The mitochondrial carboxylase PCCA interacts with Listeria monocytogenes phospholipase PlcB to modulate bacterial survival.

Listeria monocytogenes, a prominent foodborne pathogen responsible for zoonotic infections, owes a significant portion of its virulence to the presence of the phospholipase PlcB. In this study, we performed an in-depth examination of the intricate relationship between L. monocytogenes PlcB and host cell mitochondria, unveiling a novel participant in bacterial survival: the mitochondrial carboxylase propionyl-coenzyme A carboxylase (PCCA). Our investigation uncovered previously unexplored levels of interaction and colocalization between PCCA and PlcB within host cells, with particular emphasis on the amino acids 504-508 of PCCA, which play a pivotal role in this partnership. To assess the effect of PCCA expression on L. monocytogenes proliferation, PCCA expression levels were manipulated by siRNA-si-PCCA or pCMV-N-HA-PCCA plasmid transfection. Our findings demonstrated a clear inverse correlation between PCCA expression levels and the proliferation of L. monocytogenes. Furthermore, the effect of L. monocytogenes infection on PCCA expression was investigated by assessing PCCA mRNA and protein expression in HeLa cells infected with L. monocytogenes. These results indicate that L. monocytogenes infection did not significantly alter PCCA expression. These findings led us to propose that PCCA represents a novel participant in L. monocytogenes survival, and its abundance has a detrimental impact on bacterial proliferation. This suggests that L. monocytogenes may employ PlcB-PCCA interactions to maintain stable PCCA expression, representing a unique pro-survival strategy distinct from that of other intracellular bacterial pathogens. IMPORTANCE: Mitochondria represent attractive targets for pathogenic bacteria seeking to modulate host cellular processes to promote their survival and replication. Our current study has uncovered mitochondrial carboxylase propionyl-coenzyme A carboxylase (PCCA) as a novel host cell protein that interacts with L. monocytogenes PlcB. The results demonstrate that PCCA plays a negative regulatory role in L. monocytogenes infection, as heightened PCCA levels are associated with reduced bacterial survival and persistence. However, L. monocytogenes may exploit the PlcB-PCCA interaction to maintain stable PCCA expression and establish a favorable intracellular milieu for bacterial infection. Our findings shed new light on the intricate interplay between bacterial pathogens and host cell mitochondria, while also highlighting the potential of mitochondrial metabolic enzymes as antimicrobial agents.

Corrigendum: Safe and effective delivery of supplemental iron to healthy adults: a two-phase, randomized, double-blind trial - the safe iron study.

[This corrects the article DOI: 10.3389/fnut.2023.1230061.].

Safe and effective delivery of supplemental iron to healthy adults: a two-phase, randomized, double-blind trial - the safe iron study.

Introduction: The safety of novel forms of iron in healthy, iron-replete adults as might occur if used in population-based iron supplementation programs was examined. We tested the hypotheses that supplementation with nanoparticulate iron hydroxide adipate tartrate (IHAT), an iron-enriched Aspergillus oryzae product (ASP), or ferrous sulphate heptahydrate (FS) are safe as indicated by erythrocyte susceptibility to malarial infection, bacterial proliferation, and gut inflammation. Responses to FS administered daily or weekly, and with or without other micronutrients were compared. Methods: Two phases of randomized, double-blinded trials were conducted in Boston, MA. Phase I randomized 160 volunteers to six treatments: placebo, IHAT, ASP, FS, and FS plus a micronutrient powder (MNP) administrated daily at 60 mg Fe/day; and FS administered as a single weekly dose of 420 mg Fe. Phase II randomized 86 volunteers to IHAT, ASP, or FS administered at 120 mg Fe/day. Completing these phases were 151 and 77 participants, respectively. The study was powered to detect effects on primary endpoints: susceptibility of participant erythrocytes to infection by Plasmodium falciparum, the proliferation potential of selected pathogenic bacteria in sera, and markers of gut inflammation. Secondary endpoints for which the study was not powered included indicators of iron status and gastrointestinal symptoms. Results: Supplementation with any form of iron did not affect any primary endpoint. In Phase I, the frequency of gastrointestinal symptoms associated with FS was unaffected by dosing with MNP or weekly administration; but participants taking IHAT more frequently reported abdominal pain (27%, p < 0.008) and nausea (4%, p = 0.009) than those taking FS, while those taking ASP more frequently reported nausea (8%, p = 0.009). Surprisingly, only 9% of participants taking IHAT at 120 mg Fe/day (Phase II) reported abdominal pain and no other group reported that symptom. Discussion: With respect to the primary endpoints, few differences were found when comparing these forms of iron, indicating that 28 days of 60 or 120 mg/day of IHAT, ASP, or FS may be safe for healthy, iron-replete adults. With respect to other endpoints, subjects receiving IHAT more frequently reported abdominal pain and nausea, suggesting the need for further study. Clinical Trial Registration: ClinicalTrials.gov, NCT03212677; registered: 11 July 2017.

Antibacterial activity and biocompatibility of silver coating via aerosol deposition on titanium and zirconia surfaces.

PURPOSE: The purpose of this in vitro study was to investigate the antibacterial effect and biocompatibility of silver coatings via aerosol deposition on titanium and zirconia surfaces. METHODS: The surfaces of titanium and zirconia specimens were polished and coated with silver via aerosol deposition. After silver coating, the elemental composition, surface roughness and amount of silver released from the coated surfaces were measured. The bacterial growth on the silver-coated surfaces was investigated via crystal violet assay after incubation with Streptococcus gordonii for 24 h, Fusobacterium nucleatum for 72 h and Porphyromonas gingivalis for 48 h. Human gingival fibroblasts and mouse preosteoblasts were also cultured on the silver-coated specimens to examine the biocompatibility of the coating. RESULTS: After silver coating via aerosol deposition, the surface roughness increased significantly, and the released silver ranged from 0.067 to 0.110 ppm. The tested bacteria formed significantly less biofilm on the silver-coated titanium surfaces than on the uncoated titanium surfaces. In contrast, biofilm formation on the silver-coated zirconia surfaces was greater than that on the uncoated zirconia surfaces. Human gingival fibroblasts and mouse preosteoblasts proliferated on the silver-coated surfaces without significant differences from the uncoated surfaces. CONCLUSIONS: Silver coating via aerosol deposition provided an antibacterial effect against oral bacteria on titanium surfaces, whereas it promoted more bacterial growth on zirconia surfaces. The proliferation of fibroblasts and osteoblasts was not significantly affected by the silver coating on both titanium and zirconia surfaces.

Polylactide (PLA) as a Cell Carrier in Mesophilic Anaerobic Digestion-A New Strategy in the Management of PLA.

The management of waste polylactide (PLA) in various solutions of thermophilic anaerobic digestion (AD) is problematic and often uneconomical. This paper proposes a different approach to the use of PLA in mesophilic AD, used more commonly on the industrial scale, which consists of assigning the function of a microbial carrier to the biopolymer. The study involved the testing of waste wafers and waste wafers and cheese in a co-substrate system, combined with digested sewage sludge. The experiment was conducted on a laboratory scale, in a batch bioreactor mode. They were used as test samples and as samples with the addition of a carrier: WF-control and WFC-control; WF + PLA and WFC + PLA. The main objective of the study was to verify the impact of PLA in the granular (PLAG) and powder (PLAP) forms on the stability and efficiency of the process. The results of the analysis of physicochemical properties of the carriers, including the critical thermal analysis by differential scanning calorimetry (DSC), as well as the amount of cellular biomass of Bacillus amyloliquefaciens obtained in a culture with the addition of the tested PLAG and PLAP, confirmed that PLA can be an effective cell carrier in mesophilic AD. The addition of PLAG produced better results for bacterial proliferation than the addition of powdered PLA. The highest level of dehydrogenase activity was maintained in the WFC + PLAG system. An increase in the volume of the methane produced for the samples digested with the PLA granules carrier was registered in the study. It went up by c.a. 26% for WF, from 356.11 m3 Mg-1 VS (WF-control) to 448.84 m3 Mg-1 VS (WF + PLAG), and for WFC, from 413.46 m3 Mg-1 VS, (WFC-control) to 519.98 m3 Mg-1 VS (WFC + PLAG).

The grazing activity of Acrobeloides sp. drives phytate mineralisation within its trophic relationship with bacteria.

The microbial loop has been suggested as an alternative route for better utilization of phytate, a poorly available P source to plants. We hypothesized that bacterial grazer activity might dramatically enhance bacterial migration and proliferation, increasing the probability of phytate hydrolysis by bacterial phytases and, thus, phytate mineralization and release of free phosphate. We tested this hypothesis in a two-compartment system with a solid medium containing phytate or free phosphate as the source of P. Two bacterial species, B. subtilis 168 or Bradyrhizobium sp., with or without bacterial grazing nematodes belonging to Acrobeloides sp. previously fed on each of the bacterial species, were inoculated at a single point in the medium. Whatever the P source, nematode migration within both zones allowed the proliferation of bacteria. However, B. subtilis 168 was more efficient in using phytate than Bradyrhizobium sp. since the highest bacterial cell density and free phosphate concentrations were reached by Acrobeloides sp. fed on B. subtilis 168. The grazer activity seemed to be crucial to enhance phytate mineralization, despite Acrobeloides sp. showing a higher preference to feed on Bradyrhizobium sp. This study provides new insights into the effects of bacterial grazer activity on phytate mineralization.

Metallic nanoparticles induced antibiotic resistance genes attenuation of leachate culturable microbiota: The combined roles of growth inhibition, ion dissolution and oxidative stress.

The dissemination and propagation of antibiotic resistance genes (ARGs) is an emerging global health concern, and the potential effects of nanomaterials on ARGs fates have drawn much attention recently. In the current study, the effects of metallic nanoparticles on ARGs occurrence of leachate culturable microbiota were investigated by four typical metal and metal oxide nanoparticles (Cu, Zn, CuO, and ZnO). The ARGs diversity was remarkably decreased during the cultivation and enrichment of leachate microbiota, and their abundances decreased for 1.4-3.2 orders of magnitude. The presence of nanoparticles facilitated the ARGs attenuation, and the magnitude of effects depended on types of nanoparticles and ARGs. Metal oxide nanoparticles caused more remarkable effects than metal nanoparticles. Mechanism analysis indicated that bacterial growth was inhibited, and the dissolved metal ions from nanoparticles partially contributed to nanoparticles decreasing ARGs. Flow cytometry experiments further confirmed that nanoparticles could enter bacterial cells, and then induce excessive reactive oxygen species (ROS) generation and increase membrane permeability. Finally, the possible mechanisms were put forward, and the structural equation models (SEM) differentiated the contribution of different factors shaping ARGs. The dissolved metal ions and growth inhibition caused by nanoparticles decreased ARGs transfer frequencies via exerting excessive metal stress and lowering population density. On the other hand, nanoparticles were incorporated into the cells, and then induced the generation of ROS, which might facilitate ARGs horizontal transfer via increasing membrane permeability, or decrease ARGs via the damage of genomic and plasmid DNA. Therefore, nanoparticles could affect ARGs fates via several ways, and combined effects finally determined the ARGs variations.

Extending the 30-minute rule for red cell units - investigation of the bacterial risk of 60-minute exposures to ambient temperature.

BACKGROUND AND OBJECTIVES: In the UK, a significant proportion of red cell units is discarded due to the 30-min rule governing out of temperature control. Studies have shown that repeated warming to ambient temperature has little impact on red cell quality or bacterial growth. We aimed to validate extension of the rule to 60 minutes by investigation of repeated same, and different, day exposures on bacterial growth. MATERIALS AND METHODS: Red cell units were seeded individually at 100-1000 cfu/ml with Yersinia enterocolitica, Serratia liquefaciens, Pseudomonas putida, Staphylococcus epidermidis, Enterobacter cloacae and Bacillus cereus. Test units were exposed to 30°C for 30 or 60 min on a single occasion at days 15, 17 and 21, or thrice on day 15 of a 35-day storage period. A 10-fold increase in bacterial counts in tests versus controls maintained in cold storage was considered indicative of significant bacterial proliferation. RESULTS: Exposure of units to 30°C for up to 60 min had no substantial impact on the growth of bacteria and all mesophiles declined steadily in tests and controls. Only P. putida showed a near significant elevation in count on exposure for 60 min at day 35. CONCLUSIONS: Extension of the out of temperature rule for red cells to 60 min will potentially not compromise patient safety, although exposures to ambient temperatures should be minimized. Units returned to storage must not be reissued for at least 6 hours and not be exposed to ambient temperatures on more than three occasions.

Safe and effective delivery of supplemental iron to healthy older adults: The double-blind, randomized, placebo-controlled trial protocol of the Safe Iron Study.

The forms of iron currently available to correct iron deficiency have adverse effects, including infectious diarrhea, increased susceptibility to malaria, inflammation and detrimental changes to the gut microbiome. These adverse effects limit their use such that the growing burden of iron deficiency has not abated in recent decades. Here, we summarize the protocol of the "Safe Iron Study", the first clinical study examining the safety and efficacy of novel forms of iron in healthy, iron-replete adults. The Safe Iron Study is a double-blind, randomized, placebo-controlled trial conducted in Boston, MA, USA. This study compares ferrous sulfate heptahydrate (FeSO 4·H 2O) with two novel forms of iron supplements (iron hydroxide adipate tartrate (IHAT) and organic fungal iron metabolite (Aspiron™ Natural Koji Iron)). In Phase I, we will compare each source of iron administrated at a low dose (60 mg Fe/day). We will also determine the effect of FeSO 4 co-administrated with a multiple micronutrient powder and weekly administration of FeSO 4. The forms of iron found to produce no adverse effects, or adverse effects no greater than FeSO 4 in Phase I, Phase II will evaluate a higher, i.e., a therapeutic dose (120 mg Fe/day). The primary outcomes of this study include ex vivo malaria ( Plasmodium falciparum) infectivity of host erythrocytes, ex vivo bacterial proliferation (of selected species) in presence of host plasma and intestinal inflammation assessed by fecal calprotectin. This study will test the hypotheses that the novel forms of iron, administered at equivalent doses to FeSO 4, will produce similar increases in iron status in iron-replete subjects, yet lower increases in ex vivo malaria infectivity, ex vivo bacterial proliferation, gut inflammation. Ultimately, this study seeks to contribute to development of safe and effective forms of supplemental iron to address the global burden of iron deficiency and anemia. Registration: ClinicalTrials.gov identifier: NCT03212677; registered: 11 July 2017.

The potential of Loxosceles gaucho spider venom to regulate Pseudomonas aeruginosa mechanisms of virulence.

Loxosceles venom is a potential source of bioactive molecules which may be transformed into antimicrobial products against multi-resistant bacteria. Here, it was investigated whether Loxosceles gaucho spider had any influence on the proliferation, enzyme release and biofilm formation of a Pseudomonas aeruginosa strain resistant to two different classes of antibiotic. The results demonstrated that L. gaucho whole venom has no influence on P. aeruginosa proliferation. However, it increases P. aeruginosa production of gelatinase, caseinase and biofilm formation. The same effects were noted when P. aeruginosa was exposed to a L. gaucho venom molecular fraction with mass lower than 1 kDa. Separation of this molecular fraction into different subsets by RP-HPLC demonstrated that, among the molecules with the ability to increase the production of enzymes and biofilm formation, there are some with antimicrobial activities whose effects are not observed in the whole venom. In summary, the results obtained herein indicate that L. gaucho venom has a variety of low molecular mass bioactive components that influence the mechanisms of virulence of P. aeruginosa in different ways.

The potential of Loxosceles gaucho spider venom to regulate Pseudomonas aeruginosa mechanisms of virulence

Loxosceles venom is a potential source of bioactive molecules which may be transformed into antimicrobial products against multi-resistant bacteria. Here, it was investigated whether Loxosceles gaucho spider had any influence on the proliferation, enzyme release and biofilm formation of a Pseudomonas aeruginosa strain resistant to two different classes of antibiotic. The results demonstrated that L. gaucho whole venom has no influence on P. aeruginosa proliferation. However, it increases P. aeruginosa production of gelatinase, caseinase and biofilm formation. The same effects were noted when P. aeruginosa was exposed to a L. gaucho venom molecular fraction with mass lower than 1 kDa. Separation of this molecular fraction into different subsets by RP-HPLC demonstrated that, among the molecules with the ability to increase the production of enzymes and biofilm formation, there are some with antimicrobial activities whose effects are not observed in the whole venom. In summary, the results obtained herein indicate that L. gaucho venom has a variety of low molecular mass bioactive components that influence the mechanisms of virulence of P. aeruginosa in different ways.

Impact of dust exposure on mixed bacterial cultures and during eukaryotic cell co-culture infections.

On a daily basis, humans, and their colonizing microbiome, are exposed to both indoor and outdoor dust, containing both deleterious organic and inorganic contaminants, through dermal contact, inhalation, and ingestion. Recent studies evaluating the dust exposure responses of opportunistic pathogens, such as Escherichia coli and Pseudomonas aeruginosa, revealed significant increases in biofilm formation following dust exposure. In this study, the effects of dust exposure on mixed bacterial cultures as well as HT-29 co-cultures were evaluated. As it was observed in pure, single bacterial cultures earlier, neither indoor nor outdoor dust exposure (at concentrations of 100 µg/mL) influenced the growth of mixed bacterial liquid cultures. However, when in paired mixed cultures, dust exposure increased sensitivity to oxidative stress and significantly enhanced biofilm formation (outdoor dust). More specifically, mixed cultures (E. coli-Klebsiella pneumoniae, K. pneumoniae-P. aeruginosa, and E. coli-P. aeruginosa) exhibited increased sensitivity to 20 and 50 mM of H2O2 in comparison to their pure, single bacterial culture counterparts and significantly enhanced biofilm production for each mixed culture. Finally, bacterial proliferation during a eukaryotic gut cell (HT29) co-culture was significantly more robust for both K. pneumoniae and P. aeruginosa when exposed to both house and road dust; however, E. coli only experienced significantly enhanced proliferation, in HT29 co-culture, when exposed to road dust. Taken together, our findings demonstrate that bacteria respond to dust exposure differently when in the presence of multiple bacterial species or when in the presence of human gut epithelial cells, than when grown in isolation.

Hydrolysates of glycated and heat-treated peanut 7S globulin (Ara h 1) modulate human gut microbial proliferation, survival and adhesion.

AIMS: Evaluation of an effect of glycation of Ara h 1 on proliferation and survival rate and adhesion of intestinal Enterococcus faecalis, Escherichia coli and Lactobacillus acidophilus. METHODS AND RESULTS: Pure Ara h 1 heated at three different temperature conditions (G37, G60 and C145°C) in the presence or absence of glucose was subjected to enzymatic hydrolysis. Impacts of Ara h 1 hydrolysates on the bacterial proliferation, survival rate and adhesion to Caco-2 cells in mono and heterogeneous cultures were studied with fluorescent techniques: DAPI, LIVE/DEAD staining and FISH. Examined hydrolysates hindered proliferation of E. coli and Ent. faecalis with simultaneous decrease in their survival. Maillard reaction (MR, glycation) of Ara h 1 did not alter the effect of hydrolysates on bacterial proliferation rate. Hydrolysates modified at 60 and 145°C with glucose altered the profile of immobilized bacteria, mostly by lowering the number of adhering E. coli and promoting the adhesion of bacteria from genera Lactobacillus and Enterococcus. CONCLUSIONS: Ara h1 hydrolysates processed in various ways demonstrated their strong modulatory effect on bacterial proliferation, survival rate and adhesion. SIGNIFICANCE AND IMPACT OF THE STUDY: Reducing the adhesion of opportunistic bacteria by hydrolysates of Ara h 1 glycated at 60 and 145°C, together with modulation of immobilization of beneficial lactobacilli and enterococci, may be of relevance in terms of the physiological status of the intestinal barrier.