The molecular epidemiology of multidrug-resistant Acinetobacter baumannii was investigated in the medical-surgical 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 β-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.

We investigated the basis of the carbapenem resistance of 17 multidrug-resistant Acinetobacter baumannii clinical isolates collected from 2004 to 2005 at the Saint George University Hospital in Beirut, Lebanon. A. baumannii isolates were clonally related and were susceptible to colistin and trimethoprim-sulfamethoxazole, susceptible or intermediate to ampicillin-sulbactam and meropenem, and resistant to all other antimicrobials. 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 IS 18 and IS Aba3 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.

The 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. Sixty 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 bla CTX-M-1 group gene; 4 K. pneumoniae ST101 isolates were resistant to colistin. Molecular analysis of beta-lactamase genes identified bla KPC-2 and bla CTX-M-group 1 into conjugative plasmid/s assigned to IncFII incompatibility group in ST101 and ST1789 K. pneumoniae isolates, bla KPC-3 into conjugative plasmid/s assigned to IncF incompatibility group in ST512 and ST405 K. pneumoniae isolates, bla VIM-1 into conjugative plasmid/s assigned to IncN incompatibility group in ST131 E. coli isolates. The 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 bla KPC-2, bla KPC-3 and bla VIM-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.

Objectives: We evaluated the genetic and molecular basis of high-level resistance to gentamicin and amikacin in 91 clinical isolates of Enterococcus faecalis and Enterococcus faecium in a university hospital in southern Italy from 1987 to 2003. Methods: Antibiotic susceptibility was evaluated by disc diffusion and microdilution methods. Genotyping was performed by PFGE and dendrogram analysis. Aminoglycoside resistance genes were analysed by multiplex PCR. Aminoglycoside resistance gene transfer was performed by filter mating. Results: In our strain collection, 44% of E. faecalis and 52% of E. faecium were high-level-resistant to gentamicin. Fifty-two PFGE profiles were identified for E. faecalis and 15 for E. faecium. Although the majority of PFGE patterns were single isolates, four patterns (two for E. faecalis and two for E. faecium) were isolated each in 8 and 4, and 6 and 4 different patients, respectively. The aac(6′)-Ie-aph(2″)-Ia gene was responsible for high-level resistance to gentamicin and amikacin in E. faecalis and E. faecium; the aph(2″)-Id gene responsible for resistance to gentamicin was also isolated in E. faecium; the aph(3′)-IIIa and ant(4′)-Ia genes responsible for resistance to amikacin were also isolated in E. faecalis and E. faecium. High-level resistance to gentamicin, along with the aac(6′)-Ie-aph(2″)-Ia gene, was transferred at a frequency of about 10⁻⁵ to 10⁻⁸ per recipient cell in 14 of 17 E. faecalis and 3 of 4 E. faecium different genotypes. Conclusions: 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.