François Sanschagrin

Newly introduced genomic prophage islands are critical determinants of in vivo competitiveness in the Liverpool Epidemic Strain of Pseudomonas aeruginosa

Pseudomonas aeruginosa isolates have a highly conserved core genome representing up to 90% of the total genomic sequence with additional variable accessory genes, many of which are found in genomic islands or islets. The identification of the Liverpool Epidemic Strain (LES) in a childrens cystic fibrosis (CF) unit in 1996 and its subsequent observation in several centers in the United Kingdom challenged the previous widespread assumption that CF patients acquire only unique strains of P. aeruginosa from the environment. To learn about the forces that shaped the development of this important epidemic strain, the genome of the earliest archived LES isolate, LESB58, was sequenced. The sequence revealed the presence of many large genomic islands, including five prophage clusters, one defective (pyocin) prophage cluster, and five non-phage islands. To determine the role of these clusters, an unbiased signature tagged mutagenesis study was performed, followed by selection in the chronic rat lung infection model. Forty-seven mutants were identified by sequencing, including mutants in several genes known to be involved in Pseudomonas infection. Furthermore, genes from four prophage clusters and one genomic island were identified and in direct competition studies with the parent isolate; four were demonstrated to strongly impact on competitiveness in the chronic rat lung infection model. This strongly indicates that enhanced in vivo competitiveness is a major driver for maintenance and diversifying selection of these genomic prophage genes.

Structure and function of the Mur enzymes: development of novel inhibitors

One of the biggest challenges for recent medical research is the continuous development of new antibiotics interacting with bacterial essential mechanisms. The machinery for peptidoglycan biosynthesis is a rich source of crucial targets for antibacterial chemotherapy. The cytoplasmic steps of the biosynthesis of peptidoglycan precursor, catalysed by a series of Mur enzymes, are excellent candidates for drug development. There has been growing interest in these bacterial enzymes over the last decade. Many studies attempted to understand the detailed mechanisms and structural features of the key enzymes MurA to MurF. Only MurA is inhibited by a known antibiotic, fosfomycin. Several attempts made to develop novel inhibitors of this pathway are discussed in this review. Three novel inhibitors of MurA were identified recently. 4‐Thiazolidinone compounds were designed as MurB inhibitors. Many phosphinic acid derivatives and substrate analogues were identified as inhibitors of the MurC to MurF amino acid ligases.

Antimicrobial Resistance Genes in Enterotoxigenic Escherichia coli O149:K91 Isolates Obtained over a 23-Year Period from Pigs

ABSTRACT A total of 112 Escherichia coli O149:K91 strains isolated from pigs with diarrhea in Quebec, Canada, between 1978 and 2000 were characterized for their genotypic antimicrobial resistance profiles. Tests for resistance to 10 antimicrobial agents were conducted. Resistance to tetracycline and sulfonamides was found to be the most frequent, but resistance to cefotaxime and ceftiofur was absent. An increase in the number of isolates resistant to at least three antimicrobials was observed over time. The distribution of 28 resistance genes covering six antimicrobial families (beta-lactams, aminoglycosides, phenicols, tetracycline, trimethoprim, and sulfonamides) was assessed by colony hybridization. Significant differences in the distributions of tetracycline [tet(A), tet(B), tet(C)], trimethoprim (dhfrI, dhfrV, dhfrXIII), and sulfonamide (sulI, sulII) resistance genes were observed during the study period (1978 to 2000). Sixty percent of the isolates possessed a class 1 integron, illustrating the importance of integrons in the epidemiology of antibiotic resistance in E. coli strains from pigs. Amplification of the integrons variable region resulted in four distinct fragments of 1, 1.3, 1.6, and 1.8 kb, with the 1.6- and 1.8-kb fragments appearing only during the last half of the study period. Examination of linkages among the different resistance genes showed a variety of positive and negative associations. Association analysis of isolates divided into two groups, those isolated between 1978 and 1989 and those isolated between 1990 and 2000, revealed the appearance of new positive resistance gene associations. Our genotypic resistance analyses of ETEC isolates from pigs indicate that many of the antibiotic resistance genes behind phenotypic resistance are not static but, rather, are in a state of flux driven by various selection forces such as the use of specific antimicrobials.

Heterogeneity among Virulence and Antimicrobial Resistance Gene Profiles of Extraintestinal Escherichia coli Isolates of Animal and Human Origin

ABSTRACT Extraintestinal pathogenic Escherichia coli (ExPEC) isolates collected from different infected animals and from human patients with extraintestinal infections in 2001 were characterized for their phenotypic and genotypic antimicrobial resistance profiles, genotypes, and key virulence factors. Among the 10 antimicrobial agents tested, resistance to ampicillin, tetracycline, and sulfonamides was most frequent. Multiresistant strains were found in both the animal and the human groups of isolates. Resistance gene distribution was assessed by colony hybridization. Similar antibiotic resistance patterns could be observed in the animal and the human isolates. Although some resistance genes, such as blaTEM, sulI, and sulII, were equally represented in the animal and human ExPEC isolates, differences in the distributions of tetracycline [tet(D)], chloramphenicol (catI, catIII, and floR), and trimethoprim (dhfrI, dhfrV, dhfrVII, and dhfrXIII) resistance genes were observed between the animal and the human isolates. Approximately one-third of the ExPEC isolates possessed a class 1 integron. The four major different variable regions of the class 1 integron contained aminoglycoside (aadA1, aadA2, aadA5, and aadA6) and/or trimethoprim (dhfrIb, dhfrXII, and dhfrXVII) resistance genes. The ExPEC strains belonged to different phylogenetic groups, depending on their host origin. Strains isolated from animal tissues belonged to either a commensal group (group A or B1) or a virulent group (group B2 or D), while the majority of the human isolates belonged to a virulent group (group B2 or D). Although the limited number of isolates evaluated in the present study prevents firm epidemiological conclusions from being made, on a more global scale, these data demonstrate that extraintestinal isolates of E. coli can possess relatively distinct intra- and intergroup resistance gene profiles, with animal isolates presenting a more heterogeneous group than human isolates.

The sensor kinase PhoQ mediates virulence in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a ubiquitous environmental Gram-negative bacterium that is also a major opportunistic human pathogen in nosocomial infections and cystic fibrosis chronic lung infections. PhoP-PhoQ is a two-component regulatory system that has been identified as essential for virulence and cationic antimicrobial peptide resistance in several other Gram-negative bacteria. This study demonstrated that mutation of phoQ caused reduced twitching motility, biofilm formation and rapid attachment to surfaces, 2.2-fold reduced cytotoxicity to human lung epithelial cells, substantially reduced lettuce leaf virulence, and a major, 10 000-fold reduction in competitiveness in chronic rat lung infections. Microarray analysis revealed that PhoQ controlled the expression of many genes consistent with these phenotypes and with its known role in polymyxin B resistance. It was also demonstrated that PhoQ controls the expression of many genes outside the known PhoP regulon.

In Vivo Growth of Pseudomonas aeruginosa Strains PAO1 and PA14 and the Hypervirulent Strain LESB58 in a Rat Model of Chronic Lung Infection

Pseudomonas aeruginosa chronic lung infections are the major cause of morbidity and mortality in cystic fibrosis (CF) patients. The P. aeruginosa strains PAO1 and PA14 were compared with the Liverpool epidemic strain LESB58 to assess in vivo growth, infection kinetics, and bacterial persistence and localization within tissues in a rat model of chronic lung infection. The three P. aeruginosa strains demonstrated similar growth curves in vivo but differences in tissue distribution. The LESB58 strain persisted in the bronchial lumen, while the PAO1 and PA14 strains were found localized in the alveolar regions and grew as macrocolonies after day 7 postinfection. Bacterial strains were compared for swimming and twitching motility and for the production of biofilm. The P. aeruginosa LESB58 strain produced more biofilm than PAO1 and PA14. Competitive index (CI) analysis of PAO1, PA14, and LESB58 in vivo indicated CI values of 0.002, 0.0002, and 0.14 between PAO1-PA14, PAO1-LESB58, and LESB58-PA14, respectively. CI analysis comparing the in vivo growth of the PAO1 DeltaPA5441 mutant and four PA14 surface attachment-defective (sad) mutants gave CI values 10 to 1,000 times lower in competitions with their respective wild-type strains PAO1 and PA14. P. aeruginosa strains studied in the rat model of chronic lung infection demonstrated similar in vivo growth but differences in virulence as shown with a competitive in vivo assay. These differences were further confirmed with biofilm and motility in vitro assays, where strain LESB58 produced more biofilm but had less capacity for motility than PAO1 and PA14.

In Vivo-Induced Genes in Pseudomonas aeruginosa

ABSTRACT In vivo expression technology was used for testingPseudomonas aeruginosa in the rat lung model of chronic infection and in a mouse model of systemic infection. Three of the eight ivi proteins found showed sequence identity to known virulence factors involved in iron acquisition via an open reading frame (called pvdI) implicated in pyoverdine biosynthesis, membrane biogenesis (FtsY), and adhesion (Hag2).

Primary structure of OXA-3 and phylogeny of oxacillin-hydrolyzing class D beta-lactamases.

We determined the nucleotide sequence of the blaOXA-3(pMG25) gene from Pseudomonas aeruginosa. The bla structural gene encoded a protein of 275 amino acids representing one monomer of 31,879 Da for the OXA-3 enzyme. Comparisons between the OXA-3 nucleotide and amino acid sequences and those of class A, B, C, and D beta-lactamases were performed. An alignment of the eight known class D beta-lactamases including OXA-3 demonstrated the presence of conserved amino acids. In addition, conserved motifs composed of identical amino acids typical of penicillin-recognizing proteins and specific class D motifs were identified. These conserved motifs were considered for possible roles in the structure and function of oxacillinases. On the basis of the alignment and identity scores, a dendrogram was constructed. The phylogenetic data obtained revealed five groups of class D beta-lactamases with large evolutionary distances between each group.

Discovering essential and infection-related genes

Transposon-based approaches are very powerful for identification of essential and infection-related genes in bacteria, particularly in the context of microbial genomics. We describe recent progress in several of these approaches, and their underlying principles. The essential gene test (EGT) is a transposon-based technique that can rapidly identify a nucleotide sequence from a database as essential or dispensable. Also, variations of in vitro transposon mutagenesis applications, such as genomic analysis and mapping by in vitro transposition (GAMBIT), are described. The development of techniques including PCR-based signature-tagged mutagenesis is now used to find essential virulence genes in different bacterial hosts. These approaches form the basis for the identification of microbial targets in development of novel antimicrobials and vaccines by the biotechnology and pharmaceutical industry.

Thermoregulated Optical Properties of Peptidic Pseudorotaxanes

Natural peptide spine and artificial crown ether are blended together to form a hybrid structure. A dicationic benzylammonium guest threads through the crown ether pendants and the resulting noncovalent, self-assembled pseudorotaxane complex, shown schematically in the picture, is both stable and has optical properties dependant on temperature.