Intraspecific Diversity within Diaporthe Helianthi: Evidence from Rdna Intergenic Spacer (IGS) Sequence Analysis

Diaporthe helianthi is the causal agent of sunflower stem canker, a serious pathogen of sunflower in Europe but recorded sporadically in Italy. The genetic diversity of D. helianthi isolates from different geographic origins (Argentina, France, Italy, Yugoslavia, Romania) was investigated using IGS sequences. A 400 bp fragment of the portion of the IGS region flanking the 5' end of the 18S gene was amplified from each isolate. The aligned nucleotide sequences showed intraspecific sequence homology from 99-100% among French/Yugoslavian isolates to 95-100% among Italian isolates. French/Yugoslavian isolates shared 90-92% sequence homology with Italian isolates. The phylogenetic tree obtained from the aligned data revealed three separate groups. Group 1 included all isolates from France and former Yugoslavia and one isolate from Argentina; Group 2 included all Italian isolates and one isolate from Argentina. The most distantly related isolate was that from Romania (Group 3). The average genetic distances among isolates within Group 1 and within Group 2 were 0.22 and 3.29 respectively. The analysis showed that all isolates originating from countries where severe outbreaks of the disease are reported annually (France and former Yugoslavia) form a well defined taxon characterized by relatively low variability. This group is distinct from the group formed by isolates originating from Italy, whose variability is relatively much higher. Results obtained revealed a marked differentiation among pathogen isolates, and members of Group 1 seem not yet to have spread into Italian sunflower-growing areas.     

High prevalence of extended-spectrum beta lactamases among Salmonella enterica Typhimurium isolates from pediatric patients with diarrhea in China

We investigated the extended-spectrum beta lactamases among 62 Salmonella enterica Typhimurium isolates recovered from children with diarrhea in a Chinese pediatric hospital. A large proportion of S. enterica Typhimurium isolates were resistant to multiple antimicrobial agents, including ampicillin (90.3%), tetracycline (80.6%), trimethoprim/sulfamethoxazole (74.2%), chloramphenicol (66.1%), cefotaxime (27.4%). Forty-nine (79.0%) of S. enterica Typhimurium isolates were positive for bla(TEM-1b) and resistant to ampicillin. Thirteen S. enterica Typhimurium isolates (21.0%) were positive for bla(CTX-M-1-group) and bla(CTX-M-9-group), and all isolates harboring bla(CTX-M) genes were positive for ISEcp1. Two main clones (PFGE type A and D) accounted for nearly 70% of S. enterica Typhimurium isolates, and 7 CTX-M-producing isolates belonged to PFGE type D. Collectively, our data reveal multi-drug resistance and a high prevalence of extended spectrum beta lactamases among S. enterica Typhimurium isolates from children in China. In addition, we report the first identification of bla(CTX-M-55) within Salmonella spp. Our data also suggest that clonal spread is responsible for the dissemination of S. enterica Typhimurium isolates.     

Uniparental Markers of Contemporary Italian Population Reveals Details on Its Pre-Roman Heritage

Background

According to archaeological records and historical documentation, Italy has been a melting point for populations of different geographical and ethnic matrices. Although Italy has been a favorite subject for numerous population genetic studies, genetic patterns have never been analyzed comprehensively, including uniparental and autosomal markers throughout the country.

Methods/Principal Findings

A total of 583 individuals were sampled from across the Italian Peninsula, from ten distant (if homogeneous by language) ethnic communities — and from two linguistic isolates (Ladins, Grecani Salentini). All samples were first typed for the mitochondrial DNA (mtDNA) control region and selected coding region SNPs (mtSNPs). This data was pooled for analysis with 3,778 mtDNA control-region profiles collected from the literature. Secondly, a set of Y-chromosome SNPs and STRs were also analyzed in 479 individuals together with a panel of autosomal ancestry informative markers (AIMs) from 441 samples. The resulting genetic record reveals clines of genetic frequencies laid according to the latitude slant along continental Italy – probably generated by demographical events dating back to the Neolithic. The Ladins showed distinctive, if more recent structure. The Neolithic contribution was estimated for the Y-chromosome as 14.5% and for mtDNA as 10.5%. Y-chromosome data showed larger differentiation between North, Center and South than mtDNA. AIMs detected a minor sub-Saharan component; this is however higher than for other European non-Mediterranean populations. The same signal of sub-Saharan heritage was also evident in uniparental markers.

Conclusions/Significance

Italy shows patterns of molecular variation mirroring other European countries, although some heterogeneity exists based on different analysis and molecular markers. From North to South, Italy shows clinal patterns that were most likely modulated during Neolithic times.     

The occurrence and characterisation of oxy-imino-beta-lactams resistant strains among Salmonella enterica subsp. enterica isolated in Poland

Extended-spectrum beta-lactamases (ESBLs) are enzymes manifesting considerable hydrolyzing activity on a wide variety of beta-lactam antibiotics including oxyiminocephalosporins and aztreonam. In the study reported here we investigated the types of ESBL produced by Salmonella enterica subsp. enterica strains isolated from clinical samples in the microbiological laboratories of sanitary-epidemiological units in Poland from 1999 to 2004. Among 239 ampicillin-resistant Salmonella enterica subsp. enterica strains isolated from clinical samples in the microbiological laboratories of sanitary-epidemiological units in Poland, 68 isolates of oximino-beta-lactams resistant of 6 serovars were found. There were 16 epidemiological unrelated strains (6 isolates of S. Enteritidis, 5 isolates of S. Thompson, 3 isolates of S. Typhimurium, one-fold isolate of S. Muenster and S. enterica 1,9,12:-:-) coming from different areas of country and 52 epidemiologically related isolates of S. Oranienburg, coming from a prolonged outbreak in an orphanage in Lód?. All the strains were identified as the ESBLs producers. The molecular analysis revealed that most of them expressed CTX-M-3 ESBL which is widely observed in Poland and additional enzyme TEM-1. All tested isolates of S. Thompson and one of three S. Typhimurium isolates were found to produce SHV-5 ESBL. This is the first report regarding the presence of SHV-5 in the genus Salmonella in Poland.     

UDP-sugar hydrolase isozymes in Salmonella enterica and Escherichia coli: Silent alleles of ushA in related strains of Group I Salmonella isolates, and of ushB in wild-type and K12 strains of E. coli, indicate recent and early silencing events, respectively

Abstract Escherichia coli contains a single periplasmic UDP-glucose hydrolase (5'-nucleotidase) encoded by ushA. Salmonella enterica , serotype Typhimurium, also contains a single UDP-glucose hydrolase but, in contrast to E. coli , it is membrane-bound and is encoded by the non-homologous ushB gene; Salmonella enterica (Typhimurium) also contains a silent allele of the ushA gene ( ushA⁰ ). In this report, we show that nearly all natural isolates of Salmonella contain both UDP-sugar hydrolases, i.e. they are UshA⁺ UshB⁺. The only exceptions are all from sub-group I ( S. gallinarum, S. pullorum , and most Typhimurium strains), are UshA⁻ UshB⁺, and several have been shown to contain an ushA⁰ allele. These data, together with the fact that these latter strains are closely related genetically, strongly suggests a recent silencing mutation(s). We also report the presence in E. coli K-12, and in natural isolates of E. coli , of a DNA sequence which is homologous to the ushB gene of Salmonella ; since E. coli does not contain UshB activity, we tentatively refer to this sequence as ushB⁰ . Since all E. coli strains investigated are UshB⁻, we conclude that the silencing mutation(s) occured relatively eary following the divergence of Escherichia coli and Salmonella from a common ancestor that was ushA⁺ ushB⁺ .     

Prevalence and characterization of motile Salmonella in commercial layer poultry farms in Bangladesh

Salmonella is a globally widespread food-borne pathogen having major impact on public health. All motile serovars of Salmonella enterica of poultry origin are zoonotic, and contaminated meat and raw eggs are an important source to human infections. Information on the prevalence of Salmonella at farm/holding level, and the zoonotic serovars circulating in layer poultry in the South and South-East Asian countries including Bangladesh, where small-scale commercial farms are predominant, is limited. To investigate the prevalence of Salmonella at layer farm level, and to identify the prevalent serovars we conducted a cross-sectional survey by randomly selecting 500 commercial layer poultry farms in Bangladesh. Faecal samples from the selected farms were collected following standard procedure, and examined for the presence of Salmonella using conventional bacteriological procedures. Thirty isolates were randomly selected, from the ninety obtained from the survey, for serotyping and characterized further by plasmid profiling and pulsed-field gel electrophoresis (PFGE). Results of the survey showed that the prevalence of motile Salmonella at layer farm level was 18% (95% confidence interval 15-21%), and Salmonella Kentucky was identified to be the only serovar circulating in the study population. Plasmid analysis of the S. Kentucky and non-serotyped isolates revealed two distinct profiles with a variation of two different sizes (2.7 and 4.8 kb). PFGE of the 30 S. Kentucky and 30 non-serotyped isolates showed that all of them were clonally related because only one genotype and three subtypes were determined based on the variation in two or three bands. This is also the first report on the presence of any specific serovar of Salmonella enterica in poultry in Bangladesh.     

Microbiological features of acute bacterial conjunctivitis in a central Italian area

The study aims to identify bacteria causing conjunctivitis in a central Italian area and to analyze chemosusceptibility. From 2005 to 2006, 91 conjunctival swabs were collected from acute conjunctivitis cases and screened for common bacteria and fungi. Susceptibility tests were performed on isolates. Staphylococcus aureus, Streptococcus pneumoniae and Haemophilus influenzae amounted to 86.2%. Overall, 100% of strains were susceptible to chloramphenicol and 96.6% to quinolones. Conversely, 20.7% of isolates were tetracycline-resistant and, even if all Gram negative isolates were susceptible to gentamicin, the most frequently isolated pneumococci are constitutively resistant. The study provides support for a rational choice of empiric therapy.     

A tribute to Italian physiologists of Jewish descent evicted during the persecution ordered by the Fascist Regime in 1938

The present report commemorates the persecution of five renowned Italian physiologists of Jewish descent that lost their chairs in medical schools because of the anti-semitic policies of the fascist regime. In 1938, Mussolini promulgated the Racial Laws, officially with the aim of safeguarding the purity of the Italian race in conquered African colonies. However, their true intent was to persecute the Italian Jewish community in agreement with the policy of Nazi Germany. In accordance with the Racial Laws, all non-Aryans were banished from professional activities and were evicted from public, social, and academic life. As a result, 98 full professors in Italian universities were removed from their academic positions. In medical schools, physiology, more than other discipline, lost the most prominent faculty members. Of the 17 full Professors of Human Physiology, five were of Jewish descent, and all were evicted: they were Camillo Artom from Palermo, Mario Camis from Bologna, Carlo Foà from Milan, Amedeo Herlitzka from Turin, and Ugo Lombroso from Genoa. All were talented and famous scientists who were forced to leave Italy and take refuge in foreign countries. At the end of World War II, Camis, Foà, Herlitzka, and Lombroso returned to Italy and resumed their previous academic positions, whereas Artom remained in the United States. Unfortunately, Camis died later that year. During the postwar period, some of the fascists responsible for the Jewish persecution were killed or committed suicide while the survivors were imprisoned and prosecuted. However, all were soon released and resumed their former positions.     

Genetic analysis carried out on blood-spots of phenylalanine hydroxylase-deficient newborns detected by northeastern Italian neonatal screening

The aim of this work was to perform genetic analysis on 18 different blood-spot samples collected from neonates detected as hyperphenylalaninemic by Northeastern Italian screening program. DNA was extracted from blood-spots. Exons/introns of PAH gene were amplified by polymerase chain reaction (PCR), and PCR products were purified and sequenced with both forward and reverse primers. The most frequent mutations were IVS12nt1g>a (16.7%) and R408W, P281L and L48S (all together 11.1%). As expected, compound heterozygosity was the usual finding; homozygosity was found only in two patients with R158Q and IVS2nt5g>c mutations. The V230I mutation was reported for the first time in Italy. We found six previously described polymorphisms (V245V, IVS4nt47c>t, IVS2nt19t>c, IVS3nt-22c>t, IVS5nt-54a>g, and E280>Q280). To our knowledge, four genotypes were not previously described: R158Q/V230I present in one patient with classical PKU; and L48S/R408Q, A403V/IVS2nt-13t>g, and G272X/V230I present in patients showing HPA phenotype. Most of the mutations were located in the exons 12 and 7 and in exon/intron 2 (83.3% detection of total mutations in PKU or HPA patients of Northeastern Italy). From a practical viewpoint, the genetic analysis of blood-spots collected on Guthrie cards for neonatal screening for PKU could be a simple method to establish the genotype of neonates. Consequently, the genotype/phenotype correlation could lead to a more accurate diagnosis and prognosis for families.     

Acquired AmpC type beta-lactamases: an emerging problem in Italian long-term care and rehabilitation facilities

We report the multiple detection of Proteus mirabilis isolates, from 4 different long-term care and rehabilitation facilities (LTCRFs) of Northern Italy, resistant to expanded-spectrum cephalosporins and cephamycins and producing an acquired ampC-like beta-lactamase, named CMY-16. Genotyping by PFGE showed that isolates were clonally related to each other, although not identical. In all isolates the bla(CMY16) gene was not transferable by conjugation and was found to be carried on the chromosome. These results revealed multifocal spreading of a CMY-16 producing P. mirabilis clone in Northern Italy and emphasize the emergence of similar acquired resistance determinants in the LTCRFs setting.     

Additional reports for Italian moss flora

As a result of the study of both recent collections and a review of herbarium specimens, the following taxa are reported for the first time from various Italian regions: Hedwigia stellata Hedenäs, Hedwigia ciliata var. leucophaea Bruch & Schimp., Pleurozium schreberi (Willd. ex Brid.) Mitt. and Ptychostomum compactum Hornsch. var. compactum. The last taxon is new for southern Italy, while the report of Pleurozium schreberi confirms the occurrence of this species in southern Italy after more than half a century. Moreover, Crossidium laxefilamentosum Frey & Kürschner represents the second report of this species from Italy.     

Electrophoretic subtyping of phosphoglucomutase locus 1 (PGM1) polymorphism in the Italian and Czechoslovakian populations

About 3,500 subjects from Italy and Czechoslovakia have been analyzed by acid starch gel electrophoresis for the subtyping of PGM1 polymorphism. The Italian sample included three different subgroups, from Northern, Central and Southern Italy. The allele frequencies found in the three groups do not differ significantly from each other; the observed values in the pooled sample are: PGM1S1 = 0.594, PGM1F1 = 0.118, PGM2S1 = 0.231, PGM2F1 = 0.057. In the Czechoslovakian group, which differs significantly from the Italian population, the following allele frequencies were found: PGM1S1 = 0.639, PGM1F1 = 0.118, PGM2S1 = 0.180, PGM2F1 = 0.063. The analysis of 217 families did not show any exception to Mendelian inheritance of the patterns.     

A new mitochondrial haplotype confirms the distinctiveness of the Italian wolf (Canis lupus) population

In the past century the Italian wolf has been repeatedly indicated as a distinct subspecies, Canis lupus italicus, due to its unique morphology and its distinctive mtDNA control region (CR) monomorphism. However, recent studies on wolf x dog hybridization in Italy documented the presence of a second mtDNA CR haplotype (W16), previously found only in wolves from Eastern Europe, casting doubts on the genetic uniqueness of the Italian wolves. To test whether this second haplotype belongs to the Italian wolf population, we genotyped 92 wolf DNA samples from Italy, Slovenia, Greece and Bulgaria at four mtDNA regions (control-region, ATP6, COIII and ND4 genes) and at 39 autosomal microsatellites. Results confirm the presence of two mtDNA multi-fragment haplotypes (WH14 and WH19) in the Italian wolves, distinct from all the other European wolves. Network analyses of the multi-fragment mtDNA haplotypes identified two strongly differentiated clades, with the Italian wolf WH14 and WH19 multi-fragment haplotypes rooted together. Finally, Bayesian clustering clearly assigned all the wolves sampled in Italy to the Italian population, regardless of the two different multi-fragment haplotypes. These results demonstrate that the W16 CR haplotype is part of the genetic pool of the Italian wolf population, reconfirming its distinctiveness from other European wolves. Overall, considering the presence of unique mtDNA and Y-linked haplotypes, the sharply different frequencies of genome-wide autosomal alleles and the distinct morphological features of Italian wolves, we believe that this population should be considered a distinct subspecies.     

An Overview of the Genetic Structure within the Italian Population from Genome-Wide Data

In spite of the common belief of Europe as reasonably homogeneous at genetic level, advances in high-throughput genotyping technology have resolved several gradients which define different geographical areas with good precision. When Northern and Southern European groups were considered separately, there were clear genetic distinctions. Intra-country genetic differences were also evident, especially in Finland and, to a lesser extent, within other European populations. Here, we present the first analysis using the 125,799 genome-wide Single Nucleotide Polymorphisms (SNPs) data of 1,014 Italians with wide geographical coverage. We showed by using Principal Component analysis and model-based individual ancestry analysis, that the current population of Sardinia can be clearly differentiated genetically from mainland Italy and Sicily, and that a certain degree of genetic differentiation is detectable within the current Italian peninsula population. Pair-wise F_ST statistics Northern and Southern Italy amounts approximately to 0.001 between, and around 0.002 between Northern Italy and Utah residents with Northern and Western European ancestry (CEU). The Italian population also revealed a fine genetic substructure underscoring by the genomic inflation (Sardinia vs. Northern Italy = 3.040 and Northern Italy vs. CEU = 1.427), warning against confounding effects of hidden relatedness and population substructure in association studies.     

One-step triplex high-resolution melting analysis for rapid identification and simultaneous subtyping of frequently isolated Salmonella serovars

Salmonellosis is one of the most important food-borne diseases worldwide. For outbreak investigation and infection control, accurate and fast subtyping methods are essential. A triplex gene-scanning assay was developed and evaluated for serotype-specific subtyping of Salmonella enterica isolates based on specific single-nucleotide polymorphisms in fragments of fljB, gyrB, and ycfQ. Simultaneous gene scanning of fljB, gyrB, and ycfQ by high-resolution melting-curve analysis of 417 Salmonella isolates comprising 46 different serotypes allowed the unequivocal, simple, and fast identification of 37 serotypes. Identical melting-curve profiles were obtained in some cases from Salmonella enterica serotype Enteritidis and Salmonella enterica serotype Dublin, in all cases from Salmonella enterica serotype Ohio and Salmonella enterica serotype Rissen, from Salmonella enterica serotype Mbandaka and Salmonella enterica serotype Kentucky, and from Salmonella enterica serotype Bredeney, Salmonella enterica serotype Give, and Salmonella enterica serotype Schwarzengrund. To differentiate the most frequent Salmonella serotype, Enteritidis, from some S. Dublin isolates, an additional single PCR assay was developed for specific identification of S. Enteritidis. The closed-tube triplex high-resolution melting-curve assay developed, in combination with an S. Enteritidis-specific PCR, represents an improved protocol for accurate, cost-effective, simple, and fast subtyping of 39 Salmonella serotypes. These 39 serotypes represent more than 94% of all human and more than 85% of all nonhuman Salmonella isolates (including isolates from veterinary, food, and environmental samples) obtained in the years 2008 and 2009 in Austria.     

Structure of the mitochondrial control region of the Eurasian otter (Lutra lutra; Carnivora, Mustelidae): patterns of genetic heterogeneity and implications for conservation of the species in Italy

In this study we determined the complete sequence of the mitochondrial DNA (mtDNA) control region of the Eurasian otter (Lutra lutra). We then compared these new sequences with orthologues of nine carnivores belonging to six families (Mustelidae, Mephitidae, Canidae, Hyaenidae, Ursidae, and Felidae). The comparative analyses identified all the conserved regions previously found in mammals. The Eurasian otter and seven other species have a single location with tandem repeats in the right domain, while the spotted hyena (Hyaenidae) and the tiger (Felidae) have repeated sequences in both the right and left domains. To assess the degree of genetic heterogeneity of the Eurasian otter in Italy we sequenced two fragments of the gene and analyzed length polymorphisms of repeated sequences and heteroplasmy in 32 specimens. The study includes 23 museum specimens collected in northern, central, and southern Italy; most of these specimens are from extinct populations, while the southern Italian samples belong to the sole extant Italian population of the Eurasian otter. The study also includes all the captive-reared animals living in the colony "Centro Lontra, Caramanico Terme" (Pescara, central Italy). The colony is maintained for reintroduction of the species. We found a low level of genetic polymorphism; a single haplotype is dominant, but our data indicate the presence in central and southern Italy of two slightly divergent haplotypes. One haplotype belongs to an extinct population, the other is present in the single extant Italian population. Analyses of length polymorphisms and heteroplasmy indicate that the autochthonous Italian samples are characterized by a distinct array of repeated sequences from captive-reared animals.     

The Italian Twin Project: from the personal identification number to a national twin registry.

The unique opportunity given by the "fiscal code", an alphanumeric identification with demographic information on any single person residing in Italy, introduced in 1976 by the Ministry of Finance, allowed a database of all potential Italian twins to be created. This database contains up to now name, surname, date and place of birth and home address of about 1,300,000 "possible twins". Even though we estimated an excess of 40% of pseudo-twins, this still is the world's largest twin population ever collected. The database of possible twins is currently used in population-based studies on multiple sclerosis, Alzheimer's disease, celiac disease, and type 1 diabetes. A system is currently being developed for linking the database with data from mortality and cancer registries. In 2001, the Italian Government, through the Ministry of Health, financed a broad national research program on twin studies, including the establishment of a national twin registry. Among all the possible twins, a sample of 500,000 individuals are going to be contacted and we expect to enrol around 120,000 real twin pairs in a formal Twin Registry. According to available financial resources, a sub sample of the enrolled population will be asked to donate DNA. A biological bank from twins will be then implemented, guaranteeing information on future etiological questions regarding genetic and modifiable factors for physical impairment and disability, cancers, cardiovascular diseases and other age related chronic illnesses.     

Giardia microti in pet Microtus guentheri: Evidence of a parasite never detected in Italy

The genus Giardia includes several species distinguished by morphological, biological and molecular features. Currently, eight species within the genus are retained as valid. In Italy no identification of Giardia species other than Giardia duodenalis has been so far reported. Fecal samples were collected from two Günther's Voles (Microtus guentheri) positive to Giardia cysts by microscopic investigation and immunofluorescence. The voles were born in Milan (Northern Italy) from two gravid females imported from the Netherlands and kept for sale in a pet shop in Varese (Northern Italy). Positive feces were subjected to a nested PCR to amplify a 18S rRNA fragment for molecular characterization. A phylogenetic analysis was conducted to compare the obtained sequence with those of all other Giardia species available in GenBank for the 18S locus, using the Maximum Likelihood (ML) method by R software. Sequence analyses unambiguously identified the isolates as belonging to G. microti, showing 99% of identity with those of its isolates available in GenBank. A well-defined cluster, supported by significant bootstrap values and corresponding to the G. microti cluster, including sequences obtained from M. guentheri, was evidenced in the ML tree, confirming species assignment. The present finding represents the first report of G. microti from pet animals in Italy.