José Leiva

The Enterococcal Surface Protein, Esp, Is Involved in Enterococcus faecalis Biofilm Formation

ABSTRACT The enterococcal surface protein, Esp, is a high-molecular-weight surface protein of unknown function whose frequency is significantly increased among infection-derived Enterococcus faecalisisolates. In this work, a global structural similarity was found between Bap, a biofilm-associated protein of Staphylococcus aureus, and Esp. Analysis of the relationship between the presence of the Esp-encoding gene (esp) and the biofilm formation capacity in E. faecalis demonstrated that the presence of the esp gene is highly associated (P < 0.0001) with the capacity of E. faecalis to form a biofilm on a polystyrene surface, since 93.5% of the E. faecalis esp-positive isolates were capable of forming a biofilm. Moreover, none of the E. faecalis esp-deficient isolates were biofilm producers. Depending on theE. faecalis isolate, insertional mutagenesis ofesp caused either a complete loss of the biofilm formation phenotype or no apparent phenotypic defect. Complementation studies revealed that Esp expression in an E. faecalis esp-deficient strain promoted primary attachment and biofilm formation on polystyrene and polyvinyl chloride plastic from urine collection bags. Together, these results demonstrate that (i) biofilm formation capacity is widespread among clinical E. faecalis isolates, (ii) the biofilm formation capacity is restricted to the E. faecalis strains harboringesp, and (iii) Esp promotes primary attachment and biofilm formation of E. faecalis on abiotic surfaces.

Evaluation of the relatedness of Brucella spp. and Ochrobactrum anthropi and description of Ochrobactrum intermedium sp. nov., a new species with a closer relationship to Brucella spp

The relatedness of Brucella spp. and Ochrobactrum anthropi was studied by protein profiling, Western blot, immunoelectrophoresis and 16S rRNA analysis. Whole-cell and soluble proteins of brucellae and O. anthropi showed serological cross-reactivities quantitatively and qualitatively more intense than those existing with similar extracts of Agrobacterium spp. Numerical analysis of Western blot profiles of whole-cell extracts showed that O. anthropi LMG 3301 was closer to Brucella spp. than to O. anthropi LMG 3331T, a result not obtained by protein profiling. These differences were not observed by Western blot with soluble fractions, and immunoelectrophoretic analyses suggested that this was due to destruction of conformational epitopes in Western blot procedures with the subsequent simplification of antigenic profile. Analysis of the 16S rRNA sequences of strains previously used in the species definition confirmed that strain LMG 3301, and also LMG 3306, were closer to the brucellae, and that LMG 3331T was in a separate cluster. The LMG 3301 and the LMG 3331T clusters could also be separated by their different colistin sensitivity and by PCR with 16S rRNA Brucella primers, and both methods showed strains of both clusters among clinical isolates classified as O. anthropi by conventional tests. These results and those of previous DNA-DNA hybridization studies [Holmes, B., Popoff, M., Kiredjian, M. & Kersters, K. (1988). Int J Syst Bacteriol 38, 406-416] show that the LMG 3301 cluster and related clinical isolates should be given a new species status for which the name Ochrobactrum intermedium sp. nov. is proposed (type strain is LMG 3301T=NCTC 12171T = CNS 2-75T).

Biofilm testing of Staphylococcus epidermidis clinical isolates: low performance of vancomycin in relation to other antibiotics

The in vitro killing effect of widely used antibiotics (cephalothin, clindamycin, erythromycin, ofloxacin, rifampicin, teicoplanin, tetracycline, phosphomycin and vancomycin) was comparatively analyzed in this study on 24-h biofilms of 64 Staphylococcus epidermidis clinical isolates. This effect was assessed at the expected antibiotic concentration reached in serum, using ATP-bioluminescence. Erythromycin, rifampicin, tetracycline and phosphomycin presented generally a higher killing effect than vancomycin, clindamycin, cephalothin, teicoplanin and ofloxacin in these biofilms. Differences in the resistance profiles obtained in classical assays (broth microdilution and diffusion) did not help to predict differences in the killing effect of the antibiotics in biofilms. Only some antibiotics (vancomycin but not rifampicin or tetracycline) highly decreased their killing effect as the biofilm age increased (from 6 to 24 or 48 h). These studies underline the relevance of biofilm susceptibility testing and the potential danger of the indiscriminate use of vancomycin monotherapy as the ultimate resource against infections involving aged biofilms.

Structural Features Governing the Activity of Lactoferricin-Derived Peptides That Act in Synergy with Antibiotics against Pseudomonas aeruginosa In Vitro and In Vivo

ABSTRACT Pseudomonas aeruginosa is naturally resistant to many antibiotics, and infections caused by this organism are a serious threat, especially to hospitalized patients. The intrinsic low permeability of P. aeruginosa to antibiotics results from the coordinated action of several mechanisms, such as the presence of restrictive porins and the expression of multidrug efflux pump systems. Our goal was to develop antimicrobial peptides with an improved bacterial membrane-permeabilizing ability, so that they enhance the antibacterial activity of antibiotics. We carried out a structure activity relationship analysis to investigate the parameters that govern the permeabilizing activity of short (8- to 12-amino-acid) lactoferricin-derived peptides. We used a new class of constitutional and sequence-dependent descriptors called PEDES (peptide descriptors from sequence) that allowed us to predict (Spearmans ρ = 0.74; P < 0.001) the permeabilizing activity of a new peptide generation. To study if peptide-mediated permeabilization could neutralize antibiotic resistance mechanisms, the most potent peptides were combined with antibiotics, and the antimicrobial activities of the combinations were determined on P. aeruginosa strains whose mechanisms of resistance to those antibiotics had been previously characterized. A subinhibitory concentration of compound P2-15 or P2-27 sensitized P. aeruginosa to most classes of antibiotics tested and counteracted several mechanisms of antibiotic resistance, including loss of the OprD porin and overexpression of several multidrug efflux pump systems. Using a mouse model of lethal infection, we demonstrated that whereas P2-15 and erythromycin were unable to protect mice when administered separately, concomitant administration of the compounds afforded long-lasting protection to one-third of the animals.

Prevalence of extended-spectrum β-lactamase-producing Enterobacteriaceae in meat products sold in Navarra, Spain.

Patterns of resistance in β-lactamase-producing Enterobacteriaceae family were investigated in isolates from 141 meat products (beef, poultry and pork) purchased in Spain. The strains that grow in ChromID ESBL agar plates were confirmed using the paired disk diffusion method. Resistance to amoxicillin/clavulanic acid, ceftazidime, ceftriaxone, aztreonam, cefpodoxime, gentamicin, doxycycline, cotrimoxazol, norfloxacin, piperacillin/tazobactam, fosfomycin and cefoxitin were tested following CLSI recommendations. Minimum inhibitory concentrations were determined by the MicroScan® NM37 panel and β-lactamase genes were detected using multiplex PCR and sequencing. Results show poultry as the meat product having the highest prevalence (84%), with Escherichia coli being the predominant bacteria (71.3%). Predominant β-lactamase types were CTX-M (37.8%), followed by CTX-M+TEM combination (20.7%), TEM (17%), SHV (12.2%), TEM+SHV combination (10.9%) and OXA (1.2%). 93.9% of the strains were resistant to one or more β-lactam antibiotics. Results indicate a widespread distribution of ESBL-producing Enterobacteriaceae in meat products, with a high rate of β-lactam resistance and a low rate of AmpC cephalosporinase-producing strains.

Comparative analysis of selected methods for the assessment of antimicrobial and membrane-permeabilizing activity: a case study for lactoferricin derived peptides

BackgroundGrowing concerns about bacterial resistance to antibiotics have prompted the development of alternative therapies like those based on cationic antimicrobial peptides (APs). These compounds not only are bactericidal by themselves but also enhance the activity of antibiotics. Studies focused on the systematic characterization of APs are hampered by the lack of standard guidelines for testing these compounds. We investigated whether the information provided by methods commonly used for the biological characterization of APs is comparable, as it is often assumed. For this purpose, we determined the bacteriostatic, bactericidal, and permeability-increasing activity of synthetic peptides (n = 57; 9–13 amino acid residues in length) analogous to the lipopolysaccharide-binding region of human lactoferricin by a number of the most frequently used methods and carried out a comparative analysis.ResultsWhile the minimum inhibitory concentration determined by an automated turbidimetry-based system (Bioscreen) or by conventional broth microdilution methods did not differ significantly, bactericidal activity measured under static conditions in a low-ionic strength solvent resulted in a vast overestimation of antimicrobial activity. Under these conditions the degree of antagonism between the peptides and the divalent cations differed greatly depending on the bacterial strain tested. In contrast, the bioactivity of peptides was not affected by the type of plasticware (polypropylene vs. polystyrene). Susceptibility testing of APs using cation adjusted Mueller-Hinton was the most stringent screening method, although it may overlook potentially interesting peptides. Permeability assays based on sensitization to hydrophobic antibiotics provided overall information analogous – though not quantitatively comparable- to that of tests based on the uptake of hydrophobic fluorescent probes.ConclusionWe demonstrate that subtle changes in methods for testing cationic peptides bring about marked differences in activity. Our results show that careful selection of the test strains for susceptibility testing and for screenings of antibiotic-sensitizing activity is of critical importance. A number of peptides proved to have potent permeability-increasing activity at subinhibitory concentrations and efficiently sensitized Pseudomonas aeruginosa both to hydrophilic and hydrophobic antibiotics.

Effectiveness of the antibiotic lock therapy for the treatment of port-related enterococci, Gram-negative, or Gram-positive bacilli bloodstream infections

OBJECTIVE The purpose of this study is to evaluate the efficacy of antibiotic lock therapy to treat port-related enterococci, Gram-negative, or Gram-positive bacilli bloodstream infections. PATIENTS AND METHODS We conducted a prospective observational study including all patients with port-related bacteremia diagnosed at the Clinica Universitaria de Navarra, Pamplona, Spain. During a 36-month period, 110 patients were diagnosed with port-related bacteremia. Of these patients, 18 met criteria to be enrolled in the study. They were treated with a combination of systemic and antibiotic lock therapy (12-24 h/day during 7-14 days). Treatment effectiveness was assessed by clinical and microbiologic criteria. RESULTS Treatment was associated with clinical and microbiologic success in 88.8% of our patients (2/2 of the Propionibacterium acnes, 5/5 of the Corynebacterium spp., 6/7 of the Gram-negative bacillus, and 3/4 of the Enterococcus faecium port-related bloodstream infections). Mean increase of port life span for all patients after bacteremia was 288 days (range, 0-1403 days). CONCLUSION Antibiotic lock therapy combined with systemic antibiotics appears to be a safe and effective treatment of port-related bacteremia caused by enterococci, Gram-negative, or Gram-positive bacilli if the patient is stable and no septic syndrome is associated.

Lysostaphin and clarithromycin: a promising combination for the eradication of Staphylococcus aureus biofilms

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Effectiveness of teicoplanin versus vancomycin lock therapy in the treatment of port-related coagulase-negative staphylococci bacteraemia: a prospective case-series analysis

The aim of this study was to analyse the effectiveness of teicoplanin versus vancomycin lock therapy in the treatment of coagulase-negative staphylococci (CoNS) venous access port-related bloodstream infection (BSI). The study included 44 consecutive patients during a 36-month prospective case-series study. The primary endpoint was failure to cure. Treatment was successful in 39 patients. At the end of the study, the cumulative port survival rate was 100% in the teicoplanin lock group compared with 77% in the vancomycin lock group (P=0.06). In the Cox regression analysis, fever beyond 48 h of treatment was a significant predictor of treatment failure (P=0.02). Use of vancomycin or teicoplanin locks had an effectiveness of 88.6% in the treatment of CoNS port-related BSI. Teicoplanin locks reduced the failure rate from 18.5% to 0% compared with vancomycin locks. The presence of fever after beginning antimicrobial lock therapy was associated with treatment failure.

Granulicatella adiacens breast implant-associated infection

The 1st reported case of breast implant-associated infection due to Granulicatella adiacens, formerly known as nutritionally variant streptococci, Streptococcus adiacens, and Abiotrophia adiacens is presented. Microbiology and previously reported cases of infections by this organism are reviewed.