Response to Comment on "Impact of neurodegenerative diseases on human adult hippocampal neurogenesis".

Rakic and colleagues challenge the use of extensively validated adult hippocampal neurogenesis (AHN) markers and postulate an alternative interpretation of some of the data included in our study. In Terreros-Roncal et al., reconstruction of the main stages encompassed by human AHN revealed enhanced vulnerability of this phenomenon to neurodegenerative diseases. Here, we clarify points and ambiguities raised by these authors.

Response to Comment on "Impact of neurodegenerative diseases on human adult hippocampal neurogenesis".

Alvarez-Buylla and colleagues provide an alternative interpretation of some of the data included in our manuscript and question whether well-validated markers of adult hippocampal neurogenesis (AHN) are related to this phenomenon in our study. In Terreros-Roncal et al., reconstruction of the main stages of human AHN revealed its enhanced vulnerability to neurodegeneration. Here, we clarify ambiguities raised by these authors.

Impact of neurodegenerative diseases on human adult hippocampal neurogenesis.

Disrupted hippocampal performance underlies psychiatric comorbidities and cognitive impairments in patients with neurodegenerative disorders. To understand the contribution of adult hippocampal neurogenesis (AHN) to amyotrophic lateral sclerosis, Huntington's disease, Parkinson's disease, dementia with Lewy bodies, and frontotemporal dementia, we studied postmortem human samples. We found that adult-born dentate granule cells showed abnormal morphological development and changes in the expression of differentiation markers. The ratio of quiescent to proliferating hippocampal neural stem cells shifted, and the homeostasis of the neurogenic niche was altered. Aging and neurodegenerative diseases reduced the phagocytic capacity of microglia, triggered astrogliosis, and altered the microvasculature of the dentate gyrus. Thus, enhanced vulnerability of AHN to neurodegeneration might underlie hippocampal dysfunction during physiological and pathological aging in humans.

[Influence of the displacement of protein binding by ibuprofen in the activity of a third-generation cephalosporin against Streptococcus pneumoniae]. / Influencia del desplazamiento de la unión a proteínas por ibuprofeno en la actividad de una cefalosporina de tercera generación frente a Streptococcus pneumoniae.

The clinical significance of protein binding remains to be fully elucidated. The aim of this study was to evaluate the effect in the in vitro bactericidal activity of cefditoren through killing curves at Cmax concentrations against three Streptococcus pneumoniae strains (cefditoren MICs of 0.12, 0.25 and 0.5 mg/l) with or without human albumin (4 g/dl) and ibuprofen at Cmax concentrations (32.3 mg/l) and 10 times the Cmax (323 mg/l). Cefditoren was rapidly bactericidal (3 log(10) CFU/ml reduction) against the three strains at 4.2 mg/l concentration in Mueller-Hinton broth plus 5% lysed horse blood. In presence of human albumin, this effect was maintained against the most susceptible strain (MIC = 0.12 mg/l). Regrowths were observed with higher MIC values. The presence of ibuprofen (32.3 mg/l) slightly delayed regrowth while the increase of ibuprofen concentration up to 10 x Cmax recovered the bactericidal activity against all strains. The activity of an antimicrobial with high protein binding should not be linked exclusively with the theoretical unbound fraction extrapolated from the plasma concentration. The role of protein binding antagonists merits analysis due to their frequent use associated with cephalosporins in respiratory tract infections.

Genotypic and phenotypic diversity in Enterococcus faecalis: is agar invasion a pathogenicity score?

OBJECTIVES: The main objective of the present study is to analyze different genotypic and phenotypic traits related to virulence in Enterococcus faecalis, as well as evaluated the agar invasion phenotype in a collection of isolates with different clinical origins. MATERIAL AND METHODS: Seventy-nine E. faecalis isolates, with invasive and non-invasive clinical origins, have been used in this work. Presence of cytolysin activator (cylA), gelatinase (gelE), surface protein (esp), aggregation substance (asa1), endocarditis antigen (efaA), and collagen-binding protein (ace) have been analyzed by PCR. Phenotypic characterization included gelatinase activity, haemolysin production, biofilm formation and agar invasion. RESULTS: All the isolates tested harboured at least one of the virulence determinants. The 95.5% of isolates from haematologic samples were positive for agar invasion test, significantly higher than isolates from non-invasive diseases. A significant reduction in relative invasion area was observed in three selected agar-invasive strains after 15 serial passages. CONCLUSIONS: It has been observed a significant high prevalence of agar-invasion positive isolates among strains belonged to haematological samples. Agar invasiveness is reduced after adaptation of clinical isolates to laboratory conditions, showing that agar invasion phenotype can be modulate by culture conditions as other virulence factors observed in different bacterial species.

Bactericidal activity of daptomycin versus vancomycin in the presence of human albumin against vancomycin-susceptible but tolerant methicillin-resistant Staphylococcus aureus (MRSA) with daptomycin minimum inhibitory concentrations of 1-2microg/mL.

This study explored the influence of vancomycin tolerance and protein binding on the bactericidal activity of vancomycin versus daptomycin (protein binding 36.9% vs. 91.7%, respectively) against four vancomycin-tolerant methicillin-resistant Staphylococcus aureus (MRSA) [minimum inhibitory concentration/minimum bactericidal concentration (MIC/MBC)=0.5/16, 1/32, 2/32 and 1/32microg/mL for vancomycin and 1/1, 1/2, 2/2 and 2/4microg/mL for daptomycin]. Killing curves were performed with vancomycin/daptomycin concentrations equal to serum peak concentrations (C(max)) (65.70/98.60microg/mL) and trough concentrations (C(min)) (7.90/9.13microg/mL) in the presence and absence of a physiological human albumin concentration (4g/dL), controlled with curves with the theoretical free drug fraction of vancomycin/daptomycin C(max) (41.45/8.18microg/mL) and C(min) (4.98/0.76microg/mL). Vancomycin C(max) and C(min) concentrations, regardless of the media, showed a bacteriostatic profile not reaching a reduction of 99% or 99.9% of the initial inocula during the 24-h experimental time period. Daptomycin antibacterial profiles significantly differed when testing C(max) and C(min). C(max) was rapidly bactericidal (< or =4h) with >5 log(10) reduction in the initial inocula for all strains, regardless of the presence or not of albumin or the use of concentrations similar to free C(max). C(min) exhibited similar final colony counts at 0h and 24h in curves with albumin, but with >3 log colony-forming units (CFU)/mL reduction at < or =4h for strains with an MIC of 1microg/mL and ca. 2 logCFU/mL reduction at < or =6h for strains with an MIC of 2microg/mL. This activity was significantly higher than the activity of the free C(min) fraction. The results of this study reinforce the idea that pharmacodynamics using concentrations calculated using reported protein binding are unreliable. Daptomycin exhibited rapid antibacterial activity against vancomycin-tolerant MRSA isolates even against those with high daptomycin MICs in the presence of physiological albumin concentrations.

Cefditoren versus ceftazidime in inducer-substrate combinations for the evaluation of AmpC production in a disc approximation test.

OBJECTIVE: To evaluate cefditoren in inducer-substrate combinations to screen for AmpC induction. METHODS: 100 clinical isolates (25 P. aeruginosa, 25 E. cloacae, 14 M. morganii, 13 S. marcescens, 12 C. freundii, 7 P. rettgeri, and 4 E. aerogenes) were tested by the Kirby-Bauer disc approximation method using cefditoren and ceftazidime discs as substrates, and cefditoren and imipenem discs as inducers. RESULTS: None of the strains showed induction of AmpC with cefditoren-ceftazidime as inducer-substrate combination. Imipenem-cefditoren as inducer-substrate combination was not useful for evaluating strains of P. aeruginosa since no inhibition zones surrounding the cefditoren disc were found. Among evaluable enterobacteria (those showing substrate inhibition zone), inducible Amp C was detected in 48 out of 63 (76.2%) with cefditoren, and in 33 out of 68 (48.5%) isolates with ceftazidime as substrate. Significantly (p=0.013) higher number of AmpC producers were detected with cefditoren versus ceftazidime (76.2% vs. 48.5%), due to the differences found for E. cloacae (72.8% vs. 21.7%; p=0.0009) and S. marcescens (100% vs. 54.5%; p=0.03). Higher mean reductions of diameters around substrate discs were found for cefditoren (4.17 mm) vs. ceftazidime (3.79 mm), reaching statistical significance (p<0.05) for indol-positive proteae: M. morganii (5.32 mm vs. 3.92 mm) and P. rettgeri (3.47 mm vs. 2.64 mm). CONCLUSION: Cefditoren showed no induction capability, and when used as substrate (with imipenem as inducer) it offered detection rates of AmpC inducible enterobacteria higher than the imipenem-ceftazidime combination, mainly for Enterobacter spp. and Serratia spp., with higher diameter reductions for indol-positive proteae.

In vitro killing activity of crevicular concentrations of tinidazole plus common oral antibiotics against high-density mixed inocula of periodontal pathogens in strict anaerobic conditions.

BACKGROUND: Odontogenic infections are polymicrobial. This study explores the in vitro killing activity by concentrations similar to those found in crevicular fluid of tinidazole in combination with amoxicillin/clavulanic acid, clindamycin and levofloxacin against four groups of high-density mixed inocula of anaerobes (Prevotella buccae, Fusobacterium nucleatum, and Veillonella spp.) and facultative (Capnocytophaga spp. and Streptococcus spp.) isolates of periodontal pathogens. METHODS: Killing curves were assessed under strict anaerobic conditions with antibiotics alone and in combination with tinidazole at concentrations similar to those achieved in crevicular fluid against approximately 10(7) colony forming units (CFU)/ml inoculum (1:1:1:1:1 proportion of the five bacterial isolates) of the four bacterial groups. Group 1 did not include beta-lactamase-producing strains; groups 2, 3, and 4 included one, two, and three beta-lactamase-producing strains, respectively. RESULTS: In single-drug experiments, at 48 hours, tinidazole alone did not show significant killing of the entire bacterial population, whereas reductions in the initial inocula > or =2.09 log(10) CFU/ml with clindamycin, > or =3.26 log(10) CFU/ml with amoxicillin/clavulanic acid, and > or =3.83 log(10) CFU/ml with levofloxacin were obtained. When combined with tinidazole, reductions were significantly higher for all antibiotics: > or =5.28 log(10) CFU/ml with clindamycin, > or =4.78 log(10) CFU/ml with amoxicillin/clavulanic acid, and > or =6.17 log(10) CFU/ml with levofloxacin. CONCLUSION: In addition to its high activity against anaerobic periodontal pathogens, tinidazole offered synergism with other antibiotics against the large strict anaerobic subpopulation and the small facultative subpopulation of a high-density mixed inocula of odontogenic pathogens under strict anaerobic conditions, similar to those of odontogenic infections.

Exposure-response analysis of tigecycline in pharmacodynamic simulations using different size inocula of target bacteria.

This study explored tigecycline exposure-bacterial responses in pharmacodynamic simulations (in vitro kinetic model) using different inocula. One meticillin-resistant vancomycin-heteroresistant Staphylococcus aureus, one Enterococcus faecium and one extended-spectrum beta-lactamase-producing Escherichia coli with equal tigecycline minimum inhibitory concentrations/minimum bactericidal concentrations (MICs/MBCs) (0.12/0.25 microg/mL) were used. A computerised pharmacodynamic bicompartmental model simulated three tigecycline twice-daily dosing regimens over 48h: 50mg (100mg loading dose); 100mg; and 150 mg. Areas under bacterial growth curves were calculated, and differences between the growth curve used as control and the killing curve of bacteria exposed to tigecycline (ABBC) were determined. With standard inocula [ca. 1 x 10(6)colony-forming units (CFU)/mL], linear increases in area under the concentration-time curve (AUC)/MIC (25.6 for 50mg, 53.76 for 100mg and 79.52 for 150 mg) produced linear increases in activity against Gram-positive organisms (mean ABBCs of 120.60, 143.20 and 195.80 log CFU x h/mL for S. aureus and of 95.75, 172.55 and 216.90 log CFUxh/mL for E. faecium, respectively), with the activity of the 150 mg regimen being significantly higher (P<0.01) than that of the other two regimens. ABBCs obtained with the 100mg regimen using standard inocula were similar to those obtained with the 150 mg regimen when using high inocula (ca. 1 x 10(7)CFU/mL). Against E. coli, the highest dosing regimen was required to obtain significant antibacterial activity compared with control (mean ABBCs of 145.75 log CFU x h/mL with standard inocula and 63.33 log CFU x h/mL with high inocula). An increase in tigecycline dosing appears to be an interesting therapeutic option to maximise antibacterial activity owing to its linear pharmacokinetics and pharmacodynamics, especially when severe infections with high bacterial load are suspected.

Cidal activity of oral third-generation cephalosporins against Streptococcus pneumoniae in relation to cefotaxime intrinsic activity.

This study explores the killing kinetics within 12 h of four oral third-generation cephalosporins against ten Streptococcus pneumoniae strains exhibiting cefotaxime minimum inhibitory concentrations (MICs) from 0.03 to 2 microg/ml. Killing curves were performed with concentrations achievable in serum after standard doses (0.015-4 microg/ml). Reductions of 90% were achieved with all compounds at serum-achievable concentrations for strains exhibiting cefotaxime MIC < or = 0.5 microg/ml. Against strains with cefotaxime MIC > or = 1 microg/ml, only cefditoren reached a 90% reduction with concentrations of 0.5-1 microg/ml doses. At 4 microg/ml, cefditoren and cefotaxime reached 99.9% reduction in seven of the ten strains studied. At serum-achievable concentrations, cefdinir and cefixime were not bactericidal against strains exhibiting cefotaxime MIC > or = 0.25 microg/ml and > or = 0.5 microg/ml, respectively. Cefditoren showed the best killing kinetic profiles and this observation may be important when choosing an oral third-generation cephalosporin as initial or sequential therapy.

Effects of human albumin and serum on the in vitro bactericidal activity of cefditoren against penicillin-resistant Streptococcus pneumoniae.

OBJECTIVES: Attempts to interpret antibiotic pharmacodynamics using reported protein binding may underestimate true activity. To elucidate this issue we examined bacterial killing kinetics at cefditoren concentrations equal to C(max) in the presence of 90% human serum or albumin at physiological concentrations. METHODS: Killing curves (final inocula of approximately 10(7) cfu/mL, cefditoren concentration of 4.2 mg/L) were performed against Streptococcus pneumoniae strains exhibiting cefditoren MICs (mg/L) of 0.12 (strain 1), 0.25 (strain 2) and 0.5 (strain 3) in different media: (i) C(max)-MH, Mueller-Hinton broth plus 5% lysed horse blood (MH), (ii) C(max)-HS, MH broth with a final human serum concentration of 90%; and (iii) C(max)-HAlb, MH broth with 4 g/dL human albumin. Killing curves were also performed with a final cefditoren concentration of 0.5 mg/L (similar to free-drug C(max) considering 88% protein binding) in MH broth (12% C(max)). RESULTS: No significant differences were found between the different media or concentrations with strain 1 (log(10) reductions >or=4 at 24 h). Against strains 2 and 3, we observed significantly higher initial inocula decreases at 24 h for C(max)-HS as compared with C(max)-HAlb or 12% C(max). Bactericidal activity (>or=3 log(10) reduction) was obtained at 24 h against the three strains only with C(max)-HS and C(max)-MH. CONCLUSIONS: The presence of physiological concentrations of human albumin, but not 90% human serum, limited bactericidal activity as did the use of concentrations similar to free-drug C(max), suggesting that extrapolation of active drug from total drug by using the protein binding rate is not an accurate method to study antibacterial activity.

influence of TEM-1 beta-lactamase on the pharmacodynamic activity of simulated total versus free-drug serum concentrations of cefditoren (400 milligrams) versus amoxicillin-clavulanic acid (2,000/125 milligrams) against Haemophilus influenzae strains exhibiting an N526K mutation in the ftsI gene.

The aim of this study was to explore bactericidal activity of total and free serum simulated concentrations after the oral administration of cefditoren (400 mg, twice daily [bid]) versus the oral administration of amoxicillin-clavulanic acid extended release formulation (2,000/125 mg bid) against Haemophilus influenzae. A computerized pharmacodynamic simulation was performed, and colony counts and beta-lactamase activity were determined over 48 h. Three strains were used: ampicillin-susceptible, beta-lactamase-negative ampicillin-resistant (BLNAR) (also resistant to amoxicillin-clavulanic acid) and beta-lactamase-positive amoxicillin-clavulanic acid-resistant (BLPACR) strains, with cefditoren MICs of < or =0.12 microg/ml and amoxicillin-clavulanic acid MICs of 2, 8, and 8 microg/ml, respectively. Against the ampicillin-susceptible and BLNAR strains, bactericidal activity (> or =3 log(10) reduction) was obtained from 6 h on with either total and free cefditoren or amoxicillin-clavulanic acid. Against the BLPACR strain, free cefditoren showed bactericidal activity from 8 h on. In amoxicillin-clavulanic acid simulations the increase in colony counts from 4 h on occurred in parallel with the increase in beta-lactamase activity for the BLPACR strain. Since both BLNAR and BLPACR strains exhibited the same MIC, this was due to the significantly lower (P < or = 0.012) amoxicillin concentrations from 4 h on in simulations with beta-lactamase positive versus negative strains, thus decreasing the time above MIC (T>MIC). From a pharmacodynamic point of view, the theoretical amoxicillin T>MIC against strains with elevated ampicillin/amoxicillin-clavulanic acid MICs should be considered with caution since the presence of beta-lactamase inactivates the antibiotic, thus rendering inaccurate theoretical calculations. The experimental bactericidal activity of cefditoren is maintained over the dosing interval regardless of the presence of a mutation in the ftsI gene or beta-lactamase production.

In vitro effect of the presence of human albumin or human serum on the bactericidal activity of daptomycin against strains with the main resistance phenotypes in Gram-positives.

OBJECTIVES: Bactericidal activity depends on antibiotic-bacteria couples, resistance phenotype and theoretically on protein binding. This work explores the influence of protein binding on the bactericidal activity of two antibiotics, daptomycin versus vancomycin, that exhibit, respectively, different C(max) (56 versus 25.5 mg/L), protein binding (91.7% versus 36.9%) and thus theoretical free-drug fractions (4.7 versus 16.1 mg/L). METHODS: The effect of the presence of physiological concentrations of human albumin (4 g/dL) or human serum (90%) on the bactericidal activity of daptomycin was studied against Gram-positive isolates with troublesome resistance phenotypes [multidrug-resistant Streptococcus pneumoniae (MDRSP), methicillin-resistant Staphylococcus aureus (MRSA), heterogeneous vancomycin-intermediate MRSA (MRSA-hVI) and vancomycin-resistant Enterococcus faecium]. Killing curves (final inocula of approximately 10(7) cfu/mL) were performed using daptomycin and vancomycin concentrations similar to the C(max) obtained in serum. RESULTS: Daptomycin was rapidly bactericidal (> or =3 log(10) initial inocula reduction) against S. pneumoniae and S. aureus, regardless of the strain tested or the presence of albumin or human serum (that slightly delayed bactericidal activity). Against vancomycin-susceptible or -resistant enterococci, daptomycin exhibited rapid bactericidal activity, delayed to 8 and 24 h, respectively, by human albumin. Vancomycin exhibited much slower bactericidal activity against MDRSP and methicillin-susceptible or -resistant S. aureus, but was never bactericidal against MRSA-hVI and vancomycin-susceptible or -resistant E. faecium. CONCLUSIONS: Daptomycin exhibited rapid bactericidal activity against the strains of the three Gram-positive species tested, regardless of resistance phenotype or the presence of physiological concentrations of albumin.