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Summary: Serratia marcescens is an opportunistic pathogen that survives in inhospitable environments causing large outbreaks, particularly in neonatal intensive care units (NICUs). Genomic studies revealed that most S. marcescens nosocomial infections are caused by a specific clone (here “Infectious clone”). Whole genome sequencing (WGS) is the only portable method able to identify this clone, but it requires days to obtain results. We present a cultivation-free hypervariable-locus melting typing (HLMT) protocol for the fast detection and typing of S. marcescens, with 100% detection capability on mixed samples and a limit of detection that can reach the 10 genome copies. The protocol was able to identify the S. marcescens infectious clone with 97% specificity and 96% sensitivity when compared to WGS, yielding typing results portable among laboratories. The protocol is a cost and time saving method for S. marcescens detection and typing for large environmental/clinical surveillance screenings, also in low-middle income countries.

Abstract Background Infective endocarditis (IE) is associated with high rates of mortality. Prolonged treatments with high-dose intravenous antibiotics often fail to eradicate the infection, frequently leading to high-risk surgical intervention. By providing a mechanism of antibiotic tolerance, which escapes conventional antibiotic susceptibility profiling, microbial biofilm represents a key diagnostic and therapeutic challenge for clinicians. This study aims at assessing a rapid biofilm identification assay and a targeted antimicrobial susceptibility profile of biofilm-growing bacteria in patients with IE, which were unresponsive to antibiotic therapy. Results Staphylococcus aureus was the most common isolate (50%), followed by Enterococcus faecalis (25%) and Streptococcus gallolyticus (25%). All microbial isolates were found to be capable of producing large, structured biofilms in vitro. As expected, antibiotic treatment either administered on the basis of antibiogram or chosen empirically among those considered first-line antibiotics for IE, including ceftriaxone, daptomycin, tigecycline and vancomycin, was not effective at eradicating biofilm-growing bacteria. Conversely, antimicrobial susceptibility profile of biofilm-growing bacteria indicated that teicoplanin, oxacillin and fusidic acid were most effective against S. aureus biofilm, while ampicillin was the most active against S. gallolyticus and E. faecalis biofilm, respectively. Conclusions This study indicates that biofilm-producing bacteria, from surgically treated IE, display a high tolerance to antibiotics, which is undetected by conventional antibiograms. The rapid identification and antimicrobial tolerance profiling of biofilm-growing bacteria in IE can provide key information for both antimicrobial therapy and prevention strategies.

Infections caused by ESBL-producing Enterobacteriaceae and carbapenemases producing bacteria are a growing phenomenon which is one of the leading causes of death among elderly or immunosuppressed patients and is also associated with a higher cost of hospitalization. As worldwide, also in Italy a steady and alarming increase of these microorganisms is reported. With the present paper we present a brief review of the prevalence of ESBL-producing and carbapenemase strains isolated at the Hospital - University Campus - Hospital “Luigi Sacco” in Milan between January 2007 and June 2012. Samples ESBLs positive (Vitek2: bioMérieux, France) were subjected to phenotype confirmation by E-test method (bioMérieux, France). In addition 34 K. pneumoniae carbapenemases producing strains collected between 2011 and 2012, were firstly confirmed with an Hodge test and then tested with a NASBA EasyQ KPCv1.0 test (bioMérieux, France) able to detect the bla KPC gene. The data collected showed a high prevalence of ESBLs and carbapenemases producing strains. 1828 out of 15585 were positive for ESBL with the following distribution: 15.6% E. coli, 13.0% K. pneumoniae, 3.6% Enterobacter spp, 7.7% P. mirabilis and 6% P. aeruginosa. Out of 1828, 193 samples (10.5%) were confirmed as positive and respectively 89.4% were E. Coli, 80% were K. pneumoniae and 89.5% were P. mirabilis. The 3.6% of strains were positive for carbapenemase: 45.3% were A. baumannii and 41.8% were P. aeruginosa. K. pneumoniae strains carbapenemase positive were confirmed in 100% of cases by the Hodge test and in 97% of cases by molecular investigation.

In the spring of 2009, a new variant of influenza A/H1N1 virus that had never been isolated before, was identified. From April 27 to December 31, 2009 the respiratory samples of 974 patients, obtained from suspected cases of pandemic influenza A virus infection, were analyzed at the Clinical Microbiology Laboratory of the “L. Sacco” University Hospital in Milan. The diagnosis of influenza A/H1N1 infection was performed initially through the use of different molecular biological methods: Seeplex® RV12 ACE Detection (Seegene), NUCLISENS® EASYQ® INFLUENZA A/B (bioMérieux), Influenza A/B Q-PCR Alert (Nanogen) running in parallel with rRT-PCR (CDC) to confirm the positivity to the new influenza virus, then was used a single specific test, Fast set H1N1v (Arrow Diagnostics). Retrospective study of data showed that 293 (30.1%) patients were positive for the new strain of influenza A/H1N1 virus and 8 (0.8%) for influenza A other than H1N1 virus.The distribution of influenza A/H1N1 cases showed two peaks, one on July (62.9%) and the other one on October (36%), moreover we observed that 155 patients (53%) out of 293 positive for influenza A/H1N1 virus aged under 20 years old. The first positivity peak was found in travelers and the second one, occurred 2-3 months prior to the classic seasonal epidemic influenza, was attributed to autochthonous cases , by which the virus had spread worldwide. The highest proportion of cases were among subjects aged from 0 to 20 years and, over this age the positivity rate decreased proportionally with increasing age, in agreement with data reported in other countries.

Gram stain and bacterial antigen detection are economic and rapid tests for the identification of the bacteria in cerebrospinal fluid (CSF) for the diagnosis of meningitis. Even if culture is the gold standard of these diagnosis, it requires long time (growth time). The purpose of our study was to compare in the laboratory diagnosis of meningitis real time PCR (Eurospital, Italy). with standard methods. Samples were collected in patients referred to L. Sacco Universital Hospital (range of age: 16 to 70 years) from September 2008 to July 2009. 9 out of 130 CFS samples investigated were positive in PCR but they produced different results to the other methods used.

An eighteen-month study (2008-2009) was carried out on patients, aged on average 30 months, admitted with gastroenteritis to the Paediatric Unit of the L. Sacco Hospital in Milan, Italy. Faecal samples of 154 patients were investigated for Rotavirus, Norovirus,Adenovirus, Enterovirus and Bocavirus. A total of 25 norovirus positive samples were selected for nucleotide sequence analysis, that allowed identify GII.2, GII.3 and GII.4 2006 virus variants (06 a, 06 b).

Objective Aim of our study was investigate the presence of enteric viruses like Norovirus, Bocavirus and Rotavirus in hospitalized children belong to Paediatrics Clinics of L. Sacco Hospital. Material and methods 150 faecal samples were investigated from January 2008- 2009, by PCR real time for Norovirus and Bocavirus; Rotavirus was detected by enzyme immunoassay. Results We obtain a positive result on total samples in percentage of 36%: Rotaviruses are estimated to cause more than 20% of gastroenteritis cases; Noroviruses 7.3% cases; Bocaviruses 6.6% cases. Rotaviruses infections occurred in children 1-4 years of age and for 2 cases at 7 and 13 years old. Illness typically happened in the cooler months from January to March even if there were few positive cases in September/October. Noroviruses and Bocaviruses infections occurred in children 1-2 years of age and their seasonality were respectively from September to December and from January to March for Bocaviruses. Conclusions The present results point out the importance to detect new viral gastrointestinal agents associated with paediatric illness.

Surveillace of viral gastoenteritis infections is very poor in Italy, even if starting from 2004 Norovirus became one of the most causative agent of infections in all the seasons. The aim of our study was to evaluate the isolation of Norovirus both in hospitalizes patients and communitary patients. From October 2006 to March 2008 we examined 400 samples. Our results showed only 15 sporadic cases in pediatric, HIV comunitary patients. These cases were analyzed by using an ELISA screening (Biopharm) and the results were confirmed with real time PCT (Argene).