Presence of Escherichia coli O157, Salmonella spp., and Listeria monocytogenes in Raw Ovine Milk Destined for Cheese Production and Evaluation of the Equivalence Between the Analytical Methods Applied.

Italy is one of the main producers and exporters of cheese made from unpasteurized sheep milk. Since raw milk and its derived products are known sources of human infections, cheese produced from raw sheep milk could pose a microbiological threat to public health. Hence, the objectives of the study were: to characterize the potential risk of the presence of pathogens Escherichia coli O157, Listeria monocytogenes, and Salmonella in raw ovine milk destined for cheese production obtained from all the sheep farms (n = 24) in the Marches region (Central Italy) that directly transform raw milk into cheeses and to evaluate the equivalence between the analytical methods applied. A three-step molecular method (simultaneous culture enrichment, species-specific DNA magnetic isolation, and multiplex real-time polymerase chain reaction) was used for milk (n = 143) and cheese (n = 5) analysis over a 3-year period. L. monocytogenes was not detected on any of the farms, while E. coli O157 was found on three farms, although only using the molecular method. Four farms tested positive for Salmonella spp., and Salmonella enterica subsp. diarizonae serovar 61:k:1,5,7 was isolated in one of those cases. This information highlights the need to develop preventative measures to guarantee a high level of consumer safety for this specific product line, and the molecular method could be a time-saving and sensitive tool to be used in routine diagnosis.

Relationship between intended force and actual force: comparison between athletes and non-athletes.

This cross-sectional study aimed to investigate whether athletes (ATHL) and non-athletes (NON-ATHL) individuals had similar accuracy in matching intended to actual force during ballistic (BAL) and tonic (TON) isometric contractions. In this cross-sectional study, the subjects were divided into ATHL (n = 20; 22.4 ± 2.3 yrs; 73.2 ± 15.7 kg; 1.76 ± 0.08 m) and NON-ATHL (n = 20; 24.6 ± 2.4 yrs; 68.2 ± 15.0 kg; 1.73 ± 0.1 m) groups. The isometric quadriceps strength was measured with a load cell applied to a custom-built chair. For each condition, subjects performed at first three maximal voluntary isometric contractions (MVIC) as reference. Then, subjects had to match three intended force intensities expressed in percentage of the MVIC (i.e., 25%, 50%, and 75%) without any external feedback. Subjects performed three trials for each force intensity. The accuracy (AC) was calculated as the absolute difference in percentage between the intended and the actual force. A Likert scale was administered for each trial to assess the subjective matching between the intended and the actual force. Statistical analysis showed that the ATHL group was more accurate (p < 0.001) than the NON-ATHL group. In contrast, the AC (p < 0.001) was lower when the force intensities increased independently from the group. Moreover, significantly higher AC (p < 0.001) and lower aggregate Likert scores (p < 0.001) were found in BAL than TON conditions. These results suggest that (i) sports practice could enhance muscle recruitment strategies by increasing the AC in the isometric task; (ii) differences between intended and actual force appeared to be intensity-dependent with lower AC at high force intensities; (iii) different control systems act in modulating BAL and TON contractions.

Effects of self-selected versus motivational music on lower limb muscle strength and affective state in middle-aged adults.

Background: Strength training plays a crucial role in promoting healthy ageing and music might affect how individuals perform and perceive strength exercises. This study aimed to investigate the effects of self-selected music (SSM) on muscle strength and affective states during maximal isometric contractions on a customized leg extension. Methods: Twenty-six healthy middle-aged males (50.8 ± 8.4 years) performed maximal and endurance isometric strength tests under three different conditions: SSM, motivational music (MM), and control condition (CC). Peak force and Rate of Force Development (RFD) were assessed during the maximal isometric strength test. The isometric endurance test evaluated the mean force and a fatigue index. Moreover, Felt Arousal Scale (FAS) was administered before the strength protocol, whereas the Rate of Perceived Exertion (RPE) and Feeling Scale (FS) at the end of it. Results: Mean force was significantly higher in the SSM (507.3 ± 132.2 N) than MM (476.3 ± 122.4 N, p < 0.01) and CC (484.6 ± 119.2 N, p = 0.03). FAS was significantly higher in the SSM (4.0 [1.3] than MM (3.0 [2.3], p < 0.01) and CC (3.0 [1.3], p < 0.01) conditions. FS was significantly higher in the SSM (4.0 [2.0] than MM (3.0 [1.3], p < 0.01) and CC (3.0 [1.3], p < 0.01) conditions. No significant differences were found for peak force, RFD, fatigue index, and RPE. Conclusions: Listening to SSM seems to influence isometric endurance strength performance in middle-aged adults positively. Moreover, listening to SSM might improve individuals' affective states without affecting the level of perceived exertion.

Prediction equation for estimating cognitive function using physical fitness parameters in older adults.

Ageing is associated with declines in cognitive functions and physical fitness (PF). Physical exercise training and physical activity (PA) have been shown to have positive effects on cognitive functions and brain plasticity. This study aims to establish a practical equation for evaluating cognitive functions using PF parameters in healthy older adults. One-hundred and two older subjects were physically and clinically evaluated. Participants performed the Short Physical Performance Battery (SPPB) and handgrip test (HG); general cognitive functions were examined using the Mini Mental State Examination (MMSE). For all of them, a multiple regression analysis was used to predict MMSE from age, SPPB and HG variables. The new equation was cross validated to determine its prediction accuracy. Considering that SPPB and MMSE reference score are not different between genders, only one equation was developed for females and males. Age, SPPB and HG correlated significantly (p<0.01) with the MMSE score. The developed equation was MMSE = 19.479 + (1.548 x SPPB)-(0.130 x age) (R2 = 0.72 and root mean square errors of 3.6). The results of PF are useful for exercise specialists to achieve the best physical exercise training and PA in older adults. In conclusion, this study showed for the first time that our new equation can be used to predict subjects' cognitive functions based on SPPB results and subject age. We suggest its use when patients' cognitive functions or more appropriate clinical tests cannot be pursued.

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.

Rapid and simultaneous detection of Salmonella spp., Escherichia coli O157, and Listeria monocytogenes by magnetic capture hybridization and multiplex real-time PCR.

The application of rapid, specific, and sensitive methods for pathogen detection and quantification is very advantageous in diagnosis of human pathogens in several applications, including food analysis. The aim of this study was the evaluation of a method for the multiplexed detection and quantification of three significant foodborne pathogenic species (Escherichia coli O157, Salmonella spp., and Listeria monocytogenes). The assay combines specific DNA extraction by multiplex magnetic capture hybridization (mMCH) with multiplex real-time PCR. The amplification assay showed linearity in the range 106-10 genomic units (GU)/PCR for each co-amplified species. The sensitivity corresponded to 1 GU/PCR for E. coli O157 and L. monocytogenes, and 10 GU/PCR for Salmonella spp. The immobilization process and the hybrid capture of the MCH showed good efficiency and reproducibility for all targets, allowing the combination in equal amounts of the different nanoparticle types in mMCH. MCH and mMCH efficiencies were similar. The detection limit of the method was 10 CFU in samples with individual pathogens and 102 CFU in samples with combination of the three pathogens in unequal amounts (amount's differences of 2 or 3 log). In conclusion, this multiplex molecular platform can be applied to determine the presence of target species in food samples after culture enrichment. In this way, this method could be a time-saving and sensitive tool to be used in routine diagnosis.

Pch Genes Control Biofilm and Cell Adhesion in a Clinical Serotype O157:H7 Isolate.

In a previous study, induction of the Escherichia coli serotype O157:H7 SOS response decreased csgD expression in the clinical isolate PA20 at 30°C but strongly induced genes in the horizontally transferred-DNA regions (HTR), including many known virulence regulators. To determine the role of HTR regulators in the control of csgD and curli, specific regulators were plasmid-expressed in the wild-type and mutant strains of PA20 and its biofilm-forming derivative, 20R2R. At 30°C, plasmid over-expression of the O157:H7 group 3 perC homolog, pchE, strongly repressed PA20 csgD transcription (>7-fold) while the group 1 homologs, pchA and pchB, resulted in smaller reductions (<2.5-fold). However, SOS induction decreased rather than increased pchE expression (>6-fold) making group 1 pch, which are enhanced by the SOS response, the likely SOS-induced csgD repressors. Plasmid-based pchE over-expression also reduced 20R2R biofilm formation (>6-fold) and the curli-dependent, Congo red affinity of both PA20 and 20R2R. However, to properly appreciate the regulatory direction, expression patterns, and environmental consequences of these and other CsgD-controlled functions, a better understanding of natural pchE regulation will be required. The effects of HTR regulators on PA20 and 20R2R adhesion to HEp-2 cell at host temperature were also studied. Under conditions where prophage genes were not induced, curli, rather than espA, contributed to host cell adhesion in strain 20R2R. High levels of pchE expression in trans reduced curli-dependent cell adherence (>2-fold) to both 20R2R and the clinical isolate PA20, providing a host-adapting adhesion control mechanism. Expression of pchE was also repressed by induction of the SOS response at 37°C, providing a mechanism by which curli expression might complement EspA-dependent intimate adhesion initiated by the group1 pch homologs. This study has increased our understanding of the O157 pch genes at both host and environment temperatures, identifying pchE as a strong regulator of csgD and CsgD-dependent properties.

Draft Genome Sequences of Seven Strains of Shiga Toxin-Producing Escherichia coli O111 with Variation in Their Sensitivity to Novobiocin.

Inclusion of novobiocin as a selective agent for enrichment media and selective agars inhibits the growth of some Shiga toxin-producing Escherichia coli (STEC) strains, particularly non-O157 STEC, which can yield false-negative detection results. Here, we report the draft genomic sequences of seven STEC O111 isolates with different sensitivities to novobiocin.

Draft Whole-Genome Sequences of Seven Listeria monocytogenes Strains with Variations in Virulence and Stress Responses.

Listeria monocytogenes is an important foodborne pathogen that causes listeriosis. Here, we report the draft genome sequences of seven L. monocytogenes strains isolated from food, environmental, and clinical sources. Sequence differences at the genome level may help in understanding why these strains displayed different virulence and stress response characteristics.

Detection of Shiga toxin-producing Escherichia coli (STEC) in ground beef and bean sprouts: Evaluation of culture enrichment conditions.

The main purpose of this work was to evaluate culture enrichment conditions, with particular regard to those reported in ISO/TS 13136:2012, for STEC detection in food. The culture media evaluated included mTSB with novobiocin 0-16mg/l (mTSB+N0-16) or acriflavin 12mg/l (mTSB+A12); BPW; mBPWp with acriflavin 10mg/l, cefsulodin 10mg/l, vancomycin 8mg/l (mBPWp+ACV); and mBPWp with cefsulodin 10mg/l, vancomycin 8mg/l (mBPWp+CV). They were used for the growth of STEC O157, O26, O103, O111, O145 and O104 in pure cultures or in artificially contaminated food matrices (ground beef, mung bean sprouts). STEC detection was accomplished using commercially available multiplex real-time PCR assays targeting stx1-stx2 and eae, and serogroup-associated genes. More rapid multiplication of STEC in pure cultures occurred in mBPWp+CV, while an inhibitory effect of novobiocin and acriflavin was observed for some STEC serogroups in media with these selective agents. mBPWp+CV allowed the detection of all serogroups in bean sprouts when inoculated at levels as low as 1CFU/25g. A reduced novobiocin concentration of 2mg/l in mTSB was required for STEC detection in ground beef samples. A temperature of 42°C for the entire duration of the enrichment or 44°C after an initial phase of 6h at 37°C was important to limit the multiplication of non-target bacteria. Results of this study suggest that media and protocols should be adapted to the food being analyzed, since protocols provided in official reference methods may produce insufficient sensitivity.

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. PCR-related 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.