimetagenomes) of naturally-contaminated ice cream using long read (Oxford Nanopore) and short read (Illumina) sequencing data. Accuracy of ten assembly approaches, over a range of sequencing depths, was evaluated by comparing sequence similarity of genes in assemblies to a complete reference genome. Long read assemblies reconstructed a circularized genome as well as a 71 kbp plasmid after 24 h of enrichment; however, high error rates prevented high fidelity gene assembly, even at 150X depth of coverage. Short read assemblies accurately reconstructed the core genes after 28 h of enrichment but produced highly fragmented genomes. Hybrid approaches demonstrated promising results but had biases based upon the initial assembly strategy. Short read assemblies scaffolded with long reads accurately assembled the core genes after just 24 h of enrichment, but were highly fragmented. Long read assemblies polished with short reads reconstructed a circularized genome and plasmid and assembled all the genes after 24 h enrichment but with less fidelity for the core genes than the short read assemblies. CONCLUSION: The integration of long and short read sequencing of quasimetagenomes expedited the reconstruction of a high quality pathogen genome compared to either platform alone. A new and more complete level of information about genome structure, gene order and mobile elements can be added to the public health response by incorporating long read analyses with the standard short read WGS outbreak response.

Choice of library preparation affects sequence quality, genome assembly, and precise in silico prediction of virulence genes in shiga toxin-producing Escherichia coli.

Whole genome sequencing (WGS) provides essential public health information and is used worldwide for pathogen surveillance, epidemiology, and source tracking. Foodborne pathogens are often sequenced using rapid library preparation chemistries based on transposon technology; however, this method may miss random segments of genomes that can be important for accurate downstream analyses. As new technologies become available, it may become possible to achieve better overall coverage. Here we compare the sequence quality obtained using libraries prepared from the Nextera XT and Nextera DNA Prep (Illumina, San Diego, CA) chemistries for 31 Shiga toxin-producing Escherichia coli (STEC) O121:H19 strains, which had been isolated from flour during a 2016 outbreak. The Nextera DNA Prep gave superior performance metrics including sequence quality, assembly quality, uniformity of genome coverage, and virulence gene identification, among other metrics. Comprehensive detection of virulence genes is essential for making educated assessments of STECs virulence potential. The phylogenetic SNP analysis did not show any differences in the variants detected by either library preparation method which allows isolates prepared from either library method to be analysed together. Our comprehensive comparison of these chemistries should assist researchers wishing to improve their sequencing workflow for STECs and other genomic risk assessments.

Multiple Displacement Amplification as a Solution for Low Copy Number Plasmid Sequencing.

Plasmids play a major role in bacterial adaptation to environmental stress and often contribute to antibiotic resistance and disease virulence. Although the complete sequence of each plasmid is essential for studying plasmid biology, most antibiotic resistance and virulence plasmids in Salmonella are present only in a low copy number, making extraction and sequencing difficult. Long read sequencing technologies require higher concentrations of DNA to provide optimal results. To resolve this problem, we assessed the sufficiency of multiple displacement amplification (MDA) for replicating Salmonella plasmid DNA to a satisfactory concentration for accurate sequencing and multiplexing. Nine Salmonella enterica isolates, representing nine different serovars carrying plasmids for which sequence data are already available at NCBI, were cultured and their plasmids isolated using an alkaline lysis extraction protocol. We then used the Phi29 polymerase to perform MDA, thereby obtaining enough plasmid DNA for long read sequencing. These amplified plasmids were multiplexed and sequenced on one single molecule, real-time (SMRT) cell with the Pacific Biosciences (Pacbio) Sequel sequencer. We were able to close all Salmonella plasmids (sizes ranged from 38 to 166 Kb) with sequencing coverage from 24 to 2,582X. This protocol, consisting of plasmid isolation, MDA, and multiplex sequencing, is an effective and fast method for closing high-molecular weight and low-copy-number plasmids. This high throughput protocol reduces the time and cost of plasmid closure.

Genomic analysis of the diversity, antimicrobial resistance and virulence potential of clinical Campylobacter jejuni and Campylobacter coli strains from Chile.

Campylobacter jejuni and Campylobacter coli are the leading cause of human gastroenteritis in the industrialized world and an emerging threat in developing countries. The incidence of campylobacteriosis in South America is greatly underestimated, mostly due to the lack of adequate diagnostic methods. Accordingly, there is limited genomic and epidemiological data from this region. In the present study, we performed a genome-wide analysis of the genetic diversity, virulence, and antimicrobial resistance of the largest collection of clinical C. jejuni and C. coli strains from Chile available to date (n = 81), collected in 2017-2019 in Santiago, Chile. This culture collection accounts for more than one third of the available genome sequences from South American clinical strains. cgMLST analysis identified high genetic diversity as well as 13 novel STs and alleles in both C. jejuni and C. coli. Pangenome and virulome analyses showed a differential distribution of virulence factors, including both plasmid and chromosomally encoded T6SSs and T4SSs. Resistome analysis predicted widespread resistance to fluoroquinolones, but low rates of erythromycin resistance. This study provides valuable genomic and epidemiological data and highlights the need for further genomic epidemiology studies in Chile and other South American countries to better understand molecular epidemiology and antimicrobial resistance of this emerging intestinal pathogen.