12 research outputs found
Biophysical mechanisms of endotoxin neutralization by cationic amphiphilic peptides
Bacterial endotoxins (lipopolysaccharides (LPS)) are strong elicitors of the human immune system by interacting with serum and membrane proteins such as lipopolysaccharide-binding protein (LBP) and CD14 with high specificity. At LPS concentrations as low as 0.3 ng/ml, such interactions may lead to severe pathophysiological effects, including sepsis and septic shock. One approach to inhibit an uncontrolled inflammatory reaction is the use of appropriate polycationic and amphiphilic antimicrobial peptides, here called synthetic anti-LPS peptides (SALPs). We designed various SALP structures and investigated their ability to inhibit LPS-induced cytokine secretion in vitro, their protective effect in a mouse model of sepsis, and their cytotoxicity in physiological human cells. Using a variety of biophysical techniques, we investigated selected SALPs with considerable differences in their biological responses to characterize and understand the mechanism of LPS inactivation by SALPs. Our investigations show that neutralization of LPS by peptides is associated with a fluidization of the LPS acyl chains, a strong exothermic Coulomb interaction between the two compounds, and a drastic change of the LPS aggregate type from cubic into multilamellar, with an increase in the aggregate sizes, inhibiting the binding of LBP and other mammalian proteins to the endotoxin. At the same time, peptide binding to phospholipids of human origin (e.g., phosphatidylcholine) does not cause essential structural changes, such as changes in membrane fluidity and bilayer structure. The absence of cytotoxicity is explained by the high specificity of the interaction of the peptides with LPS
Caracterización de los mecanismos de resistencia de aislamientos clÃnicos de Pseudomonas aeruginosa no sensibles a carbapenems y factores de riesgo asociados a su adquisición
The epidemiology-related features of antibiotic resistant Pseudomonas aeruginosa strains (Pa) isolated in the University Hospital of Navarra from January 2002 to February 2009 were analyzed. An increase of Pa isolates not susceptible to carbapenems (Pa-nsc) was detected and the profile of antibiotic resistance of these organisms was markedly different from that of carbapenem susceptible Pa (Pasc). Both Pa-nsc and the overall Pa isolates increased their resistance to several antibiotic classes, whereas Pa-sc maintained their level of susceptibility. Phenotypic methods most commonly used for detection of metallo-beta-lactamase producing strains (MBL) including E-test MBL, simple microdilution test, double disk synergy test and disks combined test were compared and found to render 100% sensitivity. The combined disk test using imipenem and EDTA provided the greatest specificity. The susceptibility to doripenem in Pa isolates not susceptible to imipenem and meropenem was analyzed. Depending on the cut-off points established by CLSI, EUCAST and FDA, it was found that 28.6% to 11.9% of the strains not susceptible to imipenem were susceptible to doripenem, while 26.2% to 10.7% of strains not susceptible to meropenem were susceptible to doripenem. The mechanisms of resistance to carbapenems in clinical isolates of Pa-nsc were characterized. The altered expression of the porin OprD was the most common resistance mechanism (87.5%), followed by the overproduction of AmpC (33.3%). Four MBL-producing strains of the VIM-2 family were detected including the first MBL-producing strain sensitive to doripenem (MIC 1 ì/ml). The use of urinary catheter and the consumption of broad-spectrum antibiotics (piperacillin-tazobactam, carbapenems, fluoroquinolones and aminoglycosides) were established as risk factors for the acquisition of Pa-nsc. Exposure to hospital environment and the use of mechanical ventilation were identified as risk factors when the groups under comparison were Pa-nsc isolates versus isolates different from Pa. Finally, a prolonged hospital admission, the isolation of multiresistant Pa, the administration of chemotherapy and the use of immunosuppressive drugs were predictors of mortality attributable to Pa-nsc
Treatment of infections caused by carbapenemase-producing Enterobacterales
Antibiotic resistance is one of the main menaces to public and individual health worldwide. In the last two decades, an increase in the detection of carbapenem-resistant Enterobacterales has been reported. The treatment of infections caused by these strains is a therapeutic challenge. The use of carbapenems may be beneficial depending on MIC value and source of infection. New drugs, with different activity against the different classes of carbapenemases, are developed showing significant benefits
Free thiol group of MD-2 as the target for inhibition of the lipopolysaccharide-induced cell activation
MD-2 is a part of the Toll-like 4 signaling complex with an indispensable role in activation of the lipopolysaccharide (LPS) signaling pathway and thus a suitable target for the therapeutic inhibition of TLR4 signaling. Elucidation of MD-2 structure provides a foundation for rational design of inhibitors that bind to MD-2 and inhibit LPS signaling. Since the hydrophobic binding pocket of MD-2 provides little specificity for inhibitors, we have investigated targeting the solvent-accessible cysteine residue within the hydrophobic binding pocket of MD-2. Compounds with affinity for the hydrophobic pocket that contain a thiol-reactive group, which mediates covalent bond formation with the free cysteine residue of MD-2, were tested. Fluorescent compounds 2-(4'-(iodoacetamido)anilino)naphthalene-6-sulfonic acid and N-pyrene maleimide formed a covalent bond with MD-2 through Cys(133) and inhibited LPS signaling. Cell activation was also inhibited by thiol-reactive compounds JTT-705 originally targeted against cholesterol ester transfer protein and antirheumatic compound auranofin. Oral intake of JTT-705 significantly inhibited endotoxin-triggered tumor necrosis factor alpha production in mice. The thiol group of MD-2 also represents the target of environmental or endogenous thiol-reactive compounds that are produced in inflammation
Pneumococcal Serotypes Causing Acute Otitis Media Among Children in Barcelona (1992–2011): Emergence of the Multiresistant Clone ST320 of Serotype 19A
Background: There is scarce information about changes in serotypes
and clonal types of Streptococcus pneumoniae causing acute otitis media
(AOM) in recent years, particularly in European countries.
Methods: Pneumococcal serotypes and clones from S. pneumoniae strains
isolated from children with AOM who were attended at Hospital Sant Joan
de Déu, Barcelona (1992 to 2011), were studied. Heptavalent pneumococ-
cal conjugate vaccine (PCV7) was introduced in June 2001. We defined 3
periods: prevaccine period 1992 to 2001, early vaccine period 2002 to 2006
and late vaccine period 2007 to 2011.
Results: There were 376 pneumococcal strains causing AOM, and 373
(99.2%) of them were serotyped. AOM caused by PCV7 serotypes declined
significantly: 161 of 245 (65.7%) episodes in 1992 to 2001 versus 22 of
67 (32.8%) in 2002 to 2006 versus 8 of 61 (13.1%) in 2007 to 2011 P <
0.001. In the last period (2007 to 2011), the potential serotype coverage for
the PCV10 was 16.4% and for the PCV13 was 68.9% (P < 0.001). Sero-
type 19A increased from 5.7% in 1992 to 2001 to 42.6% in 2007 to 2011
(P < 0.001). Among strains with penicillin minimal inhibitory concentra-
tion ≥0.12 μg/mL (n = 241), serotype 19A rose from 2.3% in the first period
to 57.9 % in the last period (P < 0.001). The clonal-type ST320 was initially
detected in 2005, and in the period 2007 to 2011, the ST320 was found in
72.7% of nonsusceptible serotype 19A isolates.
Conclusions: Among children with AOM, a rapid expansion of the mul-
tiresistant clone ST320 expressing serotype 19A has been observed in Bar-
celona. The implementation of PCV13, which includes this serotype, may
decrease the prevalence of AOM and reduce antimicrobial resistance
Evaluation of the SediMax automated microscopy sediment analyzer and the Sysmex UF-1000i flow cytometer as screening tools to rule out negative urinary tract infections
Urinary tract infections (UTI) are highly prevalent in nosocomial and community settings, and their diagnosis is
costly and time-consuming. Screening methods represent an important advance towards the final UTI diagnosis,
diminishing inappropriate treatment or clinical complications. Automated analyzers have been developed and
commercialized to screen and rule out negative urine samples. The aim of this study was to evaluate two of
these automated analyzers (SediMax, an automatic sediment analyzer and UF-1000i a flow cytometer) to predict
negative urine cultures. A total of 1934 urine samples were analyzed. A very strong correlation for white blood
cells (WBC) (rs: 0.928) and a strong correlation for bacteria (BAC) (rs: 0.693) were obtained. We also calculated
optimal cut-off points for both autoanalyzers: 18 WBC/μL and 97 BAC/μL for SediMax (sensitivity = 96.25%,
specificity = 63.04%, negative predictive value = 97.97%), and 40 WBC/μL and 460 BAC/μL for UF-1000i
(sensitivity = 98.13%, specificity = 79.16%, negative predictive value = 99.18%). The use of SediMax and UF-
1000i resulted in a 46.33% and 57.19% reduction of all samples cultured, respectively. In conclusion, both ana-
lyzers are good UTI screening tools in our setting
Direct Identification of Urinary Tract Pathogens from Urine Samples, Combining Urine Screening Methods and Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry
Early diagnosis of urinary tract infections (UTIs) is essential to avoid inadequate or unnecessary empirical antibiotic therapy.
Microbiological confirmation takes 24 to 48 h. The use of screening methods, such as cytometry and automated microscopic
analysis of urine sediment, allows the rapid prediction of negative samples. In addition, matrix-assisted laser desorption ioniza-
tion–time of flight mass spectrometry (MALDI-TOF MS) is a widely established technique in clinical microbiology laboratories
used to identify microorganisms. We evaluated the ability of MALDI-TOF MS to identify microorganisms from direct urine
samples and the predictive value of automated analyzers for the identification of microorganisms in urine by MALDI-TOF MS. A
total of 451 urine samples from patients with suspected UTIs were first analyzed using the Sysmex UF-1000i flow cytometer, an
automatic sediment analyzer with microscopy (SediMax), culture, and then processed by MALDI-TOF MS with a simple triple-
centrifuged procedure to obtain a pellet that was washed and centrifuged and finally applied directly to the MALDI-TOF MS
plate. The organisms in 336 samples were correctly identified, mainly those with Gram-negative bacteria (86.10%). No microor-
ganisms were misidentified, and no Candida spp. were correctly identified. Regarding the data from autoanalyzers, the best bac-
teriuria cutoffs were 1,000 and 200 U/l for UF-1000i and SediMax, respectively. It was concluded that the combination of a
urine screening method and MALDI-TOF MS provided a reliable identification from urine samples, especially in those contain-
ing Gram-negative bacteria
High invasiveness of pneumococcal serotypes included in the new generation of conjugate vaccines
The implementation of the seven-valent pneumococcal conjugate vaccine, PCV7, has resulted in significant changes in the pneumococcal
population being carried and causing disease. We aimed to determine the invasive disease potential of serotypes causing invasive paediatric
disease in the era of conjugate vaccines in Catalonia, Spain, and their potential coverage by the 13-valent pneumococcal conjugate vaccine,
PCV13. As a secondary objective, we evaluated whether implementation of PCV7 had resulted in significant changes in the invasive disease
potential of the most frequent serotypes circulating in the area. Two pneumococcal collections obtained from children admitted to the
University Hospital Sant Joan de Deu (Barcelona, Spain) between 2007 and 2011 were compared: a first set of 159 invasive disease isolates,
and a second set of 209 nasopharyngeal isolates recovered from healthy children admitted for minor surgery. The most common invasive
serotypes were 1 (24.5%, n = 39), 19A (21.2%, n = 34), 5 (8.8%, n = 14), 7F (8.8%, n = 14) and 3 (5%, n = 8). The most common serotypes
in carriage were 19A (10%, n = 21), 6C (9%, n = 19), 23B (8.1%, n = 17), 6A (7.6%, n = 16) and 19F (6.2%, n = 13). A significantly higher
propensity to cause invasive disease was observed for serotypes 1, 3, 5, 7F and 19A, all of which are included in PCV13. After
false-discovery-rate correction, the results were robust for serotypes 1, 5, 7F and 19A. Non-PCV13 serotypes had a low invasive disease
potential. Our data reinforce the need for continuous surveillance and should encourage efforts to introduce universal vaccination with
PCV13 in children in our region
Recurrent Invasive Pneumococcal Disease in Children: Underlying Clinical Conditions, and Immunological and Microbiological Characteristics
Purpose
Clinical, immunological and microbiological characteristics of recurrent invasive pneumo-
coccal disease (IPD) in children were evaluated, differentiating relapse from reinfection, in
order to identify specific risk factors for both conditions.
Methods
All patients <18 years-old with recurrent IPD admitted to a tertiary-care pediatric center
from January 2004 to December 2011 were evaluated. An episode of IPD was defined as
the presence of clinical findings of infection together with isolation and/or pneumococcal
DNA detection by Real-Time PCR in any sterile body fluid. Recurrent IPD was defined as
2 or more episodes in the same individual at least 1 month apart. Among recurrent IPD, we
differentiated relapse (same pneumococcal isolate) from reinfection.
Results
593 patients were diagnosed with IPD and 10 patients died. Among survivors, 23 episodes
of recurrent IPD were identified in 10 patients (1.7%). Meningitis was the most frequent form
of recurrent IPD (10 episodes/4 children) followed by recurrent empyema (8 episodes/4 chil-
dren). Three patients with recurrent empyema caused by the same pneumococcal clone
ST306 were considered relapses and showed high bacterial load in their first episode. In
contrast, all other episodes of recurrent IPD were considered reinfections. Overall, the rate
of relapse of IPD was 0.5% and the rate of reinfection 1.2%. Five out of 7 patients with rein-
fection had an underlying risk factor: cerebrospinal fluid leak (n = 3), chemotherapytreatment (n = 1) and a homozygous mutation in MyD88 gene (n = 1). No predisposing risk
factors were found in the remainder.
Conclusions
recurrent IPD in children is a rare condition associated with an identifiable risk factor in case
of reinfection in almost 80% of cases. In contrast, recurrent IPD with pleuropneumonia is
usually a relapse of infection
Biophysical mechanisms of endotoxin neutralization by cationic amphiphilic peptides
Bacterial endotoxins (lipopolysaccharides (LPS)) are strong elicitors of the human immune system by interacting with serum and membrane proteins such as lipopolysaccharide-binding protein (LBP) and CD14 with high specificity. At LPS concentrations as low as 0.3 ng/ml, such interactions may lead to severe pathophysiological effects, including sepsis and septic shock. One approach to inhibit an uncontrolled inflammatory reaction is the use of appropriate polycationic and amphiphilic antimicrobial peptides, here called synthetic anti-LPS peptides (SALPs). We designed various SALP structures and investigated their ability to inhibit LPS-induced cytokine secretion in vitro, their protective effect in a mouse model of sepsis, and their cytotoxicity in physiological human cells. Using a variety of biophysical techniques, we investigated selected SALPs with considerable differences in their biological responses to characterize and understand the mechanism of LPS inactivation by SALPs. Our investigations show that neutralization of LPS by peptides is associated with a fluidization of the LPS acyl chains, a strong exothermic Coulomb interaction between the two compounds, and a drastic change of the LPS aggregate type from cubic into multilamellar, with an increase in the aggregate sizes, inhibiting the binding of LBP and other mammalian proteins to the endotoxin. At the same time, peptide binding to phospholipids of human origin (e.g., phosphatidylcholine) does not cause essential structural changes, such as changes in membrane fluidity and bilayer structure. The absence of cytotoxicity is explained by the high specificity of the interaction of the peptides with LPS