Influence of seasons and sampling strategy on assessment of bioaerosols in sewage treatment plants in Switzerland

An assessment of sewage workers' exposure to airborne cultivable bacteria, fungi and inhaled endotoxins was performed at 11 sewage treatment plants. We sampled the enclosed and unenclosed treatment areas in each plant and evaluated the influence of seasons (summer and winter) on bioaerosol levels. We also measured personal exposure to endotoxins of workers during special operation where a higher risk of bioaerosol inhalation was assumed. Results show that only fungi are present in significantly higher concentrations in summer than in winter (2331 +/- 858 versus 329 +/- 95 CFU m(-3)). We also found that there are significantly more bacteria in the enclosed area, near the particle grids for incoming water, than in the unenclosed area near the aeration basins (9455 +/- 2661 versus 2435 +/- 985 CFU m(-3) in summer and 11 081 +/- 2299 versus 2002 +/- 839 CFU m(-3) in winter). All bioaerosols were frequently above the recommended values of occupational exposure. Workers carrying out special tasks such as cleaning tanks were exposed to very high levels of endotoxins (up to 500 EU m(-3)) compared to routine work. The species composition and concentration of airborne Gram-negative bacteria were also studied. A broad spectrum of different species within the Pseudomonadaceae and the Enterobacteriaceae families were predominant in nearly all plants investigated. [Authors]

Use of a human-like low-grade bacteremia model of experimental endocarditis to study the role of Staphylococcus aureus adhesins and platelet aggregation in early endocarditis.

Animal models of infective endocarditis (IE) induced by high-grade bacteremia revealed the pathogenic roles of Staphylococcus aureus surface adhesins and platelet aggregation in the infection process. In humans, however, S. aureus IE possibly occurs through repeated bouts of low-grade bacteremia from a colonized site or intravenous device. Here we used a rat model of IE induced by continuous low-grade bacteremia to explore further the contributions of S. aureus virulence factors to the initiation of IE. Rats with aortic vegetations were inoculated by continuous intravenous infusion (0.0017 ml/min over 10 h) with 10(6) CFU of Lactococcus lactis pIL253 or a recombinant L. lactis strain expressing an individual S. aureus surface protein (ClfA, FnbpA, BCD, or SdrE) conferring a particular adhesive or platelet aggregation property. Vegetation infection was assessed 24 h later. Plasma was collected at 0, 2, and 6 h postinoculation to quantify the expression of tumor necrosis factor (TNF), interleukin 1α (IL-1α), IL-1β, IL-6, and IL-10. The percentage of vegetation infection relative to that with strain pIL253 (11%) increased when binding to fibrinogen was conferred on L. lactis (ClfA strain) (52%; P = 0.007) and increased further with adhesion to fibronectin (FnbpA strain) (75%; P < 0.001). Expression of fibronectin binding alone was not sufficient to induce IE (BCD strain) (10% of infection). Platelet aggregation increased the risk of vegetation infection (SdrE strain) (30%). Conferring adhesion to fibrinogen and fibronectin favored IL-1β and IL-6 production. Our results, with a model of IE induced by low-grade bacteremia, resembling human disease, extend the essential role of fibrinogen binding in the initiation of S. aureus IE. Triggering of platelet aggregation or an inflammatory response may contribute to or promote the development of IE.

NEW METHODS FOR DETECTION, SUSCEPTIBILITY TESTING AND BIOFILM ERADICATION OF DIFFICULT ORGANISMS

Aujourd'hui, les problèmes des maladies infectieuses concernent l'émergence d'infections difficiles à traiter, telles que les infections associées aux implants et les infections fongiques invasives chez les patients immunodéprimés. L'objectif de cette thèse était de développer des stratégies pour l'éradication des biofilms bactériens (partie 1), ainsi que d'étudier des méthodes innovantes pour la détection microbienne, pour l'établissement de nouveaux tests de sensibilité (partie 2). Le traitement des infections associées aux implants est difficile car les biofilms bactériens peuvent résister à des niveaux élevés d'antibiotiques. A ce jour, il n'y a pas de traitement optimal défini contre des infections causées par des bactéries de prévalence moindre telles que Enterococcus faecalis ou Propionibacterium acnés. Dans un premier temps, nous avons démontré une excellente activité in vitro de la gentamicine sur une souche de E. faecalis en phase stationnaire de croissance Nous avons ensuite confirmé l'activité de la gentamicine sur un biofilm précoce en modèle expérimental animal à corps étranger avec un taux de guérison de 50%. De plus, les courbes de bactéricidie ainsi que les résultats de calorimétrie ont prouvé que l'ajout de gentamicine améliorait l'activité in vitro de la daptomycine, ainsi que celle de la vancomycine. In vivo, le schéma thérapeutique le plus efficace était l'association daptomycine/gentamicine avec un taux de guérison de 55%. En établissant une nouvelle méthode pour l'évaluation de l'activité des antimicrobiens vis-à-vis de micro-organismes en biofilm, nous avons démontré que le meilleur antibiotique actif sur les biofilms à P. acnés était la rifampicine, suivi par la penicilline G, la daptomycine et la ceftriaxone. Les études conduites en modèle expérimental animal ont confirmé l'activité de la rifampicine seule avec un taux de guérison 36%. Le meilleur schéma thérapeutique était au final l'association rifampicine/daptomycine avec un taux de guérison 63%. Les associations de rifampicine avec la vancomycine ou la levofloxacine présentaient des taux de guérisons respectivement de 46% et 25%. Nous avons ensuite étudié l'émergence in vitro de la résistance à la rifampicine chez P. acnés. Nous avons observé un taux de mutations de 10"9. La caractérisation moléculaire de la résistance chez les mutant-résistants a mis en évidence l'implication de 5 mutations ponctuelles dans les domaines I et II du gène rpoB. Ce type de mutations a déjà été décrit au préalable chez d'autres espèces bactériennes, corroborant ainsi la validité de nos résultats. La deuxième partie de cette thèse décrit une nouvelle méthode d'évaluation de l'efficacité des antifongiques basée sur des mesures de microcalorimétrie isotherme. En utilisant un microcalorimètre, la chaleur produite par la croissance microbienne peut être-mesurée en temps réel, très précisément. Nous avons évalué l'activité de l'amphotéricine B, des triazolés et des échinocandines sur différentes souches de Aspergillus spp. par microcalorimétrie. La présence d'amphotéricine Β ou de triazole retardait la production de chaleur de manière concentration-dépendante. En revanche, pour les échinochandines, seule une diminution le pic de « flux de chaleur » a été observé. La concordance entre la concentration minimale inhibitrice de chaleur (CMIC) et la CMI ou CEM (définie par CLSI M38A), avec une marge de 2 dilutions, était de 90% pour l'amphotéricine B, 100% pour le voriconazole, 90% pour le pozoconazole et 70% pour la caspofongine. La méthode a été utilisée pour définir la sensibilité aux antifongiques pour d'autres types de champignons filamenteux. Par détermination microcalorimétrique, l'amphotéricine B s'est avéré être l'agent le plus actif contre les Mucorales et les Fusarium spp.. et le voriconazole le plus actif contre les Scedosporium spp. Finalement, nous avons évalué l'activité d'associations d'antifongiques vis-à-vis de Aspergillus spp. Une meilleure activité antifongique était retrouvée avec l'amphotéricine B ou le voriconazole lorsque ces derniers étaient associés aux échinocandines vis-à-vis de A. fumigatus. L'association échinocandine/amphotéricine B a démontré une activité antifongique synergique vis-à-vis de A. terreus, contrairement à l'association échinocandine/voriconazole qui ne démontrait aucune amélioration significative de l'activité antifongique. - The diagnosis and treatment of infectious diseases are today increasingly challenged by the emergence of difficult-to-manage situations, such as infections associated with medical devices and invasive fungal infections, especially in immunocompromised patients. The aim of this thesis was to address these challenges by developing new strategies for eradication of biofilms of difficult-to-treat microorganisms (treatment, part 1) and investigating innovative methods for microbial detection and antimicrobial susceptibility testing (diagnosis, part 2). The first part of the thesis investigates antimicrobial treatment strategies for infections caused by two less investigated microorganisms, Enterococcus faecalis and Propionibacterium acnes, which are important pathogens causing implant-associated infections. The treatment of implant-associated infections is difficult in general due to reduced susceptibility of bacteria when present in biofilms. We demonstrated an excellent in vitro activity of gentamicin against E. faecalis in stationary growth- phase and were able to confirm the activity against "young" biofilms (3 hours) in an experimental foreign-body infection model (cure rate 50%). The addition of gentamicin improved the activity of daptomycin and vancomycin in vitro, as determined by time-kill curves and microcalorimetry. In vivo, the most efficient combination regimen was daptomycin plus gentamicin (cure rate 55%). Despite a short duration of infection, the cure rates were low, highlighting that enterococcal biofilms remain difficult to treat despite administration of newer antibiotics, such as daptomycin. By establishing a novel in vitro assay for evaluation of anti-biofilm activity (microcalorimetry), we demonstrated that rifampin was the most active antimicrobial against P. acnes biofilms, followed by penicillin G, daptomycin and ceftriaxone. In animal studies we confirmed the anti-biofilm activity of rifampin (cure rate 36% when administered alone), as well as in combination with daptomycin (cure rate 63%), whereas in combination with vancomycin or levofloxacin it showed lower cure rates (46% and 25%, respectively). We further investigated the emergence of rifampin resistance in P. acnes in vitro. Rifampin resistance progressively emerged during exposure to rifampin, if the bacterial concentration was high (108 cfu/ml) with a mutation rate of 10"9. In resistant isolates, five point mutations of the rpoB gene were found in cluster I and II, as previously described for staphylococci and other bacterial species. The second part of the thesis describes a novel real-time method for evaluation of antifungals against molds, based on measurements of the growth-related heat production by isothermal microcalorimetry. Current methods for evaluation of antifungal agents against molds, have several limitations, especially when combinations of antifungals are investigated. We evaluated the activity of amphotericin B, triazoles (voriconazole, posaconazole) and echinocandins (caspofungin and anidulafungin) against Aspergillus spp. by microcalorimetry. The presence of amphotericin Β or a triazole delayed the heat production in a concentration-dependent manner and the minimal heat inhibition concentration (MHIC) was determined as the lowest concentration inhibiting 50% of the heat produced at 48 h. Due to the different mechanism of action echinocandins, the MHIC for this antifungal class was determined as the lowest concentration lowering the heat-flow peak with 50%. Agreement within two 2-fold dilutions between MHIC and MIC or MEC (determined by CLSI M38A) was 90% for amphotericin B, 100% for voriconazole, 90% for posaconazole and 70% for caspofungin. We further evaluated our assay for antifungal susceptibility testing of non-Aspergillus molds. As determined by microcalorimetry, amphotericin Β was the most active agent against Mucorales and Fusarium spp., whereas voriconazole was the most active agent against Scedosporium spp. Finally, we evaluated the activity of antifungal combinations against Aspergillus spp. Against A. jumigatus, an improved activity of amphotericin Β and voriconazole was observed when combined with an echinocandin. Against A. terreus, an echinocandin showed a synergistic activity with amphotericin B, whereas in combination with voriconazole, no considerable improved activity was observed.

Bacterial overgrowth during treatment with omeprazole compared with cimetidine: a prospective randomised double blind study.

BACKGROUND: Gastric and duodenal bacterial overgrowth frequently occurs in conditions where diminished acid secretion is present. Omeprazole inhibits acid secretion more effectively than cimetidine and might therefore more frequently cause bacterial overgrowth. AIM: This controlled prospective study compared the incidence of gastric and duodenal bacterial overgrowth in patients treated with omeprazole or cimetidine. METHODS: 47 outpatients with peptic disease were randomly assigned to a four week treatment regimen with omeprazole 20 mg or cimetidine 800 mg daily. Gastric and duodenal juice were obtained during upper gastrointestinal endoscopy and plated for anaerobic and aerobic organisms. RESULTS: Bacterial overgrowth (> or = 10(5) cfu/ml) was present in 53% of the patients receiving omeprazole and in 17% receiving cimetidine (p < 0.05). The mean (SEM) number of gastric and duodenal bacterial counts was 6.0 (0.2) and 5.0 (0.2) respectively in the omeprazole group and 4.0 (0.2) and 4.0 (0.1) in the cimetidine group (p < 0.001 and < 0.01; respectively). Faecal type bacteria were found in 30% of the patients with bacterial overgrowth. Basal gastric pH was higher in patients treated with omeprazole compared with cimetidine (4.2 (0.5) versus 2.0 (0.2); p < 0.001) and in patients with bacterial overgrowth compared with those without bacterial overgrowth (5.1 (0.6) versus 2.0 (0.1); p < 0.0001). The nitrate, nitrite, and nitrosamine values in gastric juice did not increase after treatment with either cimetidine or omeprazole. Serum concentrations of vitamin B12, beta carotene, and albumin were similar before and after treatment with both drugs. CONCLUSIONS: These results show that the incidence of gastric and duodenal bacterial overgrowth is considerably higher in patients treated with omeprazole compared with cimetidine. This can be explained by more pronounced inhibition of gastric acid secretion. No patient developed signs of malabsorption or an increase of N-nitroso compounds. The clinical significance of these findings needs to be assessed in studies with long-term treatment with omeprazole, in particular in patients belonging to high risk groups such as HIV infected and intensive care units patients.

Conjugative Transfer of Chromosomal Genes between Fluorescent Pseudomonads in the Rhizosphere of Wheat.

Bacteria released in large numbers for biocontrol or bioremediation purposes might exchange genes with other microorganisms. Two model systems were designed to investigate the likelihood of such an exchange and some factors which govern the conjugative exchange of chromosomal genes between root-colonizing pseudomonads in the rhizosphere of wheat. The first model consisted of the biocontrol strain CHA0 of Pseudomonas fluorescens and transposon-facilitated recombination (Tfr). A conjugative IncP plasmid loaded with transposon Tn5, in a CHA0 derivative carrying a chromosomal Tn5 insertion, promoted chromosome transfer to auxotrophic CHA0 recipients in vitro. A chromosomal marker (pro) was transferred at a frequency of about 10(sup-6) per donor on wheat roots under gnotobiotic conditions, provided that the Tfr donor and recipient populations each contained 10(sup6) to 10(sup7) CFU per g of root. In contrast, no conjugative gene transfer was detected in soil, illustrating that the root surface stimulates conjugation. The second model system was based on the genetically well-characterized strain PAO of Pseudomonas aeruginosa and the chromosome mobilizing IncP plasmid R68.45. Although originally isolated from a human wound, strain PAO1 was found to be an excellent root colonizer, even under natural, nonsterile conditions. Matings between an auxotrophic R68.45 donor and auxotrophic recipients produced prototrophic chromosomal recombinants at 10(sup-4) to 10(sup-5) per donor on wheat roots in artificial soil under gnotobiotic conditions and at about 10(sup-6) per donor on wheat roots in natural, nonsterile soil microcosms after 2 weeks of incubation. The frequencies of chromosomal recombinants were as high as or higher than the frequencies of R68.45 transconjugants, reflecting mainly the selective growth advantage of the prototrophic recombinants over the auxotrophic parental strains in the rhizosphere. Although under field conditions the formation of chromosomal recombinants is expected to be reduced by several factors, we conclude that chromosomal genes, whether present naturally or introduced by genetic modification, may be transmissible between rhizosphere bacteria.

A single mutation in enzyme I of the sugar phosphotransferase system confers penicillin tolerance to Streptococcus gordonii.

Tolerance is a poorly understood phenomenon that allows bacteria exposed to a bactericidal antibiotic to stop their growth and withstand drug-induced killing. This survival ability has been implicated in antibiotic treatment failures. Here, we describe a single nucleotide mutation (tol1) in a tolerant Streptococcus gordonii strain (Tol1) that is sufficient to provide tolerance in vitro and in vivo. It induces a proline-to-arginine substitution (P483R) in the homodimerization interface of enzyme I of the sugar phosphotransferase system, resulting in diminished sugar uptake. In vitro, the susceptible wild-type (WT) and Tol1 cultures lost 4.5 and 0.6 log(10) CFU/ml, respectively, after 24 h of penicillin exposure. The introduction of tol1 into the WT (WT P483R) conferred tolerance (a loss of 0.7 log(10) CFU/ml/24 h), whereas restitution of the parent sequence in Tol1 (Tol1 R483P) restored antibiotic susceptibility. Moreover, penicillin treatment of rats in an experimental model of endocarditis showed a complete inversion in the outcome, with a failure of therapy in rats infected with WT P483R and the complete disappearance of bacteria in animals infected with Tol1 R483P.

Development of glycopeptide-intermediate resistance by Staphylococcus aureus leads to attenuated infectivity in a rat model of endocarditis.

Glycopeptide-intermediate resistant Staphylococcus aureus (GISA) are characterized by multiple changes in the cell wall and an altered expression of global virulence regulators. We investigated whether GISA are affected in their infectivity in a rat model of experimental endocarditis. The glycopeptide-susceptible, methicillin-resistant S. aureus M1V2 and its laboratory-derived GISA M1V16 were examined for their ability to (i) adhere to fibrinogen and fibronectin in vitro, (ii) persist in the bloodstream after intravenous inoculation, (iii) colonize aortic vegetations in rats, and (iv) compete for valve colonization by co-inoculation. Both GISA M1V16 and M1V2 adhered similarly to fibrinogen and fibronectin in vitro. In rats, GISA M1V16 was cleared faster from the blood (P < 0.05) and required 100-times more bacteria than parent M1V2 (10(6) versus 10(4)CFU) to infect 90% of vegetations. GISA M1V16 also had 100 to 1000-times lower bacterial densities in vegetations. Moreover, after co-inoculation with GISA M1V16 and M1V2Rif, a rifampin-resistant variant of M1V2 to discriminate them in organ cultures, GISA M1V16 was out-competed by the glycopeptide-susceptible counterpart. Thus, in rats with experimental endocarditis, GISA showed an attenuated virulence, likely due to a faster clearance from the blood and a reduced fitness in cardiac vegetations. The GISA phenotype appeared globally detrimental to infectivity.

Deregulation of the arginine deiminase (arc) operon in penicillin-tolerant mutants of Streptococcus gordonii.

Penicillin tolerance is an incompletely understood phenomenon that allows bacteria to resist drug-induced killing. Tolerance was studied with independent Streptococcus gordonii mutants generated by cyclic exposure to 500 times the MIC of penicillin. Parent cultures lost 4 to 5 log(10) CFU/ml of viable counts/24 h. In contrast, each of four independent mutant cultures lost &lt; or =2 log(10) CFU/ml/24 h. The mutants had unchanged penicillin-binding proteins but contained increased amounts of two proteins with respective masses of ca. 50 and 45 kDa. One mutant (Tol1) was further characterized. The two proteins showing increased levels were homologous to the arginine deiminase and ornithine carbamoyl transferase of other gram-positive bacteria and were encoded by an operon that was &gt;80% similar to the arginine-deiminase (arc) operon of these organisms. Partial nucleotide sequencing and insertion inactivation of the S. gordonii arc locus indicated that tolerance was not a direct consequence of arc alteration. On the other hand, genetic transformation of tolerance by Tol1 DNA always conferred arc deregulation. In nontolerant recipients, arc was repressed during exponential growth and up-regulated during postexponential growth. In tolerant transformants, arc was constitutively expressed. Tol1 DNA transformed tolerance at the same rate as transformation of a point mutation (10(-2) to 10(-3)). The tolerance mutation mapped on a specific chromosomal fragment but was physically distant from arc. Importantly, arc deregulation was observed in most (6 of 10) of additional independent penicillin-tolerant mutants. Thus, although not exclusive, the association between arc deregulation and tolerance was not fortuitous. Since penicillin selection mimicked the antibiotic pressure operating in the clinical environment, arc deregulation might be an important correlate of naturally occurring tolerance and help in understanding the mechanism(s) underlying this clinically problematic phenotype.

Cefotaxime acts synergistically with levofloxacin in experimental meningitis due to penicillin-resistant pneumococci and prevents selection of levofloxacin-resistant mutants in vitro.

Cefotaxime, given in two doses (each 100 mg/kg of body weight), produced a good bactericidal activity (-0.47 Deltalog(10) CFU/ml. h) which was comparable to that of levofloxacin (-0.49 Deltalog(10) CFU/ml. h) against a penicillin-resistant pneumococcal strain WB4 in experimental meningitis. Cefotaxime combined with levofloxacin acted synergistically (-1.04 Deltalog(10) CFU/ml. h). Synergy between cefotaxime and levofloxacin was also demonstrated in vitro in time killing assays and with the checkerboard method for two penicillin-resistant strains (WB4 and KR4). Using in vitro cycling experiments, the addition of cefotaxime in sub-MIC concentrations (one-eighth of the MIC) drastically reduced levofloxacin-induced resistance in the same two strains (64-fold increase of the MIC of levofloxacin after 12 cycles versus 2-fold increase of the MIC of levofloxacin combined with cefotaxime). Mutations detected in the genes encoding topoisomerase IV (parC and parE) and gyrase (gyrA and gyrB) confirmed the levofloxacin-induced resistance in both strains. Addition of cefotaxime in low doses was able to suppress levofloxacin-induced resistance.

Leukemic cluster growth in culture is an independent risk factor for acute myeloid leukemia and short survival in patients with myelodysplastic syndrome

In patients with myelodysplastic syndrome (MDS) precursor cell cultures (colony-forming unit cells, CFU-C) can provide an insight into the growth potential of malignant myeloid cells. In a retrospective single-center study of 73 untreated MDS patients we assessed whether CFU-C growth patterns were of prognostic value in addition to established criteria. Abnormalities were classified as qualitative (i.e. leukemic cluster growth) or quantitative (i.e. strongly reduced/absent growth). Thirty-nine patients (53%) showed leukemic growth, 26 patients (36%) had strongly reduced/absent colony growth, and 12 patients showed both. In a univariate analysis the presence of leukemic growth was associated with strongly reduced survival (at 10 years 4 vs. 34%, p = 0.004), and a high incidence of transformation to AML (76 vs. 32%, p = 0.01). Multivariate analysis identified leukemic growth as a strong and independent predictor of early death (relative risk 2.12, p = 0.03) and transformation to AML (relative risk 2.63, p = 0.04). Quantitative abnormalities had no significant impact on the disease course. CFU- C assays have significant predictive value in addition to established prognostic factors in MDS. Leukemic growth identifies a subpopulation of MDS patients with poor prognosis.

Comparison of Methods for Evaluation of the Bactericidal Activity of Copper-Sputtered Surfaces against Methicillin-Resistant Staphylococcus aureus.

Bacteria can survive on hospital textiles and surfaces, from which they can be disseminated, representing a source of health care-associated infections (HCAIs). Surfaces containing copper (Cu), which is known for its bactericidal properties, could be an efficient way to lower the burden of potential pathogens. The antimicrobial activity of Cu-sputtered polyester surfaces, obtained by direct-current magnetron sputtering (DCMS), against methicillin-resistant Staphylococcus aureus (MRSA) was tested. The Cu-polyester microstructure was characterized by high-resolution transmission electron microscopy to determine the microstructure of the Cu nanoparticles and by profilometry to assess the thickness of the layers. Sputtering at 300 mA for 160 s led to a Cu film thickness of 20 nm (100 Cu layers) containing 0.209% (wt/wt) polyester. The viability of MRSA strain ATCC 43300 on Cu-sputtered polyester was evaluated by four methods: (i) mechanical detachment, (ii) microcalorimetry, (iii) direct transfer onto plates, and (iv) stereomicroscopy. The low efficacy of mechanical detachment impeded bacterial viability estimations. Microcalorimetry provided only semiquantitative results. Direct transfer onto plates and stereomicroscopy seemed to be the most suitable methods to evaluate the bacterial inactivation potential of Cu-sputtered polyester surfaces, since they presented the least experimental bias. Cu-polyester samples sputtered for 160 s by DCMS were further tested against 10 clinical MRSA isolates and showed a high level of bactericidal activity, with a 4-log(10) reduction in the initial MRSA load (10(6) CFU) within 1 h. Cu-sputtered polyester surfaces might be of use to prevent the transmission of HCAI pathogens.

Predominance of Nonculturable Cells of the Biocontrol Strain Pseudomonas fluorescens CHA0 in the Surface Horizon of Large Outdoor Lysimeters.

The persistence of the biocontrol agent Pseudomonas fluorescens CHA0 in the surface horizon of 12 large outdoor lysimeters planted with winter wheat, Phacelia tanacetifolia followed by spring wheat, or maize was monitored for 1 year. Soil was inoculated with a spontaneous rifampin-resistant mutant (CHA0-Rif) of CHA0, and the strain was studied by using colony counts, Kogure's direct viable counts, and total counts (immunofluorescence). The number of culturable cells of the inoculant decreased progressively from 8 to 2 log CFU/g of soil or lower. However, culturable cells of CHA0-Rif accounted for less than 1% of the total cells of the inoculant 8 months after release in autumn. Since viable but nonculturable cells represented less than a quarter of the latter, most cells of CHA0-Rif in soil were thus inactive-dormant or dead at that time. Nonculturable cells of the inoculant were predominant also in the surface horizon of the lysimeters inoculated in the spring, and a significant fraction of them were viable. Results suggest that the occurrence of nonculturable cells of CHA0-Rif was influenced by climatic factors (water availability and soil temperature) and the abundance of roots in soil. The fact that the inoculant persisted as mixed populations of cells of different physiological states, in which nonculturable cells were predominant, needs to be taken into account when assessing the autecology of wild-type or genetically modified pseudomonads released into the soil ecosystem.

Parenteral sparfloxacin compared with ceftriaxone in treatment of experimental endocarditis due to penicillin-susceptible and -resistant streptococci.

A new, investigational, parenteral form of sparfloxacin was compared with ceftriaxone in the treatment of experimental endocarditis caused by either of three penicillin-susceptible streptococci or one penicillin-resistant streptococcus. Both drugs have prolonged half-lives in serum, allowing single daily administration to humans. Sparfloxacin had relatively low MICs (0.25 to 0.5 mg/liter) for all four organisms and was also greater than or equal to eight times more effective than the other quinolones against 21 additional streptococcal isolates recovered from patients with bacteremia. Ceftriaxone MICs were 0.032 to 0.064 mg/liter for the penicillin-susceptible strains and 2 mg/liter for the resistant isolate. Both antibiotics resulted in moderate bacterial killing in vitro. Rats with catheter-induced aortic vegetations were inoculated with 10(7) CFU of the test organisms. Antibiotic treatment was started 48 h later and lasted either 3 or 5 days. The drugs were injected at doses which mimicked the kinetics in human serum produced by one intravenous injection of 400 mg of sparfloxacin (i.e., the daily dose expected to be given to human adults) and 2 g of ceftriaxone. Both antibiotics significantly decreased the bacterial densities in the vegetations. However, sparfloxacin was slower than ceftriaxone in its ability to eradicate valvular infection caused by penicillin-susceptible bacteria. While this difference was quite marked after 3 days of therapy, it tended to vanish when treatment was prolonged to 5 days. In contrast, sparfloxacin was very effective against the penicillin-resistant isolate, an organism against which ceftriaxone therapy failed in vivo. No sparfloxacin-resistant mutant was selected during therapy. Thus, in the present experimental setting, this new, investigational, parenteral form of sparfloxacin was effective against severe infections caused by both penicillin-susceptible and penicillin-resistant streptococci.

Ceftriaxone acts synergistically with levofloxacin in experimental meningitis and reduces levofloxacin-induced resistance in penicillin-resistant pneumococci.

Ceftriaxone acted synergistically with levofloxacin in time-killing assays in vitro over 8 h against two penicillin-resistant pneumococcal strains (WB4 and KR4; MIC of penicillin: 4 mg/L). Synergy was confirmed with the chequerboard method, showing FIC indices of 0.25. In the experimental rabbit meningitis model, ceftriaxone (1x 125 mg/kg) was slightly less bactericidal (-0.30 Deltalog(10) cfu/mL(.)h) compared with levofloxacin (-0.45 Deltalog(10) cfu/mL(.)h) against the penicillin-resistant strain WB4. The combination therapy (levofloxacin and ceftriaxone) was significantly superior (-0.64 Deltalog(10) cfu/mL(.)h) to either monotherapy. In cycling experiments in vitro, the addition of ceftriaxone at a sub-MIC concentration (1/16 MIC) reduced levofloxacin-induced resistance in the two strains KR4 and WB4. After 12 cycles with levofloxacin monotherapy, the MIC increased 64-fold in both strains versus a 16-fold increase with the combination (levofloxacin + ceftriaxone 1/16 MIC). In both strains, levofloxacin-induced resistance was confirmed by mutations detected in the genes parC and gyrA, encoding for subunits of topoisomerase IV and gyrase, respectively. The addition of ceftriaxone suppressed mutations in parC but led to a new mutation in parE in both strains.

A prospective, randomized, open label, phase iii clinical trial of novottf-100a vs best standard of care chemotherapy in patients with recurrent glioblastoma

BACKGROUND: Glioblastoma, the most common adult primary malignant brain tumor, confers poor prognosis (median survival of 15 months) notwithstanding aggressive treatment. Combination chemotherapy including carmustine (BCNU) or temozolomide (TMZ) with the MGMT inhibitor O6-benzylguanine (O6BG) has been used, but has been associated with dose-limiting hematopoietic toxicity. OBJECTIVE: To assess safety and efficacy of a retroviral vector encoding the O6BG-resistant MGMTP140K gene for transduction and autologous transplantation of hematopoietic stem cells (HSCs) in MGMT unmethylated, newly diagnosed glioblastoma patients in an attempt to chemoprotect bone marrowduring combination O6BG/TMZ therapy. METHODS: Three patients have been enrolled in the first cohort. Patients underwent standard radiation therapy without TMZ followed by G-CSF mobilization, apheresis, and conditioning with 600 mg/m2 BCNU prior to infusion of gene-modified cells. Posttransplant, patients were treated with 28-day cycles of single doseTMZ (472 mg/m2) with 48-hour intravenous O6BG (120 mg/m2 bolus, then 30 mg/m2/d). RESULTS: The BCNU dose was nonmyeloablative with ANC ,500/mL for ≤3 d and nadir thrombocytopenia of 28,000/mL. Gene marking in pre-infusion colony forming units (CFUs) was 70.6%, 79.0%, and 74.0% in Patients 1, 2, and 3, respectively, by CFU-PCR. Following engraftment, gene marking in white blood cells and sorted granulocytes ranged between 0.37-0.84 and 0.33-0.83 provirus copies, respectively, by real-time PCR. Posttransplant gene marking in CFUs from CD34-selected cells ranged from 28.5% to 47.4%. Patients have received 4, 3, and 2 cycles of O6BG/TMZ, respectively, with evidence for selection of gene-modified cells. One patient has received a single dose-escalated cycle at 590 mg/m2 TMZ. No additional extra-hematopoietic toxicity has been observed thus far and all three patients exhibit stable disease at 7-8 months since diagnosis CONCLUSIONS: We believe that these data demonstrate the feasibility of achieving significant engraftment of MGMTP140K-modified cells with a well-tolerated dose of BCNU. Further follow-up will determine whether this approach will allow for further dose escalation of TMZ and improved survival.