Acinetobacter baumannii has become a major nosocomial threat, causing infections with high rates of morbidity and mortality (1-4).Acinetobacter baumannii hospital outbreaks have been assessed with various DNA typing methods (5-8). Very few typing methods assess outbreaks in real time. Recently, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been developed for the rapid identification of pathogens (9). There are a growing number of reports showing the utility of MALDI-TOF MS to type different bacterial species for easy and early detection of nosocomial outbreaks (10-16).The aim of this study was to assess the potential utility of MALDI-TOF MS to detect the nosocomial spread of A. baumannii isolates. For this purpose, 35 multidrug-resistant strains of A. baumannii, isolated from colonized or infected patients hospitalized in a general hospital in Italy in 2010, were studied using the repetitive sequence-based PCR (rep-PCR) DiversiLab system (DL; bioMérieux, Marcy l'Etoile, France) and MALDI-TOF MS system (Bruker Daltonics, Bremen, Germany), and the results were compared. After storage at Ϫ70°C, the isolates were thawed out and cultured simultaneously on MacConkey agar.DiversiLab rep-PCR (bioMérieux) was performed according to the manufacturer's instructions (5, 17). Briefly, DNA was extracted from a bacterial culture, amplified using rep-PCR, loaded in LabChip, and run using an Agilent 2100 Bioanalyzer (Agilent Technologies). The results were analyzed by DiversiLab with the Pearson correlation (PC) coefficient to emphasize peak intensities more than peak presence or absence. The strains were considered indistinguishable, similar, or different if the similarity was Ն97.5% without differences in the fingerprinting pattern, Ͼ95% and Ͻ97.5%, or Յ95%, respectively.For MALDI-TOF MS, bacteria were cultured for 24 h at 37°C on MacConkey agar and extracts were prepared as described previously (18, 19). For isolate identification, the row spectra were compared with those in the Biotyper database and a log(score) of Ն2.3 was considered to represent a secure species identification. Spectra were acquired with a microflex LT mass spectrometer (Bruker Daltonics) and recorded in the positive linear mode at a laser frequency of 20 Hz, ion source 1 voltage of 20 kV, ion source 2 voltage of 8.5 kV, and mass range from 2,000 to 20,000 kDa, as described elsewhere (18). Reference spectra of newly created main spectra were added to the original Biotyper database. To evaluate the spectrum variation within each strain and to estimate the mass spectral variance of biological replicates, a principal component analysis (PCA) was performed; two biological replicates from six randomly selected Acinetobacter strains were tested, as described above, and spectra for each selected strain were acquired in the same experiment on the mass spectrometer. Technical mass-spectral variance was also evaluated, analyzing five technical replicates from each Acinetobacter independent culture on the same MALDI t...

In this study we report the analysis of 131 Klebsiella pneumoniae (K. pneumoniae) clinical isolates from patients hospitalized in various wards, of Perugia General Hospital, from August 2014 to January 2015. Forty two isolates (32.1 %), were resistant to at least one carbapenem antibiotic and, among these isolates, 14 (33.3 %) exhibited resistance to colistin. All isolates were carbapenemases producers and 41 (97.6 %) harboured the bla KPC gene. Carbapenem-resistant K. pneumoniae isolates (CRKPs) were, also, typed for the genotypic diversity and the results revealed the circulation of two major clusters.This surveillance study evidences the spread of CRKP isolates in Perugia General Hospital and confirms that carbapenem-resistant K. pneumoniae isolates have reached epidemic dissemination in Italy. In addition the percentage of resistance to colistin resulted to be less than that observed in other hospital laboratories across Italy. In conclusion the circulation of these isolates should be monitored and appropriate policy of surveillance must be used, in a target manner, in order to reduce the spread of carbapenem-resistant isolates.

Valutazione del sistema GeneXpert ® per la rilevazione di Staphylococcus aureus meticillinoresistente in campioni clinici SUMMARY Infections caused by methicillin-resistant Staphyloccocus aureus strains (MRSA) have reached epidemic proportions globally, being the major cause of nosocomial infections. Rapid identification of MRSA in nasal swabs or in clinical samples is considered a useful strategy for control and treatment of these infections. GeneXpert system (Cepheid Europe,Vira-Solelch, Maurence-Scopont-France) can detect by real-time PCR in approximately one hour methicillin-resistant S. aureus or coagulase-negative staphylococci (CoNS) in clinical samples, in comparison with 24 hours for the culture or 48 hours for the antimicrobial susceptibility testing. In this study GeneXpert system was compared with traditional tests for MRSA detection in nasal swabs, bloodcultures and surgical wound swabs. Materials and methods. Eighteen nasal swabs, 23 blood-cultures and 13 surgical wound swabs were tested. The samples were cultured on blood-agar and mannitol-salt agar. Identification of isolates was carried out with traditional tests (Gram staining, catalase, coagulase) and automatic Phoenix system. Methicillin-susceptibility was evaluated according to 2010 CLSI guidelines. GeneXpert system was performed according to manufacturers instructions, by using the specific kits and methicillin-resistance was detected by amplification of the genic sequences spa, SCC e mecA. Results. The results showed a 100% accordance between GeneXpert system and traditional tests for detection of methicillin-resistant staphylococci. In particular, among 18 nasal swabs, no MRSA was detected, while 1 bloodculture (4.3%) and 4 surgical wound swabs (30.7%) were positive for MRSA. Conclusions. GeneXpert system allows a rapid detection of MRSA in clinical samples and shows the same sensitivity and specificity as traditional tests. Therefore, it represents a further effective diagnostic method for prevention and treatment of nosocomial infections due to methicillin-resistant staphylococci.