Postantibiotic and bactericidal effect of imipenem againstPseudomonas aeruginosa

The postantibiotic effect of imipenem onPseudomonas aeruginosa was studied at different inocula using one ATCC strain and four clinical isolates. The postantibiotic effect was measured using two different methods: viable counts and bioluminescence assay of intracellular bacterial ATP. The postantibiotic effect could be demonstrated with both methods (viable counts 1–2 h, ATP assay 3–5 h) for all strains at an inoculum of 10⁶ CFU/ml. When the inoculum was raised to 10⁸ CFU/ml, no postantibiotic effect could be observed with either method using routine growth conditions. This disappearance of the postantibiotic effect coincided with a loss of bactericidal effect of imipenem when high inocula were used. Improved oxygenation of the cultures restored the bactericidal and postantibiotic effects of imipenem at high inocula.     

Effect of clavulanic acid and/or polymorphonuclear neutrophils on amoxicillin bactericidal activity againstStreptococcus pneumoniae

The effects of polymorphonuclear neutrophils (PMNs) and/or clavulanic acid on the bactericidal activity of amoxicillin (at human serum achievable concentrations) against a serotype 3 penicillin-susceptibleStreptococcus pneumoniae strain [minimal inhibitory concentration /minimal bactericidal concentration (MIC/MBC) values of penicillin, amoxicillin, and amoxicillin/clavulanic acid (2:1)=0.01/0.01 g/ml] and a serotype 9 penicillin-resistant strain [MIC/MBC of penicillin, amoxicillin, and amoxicillin/clavulanic acid (2:1)=1/2 g/ml] were studied. Against the penicillin-resistant strain, subinhibitory concentrations of amoxicillin reduced the growth rate; this effect was increased by the addition of clavulanic acid. A reduction of the penicillin-resistant initial inocula (3 × 10⁶ cfu/ml) at subinhibitory concentrations was obtained only with amoxicillin plus clavulanic acid and PMNs. At suprainhibitory concentrations, both clavulanic acid and PMNs increased the bactericidal activity of amoxicillin, as evidenced by an increased reduction in the penicillin-resistant initial inocula. The combined effect of these antibiotics and immune defenses may help explain the maintenance of their clinical efficacy in respiratory tract infections, despite the increase in the incidence of penicillin-resistant pneumococci.     

Rapid decrease of free vancomycin in dense staphylococcal cultures

Bacterial numbers in broth cultures were determined by bioluminescence assay of intracellular bacterial ATP. Broth MICs for strains of Staphylococcus epidermidis (ATCC 14990 and 35984) and Staphylococcus aureus (ATCC 25923, 29213 and 6538) were determined for cultures with different inocula (10⁵–10⁸ bacteria/ml) after 24 h of incubation in supplemented Mueller–Hinton broth containing vancomycin. All of the tested strains except one were susceptible to methicillin, and all of the strains were susceptible to vancomycin. Free vancomycin concentrations in the broth cultures of all strains were determined with an agar well bioassay after 24 h of incubation. Free vancomycin concentrations and bacterial numbers of ATCC 35984 and ATCC 29213 were also determined after 0.5, 2, 4, and 8 h. In a low inoculum (10⁵ bacteria/ml), the broth MICs were 1–4 μg/ml. In a high inoculum (∼10⁸ bacteria/ml), the broth MICs increased two- to fourfold to 4–8 μg/ml. In dense inocula (∼10⁹–10¹⁰ bacteria/ml), the concentrations of free vancomycin in the broth were reduced, in most cases below the detection limit of the bioassay (≤0.5 μg/ml). This reduction of free vancomycin was fast, occurring in initially dense inocula in less than 30 min. No emergence of resistance was seen. These results show a rapid reduction of free vancomycin in the broth and a simultaneous increase in broth MICs in high inocula, without development of resistance. This indicates that the dosing regimen of vancomycin is of particular importance in staphylococcal infections with dense inocula, e.g. infective endocarditis.     

Reproducible measurement of vancomycin MICs within the susceptible range in <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> by a broth microdilution method with a “quasi-continuum” gradient of antibiotic concentrations

The availability of reproducible broth microdilution (BMD) methods including inter log₂ antibiotic dilutions for measuring Staphylococcus aureus (SA) vancomycin minimum inhibitory concentrations (MICs) within the susceptible range is needed to elucidate the impact of vancomycin MICs on clinical outcomes of invasive SA infections. Here, we report on the development of a very precise BMD method that incorporates the following incremental antibiotic concentrations: 0.50, 0.62, 0.75, 0.87, 1.0, 1.25, 1.40, 1.50, 1.60, 1.75, and 2.0 μg/mL. The intra- and inter-assay coefficients of variation of this method were around 20%. The mean of the differences in MIC values for all isolates obtained across two independent runs performed at one center was 0.04 μg/mL [95% confidence interval (CI), 0.011–0.07 μg/mL] and that for ten isolates measured at two different centers was 0.04 μg/mL (95% CI, 0–13 μg/mL). Vancomycin MIC values differed by less than 0.1 μg/mL between runs for most isolates. Storage of isolates at ?20 °C for up to 3 months had no impact on the vancomycin MIC values. The mean vancomycin MIC values obtained by the Etest using a standard inoculum (0.5 McFarland) were significantly higher (p ≤ 0.001) than those measured by BMD and the MIC values measured by the two methods correlated poorly (Rho, 0.319; p = 0.148). Nevertheless, the mean MIC values measured by the Etest using lower inocula (10⁷ or 10⁶ CFU/mL) and those measured by BMD were comparable and correlated significantly (p = 0.004 for 10⁷ CFU/mL and p = 0.029 for 10⁶ CFU/mL).     

Rapid diagnosis of bacteraemia by measuring bacterial adenosine triphosphate in blood culture bottles using bioluminescence

Bacterial adenosine triphosphate (ATP) was determined in blood culture bottles using bioluminescence. Seventy bottles were inoculated with blood and bacteria. Immediately after inoculation and every 2 h thereafter for the next 10 h, bacterial ATP was measured as well as the number of colony-forming units (CFU) per millilitre of blood culture bottle. The curves for the concentration of CFUs and for the amount of bacterial ATP present are quite similar and indicate that the method described promises good results in the early detection of bacteria in blood culture bottles. ATP values > 0.1 ng/ml blood culture bottle indicate bacterial growth. This limit of detection is reached at a concentration of 4–6×10⁴ CFU/ml of theEscherichia coli ATCC 11229 strain tested.     

Comparative in vitro activity of lomefloxacin,a new difluoroquinolone

The in vitro activity of lomefloxacin, a new difluoroquinolone, was compared with that of norfloxacin, ciprofloxacin, gentamicin and ceftazidime against a total of 577 recent clinical isolates. MICs were determined by a standard agar dilution procedure, and two inocula (10⁴ and 10⁶ CFU) were used throughout. Lomefloxacin inhibited most species of theEnterobacteriaceae, Staphylococcus aureus (including methicillin-resistant strains) andHaemophilus influenzae at 1 mg/l.Pseudomonas aeruginosa (MIC 90, 4 mg/l) was somewhat more resistant, andPseudomonas maltophilia (MIC 90, 16 mg/l) and theBacteroides fragilis group (MIC 90, 32 mg/l) were considerably more resistant. Overall, lomefloxacin was as active as norfloxacin, but was two- to eightfold less active than ciprofloxacin against most species tested.     

Antimicrobial activity of heparin

Single clinical isolates of eight species of microorganisms were incubated in solutions of heparin and brain heart infusion broth at various concentrations to determine the possible antibacterial effect of heparin. At heparin concentrations ranging from 12.5 to 400 U/ml, the effect on Escherichia coli, Pseudomonas aeruginosa, Candida albicans, Staphylococcus aureus, and S. epidermidis varied with brain heart infusion broth concentrations of 1.2 to 10% and inocula of 10² to 10⁶ colony-forming units per ml; similar effects were not observed with Klebsiella pneumoniae, Enterobacter aerogenes, and Citrobacter spp. The minimal inhibitory concentrations of heparin for ten strains of each species were determined in 2.5% brain heart infusion broth with inocula of 10⁴ colony-forming units per ml. All 10 isolates of S. aureus and all 10 of S. epidermidis were inhibited by heparin concentrations of 125 to 500 U/ml. Three E. coli, four P. aeruginosa, and nine C. albicans strains were inhibited by ≤500 U of heparin per ml. None of the K. pneumoniae, E. aerogenes, Enterobacter cloacae, and Citrobacter spp. was inhibited by heparin at ≤500 U/ml. Heparin inhibition of S. aureus in 2.5% brain heart infusion broth-500 U of heparin per ml could be quantitatively overcome by addition of magnesium, calcium, or magnesium and calcium. These data suggest that the growth of microorganisms from heparin-containing material may be suppressed.     

Efficacy evaluation of iclaprim in a neutropenic rat lung infection model with methicillin-resistant <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> entrapped in alginate microspheres

The objective of this study was to demonstrate the efficacy of iclaprim in a neutropenic rat lung infection model with methicillin-resistant Staphylococcus aureus (MRSA) entrapped in alginate beads. An inoculum of 5.25?×?10⁵ colony-forming units (CFU)/mL of S. aureus strain AH1252 was administered intratracheally to rats with prepared alginate bacteria suspensions. Beginning 2 h post-infection, rats received: (1) iclaprim 80 mg/kg (n?=?16); (2) iclaprim 60 mg/kg (n?=?16), or (3) vancomycin 50 mg/kg (n?=?24), for 3 days via subcutaneous (SC) injection every 12 h. Twelve hours after the last treatment, rats were euthanized and lungs collected for CFU determination. Iclaprim administered at 80 mg/kg or 60 mg/kg or vancomycin 50 mg/kg SC twice a day for 3 days resulted in a 6.05 log₁₀ CFU reduction (iclaprim 80 mg/kg compared with control, p?<?0.0001), 5.11 log₁₀ CFU reduction (iclaprim 60 mg/kg compared with control, p?<?0.0001), and 3.42 log₁₀ CFU reduction, respectively, from the controls (p?<?0.0001). Iclaprim 80 mg/kg and 60 mg/kg resulted in 2.59 and 1.69 log₁₀ CFU reductions, respectively, from vancomycin-treated animals (80 mg/kg iclaprim vs. vancomycin, p?=?0.0005; 60 mg/kg iclaprim vs. vancomycin, p?=?0.07). Animals receiving iclaprim, vancomycin, and controls demonstrated 100%, 91.7%, and 48.3% survival, respectively. In this neutropenic rat S. aureus lung infection model, rats receiving iclaprim demonstrated a greater CFU reduction than the controls or those receiving vancomycin.     

Human polymorphonuclear leukocyte interaction with cyclosporine A.

The effects of cyclosporin A (cyA) on human polymorphonuclear leukocyte function, including phagocytosis, its associated metabolic burst, bacterial killing, and chemotaxis, were evaluated. Both Pseudomonas aeruginosa and Staphylococcus aureus were used as test particles. Polymorphonuclear leukocytes incubated in 10 and 50 micrograms of cyA per ml behaved normally with respect to phagocytosis and hexose monophosphate shunt activity at both high (10:1) and low (2:1) S. aureus/leukocyte ratios. With a small bacterial inoculum, killing of S. aureus was slightly impaired at early times only in the presence of 50 micrograms of cyA per ml. Phagocytosis and killing of P. aeruginosa with both large and small bacterial inocula were unaffected by cyA. Chemotaxis was within normal limits under all conditions. In addition, polymorphonuclear leukocytes from four renal transplant recipients receiving both cyA and prednisone demonstrated normal metabolic bursts and bacterial killing with both small and large inocula of S. aureus.     

Regulation of superoxide anion generation in bovine alveolar macrophages by bacterial lipopolysaccharide,serum proteins,and modulators of signal transduction

The respiratory burst of phagocytes in an important leukocyte function which results in generation of oxygen species that are both microbicidal and potentially damaging to host tissues. We investigated regulation of the respiratory burst of alveolar macrophages in response to lipopolysaccharide (LPS) derived from gramnegative bacteria, serum proteins, and several modulators of signal transduction. When employed as a single stimulus, LPS (E. coli 055 B5, 10 ng/ml-1g/ml) was a weak stimulus for generation of superoxide anion (O ₂ ^– ) as compared to the potent effect of the protein kinase C activator, phorbol 12-myristate 13-acetate (PMA; 500 ng/ml). However, when LPS was combined with fetal bovine serum (FBS; 0.4–1.0% vol/vol, equivalent to 128–320g protein/ml), O ₂ ^– generation was enhanced approximately two-fold over LPS alone. A chromatographically-derived bovine serum fraction which contained bovine lipopolysaccharide-binding protein (bLBP; 0.25–1.0g/ ml) was an effective substitute for FBS at a much lower protein concentration than whole FBS, and a similar synergistic effect with LPS on O ₂ ^– generation was observed. Stimulation of macrophages for generation of O ₂ ^– either with LPS alone or with LPS plus serum/serum fraction was suppressed by the protein tyrosine kinase inhibitor herbimycin A (0.2 ng/ml), and the calcium chelator BAPTA (12M), but not by modulators of G-proteins, including pertussis toxin (10 ng/ml) and cholera toxin (5g/ml protein). Essentially complete inhibition of O ₂ ^– synthesis by herbimycin A and BAPTA occurred in the presence of LPS and the bLBP-containing serum fraction (1g/ml protein), but only partial inhibition (46.7% and 64.1%, respectively) was observed in the presence of LPS plus FBS (256g/ml protein). These results indicate that when LPS is used as a sole stimulus it induces modest respiratory burst activity. However, when LPS is combined with appropriate serum components, it stimulates alveolar macrophages to generate larger amounts of O ₂ ^– . Cellular signaling pathways important in stimulation of macrophages by LPS and serum components are protein tyrosine kinase- and Ca⁺⁺-dependent, but do not relay on G-protein-mediated signaling.     

Development and Characterization of a Staphylococcus aureus Nasal Colonization Model in Mice

Staphylococcus aureus nasal carriage is a risk factor for infection in humans, particularly in the hospital environment. Attenuation of carriage has proven effective in reducing the prevalence of infection in some high-risk groups. To study staphylococcal factors that influence nasal colonization, a mouse model of S. aureus nasal colonization was developed. Mice were inoculated intranasally with S. aureus Reynolds, and nasal carriage was evaluated by quantitating cultures of the nasal tissues from mice sacrificed at various time points after inoculation. The majority of mice inoculated with 10(8) CFU of S. aureus maintained nasal carriage for at least 20 days. Nasal colonization rates were similar for inbred (BALB/c and C57BL/6) and outbred (ICR) mice. Colonization was not affected by mouse passage of strain Reynolds. Lower inoculum doses (<10(7) CFU) resulted in reduced colonization after 7 days. However, mice given streptomycin in their drinking water developed long-term carriage of S. aureus, and they were colonized with inocula as low as 10(5) CFU. Nasal colonization was also established with two other S. aureus strains (one strain each of human and murine origins). S. aureus recovered from the nares of experimentally colonized mice expressed high levels of capsule, and the ability of a capsule-defective mutant to persist in the nares was reduced in comparison to that of the parent strain. This nasal colonization model should prove useful for studies of factors that mediate S. aureus colonization and for assessment of targets for antimicrobial intervention or vaccine development.     

Immunomodulation by indoleamines: Serotonin and melatonin action on DNA and interferon-γ synthesis by human peripheral blood mononuclear cells

Different concentrations of indoleamines, serotonin and melatonin, inhibited phytohemagglutinin stimulated DNA synthesis. Thus, 10^–3 to 10^–4 M of either indoleamine acted at the optimal phytohemagglutinin concentration, while 10^–3 to 10^–7 M acted at suboptimal phytohemagglutinin levels. The serotonin effect was reversed by the serotonergic S₁-S₂ receptor antagonist methysergide but not by the S₂ antagonist ketanserin. This indicates that only the S₁ receptor is involved in the inhibitory effect. Inhibition of lymphoproliferation by indoleamines was also exerted on pokeweed mitogen and protein A fromStaphylococcus aureus stimulations. Serotonin and melatonin also inhibited phytohemagglutinin and protein A from Staphylococcus aureus induction of interferon- synthesis. The initial uptake of Ca²⁺ was not affected by indoleamines, suggesting that it is not the mechanism of their inhibitory effects. As interferon- induced tryptophan uptake by T lymphocyte- and macrophage-depleted populations, and tryptophan is the metabolic precursor of serotonin and melatonin, a new immunoregulatory circuit is postulated.     

Detection and differentiation of in vitro-spiked bacteria by real-time PCR and melting-curve analysis

We introduce a consensus real-time PCR protocol for the detection of bacterial DNA from laboratory-prepared specimens such as water, urine, and plasma. This prototype detection system enables an exact Gram stain classification and, in particular, screening for specific species of 17 intensive care unit-relevant bacteria by means of fluorescence hybridization probes and melting-curve analysis in a one-run experiment. One strain of every species was tested at a final density of 10(6) CFU/ml. All bacteria examined except Staphylococcus aureus and Staphylococcus epidermidis could be differentiated successfully; S. aureus and S. epidermidis could only be classified as "Staphylococcus species." The hands-on time for preparation of the DNA, performance of the PCR, and evaluation of the PCR results was less than 4 h. Nevertheless, this prototype detection system requires more clinical validation.     

Thiourea and dimethylthiourea decrease human neutrophil bactericidal function in vitro

Addition of thiourea (TU) or dimethylthiourea (DMTU) decreased killing ofStaphylococcus aureus, 502A, and decreased concentrations of hydrogen peroxide (H₂O₂), and hydroxyl radical (·OH), but not Superoxide anion (O₂ ^–) or lysozyme concentrations, in mixtures containing human neutrophilsin vitro. Addition of TU or DMTU also decreased concentrations of H₂O₂, · OH, or hypochlorous acid (HOCl) in neutrophil-free mixtures exposed to-D-glucose and glucose oxidase, gamma irradiation, or HOCl, respectively. Our results suggest that TU or DMTU can decrease neutrophil-mediated killing of bacteria by inhibiting O₂ metabolite-dependent bactericidal mechanisms.     

In vitro activity of the novel cephalosporin GR69153

The in vitro activity of GR69153 was compared to that of ceftazidime, ceftriaxone, imipenem and gentamicin against a total of 702 recent clinical isolates. MICs were determined by a standard agar dilution procedure and two inocula (10⁴ and 10⁸ cfu) were used throughout. GR69153 inhibited 90 % of isolates ofEscherichia coli, Klebsiella pneumonia andProteus mirabilis at 0.25 mg/l and 90 % of isolates ofPseudomonas aeruginosa at 1 mg/l.Citrobacter freundii (MIC90 16 mg/l),Morganella morganii (MIC90 128 mg/l) andEnterobacter spp. (MIC90>128 mg/l) were considerably more resistant to GR69153. GR69153 was four-fold more active than ceftazidime against methicillin-sensitiveStaphylococcus aureus but was inactive against methicillin-resistantStaphylococcus aureus, Enterococcus faecalis andBacteroides fragilis group.     

Blockade of leukocyte haptokinesis and haptotaxis by ketoprofen, diclofenac and SC-560

Background

Infections caused by community-associated strains of methicillin-resistant Staphylococcus aureus (CA-MRSA) are associated with a marked and prolonged host inflammatory response. In a sepsis simulation model, we tested whether the anesthetic ketamine inhibits the macrophage TNF response to antibiotic-exposed CA-MRSA bacteria via its antagonism of N-methyl-D-aspartate (NMDA) receptors. RAW264.7 cells were stimulated for 18 hrs with 10⁵ to 10⁷ CFU/mL inocula of either of two prototypical CA-MRSA isolates, USA300 strain LAC and USA400 strain MW2, in the presence of either vancomycin or daptomycin. One hour before bacterial stimulation, ketamine was added with or without MK-801 (dizocilpine, a chemically unrelated non-competitive NMDA receptor antagonist), APV (D-2-amino-5-phosphono-valerate, a competitive NMDA receptor antagonist), NMDA, or combinations of these agents. Supernatants were collected and assayed for TNF concentration by ELISA.

Results

RAW264.7 cells exposed to either LAC or MW2 in the presence of daptomycin secreted less TNF than in the presence of vancomycin. The addition of ketamine inhibited macrophage TNF secretion after stimulation with either of the CA-MRSA isolates (LAC, MW2) in the presence of either antibiotic. The NMDA inhibitors, MK-801 and APV, also suppressed macrophage TNF secretion after stimulation with either of the antibiotic-exposed CA-MRSA isolates, and the effect was not additive or synergistic with ketamine. The addition of NMDA substrate augmented TNF secretion in response to the CA-MRSA bacteria, and the addition of APV suppressed the effect of NMDA in a dose-dependent fashion.

Conclusions

Ketamine inhibits TNF secretion by MRSA-stimulated RAW264.7 macrophages and the mechanism likely involves NMDA receptor antagonism. These findings may have therapeutic significance in MRSA sepsis.     

Staphylococcal Enterotoxin Synthesis During the Exponential, Transitional, and Stationary Growth Phases

Small inocula (1 to 10 colony-forming units per ml of broth) of Staphylococcus aureus strains S-6, S-6R, and FRI-100 were employed to study growth and enterotoxin synthesis in 4% protein hydrolysate powder broths. For each strain, the exponential growth phase ended once the population approached 10(9) to 2 x 10(9) colony-forming units per ml. By that time, the concentrations of enterotoxins A and B reached the minimal level (1 to 2 mug/ml) at which the single gel diffusion tube method becomes applicable. By microslides and reverse passive hemagglutination, enterotoxins A and B were found to be synthesized during the exponential growth phase, but at different exponential rates.     

Gluconobacter as Well as Asaia Species,Newly Emerging Opportunistic Human Pathogens among Acetic Acid Bacteria

Acetic acid bacteria (AAB) are broadly used in industrial food processing. Among them, members of the genera Asaia, Acetobacter, and Granulibacter were recently reported to be human opportunistic pathogens. We isolated AAB from clinical samples from three patients and describe here the clinical and bacteriological features of these cases. We report for the first time (i) the isolation of a Gluconobacter sp. from human clinical samples; (ii) the successive isolation of different AAB, i.e., an Asaia sp. and two unrelated Gluconobacter spp., from a cystic fibrosis patient; and (iii) persistent colonization of the respiratory tract by a Gluconobacter sp. in this patient. We reviewed the main clinical features associated with AAB isolation identified in the 10 documented reports currently available in the literature. Albeit rare, infections as well as colonization with AAB are increasingly reported in patients with underlying chronic diseases and/or indwelling devices. Clinicians as well as medical microbiologists should be aware of these unusual opportunistic pathogens, which are difficult to detect during standard medical microbiological investigations and which are multiresistant to antimicrobial agents. Molecular methods are required for identification of genera of AAB, but the results may remain inconclusive for identification to the species level.Acetic acid bacteria (AAB) belong to the family Acetobacteraceae and oxidize alcohols or sugars, leading to the production of acetic acid. AAB, such as members of the genera Acetobacter, Asaia, Gluconobacter, and Gluconacetobacter, are commonly found in soil or are associated with plants (35). They have been used in industrial food processing throughout human history, especially to convert wine to vinegar and to produce tropical fermented products. The first report of infection with AAB in humans dates from 2004, when Snyder et al. reported a case of peritonitis associated with Asaia bogorensis in a patient with a peritoneal dialysis catheter (27). Greenberg et al. reported 2 years later that another AAB, Granulibacter bethesdensis, was the cause of infection in patients originating from geographically distinct locations and suffering from chronic granulomatous disease (CGD) (13, 14). Since then, AAB have increasingly been reported as organisms potentially infecting humans.We isolated AAB from clinical samples from three patients consulting or hospitalized in two French tertiary-care teaching hospitals. In this paper, we describe the clinical and bacteriological features of these cases and present a summarized review of the 10 documented cases of AAB isolation previously reported in humans.(This work was presented in part during the Meetings of the Three Divisions of the International Union of Microbiological Societies, Istanbul, Turkey, August 2008.)

Case 1. Gluconobacter sp. bacteremia in a nonimmunocompromised patient with a history of intravenous drug abuse.

A 29-year-old HIV-negative man (patient 1), known to be an intravenous drug abuser, was hospitalized in October 2006 at the tertiary-care teaching hospital of Nancy, France, for a progressive decrease of visual acuity in his left eye. He was afebrile upon admission. Fungal endophthalmitis was suspected, and Candida albicans was isolated from a vitreous sample. A central venous catheter was placed for amphotericin B administration. Fever (39°C) appeared after 3 days with an increase in C-reactive protein level. No ultrasonographic signs of endocarditis were detected. Two peripheral blood samples were drawn. Both aerobic culture vials (Bactec Plus Aerobic/F medium; Becton Dickinson, Le Pont de Claix, France) yielded the growth of a Gram-negative rod (strain LBN 175) after 3 days of incubation at 35°C in a Bactec 9000 system (Becton Dickinson). The organism was subcultured on chromogenic CPS-ID2 medium (bioMérieux, Marcy l''Etoile, France). Tiny colonies appeared after 48 h of incubation at 37°C. Conventional methods and commercialized systems did not allow the identification of this catalase-positive, oxidase-negative rod, which was further identified to be a Gluconobacter sp. by molecular means. The patient became afebrile after 24 h of antimicrobial treatment (ceftriaxone, 1 g/day for 10 days). The C-reactive protein level decreased progressively to normal values. To this day, no recurrence has been documented.

Case 2. Successive isolation of an Asaia sp. and of two unrelated Gluconobacter spp. in a CF patient.

A 2-year-old boy (patient 2) suffering from cystic fibrosis (CF) consulted at the CF center of the tertiary-care teaching hospital of Montpellier, France, for routine evaluation in January 2008. CF was diagnosed at birth on the basis of positive sweat tests (chloride concentration > 60 meq/liter) and an F508del homozygous genotype. He had a pancreatic insufficiency and presented with several episodes of rhinitis, but no antimicrobial therapy was recently given. Nutritional status and respiratory conditions were normal. Sputum analysis showed <25 polymorphonuclear leukocytes per microscopic field and yielded 8 × 10² CFU/ml of a catalase-positive, oxidase-negative, Gram-negative rod (strain aP75) growing in 3 days at 30°C on the Burkholderia cepacia-selective medium Cepacia agar (AES, Combourg, France) together with Haemophilus influenzae (>10⁷ CFU/ml), Moraxella catarrhalis (8 × 10⁴ CFU/ml), and a polymorphic commensal microflora. Two and 4 months later, two other isolates of atypical Gram-negative, oxidase-negative rods, strains aP78 and aP81, were recovered during routine analysis of sputum samples on Cepacia agar (3 × 10³ and 10³ CFU/ml, respectively). In the latter analysis, >25 polymorphonuclear leukocytes were observed per microscopic field, and the Gram-negative rod was the only notable isolate. In December 2009, after several sputum analyses, which did not reveal any atypical Gram-negative rod, a strain (strain aP112) presenting phenotypic characteristics similar to those of strains aP78 and aP81 was isolated from sputum at 6 × 10² CFU/ml. This strain was isolated together with >10⁶ CFU/ml each of Staphylococcus aureus, Streptococcus pneumoniae, Moraxella catarrhalis, and Haemophilus influenzae. The patient presented with increased cough, sputum production with a loss of appetite, and fatigue. On the basis of this respiratory exacerbation, a combination of amoxicillin-clavulanic acid and co-trimoxazole for 15 days was started, leading to clinical condition improvement. No significant lesions were observed on thoracic computed tomography (CT) scan. Phenotypic identification was unsuccessful for the four isolates, isolates aP75, aP78, aP81, and aP112, and molecular tools affiliated the first strain to Asaia sp. and the three others to Gluconobacter sp.

Case 3. Gluconobacter sp. in a second CF patient followed at the same CF center.

A 3-year-old girl (patient 3) with CF was hospitalized at the tertiary-care teaching hospital of Montpellier for two successive fever episodes in August 2008. CF was diagnosed at birth on the basis of positive sweat tests and an F508del homozygous genotype. She had a pancreatic insufficiency, and her medical history included a central catheter infection by a methicillin-resistant coagulase-negative Staphylococcus 8 months earlier. At first examination, no inflammatory signs were apparent around the indwelling catheter. Blood cultures were negative. Sputum analysis showed >25 polymorphonuclear leukocytes per microscopic field and numerous Gram-negative rods. Cultures yielded 8 × 10³ CFU/ml of a catalase-positive, oxidase-negative, Gram-negative rod (strain aP90) forming tiny, gray colonies in 2 days at 30°C on Cepacia agar together with Candida parapsilosis, Serratia marcescens (2 × 10² CFU/ml), and polymicrobial oral microflora. Phenotypic methods failed to identify the isolate, while molecular-based methods identified this strain as a Gluconobacter sp. Since the patient became afebrile spontaneously, no antimicrobial therapy was administered. Her clinical evolution was stable, with a normal nutritional status and no significant thoracic lesions on CT scan.     

Peptide inhibitor of interleukin-8 (IL-8) reduces staphylococcal enterotoxin-A (SEA) induced neutrophil trafficking to the lung

Staphylococcal enterotoxin A (SEA) is a superantigen, produced by some strains ofStaphylococcus aureus (S. aureus), which can cause a variety of clinical manifestations ranging from food poisoning to shock. SEA can also stimulate human alveolar macrophages to produce interleukin-8 (IL-8), a member of the -chemokine subfamily that activates and is chemotactic for neutrophils. In these studies we showed that in rabbits, intravenous SEA significantly decreased the circulating white blood cell population from a baseline value of 6409±2027×10³ cells/ml to 1943±862×10³ cells/ml in 7 h. There was a concommitent increase in IL-8 in the circulating plasma (baseline: 60±34 pg/ml, 7 h post SEA: 109±64 pg/ml). The increase in circulating IL-8 was accompanied by a much greater increase in the IL-8 concentration of the epithelial lining fluid (ELF) where the IL-8 increased from 0.05±0.08 ng/ml (control) to 13.8±9.3 ng/ml (SEA treated). The increase in IL-8 concentration in the alveolar spaces was paralleled by an increase in both the percentage of neutrophils (1.4±0.9% (control) to 26.0±10.8% (SEA treated)) and total number of neutrophils (0.04±0.02×10⁶/ml (control) to 4.8±3.3 10⁶/ml (SEA treated)) in the airspaces, and the numbers of neutrophils in the ELF correlated with the IL-8 concentration r=0.62, p=0.006). When antileukinate, a hexapeptide which inhibits the binding of IL-8 to neutrophils, was administered to animals receiving SEA, the IL-8 concentration in the ELF (14.8±10.7 ng/ml) was not significantly different from the concentration of IL-8 in those animals receiving SEA alone). However, both the percentage of neutrophils (9.5±3.2%), and the total number of neutrophils (1.3±1.0×10⁶/ml) in the ELF following SEA and antileukinate administration was significantly lower than in animals which only received SEA (p<0.05). The findings suggest that SEA released into the circulation during a Staphylococcal infection can cause an inflammatory reaction in the lung. Since this reaction is at least partially mediated by IL-8, antileukinate may have pharmacologic potential in reducing the inflammatory reaction.accepted by I. Ahnfelt-Rønne     

The dose-response relationship for ethyl methanesulfonate-induced mutations at the hypoxanthine-guanine phosphoribosyl transferase locus in Chinese hamster ovary cells

The frequency of ethyl methanesulfonate (EMS)-induced mutations to 6-thioguanine resistance in a Chinese hamster ovary cells done K ₁-BH₄ was studied at many EMS doses including the minimally lethal range (0–100 μg/ml) as well as the exponential killing portion (100–800 μg/ml) of the survival curve. The mutation frequency increases approximately in proportion with increasing EMS concentration at a fixed treatment time. The pooled data for the observed mutation frequency, f(X), as a function of EMS dose X, is adequately described by a linear function f(X)=10^?6(8.73+3.45 X), where 0≤X≤800 μg/ml. One interpretation of the linear dose-response is that, as a result of EMS treatment, ethylation of cellular constituents occurs, which is directly responsible for the mutation. Biochemical analyses demonstrate that most of the randomly isolated 6-thioguanine-resistant variants possess a highly reduced or undetectable level of HGPRT activity suggesting that the EMS-induced mutations to 6-thioguanine resistance affect primarily, if not exclusively, the HGPRT locus.