Antonino Nastasi

Snapshot of Moving and Expanding Clones of Mycobacterium tuberculosis and Their Global Distribution Assessed by Spoligotyping in an International Study

ABSTRACT The present update on the global distribution of Mycobacterium tuberculosis complex spoligotypes provides both the octal and binary descriptions of the spoligotypes for M. tuberculosis complex, including Mycobacterium bovis, from >90 countries (13,008 patterns grouped into 813 shared types containing 11,708 isolates and 1,300 orphan patterns). A number of potential indices were developed to summarize the information on the biogeographical specificity of a given shared type, as well as its geographical spreading (matching code and spreading index, respectively). To facilitate the analysis of hundreds of spoligotypes each made up of a binary succession of 43 bits of information, a number of major and minor visual rules were also defined. A total of six major rules (A to F) with the precise description of the extra missing spacers (minor rules) were used to define 36 major clades (or families) of M. tuberculosis. Some major clades identified were the East African-Indian (EAI) clade, the Beijing clade, the Haarlem clade, the Latin American and Mediterranean (LAM) clade, the Central Asian (CAS) clade, a European clade of IS6110 low banders (X; highly prevalent in the United States and United Kingdom), and a widespread yet poorly defined clade (T). When the visual rules defined above were used for an automated labeling of the 813 shared types to define nine superfamilies of strains (Mycobacterium africanum, Beijing, M. bovis, EAI, CAS, T, Haarlem, X, and LAM), 96.9% of the shared types received a label, showing the potential for automated labeling of M. tuberculosis families in well-defined phylogeographical families. Intercontinental matches of shared types among eight continents and subcontinents (Africa, North America, Central America, South America, Europe, the Middle East and Central Asia, and the Far East) are analyzed and discussed.

Global Distribution of Mycobacterium tuberculosis Spoligotypes

We present a short summary of recent observations on the global distribution of the major clades of the Mycobacterium tuberculosis complex, the causative agent of tuberculosis. This global distribution was defined by data-mining of an international spoligotyping database, SpolDB3. This database contains 11,708 patterns from as many clinical isolates originating from more than 90 countries. The 11,708 spoligotypes were clustered into 813 shared types. A total of 1,300 orphan patterns (clinical isolates showing a unique spoligotype) were also detected.

Salmonella bongori Provides Insights into the Evolution of the Salmonellae

The genus Salmonella contains two species, S. bongori and S. enterica. Compared to the well-studied S. enterica there is a marked lack of information regarding the genetic makeup and diversity of S. bongori. S. bongori has been found predominantly associated with cold-blooded animals, but it can infect humans. To define the phylogeny of this species, and compare it to S. enterica, we have sequenced 28 isolates representing most of the known diversity of S. bongori. This cross-species analysis allowed us to confidently differentiate ancestral functions from those acquired following speciation, which include both metabolic and virulence-associated capacities. We show that, although S. bongori inherited a basic set of Salmonella common virulence functions, it has subsequently elaborated on this in a different direction to S. enterica. It is an established feature of S. enterica evolution that the acquisition of the type III secretion systems (T3SS-1 and T3SS-2) has been followed by the sequential acquisition of genes encoding secreted targets, termed effectors proteins. We show that this is also true of S. bongori, which has acquired an array of novel effector proteins (sboA-L). All but two of these effectors have no significant S. enterica homologues and instead are highly similar to those found in enteropathogenic Escherichia coli (EPEC). Remarkably, SboH is found to be a chimeric effector protein, encoded by a fusion of the T3SS-1 effector gene sopA and a gene highly similar to the EPEC effector nleH from enteropathogenic E. coli. We demonstrate that representatives of these new effectors are translocated and that SboH, similarly to NleH, blocks intrinsic apoptotic pathways while being targeted to the mitochondria by the SopA part of the fusion. This work suggests that S. bongori has inherited the ancestral Salmonella virulence gene set, but has adapted by incorporating virulence determinants that resemble those employed by EPEC.

Salmonella enterica serotype Typhimurium DT 104 antibiotic resistance genomic island I in serotype paratyphi B.

We have identified Salmonella genomic island I (SGI1) in an isolate of Salmonella enterica serotype Paratyphi B. This antibiotic-resistance gene cluster, which confers multidrug resistance, has been previously identified in S. enterica serotype Typhimurium phage types DT 104 and DT 120 and in S. enterica serotype Agona.

Multidrug and Broad-Spectrum Cephalosporin Resistance among Salmonella enterica Serotype Enteritidis Clinical Isolates in Southern Italy

ABSTRACT From 1992 to 1997, only six sporadic isolates of Salmonella enterica serotype Enteritidis from patients with cases of gastroenteritis in southern Italy exhibited resistance to broad-spectrum cephalosporins. Five isolates produced SHV-12, and one isolate encoded a class C β-lactamase. The blaSHV-12 gene was located in at least two different self-transferable plasmids, one of which also carried a novel class 1 integron.

Characterization of Listeria monocytogenes Isolates from Human Listeriosis Cases in Italy

ABSTRACT The objective of this study was to characterize by serotyping, pulsed-field gel electrophoresis (PFGE), and PCR amplification of virulence genes and markers of epidemic clones I, II, and III (ECI, ECII, and ECIII) 54 human isolates from apparently sporadic cases of infection occurring in the Lombardy region and in the province of Florence, Tuscany, Italy, in the years 1996 to 2007. Listeria monocytogenes isolates were provided by the clinical microbiology laboratories of the Lombardy region and the “Careggi” Hospital of Florence, Tuscany, Italy. Serotyping, PFGE after digestion with the AscI and ApaI enzymes, and PCR amplification for the inlA, inlC, and inlJ genes and ECI, ECII, and ECIII markers were performed according to procedures described previously. Twenty-five (46.3%) L. monocytogenes isolates were assigned to serotype 1/2a, 23 (42.6%) to serotype 4b, and 6 (11.1%) to serotype 1/2b. Thirty-one AscI pulsotypes were recognized among the 54 human isolates. Eleven molecular subtype clusters, of which eight included indistinguishable pulsotypes and three included closely related pulsotypes, were shared by two to seven isolates. Fifteen isolates exhibited unique AscI pulsotypes. Three groups of clustered isolates and two apparently sporadic isolates generated EC amplicons. All strains tested positive for the inlA, inlC, and inlJ genes. Based on the results of serotyping and molecular typing, there were 11 occasions when L. monocytogenes strains with the same subtype were isolated from more than one listeriosis case. A total of 39 out of 54 isolates (72.2%) were attributed to molecular subtype clusters. The results of the study suggest that routine subtyping of L. monocytogenes strains from human listeriosis cases could allow more-timely detection of outbreaks possibly caused by food-borne isolates from a common source and could lead to control of ongoing food exposure, thus preventing the occurrence of more cases.

Genetic Diversity of Mycobacterium tuberculosis in Sicily Based on Spoligotyping and Variable Number of Tandem DNA Repeats and Comparison with a Spoligotyping Database for Population-Based Analysis

ABSTRACT In a previous study, we proposed to associate spoligotyping and typing with the variable number of tandem DNA repeats (VNTR) as an alternative strategy to IS6110-restriction fragment length polymorphism (RFLP) for molecular epidemiological studies on tuberculosis. The aim of the present study was to further evaluate this PCR-based typing strategy and to describe the population structure ofMycobacterium tuberculosis in another insular setting, Sicily. A collection of 106 DNA samples from M. tuberculosis patient isolates was characterized by spoligotyping and VNTR typing. All isolates were independently genotyped by the standard IS6110-RFLP method, and clustering results between the three methods were compared. The totals for the clustered isolates were, respectively, 15, 60, and 82% by IS6110-RFLP, spoligotyping, and VNTR typing. The most frequent spoligotype included type 42 that missed spacers 21 to 24 and spacers 33 to 36 and derived types 33, 213, and 273 that, together represented as much as 26% of all isolates, whereas the Haarlem clade of strains (types 47 and 50, VNTR allele 32333) accounted for 9% of the total strains. The combination of spoligotyping and VNTR typing results reduced the number of clusters to 43% but remained superior to the level of IS6110-RFLP clustering (ca. 15%). All but one IS6110-defined cluster were identified by the combination of spoligotyping and VNTR clustering results, whereas 9 of 15 spoligotyping-defined clusters could be further subdivided by IS6110-RFLP. Reinterpretation of previous IS6110-RFLP results in the light of spoligotyping-VNTR typing results allowed us to detect an additional cluster that was previously missed. Although less discriminative than IS6110-RFLP, our results suggest that the use of the combination of spoligotyping and VNTR typing is a good screening strategy for detecting epidemiological links for the study of tuberculosis epidemiology at the molecular level.

Persistent Endemicity of Salmonella bongori 48:z35:− in Southern Italy: Molecular Characterization of Human, Animal, and Environmental Isolates

ABSTRACT From 1984 to 1999, we collected 31 isolates of the rare serovar Salmonella bongori 48:z35:− in southern Italy. Twenty-four of the isolates were from cases of acute enteritis in humans. Pulsed-field gel electrophoresis analysis showed that all but one of our isolates were at least 80% similar. Our findings suggest that genetically related S. bongori 48:z35:− strains are endemically circulating in southern Italy.

Enhanced surveillance of invasive listeriosis in the Lombardy region, Italy, in the years 2006-2010 reveals major clones and an increase in serotype 1/2a

BackgroundInvasive listeriosis is a rare, life-threatening foodborne disease. Lombardy, an Italian region accounting for 16% of the total population, reported 55% of all listeriosis cases in the years 2006-2010. The aim of our study was to provide a snapshot of listeriosis epidemiology in this region after the implementation of a voluntary laboratory-based surveillance system.MethodsWe characterized by serotyping, pulsed-field gel electrophoresis, multilocus sequence typing and detection of epidemic clone markers, 134 isolates from 132 listeriosis cases, including 15 pregnancy-related cases, occurring in the years 2006-2010 in Lombardy. Demographic and clinical characteristics of cases have also been described.ResultsThe mean age of non pregnancy-associated cases was 64.7 years, with 55.9% of cases being older than 65 years. Cases having no underlying medical conditions accounted for 11.6%. The all-cause fatality rate of 83 cases with a known survival outcome was 25.3%.Serotypes 1/2a and 4b comprised 52.2% and 38.8% of isolates, respectively. Seventy-three AscI pulsotypes and 25 sequence types assigned to 23 clonal complexes were recognized. Moreover, 53 (39.5%) isolates tested positive for the epidemic clone markers. Twelve molecular subtype clusters including at least three isolates were detected, with cluster 11 (1/2a/ST38) including 31 isolates identified during the entire study period. No outbreaks were notified to public health authorities during this period.ConclusionsThe findings of our study proved that epidemiology of listeriosis in Lombardy is characterized by a high prevalence of major clones and the increasing role of serotype 1/2a. Molecular subtyping is an essential tool in the epidemiology and surveillance of listeriosis. Rapid molecular cluster detection could alert about putative outbreaks, thus increasing the chance of detecting and inactivating routes of transmission.

Identification of Shigella sonnei Biotype g Isolates Carrying Class 2 Integrons in Italy (2001 to 2003)

ABSTRACT Phenotyping and genotyping have been carried out on 64 epidemic and sporadic isolates of Shigella sonnei identified in Italy in the years 2001 to 2003. Class 2 integron carriage has been also investigated. Isolates from four of the five outbreaks and four of six sporadic cases were biotype g, pulsed-field gel electrophoresis type B, and class 2 integron positive, suggesting emergence and spread of an epidemic clone in Italy.