Protection against Pseudomonas aeruginosa lung infection in mice by recombinant OprF-pulsed dendritic cell immunization

BACKGROUND: The Pseudomonas aeruginosa major constitutive outer membrane porin protein F (OprF) has been shown to be a protective antigen and was previously used to activate an immunological response in a mouse model of lung pneumonia. The purpose of our study was to demonstrate the ability of mouse dendritic cells pulsed with purified or recombinant OprF to protect mice against P. aeruginosa infection and inflammation.Both native (n-OprF), isolated and purified from PAO1 bacterial strain, and recombinant (histidin-conjugated) OprF (His-OprF), obtained by cloning of the oprF gene into the pET28a expression vector, were used to stimulate dendritic cells in vitro before adoptive transfer into prospective recipient mice with P. aeruginosa pulmonary infection. RESULTS: Similar to n-OprF, His-OprF activated dendritic cells in vitro, inducing the costimulatory molecule expression as well as cytokine production. Upon adoptive transfer in vivo, porin-pulsed dendritic cells (DCs) induced Th1-mediated resistance to infection and associated inflammatory pathology caused by either the PAO1 strain or a clinically-isolated mucoid strain. CONCLUSIONS: This study highlights the pivotal contribution of DCs to vaccine-induced protection against P. aeruginosa infection and associated inflammation

MALDI-TOF mass spectrometry for the rapid identification of aetiological agents of sepsis

Introduction: The MALDI-TOF has recently become part of the methods of microbiological investigation in many laboratories of bacteriology with advantages both practical and economical.The use of this technique for the rapid identification of the causative agents of sepsis is of strategic importance to the ability to provide the clinician with useful information for a prompt and rapid establishment of an empirical antimicrobial “targeted” therapy. Methods: It was tested a total of 343 positive blood culture bottles from 211 patients. The samples after collection were incubated in the BACTEC FX (Becton Dickinson, USA). From these bottles were taken a few milliliters of broth culture and transferred into a vacutainer tube containing gel. This was centrifuged, the supernatant was decanted, and finally recovered the bacterial suspension on the gel. With micro-organisms recovered in this way, after several washes with distilled water, was prepared a slide for microscopic examination with Gram stain, and a plate for mass spectrometry (MS-Vitek, bioMérieux, France).Then, the same samples were inoculated on solid agar media according to the protocol in use in our laboratory.The next day was checked the possible bacterial growth on solid media; we then proceeded to the identification of the colonies by Vitek MS and / or with the system Vitek2 (bioMérieux, France). Results: 258 (75.2%) positive vials show concordant results between direct identification and identification after growth on agar. For 83 (24.2%) positive bottles there has been full compliance with the microscopic examination but not with culture. In particular, two bottles (0.6%) have given complete discordance between the direct identification and that after growth. Conclusions: The protocol we use for the direct identification of organisms responsible for sepsis, directly on positive bottles, seems to be a quick and inexpensive procedure, which in less than 60 minutes can give valuable feedback to the clinician

Prevalence of Clostridium difficile infections in Prato hospital in the years 2010-2011

Clostridium difficile, a Gram positive, spore-forming, anaerobic bacillus, is an important nosocomial enteric pathogen causing diarrhoea and pseudomembranous colitis. Aim of this study was to evaluate the prevalence of Clostridium difficile in Prato Hospital. Stool samples were collected from 1197 patients hospitalized from January 2010 to December 2011. In all the samples the common antigen GDH was investigated and only in samples positive for the antigen the presence of the A and B toxins was investigated. Our results showed that 170/1197 samples (14%) were positive for the antigen, and of these 170 patients, 84 (49%) were found positive also for the toxins. In addition the percentage of samples positive for toxins was higher in 2010 (8.6%) than in 2011 (5.9%)

Detection of T. vaginalis,M. hominis,M. genitalium, C. trachomatis, N. gonorrhoeae and U. urealyticum using Multiplex PCR

Intoduction. The sexually transmitted diseases include a large group of infections affecting both the sexes. In this study we evaluated the prevalence of Trichomonas vaginalis, Mycoplasma hominis, Mycoplasma genitalium, Chlamydia trachomatis, Neisseria gonorrhoeae and Ureaplasma urealyticum in the Prato area during the period September 2010 – July 2011. Methods.We analysed different kind of samples (urine, endocervical swabs, urethral swabs, seminal fluids) from hospitalized patients or referred to the Prato clinic subjects.The DNA was obtained using EZ1-DNA extraction kit and EZ1 instrument.The DNA was then amplified using the Seeplex STD6 kit (Seegene, Korea), identifying multiple pathogens simultaneously (T. vaginalis, M. hominis, M. genitalium, C. trachomatis, N. gonorrhoeae e U. urealyticum). The revelation was performed by electrophoresis on microchip (instrument Multina, Shimadzu, Japan). Results. 1136 samples from Italian and foreign patients were examined: 876 were endocervical swabs (77%), 103 urethral swabs (9%), 103 seminal fluids (9%), and 54 urines (5%). The number of females was higher than males [894 (78.7%) vs 242 (21.3%)]; the mean age of females was 37.0±11.6 years, whereas that of males was 41.5 ±12.63 years.The prevalence of urogenital pathogens was: 15 positive samples for T. vaginalis (1.3%), 56 for M. hominis (4.9%), 13 for M. genitalium (1.1%), 28 for C. trachomatis (2.5%), 8 for N. gonorrhoeae (0.7%) and 87 for U. urealyticum (7.7%).Among all positive, 25 subjects were positive for more than one pathogen and in particular: one was positive for the presence of 4 pathogens, five presented 3 pathogens simultaneously and the remaining nineteen for 2 pathogens. Conclusions. This study provides data on the prevalence of sexually transmitted diseases in the hospital of Prato

Screening for Klebsiella pneumoniae producing carbapenemase in Prato, Italy

In this study, the prevalence of carbapenemase in the area of Prato (Italy), from January 2011 to August 2013, is reported. Samples showing carbapenem resistance were tested for lactamase production with disc diffusion and molecular methods. Totally, 48 out of 1542 patients with K. pneumoniae showed carbapenem resistance (42 KPC 5 MBL and 1 OXA-48).</p