Reliable automated identification and susceptibility testing of clinically relevant bacteria is an essential routine for microbiology laboratories, thus improving patient care. Examples of automated identification systems include the Phoenix (Becton Dickinson) and the VITEK 2 (bioMérieux). However, more and more frequently, microbiologists must isolate “difficult” strains that automated systems often fail to identify. An alternative approach could be the genetic identification of isolates; this is based on 16S rRNA gene sequencing and analysis. The aim of the present study was to evaluate the possible use of MicroSeq 500 (Applera) for sequencing the 16S rRNA gene to identify isolates whose identification is unobtainable by conventional systems. We analyzed 83 “difficult” clinical isolates: 25 gram-positive and 58 gram-negative strains that were contemporaneously identified by both systems—VITEK 2 and Phoenix—while genetic identification was performed by using the MicroSeq 500 system. The results showed that phenotypic identifications by VITEK 2 and Phoenix were remarkably similar: 74% for gram-negative strains (43 of 58) and 80% for gram-positive strains were concordant by both systems and also concordant with genetic characterization. The exceptions were the 15 gram-negative and 9 gram-positive isolates whose phenotypic identifications were contrasting or inconclusive. For these, the use of MicroSeq 500 was fundamental to achieving species identification. In clinical microbiology the use of MicroSeq 500, particularly for strains with ambiguous biochemical profiles (including slow-growing strains), identifies strains more easily than do conventional systems. Moreover, MicroSeq 500 is easy to use and cost-effective, making it applicable also in the clinical laboratory.

Resistance of Helicobacter pylori to clarithromycin occurs with a prevalence ranging from 0 to 15%. This has an important clinical impact on dual and triple therapies, in which clarithromycin seems to be the better choice to achieve H. pylori eradication. In order to evaluate the possibility of new mechanisms of clarithromycin resistance, a PCR assay that amplified a portion of 23S rRNA from H. pylori isolates was used. Gastric tissue biopsy specimens from 230 consecutive patients were cultured for H. pylori isolation. Eighty-six gastric biopsy specimens yielded H. pylori -positive results, and among these 12 isolates were clarithromycin resistant. The latter were studied to detect mutations in the 23S rRNA gene. Sequence analysis of the 1,143-bp PCR product (portion of the 23S rRNA gene) did not reveal mutation such as that described at position 2142 to 2143. On the contrary, our findings show, for seven isolates, a T-to-C transition at position 2717. This mutation conferred a low level of resistance, equivalent to the MIC for the isolates, selected using the E-test as well as using the agar dilution method: 1 μg/ml. Moreover, T2717C transition is located in a highly conserved region of the 23S RNA associated with functional sites: domain VI. This fact has a strong effect on the secondary structure of the 23S RNA and on its interaction with macrolide. Mutation at position 2717 also generated an Hha I restriction site; therefore, restriction analysis of the PCR product also permits a rapid detection of resistant isolates.

Coagulase-negative staphylococci (CoNS) are a major cause of nosocomial blood stream infection, especially in critically ill and haematology patients. CoNS are usually multidrug-resistant and glycopeptide antibiotics have been to date considered the drugs of choice for treatment. The aim of this study was to characterize CoNS with reduced susceptibility to glycopeptides causing blood stream infection (BSI) in critically ill and haematology patients at the University Hospital Tor Vergata, Rome, Italy, in 2007.

Introduction: Listeria innocua is widespread in food and the environment and is considered to be a non‐pathogenic bacterium in healthy subjects. To date, this species has only been associated with human diseases in a fatal case of bacteraemia in an elderly patient. Here, we describe a case of acute meningitis infection caused by this bacterium. Case presentation: Our patient had an increased risk of infection because of treatment with etanercept and a corticosteroid given for rheumatoid arthritis. Etanercept use has been described previously as the possible cause of multiple Listeria monocytogenes infections (to date, four cases have been described, of which two were cases of arthritis and two of meningitis), but etanercept has never been associated with L. innocua meningitis. In our case, despite rapid identification of the pathogen and proper antibiotic treatment, the patient had an unfavourable outcome. Conclusion: To the best of our knowledge, this report constitutes the first documentation of a case of meningitis due to L. innocua, and our experience serves as a warning to microbiologists and clinicians that L. monocytogenes is not the only Listeria sp. that can cause human meningitis.

Background: Methods widely used to detect apoptosis do not allow us to easily distinguish between nuclei from viable or necrotic cells. Even if apoptosis and necrosis seem to occur as alternatives at the single cell level, they could be present simultaneously in a cell population much more frequently than expected. For this reason, attention was focused on attempting to recognize, by multiparameter flow cytometry, the characteristics of viable cells and of apoptotic or necrotic dead cells. Methods: Apoptosis and necrosis were induced in vitro in murine thymocytes and lymphocytes from adult peripheral blood by using dexamethasone or prostaglandin E2 treatment and heat shock at 60°C or hydrogen peroxide, respectively. Traditional methods, such as DNA gel electrophoresis and propidium iodide staining followed by single‐fluorescence analysis or annexin‐V–fluorescein isothiocyanate plus propidium iodide staining by using flow cytometry, were compared with a new method. This method consisted of combined light‐scatter and red fluorescence analysis by flow cytometry after isolation of nuclei by hypotonic solution as well as high‐dose detergent treatment and DNA staining with propidium iodide. Results: Results showed that, although traditional methods such as DNA‐gel electrophoresis and single‐parameter fluorescence flow cytometry analysis were unable, as expected, to discriminate among viability, apoptosis, and necrosis, our new method has enabled us to easily identify nuclei from viable, apoptotic, and necrotic cells. Results obtained by using our method were comparable to those obtained by using two‐color analysis of cells after propidium iodide/annexin V staining. Conclusions: A highly reproducible, inexpensive, rapid, and easily accessible method of analysis has been developed for simultaneously detecting apoptosis and necrosis. Cytometry 35:145–153, 1999.