Caterina Mammina

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.

Food safety in hospital: knowledge, attitudes and practices of nursing staff of two hospitals in Sicily, Italy

BackgroundFood hygiene in hospital poses peculiar problems, particularly given the presence of patients who could be more vulnerable than healthy subjects to microbiological and nutritional risks. Moreover, in nosocomial outbreaks of infectious intestinal disease, the mortality risk has been proved to be significantly higher than the community outbreaks and highest for foodborne outbreaks. On the other hand, the common involvement in the role of food handlers of nurses or domestic staff, not specifically trained about food hygiene and HACCP, may represent a further cause of concern.The purpose of this study was to evaluate knowledge, attitudes, and practices concerning food safety of the nursing staff of two hospitals in Palermo, Italy. Association with some demographic and work-related determinants was also investigated.MethodsThe survey was conducted, by using a semi-structured questionnaire, in March-November 2005 in an acute general hospital and a paediatric hospital, where nursing staff is routinely involved in food service functions.ResultsOverall, 401 nurses (279, 37.1%, of the General Hospital and 122, 53.5%, of the Paediatric Hospital, respectively) answered. Among the respondents there was a generalized lack of knowledge about etiologic agents and food vehicles associated to foodborne diseases and proper temperatures of storage of hot and cold ready to eat foods. A general positive attitude towards temperature control and using clothing and gloves, when handling food, was shared by the respondents nurses, but questions about cross-contamination, refreezing and handling unwrapped food with cuts or abrasions on hands were frequently answered incorrectly. The practice section performed better, though sharing of utensils for raw and uncooked foods and thawing of frozen foods at room temperatures proved to be widely frequent among the respondents. Age, gender, educational level and length of service were inconsistently associated with the answer pattern.More than 80% of the respondent nurses did not attend any educational course on food hygiene. Those who attended at least one training course fared significantly better about some knowledge issues, but no difference was detected in both the attitude and practice sections.ConclusionResults strongly emphasize the need for a safer management of catering in the hospitals, where non professional food handlers, like nursing or domestic staff, are involved in food service functions.

Rapid differentiation between livestock-associated and livestock-independent Staphylococcus aureus CC398 clades.

Staphylococcus aureus clonal complex 398 (CC398) isolates cluster into two distinct phylogenetic clades based on single-nucleotide polymorphisms (SNPs) revealing a basal human clade and a more derived livestock clade. The scn and tet(M) genes are strongly associated with the human and the livestock clade, respectively, due to loss and acquisition of mobile genetic elements. We present canonical single-nucleotide polymorphism (canSNP) assays that differentiate the two major host-associated S. aureus CC398 clades and a duplex PCR assay for detection of scn and tet(M). The canSNP assays correctly placed 88 S. aureus CC398 isolates from a reference collection into the human and livestock clades and the duplex PCR assay correctly identified scn and tet(M). The assays were successfully applied to a geographically diverse collection of 272 human S. aureus CC398 isolates. The simple assays described here generate signals comparable to a whole-genome phylogeny for major clade assignment and are easily integrated into S. aureus CC398 surveillance programs and epidemiological studies.

Genetic relatedness among isolates of Shigella sonnei carrying class 2 integrons in Tehran, Iran, 2002–2003

BackgroundShigella spp. are major cause of diarrhoeal disease in both developing and developed countries. Shigella sonnei is the serogroup of Shigella most frequently responsible for sporadic and epidemic enteritis in developed countries. In recent years the emergence and spread of S. sonnei biotype g carrying class 2 integron have been frequently reported in many countries. Recently, S. sonnei has been reported as the prevalent serogroup of Shigella in Iran.The present study was carried out to investigate phenotypic and genetic characteristics of Shigella sonnei isolates identified in the years 2002 and 2003 in Tehran, Iran.MethodsBiotyping, drug susceptibility testing, pulsed field gel electrophoresis (PFGE) and analysis of class 2 integrons have been carried out on 60 S. sonnei isolates, including 57 sporadic isolates from paediatric cases of shigellosis occurring in 2002 and 2003, two sporadic isolates recovered in 1984 and the ATCC 9290 strain.ResultsBiotype g and resistance to streptomycin, sulfamethoxazole-trimethoprim and tetracycline were exhibited by 54 of the 57 recent isolates. Of the 54 biotype g isolates, 28 exhibited a class 2 integron of 2161 bp, and 24 a class 2 integron of 1371 bp, respectively. Class 2 integrons were not detected in four isolates only, including the two endemic isolates recovered in 1984 and two strains from recent sporadic cases. PFGE divided the strains into eight pulsotypes labeled A to H, three major pulsotypes – A to C – including the large majority of the recent sporadic S. sonnei isolates. Pulsotypes A and C were the most prevalent groups, accounting for 41.6% and 35.0%, respectively, of the isolates under study.ConclusionThe results suggest that biotype g, class 2 integron carrying S. sonnei are prevalent in our geographic area. S. sonnei isolated in the years 2002 and 2003 could be attributed to a few predominant clusters including, respectively, strains with pulsotypes B and C carrying a 2161 bp class 2 integron, and those having pulsotype A and a 1371 bp class 2 integron. A few epidemic clones are responsible for the apparently endemic occurrence of shigellosis in Tehran, Iran.

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.