Italian Clones Research Articles

Aminoglycoside resistance genes in early members of the Acinetobacter baumannii ST78A (SMAL, Italian clone) reside in an IS26-bounded island in the chromosome.

The Acinetobacter baumannii isolate called SMAL, previously used to determine the structures of capsular polysaccharide and lipooligosaccharide, was recovered in Pavia, Italy in 2002 among the collection of aminoglycoside-resistant isolates designated as SMAL type. This type was later called the Italian clone, then ST78. ST78 isolates are now widely distributed. To establish the resistance gene complement and the location and structure of acquired resistance regions in early members of the Italian/ST78 clone. The draft genome of SMAL2002 was assembled from Illumina MiSeq reads. Contigs containing resistance genes were joined and located in the chromosome using PCR with custom primers. The resistance profile was determined using disc diffusion. SMAL2002 is an ST78A isolate and includes three aminoglycoside resistance genes, aadB (gentamicin, kanamycin, tobramycin) aphA1 (kanamycin, neomycin) and aac(6')-Ian (amikacin, kanamycin, tobramycin). The aadB gene cassette is incorporated at a secondary site in a relative of the aphA1-containing, IS26-bounded pseudo-compound transposon, PTn6020. The aac(6')-Ian gene is in an adjacent IS26-bounded structure that includes sul2 (sulphonamide) and floR (florfenicol) resistance genes. The two pseudo-compound transposons overlap and are in the chromosomal hutU gene flanked by an 8 bp target site duplication. Although aac(6')-Ian was not noticed previously, the same genes and structures were found in several available draft genomes of early ST78A isolates. This study highlights the importance of correlating resistance profiles with resistance gene content. The location of acquired resistance genes in the SMAL2002 chromosome represents the original location in the ST78A lineage of ST78.

Genotypic analysis of Italian MRSA strains exhibiting low-level ceftaroline and ceftobiprole resistance

The aim of this study was to address the involvement of PBP mutations in the bactericidal activity to novel cephalosporins, alone and in combination with daptomycin, in not-related multidrug-resistant methicillin-resistant Staphylococcus aureus strains isolated during a nationwide Italian survey. MICs determination and time-killing assays were performed and mecA, pbp1, pbp2, pbp3, pbp4, and gdpP genes were sequenced. Ten strains showed low-level resistance to ceftaroline and ceftobiprole. PBP2a sequence analysis identified four different mutations (N146K; N204K; T235I; E239K) uniquely present in the non-penicillin-binding domain (nPBD). Epidemiologically, this resistance was associated with the most widespread MDR Italian clone ST228-SCCmecI-t001/t041, confirming its proclivity to accumulate mutations, and it is also associated to substitutions in the GdpP signaling protein, involved in the maintenance of di-AMP balance, recently associated with resistance to beta-lactams. Despite these mutations, both drugs retained their potent in vitro bactericidal activity and showed a synergistic effect towards difficult-to-treat isolates.

Fruit production from Opuntia ficus-indica ecotypes in comparison to commercial Italian clones

Fruit production, as an elementary chemical characteristic of the fruit, was evaluated in 16 Opuntia ficus-indica Portuguese ecotypes cultivated in a marginal soil without tillage, in the second and third years after plantation. The O. ficus-indica ecotypes were compared with the Italian cultivars ‘Bianca’ and ‘Gialla’. Significant differences were found among the O. ficus-indica ecotypes in biomass-related parameters and fruit yield, and different groups were established. Two spineless ecotypes (OFI-12 and OFI-13) had highest biomass production, with 9.9 Mg/ha dry matter on average. This was not significantly different from the ‘Gialla’ cultivar, which averaged 11.9 Mg/ha, for a density of 2,667 plants/ha,in the third year after plantation. Among Portuguese ecotypes, the fruit yields ranged from 2.4 to 10.1 Mg/ha fresh weight. The cultivars ‘Gialla’ and ‘Bianca’ had the highest fruit yield (13.8 and 13.6 Mg/ha fresh weight, respectively). The ‘Gialla’ cultivar and the group of ecotypes with orange pulp produced fruits of larger size and weight compared to the ‘Bianca’ cultivar and the group of ecotypes with white pulp.

Factors Contributing to Epidemic MRSA Clones Replacement in a Hospital Setting

The mechanisms governing the epidemiology dynamics and success determinants of a specific healthcare-associated methicillin-resistant S. aureus (HA-MRSA) clone in hospital settings are still unclear. Important epidemiological changes have occurred in Europe since 2000 that have been related to the appearance of the ST22-IV clone. Between 2006 and 2010, we observed the establishment of the ST22-IV clone displacing the predominant Italian clone, ST228-I, in a large Italian university hospital. To investigate the factors associated with a successful spread of epidemic MRSA clones we studied the biofilm production, the competitive behavior in co-culture, the capacity of invasion of the A549 cells, and the susceptibility to infection in a murine model of acute pneumonia of the two major HA-MRSA clones, ST22-IV and ST228-I. We showed that persistence of ST22-IV is associated with its increased biofilm production and capacity to inhibit the growth of ST228-I in co-culture. Compared to ST228-I, ST22-IV had a significantly higher capacity to invade the A549 cells and a higher virulence in a murine model of acute lung infection causing severe inflammation and determining death in all the mice within 60 hours. On the contrary, ST228-I was associated with mice survival and clearance of the infection. ST22-IV, compared with ST228-I, caused a higher number of persistent, long lasting bacteremia. These data suggest that ST22-IV could have exploited its capacity to i) increase its biofilm production over time, ii) maintain its growth kinetics in the presence of a competitor and iii) be particularly invasive and virulent both in vitro and in vivo, to replace other well-established MRSA clones, becoming the predominant European clone.

Epidemiological characterization and distribution of carbapenem-resistant Acinetobacter baumannii clinical isolates in Italy

This study was aimed at tracing the molecular characteristics of carbapenem-resistant Acinetobacter baumannii (CRAB) clinical isolates in Italy with both pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Two hundred and two CRAB isolates were collected during 2004–2009, in two different surveillance periods, from 22 Italian hospitals that were representative for both distribution and infection. PFGE was performed, and the MLST scheme used was based on the gene sequence as published on the MLST Pasteur website http://www.pasteur.fr/mlst. Representatives of the major European clones I (RUH 875) and II (RUH 134) were used as controls. The two groups of isolates were characterized for their carbapenem resistance genes: 154 of 202 carried blaOXA-58 alone, 21 of 202 also carried blaOXA-23, and 27 of 202 carried blaOXA-23 alone. No isolates were positive for blaOXA-24. Genotype analysis of all isolates identified four distinct patterns by PFGE, which correlated with four distinct sequence types (STs) by MLST. The distribution of these four clusters in Italy confirmed the propensity of A. baumannii for nosocomial cross-transmission in a vast geographical area. We observed that clones A and B had similarities with European clone II and I respectively. By MLST, clone A was ST2, like European clone II, and clone B was ST1, like European clone I. PFGE and MLST showed the same discriminatory power and reproducibility. In addition, the two methods were concordant in defining CRAB Italian clones and in correlating them with the two pan-European clones.

Inter- and Intra-Varietal Genetic Variability in Malvasia Cultivars

The DNA molecular analyses together with ampelography, ampelometry, and biochemistry are essential for grapevine identification and investigation of genetic differences among the Vitis vinifera L. cultivars and clones. Ten Malvasia cultivars (i.e., Istrian Malvasia; M. delle Lipari; M. bianca di Candia; M. di Candia Aromatica; M. del Lazio; M. bianca lunga, also known as Malvasia del Chianti; M. nera di Brindisi/Lecce; M. di Casorzo; M. di Schierano, and M. nera di Bolzano) were analyzed using molecular approaches to study the genetic inter-varietal variability. Thirty Istrian Malvasia genotypes (i.e., 8 Italian clones, such as ISV 1, ISV F6, VCR 4, VCR 113, VCR 114, VCR 115, ERSA 120, ERSA 121, and 22 autochthonous grapevine accessions grown in Istrian Peninsula, Croatia) were investigated to evaluate the morphological and genetic intra-varietal variability. DNA analysis allowed discrimination of all Malvasia genotypes at molecular level using AFLP, SAMPL, and M-AFLP markers. Italian clones and autochthonous Croatian accessions of Istrian Malvasia were grouped according to their different geographic origins. These results showed the great genetic variability of Malvasia genotypes suggesting the need for the preservation of autochthonous grapevine biotypes found on different areas to approve the correct choice and selection of the grape multiplication materials.

Widespread carbapenem resistant Acinetobacter baumannii clones in Italian hospitals revealed by a multicenter study

Population diversity, susceptibility to antibiotics including carbapenems of 277 Acinetobacter baumannii strains collected in 17 Italian hospitals over a 6-months’ period was assessed. Semi-automated rep-PCR was used for screening strains for genotypic relatedness. AFLP analysis and MLST were used as definitive methods for strain, species and/or clone identification.Among the 277 strains, 49 rep-PCR types were distinguished with four types (1–4) predominant, indicating both intra- and interhospital spread. AFLP analysis allowed to distinguish 51 types and largely confirmed rep-typing results. Isolates with predominant rep-types 1 and 2 (in 3 and 9 hospitals) were allocated to EU clones I and II, respectively. Rep-type 3 (8 hospitals) belonged to a new clone (“Italian clone”). Rep-type 4 was found in 2 neighbouring hospitals. Two isolates from 2 locations belonged to EU clone III. Twenty-five isolates were identified by AFLP-analysis to A. pittii, emphasizing misidentification by phenotypic methods. MLST confirmed clone identification by AFLP; demonstrating also that the “Italian clone” was ST78, recently detected in different Mediterranean countries.Multidrug resistance, defined as resistance to 9 out of the 11 drugs tested, was common in 10 out of 17 hospitals. The high prevalence of carbapenem resistance was associated with OXA-58 found in 9 out of the 10 hospitals. A high percentage of noted very major errors in susceptibility testing, especially for amikacin and meropenem, was probably due to heteroresistant strains. The occurrence of carbapenem and multidrug resistance in A. baumannii was mainly confined to a limited number of clonal lineages of A. baumannii.

First year development of poplar clones in biomass short rotation coppiced experimental cultures

Poplar and willows are the main fast growing tree species used for biomass production in short rotation coppiced (SRC) cultures. The main characteristics for biomass production (plant survival, basal diameter and height increment) after first growing season are presented. Four Italian clones - AF2, AF6, AF8 and Monviso (Alasia New Clones) and two controls - Turcoaia and Sacrau 79’ (ICAS, România) have been tested in five experimental cultures, in different locations. Italian energetic clones have adapted very well to the pedoclimatic conditions of South-Eastern Romania, being more competitive than control clones in terms of biomass production and resistance to leaf diseases. AF8 and Monviso have registered the most active growth.

Hospital-associated methicillin-resistant Staphylococcus aureus (HA-MRSA) in Italy

The aim of our study was to trace the dynamic changes of hospital-associated methicillin-resistant Staphylococcus aureus (HA-MRSA) lineages in Italy, comparing the genotypic backgrounds of contemporary isolates over a period of 17 years, with those of a sample of early MRSA strains from 1980.In total, 301 non-repetitive MRSA clinical isolates, recovered from 19 Italian hospitals between 1990 and 2007 were selected and analyzed for their antibiotic resistance, typed by PFGE and SCCmec, grouped into clonal-types and further characterized using Multi Locus Sequence Typing (MLST). A sample of fifteen early MRSA strains from 1980 was also used for comparison.The most interesting feature was the recent increase of ST228-MRSA-I (formerly the Italian clone; PFGE E) over the period 2000–2007 (57%), when compared to the period 1990–1999 (29%), and its stability to date, associated with a decrease of the highly epidemic ST247-MRSA-IA (formerly the Iberian clone; PFGE A), (23% from 1990 to 1999, 6% from 2000 to 2007). ST1-MRSA-I (1 out of 2 strains carrying ccrA2B2), ST8-MRSA-I (4 strains), ST15-MRSA-I (1 out of 4 carrying ccrA2B2) and ST30-MRSA-I (2 out of 5 carrying no ccrAB-types and ccrC) were the predominant earliest STs among the MRSA strains in 1980.A temporal shift in the susceptibility levels to glycopeptides was observed: strains with vancomycin MIC of ≥ 2 mg/L increased from 19.4% to 35.5%.In conclusion, we describe the alternation of MRSA clones that occurred in hospitals from 1990 to 2007 and the increase of the glycopeptide MIC levels, reflecting a worldwide trend. We document the detection of ST1, ST8, ST15 and ST30 in the 1980 isolates; we hypothesize their possible latency and their appearance as the current CA-MRSA clones.

AFLP reveals structural details of genetic diversity within cultivated olive germplasm from the Eastern Mediterranean

Amplified fragment length polymorphism (AFLP) analysis was used to assess genetic inter-relationships among olive varieties cultivated in the Eastern Mediterranean Basin. The genotypes sampled included most of the important cultivars from Turkey, Greece and the Middle East and selected genotypes from the Western Mediterranean area. A total of 119 polymorphic markers were generated from five selective primer-pair combinations. The combined data sets generated by just two primer-pairs were adequate to discriminate between all 65 genotypes, while each primer-pair could individually identify up to 64 genotypes. A factorial correspondence analysis (FCA) plot indicated that the cultivars clustered into two relatively modestly defined groups. The first broad group was dominated by cultivars from Turkey but also included genotypes originating from the Middle East (Syria and Lebanon) that collectively formed a tight subcluster. The second group comprised Greek cultivars and those originating from the Western Mediterranean. A significant genetic distance value between Greek and Turkish cultivars was provided by an analysis of molecular variance (AMOVA). There was also evidence of substructure here, with an apparent separation of most Spanish and Italian clones. These findings are in general accordance to previous suggestions of an East-West divergence of olive cultivars, although the dichotomy is less extensive than reported previously and complicated by regional variation within each group.

Clonal types and multidrug resistance patterns of methicillin-resistant Staphylococcus aureus (MRSA) recovered in Italy during the 1990s.

A large number (272) of methicillin-resistant Staphylococcus aureus (MRSA) isolates recovered from Italian hospitals during the early and late 1990s were characterized for multidrug resistance pattern and clonal type using a combination of genotyping methods, including pulsed-field gel electrophoresis (PFGE), spaA typing, multilocus sequence typing (MLST), determination of SCC mec type, and hybridization pattern with Tn 554. The majority of MRSA belonged to four genetic lineages: the pandemic Iberian and Brazilian clones, and two unique clonal types-the "Italian" and "Rome" clones of MRSA. The Italian clone carried the SCC mec type I in the genetic background of ST228, which is a double-locus variant of the sequence type of the multidrug-resistant New York/Japanese clone (ST5). The properties of the Rome clone showed several striking similarities to those of the Archaic clone of MRSA that was dominant among MRSA isolates in the mid-1960s to 1970s, but has not been detected since then in recent global surveillance studies.

Elm breeding for DED resistance, the Italian clones and their wood properties

In Italy, an elm breeding program to develop Dutch elm disease-resistant trees has been established by cross-breeding Asian elms with indigenous species, in order to produce individuals that combine resistance of the first with growth characteristics and higher degree of environmental adaptability of the second. Thus, the favourable adaptation of Ulmus pumila in Italy has been explored: «San Zanobi» and «Plinio» are two recent results of this research and many others promising clones as «FL090», «FL146», «FL441», «FL568», «FL634», are in the final stage of field testing. The resistance levels of all these clones were significantly higher level of resistance than «Lobel» and «Urban» in several assessments during different years. First technological tests indicate that the wood characteristics of «San Zanobi» and «FL090» clones are comparable to those of elm wood traditionally present on the market, thus highlighting that those clones is likely to satisfy the elm wood demand. The present characterisation needs to be confirmed by further evaluations of mature trees having bigger diameters and allowing industrial trials, still not available so far.

Epidemiology of methicillin-resistant staphylococci in Europe

Methicillin-resistant staphylococci are mostly resistant not only to all beta-lactams but also to a wide range of other antibiotics, and have emerged as major nosocomial pathogens during the past two decades. Considerable variations in the prevalence of methicillin-resistant Staphylococcus aureus (MRSA) exist between institutions and between geographic areas. In Europe, in general, a north-south gradient is observed, MRSA strains being rare in Scandinavian hospitals (<2%) and far more prevalent in Mediterranean hospitals (>40%). Whether low or high, the rates of MRSA prevalence in European countries have remained approximately the same during the last decade. Recent findings suggest that MRSA might also be emerging as a community-acquired pathogen. The first stage in the emergence of MRSA is its acquisition by methicillin-susceptible S. aureus, and the integration into its chromosome, of the mecA gene, which, together with the other mec genes, is carried on a mobile genetic element, the staphylococcal chromosomal cassette mec (SCCmec). The origin of SCCmec elements as well as the mechanisms of their acquisition remain unknown. Molecular epidemiology studies using different techniques clearly indicate that the massive geographic spread of MRSA results from the dissemination of relatively few highly epidemic clones. Five major lineages (the so-called Iberian, Brazilian, Hungarian, New York/Japan and pediatric pandemic MRSA clones) have been defined. In Europe, the Iberian clone has been reported in several countries; the Brazilian, pediatric and Hungarian clones have also been detected, but less frequently. A unique Italian clone is predominant in Italy. As with S. aureus, coagulase-negative staphylococci (CNS) represent a serious concern in hospital-acquired infections. Despite marked geographic variations, in some areas of Europe high proportions (60-70%) of CNS are methicillin resistant. The formation of biofilm is a key virulence factor of S. epidermidis, the prominent CNS pathogen, which is the most common cause of bacteremia in device-related infections. Another emerging nosocomial pathogen, S. hemolyticus, is characterized by a tendency to develop multiple antibiotic resistances, with a unique predisposition to glycopeptide resistance.

Aspects of forestry in Hungary

In central Hungary forest managers have achieved notable success in afforestation of both excessively dry and frequently flooded sites with valuable tree species. Planting of pines in predug trenches and angle-planting of specially-grown pine seedlings in furrows have successfully afforested dry sand areas. More moist sites have been afforested by planting poplar shoots in boreholes. Inundated areas have been planted with poplars of the Italian Clone No. 214 and the "Robusta" variety. Management practices are modified to enable forests to support dense game populations. Afforestation of difficult sites is justified not only on the basis of future wood production but also for amenity purposes and for game production. The successful establishment of plantations is the product of local ecological knowledge and an innovative approach to silvicultural techniques.