Yeasts Inoculation Effect on Bacterial Development in Carbonic Maceration Wines Elaboration.

Carbonic maceration (CM) vinification is a very traditional method that allows saving energy without great equipment investment, obtaining high-quality wines. However, due to its particularities, CM winemaking implies a higher risk of microbial alteration. This work studies the evolution of bacterial population along carbonic maceration wines elaboration with and without yeast inoculation. In the same way, two strategies of yeast inoculation were studied: "pied de cuve" and Active Dry Yeasts (ADY) seed. For this purpose, three conditions were assayed: spontaneous fermentation (without inoculation), "pied de cuve" technology, and ADY inoculation. For each condition, two winemaking methods were compared: carbonic maceration and the standard method of destemming and crushing (DC). The bacterial evolution (lactic acid and acetic acid bacteria) was followed in different fermentation stages. Finally, the wines obtained were analysed (pH and volatile acidity). In the non-inoculated wines produced by CM, high development of the bacterial population was observed (counts of acetic acid bacteria around 4.3 log cfu/mL), and finished wines presented high values of volatile acidity (>1.5 g/L), which did not occur in the inoculated vinifications (counts of acetic acid bacteria around 1.5 log cfu/mL and 0.5 g/l of volatile acidity). Thus, the control of yeast population, as a "pied de cuve" as ADY seed, seems to be an effective tool to avoid bacterial alterations in CM vinifications.

Combined Effect of High Hydrostatic Pressure, Sous-Vide Cooking, and Carvacrol on the Quality of Veal, Plant-Based, and Hybrid Patties during Storage.

The effect of carvacrol added to patties stored at 4 °C for 14 days, previously pressurized and vacuum-cooked (HPP-SVCOOK), was investigated. Three formulations were prepared (veal, plant-based product, and hybrid product). An emulsion made with olive and linseed oils was added. The physicochemical and microbiological qualities were assessed. Microbial tests indicated negligible growth of spoilage organisms in treated patties. No significant effect of carvacrol on the microbial loads of patties was noticed. Sulfite-reducing clostridia and Enterobacteriaceae were absent in the treated patties, whereas, in the treated veal and hybrid samples, 3 and 2 units of log cfu/g reduction for lactic acid bacteria and molds and yeasts were noted, respectively. On day 7 of storage, veal patties exhibited a significant reduction (p < 0.05) in the L* (53.9−49.3), hardness (32.3−21.4 N), springiness (0.8−0.7 N), cohesiveness (0.49−0.46), and chewiness (12.2−7.1) and a hike in the a* value (5.3−9.4). No significant changes in L* (59.1−58.6), a* (8.57−8.61), hardness (11.6−10.6 N), or cohesiveness (0.27−0.26) were observed in plant-based patties over the storage times, whereas reductions in springiness (0.5−0.4), chewiness (1.9−1.3), and b* (26.6−29.1) were noted in them. In hybrid patties, the L* (53.9−52.5) and b* values (24.9−24.3) were consistent but had a significant decrease in a* value (5.9−3.5) along the days of storage under study. The texture parameters of the hybrid patties altered were similar to those of veal patties during the 14-day storage time. In all samples, pH decreased with storage time. HPP-SVCOOK was effective on rendering safe and shelf-stable, ready-to-eat patties regardless of their matrix formulation. The addition of carvacrol had limited effects on the textural qualities of the HPP-SVCOOK products. Future studies need to be undertaken to assess the treated patties' consumer acceptability and sensory profile. The study provides the basis for the development of novel meat-based and plant-based products that are microbiologically safe, with minimum physicochemical alterations during storage.

Antimicrobial Resistance in Escherichia coli from the Broiler Farm Environment, with Detection of SHV-12-Producing Isolates.

Antimicrobial resistance is an important One Health challenge that encompasses the human, animal, and environmental fields. A total of 111 Escherichia coli isolates previously recovered from manure (n = 57) and indoor air (n = 54) samples from a broiler farm were analyzed to determine their phenotypes and genotypes of antimicrobial resistance and integron characterization; in addition, plasmid replicon analysis and molecular typing were performed in extended-spectrum-beta-lactamase (ESBL) producer isolates. A multidrug-resistance phenotype was detected in 46.8% of the isolates, and the highest rates of resistance were found for ampicillin, trimethoprim−sulfamethoxazole, and tetracycline (>40%); moreover, 15 isolates (13.5%) showed susceptibility to all tested antibiotics. None of the isolates showed imipenem and/or cefoxitin resistance. Twenty-three of the one hundred and eleven E. coli isolates (20.7%) were ESBL producers and carried the blaSHV-12 gene; one of these isolates was recovered from the air, and the remaining 22 were from manure samples. Most of ESBL-positive isolates carried the cmlA (n = 23), tet(A) (n = 19), and aac(6')-Ib-cr (n = 11) genes. The following genetic lineages were identified among the ESBL-producing isolates (sequence type-phylogroup-clonotype): ST770-E-CH116−552 (n = 12), ST117-B2-CH45−97 (n = 4), ST68-E-CH26−382/49 (n = 3), ST68-E-CH26−49 (n = 1), and ST10992-A/B1-CH11−23/41/580 (n = 4); the latter two were detected for the first time in the poultry sector. At least two plasmid replicon types were detected in the ESBL-producing E. coli isolates, with IncF, IncF1B, IncK, and IncHI1 being the most frequently found. The following antimicrobial resistance genes were identified among the non-ESBL-producing isolates (number of isolates): blaTEM (58), aac(6')-Ib-cr (6), qnrS (2), aac(3)-II (2), cmlA (6), tet(A)/tet(B) (22), and sul1/2/3 (51). Four different gene-cassette arrays were detected in the variable region of class 1 (dfrA1-aadA1, dfrA12-aadA2, and dfrA12-orf-aadA2-cmlA) and class 2 integrons (sat2-aadA1-orfX). This work reveals the worrying presence of antimicrobial-resistant E. coli in the broiler farm environment, with ESBL-producing isolates of SHV-12 type being extensively disseminated.

Airborne Dissemination of Bacteria (Enterococci, Staphylococci and Enterobacteriaceae) in a Modern Broiler Farm and Its Environment.

The role of the air as a vehicle of bacteria dissemination in the farming environment has been previously reported, but still scarcely studied. This study investigated the bacteria density/diversity of the inside and outside air and of litter samples at a broiler farm. Samples were collected considering two seasons, three outside air distances (50/100/150 m) and the four cardinal directions. Selective media was used for staphylococci, enterococci, and Enterobacteriaceae recovery. A high number of bacteria was detected in the litter (2.9 × 105-5.8 × 107 cfu/g) and in the inside air (>105 cfu/m3), but a low emission of bacteria was evidenced in the outside air (<6 cfu/m3). Moreover, the bacteria detected in the farm's outside air decreased the further from the farm the sample was taken. A total of 544 isolates were identified by MALDI-TOF (146 from the litter, 142 from inside air and 256 from outside air). From these, 162 staphylococci (14 species; S. saprophyticus 40.7%), 176 Enterobacteriaceae (4 species; E. coli 66%) and 190 enterococci (4 species; E. hirae 83%) were detected. E. hirae was the predominant species, and identical PFGE clones were detected in inside and outside samples. The detection of identical DNA profiles in E. hirae isolates from inside and outside samples suggests the role of the air in bacterial dissemination from the inside of the broiler farm to the immediate environment.

Coagulase-negative staphylococci carrying cfr and PVL genes, and MRSA/MSSA-CC398 in the swine farm environment.

This work aimed at characterizing four Staphylococcus aureus and 68 coagulase-negative staphylococci (CoNS), recovered from the air and liquid manure tank of two swine farms with intensive- and semi-extensive-production types, for their antimicrobial resistance pheno-/genotypes and their virulence gene content. Molecular typing was performed by spa typing, MLST, agr typing, and SCCmec typing, where applicable. Conjugation experiments were performed to assess the transferability of the linezolid resistance gene cfr, and its genetic environment was determined by Whole-Genome-Sequencing. The four S. aureus (intensive-production farm, IP-farm) were typed as t011-agrI-CC398-ST398, were scn-negative and two of them were methicillin-resistant (MRSA) with the mecA gene (SCCmec-V). Multidrug resistance was seen in 87 % of the CoNS. Statistically significant differences among the antimicrobial resistance rates of CoNS from the two farms were observed for cefoxitin, aminoglycosides, tetracycline, ciprofloxacin and trimethoprim-sulfamethoxazole. Eight methicillin-resistant CoNS, which were recovered from the IP-farm, carried the mecA gene. One S. simulans isolate was PVL-positive and three S. cohnii eta-positive. One S. equorum and one S. arlettae showed linezolid resistance and carried the cfr gene (IP-farm), which was non-transferable by conjugation into S. aureus. The cfr genetic context in both isolates was identical, with the lsa(B) gene located upstream of cfr. The environment of swine farms might contribute to the dissemination of CoNS that show multidrug resistance and harbor important virulence factors.

Detection of poxtA- and optrA-carrying E. faecium isolates in air samples of a Spanish swine farm.

OBJECTIVE: Two linezolid-resistant Enterococcus faecium isolates, C10004 and C10009, were recovered from air samples of a Spanish swine farm and comprehensively characterized. METHODS: Detection of linezolid resistance mechanisms (mutations and acquisition of resistance genes) was performed by PCR/sequencing. Isolates were characterized by multilocus sequence typing (MLST), antimicrobial susceptibility testing, detection of antimicrobial resistance and virulence genes, and analysis of the genetic environment of the linezolid resistance genes. The characterization of isolate C10009 was performed by Whole-Genome-Sequencing and of isolate C10004 by PCR and amplicon sequencing, where applicable. Conjugation experiments to assess the transferability of the optrA and poxtA genes implicated in linezolid resistance were performed. RESULTS: The linezolid-resistant E. faecium isolates C10004 and C10009, assigned to ST128 and ST437, respectively, harbored the optrA and poxtA genes. Neither mutations in the 23S rRNA nor in the genes for the ribosomal proteins L3, L4 and L22 were detected. C10004 and C10009 carried fourteen and thirteen antimicrobial resistance genes, respectively. The sequence alignment indicated that the genetic environment of the poxtA gene was identical in both isolates, with a downstream-located fexB gene. The poxtA gene was transferred by conjugation together with the fexB gene, and also with tet(M) and tet(L) in the case of isolate C10004. The optrA gene could not be transferred. CONCLUSIONS: This is the first report of the poxtA gene in Spain. The presence of poxtA- and optrA-carrying E. faecium isolates in air samples represents a public health concern, indicating an involvement of swine farms in the spread of linezolid-resistant bacteria.

Identification of Enterococci, Staphylococci, and Enterobacteriaceae from Slurries and Air in and around Two Pork Farms.

In this study, we investigated the airborne dissemination of bacteria from the inside of two very different pork farms (an intensively confined farm and an open-range farm) to the immediate environment. Samples were taken from the slurry, from the air inside the farms (area 0), and from their immediate surroundings at a distance of 50, 100, and 150 m in four directions (north, south, east, and west). A control sample in the air of a zone far away from human or animal activity was also taken. Identification of isolates was made by means of the matrix-assisted laser desorption-ionization time of flight system. A total of 1,063 isolates were obtained, of which a mere 7 came from the air of the control area. Staphylococci, enterococci, and Enterobacteriaceae were selectively targeted for isolation and represented 48.6, 27.2, and 21.6% of the isolates, respectively. The species identified from the air of surrounding areas ( Enterococcus faecalis, Enterococcus hirae, and Staphylococcus arlettae, mainly) were also present inside the farms studied. The results suggest that air is involved in bacterial dissemination, and pork farms should be considered a potential source of foodborne bacteria that might contaminate surrounding areas, including vegetable orchards. Wind direction appears as a factor involved in bacterial dispersion through the air, but its effect may be conditioned by existing vegetation and orographic conditions.

Molecular characterization of antibiotic resistance in Escherichia coli strains from a dairy cattle farm and its surroundings.

BACKGROUND: This study describes the phenotypic and genotypic characteristics of 78 genetically different Escherichia coli recovered from air and exudate samples of a dairy cattle farm and its surroundings in Spain, in order to gain insight into the flow of antimicrobial resistance through the environment and food supply. RESULTS: Antimicrobial resistance was detected in 21.8% of the 78 E. coli isolates analyzed (resistance for at least one of the 14 agents tested). The highest resistance rates were recorded for ampicillin, nalidixic acid, trimethoprim/sulfamethoxazole and tetracycline. The resistance genes detected were as follows (antibiotic (number of resistant strains), gene (number of strains)): ampicillin (9), blaTEM-1 (6); tetracycline (15), tet(A) (7), tet(B) (4), tet(A) + tet(B) (1); chloramphenicol (5), cmlA (2), floR (2); trimethoprim/sulfamethoxazole (10), sul2 (4), sul1 (3), sul3 (2), sul1 + sul2 (1); gentamicin-tobramycin (1), ant(2″) (1). About 14% of strains showed a multidrug-resistant phenotype and, of them, seven strains carried class 1 integrons containing predominantly the dfrA1-aadA1 array. One multidrug-resistant strain was found in both inside and outside air, suggesting that the airborne spread of multidrug-resistant bacteria from the animal housing facilities to the surroundings is feasible. CONCLUSIONS: This study gives a genetic background of the antimicrobial resistance problem in a dairy cattle farm and shows that air can act as a source for dissemination of antimicrobial-resistant bacteria. © 2016 Society of Chemical Industry.

Airborne dissemination of Escherichia coli in a dairy cattle farm and its environment.

There are multiple ways bacteria can be transported from its origin to another area or substrate. Water, food handlers, insects and other animals are known to serve as a vehicle for bacterial dispersion. However, the importance of the air in open areas as a possible way of bacterial dissemination has not been so well analyzed. In this study, we investigated the airborne dissemination of Escherichia coli from the inside of a dairy cattle farm to the immediate environment. The air samples were taken inside the farm (area 0) and from the immediate outside farm surroundings at distance of 50, 100 and 150m in four directions (north, south, east, and west). At each point, the air was collected at different heights: 40cm, 70cm and 1m. The sampling was carried out in two weather seasons (November and July). E. coli was isolated in both inside and outside air, even in samples taken 150m from the farm. A seasonal effect was observed with more bacterial isolates when temperature was higher. Regarding the distribution of the isolates, wind direction appeared as a determining factor. In order to verify that E. coli strains isolated from animal housing facilities were identical to those isolated from the air of the immediate farm environment, their genomic DNA profiles were analyzed by pulsed-field gel electrophoresis (PFGE) after digestion with the endonuclease XbaI. The comparison of genetic profiles suggested that the strains isolated from inside and outside the farm were related, leading to the conclusion that the air is an important vehicle for E. coli dissemination.

The occurrence of fungi, yeasts and bacteria in the air of a Spanish winery during vintage.

This research studies the presence of microorganisms of enological interest (yeasts, bacteria and molds) and their evolution in the air of a wine cellar. The samples were taken throughout the winemaking campaign (September-December) in a winery of the D.O.Ca. Rioja, Spain. They were collected using an airIDEAL atmosphere sampler from Biomerieux. For the isolation, specific selective media were used for each group of microorganisms. The results obtained indicate that the presence in the winery air of the various different microorganisms studied is directly related to the winemaking processes that are taking place in the winery. Thus, the number of molds present decreases once grapes have ceased to be brought into the winery. The maximum number of yeasts in the air is found when all the vats in the cellar are fermenting, while the lactic bacteria are not detected until the first malolactic fermentation begins. The species of yeasts and molds identified are also related to the winemaking processes. The coincidence of strains of Saccharomyces cerevisiae among those present in the vats during alcoholic fermentation and those isolated from the air, confirms the role of the latter as a transmitter of microorganisms.

Influence of exposure to light on the sensorial quality of minimally processed cauliflower.

The impact of lighting on minimally processed cauliflower packaged in 4 different film types (PVC and 3 P-Plus) has been measured and quantified. The effect on the sensorial quality of storage at 4 degrees C in darkness and partial or continuous lighting was evaluated. The gas concentrations in the packages and the weight losses were also determined. Atmosphere composition inside the packages depended on both the permeability of the film used for the packaging and exposure to light. Samples stored with lighting maintained the gaseous exchange between plant tissue and the atmosphere inside the packages for longer periods than in samples kept in darkness. This prompted a greater loss of water vapor as well as the development of atmospheres with low levels of O2 and high levels of CO2 in the samples packed with less permeable films. The most important aspect in sensory evaluation was color. In instrumental color evaluation, coordinates h* and L* were the main means for estimating color evolution. The presence of light accelerated browning in the cut zones. The development of abnormal coloring in these areas marked the end of shelf life for minimally processed cauliflower. Among the sensory attributes studied, color was the most affected by exposure to light. Samples packed in P-Plus 120 film displayed the lowest level of color deterioration in the cut zones. However, under lit conditions, the low permeability of this film caused atmospheres with very low O2 contents and high CO2 contents. These atmospheres produced a loss of texture and the development of off-odors.

Survival and growth of Listeria monocytogenes and enterohemorrhagic Escherichia coli O157:H7 in minimally processed artichokes.

The ability of Listeria monocytogenes and Escherichia coli O157:H7 inoculated by immersion (at 4.6 and 5.5 log CFU/ g, respectively) to survive on artichokes during various stages of preparation was determined. Peeling, cutting, and disinfecting operations (immersion in 50 ppm of a free chlorine solution at 4 degrees C for 5 min) reduced populations of L. monocytogenes and E. coli O157:H7 by only 1.6 and 0.8 log units, respectively. An organic acid rinse (0.02% citric acid and 0.2% ascorbic acid) was more effective than a tap water rinse in removing these pathogens. Given the possibility of both pathogens being present on artichokes at the packaging stage, their behavior during the storage of minimally processed artichokes was investigated. For this purpose, batches of artichokes inoculated with L. monocytogenes or E. coli O157:H7 (at 5.5 and 5.2 log CFU/g, respectively) were packaged in P-Plus film bags and stored at 4 degrees C for 16 days. During this period, the equilibrium atmosphere composition and natural background microflora (mesophiles, psychrotrophs, anaerobes, and fecal coliforms) were also analyzed. For the two studied pathogens, the inoculum did not have any effect on the final atmospheric composition (10% O2, 13% CO2) or on the survival of the natural background microflora of the artichokes. L. monocytogenes was able to survive during the entire storage period in the inoculated batches, while the E. coli O157:H7 level increased by 1.5 log units in the inoculated batch during the storage period. The modified atmosphere was unable to control the behavior of either pathogen.