Jean-Claude Pechère

Swarming of Pseudomonas aeruginosa Is Dependent on Cell-to-Cell Signaling and Requires Flagella and Pili

We describe swarming in Pseudomonas aeruginosa as a third mode of surface translocation in addition to the previously described swimming and twitching motilities. Swarming in P. aeruginosa is induced on semisolid surfaces (0.5 to 0.7% agar) under conditions of nitrogen limitation and in response to certain amino acids. Glutamate, aspartate, histidine, or proline, when provided as the sole source of nitrogen, induced swarming, while arginine, asparagine, and glutamine, among other amino acids, did not sustain swarming. Cells from the edge of the swarm were about twice as long as cells from the swarm center. In both instances, bacteria possessing two polar flagella were observed by light and electron microscopy. While a fliC mutant of P. aeruginosa displayed slightly diminished swarming, a pilR and a pilA mutant, both deficient in type IV pili, were unable to swarm. Furthermore, cells with mutations in the las cell-to-cell signaling system showed diminished swarming behavior, while rhl mutants were completely unable to swarm. Evidence is presented for rhamnolipids being the actual surfactant involved in swarming motility, which explains the involvement of the cell-to-cell signaling circuitry of P. aeruginosa in this type of surface motility.

Characterization of MexE–MexF–OprN, a positively regulated multidrug efflux system of Pseudomonas aeruginosa

Antibiotic‐resistant mutants of Pseudomonas aeruginosa were generated using chloramphenicol and ciprofloxacin as selective agents. These mutants displayed a multidrug phenotype and overexpressed an outer membrane protein of 50kDa, which was shown by Western blot analysis to correspond to OprN. A cosmid clone harbouring the oprN gene was isolated by partial complementation of a mutant deficient in OprM, the outer membrane component of the mexAB–oprM efflux operon. Antibiotic‐accumulation studies indicated that OprN was part of an energy‐dependent antibiotic‐efflux system. Sequencing of a 6180bp fragment from the complementing cosmid revealed the presence of three open reading frames (ORFs), which exhibited amino acid similarity to the components of the mexAB–oprM and mexCD–oprJ efflux operons of P. aeruginosa. The ORFs were designated MexE, MexF and OprN. Mutation of the mexE gene eliminated the multidrug‐resistance phenotype in an OprN‐overexpressing strain, but did not affect the susceptibility profile of the wild‐type strain. Expression of the mexEF–oprN operon was shown to be positively regulated by a protein encoded on a 1.5 kb DNA fragment located upstream of mexE and belonging to the LysR family of transcriptional activators. The presence of a plasmid containing this DNA fragment was sufficient to confer a multidrug phenotype onto the wild‐type strain but not onto the mexE mutant. Evidence is provided to show that the mexEF–oprN operon may be involved in the excretion of intermediates for the biosynthesis of pyocyanin, a typical secondary metabolite of P. aeruginosa.

Azithromycin Inhibits Quorum Sensing in Pseudomonas aeruginosa

ABSTRACT We report that 2 μg of azithromycin/ml inhibits the quorum-sensing circuitry of Pseudomonas aeruginosa strain PAO1. Addition of synthetic autoinducers partially restored the expression of the trancriptional activator-encoding geneslasR and rhlR but not that of the autoinducer synthase-encoding gene lasI. We propose that azithromycin interferes with the synthesis of autoinducers, by an unknown mechanism, leading to a reduction of virulence factor production.

Overexpression of the MexEF-OprN Multidrug Efflux System Affects Cell-to-Cell Signaling in Pseudomonas aeruginosa

Intrinsic and acquired antibiotic resistance of the nosocomial pathogen Pseudomonas aeruginosa is mediated mainly by the expression of several efflux pumps of broad substrate specificity. Here we report that nfxC type mutants, overexpressing the MexEF-OprN efflux system, produce lower levels of extracellular virulence factors than the susceptible wild type. These include pyocyanin, elastase, and rhamnolipids, three factors controlled by the las and rhl quorum-sensing systems of P. aeruginosa. In agreement with these observations are the decreased transcription of the elastase gene lasB and the rhamnosyltransferase genes rhlAB measured in nfxC type mutants. Expression of the lasR and rhlR regulator genes was not affected in the nfxC type mutant. In contrast, transcription of the C4-homoserine lactone (C4-HSL) autoinducer synthase gene rhlI was reduced by 50% in the nfxC type mutant relative to that in the wild type. This correlates with a similar decrease in C4-HSL levels detected in supernatants of the nfxC type mutant. Transcription of an rhlAB-lacZ fusion could be partially restored by the addition of synthetic C4-HSL and Pseudomonas quinolone signal (PQS). It is proposed that the MexEF-OprN efflux pump affects intracellular PQS levels.

Effect of Clarithromycin in Patients with Sepsis and Ventilator-Associated Pneumonia

BACKGROUND Because clarithromycin provided beneficiary nonantibiotic effects in experimental studies, its efficacy was tested in patients with sepsis and ventilator-associated pneumonia (VAP). METHODS Two hundred patients with sepsis and VAP were enrolled in a double-blind, randomized, multicenter trial from June 2004 until November 2005. Clarithromycin (1 g) was administered intravenously once daily for 3 consecutive days in 100 patients; another 100 patients were treated with placebo. Main outcomes were resolution of VAP, duration of mechanical ventilation, and sepsis-related mortality within 28 days. RESULTS The groups were well matched with regard to demographic characteristics, disease severity, pathogens, and adequacy of the administered antimicrobials. Analysis comprising 141 patients who survived revealed that the median time for resolution of VAP was 15.5 days and 10.0 days among placebo- and clarithromycin-treated patients, respectively (P = .011); median times for weaning from mechanical ventilation were 22.5 days and 16.0 days, respectively (p = .049). Analysis comprising all enrolled patients showed a more rapid decrease of the clinical pulmonary infection score and a delay for advent of multiple organ dysfunction in clarithromycin-treated patients, compared with those of placebo-treated patients (p = .047). Among the 45 patients who died of sepsis, time to death was significantly prolonged in clarithromycin-treated compared with placebo-treated patients (p = .004). Serious adverse events were observed in 0% and 3% of placebo- and clarithromycin-treated patients, respectively (P = .25). CONCLUSIONS Clarithromycin accelerated the resolution of VAP and weaning from mechanical ventilation in surviving patients and delayed death in those who died of sepsis. The mortality rate at day 28 was not altered. Results are encouraging and render new perspectives on the management of sepsis and VAP.

Expression of host resistance to Salmonella typhi and Salmonella typhimurium: bacterial survival within macrophages of murine and human origin☆

Cell-association of various strains of Salmonella typhi and Salmonella typhimurium with different populations of macrophages was studied. Macrophages were infected, exposed to gentamicin, washed, and counts of viable bacteria protected from gentamicin killing were made. J774A.1 cells, a continuous macrophage-like cell line, were the most permissive, all strains tested achieving similar high recoveries. Virulent S. typhimurium 779C-Sms, but not avirulent S. typhimurium 779C-SmD, survived well in mouse peritoneal macrophages and human monocyte-derived macrophages. Virulent S. typhi Ty2 were killed by mouse peritoneal macrophages, but were able to survive within human monocyte-derived macrophages. Viable counts of clinical isolates of S. typhi within the human monocyte-derived phagocytes were lower as compared with those of S. typhi Ty2. Phagocytosis of opsonized and non-opsonized virulent S. typhi Ty2 and S. typhimurium 779C-SmS by mouse peritoneal macrophages failed to trigger their respiratory burst as assessed by the intracellular reduction of nitroblue tetrazolium dye (NBT). These experiments support the view that the intracellular survival of Salmonella is in part host dependent and specific in nature. They also suggest that virulence influences the survival and intracellular multiplication of Salmonella within macrophages, and that their ultimate fate within macrophages may not be related to oxygen-dependent mechanisms.

C-Terminal Region of Pseudomonas aeruginosa Outer Membrane Porin OprD Modulates Susceptibility to Meropenem

ABSTRACT We investigated the unusual susceptibility to meropenem observed for seven imipenem-resistant clinical isolates of Pseudomonas aeruginosa. These strains were genetically closely related, expressed OprD, as determined by Western blot analyses, and were resistant to imipenem (>5 μg/ml) but susceptible to meropenem (<1 μg/ml). The oprD genes from two isolates were entirely sequenced, and their deduced protein sequences showed 93% identity with that of OprD of strain PAO1. The major alteration consisted of the replacement of a stretch of 12 amino acids, located in putative external loop L7 of OprD, by a divergent sequence of 10 amino acid residues. The oprD gene variants and the wild-typeoprD gene were cloned and expressed in a definedoprD mutant. The meropenem MICs for strains carrying theoprD genes from clinical isolates were four times lower than that for the strain carrying the wild-type oprDgene. Imipenem activities, however, were comparable for all strains. Furthermore, meropenem hypersusceptibility was obtained with a hybrid OprD porin that consisted of the PAO1 oprD gene containing loop L7 from a clinical isolate. These results show that the C-terminal portion of OprD, in particular, loop L7, was responsible for the unusual meropenem hypersusceptibility. Competition experiments suggested that the observed OprD modifications in the clinical isolates did not affect antagonism between imipenem and the basic amino acidl-lysine. We further propose that shortening of putative loop L7 of the OprD porin by 2 amino acid residues sufficiently opens the porin channel to allow optimal penetration of meropenem and increase its activity. In contrast, this alteration would not affect susceptibility to a smaller carbapenem molecule, such as imipenem.

Treatment of vascular graft infection by in situ replacement with cryopreserved aortic allografts: An experimental study

PURPOSE The purposes of this study were to prove the efficacy of cryopreserved aortic allografts to treat an established vascular graft infection by in situ replacement in an animal model and to evaluate the role of the antibiotics normally used to decontaminate the allografts. METHODS Twenty-three dogs underwent infrarenal aortic replacement with a gelatin-sealed knitted polyester graft contaminated in vitro by Staphylococcus epidermidis RP-62. One week later, the 18 surviving animals underwent reoperation for graft removal and were randomized into three groups for in situ replacement: group I (control, n = 6) received a new gelatin-sealed graft; group II (n = 6) received a non-antibiotic-treated cryopreserved allograft; and group III (n = 6) received an antibiotic-treated cryopreserved allograft. Control grafts and allografts were removed 4 weeks after the initial intervention for quantitative bacteriologic analysis and histologic analysis. Bacteriologic results were expressed as colony-forming units per square centimeter of graft material. Qualitative bacteriologic analysis was also obtained from perigraft fluid and tissue. RESULTS All of the initially implanted grafts and all of the control grafts (group I) were infected at the time of removal. In group II, three out of six allografts were not totally incorporated, whereas in group III incorporation was always complete, with a significantly decreased inflammatory reaction. All of the antibiotic-treated allografts were sterile, whereas three untreated allografts grew bacteria. CONCLUSIONS In this model, cryopreserved aortic allografts were more resistant to reinfection than synthetic grafts after in situ replacement of an infected prosthetic graft. However, the antibiotic loading of the cryopreserved aortic allograft appears to be essential to obtain optimal therapeutic effects.

Combination Therapy: A Way to Limit Emergence of Resistance?

The ability of antibiotic combinations to limit the emergence of resistance during therapy was evaluated in a murine model. Peritonitis was produced by injecting a mixture containing 10(8) colony-forming units of bacteria and sterilized talcum into the peritoneum. Two hours later, a single antibiotic dose was administered subcutaneously. The next day, peritoneal bacterial populations were analyzed on Szybalskis gradients. Acquired resistance was recorded when there was at least a fourfold increase in minimum inhibitory concentrations compared with untreated animals. No resistance emerged after amikacin monotherapy (15 mg/kg); however, resistance was frequently observed after monotherapy with ceftriaxone (50 mg/kg) or pefloxacin (25 mg/kg). Resistance to ceftriaxone and pefloxacin emerged, respectively, in 15 percent and 83 percent of animals with Klebsiella pneumoniae, 71 percent and 54 percent with Enterobacter cloacae, 0 percent and 83 percent with Serratia marcescens, 25 percent and 100 percent with Pseudomonas aeruginosa, and 0 percent with both Escherichia coli and Staphylococcus aureus. In mice with K. pneumoniae or E. cloacae infections, any dual combination of amikacin, pefloxacin, and ceftriaxone produced less acquired resistance than did monotherapy. In these animals, the combination of ceftriaxone and pefloxacin abolished all resistance, whereas the combinations of amikacin plus ceftriaxone or amikacin plus pefloxacin reduced the frequency of resistance by more than half. In animals with P. aeruginosa or S. marcescens infections, resistance to pefloxacin diminished or disappeared after treatment with the combinations of pefloxacin plus ceftriaxone or pefloxacin plus amikacin. However, combinations with ceftriaxone resulted in more frequent resistance to ceftriaxone than did ceftriaxone alone. This was the case in P. aeruginosa infections treated with ceftriaxone plus amikacin (p less than 0.01), and in S. marcescens infections treated with ceftriaxone plus pefloxacin (p less than 0.05). Despite these certain notable exceptions, our data confirm that in most cases combination therapy does limit the emergence of resistance.

Streptogramins. A unique class of antibiotics.

Streptogramin antibiotics represent a unique class of antibacterials in that each member of the class consists of at least 2 structurally unrelated molecules: group A streptogramins (macrolactones) and group B streptogramins (cyclic hexadepsipeptides). Both group A and group B streptogramins inhibit protein synthesis at the ribosomal level, and they act synergistically against many isolates, their combination generating bactericidal activities and reducing the possibility of emergence of resistant strains. The mechanisms of acquired resistance to group B streptogramins are similar to those induced by erythromycin, but group A streptogramins remain unaffected by target modifications and active efflux. The pharmacokinetic parameters of group A and group B streptogramins in blood are quite similar. In addition, both the A and B groups penetrate and accumulate in macrophages and in the bacterial vegetations of experimental endocarditis. There are important structural and biological differences between the streptogramins and the macrolides. The main differentiating features are the rapid anti-bacterial killing of streptogramins and the rarity of cross-resistance between the 2 groups of antibiotics.