The molecular epidemiology of multidrug-resistant Acinetobacter baumannii was investigated in the medicalsurgical intensive care unit (ICU) of a university hospital in Italy during two window periods in which two sequential A. baumannii epidemics occurred. Genotype analysis by pulsed-field gel electrophoresis (PFGE) of A. baumannii isolates from 131 patients identified nine distinct PFGE patterns. Of these, PFGE clones B and I predominated and occurred sequentially during the two epidemics. A. baumannii epidemic clones showed a multidrug-resistant antibiotype, being clone B resistant to all antimicrobials tested except the carbapenems and clone I resistant to all antimicrobials except ampicillin-sulbactam and gentamicin. Type 1 integrons of 2.5 and 2.2 kb were amplified from the chromosomal DNA of epidemic PFGE clones B and I, respectively, but not from the chromosomal DNA of the nonepidemic clones. Nucleotide analysis of clone B integron identified four gene cassettes: aacC1, which confers resistance to gentamicin; two open reading frames (ORFs) coding for unknown products; and aadA1a, which confers resistance to spectinomycin and streptomycin. The integron of clone I contained three gene cassettes: aacA4, which confers resistance to amikacin, netilmicin, and tobramycin; an unknown ORF; and bla OXA-20 , which codes for a class D ␤-lactamase that confers resistance to amoxicillin, ticarcillin, oxacillin, and cloxacillin. Also, the bla IMP allele was amplified from chromosomal DNA of A. baumannii strains of PFGE type I. Class 1 integrons carrying antimicrobial resistance genes and bla IMP allele in A. baumannii epidemic strains correlated with the high use rates of broad-spectrum cephalosporins, carbapenems, and aminoglycosides in the ICU during the study period.

Conjugation experiments demonstrated that resistance to imipenem could be transferred along with a plasmid containing the carbapenem-hydrolyzing oxacillinase bla OXA-58 gene. The plasmid that we called pABIR was 29,823 bp in size and showed a novel mosaic structure composed of two origins of replication, four insertion sequence (IS) elements, and 28 open reading frames. The bla OXA-58 gene was flanked by IS18 and ISAba3 elements at the 5 and 3 ends, respectively. The production of the carbapenem-hydrolyzing oxacillinase OXA-58 was apparently the only mechanism for carbapenem resistance in A. baumannii isolates causing the outbreak at the Lebanese Hospital.Acinetobacter baumannii is an important opportunistic pathogen responsible for a variety of nosocomial infections, especially in intensive care unit (ICU) patients (11,18). These organisms are frequently resistant to multiple antimicrobial agents including broad-spectrum ␤-lactams, carbapenems, aminoglycosides, and fluoroquinolones (11,18,31). A. baumannii may develop resistance to carbapenems through various mechanisms including decreased permeability because of porin modifications or reduced expression, the overexpression of efflux pumps, and the production of carbapenemases, such as metallo-␤-lactamase or carbapenem-hydrolyzing oxacillinases (CHDLs) (3,(17)(18)(19)(20)(21)(22)(23)(24).The emergence of carbapenem resistance in A. baumannii has been reported worldwide (11,18) and has been correlated in Europe with the acquisition of CHDLs (10,(18)(19)(20)(21)(22)(23). Three main acquired CHDL gene clusters have been identified in A. baumannii, represented by the bla OXA-23 -, bla OXA-24 -, and bla OXA-58 -like genes (13). Plasmid-borne bla OXA-23 and bla OXA-58 genes have been shown to contribute significantly to carbapenem resistance in A. baumannii (3,10,13,18,21,22). In particular, the bla OXA-58 gene has been identified in carbapenem-resistant A. baumannii isolates worldwide (3,13,20,23,30). Recent studies have shown that the flanking insertion sequence (IS) elements ISAba1, ISAba2, ISAba3, and IS18 regulate bla OXA-58 gene expression (22), and ISAba3 possibly regulates its acquisition (19).An outbreak of multidrug-resistant Acinetobacter baumannii was observed between November 2004 and October 2005 in the Saint George University Hospital of Beirut, Beirut, Lebanon. The aim of the present study was to (i) assess the genetic relatedness and the antimicrobial susceptibility of A. baumannii isolates in the hospital, (ii) study the horizontal gene transfer of the carbapenem resistance of the A. baumannii isolates, and (iii) analyze plasmid DNA sequences involved in the acquisition of carbapenem resistance of the A. baumannii isolates. MATERIALS AND METHODS Microbiological methods.A. baumannii isolates were identified as being A. baumannii spp. by using the Vitek 2 automatic system with an ID-GNB card for the identification of gram-negative bacilli according to the manufacturer's instructions (bioMerieux, Marcy-l'Etoile, France). Species identification was conf...

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This study investigated the molecular epidemiology of a clonal outbreak of multidrug-resistant Acinetobacter baumannii that occurred between June 2003 and June 2004 in a tertiary-care hospital in Naples, Italy. A. baumannii was isolated from 74 patients, of whom 38 were infected and 36 were colonised. Thirty-three patients had ventilator-associated pneumonia, three had hospital-acquired pneumonia, and two had sepsis. Genotypic analysis of 45 available A. baumannii isolates revealed two distinct pulsed-field gel electrophoresis (PFGE) patterns. Of these, PFGE pattern 1 was represented by isolates from 44 patients and was identical to that of an epidemic A. baumannii clone isolated in another hospital of Naples during 2002. All A. baumannii isolates of PFGE type 1 showed identical multiresistant antibiotypes, characterised by resistance to all antimicrobial agents tested, including carbapenems, with the exception of colistin. In these isolates, inhibition of OXA enzymes by 200 mM NaCl reduced the imipenem MIC by up to four-fold. Molecular analysis of antimicrobial resistance genes showed that all A. baumannii isolates of PFGE type 1 harboured a class 1 integron containing the aacA4, orfX and bla(OXA-20) gene cassettes, an ampC gene and a bla(OXA-51)-like allele. Moreover, a bla(OXA-58)-like gene surrounded by the regulatory elements ISAba2 and ISAba3 was identified in a 30-kb plasmid from A. baumannii isolates of PFGE type 1, but not PFGE type 2. Thus, selection of a single A. baumannii clone producing an OXA-58-type carbapenem-hydrolysing oxacillinase was responsible for the increase in the number of A. baumannii infections that occurred in this hospital.

BackgroundThe spread of carbapenem resistant Enterobacteriaceae (CRE) is an emerging clinical problem, of great relevance in Europe and worldwide. The aim of this study was the molecular epidemiology of CRE isolates in Valle d’Aosta region, Italy, and the mechanism of carbapenem resistance.ResultsSixty consecutive CRE samples were isolated from 52 hospital inpatients and/or outpatients from November 2013 to August 2014. Genotyping of microbial isolates was done by pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST), carbapenemases were identified by PCR and sequencing. Carbapenem resistance gene transfer was performed by filter mating, plasmids from parental and transconjugant strains were assigned to incompatibility groups by PCR-based replicon typing. Molecular characterization of CRE isolates assigned 25 Klebsiella pneumoniae isolates to PFGE types A1-A5 and sequencing type (ST) 101, 17 K. pneumoniae isolates to PFGE type A and ST1789 (a single locus variant of ST101), 7 K. pneumoniae isolates to PFGE types B or C and ST512, 2 K. pneumoniae isolates to PFGE type D and ST405, and 5 Escherichia coli isolates to PFGE type a and ST131. All K. pneumoniae ST101 and ST1789 isolates were extended-spectrum beta-lactamase (ESBL) producers and carried blaCTX-M-1 group gene; 4 K. pneumoniae ST101 isolates were resistant to colistin. Molecular analysis of beta-lactamase genes identified blaKPC-2 and blaCTX-M-group 1 into conjugative plasmid/s assigned to IncFII incompatibility group in ST101 and ST1789 K. pneumoniae isolates, blaKPC-3 into conjugative plasmid/s assigned to IncF incompatibility group in ST512 and ST405 K. pneumoniae isolates, blaVIM-1 into conjugative plasmid/s assigned to IncN incompatibility group in ST131 E. coli isolates.ConclusionsThe spread of CRE in Valle d’Aosta region was caused by the selection of KPC-2 producing K. pneumoniae ST101 and ST1789 epidemic clones belonging to clonal complex 101, KPC-3 producing K. pneumoniae epidemic clones assigned to ST512 and ST405, and VIM-1 producing E.coli ST131 epidemic clone. Carbapenem resistance, along with blaKPC-2, blaKPC-3 and blaVIM-1 carbapenemase genes, was transferred by conjugative plasmids assigned to IncFII, IncF, and IncN incompatibility groups, respectively, in filter mating experiments. The emergence of colistin resistance was observed in KPC-2 producing K. pneumoniae ST101 isolates.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-015-0597-z) contains supplementary material, which is available to authorized users.

In order to compare oral and high-dose iv corticosteroid therapy for Graves' disease, 25 patients with Graves' ophthalmopathy were treated with two weekly iv injections of 1 g of methylprednisolone diluted in 250-500 ml of physiological solution for 6 weeks, and were compared to a group of 26 patients treated with oral prednisone at a dose of 60-80 mg/day progressively reduced every 2 weeks for a total duration of 4-6 months. The efficacy of treatment was evaluated using the ophthalmopathy index score. Patients were followed at 3, 6, 12 months, and afterwards yearly. All patients showed a significant improvement in signs and symptoms of orbital inflammation and a slight improvement in proptosis and diplopia. Relevant side-effects were reported from patients receiving oral therapy, but no significant side-effects were observed in patients treated with high iv doses; a few cases presented with gastric pain (highly sensitive to aluminium oxide or ranitidine), while most of the patients referred to cutaneous rashes and a metal taste that disappeared some hours after the infusion. Improvements observed after treatment have been stable in both groups. In conclusion, in addition to a lower incidence of side-effects compared to the classic oral therapy, the high-dose iv steroid therapy provides efficient and stable improvement in Graves' ophthalmopathy.

The spread of the aac(6')-Ie-aph(2'')-Ia gene was responsible for high-level resistance to gentamicin and amikacin among enterococci isolated from patients in our geographical area.

BackgroundHealthcare-associated infections (HAIs) are a frequent complication associated with hospitalization of infants in neonatal intensive care units (NICUs). The aim of this study was to evaluate and describe the results of surveillance of HAIs in a III level NICU in Naples, Italy during 2006–2010.MethodsThe surveillance covered 1,699 neonates of all birth weight (BW) classes with > 2 days NICU stay. Infections were defined using standard Centers for Disease Control and Prevention definitions adapted to neonatal pathology and were considered to be healthcare-associated if they developed > 2 days after NICU admission.ResultsOne hundred-fifty-three HAIs were diagnosed with a frequency of 9% and an incidence density of 3.5 per 1000 days of hospital stay. HAIs developed in all BW classes, but patients weighing ≤ 1000 g at birth were more affected with a decreasing trend from the lowest to the highest BW classes. Sepsis proved to be the most frequent infection (44.4%), followed by urinary tract infection (UTI) (28.8%), pneumonia (25.5%) and meningitis (1.3%). Device associated infections (i.e. central line-associated bloodstream infections (BSIs), umbilical catheter-associated BSI and ventilator associated pneumonias (VAPs) represented 64.1% of all HAIs. Most frequent pathogens responsible for all types of infections were: P. aeruginosa (17%), C. parapsilosis (16.3%), E. coli (13.1%), C. albicans (10.5%), non- extended spectrum beta-lactamase (ESBL) K. pneumoniae (7.8%), and coagulase-negative Staphylococci (5.2%). No microbiological diagnosis was achieved for 6.5% of infections.ConclusionsHAIs developed in all BW classes but low BW neonates were at major risk to acquire HAIs in our NICU. Use of central line-, umbilical-catheter and mechanical ventilation was associated with higher risk of infection. Our findings highlight the importance of an extensive surveillance approach in the NICU setting, which includes all BW classes of neonates and monitors infections associated with the use of medical devices.