Maria Luisa Marziano

Waterborne Outbreak of Norwalk-Like Virus Gastroenteritis at a Tourist Resort, Italy

In July 2000, an outbreak of gastroenteritis occurred at a tourist resort in the Gulf of Taranto in southern Italy. Illness in 344 people, 69 of whom were staff members, met the case definition. Norwalk-like virus (NLV) was found in 22 of 28 stool specimens tested. The source of illness was likely contaminated drinking water, as environmental inspection identified a breakdown in the resort water system and tap water samples were contaminated with fecal bacteria. Attack rates were increased (51.4%) in staff members involved in water sports. Relative risks were significant only for exposure to beach showers and consuming drinks with ice. Although Italy has no surveillance system for nonbacterial gastroenteritis, no outbreak caused by NLV has been described previously in the country.

Production of the Subtilase AB5 Cytotoxin by Shiga Toxin-Negative Escherichia coli

ABSTRACT The subtilase cytotoxin (SubAB) is an AB5 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.

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.

Alteration of organized structure of biofilm formed by Staphylococcus epidermidis on soft contact lenses

The effect of a nonsteroidal antiinflammatory drug commercially available in eye drop form (sodium diclofenac) was assayed for its ability to affect biofilms formed by clinical Staphylococcus epidermidis isolates. Biofilms produced by one strain positive for a slime-associated antigen, suggested to be expressed by more virulent strains, was not affected by sodium diclofenac treatment. On the other hand, biofilm produced by the slime-positive, antigen-negative strain showed dramatic alterations already after short treatments with sodium diclofenac as reported for salicylate and other nonsteroidal drugs. Such results suggest further investigation of the possible use of sodium diclofenac drops in the treatment of ophthalmic infections in soft contact lens wearers.

SA-11 rotavirus binding to human serum lipoproteins

An investigation of SA-11 rotavirus binding to human serum lipoproteins was carried out. Various subclasses of lipoproteins, purified by ultracentrifugal flotation, and apoproteins were tested for their activity in inhibiting viral infectivity and hemagglutination. All tested lipoprotein subclasses (very low, low and high density, lipoproteins: VLDL, LDL, HDL, HDL1) were shown to interact with SA-11 rotavirus: VLDL and LDL were the most active in preventing rotavirus replication, whereas HDL and HDL1 inhibited viral hemagglutination to a greater extent. Moreover, A1 and A2 apoproteins were effective towards both viral infectivity and hemagglutination. Results obtained are in agreement with a preferential interaction of VP7 or VP4 proteolytic products with low density lipoproteins and of VP8* with high density lipoproteins. Binding of SA-11 to lipoproteins or apoproteins was also quantified by an enzyme-linked immunosorbent assay procedure and lipoproteins-virus interaction was visualized by electron microscopy.

Effect of polyions on the infectivity of SA-11 rotavirus in LCC-MK2 cells

We investigated the effect of different polyions on the early phases of SA-11 rotavirus infection in susceptible LLC-MK2 cells in order to clarify the influence of electrostatic interactions in rotavirus binding to cell membranes and to select antiviral compounds able to prevent viral attachment. When added during the viral attachment step, polymers having positive charge (protamine, protamine sulphate, DEAE-dextran, histone and poly-L-lysine hydrobromide) enhanced virus infection whereas those having negative charge (mucin, heparin, heparan sulphate, alpha-1-acid glycoprotein and dextran sulphate) inhibited the viral replication. The effect of polyanions on SA-11 rotavirus and on cell membrane receptors has also been examined. Results obtained indicated that while mucin and alpha-1-acid glycoprotein act directly on virus particles, the target of heparin, heparan sulphate and dextran sulphate is the host cell membrane.

In vitro effect of synthetic flavanoids on astrovirus infection

In this study we investigated the activity of halogeno-, cyano- and amidino-isoflavenes, isoflavans and flavans on the multiplication of human astroviruses. These are naked small round viruses which have been recognized as causative agents of human gastroenteritis, and whose capsid proteins are similar to those of picornaviruses. Although all drugs tested caused a dose-dependent reduction of viral antigen synthesis as monitored by immunofluorescence, the chloro derivatives were the most effective.

Enhancement of rotavirus infectivity by saturated fatty acids

The effect of different saturated fatty acids from 10 to 16 carbon atom chains and some derivatives on the infectivity of SA-11 rotavirus was examined. Both fatty acids and derivatives induced an increase of rotavirus infected LLC-MK2 cells when present during viral absorption to host cells. Capric acid and palmitic acid were the most effective with a dose-dependent relationship. These last lipids, in the same experimental conditions, failed to restore the susceptibility to infection of LLC-MK2 cells made resistant by neuraminidase treatment or to allow cell infection by non-infectious single-shelled viral particles. Results obtained suggest that the enhancing effect on viral infectivity by saturated fatty acids requires previous binding of rotaviral outer capsid proteins to sialic acid containing cell receptors.