Validation and application of a quantitative real-time PCR assay to detect common wheat adulteration of durum wheat for pasta production

Pasta is the Italian product par excellence and it is now popular worldwide. Pasta of a superior quality is made with pure durum wheat. In Italy, addition of Triticum aestivum (common wheat) during manufacturing is not allowed and, without adequate labeling, its presence is considered an adulteration. PCRrelated techniques can be employed for the detection of common wheat contaminations. In this work, we demonstrated that a previously published method for the detection of T. aestivum, based on the gliadin gene, is inadequate. Moreover, a new molecular method, based on DNA extraction from semolina and real-time PCR determination of T. aestivum in Triticum spp., was validated. This multiplex real-time PCR, based on the dual-labeled probe strategy, guarantees target detection specificity and sensitivity in a short period of time. Moreover, the molecular analysis of common wheat contamination in commercial wheat and flours is described for the first time

Comparison of the Diatheva STEC FLUO with BAX System Kits for Detection of O157:H7 and Non-O157 Shiga Toxin-Producing Escherichia coli (STEC) in Ground Beef and Bean Sprout Samples Using Different Enrichment Protocols

The aim of this study was to assess the performance of the Diatheva STEC FLUO and BAX System real-time PCR assays for detection of Shiga toxin-producing Escherichia coli (STEC) (stx1/stx2 and eae target genes) and O-group identification in ground beef and bean sprout samples. Ground beef (325 g or 25 g) and mung bean sprout (25 g) samples were inoculated with ~ 10 CFU of the Btop five^ STEC (O157:H7, O26, O103, O111, and O145 as specified in EU regulation ISO13136:2012), enriched using different broths and incubation temperatures, and tested using the Diatheva and BAX real-time PCR assays. In ground beef, both molecular methods were able to detect the Btop five^ STEC, and lower Ct values were observed for the Diatheva kits compared to BAX System. The O111-contaminated samples gave negative results with both methods using mTSB + novobiocin for enrichment. In bean sprouts, both methods provided positive results, although detection was not possible using mTSB + acriflavin/ cefsulodin/vancomycin for enrichment. In conclusion, the Diatheva and BAX methods detected the Btop five^ STEC in ground beef and bean sprouts when inoculated at low levels. Both assays provided equivalent results in terms of performance and reliability. Thus, the Diatheva kits are comparable to reference STEC-detection methods and could be used by the food industry to reliably detect the Btop five^ STEC

Salmonella Abortusovis: An Epidemiologically Relevant Pathogen

The ovine pathogen Salmonella enterica serovar Abortusovis (SAO), a pathogen strictly adapted to ovine hosts, is endemic in several European and Asian countries, where it causes significant economic losses due to the high rates of abortion in infected flocks. In some countries (i.e. Switzerland and Croatia), re-emergence of infection by SAO occurred after decades during which the disease has not been reported. The introduction of (SAO) epidemic strains in new areas is difficult to control due to the asymptomatic behaviors in infected adult lambs, rams, and nonpregnant ewes. Culture-based diagnosis may provide false-negative results. Moreover, the retrospective identification of Salmonella infection in ewes is challenging as excretion of the causative agent is transient and the serum antibodies fall to low titres soon after the abortion. Therefore, regular monitoring of pathogen exposure, mainly through seroconversion assessment, is advisable to prevent disease introduction and spread in SAO-free areas, especially in case of animal export, and to reduce abortion risk

Evaluation of PCR-based methods for the identification of enteroaggregative hemorrhagic Escherichia coli in sprouts

In this study real-time PCR assays were evaluated for the detection of enteroaggregative hemorrhagic Escherichia coli (EAHEC) O104:H4 in artificially contaminated mung bean and/alfalfa sprouts inoculated with 1, 10, and 100 CFU of EAHEC O104:H4 per 25 g sample (20, 10, and 2 replicates respectively). After selective culture enrichment the samples were tested using commercial real-time PCR kits detecting aggR/aaiC, stx/eae, and wzxO104. Using the commercial real-time PCR kits, the artificially contaminated samples were detected in the range of 75–80% positive results when contaminated with approximately 1 CFU, and 100% at 10 and 100 CFU. Microbiological detection employing O104-specific immunomagnetic capture and plating onto chromogenic media (modified Rainbow Agar and CHROMagar STEC) and confirmation by latex agglutination and PCR gave similar results (Cohen's kappa value between 0.61 and 1). In addition, the real-time PCR assay targeting the aggR and aaiC genes, indicative of enteroaggregative Escherichia coli (EAggEC), was tested against a panel of 60 bacterial strains and demonstrated 100% exclusivity (54 strains) and 100% inclusivity (6 strains). This study demonstrates the efficacy of the real-time PCR assays for the specific and sensitive detection of EAHEC from spouts

Epidemiological surveillance of circulating clones among carbapenem-resistant Enterobacteriaceae strains in hospitalised patients

Introduction The emerging spread of carbapenemase-producing Enterobacteriaceae strains (CPE), in particular Klebsiella pneumoniae (KPCKp) and Escherichia coli (CP-Ec), has become a significant threat for hospitalised patients. It is known as KPC-Kp strains are currently responsible for hospital-acquired infections whereas CP-Ec represent an important risk for spread in the community 1. Genes encoding for carbapenemase enzymes are frequently located on plasmids than can be exchanged among clonal strains increasing the antibiotic resistance rate. Their adaptation ability allows such clones to colonise and persist in clinical niches contributing to the global expansion of certain high-risk sequence types (STs) 2. This study was aimed to provide an epidemiological survey of CPE isolates collected from hospitalised patients in order to evaluate the potential risk associated to the circulating clones. Materials and methods Isolates were sampled during 2018 as part of routine clinical microbiology screening of patients admitted to the Regional Hospital of Ancona. Antimicrobial susceptibility was determined in accordance with EUCAST guidelines and carbapenem-resistance was assessed through PCR amplification of determinant genes (blaKPC, blaVIM, blaNDM, blaOXA-48, blaIMP). Plasmid incompatibility (Inc) group was established by PCR-based replicon typing using the PBRT 2.0 kit (Diatheva Srl). A representative strain of PBRT profile for each species was finally characterized by multilocus sequence typing (MLST) to identify the circulating STs. Results A total of 48 strains were collected from the rectal swabs of adult patients. The major part was identified as KPC-Kp (94%) whereas a low percentage was CP-Ec (6%) and more than 50% were multidrug-resistant strains (MDR). In all isolates, with the exception of a CP-Ec strain, blaKPC gene was detected and only once in combination with blaVIM. PBRT revealed that IncFIB KQ was the most predominant Inc group observed in 81% of strains and a multireplicon status was observed in 39 strains with the prevalence of FIIK, FIB KQ (33%) profile. MLST analysis distinguished 5 different STs among the 14 strains representative of each PBRT profile. In 11 strains of K. pneumoniae, 3 STs were determined with the prevalence of ST512, an endemic Italian variant of ST258, and the emerging of ST101 and ST307. The latter is slowly becoming the main competitor of the clonal group CG258 3. On the other hand, 2 STs were detected among the 3 strains of E. coli: the well-known ST131 and the sporadic ST405. All of them, designed as MDR, are able to persist in hosts for long period of time 2. Conclusions Despite the modest size, this study offers a general but relevant view of the epidemiological situation within an Italian hospital. The widespread dissemination of CPE strains as well as the identification of high-risk clones poses a threat for public health. These data emphasise the need to implement infection control measures in hospitals based on a multidisciplinary approach. An adequate cooperation between healthcare and infection control staffing supported by the routinely application of advanced molecular methods might be helpful to monitor and contain the further diffusion of clonal strains among different hospital wards

Carriage of Carbapenem-Resistant Enterobacterales in Adult Patients Admitted to a University Hospital in Italy

none14The emerging spread of carbapenemase-producing Enterobacterales (CPE) strains, in particular, Klebsiella pneumoniae and Escherichia coli, has become a significant threat to hospitalized patients. Carbapenemase genes are frequently located on plasmids than can be exchanged among clonal strains, increasing the antibiotic resistance rate. The aim of this study was to determine the prevalence of CPE in patients upon their admission and to analyze selected associated factors. An investigation of the antibiotic resistance and genetic features of circulating CPE was carried out. Phenotypic tests and molecular typing were performed on 48 carbapenemase-producing strains of K. pneumoniae and E. coli collected from rectal swabs of adult patients. Carbapenem-resistance was confirmed by PCR detection of resistance genes. All strains were analyzed by PCR-based replicon typing (PBRT) and multilocus sequence typing (MLST) was performed on a representative isolate of each PBRT profile. More than 50% of the strains were found to be multidrug-resistant, and the blaKPC gene was detected in all the isolates with the exception of an E. coli strain. A multireplicon status was observed, and the most prevalent profile was FIIK, FIB KQ (33%). MLST analysis revealed the prevalence of sequence type 512 (ST512). This study highlights the importance of screening patients upon their admission to limit the spread of CRE in hospitals.nonePamela Barbadoro, Daniela Bencardino, Elisa Carloni, Enrica Omiccioli, Elisa Ponzio, Rebecca Micheletti, Giorgia Acquaviva, Aurora Luciani, Annamaria Masucci , Antonella Pocognoli, Francesca Orecchioni, Marcello Mario D'Errico, Mauro Magnani, Francesca AndreoniBarbadoro, Pamela; Bencardino, Daniela; Carloni, Elisa; Omiccioli, Enrica; Ponzio, Elisa; Micheletti, Rebecca; Acquaviva, Giorgia; Luciani, Aurora; Masucci, Annamaria; Pocognoli, Antonella; Orecchioni, Francesca; D'Errico, Marcello Mario; Magnani, Mauro; Andreoni, Francesc