Louis Garry

Organised Genome Dynamics in the Escherichia coli Species Results in Highly Diverse Adaptive Paths

The Escherichia coli species represents one of the best-studied model organisms, but also encompasses a variety of commensal and pathogenic strains that diversify by high rates of genetic change. We uniformly (re-) annotated the genomes of 20 commensal and pathogenic E. coli strains and one strain of E. fergusonii (the closest E. coli related species), including seven that we sequenced to completion. Within the ∼18,000 families of orthologous genes, we found ∼2,000 common to all strains. Although recombination rates are much higher than mutation rates, we show, both theoretically and using phylogenetic inference, that this does not obscure the phylogenetic signal, which places the B2 phylogenetic group and one group D strain at the basal position. Based on this phylogeny, we inferred past evolutionary events of gain and loss of genes, identifying functional classes under opposite selection pressures. We found an important adaptive role for metabolism diversification within group B2 and Shigella strains, but identified few or no extraintestinal virulence-specific genes, which could render difficult the development of a vaccine against extraintestinal infections. Genome flux in E. coli is confined to a small number of conserved positions in the chromosome, which most often are not associated with integrases or tRNA genes. Core genes flanking some of these regions show higher rates of recombination, suggesting that a gene, once acquired by a strain, spreads within the species by homologous recombination at the flanking genes. Finally, the genomes long-scale structure of recombination indicates lower recombination rates, but not higher mutation rates, at the terminus of replication. The ensuing effect of background selection and biased gene conversion may thus explain why this region is A+T-rich and shows high sequence divergence but low sequence polymorphism. Overall, despite a very high gene flow, genes co-exist in an organised genome.

Pathogenicity-associated islands in extraintestinal pathogenic Escherichia coli are fitness elements involved in intestinal colonization.

The virulence of many human pathogens does not seem to be an evolutionarily selected trait, but an accidental by-product of the selection that operates in another ecological context. We investigated the possibility that virulence of the extraintestinal pathogenic Escherichia coli (ExPEC) strains, which frequently cause disease in the host in which they asymptomatically colonize the intestine, is the consequence of commensalism. Most of the ExPEC virulence factors are clustered on genomic islands called pathogenicity-associated islands (PAIs). We constructed and characterized several mutants of the ExPEC 536 strain with either (i) deletions of each single PAI or (ii) a complete deletion of all seven PAIs. In vitro phenotypic characterization of 536 mutants showed that the seven PAIs were dispensable for growth in the absence of external stress, as well as under a range of biologically relevant stressors, i.e., serum, bile, and oxidative, nitrosative, hyperosmotic, and acidic stress. However, challenge against the wild-type (WT) strain in a murine model shows that the deletion of all seven PAIs drastically reduces the fitness of 536 during persistent intestinal colonization. This defect seems to be linked to the hypermotility observed for mutants devoid of all seven PAIs. In addition, we show that PAIs diminish fitness of their carrier during growth in urine, suggesting that urinary tract infections are unlikely to provide selective pressure for the maintenance of ExPEC PAIs. Our results are in accordance with the coincidental-evolution hypothesis postulating that extraintestinal E. coli virulence is a by-product of commensalism.

Activity of LY333328 Combined with Gentamicin In Vitro and in Rabbit Experimental Endocarditis Due to Vancomycin-Susceptible or -Resistant Enterococcus faecalis

ABSTRACT We investigated the activity of LY333328 alone and combined with gentamicin, both in vitro and in a rabbit model of experimental endocarditis, against the susceptible strain Enterococcus faecalis JH2-2 and its two glycopeptide-resistant transconjugants, BM4316 (VanA) and BM4275 (VanB). MICs of LY333328 and gentamicin were 2 and 16 μg/ml, respectively, for the three strains. In vitro, LY333328 alone was bactericidal at 24 h against JH2-2 at a concentration of 2 μg/ml and against BM4316 and BM4275 at a concentration of 30 μg/ml. The combination of LY333328 and gentamicin (4 μg/ml) was synergistic and bactericidal after 24 h of incubation against the three strains at LY333328 concentrations of 2 μg/ml for JH2-2 and 8 μg/ml for BM4275 and BM4316. The combination of LY333328 and gentamicin was the only regimen demonstrating in vitro bactericidal activity against BM4316. In vivo, intravenous treatment with LY333328 alone, providing peak and trough serum levels of 83.3 ± 1.3 and 3.8 ± 0.2 μg/ml, respectively, was inactive against BM4316 and BM4275 and selected mutants resistant to LY333328 in half of the rabbits infected with the VanA-type strain (MICs, 8 to 20 μg/ml). However, the LY333328-gentamicin combination was active against the three strains and prevented the emergence of mutants resistant to both components of the combination. We conclude that the LY333328-gentamicin combination might be of interest for the treatment of enterococcal infections, particularly against VanA-type strains.

Activities of Dalbavancin In Vitro and in a Rabbit Model of Experimental Endocarditis Due to Staphylococcus aureus with or without Reduced Susceptibility to Vancomycin and Teicoplanin

ABSTRACT For the treatment of rabbit endocarditis, dalbavancin given once daily (10 mg/kg of body weight for 4 days) or as a single 40-mg/kg dose was active against Staphylococcus aureus with or without reduced susceptibility to glycopeptides, as expected from its good in vitro activity, even in broth supplemented with 90% serum and given its prolonged elimination half-life.

The CTX-M-15-producing Escherichia coli clone O25b: H4-ST131 has high intestine colonization and urinary tract infection abilities.

Increasing numbers of pyelonephritis-associated uropathogenic Escherichia coli (UPEC) are exhibiting high resistance to antibiotic therapy. They include a particular clonal group, the CTX-M-15-producing O25b:H4-ST131 clone, which has been shown to have a high dissemination potential. Here we show that a representative isolate of this E. coli clone, referred to as TN03, has enhanced metabolic capacities, acts as a potent intestine- colonizing strain, and displays the typical features of UPEC strains. In a modified streptomycin-treated mouse model of intestinal colonization where streptomycin was stopped 5 days before inoculation, we show that TN03 outcompetes the commensal E. coli strains K-12 MG1655, IAI1, and ED1a at days 1 and 7. Using an experimental model of ascending UTI in C3H/HeN mice, we then show that TN03 colonized the urinary tract. One week after the transurethral inoculation of the TN03 isolates, the bacterial loads in the bladder and kidneys were significantly greater than those of two other UPEC strains (CFT073 and HT7) belonging to the same B2 phylogenetic group. The differences in bacterial loads did not seem to be directly linked to differences in the inflammatory response, since the intrarenal expression of chemokines and cytokines and the number of polymorphonuclear neutrophils attracted to the site of inflammation was the same in kidneys colonized by TN03, CFT073, or HT7. Lastly, we show that in vitro TN03 has a high maximum growth rate in both complex (Luria-Bertani and human urine) and minimum media. In conclusion, our findings indicate that TN03 is a potent UPEC strain that colonizes the intestinal tract and may persist in the kidneys of infected hosts.

Activities of the Combination of Quinupristin-Dalfopristin with Rifampin In Vitro and in Experimental Endocarditis Due to Staphylococcus aureus Strains with Various Phenotypes of Resistance to Macrolide-Lincosamide-Streptogramin Antibiotics

ABSTRACT We evaluated the activities of quinupristin-dalfopristin (Q-D), alone or in combination with rifampin, against three strains ofStaphylococcus aureus susceptible to rifampin (MIC, 0.06 μg/ml) and to Q-D (MICs, 0.5 to 1 μg/ml) but displaying various phenotypes of resistance to macrolide-lincosamide-streptogramin antibiotics: S. aureus HM1054 was susceptible to quinupristin and dalfopristin (MICs of 8 and 4 μg/ml, respectively); for S. aureus RP13, the MIC of dalfopristin was high (MICs of quinupristin and dalfopristin for strain RP13, 8 and 32 μg/ml, respectively); and S. aureus HM1054R was obtained after conjugative transfer of macrolide-lincosamide-streptogramin B constitutive resistance to HM1054, and the MIC of quinupristin for this strain was high (MICs of quinupristin and dalfopristin, 64 and 4 μg/ml, respectively). In vitro time-kill curve studies showed no difference between Q-D and rifampin, at a concentration of four times the MIC, against the three strains. Rabbits with aortic endocarditis were treated 4 days with Q-D, rifampin, or their combination. In vivo, the combination was highly bactericidal and synergistic against strains susceptible to quinupristin (HM1054 and RP13) and sterilized 94% of the animals. In contrast, the combination was neither synergistic nor bactericidal against the quinupristin-resistant strain (HM1054R) and did not prevent the emergence of mutants resistant to rifampin. We conclude that the in vivo synergistic and bactericidal activity of the combination of Q-D and rifampin against S. aureus is predicted by the absence of resistance to quinupristin but not by in vitro combination studies.

Effects of diclofenac on experimental streptococcal necrotizing fasciitis (NF) in rabbit

Abstract Aggravation of necrotizing fasciitis (NF) by the administration of non-steroidal antiinflammatory drugs (NSAIDs) has recently been suggested. A rabbit model of streptococcal NF was used to study the effects of parenteral administration of an NSAID on NF evolution and outcome. Of 16 rabbits inoculated with a Streptococcus pyogenes suspension together with staphylococcal alpha toxin, 8 were treated with two doses of 4 mg/kg diclofenac on day 1 after inoculation. Clinical, bacteriological and histological studies were performed until day 10. Under our experimental conditions, NSAID treatment significantly limited NF extension. A specific inverse relationship between the extent of inflammation and bacterial density in NF lesions was observed on day 1 after inoculation in the treated group suggesting that the greater severity of NF in humans treated with an NSAID could be due to the therapeutic delay induced by the misleading clinical effects of the NSAID, and not to inhibition of antibacterial defence.

Efficacies of Quinupristin-Dalfopristin Combined with Vancomycin In Vitro and in Experimental Endocarditis Due to Methicillin-Resistant Staphylococcus aureus in Relation to Cross-Resistance to Macrolides, Lincosamides, and Streptogramin B- Type Antibiotics

ABSTRACT A beneficial effect of the combination of quinupristin-dalfopristin and vancomycin was observed against two methicillin-resistant strains of Staphylococcus aureus harboring or not harboring the ermC gene, which codes for constitutive macrolide, lincosamide, and streptogramin B resistance. The beneficial effect was observed in time-kill studies, in which the drugs were used at inhibitory concentrations, and in a rabbit model of endocarditis, in which the combination was highly bactericidal and more active than monotherapies.

Influence of Reduced Susceptibility to Glycopeptides on Activities of Vancomycin and Teicoplanin against Staphylococcus aureus in Experimental Endocarditis

ABSTRACT The influence of reduced susceptibilities to glycopeptides on the activities of vancomycin and teicoplanin against an isogenic pair of clinical Staphylococcus aureus strains in experimental endocarditis was investigated. While vancomycin was similarly active against both strains, teicoplanin was approximately 100-fold less active against the resistant strain and selected for the emergence of more resistant subpopulations.

Effects of single and multiple pathogenicity island deletions on uropathogenic Escherichia coli strain 536 intrinsic extra-intestinal virulence.

BACKGROUND Escherichia coli strain 536 is a uropathogenic strain harboring 7 pathogenicity islands (PAIs). Whether or not these PAIs additively contribute to extra-intestinal virulence is unknown. METHODS We tested 7 single and several multiple-PAI deletion mutants in a mouse septicemia model by monitoring mouse survival. RESULTS E. coli 536 mutants in which PAIs II or III were deleted showed a significant decrease in virulence compared to the wild type (WT). All other single-PAI deletion mutants were as lethal to mice as was the WT. The mutant in which all seven PAIs were deleted showed milder virulence than the mutants in which PAI III or PAIs III and IV were deleted. The mutant in which PAIs II, III, IV, V, and VII were deleted tended to be less virulent than the mutant with deletion of PAI III only. All together, these results indicate a rough additive effect of PAIs in extra-intestinal virulence. CONCLUSION All PAIs of E. coli 536 do not play the same role in extra-intestinal virulence estimated in a mouse septicemia model and PAIs cooperate in an additive manner to achieve extra-intestinal virulence.