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July 7, 2019

Complete genomic sequences of two Salmonella enterica subsp. enterica serogroup C2 (O:6,8) strains from Central California.

Salmonella enterica subsp. enterica strains RM11060, serotype 6,8:d:-, and RM11065, serotype 6,8:-:e,n,z15, were isolated from environmental samples collected in central California in 2009. We report the complete genome sequences of these two strains. These genomic sequences are distinct and will provide additional data to our understanding of S. enterica genomics.


July 7, 2019

An integrative strategy to identify the entire protein coding potential of prokaryotic genomes by proteogenomics.

Accurate annotation of all protein-coding sequences (CDSs) is an essential prerequisite to fully exploit the rapidly growing repertoire of completely sequenced prokaryotic genomes. However, large discrepancies among the number of CDSs annotated by different resources, missed functional short open reading frames (sORFs), and overprediction of spurious ORFs represent serious limitations. Our strategy toward accurate and complete genome annotation consolidates CDSs from multiple reference annotation resources, ab initio gene prediction algorithms and in silico ORFs (a modified six-frame translation considering alternative start codons) in an integrated proteogenomics database (iPtgxDB) that covers the entire protein-coding potential of a prokaryotic genome. By extending the PeptideClassifier concept of unambiguous peptides for prokaryotes, close to 95% of the identifiable peptides imply one distinct protein, largely simplifying downstream analysis. Searching a comprehensive Bartonella henselae proteomics data set against such an iPtgxDB allowed us to unambiguously identify novel ORFs uniquely predicted by each resource, including lipoproteins, differentially expressed and membrane-localized proteins, novel start sites and wrongly annotated pseudogenes. Most novelties were confirmed by targeted, parallel reaction monitoring mass spectrometry, including unique ORFs and single amino acid variations (SAAVs) identified in a re-sequenced laboratory strain that are not present in its reference genome. We demonstrate the general applicability of our strategy for genomes with varying GC content and distinct taxonomic origin. We release iPtgxDBs for B. henselae, Bradyrhizobium diazoefficiens and Escherichia coli and the software to generate both proteogenomics search databases and integrated annotation files that can be viewed in a genome browser for any prokaryote.© 2017 Omasits et al.; Published by Cold Spring Harbor Laboratory Press.


July 7, 2019

Genomic comparison between Staphylococcus aureus GN strains clinically isolated from a familial infection case: IS1272 transposition through a novel inverted repeat-replacing mechanism.

A bacterial insertion sequence (IS) is a mobile DNA sequence carrying only the transposase gene (tnp) that acts as a mutator to disrupt genes, alter gene expressions, and cause genomic rearrangements. “Canonical” ISs have historically been characterized by their terminal inverted repeats (IRs), which may form a stem-loop structure, and duplications of a short (non-IR) target sequence at both ends, called target site duplications (TSDs). The IS distributions and virulence potentials of Staphylococcus aureus genomes in familial infection cases are unclear. Here, we determined the complete circular genome sequences of familial strains from a Panton-Valentine leukocidin (PVL)-positive ST50/agr4 S. aureus (GN) infection of a 4-year old boy with skin abscesses. The genomes of the patient strain (GN1) and parent strain (GN3) were rich for “canonical” IS1272 with terminal IRs, both having 13 commonly-existing copies (ce-IS1272). Moreover, GN1 had a newly-inserted IS1272 (ni-IS1272) on the PVL-converting prophage, while GN3 had two copies of ni-IS1272 within the DNA helicase gene and near rot. The GN3 genome also had a small deletion. The targets of ni-IS1272 transposition were IR structures, in contrast with previous “canonical” ISs. There were no TSDs. Based on a database search, the targets for ce-IS1272 were IRs or “non-IRs”. IS1272 included a larger structure with tandem duplications of the left (IRL) side sequence; tnp included minor cases of a long fusion form and truncated form. One ce-IS1272 was associated with the segments responsible for immune evasion and drug resistance. Regarding virulence, GN1 expressed cytolytic peptides (phenol-soluble modulin a and d-hemolysin) and PVL more strongly than some other familial strains. These results suggest that IS1272 transposes through an IR-replacing mechanism, with an irreversible process unlike that of “canonical” transpositions, resulting in genomic variations, and that, among the familial strains, the patient strain has strong virulence potential based on community-associated virulence factors.


July 7, 2019

Complete genome sequence of Salmonella enterica subsp. enterica serovar Minnesota strain

Mango has been implicated as food vehicle in several Salmonella-causing foodborne outbreaks. Here, Salmonella enterica subsp. enterica serovar Minnesota was isolated from fresh mango fruit imported from Mexico in 2014. The complete genome sequence of S. Minnesota CFSAN017963 was sequenced using single-molecule real-time DNA sequencing. Distinct prophage regions, Salmonella pathogenicity islands, and fimbrial gene clusters were observed in comparative genomic analysis on S. Minnesota CFSAN017963 with other phylogenetically closely related Salmonella serovars. Core genome multilocus sequencing typing analysis of all the S. Minnesota isolates in the Genbank and Enterobase also revealed a high genomic diversity among the genomes analyzed.


July 7, 2019

Complete genome sequence of multidrug-resistant Staphylococcus sciuri strain SNUDS-18 isolated from a farmed duck in South Korea.

This study aimed to determine the complete genome sequence of multidrug-resistant Staphylococcus sciuri strain SNUDS-18 isolated from a farmed duck in South Korea.Genomic DNA was sequenced using a PacBio RS II system. The obtained genome was annotated and antimicrobial resistance and virulence genes were identified.The sequenced genome possessed a mecA homologue (mecA1) that was almost identical to that of other oxacillin-susceptible S. sciuri strains, whereas the staphylococcal cassette chromosome mec (SCCmec) was not detected. Moreover, various antimicrobial resistance genes conferring resistance to ß-lactams, aminoglycosides, phenicols, tetracycline and macrolide-lincosamide-streptogramin B (MLSB) antimicrobials were identified.The SNUDS-18 genome and its associated genomic data will provide important insights into the biodiversity of the S. sciuri group as well as valuable information for the control of this potential pathogen. Copyright © 2017 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.


July 7, 2019

Genomic analysis of Bacillus licheniformis CBA7126 isolated from a human fecal sample.

Bacillus licheniformis is a Gram-positive, endospore-forming, saprophytic organism that occurs in plant and soil (Veith et al., 2004). A taxonomical approach shows that it is closely related to Bacillus subtilis (Lapidus et al., 2002; Xu and Côte, 2003; Rey et al., 2004). Generally, most bacilli are predominantly aerobic; however, B. licheniformis is a facultative anaerobe compared to other bacilli in ecological niches (Alexander, 1977). The commercial utility of the extracellular products of B. licheniformis makes this microorganism an economically interesting species (Kovács et al., 2009). For example, B. licheniformis is used industrially for manufacturing biochemicals, enzymes, antibiotics, and aminopeptidase. Several proteases such as a-amylase, penicillinase, pentosanase, cycloglucosyltransferase, ß-mannanase, and certain pectinolytic enzymes are synthesized industrially using B. licheniformis (Rodríguez-Absi and Prescott, 1978; Rey et al., 2004). The proteases are used in the detergent industry and the amylases are utilized for starch hydrolysis, desizing of textiles, and sizing of paper (Erickson, 1976). In addition, certain strains are utilized to produce peptide antibiotics, specialty chemicals, and poly-?-glutamic acid (Nierman and Maglott, 1989; Rey et al., 2004).


July 7, 2019

RNA-seq and Tn-seq reveal fitness determinants of vancomycin-resistant Enterococcus faecium during growth in human serum.

The Gram-positive bacterium Enterococcus faecium is a commensal of the human gastrointestinal tract and a frequent cause of bloodstream infections in hospitalized patients. The mechanisms by which E. faecium can survive and grow in blood during an infection have not yet been characterized. Here, we identify genes that contribute to growth of E. faecium in human serum through transcriptome profiling (RNA-seq) and a high-throughput transposon mutant library sequencing approach (Tn-seq).We first sequenced the genome of E. faecium E745, a vancomycin-resistant clinical isolate, using a combination of short- and long read sequencing, revealing a 2,765,010 nt chromosome and 6 plasmids, with sizes ranging between 9.3 kbp and 223.7 kbp. We then compared the transcriptome of E. faecium E745 during exponential growth in rich medium and in human serum by RNA-seq. This analysis revealed that 27.8% of genes on the E. faecium E745 genome were differentially expressed in these two conditions. A gene cluster with a role in purine biosynthesis was among the most upregulated genes in E. faecium E745 upon growth in serum. The E. faecium E745 transposon mutant library was then used to identify genes that were specifically required for growth of E. faecium in serum. Genes involved in de novo nucleotide biosynthesis (including pyrK_2, pyrF, purD, purH) and a gene encoding a phosphotransferase system subunit (manY_2) were thus identified to be contributing to E. faecium growth in human serum. Transposon mutants in pyrK_2, pyrF, purD, purH and manY_2 were isolated from the library and their impaired growth in human serum was confirmed. In addition, the pyrK_2 and manY_2 mutants were tested for their virulence in an intravenous zebrafish infection model and exhibited significantly attenuated virulence compared to E. faecium E745.Genes involved in carbohydrate metabolism and nucleotide biosynthesis of E. faecium are essential for growth in human serum and contribute to the pathogenesis of this organism. These genes may serve as targets for the development of novel anti-infectives for the treatment of E. faecium bloodstream infections.


July 7, 2019

The complete genome sequence of Ensifer meliloti strain CCMM B554 (FSM-MA), a highly effective nitrogen-fixing microsymbiont of Medicago truncatula Gaertn.

Strain CCMM B554, also known as FSM-MA, is a soil dwelling and nodule forming, nitrogen-fixing bacterium isolated from the nodules of the legume Medicago arborea L. in the Maamora Forest, Morocco. The strain forms effective nitrogen fixing nodules on species of the Medicago, Melilotus and Trigonella genera and is exceptional because it is a highly effective symbiotic partner of the two most widely used accessions, A17 and R108, of the model legume Medicago truncatula Gaertn. Based on 16S rRNA gene sequence, multilocus sequence and average nucleotide identity analyses, FSM-MA is identified as a new Ensifer meliloti strain. The genome is 6,70 Mbp and is comprised of the chromosome (3,64 Mbp) harboring 3574 predicted genes and two megaplasmids, pSymA (1,42 Mbp) and pSymB (1,64 Mbp) with respectively 1481 and 1595 predicted genes. The average GC content of the genome is 61.93%. The FSM-MA genome structure is highly similar and co-linear to other E. meliloti strains in the chromosome and the pSymB megaplasmid while, in contrast, it shows high variability in the pSymA plasmid. The large number of strain-specific sequences in pSymA as well as strain-specific genes on pSymB involved in the biosynthesis of the lipopolysaccharide and capsular polysaccharide surface polysaccharides may encode novel symbiotic functions explaining the high symbiotic performance of FSM-MA.


July 7, 2019

Streptococcal toxic shock syndrome caused by the dissemination of an invasive emm3/ST15 strain of Streptococcus pyogenes.

Streptococcus pyogenes (group A Streptococcus [GAS]) is a major human pathogen that causes a wide spectrum of clinical manifestations. Although invasive GAS (iGAS) infections are relatively uncommon, emm3/ST15 GAS is a highly virulent, invasive, and pathogenic strain. Global molecular epidemiology analysis has suggested that the frequency of emm3 GAS has been recently increasing.A 14-year-old patient was diagnosed with streptococcal toxic shock syndrome and severe pneumonia, impaired renal function, and rhabdomyolysis. GAS was isolated from a culture of endotracheal aspirates and designated as KS030. Comparative genome analysis suggested that KS030 is classified as emm3 (emm-type) and ST15 (multilocus sequencing typing [MLST]), which is similar to iGAS isolates identified in the UK (2013) and Switzerland (2015).We conclude that the global dissemination of emm3/ST15 GAS strain has the potential to cause invasive disease.


July 7, 2019

Complete genome sequences of two strains of the meat spoilage bacterium Brochothrix thermosphacta isolated from ground chicken.

Brochothrix thermosphacta is an important meat spoilage bacterium. Here we report the genome sequences of two strains of B. thermosphacta isolated from ground chicken. The genome sequences were determined using long-read PacBio single-molecule real-time (SMRT) technology and are the first complete genome sequences reported for B. thermosphacta.


July 7, 2019

Genomic analysis of a pathogenic bacterium, Paeniclostridium sordellii CBA7122 containing the highest number of rRNA operons, isolated from a human stool sample.

Paeniclostridium sordellii was first isolated by Alfredo Sordelli in 1922 under the proposed name Bacillus oedematis, and was then renamed Bacillus sordellii in 1927 (Hall and Scott, 1927). Two years later, it was classified as Clostridium sordellii (Hall et al., 1929). Recently, this bacterium was reclassified as a species of the genus Paeniclostridium, named P. sordellii comb. nov. (Sasi Jyothsna et al., 2016). P. sordellii is an anaerobic, Gram-stain-positive, spore-forming rod bacterium with flagella. Most strains are non-pathogenic, but some strains have been associated with severe infections of humans and animals. In humans, P. sordellii is mainly associated with trauma, toxic shock, soft tissue skin infections, and gynecologic infections. Despite the serious consequences of infection with P. sordellii, treatment is difficult because of the rapid progression from recognition of the first symptoms to death (Aldape et al., 2006).


July 7, 2019

Comparative genomics reveals specific genetic architectures in nicotine metabolism of Pseudomonassp. JY-Q.

Microbial degradation of nicotine is an important process to control nicotine residues in the aqueous environment. In this study, a high active nicotine degradation strain namedPseudomonassp. JY-Q was isolated from tobacco waste extract (TWE). This strain could completely degrade 5.0 g l-1nicotine in 24 h under optimal culture conditions, and it showed some tolerance even at higher concentrations (10.0 g l-1) of nicotine. The complete genome of JY-Q was sequenced to understand the mechanism by which JY-Q could degrade nicotine and tolerate such high nicotine concentrations. Comparative genomic analysis indicated that JY-Q degrades nicotine through putative novel mechanisms. Two candidate gene cluster duplications located separately at distant loci were predicted to be responsible for nicotine degradation. These two nicotine (Nic) degradation-related loci (AA098_21325-AA098_21340, AA098_03885-AA098_03900) exhibit nearly completely consistent gene organization and component synteny. The nicotinic acid(NA)degradation gene cluster (AA098_17770-AA098_17790) andNic-like clusters were both predicted to be flanked by mobile genetic elements (MGE). Furthermore, we analyzed the regions of genomic plasticity (RGP) in the JY-Q strain and found a dynamic genome carrying a type VI secretion system (T6SS) that promotes nicotine metabolism and tolerance based on transcriptomics and usedin silicomethods to identify the T6SS effector protein. Thus, a novel nicotine degradation mechanism was elucidated forPseudomonassp. JY-Q, suggesting its potential application in the bioremediation of nicotine-contaminated environments, such as TWEs.


July 7, 2019

Probing genomic aspects of the multi-host pathogen Clostridium perfringens reveals significant pangenome diversity, and a diverse array of virulence factors.

Clostridium perfringens is an important cause of animal and human infections, however information about the genetic makeup of this pathogenic bacterium is currently limited. In this study, we sought to understand and characterise the genomic variation, pangenomic diversity, and key virulence traits of 56 C. perfringens strains which included 51 public, and 5 newly sequenced and annotated genomes using Whole Genome Sequencing. Our investigation revealed that C. perfringens has an “open” pangenome comprising 11667 genes and 12.6% of core genes, identified as the most divergent single-species Gram-positive bacterial pangenome currently reported. Our computational analyses also defined C. perfringens phylogeny (16S rRNA gene) in relation to some 25 Clostridium species, with C. baratii and C. sardiniense determined to be the closest relatives. Profiling virulence-associated factors confirmed presence of well-characterised C. perfringens-associated exotoxins genes including a-toxin (plc), enterotoxin (cpe), and Perfringolysin O (pfo or pfoA), although interestingly there did not appear to be a close correlation with encoded toxin type and disease phenotype. Furthermore, genomic analysis indicated significant horizontal gene transfer events as defined by presence of prophage genomes, and notably absence of CRISPR defence systems in >70% (40/56) of the strains. In relation to antimicrobial resistance mechanisms, tetracycline resistance genes (tet) and anti-defensins genes (mprF) were consistently detected in silico (tet: 75%; mprF: 100%). However, pre-antibiotic era strain genomes did not encode for tet, thus implying antimicrobial selective pressures in C. perfringens evolutionary history over the past 80 years. This study provides new genomic understanding of this genetically divergent multi-host bacterium, and further expands our knowledge on this medically and veterinary important pathogen.


July 7, 2019

Safety evaluation of HOWARU®Restore (Lactobacillus acidophilus NCFM, Lactobacillus paracasei Lpc-37, Bifidobacterium animalis subsp. lactis Bl-04 and B. lactis Bi-07) for antibiotic resistance, genomic risk factors, and acute toxicity.

Although probiotic lactobacilli and bifidobacteria are generally considered safe by various regulatory agencies, safety properties, such as absence of transferable antibiotic resistance, must still be determined for each strain prior to market introduction as a probiotic. Safety requirements for probiotics vary regionally and evaluation methods are not standardized, therefore methodologies are often adopted from food ingredients or chemicals to assess microbial safety. Four individual probiotic strains, Lactobacillus acidophilus NCFM®, Lactobacillus paracasei Lpc-37®, Bifidobacterium animalis subsp. lactis strains Bl-04®, and Bi-07®, and their combination (HOWARU®Restore) were examined for antibiotic resistance by broth microdilution culture, toxin genes by PCR and genome mining, and acute oral toxicity in rats. Only B. lactis Bl-04 exhibited antibiotic resistance above a regulated threshold due to a tetW gene previously demonstrated to be non-transferable. Genomic mining did not reveal any bacterial toxin genes known to harm mammalian hosts in any of the strains. The rodent studies did not indicate any evidence of acute toxicity following a dose of 1.7-4.1 × 1012 CFU/kg body weight. Considering a 100-fold safety margin, this corresponds to 1.2-2.8 × 1012 CFU for a 70 kg human. Our findings demonstrate a comprehensive approach of in vitro, in silico, and in vivo safety testing for probiotics. Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.


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