In Vitro Reduced Susceptibility to Pentavalent Antimonials of a Leishmania infantum Isolate from a Human Cutaneous Leishmaniasis Case in Central Italy

Cutaneous leishmaniasis (CL) caused by Leishmania (Leishmania) infantum is endemic in the Mediterranean basin. Here we report an autochthonous case of CL in a patient living in central Italy with an unsatisfactory response to treatment with intralesional Meglumine Antimoniate and in vitro demonstration of reduced susceptibility to SbIII. Parasitological diagnosis was first achieved by histopathology on tissue biopsy and the patient was treated with a local infiltration of Meglumine Antimoniate. Since the clinical response at 12 weeks from the treatment’s onset was deemed unsatisfactory, two further skin biopsies were taken for histopathological examination, DNA extraction and parasite isolation. L. (L.) infantum was identified by molecular typing. The low susceptibility to Meglumine Antimoniate was confirmed in vitro: the promastigotes from the patient strain showed significantly lower susceptibility to SbIII (the active trivalent form of antimonial) compared to the reference strain MHOM/TN/80/IPT1. The patient underwent a new treatment course with intravenous liposomal Amphotericin B, reaching complete healing of the lesion. Additional studies are needed to confirm the epidemiological and clinical relevance of reduced susceptibility to SbIII of human L. (L.) infantum isolate in Italy

Inhibition of Listeria monocytogenes by a formulation of selected dairy starter cultures and probiotics in an in vitro model

Three strains of lactic acid bacteria (LAB) and a commercial probiotic were selected to evaluate their in vitro activity towards Listeria monocytogenes. The strains Lactococcus lactis ssp. lactis, strain 340, L. lactis ssp. lactis, strain 16; Lactobacillus casei ssp. casei, strain 208 and Enterococcus faecium UBEF-41 were inoculated into skim milk and brain heart infusion broth (BHI) to get an initial Lactococcus: Lactobacillus: E. faecium UBEF-41 ratio of 2:1:1 and a concentration of approximately 7 log cfu mL−1 until challenge vs. pathogen. L. monocytogenes ATCC 7644 was also inoculated in same media to get approximately 4 log cfu mL−1. Growth curves in skim milk and BHI at 4, 10 and 30 °C, respectively were studied for: (i) LAB formulation; (ii) L. monocytogenes and (iii) LAB vs. L. monocytogenes. When challenged with LAB, at 30 °C in milk, L. monocytogenes was not detectable after day-3 and in BHI it decreased below log cfu mL−1 after day-5. At 10 and 4 °C, in both media, L. monocytogenes counts were always significantly lower (p < .001) than the counts of L. monocytogenes alone from day-2 for milk at 4 °C and BHI at 10 °C and from day-7 for BHI at 4 °C and milk at 10 °C. In conclusion, the proposed formulation was able to limit L. monocytogenes in vitro growth, even at refrigeration temperature

Fate of selected pathogens in spiked «SALAME NOSTRANO» produced without added nitrates following the application of NONIT™ technology

This study evaluated the effect of a novel formulation for starter culture associated with specific ripening conditions (NoNit™ technology) vs. a commercial» starter on the fate of selected pathogens and hygiene indicators during the fermentation and ripening of experimentally spiked salame nostrano (Italian dry sausage). Selected strains of Staphylococcus aureus 27R, Escherichia coli CSH26 K 12, Listeria innocua ATCC 33090 and Salmonella Derby 27 were inoculated into salami batter and challenged with two formulations of starter cultures (a commercial formulation and the NoNit™ formulation, consisting of Lactococcus lactis ssp. lactis, strain 340; L. lactis ssp. lactis, strain 16; Lactobacillus casei ssp. casei, strain 208 and Enterococcus faecium strain 614) with ripening at a low temperature. The proposed technology (NoNit™) performed better than the commercial formulation and limited the growth of spiked Escherichia coli, Staphylococcus aureus (including the production of enterotoxin), Salmonella Derby and Listeria innocua, yet maintained the basic product appearance and texture.Supplementary Data 1 : Reduction of selected microorganisms challenged vs. NoNitTM in milk.Supplementary Data 2 : Reduction of selected microorganisms challenged vs. NoNitTM in salami.Supplementary Data 3 : Curve for acidification kinetic for NoNitTM alone and in associations with selected pathogens.A grant from the Sviluppumbria S.p.A., programme iStart 2014, grant n. 28/2014.http://www.elsevier.com/locate/meatsci2019-05-01hj2019Paraclinical Science

Short communication : Characterization of enterotoxin producing Staphylococcus aureus isolated from mastitic cows

Staphylococcus aureus is not only a common cause of bovine mastitis, but also an agent of food poisoning in humans. In an attempt to determine whether staphylococci causing bovine mastitis could also cause food poisoning, 60 isolates of presumed S. aureus were isolated in the period between March and August 2017 from 3,384 routine, composite, quarter milk samples of individual cows raised on 12 dairy farms in central Italy. Seventeen out of 60 isolates were confirmed as S. aureus after coagulase, thermonuclease, and biochemical tests. These isolates were analyzed by PCR for the presence of the nuc, sea, seb, sec, sed, and see genes. The positive isolates were nuc, 100% (17); sea, 35.29% (6); seb, 5.88% (1); sec, 5.88% (1); sed, 29.41% (5); and see, 47.06% (8). The isolates were also tested with 2 enzyme immunoassay diagnostic kits, one for the screening detection of the production of staphylococcal enterotoxins (SEA, SEB, SEC, SED, SEE) and one for the detection of specific enterotoxin produced by each isolate. Seven out of 17 (41.18%) were enterotoxin producers: 7 produced SEA (41.18%), 1 SEB (5.88%), 1 SEC (5.88%), 5 SED (29.41%), and 6 SEE (35.29%). To further characterize the isolates, they were analyzed by the Kirby Bauer test for susceptibility to 13 antimicrobials (ampicillin, ciprofloxacin, kanamycin, tetracycline, gentamicin, methicillin, nalidixic acid, erythromycin, amoxicillin/clavulanic acid, streptomycin, vancomycin, neomycin, and enrofloxacin), and we detected resistance to ampicillin (52.94%), nalidixic acid (70.59%), erythromycin (5.88%), and amoxicillin/clavulanic acid (17.65%). The isolates were sensitive to the main classes of antimicrobials used for the treatment of bovine subclinical mastitis. The presence of enterotoxin-producing isolates of S. aureus in bovine milk means that a temperature abuse or a breakdown in the thermal treatment of the milk could present a food safety risk, particularly if all enterotoxigenic isolates could potentially produce SEA in milk.http://www.journals.elsevier.com/journal-of-dairy-science2020-12-24hj2019Paraclinical Science

Good survival outcome of metastatic SDH-deficient gastrointestinal stromal tumors harboring SDHA mutations

Purpose:A subset of patients with KIT/PDGFRA wild-type gastrointestinal stromal tumors show loss of function of succinate dehydrogenase, mostly due to germ-line mutations of succinate dehydrogenase subunits, with a predominance of succinate dehydrogenase subunit A. The clinical outcome of these patients seems favorable, as reported in small series in which patients were individually described. This work evaluates a retrospective survival analysis of a series of patients with metastatic KIT/PDGFRA wild-type succinate dehydrogenase-deficient gastrointestinal stromal tumors.Methods:Sixty-nine patients with metastatic gastrointestinal stromal tumors were included in the study (11 KIT/PDGFRA wild-type, of whom 6 were succinate dehydrogenase deficient, 5 were non-succinate dehydrogenase deficient, and 58 were KIT/PDGFRA mutant). All six succinate dehydrogenase-deficient patients harbored SDHA mutations. Kaplan-Meier curves and log-rank tests were used to compare the survival of patients with succinate dehydrogenase subunit A-mutant gastrointestinal stromal tumors with that of KIT/PDGFRA wild-type patients without succinate dehydrogenase deficiency and patients with KIT/PDGFRA-mutant gastrointestinal stromal tumors.Results:Follow-up ranged from 8.5 to 200.7 months. The difference between succinate dehydrogenase subunit A-mutant gastrointestinal stromal tumors and KIT/PDGFRA-mutant or KIT/PDGFRA wild-type non-succinate dehydrogenase deficient gastrointestinal stromal tumors was significant considering different analyses (P = 0.007 and P = 0.033, respectively, from diagnosis of gastrointestinal stromal tumor for the whole study population; P = 0.005 and P = 0.018, respectively, from diagnosis of metastatic disease for the whole study population; P = 0.007 for only patients who were metastatic at diagnosis).Conclusion:Patients with metastatic KIT/PDGFRA wild-type succinate dehydrogenase-deficient gastrointestinal stromal tumors harboring succinate dehydrogenase subunit A mutations present an impressively long survival. These patients should be identified in clinical practice to better tailor treatments and follow-up over time A subset of patients with KIT/PDGFRA wild-type gastrointestinal stromal tumors show loss of function of succinate dehydrogenase, mostly due to germ-line mutations of succinate dehydrogenase subunits, with a predominance of succinate dehydrogenase subunit A. The clinical outcome of these patients seems favorable, as reported in small series in which patients were individually described. This work evaluates a retrospective survival analysis of a series of patients with metastatic KIT/PDGFRA wild-type succinate dehydrogenase-deficient gastrointestinal stromal tumors.Methods:Sixty-nine patients with metastatic gastrointestinal stromal tumors were included in the study (11 KIT/PDGFRA wild-type, of whom 6 were succinate dehydrogenase deficient, 5 were non-succinate dehydrogenase deficient, and 58 were KIT/PDGFRA mutant). All six succinate dehydrogenase-deficient patients harbored SDHA mutations. Kaplan-Meier curves and log-rank tests were used to compare the survival of patients with succinate dehydrogenase subunit A-mutant gastrointestinal stromal tumors with that of KIT/PDGFRA wild-type patients without succinate dehydrogenase deficiency and patients with KIT/PDGFRA-mutant gastrointestinal stromal tumors.Results:Follow-up ranged from 8.5 to 200.7 months. The difference between succinate dehydrogenase subunit A-mutant gastrointestinal stromal tumors and KIT/PDGFRA-mutant or KIT/PDGFRA wild-type non-succinate dehydrogenase deficient gastrointestinal stromal tumors was significant considering different analyses (P = 0.007 and P = 0.033, respectively, from diagnosis of gastrointestinal stromal tumor for the whole study population; P = 0.005 and P = 0.018, respectively, from diagnosis of metastatic disease for the whole study population; P = 0.007 for only patients who were metastatic at diagnosis).Conclusion:Patients with metastatic KIT/PDGFRA wild-type succinate dehydrogenase-deficient gastrointestinal stromal tumors harboring succinate dehydrogenase subunit A mutations present an impressively long survival. These patients should be identified in clinical practice to better tailor treatments and follow-up over time

Bovine lymph nodes as a source of Escherichia coli contamination of the meat

Ground beef contamination with Escherichia coli is usually a result of carcass faecal contamination during the slaughter process. Carcasses are contaminated when they come into contact with soiled hides or intestinal leakage content during dressing and the evisceration processes. A more recent and compelling hypothesis is that, when lymph nodes are present in manufacturing beef trimmings, they can be a potential source of Enterobacteriaceae contamination of ground beef. The aim of this study was to investigate the occurrence of E. coli in lymph nodes from beef carcasses used for ground meat production, in six slaughter plants situated in central Italy A total of 597 subiliac (precrural) lymph nodes were obtained from 597 cattle carcasses and screened for E. coli by culture. Furthermore, E. coli isolates (one per positive carcass) were tested for stx1, stx2 eaeA and hlyA genes that are commonly used to identify and characterise shiga toxin-producing E. coli (STEC). In addition, the E. coli isolates were profiled for antimicrobial susceptibility. A proportion of 34.2% (204/597) carcasses were positive for E. coli. PCR revealed that 29% (59/204) of E. coli possessed stx1 or stx2 which corresponded to 9.9% of the cattle sampled. Moreover, a combination of stx1 or stx2 and eaeA was found in in 4 isolates (2% among E. coli positive samples and 1% among cattle sampled) and a combination of stx1 or stx2 and eaeA and hly in 1 isolate (0.5% and 0.2%). More than 95% of isolates were susceptible to gentamicin, ceftriaxone, cyprofloxacin and cefotaxime while high rates of resistance were recorded for cephalotin, ampicillin, tetracycline, tripe sulfa and streptomycin. The multivariate analysis identified “age” as the factor most closely related to E. coli positivity (either generic E. coli or STEC) in bovine lymph nodes. In conclusion, subiliac lymph nodes represent a source of E. coli for ground beef. These results are of major importance for risk assessment and improving good manufacturing practices during animal slaughter and ground meat production.Data for: Cross-sectional study to identify risk factors associated with the occurrence of Escherichia coli in bovine lymph nodes at slaughter. DOI: 10.17632/4v6dcg4j2d.1 available at https://data.mendeley.com/datasets/4v6dcg4j2d/1European Food Safety Authorityhttp://www.elsevier.com/ locate/ijfoodmicroParaclinical Science

Integrated genomic study of quadruple-WT GIST (KIT/PDGFRA/SDH/RAS pathway wild-type GIST)

BACKGROUND: About 10-15% of adult gastrointestinal stromal tumors (GIST) and the vast majority of pediatric GIST do not harbour KIT or platelet-derived growth factor receptor alpha (PDGFRA) mutations (J Clin Oncol 22:3813-3825, 2004; Hematol Oncol Clin North Am 23:15-34, 2009). The molecular biology of these GIST, originally defined as KIT/PDGFRA wild-type (WT), is complex due to the existence of different subgroups with distinct molecular hallmarks, including defects in the succinate dehydrogenase (SDH) complex and mutations of neurofibromatosis type 1 (NF1), BRAF, or KRAS genes (RAS-pathway or RAS-P).In this extremely heterogeneous landscape, the clinical profile and molecular abnormalities of the small subgroup of WT GIST suitably referred to as quadruple wild-type GIST (quadrupleWT or KITWT/PDGFRAWT/SDHWT/RAS-PWT) remains undefined. The aim of this study is to investigate the genomic profile of KITWT/PDGFRAWT/SDHWT/RAS-PWT GIST, by using a massively parallel sequencing and microarray approach, and compare it with the genomic profile of other GIST subtypes. METHODS: We performed a whole genome analysis using a massively parallel sequencing approach on a total of 16 GIST cases (2 KITWT/PDGFRAWT/SDHWT and SDHBIHC+/SDHAIHC+, 2 KITWT/PDGFRAWT/SDHAmut and SDHBIHC-/SDHAIHC- and 12 cases of KITmut or PDGFRAmut GIST). To confirm and extend the results, whole-genome gene expression analysis by microarray was performed on 9 out 16 patients analyzed by RNAseq and an additional 20 GIST patients (1 KITWT/PDGFRAWTSDHAmut GIST and 19 KITmut or PDGFRAmut GIST). The most impressive data were validated by quantitave PCR and Western Blot analysis. RESULTS: We found that both cases of quadrupleWT GIST had a genomic profile profoundly different from both either KIT/PDGFRA mutated or SDHA-mutated GIST. In particular, the quadrupleWT GIST tumors are characterized by the overexpression of molecular markers (CALCRL and COL22A1) and of specific oncogenes including tyrosine and cyclin- dependent kinases (NTRK2 and CDK6) and one member of the ETS-transcription factor family (ERG). CONCLUSION: We report for the first time an integrated genomic picture of KITWT/PDGFRAWT/SDHWT/RAS-PWT GIST, using massively parallel sequencing and gene expression analyses, and found that quadrupleWT GIST have an expression signature that is distinct from SDH-mutant GIST as well as GIST harbouring mutations in KIT or PDGFRA. Our findings suggest that quadrupleWT GIST represent another unique group within the family of gastrointestintal stromal tumors