Giorgio Piccinelli

Nosocomial outbreak of the pandemic Influenza A (H1N1) 2009 in critical hematologic patients during seasonal influenza 2010-2011: detection of oseltamivir resistant variant viruses

BackgroundThe pandemic influenza A (H1N1) 2009 (H1N1pdm09) virus infection caused illness and death among people worldwide, particularly in hematologic/oncologic patients because influenza infected individuals can shed virus for prolonged periods, thus increasing the chances for the development of drug-resistant strains such as oseltamivir-resistant (OST-r) variant.MethodsThe aim of our study was to retrospectively evaluate the clinical importance of OST-r variant in circulating strains of the pandemic H1N1pdm09 virus. By means of RT-PCR and Sanger sequencing we analysed the presence of OST-r variant in 76 H1N1pdm09 laboratory-confirmed cases, hospitalized at the hematologic/oncologic ward at Spedali Civili of Brescia –Italy.ResultsOut of 76 hospitalized hematologic/oncologic patients, 23 patients (30.2%) were infected by H1N1pdm09 virus. Further investigation revealed that 3 patients were positive for the OST-r variant carrying the H275Y mutation. All the 23 infected patients were immuno-compromised, and were under treatment or had been treated previously with oseltamivir. Three patients died (13%) after admission to intensive care unit and only one of them developed H275Y mutation.ConclusionsOur retrospective observational study shows that pandemic influenza A (H1N1) 2009 virus can cause significant morbidity and even mortality in hematologic/oncologic patients and confirms the high rate of nosocomial transmission of pandemic H1N1pdm09 virus in these critical subjects. Indeed, the reduction in host defences in these hospitalized patients favoured the prolonged use of antiviral therapy and permitted the development of OST-r strain. Strategies as diagnostic vigilance, early isolation of patients and seasonal influenza A(H1N1) vaccination may prevent transmission of influenza in high risk individuals.

In Vitro Activity of Solithromycin against Erythromycin-Resistant Streptococcus agalactiae

ABSTRACT The in vitro antibacterial activity of solithromycin (CEM-101) against macrolide-resistant isolates (n = 62) of Streptococcus agalactiae (group B streptococcus [GBS]) was determined. Phenotypic characterization of macrolide-resistant strains was performed by double-disc diffusion testing. A multiplex PCR was used to identify the erm(B), erm(TR), and mef(A/E) genes, capsular genotypes, and alpha-like (Alp) protein genes from the GBS strains. Determination of MIC was carried out using the microdilution broth method. The Etest method was used for penicillin, azithromycin, clarithromycin, and erythromycin. Solithromycin had a MIC50 of ≤0.008 μg/ml and a MIC90 of 0.015 μg/ml against macrolide-susceptible S. agalactiae. These MICs were lower than those displayed by penicillin (MIC50 of 0.032 μg/ml and MIC90 of 0.047 μg/ml), the antibiotic agent of choice for prophylaxis and treatment of GBS infections. Against macrolide-resistant S. agalactiae, solithromycin had a MIC50 of 0.03 μg/ml and a MIC90 of 0.125 μg/ml. Against erm(B) strains, solithromycin had a MIC50 of 0.03 μg/ml and a MIC90 of 0.06 μg/ml, while against mef(A) strains, it had a MIC50 of 0.03 μg/ml and a MIC90 of 0.125 μg/ml. Most erythromycin-resistant GBS strains were of serotype V (64.5%) and associated significantly with alp2-3. Moreover, a statistically significant association was observed between the constitutive macrolide-lincosamide-streptogramin B resistance (cMLSB) phenotype and the erm(B) gene-carrying strains, the alp2-3 gene and the M phenotype, and the mef(A/E) gene and epsilon. Overall, our results show that solithromycin had lower or similar MICs than penicillin and potent activity against macrolide-resistant strains independent of their genotype or phenotype, representing a valid therapeutic alternative where β-lactams cannot be used.

Emergence of the first levofloxacin-resistant strains of Streptococcus agalactiae isolated in Italy

ABSTRACT Of 901 group B streptococcus strains analyzed, 13 (1.4%) were resistant to levofloxacin (MICs of >32 μg/ml for seven isolates, 2 μg/ml for four isolates, and 1.5 μg/ml for four isolates). Mutations in the quinolone resistance-determining regions (QRDRs) of gyrase and topoisomerase IV were identified. A double mutation involving the Ser-81 change to Leu for gyrA and the Ser-79 change to Phe or to Tyr for parC was linked to a high level of fluoroquinolone resistance. In addition, two other mutational positions in parC were observed, resulting in an Asp-83-to-Tyr substitution and an Asp-83-to-Asn substitution. Different mutations were also observed in gyrB, with unknown significance. Most levofloxacin-resistant GBS strains were of serotype Ib and belonged to sequence type 19 (ST19) and clonal complex 19 (CC-19). Most of them exhibited the epsilon gene.

Characterization and antibiotic susceptibility of Streptococcus agalactiae isolates causing urinary tract infections

Streptococcus agalactiae (GBS) has been implicated in urinary tract infections but the microbiological characteristics and antimicrobial susceptibility of these strains are poorly investigated. In this study, 87 isolates recovered from urine samples of patients who had attended the Spedali Civili of Brescia (Italy) and had single organism GBS cultured were submitted to antimicrobial susceptibility testing, molecular characterization of macrolide and levofloxacin resistance, PCR-based capsular typing and analysis of surface protein genes. By automated broth microdilution method, all isolates were susceptible to penicillin, cefuroxime, cefaclor, and ceftriaxone; 80%, 19.5% and 3.4% of isolates were non-susceptible to tetracycline, erythromycin, and levofloxacin, respectively. Macrolide resistance determinants were iMLS(B) (n=1), cMLS(B) (n=10) and M (n=5), associated with ermTR, ermB and mefA/E. Levofloxacin resistance was linked to mutations in gyrA and parC genes. Predominant capsular types were III, Ia, V, Ib and IX. Type III was associated with tetracycline resistance, while type Ib was associated with levofloxacin resistance. Different capsular type-surface protein gene combinations (serotype V-alp2, 3; serotype III-rib; serotype Ia-epsilon) were detected. A variety of capsular types are involved in significant bacteriuria. The emergence of multidrug resistant GBS may become a significant public health concern and highlights the importance of careful surveillance to prevent the emergence of these virulent GBS.

Detection and molecular characterization of enteric viruses in children with acute gastroenteritis in Northern Italy

Enteric viral infections are a major concern for public health, and viral acute gastroenteritis is the principal cause of pediatric morbidity and mortality worldwide, mostly in developing countries. The purpose of this study was to determine the prevalence of different enteric viruses detected in a pediatric population with acute gastroenteritis symptoms, and to characterize the strains detected. Stools were collected from children, aged from 2 months to 15 years old, admitted to one of the main hospitals of Northern Italy, between November 2015 and October 2016. Stools were tested for nine enteric viruses (adenovirus, rotavirus A, norovirus, astrovirus, sapovirus, enterovirus, parechovirus, bocavirus and aichivirus) by molecular methods. Furthermore, rotavirus, norovirus and adenovirus were deeply characterized by nucleotide sequencing and phylogenetic analysis. A total of 151 out of 510 (29.6%) stools analyzed resulted positive for at least one of the enteric virus investigated. The most common virus detected was rotavirus A (53/151, 35.1%), followed by norovirus (39/151, 25.8%), adenovirus (35/151, 23.1%), sapovirus (9/151, 6%), enterovirus (5/151, 3.3%), astrovirus (5/151, 3.3%), parechovirus (4/151, 2.6%) and bocavirus (1/151, 0.6%). Aichi virus was not detected in any sample. Co-infections were detected in 12 out of 510 faecal samples (2.3%). These data improved the knowledge of the enteric viruses circulating in children in Northern Italy. In fact, besides rotavirus, adenovirus and norovirus, several viruses circulated across the whole year in the pediatric population object of this study. The introduction of specific viral diagnosis in our clinical setting will improve patient care by reducing unnecessary use of antibiotics addressing the right etiologic diagnosis.

Invasive Candidiasis in Brescia, Italy: Analysis of Species Distribution and Antifungal Susceptibilities During Seven Years

The aims of this study were to evaluate the epidemiology of nosocomial candidemia in a large teaching hospital in Brescia, Italy, and the in vitro antifungal susceptibility of isolates. We analyzed 196 isolates causing fungemia in patients admitted in our hospital, between January 2009 and December 2015. Strains were identified by VITEK 2 and MALDI-TOF MS. MICs were determined by Sensititre Yeast OneTM. The resistance was defined by using the revised CLSI breakpoints/epidemiological cutoff values to assign susceptibility or wild type to systemic antifungal agents. Most infections were caused by Candida albicans (60%), Candida parapsilosis (15%), Candida glabrata (12%) and Candida tropicalis (6%). The susceptibility rate for fluconazole was 96.5%. Non-Candida species isolates exhibited full susceptibilities to echinocandins according to CLSI breakpoints. Amphotericin B demonstrated excellent activity against all Candida species. Local epidemiological and antifungal susceptibility studies are necessary in order to improve empirical treatment guidelines.

A cluster of invasive listeriosis in Brescia, Italy.

Clinical forms were indicated as bacteraemia, miscarriage and peritonitis. Listeriosis was considered hospital-acquired when signs of infection occurred ≥72 h after admission. For each patient with listeriosis, different data were analyzed retrospectively using a medical chart review: demographic information, underlying diseases preceding the onset of listeriosis; days from admission to onset of listeriosis, type of infection and outcome, antibiotic used for therapy. Data on the type of food consumed could not be collected because of insufficient information in medical charts for both the period before admission and during the hospital stay. Before the organism was isolated from microbiological specimens, Listeria had not been considered as being the causative organism in any of these cases. Gram-positive, nonspore forming rods, facultative anaerobes, which produced catalase and a narrow hemolysis, were presumptively identified as Listeria. Further identification was attained using VITEK (bioMérieux, St. Louis, MO, USA) and real-time PCR (Eusepscreen, Eurospital SpA, Trieste, Italy). Genotyping was performed by a multiplex PCR that separates the four major serovars (1/2a, 1/2b, 1/2c, and 4b) of L. monocytogenes strains into four distinct groups as previously described [7]. DNA was extracted from each isolate by the NucliSENS easyMAG (Biomèrieux, Florence, Italy), according to the manufacturer’s instructions. Approximately 1 ng of DNA was used in the PCRs with primers and conditions as described elsewhere [7]. The PCR products were identified by analyzing the unique banding pattern following agarose gel 2 % (wt/vol) electrophoresis. An automated DiversiLab system (BioMérieux), which uses repetitive extragenic palindromic sequencebased PCR (rep-PCR), was performed to further characterize the Listeria isolates. Listeria monocytogenes is a gram-positive facultative intracellular organism that frequently contaminates foodprocessing environments. Increasing incidence rates of invasive listeriosis have been reported over the last few decades by several European countries, with an incidence of listeriosis ranging from 0.3 to 0.8/100,000 people/year in France and Italy, respectively [1]. L. monocytogenes causes an invasive disease with the involvement of central nervous system and bactaeremia with a high case fatality (20– 30 %). Various risk factors have been related to invasive listeriosis. Older adults, where there is an immunosenescence of T-cell-mediated immunity, patients receiving immunosuppressive therapies and people with impaired cell-mediated immunity or autoimmune diseases are at higher risk of invasive listeriosis. Maternal infections are often asymptomatic or mild, but pregnant women can transmit the infection to the fetus, for whom the infection can be fatal [2]. Listeriosis is mainly transmitted through the consumption of contaminated food in the community, and vertical transmission from mother to child. Hospital-associated infections usually occur as clustered outbreaks from environmental contact [3, 4] or contaminated food [5, 6]. Here, we describe a cluster of six cases of invasive listeriosis identified at the Spedali Civili Hospital in Brescia, Italy, between July 2013 and February 2014. A listeriosis case was defined as isolation of L. monocytogenes from a normally sterile site such as blood or cerebrospinal fluid (CSF) or from placental or fetal tissue.

Analysis of mutations in DNA gyrase and topoisomerase IV of Ureaplasma urealyticum and Ureaplasma parvum serovars resistant to fluoroquinolones.

This study aims to determine the prevalence of fluoroquinolone resistance of Ureaplasma biovars and serovars isolated from urogenital clinical samples and determine the underlying molecular mechanism for quinolone resistance for all resistant isolates. Of 105 samples confirmed as positive for U. urealyticum/U. parvum, 85 were resistant to quinolones by the Mycoplasma-IST2 kit. However, only 43 out of 85 quinolone resistant isolates had amino acid substitutions in GyrA, GyrB, ParC and ParE proteins underlining that this assay have mis-identified as fluoroquinolone resistant 42 isolates. The known ParC E87K and ParC S83L mutations were found in 1 and 10 isolates, respectively. An original mutation of ureaplasmal ParC (E87Q, 1 isolate) was found. Furthermore, we found a ParE R448K mutation in one isolate, already described. Among the additional alterations detected, the most prevalent mutation found was L176F in GyrA protein in 18 isolates with single infection and in 3 isolates with mixed ureaplasma infections. Mutations in GyrB (E502Q, 4 isolates), ParE (Q412K, Q412P, Q412T, 3 independent isolates), whose role is unknown, were also found. Other sporadic mutations in the four genes were identified. This investigation is the result of monitoring the data for molecular fluoroquinone resistance in Ureaplasma spp. in Italy. Resulting that this acquired resistance is high and that continued local epidemiological studies are essential to monitor and document their antimicrobial resistance trends.

Fulminant septic shock caused by Capnocytophaga canimorsus in Italy: case report

Capnocytophaga canimorsus infection was recently recognized as a zoonosis. We report the first case of fulminant septic shock in Italy caused by this pathogen. The patient, with a history of splenectomy, died at the main hospital in Brescia with a presumptive diagnosis of sepsis. PCR and sequencing on post mortem samples confirmed C. canimorsus as a causative organism. Our purpose is to alert medical professionals to the virulence of C. canimorsus in asplenic and immunocompromised patients.

Myroides odoratimimus urinary tract infection in an immunocompromised patient: an emerging multidrug-resistant micro-organism

BackgroundMyroides spp. are common environmental organisms and they can be isolated predominantly in water, soil, food and in sewage treatment plants. In the last two decades, an increasing number of infections such as urinary tract infections and skin and soft tissue infections, caused by these microorganisms has been reported. Selection of appropriate antibiotic therapy to treat the infections caused by Myroides spp. is difficult due to the production of a biofilm and the organism’s intrinsic resistance to many antibiotic classes.Case presentationWe report the case of a 69-year-old immunocompromised patient who presented with repeated episodes of macroscopic haematuria, from Northern Italy.A midstream urine sample cultured a Gram negative rod in significant amounts (> 105 colony-forming units (cfu)/mL), which was identified as Myroides odoratimimus. The patient was successfully treated with trimethoprim/sulfamethoxazole after antibiotic susceptibility testing confirmed its activity.ConclusionThis case underlines the emergence of multidrug resistant Myroides spp. which are ubiquitous in the environment and it demands that clinicians should be more mindful about the role played by atypical pathogens, which may harbour or express multidrug resistant characteristics, in immunocompromised patients or where there is a failure of empiric antimicrobial therapy.