Outcomes of the PIRASOA programme, an antimicrobial stewardship programme implemented in hospitals of the Public Health System of Andalusia, Spain: an ecologic study of time-trend analysis.

OBJECTIVES: Inappropriate antimicrobial use favours the spread of resistance, and multidrug-resistant microorganisms (MDR) are currently of major concern. Antimicrobial stewardship programmes (ASPs) are essential for improving antibiotic use in hospitals. However, their impact on entire healthcare systems has not been thoroughly assessed. Our objective was to provide the results of an institutionally supported ASP involving 31 public hospitals in Andalusia, Spain. METHODS: We designed an ecologic time-series study from 1 January 2014 to 31 December 2017. Quarterly, data on indicators were collected prospectively, and feedback reports were provided. PIRASOA is an ongoing clinically based quality-improvement programme whose key intervention is the educational interview, regular peer-to-peer interventions between advisors and prescribers to reinforce the appropriate use of antibiotics. Seventy-two indicators were monitored to measure prescribing quality (inappropriate treatments), antimicrobial consumption (defined daily doses per 1000 occupied bed-days), incidence density of MDR per 1000 occupied bed-days and crude mortality rate associated with bloodstream infections. We used Joinpoint regression software to analyse the trends. RESULTS: The quality of antimicrobial prescribing improved markedly, and the inappropriate treatment rate was significantly lower, with quarterly percentage change (QPC) = -3.0%, p < 0.001. Total antimicrobial consumption decreased (QPC = -0.9%, p < 0.001), specifically carbapenems, amoxicillin/clavulanic acid, quinolones and antifungal agents, whereas antipseudomonal cephalosporin use increased. While the incidence of MDR showed a sustained decreasing trend (QPC = -1.8%; p 0.002), the mortality of patients with bloodstream infections remained stable (QPC = -0.2%, p 0.605). CONCLUSIONS: To date, the PIRASOA programme has succeeded in optimizing the use of antimicrobial agents and has had a positive ecologic result on bacterial resistance at level of an entire healthcare system.

Glycerol metabolism and bitterness producing lactic acid bacteria in cidermaking.

Several lactic acid bacteria were isolated from bitter tasting ciders in which glycerol was partially removed. The degradation of glycerol via glycerol dehydratase pathway was found in 22 out of 67 isolates. The confirmation of glycerol degradation by this pathway was twofold: showing their glycerol dehydratase activity and detecting the presence of the corresponding gene by a PCR method. 1,3-propanediol (1,3-PDL) and 3-hydroxypropionic acid (3-HP) were the metabolic end-products of glycerol utilization, and the accumulation of the acrolein precursor 3-hydroxypropionaldehyde (3-HPA) was also detected in most of them. The strain identification by PCR-DGGE rpoB showed that Lactobacillus collinoides was the predominant species and only 2 belonged to Lactobacillus diolivorans. Environmental conditions conducting to 3-HPA accumulation in cidermaking were studied by varying the fructose concentration, pH and incubation temperature in L. collinoides 17. This strain failed to grow with glycerol as sole carbon source and the addition of fructose enhanced both growth and glycerol degradation. Regarding end-products of glycerol metabolism, 1,3-PDL was always the main end-product in all environmental conditions assayed, the only exception being the culture with 5.55 mM fructose, where equimolar amounts of 1,3-PDL and 3-HP were found. The 3-HPA was transitorily accumulated in the culture medium under almost all culture conditions, the degradation rate being notably slower at 15 degrees C. However, no disappearance of 3-HPA was found at pH 3.6, a usual value in cider making. After sugar exhaustion, L. collinoides 17 oxidated lactic acid and/or mannitol to obtain energy and these oxidations were accompanied by the removal of the toxic 3-HPA increasing the 1,3-PDL, 3-HP and acetic acid contents.

Biogenic amine production by lactic acid bacteria isolated from cider.

AIMS: To study the occurrence of histidine, tyrosine and ornithine decarboxylase activity in lactic acid bacteria (LAB) isolated from natural ciders and to examine their potential to produce detrimental levels of biogenic amines. METHODS AND RESULTS: The presence of biogenic amines in a decarboxylase synthetic broth and in cider was determined by reversed-phase high-performance liquid chromatography (RP-HPLC). Among the 54 LAB strains tested, six (five lactobacilli and one oenococci) were biogenic amine producers in both media. Histamine and tyramine were the amines formed by the LAB strains investigated. Lactobacillus diolivorans were the most intensive histamine producers. This species together with Lactobacillus collinoides and Oenococcus oeni also seemed to produce tyramine. No ability to form histamine, tyramine or putrescine by Pediococus parvulus was observed, although it is a known biogenic amine producer in wines and beers. CONCLUSIONS: This study demonstrated that LAB microbiota growing in ciders had the ability to produce biogenic amines, particularly histamine and tyramine, and suggests that this capability might be strain-dependent rather than being related to a particular bacterial species. SIGNIFICANCE AND IMPACT OF THE STUDY: Production of biogenic amines by food micro-organisms has continued to be the focus of intensive study because of their potential toxicity. The main goal was to identify the microbial species capable of producing these compounds in order to control their presence and metabolic activity in foods.

Growth and exopolysaccharide (EPS) production by Oenococcus oeni I4 and structural characterization of their EPSs.

AIMS: To study the influence of medium constituents on growth, and exopolysaccharide (EPS) production by a strain of Oenococcus oeni. The structure of one of the EPSs has also been characterized. METHODS AND RESULTS: EPS concentration was estimated by the phenol/sulfuric acid method. After purification and fractionation of crude EPSs, the sugar composition was determined by GLC-MS of the TMS methyl glycosides. The major polysaccharide is 2-substituted-(1-3)-beta-D-glucan. This structure was determined by methylation analysis and conventional (1)H- and (13)C-nuclear magnetic resonance spectroscopy. In addition, O. oeni synthesized two heteropolysaccharides, although a lesser proportion, constituted by galactose and glucose, and one of them also showed rhamnose. The sugar source has a clear influence on growth and EPS synthesis, and EPS production was not enhanced by adding ethanol or increasing the nitrogen source. EPS biosynthesis starts in the exponential growth phase, and continued during the stationary growth phase. CONCLUSIONS: Higher EPS yields were obtained on cultures grown on glucose + fructose. O. oeni produces a beta-glucan, as the predominant EPS, and it is also able to produce two heteropolysaccharides. SIGNIFICANCE AND IMPACT OF THE STUDY: This work provides a better understanding of EPS synthesis by O. oeni and shows the first EPS structure described for this species.

Influence of the carbohydrate source on beta-glucan production and enzyme activities involved in sugar metabolism in Pediococcus parvulus 2.6.

The influence of carbohydrate source on growth, exopolysaccharide (EPS) production and on the activity of the enzymes implicated in energy generation and UDP-glucose synthesis in Pediococcus parvulus 2.6 was evaluated. The highest EPS production was obtained on glucose, while fructose was a poor substrate for EPS synthesis. HPLC and NMR analysis on monomer composition and structure of the EPS showed that this strain produced the same beta-glucan, regardless of the carbohydrate source. The alpha-phosphoglucomutase specific activities were dependent on the carbohydrate source and a high correlation between the activity of this enzyme and the amount of EPS was found in glucose- and maltose-grown cultures. alpha-UDP-glucose pyrophosphorylase activity, necessary for the activation of glucose, was very low, but significantly higher on glucose as sugar source. In vitro phosphorylation assays and transport activities showed that glucose is taken up by a proton motive force-dependent permease, while fructose is internalized by an inducible phosphotransferase system, which renders fructose-6-phosphate. The levels of 6-phosphofructokinase activity and alpha-phosphoglucomutase activities determined on fructose were higher and lower, than those found on glucose or maltose, respectively. This suggests that fructose-6-phosphate is mainly diverted to glycolysis and explains the low EPS synthesis on fructose. Results indicate that alpha-phosphoglucomutase and/or alpha-UDP-glucose pyrophosphorylase might be the bottlenecks for EPS biosynthesis, opening the field for metabolic-engineering strategies aimed to improve EPS production.

Biogenic amines in natural ciders.

Biogenic amines play an important physiological role in mammals, and high amounts of some exogenous amines in human diet may contribute to a wide variety of toxic effects. These amines are commonly found in many foodstuffs, particularly in fermented products such as cheese, meat products, beer, wine, and ciders. Here, the level of biogenic amines in some natural ciders was examined. Twenty-four samples of cider purchased from commercial sources were analyzed by reverse-phase high-performance liquid chromatography and fluorescence detection after precolumn derivatization with o-phthaldialdehyde. Amine levels were variable, ranging from not detected to 23 mg/liter. The average level of total biogenic amines in ciders was 5.94 +/- 8.42 mg/liter. Putrescine, histamine, and tyramine were the prevailing amines being present in 50.0, 37.5, and 33.3% of the ciders studied; very small amounts of ethylamine and phenylethylamine were observed in only one sample. Other cider parameters were analyzed to determine whether they affect the biogenic amine content in ciders, and the results were evaluated by applying cluster analysis and principal component analysis. Ciders that showed lower glycerol contents and higher amounts of 1,3-propanediol had much higher levels of histamine, tyramine, and putrescine, suggesting a high activity of lactic acid bacteria during cider making and thus the need for effective control of lactic acid bacteria.

Environmental factors influencing growth of and exopolysaccharide formation by Pediococcus parvulus 2.6.

Natural exopolysaccharides (EPSs) from food-grade lactic acid bacteria have potential for development and exploitation as food additives and functional food ingredients with both health and economic benefits. In this study, we have examined the physiological capacity of EPS production in Pediococcus parvulus 2.6. EPS formation by P. parvulus 2.6 was found to be linked to biomass yields, provided that glucose was not limiting. Higher biomass yields and EPS productions were obtained when cultures were pH-controlled at pH 5.2. Various compounds have been tested for their influence on growth rate and EPS formation. Of those, only glucose (up to 75 g l(-1)), ethanol (up to 4.9%, w/v) and glycerol (up to 6.6%, w/v) had positive effects on EPS production. EPS production was not directly linked to growth, because its production continued in the stationary phase provided that glucose was present. According to an empirical model, the growth of P. parvulus 2.6 was completely inhibited by 58.9+/-18.1 g l(-1) lactate. Lactate, the sole fermentation product, was suggested to affect growth by chelation of manganese. The organism grew in an apparent linear fashion due to this imposed manganese limitation. This could be overcome by increasing the manganese concentration to at least 2 mg l(-1) in the medium. The excretion of Mn(2+) upon depletion of glucose indicated that maintenance of the high Mn(2+) gradient over the cell membrane is an energy requiring process. EPS production was increased from 0.12 g l(-1) to 4.10 g l(-1) in an improved medium that is based on the results from this study.

The effect of temperature on the growth of strains of Kloeckera apiculata and Saccharomyces cerevisiae in apple juice fermentation.

The influence of temperature (10 degrees C and 25 degrees C) on the survival and growth of Saccharomyces cerevisiae and Kloeckera apiculata was examined in mixed and pure cultures during fermentation in apple juice. The growth reached by S. cerevisiae did not seem to be affected by temperature and the presence of K. apiculata. However, the growth and survival of K. apiculata, both in single and mixed cultures, were substantially enhanced at 10 degrees C. The highest amount of ethyl acetate was produced by K. apiculata in pure culture at 10 degrees C. Nevertheless, this concentration was lowest when both yeasts were fermented together at 10 degrees C and 25 degrees C.

Characterization and DNA Plasmid Analysis of Ropy Pediococcus spp. Strains Isolated from Basque Country Ciders.

Ten strains of Pediococcus spp. causing ropiness were isolated from Basque Country spoiled ciders and characterized with regards to growth at different pHs, temperatures, and ethanol and SO2 concentrations. They grew well at pHs above 3.7 and failed to grow at temperatures over 37°C. In addition, they were tolerant to 10% ethanol and to 15, 25, and 50 mg/l total SO2 (pH 3.8). A selected ropy strain was used to carry out plasmid DNA analysis; the agarose gel pattern showed the presence of 6 bands of plasmid DNA. Plasmid-curing experiments with ethidium bromide and novobiocin suggested that the ropy phenotype was encoded by plasmid DNA. In addition, this nonropy strain lost its resistance to oleandomycin after curing trials.

Heterofermentative Lactobacilli Causing Ropiness in Basque Country Ciders.

Ropy strains of heterolactic Lactobacillus spp. were isolated from bottled spoiled ciders produced in the Basque Country. The isolates were able to produce slime experimentally on glucose containing medium and apple must. The production of ropy slime occurred during exponential growth and the increase in viscosity was correlated with production of polysaccharide. They were characterized with regards to growth at different pH, sulfur dioxide (SO2), ethanol and temperatures and showed similar behavior. All ropy strains grew in the presence of high ethanol concentrations (15%) and at low temperatures (10 and 15°C). In addition, these were tolerant to 10, 25 and 50 mg/l of total SO2 (pH 3.8), but with longer lag times as the concentration of SO2, increased. However, the ropy lactobacilli studied were affected by the pH of the medium and in general pH below 3.7 limited their growth seriously.

Calcium-induced conidiation in Penicillium cyclopium: calcium triggers cytosolic alkalinization at the hyphal tip.

Addition of Ca2+ (1 to 10 mM) to submerged cultures of Penicillium cyclopium induces conidiation. Ca2+ induced an increase in cytosolic pH from approximately 7.00 to > 7.60 in less than 10 min, as determined with the fluorescent pH probe fluorescein. Measurement of the H(+)-ATPase activity in total membrane fractions did not show any stable activation in vivo as a result of Ca2+ treatment. By fluorescence ratio imaging microscopy, it was observed that vegetative hyphae exhibit a tip-to-base pH gradient, with the tip being more acidic. Ca2+ caused this gradient to dissipate within 10 min. The effect of several agents that are supposed to cause internal acidification, by different means, on conidiation was tested. Concentrations of these agents that did not significantly affect growth but inhibited Ca(2+)-induced conidiation also prevented the intracellular alkalinization observed after exposure to the cation. Calcium channel blockers (lanthanum, cobalt, verapamil, and nifedipine) were not able to inhibit Ca(2+)-induced conidiation, although their effect on calcium uptake was not evaluated. However, the combined results point towards externally bound Ca2+ as the primary agent of conidiation induction, causing changes in plasma membrane function which disrupt the pH gradient observed during apical growth.