Nouri L. Ben Zakour

BLAST Ring Image Generator (BRIG): simple prokaryote genome comparisons

BackgroundVisualisation of genome comparisons is invaluable for helping to determine genotypic differences between closely related prokaryotes. New visualisation and abstraction methods are required in order to improve the validation, interpretation and communication of genome sequence information; especially with the increasing amount of data arising from next-generation sequencing projects. Visualising a prokaryote genome as a circular image has become a powerful means of displaying informative comparisons of one genome to a number of others. Several programs, imaging libraries and internet resources already exist for this purpose, however, most are either limited in the number of comparisons they can show, are unable to adequately utilise draft genome sequence data, or require a knowledge of command-line scripting for implementation. Currently, there is no freely available desktop application that enables users to rapidly visualise comparisons between hundreds of draft or complete genomes in a single image.ResultsBLAST Ring Image Generator (BRIG) can generate images that show multiple prokaryote genome comparisons, without an arbitrary limit on the number of genomes compared. The output image shows similarity between a central reference sequence and other sequences as a set of concentric rings, where BLAST matches are coloured on a sliding scale indicating a defined percentage identity. Images can also include draft genome assembly information to show read coverage, assembly breakpoints and collapsed repeats. In addition, BRIG supports the mapping of unassembled sequencing reads against one or more central reference sequences. Many types of custom data and annotations can be shown using BRIG, making it a versatile approach for visualising a range of genomic comparison data. BRIG is readily accessible to any user, as it assumes no specialist computational knowledge and will perform all required file parsing and BLAST comparisons automatically.ConclusionsThere is a clear need for a user-friendly program that can produce genome comparisons for a large number of prokaryote genomes with an emphasis on rapidly utilising unfinished or unassembled genome data. Here we present BRIG, a cross-platform application that enables the interactive generation of comparative genomic images via a simple graphical-user interface. BRIG is freely available for all operating systems at http://sourceforge.net/projects/brig/.

Recent human-to-poultry host jump, adaptation, and pandemic spread of Staphylococcus aureus

The impact of globalization on the emergence and spread of pathogens is an important veterinary and public health issue. Staphylococcus aureus is a notorious human pathogen associated with serious nosocomial and community-acquired infections. In addition, S. aureus is a major cause of animal diseases including skeletal infections of poultry, which are a large economic burden on the global broiler chicken industry. Here, we provide evidence that the majority of S. aureus isolates from broiler chickens are the descendants of a single human-to-poultry host jump that occurred approximately 38 years ago (range, 30 to 63 years ago) by a subtype of the worldwide human ST5 clonal lineage unique to Poland. In contrast to human subtypes of the ST5 radiation, which demonstrate strong geographic clustering, the poultry ST5 clade was distributed in different continents, consistent with wide dissemination via the global poultry industry distribution network. The poultry ST5 clade has undergone genetic diversification from its human progenitor strain by acquisition of novel mobile genetic elements from an avian-specific accessory gene pool, and by the inactivation of several proteins important for human disease pathogenesis. These genetic events have resulted in enhanced resistance to killing by chicken heterophils, reflecting avian host-adaptive evolution. Taken together, we have determined the evolutionary history of a major new animal pathogen that has undergone rapid avian host adaptation and intercontinental dissemination. These data provide a new paradigm for the impact of human activities on the emergence of animal pathogens.

Global dissemination of a multidrug resistant Escherichia coli clone

Significance Escherichia coli sequence type 131 (ST131) is a globally disseminated multidrug-resistant clone associated with human urinary tract and bloodstream infections. Here, we have used genome sequencing to map the temporal and spatial relationship of a large collection of E. coli ST131 strains isolated from six distinct geographical regions across the world. We show that E. coli ST131 strains are distinct from other extraintestinal pathogenic E. coli and arose from a single progenitor strain prior to the year 2000. Escherichia coli sequence type 131 (ST131) is a globally disseminated, multidrug resistant (MDR) clone responsible for a high proportion of urinary tract and bloodstream infections. The rapid emergence and successful spread of E. coli ST131 is strongly associated with several factors, including resistance to fluoroquinolones, high virulence gene content, the possession of the type 1 fimbriae FimH30 allele, and the production of the CTX-M-15 extended spectrum β-lactamase (ESBL). Here, we used genome sequencing to examine the molecular epidemiology of a collection of E. coli ST131 strains isolated from six distinct geographical locations across the world spanning 2000–2011. The global phylogeny of E. coli ST131, determined from whole-genome sequence data, revealed a single lineage of E. coli ST131 distinct from other extraintestinal E. coli strains within the B2 phylogroup. Three closely related E. coli ST131 sublineages were identified, with little association to geographic origin. The majority of single-nucleotide variants associated with each of the sublineages were due to recombination in regions adjacent to mobile genetic elements (MGEs). The most prevalent sublineage of ST131 strains was characterized by fluoroquinolone resistance, and a distinct virulence factor and MGE profile. Four different variants of the CTX-M ESBL–resistance gene were identified in our ST131 strains, with acquisition of CTX-M-15 representing a defining feature of a discrete but geographically dispersed ST131 sublineage. This study confirms the global dispersal of a single E. coli ST131 clone and demonstrates the role of MGEs and recombination in the evolution of this important MDR pathogen.

Population Genetic Structure of the Staphylococcus intermedius Group: Insights into agr Diversification and the Emergence of Methicillin-Resistant Strains

The population genetic structure of the animal pathogen Staphylococcus intermedius is poorly understood. We carried out a multilocus sequence phylogenetic analysis of isolates from broad host and geographic origins to investigate inter- and intraspecies diversity. We found that isolates phenotypically identified as S. intermedius are differentiated into three closely related species, S. intermedius, Staphylococcus pseudintermedius, and Staphylococcus delphini. S. pseudintermedius, not S. intermedius, is the common cause of canine pyoderma and occasionally causes zoonotic infections of humans. Over 60 extant STs were identified among the S. pseudintermedius isolates examined, including several that were distributed on different continents. As the agr quorum-sensing system of staphylococci is thought to have evolved along lines of speciation within the genus, we examined the allelic variation of agrD, which encodes the autoinducing peptide (AIP). Four AIP variants were encoded by S. pseudintermedius isolates, and identical AIP variants were shared among the three species, suggesting that a common quorum-sensing capacity has been conserved in spite of species differentiation in largely distinct ecological niches. A lack of clonal association of agr alleles suggests that assortive recombination may have contributed to the distribution of agr diversity. Finally, we discovered that the recent emergence of methicillin-resistant strains was due to multiple acquisitions of the mecA gene by different S. pseudintermedius clones found on different continents. Taken together, these data have resolved the population genetic structure of the S. intermedius group, resulting in new insights into its ancient and recent evolution.

Evolutionary Genomics of Staphylococcus aureus Reveals Insights into the Origin and Molecular Basis of Ruminant Host Adaptation

Phenotypic biotyping has traditionally been used to differentiate bacteria occupying distinct ecological niches such as host species. For example, the capacity of Staphylococcus aureus from sheep to coagulate ruminant plasma, reported over 60 years ago, led to the description of small ruminant and bovine S. aureus ecovars. The great majority of small ruminant isolates are represented by a single, widespread clonal complex (CC133) of S. aureus, but its evolutionary origin and the molecular basis for its host tropism remain unknown. Here, we provide evidence that the CC133 clone evolved as the result of a human to ruminant host jump followed by adaptive genome diversification. Comparative whole-genome sequencing revealed molecular evidence for host adaptation including gene decay and diversification of proteins involved in host–pathogen interactions. Importantly, several novel mobile genetic elements encoding virulence proteins with attenuated or enhanced activity in ruminants were widely distributed in CC133 isolates, suggesting a key role in its host-specific interactions. To investigate this further, we examined the activity of a novel staphylococcal pathogenicity island (SaPIov2) found in the great majority of CC133 isolates which encodes a variant of the chromosomally encoded von Willebrand-binding protein (vWbpSov2), previously demonstrated to have coagulase activity for human plasma. Remarkably, we discovered that SaPIov2 confers the ability to coagulate ruminant plasma suggesting an important role in ruminant disease pathogenesis and revealing the origin of a defining phenotype of the classical S. aureus biotyping scheme. Taken together, these data provide broad new insights into the origin and molecular basis of S. aureus ruminant host specificity.

Tandem repeat sequence analysis of staphylococcal protein A (spa) gene in methicillin-resistant Staphylococcus pseudintermedius.

A putative staphylococcal protein A (spa) gene was discovered in the genome of Staphylococcus pseudintermedius and used for developing a species-specific spa typing protocol. Thirty-one clinical methicillin-resistant S. pseudintermedius (MRSP) isolates from dogs and cats in four countries were characterized by spa typing, pulsed-field gel electrophoresis (PFGE) and staphylococcal cassette chromosome (SCCmec) typing. The results indicated the occurrence of two MRSP clones that acquired distinct SCCmec elements in Europe (t02, PFGE type A, SCCmec type III,) and California (t06, PFGE type B, SCCmec type V). Sequence analysis of mecA revealed the occurrence of four alleles (mecA1 to mecA4), which correlated with the geographical origin of the isolates and enabled discrimination of two distinct subtypes within the European clone. The newly developed spa typing method appeared to be a promising tool for easy and rapid typing of MRSP, either alone or in combination with SCCmec and mecA typing for fine-structure epidemiological analysis.

Low occurrence of safety hazards in coagulase negative staphylococci isolated from fermented foodstuffs

Some coagulase negative staphylococci (CNS) species play an important role in the fermentation of meat and milk products and are considered as food-grade. However, the increasing clinical significance of CNS and the presence of undesirable and unsafe properties in CNS question their presence or use in food. Our goal was to assess the safety of CNS by developing a diagnostic microarray targeting 268 genes corresponding to safety hazards in a food context i.e. toxins (especially enterotoxins) and determinants of antibiotic resistance and biogenic amine production. Target genes were selected among staphylococci and Gram-positive species that may be in contact with CNS in foodstuffs. The diagnostic microarray was used to screen 129 strains belonging to the 2 dominant species isolated from foodstuffs (S. equorum and S. xylosus) and the 2 main species isolated both in foodstuffs and clinical samples (S. epidermidis and S. saprophyticus). Microarray data were further completed by antibiograms and measurement of biogenic amine production. Safety hazards associated with CNS were mostly limited to the presence of antibiotic resistance. Seventy-one percent of the strains possessed at least one gene encoding antibiotic resistance, while only one strain carried an enterotoxin gene. Most strains did not carry any genes encoding staphylococcal toxins (68%), non-staphylococcal toxins (95%) or decarboxylases involved in biogenic amine production (78%). Food safety hazards were more pronounced in S. epidermidis than in the three other species regardless the food or clinical origin of the strains. Seventy-six percent of the strains carrying genes encoding staphylococcal toxin and 69% of strains carrying 5 or more antibiotic determinants belonged to S. epidermidis species. The dominant antibiotic resistance targeted erythromycin, tetracycline and penicillin and were generally traced back to the presence of tetK and blaZ in the two latest cases. Six percent of the food-related strains produced significant amounts of biogenic amines in vitro without any of the corresponding genes detected, reflecting a lack of knowledge on genetic determinants of such production in staphylococci. This work gives a first picture of safety hazards within four species of CNS frequently isolated from food or clinical environment.

Emergence of scarlet fever Streptococcus pyogenes emm12 clones in Hong Kong is associated with toxin acquisition and multidrug resistance

A scarlet fever outbreak began in mainland China and Hong Kong in 2011 (refs. 1–6). Macrolide- and tetracycline-resistant Streptococcus pyogenes emm12 isolates represent the majority of clinical cases. Recently, we identified two mobile genetic elements that were closely associated with emm12 outbreak isolates: the integrative and conjugative element ICE-emm12, encoding genes for tetracycline and macrolide resistance, and prophage ΦHKU.vir, encoding the superantigens SSA and SpeC, as well as the DNase Spd1 (ref. 4). Here we sequenced the genomes of 141 emm12 isolates, including 132 isolated in Hong Kong between 2005 and 2011. We found that the introduction of several ICE-emm12 variants, ΦHKU.vir and a new prophage, ΦHKU.ssa, occurred in three distinct emm12 lineages late in the twentieth century. Acquisition of ssa and transposable elements encoding multidrug resistance genes triggered the expansion of scarlet fever–associated emm12 lineages in Hong Kong. The occurrence of multidrug-resistant ssa-harboring scarlet fever strains should prompt heightened surveillance within China and abroad for the dissemination of these mobile genetic elements.

Staphylococcus aureus Virulence Expression Is Impaired by Lactococcus lactis in Mixed Cultures

ABSTRACT Staphylococcus aureus is responsible for numerous food poisonings due to the production of enterotoxins by strains contaminating foodstuffs, especially dairy products. Several parameters, including interaction with antagonistic flora such as Lactococcus lactis, a lactic acid bacterium widely used in the dairy industry, can modulate S. aureus proliferation and virulence expression. We developed a dedicated S. aureus microarray to investigate the effect of L. lactis on staphylococcal gene expression in mixed cultures. This microarray was used to establish the transcriptomic profile of S. aureus in mixed cultures with L. lactis in a chemically defined medium held at a constant pH (6.6). Under these conditions, L. lactis hardly affected S. aureus growth. The expression of most genes involved in the cellular machinery, carbohydrate and nitrogen metabolism, and stress responses was only slightly modulated: a short time lag in mixed compared to pure cultures was observed. Interestingly, the induction of several virulence factors and regulators, including the agr locus, sarA, and some enterotoxins, was strongly affected. This work clearly underlines the complexity of L. lactis antagonistic potential for S. aureus and yields promising leads for investigations into nonantibiotic biocontrol of this major pathogen.

Genome-Wide Analysis of Ruminant Staphylococcus aureus Reveals Diversification of the Core Genome

Staphylococcus aureus causes disease in humans and a wide array of animals. Of note, S. aureus mastitis of ruminants, including cows, sheep, and goats, results in major economic losses worldwide. Extensive variation in genome content exists among S. aureus pathogenic clones. However, the genomic variation among S. aureus strains infecting different animal species has not been well examined. To investigate variation in the genome content of human and ruminant S. aureus, we carried out whole-genome PCR scanning (WGPS), comparative genomic hybridizations (CGH), and the directed DNA sequence analysis of strains of human, bovine, ovine, and caprine origin. Extensive variation in genome content was discovered, including host- and ruminant-specific genetic loci. Ovine and caprine strains were genetically allied, whereas bovine strains were heterogeneous in gene content. As expected, mobile genetic elements such as pathogenicity islands and bacteriophages contributed to the variation in genome content between strains. However, differences specific for ruminant strains were restricted to regions of the conserved core genome, which contained allelic variation in genes encoding proteins of known and unknown function. Many of these proteins are predicted to be exported and could play a role in host-pathogen interactions. The genomic regions of difference identified by the whole-genome approaches adopted in the current study represent excellent targets for studies of the molecular basis of S. aureus host adaptation.