Regulation of Cysteine Homeostasis and Its Effect on Escherichia coli Sensitivity to Ciprofloxacin in LB Medium.

Cysteine and its derivatives, including H2S, can influence bacterial virulence and sensitivity to antibiotics. In minimal sulfate media, H2S is generated under stress to prevent excess cysteine and, together with incorporation into glutathione and export into the medium, is a mechanism of cysteine homeostasis. Here, we studied the features of cysteine homeostasis in LB medium, where the main source of sulfur is cystine, whose import can create excess cysteine inside cells. We used mutants in the mechanisms of cysteine homeostasis and a set of microbiological and biochemical methods, including the real-time monitoring of sulfide and oxygen, the determination of cysteine and glutathione (GSH), and the expression of the Fur, OxyR, and SOS regulons genes. During normal growth, the parental strain generated H2S when switching respiration to another substrate. The mutations affected the onset time, the intensity and duration of H2S production, cysteine and glutathione levels, bacterial growth and respiration rates, and the induction of defense systems. Exposure to chloramphenicol and high doses of ciprofloxacin increased cysteine content and GSH synthesis. A high inverse relationship between log CFU/mL and bacterial growth rate before ciprofloxacin addition was revealed. The study points to the important role of maintaining cysteine homeostasis during normal growth and antibiotic exposure in LB medium.

Development of a functional Greek sheep yogurt incorporating a probiotic Lacticaseibacillus rhamnosus wild-type strain as adjunct starter culture.

Greek yogurt is a fermented dairy product of high nutritional value that can be used as a matrix for the delivery of probiotics. The aim of this study was to develop a new probiotic Greek sheep yogurt with upgraded quality and functional characteristics. To do this, yogurt was manufactured by fermenting pasteurized milk with the commercial starter culture (Streptococcus thermophilus (ST), Lactobacillus bulgaricus (LB)) together with a probiotic Lacticaseibacillus rhamnosus (LR) wild-type strain (probiotic yogurt; PY). As a control, yogurt manufactured with only the starter culture (ST, LB) was used (conventional yogurt; CY) The survival of all three lactic acid bacteria (LAB) species (ST, LB, and LR) was monitored throughout the products' shelf life (storage at 4 °C for 25 days), and also following exposure to a static in vitro digestion model (SIVDM). The population dynamics of total aerobic plate count (APC), Enterobacteriaceae, yeasts and molds grown in both yogurts were also determined. The total antioxidant activity (AA) of yogurts was comparatively determined using in parallel two different assays, whereas the Folin-Ciocalteu assay was used to determine their total phenolic content (TPC). At each sampling day, yogurts were also evaluated for their pH, titratable acidity (TA) and main sensory characteristics. The population of probiotic LR remained stable during the shelf life (and above 108 CFU/g). Yogurt starters (ST, LB) were not detected following SIVDM, whereas LR (in PY) presented a reduction of about only one log. The AA and TPC of PY were found significantly higher than that of CY (P < 0.05). At the end of storage (25th day), neither pH nor TA differed significantly between the two yogurt types, while no fungal growth was observed in the PY. Consumer sensory analysis did not reveal important differences between the two yogurt types during their shelf life. To sum up, the novel yogurt was able to deliver to consumers a high number of probiotic cells (>108 CFU/g), presented increased antioxidant power, had an expanded shelf life, and maintained its good sensory attributes.

Susceptible bacteria can survive antibiotic treatment in the mammalian gastrointestinal tract without evolving resistance.

Antibiotic resistance and evasion are incompletely understood and complicated by the fact that murine interval dosing models do not fully recapitulate antibiotic pharmacokinetics in humans. To better understand how gastrointestinal bacteria respond to antibiotics, we colonized germ-free mice with a pan-susceptible genetically barcoded Escherichia coli clinical isolate and administered the antibiotic cefepime via programmable subcutaneous pumps, allowing closer emulation of human parenteral antibiotic dynamics. E. coli was only recovered from intestinal tissue, where cefepime concentrations were still inhibitory. Strikingly, "some" E. coli isolates were not cefepime resistant but acquired mutations in genes involved in polysaccharide capsular synthesis increasing their invasion and survival within human intestinal cells. Deleting wbaP involved in capsular polysaccharide synthesis mimicked this phenotype, allowing increased invasion of colonocytes where cefepime concentrations were reduced. Additionally, "some" mutant strains exhibited a persister phenotype upon further cefepime exposure. This work uncovers a mechanism allowing "select" gastrointestinal bacteria to evade antibiotic treatment.

Relationship between Desiccation Tolerance and Biofilm Formation in Shiga Toxin-Producing Escherichia coli.

Shiga toxin-producing Escherichia coli (STEC) is a major concern in the food industry and requires effective control measures to prevent foodborne illnesses. Previous studies have demonstrated increased difficulty in the control of biofilm-forming STEC. Desiccation, achieved through osmotic stress and water removal, has emerged as a potential antimicrobial hurdle. This study focused on 254 genetically diverse E. coli strains collected from cattle, carcass hides, hide-off carcasses, and processing equipment. Of these, 141 (55.51%) were STEC and 113 (44.48%) were generic E. coli. The biofilm-forming capabilities of these isolates were assessed, and their desiccation tolerance was investigated to understand the relationships between growth temperature, relative humidity (RH), and bacterial survival. Only 28% of the STEC isolates had the ability to form biofilms, compared to 60% of the generic E. coli. Stainless steel surfaces were exposed to different combinations of temperature (0 °C or 35 °C) and relative humidity (75% or 100%), and the bacterial attachment and survival rates were measured over 72 h and compared to controls. The results revealed that all the strains exposed to 75% relative humidity (RH) at any temperature had reduced growth (p < 0.001). In contrast, 35 °C and 100% RH supported bacterial proliferation, except for isolates forming the strongest biofilms. The ability of E. coli to form a biofilm did not impact growth reduction at 75% RH. Therefore, desiccation treatment at 75% RH at temperatures of 0 °C or 35 °C holds promise as a novel antimicrobial hurdle for the removal of biofilm-forming E. coli from challenging-to-clean surfaces and equipment within food processing facilities.

Effect of Lyoprotective Agents on the Preservation of Survival of a Bacillus cereus Strain PBG in the Freeze-Drying Process.

Lyophilization is a widely employed long-term preservation method in which the bacterial survival rate largely depends on the cryoprotectant used. Bacillus cereus strain PBC was selected for its ability to thrive in environments contaminated with arsenic, lead, and cadmium, tolerate 500 ppm of free cyanide, and the presence of genes such as ars, cad, ppa, dap, among others, associated with the bioremediation of toxic compounds and enterotoxins (nheA, nheB, nheC). Following lyophilization, the survival rates for Mannitol 2.5%, Mannitol 10%, and Glucose 1% were 98.02%, 97.12%, and 96.30%, respectively, with the rates being lower than 95% for other sugars. However, during storage, for the same sugars, the survival rates were 78.71%, 97.12%, and 99.97%, respectively. In the cake morphology, it was found that the lyophilized morphology showed no relationship with bacterial survival rate. The best cryoprotectant for the PBC strain was 1% glucose since it maintained constant and elevated bacterial growth rates during storage, ensuring that the unique characteristics of the bacterium were preserved over time. These findings hold significant implications for research as they report a new Bacillus cereus strain with the potential to be utilized in bioremediation processes.

The mef(A)/msr(D)-carrying streptococcal prophage Φ1207.3 encodes an SOS-like system, induced by UV-C light, responsible for increased survival and increased mutation rate.

Bacterial SOS response is an inducible system of DNA repair and mutagenesis. Streptococci lack a canonical SOS response, but an SOS-like response was reported in some species. The mef(A)-msr(D)-carrying prophage Ф1207.3 of Streptococcus pyogenes contains a region, spanning orf6 to orf11, showing homology to characterized streptococcal SOS-like cassettes. Genome-wide homology search showed the presence of the whole Φ1207.3 SOS-like cassette in three S. pyogenes prophages, while parts of it were found in other bacterial species. To investigate whether this cassette confers an SOS-mutagenesis phenotype, we constructed Streptococcus pneumoniae R6 isogenic derivative strains: (i) FR172, streptomycin resistant, (ii) FR173, carrying Φ1207.3, and (iii) FR174, carrying a recombinant Φ1207.3, where the SOS-like cassette was deleted. These strains were used in survival and mutation rate assays using a UV-C LED instrument, for which we designed and 3D-printed a customized equipment, constituted of an instrument support and swappable-autoclavable mini-plates and lids. Upon exposure to UV fluences ranging from 0 to 6,400 J/m2 at four different wavelengths, 255, 265, 275, and 285 nm, we found that the presence of Φ1207.3 SOS-like cassette increases bacterial survival up to 34-fold. Mutation rate was determined by measuring rifampicin resistance acquisition upon exposure to UV fluence of 50 J/m2 at the four wavelengths by fluctuation test. The presence of Φ1207.3 SOS-like cassette resulted in a significant increase in the mutation rate (up to 18-fold) at every wavelength. In conclusion, we demonstrated that Φ1207.3 carries a functional SOS-like cassette responsible for an increased survival and increased mutation rate in S. pneumoniae. IMPORTANCE Bacterial mutation rate is generally low, but stress conditions and DNA damage can induce stress response systems, which allow for improved survival and continuous replication. The SOS response is a DNA repair mechanism activated by some bacteria in response to stressful conditions, which leads to a temporary hypermutable phenotype and is usually absent in streptococcal genomes. Here, using a reproducible and controlled UV irradiation system, we demonstrated that the SOS-like gene cassette of prophage Φ1207.3 is functional, responsible for a temporary hypermutable phenotype, and enhances bacterial survival to UV irradiation. Prophage Φ1207.3 also carries erythromycin resistance genes and can lysogenize different pathogenic bacteria, constituting an example of a mobile genetic element which can confer multiple phenotypes to its host.

Polyphosphate uses mTOR, pyrophosphate, and Rho GTPase components to potentiate bacterial survival in Dictyostelium.

IMPORTANCE: Although most bacteria are quickly killed after phagocytosis by a eukaryotic cell, some pathogenic bacteria escape death after phagocytosis. Pathogenic Mycobacterium species secrete polyP, and the polyP is necessary for the bacteria to prevent their killing after phagocytosis. Conversely, exogenous polyP prevents the killing of ingested bacteria that are normally killed after phagocytosis by human macrophages and the eukaryotic microbe Dictyostelium discoideum. This suggests the possibility that in these cells, a signal transduction pathway is used to sense polyP and prevent killing of ingested bacteria. In this report, we identify key components of the polyP signal transduction pathway in D. discoideum. In cells lacking these components, polyP is unable to inhibit killing of ingested bacteria. The pathway components have orthologs in human cells, and an exciting possibility is that pharmacologically blocking this pathway in human macrophages would cause them to kill ingested pathogens such as Mycobacterium tuberculosis.

Probiotic Fermented Goat's and Sheep's Milk: Effect of Type and Dose of Collagen on Survival of Four Strains of Probiotic Bacteria during Simulated In Vitro Digestion Conditions.

Microbial tolerance of digestive stresses depends not only on the bacterial strain but also on the structure and physicochemical properties of the supply chain and the foods that contain it. In the present study, we aimed to evaluate the effects of the type of milk (ovine, caprine) and the type and dose of collagen on the viability of four probiotic strains, Lacticaseibacillus paracasei L-26, Lacticaseibacillus casei 431, Lactobacillus acidophilus LA-5, and Lacticaseibacillus rhamnosus Lr-32, during in vitro gastrointestinal digestion. The highest survival rate under simulated in vitro digestion conditions compared to the number of cells before digestion was found in two strains, L. casei and L. paracasei, where survival rates were greater than 50% in each batch. The survival rate of the L. rhamnosus strain ranged from 41.05% to 64.23%. In caprine milk fermented by L. acidophilus, a higher survival rate was found in milk with 1.5% hydrolysate than the control, by about 6%. Survival of the L. rhamnosus strain was favorably affected by the 3% addition of bovine collagen in caprine milk, which increased survival by about 14% compared to the control sample. Adding 3% of hydrolysate to sheep's and goat's milk enhanced the survival of the L. rhamnosus strain by 3% and 19%, respectively. This study reports that fermented caprine and ovine milk may be suitable matrices for the probiotic supply of commercial dairy starter cultures and promote gut homeostasis.

Escherichia coli ST2797 Is Abundant in Wastewater and Might Be a Novel Emerging Extended-Spectrum Beta-Lactamase E. coli.

The increasing prevalence of antibiotic-resistant bacteria is an emerging threat to global health. The analysis of antibiotic-resistant enterobacteria in wastewater can indicate the prevalence and spread of certain clonal groups of multiresistant bacteria. In a previous study of Escherichia coli that were isolated from a pump station in Norway over 15 months, we found a recurring E. coli clone that was resistant to trimethoprim, ampicillin, and tetracycline in 201 of 3,123 analyzed isolates (6.1%). 11 representative isolates were subjected to whole-genome sequencing and were found to belong to the MLST ST2797 E. coli clone with plasmids carrying resistance genes, including blaTEM-1B, sul2, dfrA7, and tetB. A phenotypic comparison of the ST2797 isolates with the uropathogenic ST131 and ST648 that were repeatedly identified in the same wastewater samples revealed that the ST2797 isolates exhibited a comparable capacity for temporal survival in wastewater, greater biofilm formation, and similar potential for the colonization of mammalian epithelial cells. ST2797 has been isolated from humans and has been found to carry extended spectrum ß-lactamase (ESBL) genes in other studies, suggesting that this clonal type is an emerging ESBL E. coli. Collectively, these findings show that ST2797 was more ubiquitous in the studied wastewater than were the infamous ST131 and ST648 and that ST2797 may have similar abilities to survive in the environment and cause infections in humans. IMPORTANCE The incidence of drug-resistant bacteria found in the environment is increasing together with the levels of antibiotic-resistant bacteria that cause infections. The COVID-19 pandemic has shed new light on the importance of monitoring emerging threats and finding early warning systems. Therefore, to mitigate the antimicrobial resistance burden, the monitoring and early identification of antibiotic-resistant bacteria in hot spots, such as wastewater treatment plants, are required to combat the occurrence and spread of antibiotic-resistant bacteria. Here, we applied a PhenePlate system as a phenotypic screening method for genomic surveillance and discovered a dominant and persistent E. coli clone ST2797 with a multidrug resistance pattern and equivalent phenotypic characteristics to those of the major pandemic lineages, namely, ST131 and ST648, which frequently carry ESBL genes. This study highlights the continuous surveillance and report of multidrug resistant bacteria with the potential to spread in One Health settings.

DNA-Binding Protein Dps Protects Escherichia coli Cells against Multiple Stresses during Desiccation.

Gradual dehydration is one of the frequent lethal yet poorly understood stresses that bacterial cells constantly face in the environment when their micro ecotopes dry out, as well as in industrial processes. Bacteria successfully survive extreme desiccation through complex rearrangements at the structural, physiological, and molecular levels, in which proteins are involved. The DNA-binding protein Dps has previously been shown to protect bacterial cells from many adverse effects. In our work, using engineered genetic models of E. coli to produce bacterial cells with overproduction of Dps protein, the protective function of Dps protein under multiple desiccation stresses was demonstrated for the first time. It was shown that the titer of viable cells after rehydration in the experimental variants with Dps protein overexpression was 1.5-8.5 times higher. Scanning electron microscopy was used to show a change in cell morphology upon rehydration. It was also proved that immobilization in the extracellular matrix, which is greater when the Dps protein is overexpressed, helps the cells survive. Transmission electron microscopy revealed disruption of the crystal structure of DNA-Dps crystals in E. coli cells that underwent desiccation stress and subsequent watering. Coarse-grained molecular dynamics simulations showed the protective function of Dps in DNA-Dps co-crystals during desiccation. The data obtained are important for improving biotechnological processes in which bacterial cells undergo desiccation.

Influence of Growth Medium Composition on Physiological Responses of Escherichia coli to the Action of Chloramphenicol and Ciprofloxacin.

The ability of hydrogen sulfide (H2S) to protect bacteria from bactericidal antibiotics has previously been described. The main source of H2S is the desulfurization of cysteine, which is either synthesized by cells from sulfate or transported from the medium, depending on its composition. Applying electrochemical sensors and a complex of biochemical and microbiological methods, changes in growth, respiration, membrane potential, SOS response, H2S production and bacterial survival under the action of bactericidal ciprofloxacin and bacteriostatic chloramphenicol in commonly used media were studied. Chloramphenicol caused a sharp inhibition of metabolism in all studied media. The physiological response of bacteria to ciprofloxacin strongly depended on its dose. In rich LB medium, cells retained metabolic activity at higher concentrations of ciprofloxacin than in minimal M9 medium. This decreased number of surviving cells (CFU) by 2-3 orders of magnitude in LB compared to M9 medium, and shifted optimal bactericidal concentration (OBC) from 0.3 µg/mL in M9 to 3 µg/mL in LB. Both drugs induced transient production of H2S in M9 medium. In media containing cystine, H2S was produced independently of antibiotics. Thus, medium composition significantly modifies physiological response of E. coli to bactericidal antibiotic, which should be taken into account when interpreting data and developing drugs.

Sheep Milk Symbiotic Ice Cream: Effect of Inulin and Apple Fiber on the Survival of Five Probiotic Bacterial Strains during Simulated In Vitro Digestion Conditions.

We conducted a study to determine the survival of bacterial cells under in vitro digestion. For this purpose, ice cream mixes were prepared: control, with 4% inulin, 2.5% inulin and 1.5% apple fiber and 4% apple fiber. Each inoculum (pH = 4.60 ± 0.05), containing 9 log cfu g-1 bacteria, at 5% (w/w) was added to the ice cream mixes (Lacticaseibacilluscasei 431, Lactobacillus acidophilus LA-5, Lacticaseibacillus paracasei L-26, Lacticaseibacillusrhamnosus, Bifidobacterium animalis ssp. lactis BB-12) and fermentation was carried out to pH 4.60 ± 0.05. The in vitro digestion method simulated the stages of digestion that occur in the mouth, stomach and small intestine under optimal controlled conditions (pH value, time and temperature). At each stage of digestion, the survival rate of probiotic bacteria was determined using the plate-deep method. As expected, in the oral stage, there was no significant reduction in the viability of the probiotic bacteria in any ice cream group compared to their content before digestion. In the stomach stage, Bifidobacterium animalis ssp. lactis BB-12 strain had the highest viable counts (8.48 log cfu g-1) among the control samples. Furthermore, a 4% addition of inulin to ice cream with Bifidobacterium BB-12 increased gastric juice tolerance and limited strain reduction by only 16.7% compared to the number of bacterial cells before digestion. Regarding ice cream samples with Bifidobacterium BB-12, replacing part of the inulin with apple fiber resulted in increased survival at the stomach stage and a low reduction in the bacterial population of only 15.6% compared to samples before digestion. At the stomach stage, the positive effect of the addition of inulin and apple fiber was also demonstrated for ice cream samples with Lacticaseibacilluscasei 431 (9.47 log cfu g-1), Lactobacillus acidophilus LA-5 (8.06 log cfu g-1) and Lacticaseibacillus paracasei L-26 (5.79 log cfu g-1). This study showed the highest sensitivity to simulated gastric stress for ice cream samples with Lacticaseibacillusrhamnosus (4.54 log cfu g-1). Our study confirmed that the 4% addition of inulin to ice cream increases the survival rate of L. casei and Bifidobacterium BB-12 in simulated intestinal juice with bile by 0.87 and 2.26 log cfu g-1, respectively. The highest viable count in the small intestine stage was observed in ice cream with L. acidophilus. The addition of inulin increased the survival of L. rhamnosus by 10.8% and Bifidobacterium BB-12 by about 22% under conditions of simulated in vitro digestion compared to their control samples. The survival rates of L. casei and L. paracasei were also highly affected by the 4% addition of apple fiber, where the increase under gastrointestinal passage conditions was determined to range from 7.86-11.26% compared to their control counterparts. In comparison, the lowest survival rate was found in the control ice cream with L. rhamnosus (47.40%). In our study at the intestinal stage, only five ice cream groups: a sample with 4% inulin and L. acidophilus, a control sample with Bifidobacterium BB12, a sample with 2.5% inulin and 1.5% apple fiber with Bifidobacterium BB12, a control sample with L. rhamnosus, a sample with 4% fiber and L. rhamnosus reported bacterial cell counts below 6 log cfu g-1 but higher than 5 log cfu g-1. However, in the remaining ice cream groups, viable counts of bacterial cells ranged from 6.11 to 8.88 log cfu g-1, ensuring a therapeutic effect. Studies have clearly indicated that sheep milk ice cream could provide a suitable matrix for the delivery of probiotics and prebiotics and contribute to intestinal homeostasis. The obtained results have an applicative character and may play an essential role in developing new functional sheep milk ice cream.

Studying the Interaction of Neutrophils and Glaesserella Parasuis Indicates a Serotype Independent Benefit from Degradation of NETs.

Glaesserella (G.) parasuis is one of the most important porcine pathogens causing Glaesser's disease. Neutrophil granulocytes are the major counteracting cell type of the innate immune system, which contribute to the host defense by phagocytosis or the formation of neutrophil extracellular traps (NETs). Recently, NET-formation has been shown to facilitate the survival of bacteria from the Pasteurellaceae family. However, the interaction of NETs and G. parasuis is unclear so far. In this study, we investigated the interplay of three G. parasuis serotypes with porcine neutrophils. The production of reactive oxygen species by neutrophils after G. parasuis infection varied slightly among the serotypes but was generally low and not significantly influenced by the serotypes. Interestingly, we detected that independent of the serotype of G. parasuis, NET formation in neutrophils was induced to a small but significant extent. This phenomenon occurred despite the ability of G. parasuis to release nucleases, which can degrade NETs. Furthermore, the growth of Glaesserella was enhanced by external DNases and degraded NETs. This indicates that Glaesserella takes up degraded NET components, supplying them with nicotinamide adenine dinucleotide (NAD), as this benefit was diminished by inhibiting the 5'-nucleotidase, which metabolizes NAD. Our results indicate a serotype-independent interaction of Glaesserella with neutrophils by inducing NET-formation and benefiting from DNA degradation.

Selective Quantification of Erwinia amylovora Live Cells in Pome Fruit Tree Cankers by Viability Digital PCR.

The accurate assessment of Erwinia amylovora live cell populations in fire blight cankers by classic microbiology methods has major limitations. Some of them are the presence of competitive microbiota in samples that inhibit E. amylovora's growth and the release of toxic compounds by plant material during sample processing, which may hamper the pathogen's ability to form colonies on solid media. Digital PCR (dPCR) combined with the photo-reactive DNA-binding dye propidium monoazide (PMA) allows selective detection and quantification of live E. amylovora cells in woody samples while overcoming the constraints of culture-dependent methods. This work describes a reliable viability dPCR procedure to determine E. amylovora live cell concentrations in fire blight cankers from pome fruit trees. This protocol can be adapted for the analysis of other types of plant material and enables investigation of ecological, epidemiological, and management significance of cankers as a relatively underexplored part of the fire blight disease cycle.

Impact of seed-applied fungicide and insecticide on Azospirillum brasilense survival and wheat growth-promoting ability.

The use of Azospirillum brasilense as a crop inoculant has increased in recent years. Thus, the compatibility of the inoculation technology with seed treatments using pesticides needs to be evaluated. In this study, we evaluated the effect of an insecticide and fungicide formulation on A. brasilense strain FP2 population by culturing and culture-independent approaches. In addition, we evaluated the impact of these pesticides on the ability of A. brasilense to promote plant growth by monitoring biometric traits (root and shoot dry mass and length) of wheat grown in Greenhouse conditions. Seed pesticide dressings, mainly fungicide, led to a significant mortality of A. brasilense over time. The ability of A. brasilense to promote wheat growth also decreased due to pesticide treatments combined with sowing delay. Considering that pesticides confer fitness advantages to the wheat in field condition, our results suggest that sowing within the first 4 h after inoculation maintain the beneficial effects of A. brasilense on wheat growth promotion. Furthermore, we conclude that inoculation and treatment of seeds with pesticides may be compatible techniques when carried out immediately before sowing.

Effect of Pseudomonas putida-producing pyoverdine on copper uptake by Helianthus annuus cultivated on vineyard soils.

Bioaugmentation-assisted phytoextraction was used to reduce the Cu load in vineyard soils. While performance is usually the endpoint of such studies, here we identified some mechanisms underlying Cu soil to plant transfer, particularly the role of siderophores in the extraction of Cu from the soil-bearing phases and its phytoavailability. Carbonated vs. non­carbonated vineyard soils were cultivated with sunflower in rhizoboxes bioaugmented with Pseudomonas putida. gfp-Tagged P. putida was monitored in the soil and pyoverdine (Pvd), Cu, Fe, Mn, and Zn were measured in the soil solution. Trace elements (TE) were analysed in the roots and shoots. Plant growth and nutritional status were also measured. With bioaugmentation, the concentration of total Cu (vs. Cu2+) in the soil solution increased (decreased) by a factor of 1.6 to 2.6 (7 to 13) depending on the soil. The almost 1:1 relationship between the excess of Fe + Cu mobilized from the solid phase and the amount of Pvd in the soil solution in bioaugmented treatments suggests that Pvd mobilized Fe and Cu mainly by ligand-controlled dissolution via a 1:1 metal-Pvd complex. Bioaugmentation increased the Cu concentration by 17% in the shoots and by 93% in the roots, and by 30% to 60% the sunflower shoot biomass leading to an increase in the amount of Cu phytoextracted by up to 87%. The amount of Fe, Mn, Zn, and P also increased in the roots and shoots. Contrary to what was expected, carbonated soil did not increase the mobilization of TE. Our results showed that bioaugmentation increased phytoextraction, and its performance can be further improved by promoting the dissociation of Pvd-Cu complex in the solution at the soil-root interface.

Viability of hospital pathogens on mobile phone.

INTRODUCTION: Clinical use of mobile phones have increased exponentially. Whilst evidence of contamination is documented, a key factor when determining potential risks of contamination, is establishing the duration the organism remains viable on the device. If pathogens are found to persist for extended duration, healthcare mobile phones may become fomites for cross departmental transmission. AIM: Determine the duration pathogenic bacteria, Acinetobacter baumannii, Escherichia coli, two Pseudomonas sp. Bacillus cereus, Enterococcus faecalis susceptible and resistant to vancomycin (VSE and VRE) Staphylococcus aureus susceptible and resistant to methicillin (MSSA and MRSA), and a coagulase negative Staphylococcus (CoNs) can remain viable on a mobile phone under controlled conditions. METHOD: Phones were inoculated with 106 - 107 of each bacterium. The duration of viability was measured from the point the inoculum had dried and CFUs retrieved at timed intervals over 28 days. RESULTS: The mean percentage of bacteria viable at each time point was significantly different (20mins, P = .004, 1 hour P = .014, 6 hours P = .006, 24 hours P = .004, 7 days P = .007, 14 days P = .003, 21 days P = .002- and 28 days P = .004). Gram-positive bacteria remained viable longer than gram-negative bacteria (P = .010). MSSA declined faster than MRSA within the first 6 hours (P = .036). CONCLUSIONS: The extended duration of bacterial viability indicates the ability for pathogens to persist on a device and remain viable long enough to be transmitted to new areas both within the hospital and out to the community. Mobile phone decontamination should occur in combination of hand hygiene.

Effects of NaCl Concentrations on Growth Patterns, Phenotypes Associated With Virulence, and Energy Metabolism in Escherichia coli BW25113.

According to the sit-and-wait hypothesis, long-term environmental survival is positively correlated with increased bacterial pathogenicity because high durability reduces the dependence of transmission on host mobility. Many indirectly transmitted bacterial pathogens, such as Mycobacterium tuberculosis and Burkhoderia pseudomallei, have high durability in the external environment and are highly virulent. It is possible that abiotic stresses may activate certain pathways or the expressions of certain genes, which might contribute to bacterial durability and virulence, synergistically. Therefore, exploring how bacterial phenotypes change in response to environmental stresses is important for understanding their potentials in host infections. In this study, we investigated the effects of different concentrations of salt (sodium chloride, NaCl), on survival ability, phenotypes associated with virulence, and energy metabolism of the lab strain Escherichia coli BW25113. In particular, we investigated how NaCl concentrations influenced growth patterns, biofilm formation, oxidative stress resistance, and motile ability. In terms of energy metabolism that is central to bacterial survival, glucose consumption, glycogen accumulation, and trehalose content were measured in order to understand their roles in dealing with the fluctuation of osmolarity. According to the results, trehalose is preferred than glycogen at high NaCl concentration. In order to dissect the molecular mechanisms of NaCl effects on trehalose metabolism, we further checked how the impairment of trehalose synthesis pathway (otsBA operon) via single-gene mutants influenced E. coli durability and virulence under salt stress. After that, we compared the transcriptomes of E. coli cultured at different NaCl concentrations, through which differentially expressed genes (DEGs) and differential pathways with statistical significance were identified, which provided molecular insights into E. coli responses to NaCl concentrations. In sum, this study explored the in vitro effects of NaCl concentrations on E. coli from a variety of aspects and aimed to facilitate our understanding of bacterial physiological changes under salt stress, which might help clarify the linkages between bacterial durability and virulence outside hosts under environmental stresses.

Bacterial survival strategies in sludge alkaline fermentation for volatile fatty acids production: Study on the physiological properties, temporal evolution and spatial distribution of bacterial community.

This study investigated the dynamics of ATP synthase activity, phospholipid fatty acid (PLFA) profile, and temporal evolution and spatial distribution of bacterial community to analyze bacterial survival strategies in sludge alkaline fermentation (SAF) for volatile fatty acids (VFAs) production. The results revealed a significant increase in ATP synthase activity at pH 9 and 10 (p < 0.05), which could contribute to proton entry into cells and benefit bacterial survival. PLFA analysis indicated that the unsaturated fatty acids content increased with the increase of pH. Firmicutes were the dominant microorganisms in the running stage of the pH 10 reactor (35.81-62.34%) and might have been the key microbes that influenced VFAs production. Further analysis of the spatial distribution of microbial community suggested that Firmicutes mainly lived inside flocs during SAF. These findings provide an understanding for bacterial survival strategies in SAF, which could help to develop methods to further improve VFAs yield.

Response of Controlled Cell Load Biofilms to Cold Atmospheric Plasma Jet: Evidence of Extracellular Matrix Contribution.

AIM: Study of the biocidal effect of a cold atmospheric-pressure plasma in ambient air on single-species bacterial biofilms with controlled cell density, characterized by different extracellular matrices. METHODS AND RESULTS: Two bacterial strains were chosen to present different Gram properties and contrasted extracellular matrices: Pseudomonas aeruginosa ATCC 15442 (Gram-negative), and Leuconostoc citreum NRRL B-1299 (Gram-positive). P. aeruginosa biofilm exhibits a complex matrix, rich in proteins while L. citreum presents the specificity to produce glucan-type exopolysaccharides when grown in the presence of sucrose. Plasma was applied on both surface-spread cells and 24-h grown biofilms with controlled cell loads over 5, 10, or 20 min. Surface-spread bacteria showed a time dependent response, with a maximal bacterial reduction of 2.5 log after 20 min of treatment. On the other hand, in our experimental conditions, no bactericidal effect could be observed when treating biofilms of P. aeruginosa and glucan-rich L. citreum. CONCLUSIONS: For biofilms presenting equivalent cell loads, the response to plasma treatment seemed to depend on the properties of the extracellular matrix characterized by infrared spectroscopy, scanning electron microscopy, or dry weight. SIGNIFICANCE AND IMPACT OF STUDY: Both cell load standardization and biofilm characterization are paramount factors to consider the biocide effect of plasma treatments. The extracellular matrix could affect the plasma efficacy by physical and/or chemical protective effects.