Impact on disinfection efficiency of cell load and of planktonic/adherent/detached state: case of Hafnia alvei inactivation by Plasma Activated Water

This paper describes the effects of initial microbial concentration and planktonic/adherent/detached states on the efficiency of plasma-activated water. This disinfecting solution was obtained by treating distilled water with an atmospheric pressure plasma produced by gliding electric discharges in humid air. The inactivation kinetics of planktonic cells of Hafnia alvei (selected as a bacterial model) were found to be of the first order. They were influenced by the initial microbial concentration. Efficiency decreased when the initial viable population N ₀ increased, and the inactivation rate k _max was linearly modified as a function of Log₁₀ (N ₀). This relation was used to compare planktonic, adherent, and detached cells independently from the level of population. Bacteria adhering to stainless steel and high-density polyethylene were also sensitive to treatment, but at a lower rate than their free-living counterparts. Moreover, cells detached from these solid substrates exhibited an inactivation rate lower than that of planktonic cells but similar to adherent bacteria. This strongly suggests the induction of a physiological modification to bacteria during the adhesion step, rendering adherent—and further detached—bacteria less susceptible to the treatment, when compared to planktonic bacteria.     

Yeast protein–surfactant complexes uncouple microbial electron transfer and increase transmembrane leak of protons

Aims:  To explore the combined effect of yeast proteins and surfactants on bacterial metabolism.
Methods and Results:  Protein-rich cell-free supernatant from heat-shocked yeast Saccharomyces cerevisiae was combined with certain synthetic surfactants. These blends affected the metabolism of a Polyseed inoculum of aerobic bacteria, accelerating CO₂ production and consumption of nutrients from a sterile nutrient broth solution, without a concomitant accumulation of biomass. It is suggested that in the presence of the yeast protein–surfactant complexes, bacterial electron transport is uncoupled from biomass accumulation. The 'uncoupling hypothesis' is supported by experiments with model membranes, in which the same complexes induced proton leak similar to standard chemical uncouplers, such as dinitrophenol, indicating that uncoupling may occur at the stage of generation of the transmembrane pH gradient as the driving force for ATP production.
Conclusions:  Yeast protein–surfactant complexes behave as uncouplers of oxidative metabolism in bacteria and appear to do so by increasing proton permeability of membranes.
Significance and Impact of the Study:  Yeast proteins may be of interest as nontoxic, environmentally benign and economically sound agents accelerating oxidative bacterial metabolism while uncoupling it from biomass accumulation. There are actual and potential implications in waste water/soil decontamination, degreasing and other environmental technologies.     

Rapid detection of lactobacillus and yeast concentrations using a particle size distribution analyser

Aims:  To see the possibility of particle size distribution analyser (PSDA) in detecting concentration of lactobacillus contaminants in yeast fermentation.
Methods and Results:  A PSDA was used to rapidly determine the size and concentration of lactobacillus and Saccharomyces cerevisiae . Data showed that the aerodynamic diameters of Lactobacillus casei and S. cerevisiae cells were around 0·63 and 2·9 μm, respectively, with both cultures showing a linear relationship between cell density and particle count on a size distribution curve of PSDA. In addition, Lactobacillus fermentum showed high similarity in bacterial size distribution and particle count numbers with L. casei . The PSDA also rapidly detected (within 1 min) the cell concentrations of S. cerevisiae and L. casei in a mixed sample with different concentration ratios with 10⁷–10⁹ cells ml⁻¹ of detection range.
Conclusions:  PSDA was demonstrated to be useful for the rapid detection of lactobacillus and S. cerevisiae concentrations.
Significance and Impact of the Study:  This is the first report concerning PSDA to detect the concentration of bacteria and yeast. This method can be useful in the actual field during ethanol fermentation because of relatively easy handling and rapid detection.     

The improvement of amorpha-4,11-diene production by a yeast-conform variant

Aims:  To investigate the effect of the yeast-conform variant of the Artemisia annua gene encoding for amorpha-4,11-diene synthase (ADS) on the production of amorpha-4,11-diene in a transformed yeast.
Methods and Results:  The ADS gene was mutated to the yeast-conform variant ADSm . The ADSm synthesis was performed based on step-by-step extension of a short region of the gene through a series of polymerase chain reactions (PCR). The artificial ADSm gene contained codons preferred by the yeast translation machinery. The sequence was then integrated into a yeast expression vector pYeDP60. The fusion construct was active and the transformed yeast cells produced higher level of amorpha-4,11-diene compared with the plant gene-transformed yeast cells.
Conclusions:  Strains transformed with the yeast-conform allele ( ADSm ) were more efficient in terms of production of amorpha-4,11-diene than those transformed with the plant gene.
Significance and Impact of the Study:  We demonstrated that yeast-conform allele of foreign genes by serial PCR reactions can be a solution to low efficiency of heterologous gene expression in Saccharomyces cerevisiae cells.     

The presence of Saccharomyces cerevisiae DNA in various media used to propagate yeasts and its removal by ethidium monoazide

Aims:  In this study we demonstrate the interference of yeast extract in enumeration of Saccharomyces cerevisiae using real-time PCR and develop a method for its removal from the media using ethidium monoazide (EMA).
Methods and Results:  Using real-time PCR and primers to S. cerevisiae we demonstrate the presence of yeast DNA in various media as well as the media impact on S. cerevisiae real-time PCR standard curves. By pretreatment with EMA, we were able to remove this interference.
Conclusions:  Saccharomyces cerevisiae DNA can be found in a number of common laboratory media and may impact the enumeration of this yeast by real-time PCR. However, pretreatment with EMA eliminates this concern.
Significance and Impact of the Study:  We have developed a method for removal of contaminating DNA in yeast growth media.     

Identification of yeast and bacteria involved in the mezcal fermentation of Agave salmiana

Aims:  To identify the yeast and bacteria present in the mezcal fermentation from Agave salmiana .
Methods and Results:  The restriction and sequence analysis of the amplified region, between 18S and 28S rDNA and 16S rDNA genes, were used for the identification of yeast and bacteria, respectively. Eleven different micro-organisms were identified in the mezcal fermentation. Three of them were the following yeast: Clavispora lusitaniae , Pichia fermentans and Kluyveromyces marxianus. The bacteria found were Zymomonas mobilis subsp. mobilis and Zymomonas mobilis subsp. pomaceae, Weissella cibaria , Weissella paramesenteroides , Lactobacillus pontis , Lactobacillus kefiri , Lactobacillus plantarum and Lactobacillus farraginis .
Conclusions:  The phylogenetic analysis of 16S rDNA and ITS sequences showed that microbial diversity present in mezcal is dominated by bacteria, mainly lactic acid bacteria species and Zymomonas mobilis . Pichia fermentans and K. marxianus could be micro-organisms with high potential for the production of some volatile compounds in mezcal.
Significance and Impact of the Study:  We identified the community of bacteria and yeast present in mezcal fermentation from Agave salmiana.     

PCR-DGGE analysis of lactic acid bacteria and yeast dynamics during the production processes of three varieties of Panettone

Aims:  To study lactic acid bacteria (LAB) and yeast dynamics during the production processes of sweet-leavened goods manufactured with type I sourdoughs.
Methods and Results:  Fourteen sourdough and dough samples were taken from a baking company in central Italy during the production lines of three varieties of Panettone. The samples underwent pH measurements and plating analysis on three solid media. The microbial DNA was extracted from both the (sour)doughs and the viable LAB and yeast cells collected in bulk, and subjected to PCR-denaturing gradient gel electrophoresis (DGGE) analysis. The molecular fingerprinting of the cultivable plus noncultivable microbial populations provide evidence of the dominance of Lactobacillus sanfranciscensis , Lactobacillus brevis and Candida humilis in the three fermentation processes. The DGGE profiles of the cultivable communities reveal a bacterial shift in the final stages of two of the production processes, suggesting an effect of technological parameters on the selection of the dough microflora.
Conclusions:  Our findings confirm the importance of using a combined analytical approach to explore microbial communities that develop during the leavening process of sweet-leavened goods.
Significance and Impact of the Study:  In-depth studies of sourdough biodiversity and population dynamics occurring during sourdough fermentation are fundamental for the control of the leavening process and the manufacture of standardized, high-quality products.     

Further Contribution to the Chemistry of Plasma-Activated Water: Influence on Bacteria in Planktonic and Biofilm Forms

This article is a continuation of the authors’ previous contribution. Plasma-activated water (PAW) prepared by exposure to the point-to-plane dc corona discharge was analyzed, and its biological effect tested on bacteria in planktonic and biofilm forms. Hydrogen peroxide and nitric acid were found as active components of PAW, although the presence of another unknown compound cannot be excluded unambiguously. PAW inhibits rapidly planktonic Gram-positive bacteria, whereas the inhibition of Gram-negative ones is somewhat slower. In biofilm form, this activity was not observed, so that PAW is not able to disinfect bacterial biofilms.     

Scanning electron microscope imaging of bacteria based on DNA sequence

Aims:  To develop a scanning electron microscopic approach using in situ hybridization (SEM–ISH) for gaining both genetic and morphological information about target bacteria.
Methods and Results:  Target cells were hybridized with DNA-targeted polynucleotide probes, and a tyramide signal amplification system was used to increase the sensitivity. The protocol of SEM–ISH enabled to detect low copy number target DNA sequences in individual cells.
Conclusions:  SEM–ISH allowed the in situ detection of bacteria carrying a specific gene.
Significance and Impact of the Study:  Combining morphological study with SEM and ISH techniques appears to be a valuable tool to understand the spatial distribution of target cells in complex microbial communities on various materials.     

Model systems allowing quantification of sensitivity to disinfectants and comparison of disinfectant susceptibility of persistent and presumed nonpersistent Listeria monocytogenes

Aims:  To determine if Listeria monocytogenes persistent strains differ from presumed nonpersistent strains in disinfection susceptibility and to examine the influence of attachment and NaCl on susceptibility.
Methods and Results:  Two model-systems that allowed quantitative inter-strain comparison of disinfectant sensitivity were developed. Persistent L. monocytogenes were not more tolerant to the disinfectants Incimaxx DES and Triquart SUPER than presumed nonpersistent isolates. When calibrating the systems with respect to presence of biological material and cell density, attached bacteria were as sensitive to disinfectants as were planktonic bacteria. Growth with 5% NaCl increased the tolerance of planktonic cells to Incimaxx DES. All strains of spot inoculated L. monocytogenes survived well 20 h of drying when protected by growth media and 5% NaCl, but were not protected by NaCl against disinfection.
Conclusions:  Persistent strains of L. monocytogenes are as susceptible to disinfectants as are presumed nonpersistent strains and attachment does not render the strains more tolerant to disinfectants. Growth with NaCl affected the susceptibility of the planktonic L. monocytogenes to Incimaxx DES and protected spot inoculated cells during drying.
Significance and Impact of the Study:  Attachment to surfaces does not per se offer protection to L. monocytogenes against disinfectants and disinfection tolerances do not appear to influence the ability of a strain to persist.     

The efficacy of biocides and other chemical additives in cooling water systems in the control of amoebae

Aims:  In vitro experiments were undertaken to evaluate biocide formulations commonly used in cooling water systems against protozoa previously isolated from cooling towers. The investigations evaluated the efficacy of these formulations against amoebic cysts and trophozoites.
Methods and Results:  Laboratory challenges against protozoa isolated from cooling towers using chlorine, bromine and isothiazolinone biocides showed that all were effective after 4 h. The presence of molybdate and organic phosphates resulted in longer kill times for bromine and isothiazolinones. All treatments resulted in no detectable viable protozoa after 4 h of exposure.
Conclusions:  The chemical disinfection of planktonic protozoa in cooling water systems is strongly influenced by the residence time of the formulation and less so by its active constituent. Bromine and isothiazolinone formulations may require higher dosage of concentrations than currently practiced if used in conjunction with molybdate- and phosphate-based scale/corrosion inhibitors.
Significance and Impact of the Study:  Cooling water systems are complex microbial ecosystems in which predator–prey relationships play a key role in the dissemination of Legionella . This study demonstrated that at recommended dosing concentrations, biocides had species-specific effects on environmental isolates of amoebae that may act as reservoirs for Legionella multiplication in cooling water systems.     

Polaromonas and Hydrogenophaga species are the predominant bacteria cultured from granular activated carbon filters in water treatment

Aim:  Identification of the predominating cultivable bacteria in granular activated carbon (GAC) filters used in a variety of water treatment plants for selecting representative strains to study the role of bacteria in the removal of dissolved organic matter.
Methods and Results:  Bacterial isolates were collected from 21 GAC filters in nine water treatment plants treating either ground water or surface water with or without oxidative pretreatment. Enrichment of samples in dilute liquid medium improved culturability of the bacteria by approximately log unit, to 9% up to 70% of the total cell counts. Genomic fingerprinting and 16S rDNA sequence analysis revealed that most (68%) of the isolates belonged to the Betaproteobacteria and 25% were identified as Alphaproteobacteria . The number of different genera within the Betaproteobacteria was higher in the GAC filters treating ozonated water than in the filters treating nonozonated water. Polaromonas was observed in nearly all of the GAC filters (86%), and the genera Hydrogenophaga , Sphingomonas and Afipia were observed in 43%, 33% and 29% of the filter beds, respectively. AFLP analysis revealed that the predominating genus Polaromonas included a total of 23 different genotypes.
Conclusions:  This study is the first to demonstrate that Polaromonas , which has mainly been observed in ultraoligotrophic freshwater environments, is a common component of the microbial community in GAC filters used in water treatment.
Significance and Impact of the Study:  The predominance of ultraoligotrophic bacteria in the GAC filters indicates that very low concentrations of substrates are available for microbial growth. Polaromonas species are suited for further studies on the nutritional versatility and growth kinetics enabling the modelling of biodegradation processes in GAC filters.     

Chimeric types of chromosome X in bottom-fermenting yeasts

Aim:  To determine the structure of the chimeric chromosome X of bottom-fermenting yeasts.
Methods and Results:  Eight cosmid clones carrying DNA from chromosome X of bottom-fermenting yeasts were selected by end-sequencing. Four of the cosmid clones had Saccharomyces cerevisiae (SC)-type and Saccharomyces bayanus (SB)-type chimeric ends, two had SC-type ends and two had SB-type ends. Sequencing revealed that the bottom-fermenting yeast strains in this study had four types of chromosome X: SC–SC, SC–SB, SB–SC and SB–SB. The translocation site in the chimeric chromosome is conserved among bottom-fermenting yeast strains, and was created by homologous recombination within a region of high sequence identity between the SC-type sequence and the SB-type sequence.
Conclusions:  Existing bottom-fermenting yeast strains share a common ancestor in which the chimeric chromosome X was generated.
Significance and Impact of the Study:  The knowledge gained in this study sheds light on the evolution of bottom-fermenting yeasts.     

Protective effect of salicylates against hydrogen peroxide stress in yeast

Aims:  To investigate the effects of salicylates in Saccharomyces cerevisiae exposed to oxidative stress induced by hydrogen peroxide (H₂O₂).
Methods and Results:  Saccharomyces cerevisiae was cultured through to the postlogarithmic phase of growth. Stress was induced by the addition of 1·5 mmol l⁻¹ H₂O₂ for 1 h, while N-acetyl-l-cysteine (NAC) and glutathione (GSSG) were used as control agents that affect the redox balance. Sodium salicylate, at 0·01–10 mmol l⁻¹or acetylsalicylic acid, at 0·02–2·5 mmol l⁻¹ was administered at various times before hydrogen peroxide stress. Both agents conferred resistance to a subsequent hydrogen peroxide stress, similarly to the induction of the adaptive response observed upon pretreatment with NAC and GSSG. Sodium salicylate was more potent as a short-term, but not as a long-term pretreatment agent, compared to acetylsalicylic acid.
Conclusions:  Pharmacological pretreatment with salicylates resulted in dose related increases in cell survival, indicating the induction of the protective response in yeast.
Significance and Impact of the study:  The possible role of salicylates in the modulation of the hydrogen peroxide stress response in eukaryotic cells address questions on the effects of these commonly used therapeutic agents in a number of disorders exhibiting an oxidative stress component.     

Effects of yeast probiotic formulation on viability, revival and protection against infection with Salmonella enterica ssp. enterica serovar Typhimurium in mice

Aims:  To compare the effects of five yeast probiotic formulations on viability, revival and washout kinetic in the digestive tract of mice, and the protection against an experimental infection with Salmonella enterica serovar Typhimurium.
Methods and Results:  The number of viable cells in five commercial probiotic products codified as A, B, C and D ( Saccharomyces boulardii – lyophilized) and E ( Saccharomyces cerevisiae – aqueous suspension) was determined, as well as revival and washout kinetic in mouse intestine. Protective capacity was evaluated by survival rate and histopathology of liver and intestine of mice treated with each product and then challenged with Salm . Typhimurium.
Conclusions:  Product A contained the highest number of viable cells and, fed to mice, gave the highest counts of viable yeasts and the longest persistence in faeces. Probably as a consequence, the highest survival and protection of intestinal and hepatic tissues were observed when product A was used for mouse treatment. Product E showed low counts in the formulation and was not recovered from mouse intestine.
Significance and Impact of the Study:  Formulation (lyophilization or aqueous suspension) is an important factor for revival and survival of a probiotic product in vivo and consequently for its protective properties.     

A comparative study of the wine fermentation performance of Saccharomyces paradoxus under different nitrogen concentrations and glucose/fructose ratios

Aims:  The main goal of the present study is to determine the effects of different nitrogen concentrations and glucose/fructose ratios on the fermentation performance of Saccharomyces paradoxus , a nonconventional species used for winemaking.
Methods and Results:  Ethanol yield, residual sugar concentration, as well as glycerol and acetic acid production were determined for diverse wine fermentations conducted by S. paradoxus . Experiments were also carried out with a commercial Saccharomyces cerevisiae wine strain used as control. The values obtained were compared to test significant differences by means of a factorial anova and the Scheffé test. Our results show that S. paradoxus strain was able to complete the fermentation even in the nonoptimal conditions of low nitrogen content and high fructose concentration. In addition, the S. paradoxus strain showed significant higher glycerol synthesis and lower acetic acid production than S. cerevisiae in media enriched with nitrogen, as well as a lower, but not significant, ethanol yield.
Conclusions:  The response of S. paradoxus was different with respect to the commercial S. cerevisiae strain, especially to glycerol and acetic acid synthesis.
Significance and Impact of the Study:  The present study has an important implication for the implementation of S. paradoxus strains as new wine yeast starters exhibiting interesting enological properties.     

Molecular ecology of a facultative swine waste lagoon

Aims:  To investigate the microbial ecology of three facultative swine waste lagoons.
Methods and Results:  Phylogenetic analysis of sequences in a 16S rRNA gene clone library and fluorescence in situ hybridization (FISH) analyses were used to assess bacterial diversity in a swine waste lagoon. FISH analysis and Gram-staining were used to compare the microbial communities of all three swine waste lagoons. Six operational taxonomic units were in high relative abundance and corresponded to the following phylotypes; Thiolamprovum , Verrucomicrobia , Acholeplasma , Turicibacter , Clostridium and Bacteroides . PCR was employed to detect the genes apsA and dsrAB which encode for enzymes specifically associated with dissimilatory sulfate-reduction within sulfate-reducing bacteria (SRB). Amplification of these genes confirmed their presence within the lagoons.
Conclusions:  All lagoons were dominated by purple sulfur bacteria, affiliated to Thiolamprovum pedioforme . The molecular identification of fermentative bacteria and SRB indicate the following metabolic processes within such facultative ponds: sulfur-cycling, fermentation, inter-species hydrogen transfer and carbon cycling.
Significance and Impact of the Study:  This study provides the first molecular evidence for the existence of a sulfur cycle which is linked to phototrophic sulfide oxidation by purple bacteria and organotrophic sulfate-reduction by SRB.     

Control of microbial contamination in dental unit water systems using tetra-sodium EDTA

Aim:  To examine the efficacy of tetra-sodium EDTA in controlling microbial contamination of dental unit water systems (DUWS).
Methods and Results:  Ten dental units were treated once a week with either 4% or 8% tetra-sodium EDTA for four or two consecutive weeks, respectively. Before treatment, 43% and 60% of the water samples from the air/water triple syringe and high-speed hand-pieces, respectively, exceeded the American Dental Association (ADA) guidelines of 200 CFU ml⁻¹ water during a 6-week baseline period. After each weekend treatment, the levels of microbial contamination in all DUWS fell significantly ( P  < 0·001) to below the ADA guideline. By the end of the week, microbial counts in the outflowing water had returned to baseline levels indicating a transient effect of single doses of tetra-sodium EDTA, and the need for multiple applications. The biofilms were virtually eliminated after a single weekend treatment.
Conclusions:  Tetra-sodium EDTA is effective in controlling microbial contamination in DUWS.
Significance and Impact of the Study:  Inexpensive, effective and safe products for reducing the microbial load of water from DUWS are needed to meet ADA and other national guidelines. Tetra-sodium EDTA can significantly reduce microbial biofilms and bacterial counts in outflowing water, and is compatible for use in DUWS.