C10 | Study of virulence factors in strains of Bacillus cereus identified in vending machines in the Campania region: preliminary investigations

M. Astuni¹, M. Ceruso*¹, P. Bussone¹, M. Esposito², G. Varcasia², T. Pepe¹. | ¹ Dipartimento di Medicina Veterinaria e Produzioni Animali, Università degli Studi di Napoli “Federico II”; ²Centro di Riferimento Regionale per la Sicurezza della Ristorazione Pubblica e Collettiva e delle Produzioni Agroalimentari Tradizionali (C.Ri.P.A.T.) – Regione Campania, Napoli.

Purpose. Bacillus cereus is a ubiquitous spore-forming microorganism responsible for foodborne illness. Its pathogenicity is linked to its ability to produce different toxins, both emetic and diarrhoeic. Its persistence in the environment is favoured by its ability to form spores and colonise surfaces. In a previous study, the hygienic-sanitary conditions of some vending machines (VMs) located in the Campania Region were analysed. B. cereus was the pathogen most frequently found. For this bacterium, the simultaneous presence of multiple virulence genes can significantly influence its pathogenic potential. The aim of this study was to use molecular methods to search for virulence genes present in previously identified strains of B. cereus in order to estimate the pathogenicity of the circulating strains and the potential risk to consumers. Methods Sampling was carried out on 30 VMs. From each VM, 8 samples were taken and analysed as follows: 1 sample of finished product (cappuccino), 4 powder samples (milk, coffee, ginseng and chocolate), 3 surface swabs (taken from the inside wall of the water suction tube, the inside wall of the tray where the powders are mixed, and the inside wall of the beverage dispensing spout), for a total of 240 samples. B. cereus was identified by amplifying a fragment of the NheB/C gene, specific to the bacterial species, which codes for non-haemolytic enterotoxin. To highlight the presence of virulence genes, a fragment of the following genes was amplified: CwpFM, which encodes a peptidase involved in cell wall structure and adhesion; CytK, which encodes cytotoxin K, responsible for haemolytic and necrotic effects; HblD, part of the haemolytic Hbl complex, which contributes to cell lysis and increased intestinal permeability; Ces, which encodes the emetic and thermostable cereulide toxin. The analyses were performed using end-point PCR. Results B. cereus was identified in 30 vending machines. Of the 240 samples taken, the presence of this microorganism was 48% (114/240). The results revealed the following percentages of positivity for virulence genes: 66% (76/114) for CwpFM, 54% (61/114) for CytK, 53% (60/114) for HblD, and 11% (13/114) for Ces. Conclusions The high percentage of multiple virulence genes in the isolated bacterial strains suggests a potential risk to consumers. In order to increase the safety of products supplied by VMs, it is necessary to encourage the application of correct good manufacturing practices (GMP), make corrections to sanitation methods and times, and optimise official control activities. Monitoring activities should be integrated into planned documents, such as “monitoring plans”, with a risk-based approach. This approach is an effective strategy for ensuring the hygiene of VMs, the safety of the products dispensed and the protection of the health of the end consumer.