Gaia Scavia

Enteroaggregative Escherichia coli associated with a foodborne outbreak of gastroenteritis.

This study investigated two foodborne outbreaks of gastroenteritis that occurred 10 days apart among individuals who had meals at the restaurant of a farm holiday resort. Mild gastrointestinal symptoms were reported and none of the patients needed hospitalization. Mean incubation times were 45 and 33 h, and the overall attack rates were 43.5 and 58.3%, respectively. Stool sample examination was negative for common enteric pathogens in both outbreaks. Specimens from 13 people involved in the second outbreak and 3 restaurant staff were examined for diarrhoeagenic Escherichia coli. An enteroaggregative E. coli (EAEC) strain of serotype O92:H33 was isolated from six participants and one member of staff. In particular, the EAEC strain was isolated from five of the six cases of diarrhoea examined. The strain showed an aggregative pattern of adherence to HEp-2 cells, did not produce a biofilm and possessed the virulence-related genes aat, aggR, aap and set1A, but not the astA gene. A retrospective cohort study indicated a pecorino cheese made with unpasteurized sheep milk as the possible source (P<0.001). Samples of the cheese had E. coli counts higher than 10(6) c.f.u. g(-1), but the outbreak EAEC strain was not isolated. This report confirms that EAEC infections are probably underdiagnosed because of the limited availability of laboratories capable of identifying this group of pathogenic E. coli.

Clinical relevance of shiga toxin concentrations in the blood of patients with hemolytic uremic syndrome

Background: Intestinal infections with Shiga toxin-producing Escherichia coli (STEC) in children can lead to the hemolytic uremic syndrome (HUS). Shiga toxins (Stx) released in the gut by bacteria enter the blood stream and target the kidney causing endothelial injury. Free toxins have never been detected in the blood of HUS patients, but they have been found on the surface of polymorphonuclear leukocytes (PMN). Methods: With respect to their clinical features, the clinical relevance of the amounts of serum Stx (cytotoxicity assay with human endothelial cells) and PMN-bound Stx (cytofluorimetric assay) in 46 patients with STEC-associated HUS was evaluated. Results: Stx-positive PMN were found in 60% of patients, whereas negligible amounts of free Stx were detected in the sera. Patients with high amounts of Stx on PMN showed preserved or slightly impaired renal function (incomplete form of HUS), whereas cases with low amounts of Stx usually presented evidence of acute renal failure. Conclusions: These observations suggest that the extent of renal damage in children with STEC-associated HUS could depend on the concentration of Stx present on their PMN and presumably delivered by them to the kidney. As previously shown by experimental models from our laboratory, high amounts of Stx could induce a reduced release of cytokines by the renal endothelium, with a consequent lower degree of inflammation. Conversely, low toxin amounts can trigger the cytokine cascade, provoking inflammation, thereby leading to tissue damage.

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.

Protective effect of the AT137RQ and ARQK176 PrP allele against classical scrapie in Sarda breed sheep.

The susceptibility of sheep to scrapie is under the control of the host’s prion protein (PrP) gene and is also influenced by the strain of the agent. PrP polymorphisms at codons 136 (A/V), 154 (R/H) and 171 (Q/R/H) are the main determinants of susceptibility/resistance of sheep to classical scrapie. They are combined in four main variants of the wild-type ARQ allele: VRQ, AHQ, ARH and ARR. Breeding programmes have been undertaken on this basis in the European Union and the USA to increase the frequency of the resistant ARR allele in sheep populations. Herein, we report the results of a multi-flock study showing the protective effect of polymorphisms other than those at codons 136, 154 and 171 in Sarda breed sheep. All ARQ/ARQ affected sheep (n = 154) and 378 negative ARQ/ARQ controls from four scrapie outbreaks were submitted to sequencing of the PrP gene. The distribution of variations other than those at the standard three codons, between scrapie cases and negative controls, was statistically different in all flocks. In particular, the AT137RQ and ARQK176 alleles showed a clear protective effect. This is the first study demonstrating a protective influence of alleles other than ARR under field conditions. If further investigations in other sheep breeds and with other scrapie sources confirm these findings, the availability of various protective alleles in breeding programmes of sheep for scrapie resistance could be useful in breeds with a low frequency of the ARR allele and would allow maintaining a wider variability of the PrP gene.

Ongoing outbreak of hepatitis A in Italy: preliminary report as of 31 May 2013

Since January 2013, an unusual increase in hepatitis A cases has been detected in northern Italy. A total number of 352 cases were reported to the integrated surveillance system between January and the end of May 2013 and this represents a 70% increase compared to the same period of the previous year. The outbreak is ongoing and the public health authorities are continuing their investigations to establish the transmission vehicle and to control the outbreak.

Endothelial damage induced by Shiga toxins delivered by neutrophils during transmigration

The endothelial damage induced by Stx represents the main pathogenic event in the HUS associated with STEC infections in humans. Stx, released in the gut by bacteria, enter the bloodstream and are targeted to renal endothelia. The role of PMN as a toxin carrier has been the object of controversy. In this paper, we confirm the binding of Stx1 to PMN, also showing its degranulating effects on full‐loaded leukocytes, and support the carrier role of PMN by using a two‐chamber transmigration device, in which PMN, loaded in vitro with different amounts of Stx1, transmigrated through confluent monolayers of endothelial cells, mimicking the toxin‐induced renal endothelial injury. Stx1 was transferred during PMN transmigration, impairing protein synthesis and triggering production of proinflammatory cytokines in endothelial cells. PMN, carrying low toxin amounts, induced the release of high levels of cytokines in viable endothelial cells, whereas cytokine production was blocked in cells challenged with PMN fully loaded with Stx as a result of an almost total impairment of translation and of the activation of the apoptotic program. In agreement with previous unexplained observations in animal models, the results obtained with our experimental setting suggest that a self‐amplifying circle triggered by low doses of toxin may lead to the production of proinflammatory mediators of renal damage in HUS.

Similarity of Shiga Toxin–producing Escherichia coli O104:H4 Strains from Italy and Germany

To the Editor: Since the beginning of May 2011, a large outbreak of infections associated with Shiga toxin (Stx)–producing Escherichia coli (STEC) O104:H4 has occurred in Germany (1). The outbreak showed 3 unusual features: 1) a large proportion of case-patients with hemolytic uremic syndrome (HUS); 2) HUS in adults, although it usually affects children; and 3) frequent development of neurologic symptoms in patients when clinical and laboratory markers of HUS were improving (1,2). A second point-source outbreak caused by the same STEC O104 strain was reported in June 2011 in France (3). Both outbreaks were linked to eating fenugreek sprouts obtained from seeds produced in Egypt and distributed in Germany and other European countries (4). Instead of the attaching–effacing mechanism of adhesion to intestinal mucosa that is typical of STEC associated with severe human disease (5), the STEC O104 epidemic strain had genetic markers and an adhesion pattern (6) typical of enteroaggregative E. coli (EAEC), another group of diarrheagenic strains found frequently in developing countries (5). On basis of these findings, we reviewed our culture collection and found that an STEC strain (ED-703) from a case-patient with HUS in 2009 in Italy had the same combination of virulence factors as the strain from Germany: Stx2 production and enteroaggregative adhesion genetic markers. This strain, which had not been typed when it was isolated, showed positive PCR results for O104 (7) and H4 (8) antigen–associated genes and was agglutinated by an O104 antiserum (Statens Serum Institut, Copenhagen, Denmark). Pulsed-field gel electrophoresis showed a high degree of similarity (94.7%) with the outbreak strain from Germany (provided by M. Mielke, Robert Koch Institute, Berlin, Germany). In contrast with the outbreak strain, ED-703 did not produce extended-spectrum β-lactamases. The strain from our culture collection had been isolated from a 9-year-old girl admitted to the pediatric nephrology unit of the Ospedale Maggiore (Milan, Italy) on August 5, 2009, after 5 days of bloody diarrhea, vomiting, and abdominal pain. Diagnosis of HUS was based on the presence of hemolytic anemia, thrombocytopenia, and anuria. Neurologic symptoms (e.g., lethargy, diplopia, and nystagmus) occurred during hospitalization; magnetic resonance imaging showed signal abnormalities in the lenticular nuclei. Because of severe cardiac impairment with ejection fraction reduction and troponin increase, inotropic support and mechanical ventilation were temporarily needed. After improvement of clinical conditions, the patient was discharged, but she was readmitted a few days later because of headache, vomiting, confusion, dysarthria, hypertension, and visual impairment. Ischemic lesions were found by magnetic resonance imaging at fundus oculi. Neurologic status improved the next day, but the visual deficit persisted. Hemodialysis was needed for 2 months. Long-term sequelae of the disease were stage IV chronic kidney disease, hypertension, and severe visual impairment. Informed consent and an epidemiologic interview were obtained from the patient’s parents. The household, including her mother and 2 siblings (4 and 5 years of age), had traveled for 1 week to a resort in Tunisia; they had returned 3 weeks before the onset of the prodromal symptoms of HUS. Four days after their return, the youngest sister was hospitalized for 3 days because of bloody diarrhea, but no laboratory diagnosis was established. The mother reported having had watery diarrhea and abdominal pain on August 2. The patient history did not show any other usual risk factor for STEC infection, such as consumption of unpasteurized milk or dairy products, undercooked meat, or raw sprouts or direct exposure to ruminants or their manure. This finding suggests that the infection was probably acquired through person-to-person transmission. This case report confirms that strains of STEC O104 strictly related to the epidemic strain in Germany had already caused sporadic infections in Europe (9). Other cases have been documented in 2001 in Germany (6,9), in 2004 in France (9), and in 2010 in Finland in a patient with diarrhea who had traveled to Egypt (9). Both of the cases for which the information on the origin of the infection was available were related to travel to northern Africa, from which the seeds associated with both outbreaks could be traced (4). The history of this patient supports the hypothesis that ruminants would not have had a specific role in the transmission of STEC O104:H4, as already suggested by the epidemiologic features of the recent outbreaks (1,3). In fact, STEC O104 cannot be considered true STEC but rather EAEC strains that acquired the Stx2-coding phages by horizontal gene transfer, and EAEC is considered to be a human pathogen usually transmitted by the oral–fecal route (5). The clinical course of our patient closely resembles those of persons who had HUS associated with the German outbreak (1,2). The unusual combination of virulence factors of STEC and EAEC, already described in a group of STEC O111:H2 from an outbreak of HUS in France in 1996 (10), might confer a high degree of virulence to these strains. It also might explain the severity of the clinical findings associated with STEC O104:H4 infections.

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 β-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.

Assessment of the Genetic Susceptibility of Sheep to Scrapie by Protein Misfolding Cyclic Amplification and Comparison with Experimental Scrapie Transmission Studies

ABSTRACT The susceptibility of sheep to scrapie is influenced mainly by the prion protein polymorphisms A136V, R154H, and Q171R/H. Here we analyzed the ability of protein misfolding cyclic amplification (PMCA) to model the genetic susceptibility of sheep to scrapie. For this purpose, we studied the efficiency of brain homogenates from sheep with different PrP genotypes to support PrPSc amplification by PMCA using an ARQ/ARQ scrapie inoculum. The results were then compared with those obtained in vivo using the same sheep breed, genotypes, and scrapie inoculum. Genotypes associated with susceptibility (ARQ/ARQ, ARQ/AHQ, and AHQ/ARH) were able to sustain PrPSc amplification in PMCA reactions, while genotypes associated with resistance to scrapie (ARQ/ARR and ARR/ARR) were unable to support the in vitro conversion. The incubation times of the experimental infection were then compared with the in vitro amplification factors. Linear regression analysis showed that the efficiency of in vitro PrPSc amplification of the different genotypes was indeed inversely proportional to their incubation times. Finally, the rare ARQK176/ARQK176 genotype, for which no in vivo data are available, was studied by PMCA. No amplification was obtained, suggesting ARQK176/ARQK176 as an additional genotype associated with resistance, at least to the isolate tested. Our results indicate a direct correlation between the ability of different PrP genotypes to undergo PrPC-to-PrPSc conversion by PMCA and their in vivo susceptibility and point to PMCA as an alternative to transmission studies and a potential tool to test the susceptibility of numerous sheep PrP genotypes to a variety of prion sources.

A severe foodborne outbreak of diarrhoea linked to a canteen in Italy caused by enteroinvasive Escherichia coli , an uncommon agent

We describe a foodborne outbreak in Italy caused by enteroinvasive Escherichia coli (EIEC), an enteric pathogen uncommon in industrialized countries. On 14 April 2012 a number of employees of the city of Milan Fire Brigade (FB) were admitted to hospital with severe diarrhoea after attending their canteen. Thirty-two patients were hospitalized and a total of 109 cases were identified. A case-control study conducted on 83 cases and 32 controls attending the canteen without having symptoms identified cooked vegetables to be significantly associated with the disease. Stool samples collected from 62 subjects were screened for enteric pathogens using PCR-based commercial kits: 17 cases and two asymptomatic kitchen-workers were positive for the Shigella marker gene ipaH; an ipaH-positive EIEC strain O96:H19 was isolated from six cases. EIEC may cause serious dysentery-like outbreaks even in Western European countries. Microbiologists should be aware of microbiological procedures to detect EIEC, to be applied especially when no common enteric pathogens are identified.