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Pest infestation in any stage can lead to a quality reduction in the finished products. This study aimed to detect Escherichia coli, Salmonella spp., Campylobacter spp., and Staphylococcus aureus in Alphitobius diaperinus adults, and in samples from broiler swabs, administered water and feed collected in a single house from a broiler production facility in central Italy. Three samplings were carried out, each collecting ninety adult beetles for microbial detection in the external, faecal and internal content; ten cloacal swab samples; and one sample of both administered feed and water. Microbiological cultures and biochemical identification were performed on suspected cultures and confirmed by species‐specific PCRs. A. diaperinus was abundantly found near the windows, under the manger and in the corners of the facility. Salmonella enterica serovar Cholerasuis was found at the external surface of the beetles, while Staphylococcus xylosus and E. coli were in the faecal content. The latter micro‐organism together with Staphylococcus lentus, S. xylosus and other staphylococcal species were detected in the internal microbiota. E. coli and Campylobacter spp. were observed in cloacal swabs, and S. xylosus in one feed sample. The study findings support evidence for Salmonella spp. and E. coli, and remark that adherence to sanitation rules and biosecurity procedures are required. [ABSTRACT FROM AUTHOR]

The emergence of novel resistant markers hampers the efficacy of beta‐lactam antibiotics to treat infections caused by micro‐organisms carrying such resistances. This study investigated the antimicrobial susceptibility pattern, the carpapenem‐associated determinants and the molecular epidemiology of Klebsiella pneumoniae showing a New Delhi (NDM) metallo‐β‐lactamase phenotype, isolated from a patient admitted to intensive care unit of the main hospital for acute care of Molise region, central Italy. Antimicrobial susceptibility was assessed for nineteen antibiotics by disc diffusion and agar dilution methods. Carbapenem‐associated resistance determinants were detected through gene‐specific amplifications, targeting blaNDM‐1, blaSHV and blaTEM, blaCTX‐M, blaKPC, blaVIM, blaIMP, blaGES and blaOXA‐48‐lixe. Molecular characterization was carried out through multilocus sequence typing. The strain showed a multidrug resistant profile, and PCR and sequencing confirmed the presence of blaNDM‐1 gene. Among the multiple resistance‐associated determinants tested, the isolate, which was assigned to the sequence type ST11, only harboured blaSHV and blaTEM genes. This is the first report of NDM‐1 variant in the regional healthcare setting for acute patients, raising significant concerns about the increase in the antimicrobials resistance spread through a different mechanism from the endemic KPC carbapenemase, and underlining the circulation of a virulent clone never identified before in this area. [ABSTRACT FROM AUTHOR]

Aims: A high resolution melting (HRM) assay was developed for characterizing lineage II Listeria monocytogenes based on the amplification and the melting profiles analysis of 81 fragments targeting the region from the prs to ldh loci, including the Listeria Pathogenicity Island‐1 (LIPI‐1) genes and the inlAB operon. Methods and Results: Real‐time PCR and HRM protocols were standardized using 10 replicate assays from L. monocytogenes EGD‐e reference strain (serovar 1/2a). Twenty wild‐type isolates of serovar 1/2a and two of serovar 1/2c were tested, and differences between EGD‐e strain and the wild‐type isolates were defined if the melting temperature (Tm) of an amplicon was not within the lower and the upper limits calculated from replicate testing on EGD‐e. The analysis revealed 17 and 19 HRM profiles with respect to prs/LIPI‐1/ldh and inlAB target regions (Simpson's Index of Diversity 0·979 and 0·983) respectively. The 1/2c cultures showed 98·1% similarity to melting characteristics with EGD‐e, whilst 1/2a isolates had the greatest heterogeneity that was related to inlA, inlB and actA genes. Sequencing of amplicons generating different Tm values from EGD‐e confirmed the presence of point mutations. Conclusions: This method was useful for L. monocytogenes subtyping based on single nucleotide polymorphisms detection through the melting behaviour analysis of main virulence genes. Significance and Impact of the Study: The study underlines the effectiveness of HRM in differentiating L. monocytogenes strains with high discriminatory power, thus rendering it useful for epidemiological surveillance. [ABSTRACT FROM AUTHOR]

The microbiological and toxicological quality of 51 samples of dried herbs ( Melissa officinalis, Salvia officinalis, Malva sylvestris, Matricaria chamomilla, Alchemilla vulgaris and Centaurea cyanus) cultivated in family-managed farms in Molise Region (Italy) was evaluated. All the samples were analysed by using conventional methods, and for samples preparation, an alternative Washing and Shaking (WaS) protocol was developed to reduce release of antimicrobial compounds. None of the samples were of unsatisfactory quality with respect to aflatoxin B1, and only three samples from Malva sylvestris exceeded the limit of total aflatoxins according to Recommendation 2004/24/EC. The International Commission on Microbiological Specifications for Foods limits for mesophilic bacteria and total coliforms were exceeded in the 29·4 and 3·9% of samples, respectively: 7·8% of samples also exceeded the limit for Escherichia coli established by European Spice Association. When the 'WaS' method was used, higher microbial counts were obtained, especially for A. vulgaris, S. officinalis and M. officinalis. Herbs cultivated in family-managed small agricultural areas showed a good microbiological and toxicological quality, irrespectively of preliminary washing or selection procedures. Herb matrices may contain antimicrobial activity which should be considered when applying the conventional microbiological methods for sample preparation. Alternative preparation protocols may have advantages to reduce antimicrobial effects and should be further evaluated. [ABSTRACT FROM AUTHOR]

Aims: In this study, 105 Listeria monocytogenes strains isolated from humans, foods and environmental samples were characterized using several typing methods. Moreover, serotyping procedure was evaluated, and a cost-effective methodological approach based on preliminary PCRs screening was proposed. Methods and Results: The isolates were analysed by conventional serotyping, multiplex-PCRs for serogroup and lineage identification and PCR–RFLP of inlA gene to identify potentially noninvasive L. monocytogenes. Among the strains, only the serotypes 1/2a, 1/2c, 1/2b, 4b and 3a were identified. The isolates were classified into serogroups I (58·10%), II (22·85%), III (12·38%) and IV (6·67%). Among clinical strains, lineage I was more represented (68·75%) than lineage II; whereas, lineage II was more associated with food (90·24%) and environmental (85·72%) isolates. Most of food (89·02%) and environmental (85·71%) isolates were classified into truncated InlA profiles, whereas the 93·75% of clinical strains were associated with a complete form of the protein. Conclusion: Molecular techniques were sensitive and specific for classifying strains into serogroup and lineage and in agreement with the serotyping. Moreover, a preliminary PCRs-based screening was proposed to select only the necessary antisera by a flow chart; this methodological approach allows cost saving up to 42%. Our results further suggest the role of InlA protein in human listeriosis, particularly in immunocompetent individuals, and a correlation between truncated protein and serotype. Significance and Impact of the Study: This study further validates molecular methods for L. monocytogenes analysis and proposed a new cost-effective approach for serotyping. It could help to improve a national surveillance network for L.  monocytogenes infections in Italy. [ABSTRACT FROM AUTHOR]