From phenotyping to the study of clonal relationship of microbial isolates

The term "typing" is generally used with two meanings: a methods to establish the correct taxonomic collocation of a genus/specie/biotype, b methods for discriminating different bacterial isolates of the same species in order to establish the genetic relationship among the microorganisms involved in a possible outbreak. In this paper we focus our attention on the second aspect, that represents a relevant epidemiological tools in infection prevention and control. Typing systems are traditionally based on two steps workup: the first is the study of phenotypes such as serotype, biotype, phage-type, or antimicrobial susceptibilities of the isolates and this can be easily performed in every microbiology laboratory; the second, examines the relatedness of isolates at a molecular level. Over the years many molecular methods have been developed and efficiently applied in several hospital settings. The large panorama of methods put the microbiologists in trouble to operate the proper choice. Thus, in the present paper, we have reviewed old as well new molecular typing methods in order to provide a useful guide that can represent an overview on molecular methods and particularly of their specific pro and cons

Naphthoquinone Derivatives Exert Their Antitrypanosomal Activity via a Multi-Target Mechanism

BACKGROUND AND METHODOLOGY: Recently, we reported on a new class of naphthoquinone derivatives showing a promising anti-trypanosomatid profile in cell-based experiments. The lead of this series (B6, 2-phenoxy-1,4-naphthoquinone) showed an ED(50) of 80 nM against Trypanosoma brucei rhodesiense, and a selectivity index of 74 with respect to mammalian cells. A multitarget profile for this compound is easily conceivable, because quinones, as natural products, serve plants as potent defense chemicals with an intrinsic multifunctional mechanism of action. To disclose such a multitarget profile of B6, we exploited a chemical proteomics approach. PRINCIPAL FINDINGS: A functionalized congener of B6 was immobilized on a solid matrix and used to isolate target proteins from Trypanosoma brucei lysates. Mass analysis delivered two enzymes, i.e. glycosomal glycerol kinase and glycosomal glyceraldehyde-3-phosphate dehydrogenase, as potential molecular targets for B6. Both enzymes were recombinantly expressed and purified, and used for chemical validation. Indeed, B6 was able to inhibit both enzymes with IC(50) values in the micromolar range. The multifunctional profile was further characterized in experiments using permeabilized Trypanosoma brucei cells and mitochondrial cell fractions. It turned out that B6 was also able to generate oxygen radicals, a mechanism that may additionally contribute to its observed potent trypanocidal activity. CONCLUSIONS AND SIGNIFICANCE: Overall, B6 showed a multitarget mechanism of action, which provides a molecular explanation of its promising anti-trypanosomatid activity. Furthermore, the forward chemical genetics approach here applied may be viable in the molecular characterization of novel multitarget ligands

Addition of the Aldose Reductase Inhibitor Benzofuroxane Derivative BF-5m to Prolonged and Moderate Exercise Training Enhanced Protection of the Rat Heart From Type-1 Diabetes

Moderate exercise training may not be sufficient to exert beneficial effects on the cardiovascular system because of the long-term multifactorial etiology of diabetic complications. The addition of a proper pharmacological tool to the physical exercise should improve the outcomes of the diabetic damage. Here it is shown that 8 weeks exercise training of type 1 diabetic Sprague-Dawley (SD) rats resulted in a significantly increased heart rate, a 14% increase in the left ventricular ejection fraction (LVEF) increased plasma insulin levels and a 13% decrease in plasma glucose with respect to sedentary animals. The training also resulted in a 22% reduction in cardiac QT interval from a diabetic sedentary value of 185 ± 19 ms. Treatment of trained rats with the new antioxidant and NO-releasing aldose reductase 2 inhibitor 5(6)-(benzo[d]thiazol-2-ylmethoxy) benzofuroxane BF-5m, 20 mg/kg/day, added a further and significant (P < 0.01 vs. sedentary) increase of the LVEF up to 38% at 8 week time point. The long QT interval recorded in trained rats was reduced to further 12% by addition to the training of pharmacological treatment with 20 mg/kg/day BF-5m. At this time, the association of the two treatments improved the expression into the cardiac tissue of sarcoplasmic reticulum Ca2+ ATPase 2 (SERCA2) and manganese superoxide dismutase (MnSOD), and reduced the fibrosis

Drug-resistant Streptococcus pyogenes: a case report of pyoderma and cellulitis

In the last years there has been worldwide an increase in the incidence of Streptococcus pyogenes infections and its sequelae. The purpose of the present study is to describe a clinical case of pyoderma and cellulitis by S. pyogenes with a pattern of resistance to penicillin, erythromycin and levofloxacin. Antimicrobial susceptibility was analysed by automated method of disk diffusion while E-test method was used to verify the minimum inhibitory concentration to penicillin, erythromycin and levofloxacin. Although penicillin remains the first-choice treatment for S. pyogenes infection, worldwide antibiotic resistance and the associated phenotypes are highly variable across countries. This case report strengthens the need for continued surveillance to obtain further insights into the forces governing resistance in S. pyogenes.</em

Serological diagnosis of infections sustained by Toxoplasma gondii, Rubella virus and Citomegalovirus, a comparison between two methods

The serological diagnosis of infections from Toxoplasma gondii, Rubella virus and Cytomegalovirus is of a great importance especially during pregnancy for the risk of maternal-fetal infections. Serological testing have evolved with time and are many on the market with different levels of specificity and sensivity. The aim of the study was to determine the analytical and clinical concordance between two methods for the determination of specific IgG and IgM antibodies against T. gondii, Rubella virus and Cytomegalovirus. The total numbers of samples evaluated for T. gondii, Rubella virus and Cytomegalovirus IgG and IgM were 124 and 127, 83 and 87, 129 and 103, respectively. The comparison methods used for this study were Diamedix® enzime imunoassay run in MAGO-Plus Delta Biologicals® Immunodiagnostics System (routinely used) and Vitros© Eci chemiluminescence immunoassay run in Vitros ECiQ Immunodiagnostics System ORTHO-Clinical Diagnostics Johnson &amp; Johnson company.Vidas bioMérieux was take as referee method to evaluate discrepant results. Analytical concordance ranged between 87 and 97%, clinical concordance ranged between 93 and 99%. Despite differences in detection antibodies (specific IgG and IgM) and revelation of signal the methods evaluated show generally good levels of clinical concordance.The results are consistent with references and External Quality Assurance Programs

Role of Signal-to-Cutoff Ratios in Hepatitis C Virus Antibody Detection

none6noWe reviewed results from 12,800 samples tested for hepatitis C virus (HCV) antibody detection in our laboratory by screening (Ortho chemiluminescence immunoassay [CIA]) and supplemental tests (Chiron recombinant immunoblot assay [RIBA]). We found that a signal-to-cutoff (S/Co) ratio of 10.3 was, in our setting, the most efficient cutoff point to improve the diagnostic algorithm of HCV infection.mixedM.Moretti; B.Pieretti; A.Masucci; D.Sisti; M.Rocchi; E.DelpreteM., Moretti; B., Pieretti; A., Masucci; Sisti, Davide; Rocchi, MARCO BRUNO LUIGI; E., Delpret

“Alert” microrganisms: role of microbiology laboratory

The phenomenom of antibiotic resistance represents today an important problem especially in nosocomial settings with a considerable impact in clinical treatment of infections. We have draft in collaboration with C.I.O. (Comitato infezioni Ospedaliere) and applied in the routine of microbiology laboratory the “Procedura per la gestione dei microrganismi sentinella” (Protocol for the management of “alert microrganisms”). In this study we have investigated the isolation of “alert microrganisms” in our hospital in a period of fifteen months and we have evaluated in time a relative antibiotic resistance.The term “alert microrganisms” is refered to microrganisms with particular antibiotic resistance profiles that are responsible of difficult to treat hospital infections. The study suggests the role of microbiology laboratory in the systematic research of “alert microrganisms” as excellent and economic information source for identify they to level of species and following the antibiotic resistance in time

Diagnosis of Bacteriuria and Leukocyturia by Automated Flow Cytometry Compared with Urine Culture▿

Urinary tract infection (UTI) is a widespread disease, and thus, the most common samples tested in diagnostic microbiology laboratories are urine samples. The “gold standard” for diagnosis is still bacterial culture, but a large proportion of samples are negative. Unnecessary culture can be reduced by an effective screening test. We evaluated the performance of a new urine cytometer, the Sysmex UF-1000i (Dasit), on 703 urine samples submitted to our laboratory for culture. We compared bacteria and leukocyte (WBC) counts performed with the Sysmex UF-1000i to CFU-per-milliliter quantification on CPS agar to assess the best cutoff values. Different cutoff values of bacteria/ml and WBC/ml were compared to give the best discrimination. On the basis of the results obtained in this study, we suggest that when the Sysmex UF-1000i analyzer is used as a screening test for UTI the cutoff values should be 65 bacteria/ml and 100 WBC/ml. Diagnostic performance in terms of sensitivity (98.2%), specificity (62.1%), negative predictive value (98.7%), positive predictive value (53.7%), and diagnostic accuracy (73.3%) were satisfactory. Screening with the Sysmex UF-1000i is acceptable for routine use. In our laboratory, we have reduced the number of bacterial cultures by 43%, speeded up their reporting, and decreased the inappropriate use of antibiotics

Epidemiology, diagnosis and management of superficial mycoses: ours three years retrospective analysis

Mycotic infections of the skin, hair and nails are disorder with high prevalence. Dermatophytes are the most frequently etiologic agents followed by yeasts and non-dermatophyte moulds. The geographic distribution is variable because of migration, lifestyle and socio-economics conditions. In this study, based on ours three years retrospective data analysis, we evaluate the epidemiology of etiologic agents, diagnosis and management of superficial mycoses. Our data and our experience suggest the importance of this microbiological diagnosis and the role of the Clinical Microbiologist to assess with Dermatologist and Primary Care Phyisician an appropriate diagnostic and therapeutic plan