The Gene tia, Harbored by the Subtilase-Encoding Pathogenicity Island, Is Involved in the Ability of Locus of Enterocyte Effacement-Negative Shiga Toxin-Producing Escherichia coli Strains To Invade Monolayers of Epithelial Cells.

Locus of enterocyte effacement (LEE)-negative Shiga toxin (Stx)-producing Escherichia coli (STEC) strains are human pathogens that lack the LEE locus, a pathogenicity island (PAI) involved in the intimate adhesion of LEE-positive strains to the host gut epithelium. The mechanism used by LEE-negative STEC strains to colonize the host intestinal mucosa is still not clear. The cell invasion determinant tia, previously described in enterotoxigenic E. coli strains, has been identified in LEE-negative STEC strains that possess the subtilase-encoding pathogenicity island (SE-PAI). We evaluated the role of the gene tia, present in these LEE-negative STEC strains, in the invasion of monolayers of cultured cells. We observed that these strains were able to invade Caco-2 and HEp-2 cell monolayers and compared their invasion ability with that of a mutant strain in which the gene tia had been inactivated. Mutation of the gene tia resulted in a strong reduction of the invasive phenotype, and complementation of the tia mutation with a functional copy of the gene restored the invasion activity. Moreover, we show that the gene tia is overexpressed in bacteria actively invading cell monolayers, demonstrating that tia is involved in the ability to invade cultured monolayers of epithelial cells shown by SE-PAI-positive E. coli, including STEC, strains. However, the expression of the tia gene in the E. coli K-12 strain JM109 was not sufficient, in its own right, to confer to this strain the ability to invade cell monolayers, suggesting that at least another factor must be involved in the invasion ability displayed by the SE-PAI-positive strains.

Whole-Genome Characterization and Strain Comparison of VT2f-Producing Escherichia coli Causing Hemolytic Uremic Syndrome.

Verotoxigenic Escherichia coli infections in humans cause disease ranging from uncomplicated intestinal illnesses to bloody diarrhea and systemic sequelae, such as hemolytic uremic syndrome (HUS). Previous research indicated that pigeons may be a reservoir for a population of verotoxigenic E. coli producing the VT2f variant. We used whole-genome sequencing to characterize a set of VT2f-producing E. coli strains from human patients with diarrhea or HUS and from healthy pigeons. We describe a phage conveying the vtx2f genes and provide evidence that the strains causing milder diarrheal disease may be transmitted to humans from pigeons. The strains causing HUS could derive from VT2f phage acquisition by E. coli strains with a virulence genes asset resembling that of typical HUS-associated verotoxigenic E. coli.

Community-wide outbreak of haemolytic uraemic syndrome associated with Shiga toxin 2-producing Escherichia coli O26:H11 in southern Italy, summer 2013.

In summer 2013, an excess of paediatric cases of haemolytic uraemic syndrome (HUS) in a southern region of Italy prompted the investigation of a community-wide outbreak of Shiga toxin 2-producing Escherichia coli (STEC) O26:H11 infections. Case finding was based on testing patients with HUS or bloody diarrhoea for STEC infection by microbiological and serological methods. A case-control study was conducted to identify the source of the outbreak. STEC O26 infection was identified in 20 children (median age 17 months) with HUS, two of whom reported severe neurological sequelae. No cases in adults were detected. Molecular typing showed that two distinct STEC O26:H11 strains were involved. The case-control study showed an association between STEC O26 infection and consumption of dairy products from two local plants, but not with specific ready-to-eat products. E.coli O26:H11 strains lacking the stx genes were isolated from bulk milk and curd samples, but their PFGE profiles did not match those of the outbreak isolates. This outbreak supports the view that infections with Stx2-producing E. coli O26 in children have a high probability of progressing to HUS and represent an emerging public health problem in Europe.

Identification and characterization of a peculiar vtx2-converting phage frequently present in verocytotoxin-producing Escherichia coli O157 isolated from human infections.

Certain verocytotoxin-producing Escherichia coli (VTEC) O157 phage types (PTs), such as PT8 and PT2, are associated with severe human infections, while others, such as PT21, seem to be restricted to cattle. In an attempt to delve into the mechanisms underlying such a differential distribution of PTs, we performed microarray comparison of human PT8 and animal PT21 VTEC O157 isolates. The main differences observed were in the vtx2-converting phages, with the PT21 strains bearing a phage identical to that present in the reference strain EDL933, BP933W, and all the PT8 isolates displaying lack of hybridization in some regions of the phage genome. We focused on the region spanning the gam and cII genes and developed a PCR tool to investigate the presence of PT8-like phages in a panel of VTEC O157 strains belonging to different PTs and determined that a vtx2 phage reacting with the primers deployed, which we named Φ8, was more frequent in VTEC O157 strains from human disease than in bovine strains. No differences were observed in the production of the VT2 mRNA when Φ8-positive strains were compared with VTEC O157 possessing BP933W. Nevertheless, we show that the gam-cII region of phage Φ8 might carry genetic determinants downregulating the transcription of the genes encoding the components of the type III secretion system borne on the locus of enterocyte effacement pathogenicity island.

Whole genome sequence comparison of vtx2-converting phages from Enteroaggregative Haemorrhagic Escherichia coli strains.

BACKGROUND: Enteroaggregative Haemorrhagic E. coli (EAHEC) is a new pathogenic group of E. coli characterized by the presence of a vtx2-phage integrated in the genomic backbone of Enteroaggregative E. coli (EAggEC). So far, four distinct EAHEC serotypes have been described that caused, beside the large outbreak of infection occurred in Germany in 2011, a small outbreak and six sporadic cases of HUS in the time span 1992-2012. In the present work we determined the whole genome sequence of the vtx2-phage, termed Phi-191, present in the first described EAHEC O111:H2 isolated in France in 1992 and compared it with those of the vtx-phages whose sequences were available. RESULTS: The whole genome sequence of the Phi-191 phage was identical to that of the vtx2-phage P13374 present in the EAHEC O104:H4 strain isolated during the German outbreak 20 years later. Moreover, it was also almost identical to those of the other vtx2-phages of EAHEC O104:H4 strains described so far. Conversely, the Phi-191 phage appeared to be different from the vtx2-phage carried by the EAHEC O111:H21 isolated in the Northern Ireland in 2012.The comparison of the vtx2-phages sequences from EAHEC strains with those from the vtx-phages of typical Verocytotoxin-producing E. coli strains showed the presence of a 900 bp sequence uniquely associated with EAHEC phages and encoding a tail fiber. CONCLUSIONS: At least two different vtx2-phages, both characterized by the presence of a peculiar tail fiber-coding gene, intervened in the emergence of EAHEC. The finding of an identical vtx2-phage in two EAggEC strains isolated after 20 years in spite of the high variability described for vtx-phages is unexpected and suggests that such vtx2-phages are kept under a strong selective pressure.The observation that different EAHEC infections have been traced back to countries where EAggEC infections are endemic and the treatment of human sewage is often ineffective suggests that such countries may represent the cradle for the emergence of the EAHEC pathotype. In these regions, EAggEC of human origin can extensively contaminate the environment where they can meet free vtx-phages likely spread by ruminants excreta.

Identification of two allelic variants of toxB gene and investigation of their distribution among Verocytotoxin-producing Escherichia coli.

Verocytotoxin-producing Escherichia coli (VTEC) are food borne pathogens causing severe human infections. The virulence genes asset of VTEC is complex and has not been completely defined yet. Nonetheless, all the virulence genes described so far have been described as conveyed by mobile genetic elements. A gene, termed toxB, has been identified in a large virulence plasmid of VTEC O157, later described in similar plasmids carried by VTEC O26 and O145. In this study we identified for the first time an intact copy of toxB gene in a plasmid present in a VTEC O111 strain and observed the existence of two allelic variants of the gene, that we termed toxB1 and toxB2. We investigated the distribution of the two alleles in a panel of VTEC strains belonging to different serogroups and demonstrated that this gene is present only in VTEC serogroups associated with the most severe forms of the infections such as those belonging to the five serogroups O157, O26, O111, O103 and O145 and that the two alleles segregate with the serogroup of the hosting strains. In particular the toxB1 variant was only present in VTEC O157 while the toxB2 allele was present in the remaining four VTEC serogroups.

Enterohemorrhagic Escherichia coli O26:H11/H-: a new virulent clone emerges in Europe.

BACKGROUND: Enterohemorrhagic Escherichia coli (EHEC) O26 causes diarrhea and hemolytic uremic syndrome (HUS). Strains harboring the stx1a gene prevail, but strains with stx2a as the sole Shiga toxin-encoding gene are now emerging. The traits and virulence of the latter set of strains are unknown. We correlated stx genotypes of 272 EHEC O26 strains isolated in 7 European countries between 1996 and 2012 with disease phenotypes. We determined phylogeny, clonal structure, and plasmid gene profiles of the isolates and portray geographic and temporal distribution of the different subgroups. METHODS: The stx genotypes and plasmid genes were identified using polymerase chain reaction, phylogeny was assigned using multilocus sequence typing, and clonal relatedness was established using pulsed-field gel electrophoresis. RESULTS: Of the 272 EHEC O26 isolates, 107 (39.3%), 139 (51.1%), and 26 (9.6%) possessed stx1a, stx2a, or both genes, respectively. Strains harboring stx2a only were significantly associated with HUS (odds ratio, 14.2; 95% confidence interval, 7.9-25.6; P < .001) compared to other stx genotypes. The stx2a-harboring strains consist of 2 phylogenetically distinct groups defined by sequence type (ST) 21 and ST29. The ST29 strains are highly conserved and correspond by plasmid genes to the new virulent clone of EHEC O26 that emerged in Germany in the 1990s. This new clone occurred in 6 of the 7 countries and represented approximately 50% of all stx2a-harboring EHEC O26 strains isolated between 1996 and 2012. CONCLUSIONS: A new highly virulent clone of EHEC O26 has emerged in Europe. Its reservoirs and sources warrant identification.

Pathogenic potential to humans of bovine Escherichia coli O26, Scotland.

Escherichia coli O26 and O157 have similar overall prevalences in cattle in Scotland, but in humans, Shiga toxin-producing E. coli O26 infections are fewer and clinically less severe than E. coli O157 infections. To investigate this discrepancy, we genotyped E. coli O26 isolates from cattle and humans in Scotland and continental Europe. The genetic background of some strains from Scotland was closely related to that of strains causing severe infections in Europe. Nonmetric multidimensional scaling found an association between hemolytic uremic syndrome (HUS) and multilocus sequence type 21 strains and confirmed the role of stx(2) in severe human disease. Although the prevalences of E. coli O26 and O157 on cattle farms in Scotland are equivalent, prevalence of more virulent strains is low, reducing human infection risk. However, new data on E. coli O26-associated HUS in humans highlight the need for surveillance of non-O157 enterohemorrhagic E. coli and for understanding stx(2) phage acquisition.

Multicenter evaluation of a sequence-based protocol for subtyping Shiga toxins and standardizing Stx nomenclature.

When Shiga toxin-producing Escherichia coli (STEC) strains emerged as agents of human disease, two types of toxin were identified: Shiga toxin type 1 (Stx1) (almost identical to Shiga toxin produced by Shigella dysenteriae type 1) and the immunologically distinct type 2 (Stx2). Subsequently, numerous STEC strains have been characterized that express toxins with variations in amino acid sequence, some of which confer unique biological properties. These variants were grouped within the Stx1 or Stx2 type and often assigned names to indicate that they were not identical in sequence or phenotype to the main Stx1 or Stx2 type. A lack of specificity or consistency in toxin nomenclature has led to much confusion in the characterization of STEC strains. Because serious outcomes of infection have been attributed to certain Stx subtypes and less so with others, we sought to better define the toxin subtypes within the main Stx1 and Stx2 types. We compared the levels of relatedness of 285 valid sequence variants of Stx1 and Stx2 and identified common sequences characteristic of each of three Stx/Stx1 and seven Stx2 subtypes. A novel, simple PCR subtyping method was developed, independently tested on a battery of 48 prototypic STEC strains, and improved at six clinical and research centers to test the reproducibility, sensitivity, and specificity of the PCR. Using a consistent schema for nomenclature of the Stx toxins and stx genes by phylogenetic sequence-based relatedness of the holotoxin proteins, we developed a typing approach that should obviate the need to bioassay each newly described toxin and that predicts important biological characteristics.

Use of antimicrobials in companion animal practice: a retrospective study in a veterinary teaching hospital in Italy.

OBJECTIVES: To describe the use of antimicrobials in a veterinary teaching hospital for companion animals in Italy, with particular regard to the agreement with recommendations of prudent use METHODS: The study was conducted with a retrospective, cross-sectional design. The population under investigation included 18,905 cats and dogs that were referred to the hospital between 2000 and 2007. Two different samples of the clinical paper forms were randomly selected to estimate the prevalence of animals receiving an antimicrobial prescription and to describe the pattern of antimicrobials used in relation to the condition being treated. The proportion of antimicrobials prescribed accomplishing recommendations of prudent use was also estimated, as well as the level of agreement with specific, diagnosis-based guidelines for antimicrobial use. RESULTS: Broad-spectrum antimicrobials, including penicillins with ß-lactamase inhibitors, first-generation cephalosporins and fluoroquinolones, were the most frequently prescribed compounds. Antimicrobials prescribed with the support of microbiological analyses and susceptibility testing were less than 5%. Among the recommendation of prudent use, the availability of information from laboratory testing had the poorest degree of agreement, while the other evaluated items were accomplished in most of the cases. CONCLUSIONS: Our results highlight the need to improve the procedures of antimicrobial prescription in the study setting. This can be achieved by supporting the guidance for antimicrobial use at the local level, with the adoption of specific guidelines, and at the national level with a further implementation of the policies of prudent prescriptions.

The complete DNA sequence and analysis of the virulence plasmid and of five additional plasmids carried by Shiga toxin-producing Escherichia coli O26:H11 strain H30.

Shiga toxin-producing Escherichia coli (STEC) strains belonging to serogroup O26 have been associated with sporadic cases and outbreaks of hemorrhagic colitis and hemolytic uremic syndrome. In addition to chromosomal virulence genes, STEC strains usually harbor a large plasmid that carries genes associated with pathogenicity. The complete nucleotide sequence and genetic organization of 6 plasmids carried by STEC O26:H11 strain H30 were determined. The large virulence plasmid (pO26-Vir) was approximately 168 kb in size and contained 196 open reading frames (ORFs). pO26-Vir possesses a mosaic structure and shows similarity to the virulence plasmids in locus of enterocyte effacement (LEE)-negative STEC O113:H21 EH41 (pO113), in E. coli clinical strain C1096 (pSERB1), and in E. coli O157:H7 RIMD 0509952 (pO157). Plasmid pO26-Vir shares several highly conserved regions with pO157 and carries important virulence genes, including toxB, katP, espP, and the hly gene cluster. In addition, pO26-Vir possesses genes encoding for type IV pili (pilL-V). The second largest plasmid, pO26-L (73 kb) contains 101 ORFs. pO26-L carries the tetracycline resistance gene and has regions that show similarity to the E. coli conjugative resistance plasmid NR1. The third largest plasmid, pO26-S4 (5.8 kb), is homologous to the ColE2 colicinogenic plasmid that encodes for colicin E2. The remaining 3 plasmids, pO26-S1 (1.5 kb), pO26-S2 (3.1 kb), and pO26-S3 (4.2 kb), carry very little genetic information except for putative proteins involved in plasmid replication and DNA maintenance. The data presented underscore the diversity among the STEC virulence plasmids and provide insights into the evolution of these plasmids in STEC strains that cause serious human illness.

Virulotyping of Salmonella enterica serovar Napoli strains isolated in Italy from human and nonhuman sources.

Salmonella enterica serovar Napoli is an emerging serovar in Italy, France, and Switzerland, but little is known about its pathogenicity to humans. A collection of 112 strains of Salmonella Napoli isolated in Italy from human cases, foods of animal origin, and the environment have been characterized by the detection of a set of virulence genes, pulsed-field gel electrophoresis (PFGE), and antibiotic susceptibility. All the strains examined were susceptible to all the antimicrobials tested. The Salmonella pathogenicity islands genes ssaQ, mgtC spi_4D, and sopB were present from 75.0% to 100% of the tested strains. Only one human and four environmental strains showed the avrA gene. The phage-related sopE1 gene was present in 93% of the strains, whereas sodC1 and gipA genes were only in four and two environmental strains, respectively. The bcfC fimbrial gene was present in all the animal/food strains, in the 71.4% of environmental strains, and in 46.8% of the human strains, respectively. Overall, we observed 10 distinct virulence profiles (VP), but VP1-2-3 included 99 out of 112 strains. PFGE showed that 103 out of 111 isolates were grouped in four major clusters and three minor clusters, whereas two strains were totally unrelated. The most represented PFGE clusters mainly correlated with the virulotypes are VP1, VP2, and VP3. Salmonella Napoli shows an array of virulence genes similar to other serovars considered of public health importance and confirming its capability to cause infection in human. Concerning possible source of infection or reservoirs, the results did not point out any, but our hypothesis is that the environment can act as the main reservoir for Salmonella Napoli, and from there it can spill over to animals and humans. Further studies are needed to increase the knowledge on the ecology of Salmonella Napoli serovar and on the main risk factors for human infection.

OI-57, a genomic island of Escherichia coli O157, is present in other seropathotypes of Shiga toxin-producing E. coli associated with severe human disease.

Strains of Shiga toxin-producing Escherichia coli (STEC) are a heterogeneous E. coli group that may cause severe disease in humans. STEC have been categorized into seropathotypes (SPTs) based on their phenotypic and molecular characteristics and the clinical features of the associated diseases. SPTs range from A to E, according to a decreasing rank of pathogenicity. To define the virulence gene asset ("virulome") characterizing the highly pathogenic SPTs, we used microarray hybridization to compare the whole genomes of STEC belonging to SPTs B, C, and D with that of STEC O157 (SPT A). The presence of the open reading frames (ORFs) associated with SPTs A and B was subsequently investigated by PCR in a larger panel of STEC and in other E. coli strains. A genomic island termed OI-57 was present in SPTs A and B but not in the other SPTs. OI-57 harbors the putative virulence gene adfO, encoding a factor enhancing the adhesivity of STEC O157, and ckf, encoding a putative killing factor for the bacterial cell. PCR analyses showed that OI-57 was present in its entirety in the majority of the STEC genomes examined, indicating that it represents a stable acquisition of the positive clonal lineages. OI-57 was also present in a high proportion of the human enteropathogenic E. coli genomes assayed, suggesting that it could be involved in the attaching-and-effacing colonization of the intestinal mucosa. In conclusion, OI-57 appears to be part of the virulome of pathogenic STEC and further studies are needed to elucidate its role in the pathogenesis of STEC infections.

Production of the subtilase AB5 cytotoxin by Shiga toxin-negative Escherichia coli.

The subtilase cytotoxin (SubAB) is an AB(5) toxin described in certain Shiga toxin (Stx)-producing Escherichia coli (STEC) strains that usually lack the locus for enterocyte effacement (LEE). We report for the first time the production of SubAB by two Stx-negative E. coli strains, isolated from unrelated cases of childhood diarrhea. The characterization of the SubAB-coding genes showed a 90% nucleotide sequence similarity with that of the prototype subAB, located on the virulence plasmid of the STEC O113 strain 98NK2 (pO113). In both strains, subAB was physically associated with tia, an invasion genetic determinant of enterotoxigenic E. coli. The strains were negative for the saa gene, encoding an adhesin located on pO113 and present in many of the SubAB-positive strains described so far. PCR screening of 61 STEC and 100 Stx-negative E. coli strains in our collection revealed the presence of subAB in five LEE-negative STEC strains but not in the Stx-negative strains. subAB was contiguous to tia in three of the positive strains, which were all negative for saa. These results indicate that SubAB production is not restricted to STEC and suggest that a subAB-tia putative pathogenicity island is involved in the dissemination of subAB genes, as an alternative to plasmid pO113.

Evidence for a second genomic island conferring multidrug resistance in a clonal group of strains of Salmonella enterica serovar Typhimurium and its monophasic variant circulating in Italy, Denmark, and the United Kingdom.

During the 2000s, a new clonal group with resistances to ampicillin, streptomycin, sulfonamides, and tetracycline (ASSuT) emerged in Italy among strains of Salmonella enterica serovar Typhimurium and its monophasic variant, Salmonella enterica subspecies enterica serovar 4,[5],12:i:-. The PulseNet Europe database allowed us to identify ASSuT strains of both S. Typhimurium and its monophasic variant, isolated in Denmark and the United Kingdom, with the same or very closely related pulsed-field gel electrophoresis (PFGE) patterns as the Italian strains, suggesting that the ASSuT clonal group is circulating in different European countries. With the aim of analyzing the molecular basis of antibiotic resistance, resistance genes were identified and their localization was investigated in 66 ASSuT strains and, as controls, in 11 strains with different resistance patterns and PFGE profiles, belonging both to S. Typhimurium and to its monophasic variant, isolated from humans in Italy, Denmark, and the United Kingdom. All the ASSuT strains were positive for the following resistance genes: bla(TEM-1), strA-strB, sul2, and tet(B). A localization experiment demonstrated that the ASSuT resistance genes are chromosomally located. This study confirms that a multidrug-resistant clonal group, ASSuT, of S. Typhimurium and its monophasic variant has emerged and is circulating in Italy, Denmark, and the United Kingdom. Moreover, the results of this work demonstrate that the multidrug resistance in this clonal group of Salmonella strains is conferred by a new genomic island.

Particulate Shiga Toxin 2 in Blood is Associated to the Development of Hemolytic Uremic Syndrome in Children.

Hemolytic uremic syndrome (HUS), the leading cause of acute renal failure in children (< 3 years), is mainly related to Shiga toxins (Stx)-producing Escherichia coli (STEC) infections. STEC are confined to the gut resulting in hemorrhagic colitis, whereas Stx are delivered in blood to target kidney and brain, with unclear mechanisms, triggering HUS in 5 to 15% of infected children. Stx were found on circulating cells, free in sera (soluble Stx) or in blood cell-derived microvesicles (particulate Stx), whereby the relationship between these forms of circulating toxins is unclear. Here, we have examined 2,846 children with bloody diarrhea and found evidence of STEC infection in 5%. Twenty patients were enrolled to study the natural course of STEC infections before the onset of HUS. In patients, Stx were found to be associated to circulating cells and/or free and functionally active in sera. In most children, Stx were bound to neutrophils when high amounts of toxins were found in feces. Time-course analysis showed that Stx increased transiently in patients' sera while the decrease of toxin amount on leukocytes was observed. Notably, patients who recovered (85%) displayed different settings than those who developed HUS (15%). The distinctive feature of the latter group was the presence in blood of particulate Stx2 (Stx2 sedimented at g-forces corresponding to 1 µm microvesicles) the day before diagnosis of HUS, during the release phase of toxins from circulating cells. This observation strongly suggests the involvement of blood cell-derived particulate Stx2 in the transition from hemorrhagic colitis to HUS.

First report of plasmid-mediated quinolone resistance determinant qnrS1 in an Escherichia coli strain of animal origin in Italy.

A qnrS1-positive strain of Escherichia coli was detected among 73 poultry isolates showing ciprofloxacin MICs of > or =0.125 microg/ml. The qnrS1 gene was associated with a Tn3-like transposon, as previously described to occur in a Salmonella enterica serovar Infantis strain of animal origin, but the plasmid scaffold carrying this element resembled that of a plasmid previously identified in Salmonella enterica serovar Dublin. These elements suggest genetic exchanges among Salmonella and E. coli and a potential animal reservoir for the qnr genes.

Interactions between Shiga toxins and human polymorphonuclear leukocytes.

Human intestinal infections by Shiga toxin (Stx)-producing Escherichia coli cause hemorrhagic colitis and hemolytic uremic syndrome (HUS), which represents the main cause of acute renal failure in early childhood. In HUS, Stx released in the gut enter the bloodstream and are targeted to renal endothelium. The mechanism of toxin delivery is still a matter of debate, although the role of polymorphonuclear leukocytes (PMN) as a Stx carrier has been indicated. The aim of this paper was to better define the interactions between Stx and human PMN. Direct and indirect flow cytometric analysis and binding experiments with radiolabeled toxins demonstrated that Stx bind to the surface of human mature PMN but not to immature PMN from G-CSF-treated donors. The use of the human myeloid leukemia cell (HL-60) model for inducible cell differentiation confirmed that the toxin binding occurs only after granulocytic differentiation. Stx binding caused a delay of the spontaneous apoptosis of PMN, as shown by the delayed appearance of apoptotic nuclei and activation of caspase 3 and by the higher number of cells negative to the annexin V-binding assay after 48 h. Moreover, flow cytometric analysis of mixed Stx-positive and Stx-negative PMN populations showed that the toxins were transferred from positive to negative PMN. The delayed, spontaneous apoptosis and the passage of the toxic ligand from older PMN to new, mature cells entering the circulation from the bone marrow may explain the previously reported persistence of Stx in the blood of children with HUS.

Molecular characterization of multidrug-resistant strains of Salmonella enterica serotype Typhimurium and Monophasic variant (S. 4,[5],12:i:-) isolated from human infections in Italy.

Salmonella enterica serovar Typhimurium (STM) represents the prevalent cause of foodborne gastroenteritis in Italy with the majority of isolates exhibiting multidrug resistance. A resistant pattern that includes ampicillin (A), streptomycin (S), sulfonamide (Su), and tetracycline (T) (ASSuT) but lacks resistance to chloramphenicol (C) has recently emerged in Italy among strains of STM and of its monophasic variant, S. enterica subspecies enterica serovar S. 4,[5],12:i:-. With the aim to evaluate their clonal relationships, 553 strains of STM and S. 4,[5],12:i:- with the ASSuT and ACSSuT resistance patterns isolated in Italy from human infections between 2003 and 2006 were characterized by pulsed-field gel electrophoresis (PFGE) according to the PulseNet-Europe protocol and nomenclature. Among both the STM and S. 4,[5],12:i:- ASSuT strains, the predominant PFGE profile was STYMXB.0079 (53.2-73.0% of strains, respectively), while the STM ACSSuT strains belonged to the STYMXB.0061 (37.2% of strains) and STYMXB.0067 (29.9% of strains). Bionumerics cluster analysis of the nonunique PFGE profiles showed that more than 90% of ASSuT and ACSSuT-resistant strains were included in two distinct clusters with a genetic homology of 73% each other, suggesting that the ASSuT-resistant strains belong to a same clonal lineage different from that of the ACSSuT strains. Phage typing showed that 23% of the ASSuT STM strains were not typeable and 22.3% were U302. The same phage types were observed among the ASSuT strains of S. 4,[5],12:i:-. A different figure was observed for the ACSSuT strains: the STM isolates mostly belonged to DT104 (70.2%), while none of the S. 4,[5],12:i:- strains belonged to this phage type. This study indicates that the tetra-resistant ASSuT strains of STM and S. 4,[5],12:i:-, increasingly isolated in Italy, belong to a same clonal lineage and that the S. 4,[5],12:i:- strains circulating in our country mainly derive from this STM clonal lineage.

Validation on milk and sprouts of EN ISO 16654:2001 - Microbiology of food and animal feeding stuffs - Horizontal method for the detection of Escherichia coli O157.

In 2006, the European Committee for standardisation (CEN)/Technical Committee 275 - Food analysis - Horizontal methods/Working Group 6 - Microbiology of the food chain (TC275/WG6), launched the project of validating the method ISO 16654:2001 for the detection of Escherichia coli O157 in foodstuff by the evaluation of its performance, in terms of sensitivity and specificity, through collaborative studies. Previously, a validation study had been conducted to assess the performance of the Method No 164 developed by the Nordic Committee for Food Analysis (NMKL), which aims at detecting E. coli O157 in food as well, and is based on a procedure equivalent to that of the ISO 16654:2001 standard. Therefore, CEN established that the validation data obtained for the NMKL Method 164 could be exploited for the ISO 16654:2001 validation project, integrated with new data obtained through two additional interlaboratory studies on milk and sprouts, run in the framework of the CEN mandate No. M381. The ISO 16654:2001 validation project was led by the European Union Reference Laboratory for Escherichia coli including VTEC (EURL-VTEC), which organized the collaborative validation study on milk in 2012 with 15 participating laboratories and that on sprouts in 2014, with 14 participating laboratories. In both studies, a total of 24 samples were tested by each laboratory. Test materials were spiked with different concentration of E. coli O157 and the 24 samples corresponded to eight replicates of three levels of contamination: zero, low and high spiking level. The results submitted by the participating laboratories were analyzed to evaluate the sensitivity and specificity of the ISO 16654:2001 method when applied to milk and sprouts. The performance characteristics calculated on the data of the collaborative validation studies run under the CEN mandate No. M381 returned sensitivity and specificity of 100% and 94.4%, respectively for the milk study. As for sprouts matrix, the sensitivity resulted in 75.9% in the low level of contamination samples and 96.4% in samples spiked with high level of E. coli O157 and specificity was calculated as 99.1%.