In this study, the possibility of an auto-aggregating bacterium Pseudomonas strain XL-2 with heterotrophic nitrification-aerobic denitrification capacity for improving granulation and nitrogen removal was evaluated. The results showed that the supplementation of strain XL-2 promoted granulation, making R1 (experimental group with strain XL-2) dominated by granules at 14 d, which was 12 days earlier than R2 (control group without strain XL-2). This was attributed to the promotion of extracellular polymeric substances (EPS) secretion, particularly proteins by adding strain XL-2, thereby improving the hydrophobicity of sludge and altering the proteins secondary structures to facilitate aggregation. Meanwhile, adding strain XL-2 improved simultaneous nitrification and denitrification efficiency of R1. Microbial community analysis indicated that strain XL-2 successfully proliferated in aerobic granule sludge and might induce the enrichment of genera such as Flavobacterium and Paracoccus that were favorable for EPS secretion and denitrification, jointly promoting granulation and enhancing nitrogen removal efficiency.

Pathogenic bacterial biofilms present significant challenges, particularly in food safety and material deterioration. Therefore, using Enterococcus mundtii A2, known for its antagonistic activity against pathogen adhesion, could serve as a novel strategy to reduce pathogenic colonization within the food sector. This study aimed to investigate the biofilm-forming ability of E. mundtii A2, isolated from camel milk, on two widely used stainless steels within the agri-food domain and to assess its anti-adhesive properties against various pathogens, especially on stainless steel 316L. Additionally, investigations into auto-aggregation and co-aggregation were also conducted. Plate count methodologies revealed increased biofilm formation by E. mundtii A2 on 316L, followed by 304L. Scanning electron microscopy (SEM) analysis revealed a dense yet thin biofilm layer, playing a critical role in reducing the adhesion of L. monocytogenes CECT 4032 and Staphylococcus aureus CECT 976, with a significant reduction of ≈ 2 Log CFU/cm2. However, Gram-negative strains, P. aeruginosa ATCC 27853 and E. coli ATCC 25922, exhibit modest adhesion reduction (~ 0.7 Log CFU/cm2). The findings demonstrate the potential of applying E. mundtii A2 biofilms as an effective strategy to reduce the adhesion and propagation of potentially pathogenic bacterial species on stainless steel 316L.

Clostridioides difficile (C. difficile) is responsible for one of the most common nosocomial infections worldwide. This work assessed associations between biofilm-formation capacity levels of C. difficile and cell viability, motility, flagella, motility and auto-aggregation in 118 clinical isolates. Biofilm production was assessed by the crystal violet method. Cell viability was determined by BacTiter-Glo™ Microbial Cell Viability Assay and live-imaging microscopy. Expression levels of LuxS, Cwp84, Spo0A, PilA, and FliC were measured by real-time PCR. Motility was visually assessed in agar tubes. Auto-aggregation levels were determined by OD600 measurements. Out of 118 isolates, 66 (56 %) were biofilm producers, with most being strong or moderate producers. Cell viability, motility and auto-aggregation positively correlated with biofilm-production capacity (p = 0.0001, p = 0.036 and p < 0.0001, respectively). Positive associations were found between pilA, fliC and luxS expression levels and biofilm-production capacity (p = 0.04, p = 0.01, p = 0.036, respectively). This is the first report of associations between biofilm-formation capacity and cell viability, pilA, fliC, and luxS gene expression, auto-aggregation and motility. These correlations should be further explored to expand knowledge on the regulation of C. difficile biofilm formation, and pathogenesis, which will have notable implications on treatment options.

The properties of intestinal bacteria/probiotics, such as cell surface hydrophobicity (CSH), auto-aggregation, and biofilm formation ability, play an important role in shaping the relationship between the bacteria and the host. The current study aimed to investigate the cell surface properties of fish intestinal bacteria and probiotics. Microbial adhesion to hydrocarbons was tested according to Kos and coauthors. The aggregation abilities of the investigated strains were studied as described by Collado and coauthors. The ability of bacterial isolates to form a biofilm was determined by performing a qualitative analysis using crystal violet staining based on the attachment of bacteria to polystyrene. These studies prove that bacterial cell surface hydrophobicity (CSH) is associated with the growth medium, and the effect of the growth medium on CSH is species-specific and likely also strain-specific. Isolates of intestinal lactobacilli from fish (Salmo ischchan) differed from isolates of non-fish/shrimp origin in the relationship between auto-aggregation and biofilm formation. Average CSH levels for fish lactobacilli and E. coli might were lower compared to those of non-fish origin, which may affect the efficiency of non-fish probiotics use in fisheries due to the peculiarities of the hosts\' aquatic lifestyles.

The study aimed to evaluate the effect of thermal inactivation of potentially probiotic lactic acid bacteria (LAB) strains isolated from food on their ability to compete with pathogenic microorganisms. Five strains of LAB, previously isolated from food and characterized, one commercial reference strain of Lactiplantibacillus plantarum 299v, and two indicator strains of Staphylococcus aureus 25923 and Listeriamonocytogenes 15313 were used in the study. The experiment consisted in applying a stress factor (high temperature: 80 °C, at a different time: 5, 15, and 30 min) to the tested LAB cells to investigate the in vitro properties such as hydrophobicity abilities (against p-xylene and n-hexadecane), auto-aggregation, co-aggregation with pathogens, and inhibition of pathogens adhesion to the porcine gastric mucin. The bacterial strains showed various hydrophobicity to p-xylene (36-73%) and n-hexadecane (11-25%). The affinity for solvents expanded with increasing thermal inactivation time. All LAB isolates were able to auto-aggregate (ranging from 17 to 49%). Bacterial strains subjected to 5 and 15 min of thermal inactivation had the highest auto-aggregation ability in comparison to viable and heat-killed cells for 30 min. The LAB strains co-aggregated with pathogens to different degrees; among them, the highest scores of co-aggregation were observed for L. monocytogenes, reaching 27% (with 15 min of heat-killed LAB cells). All LAB strains reduced the adherence of pathogenic bacteria in the competition test, moreover, heat-killed cells (especially 15 min inactivated) were more efficient than viable cells. The properties of selected LAB strains as moderately heat-stressed forms analyzed in the study increased the prevention of colonization and elimination of pathogenic bacteria in the in vitro model of gastrointestinal tract. The thermal inactivation process may therefore preserve and modifies some characteristics of bacterial cells.

The impact of high concentrations of heavy metals and the loss of functional microorganisms usually affect the nitrogen removal process in wastewater treatment systems. In the study, a unique auto-aggregating aerobic denitrifier (Pseudomonas stutzeri strain YC-34) was isolated with potential applications for Cr(VI) biosorption and reduction. The nitrogen removal efficiency and denitrification pathway of the strain were determined by measuring the concentration changes of inorganic nitrogen during the culture of the strain and amplifying key denitrification functional genes. The changes in auto-aggregation index, hydrophobicity index, and extracellular polymeric substances (EPS) characteristic index were used to evaluate the auto-aggregation capacity of the strain. Further studies on the biosorption ability and mechanism of cadmium in the process of denitrification were carried out. The changes in tolerance and adsorption index of cadmium were measured and the micro-characteristic changes on the cell surface were analyzed. The strain exhibited excellent denitrification ability, achieving 90.58% nitrogen removal efficiency with 54 mg/L nitrate-nitrogen as the initial nitrogen source and no accumulation of ammonia and nitrite-nitrogen. Thirty percentage of the initial nitrate-nitrogen was converted to N2, and only a small amount of N2O was produced. The successful amplification of the denitrification functional genes, norS, norB, norR, and nosZ, further suggested a complete denitrification pathway from nitrate to nitrogen. Furthermore, the strain showed efficient aggregation capacity, with the auto-aggregation and hydrophobicity indices reaching 78.4 and 75.5%, respectively. A large amount of protein-containing EPS was produced. In addition, the strain effectively removed 48.75, 46.67, 44.53, and 39.84% of Cr(VI) with the initial concentrations of 3, 5, 7, and 10 mg/L, respectively, from the nitrogen-containing synthetic wastewater. It also could reduce Cr(VI) to the less toxic Cr(III). FTIR measurements and characteristic peak deconvolution analysis demonstrated that the strain had a robust hydrogen-bonded structure with strong intermolecular forces under the stress of high Cr(VI) concentrations. The current results confirm that the novel denitrifier can simultaneously remove nitrogen and chromium and has potential applications in advanced wastewater treatment for the removal of multiple pollutants from sewage.

The coexistence of multiple pollutants has become a distinctive feature of water pollution. However, there are a few strains that can remove nitrate and tetracycline (TC). Here, the efficiency of strain XS-18 in removing nitrate and TC was analyzed, and the mechanism of tolerance and removal of TC was investigated by infrared spectroscopy, three-dimensional fluorescence spectroscopy, and genome analysis. XS-18 could efficiently remove TC (0.40 mg·L-1·h-1) at pH 7.0-11.0 with auto-aggregation. TC was removed via extracellular polymeric substance (EPS) (55.90%) and cell surface (44.10%) adsorption. TC (10 mg/L) could stimulate XS-18 to secrete more polysaccharides and hydrophobic proteins to improve its auto-aggregation ability. The findings also confirmed that TC resistance genes were present. Furthermore, the bacterial flagellum, signal transduction of the chemotactic system and regulatory genes were shown to be related to the auto-aggregation of the strain. XS-18 has potential applications in the treatment of wastewater containing nitrate and TC.

High concentrations of heavy metals and other pollutants affect microbial activity in the wastewater treatment system and impede biological denitrification process. In this study, a novel Zn(II)-resistant aerobic denitrifier (Pseudomonas stutzeri KY-37) was isolated with potential in Bisphenol A (BPA) biodegradation and removal. The capability of this denitrifier in removing nitrogen, zinc, and BPA was tested. Using 56 mg/L nitrate as the sole nitrogen source, its removal efficiency achieved 98.5% in 12 h. This novel denitrifier had a strong auto-aggregation (maximum 65.8%), a high hydrophobicity rate (maximum 88.2%), and a massive amount (maximum 41.1 mg/g cell dry weight) of extracellular polymeric substances (EPS) production. Moreover, Zn(II) removal efficiency reached more than 95% with the initial high concentrations of 200 mg/L. The maximum BPA removal efficiency reached 88.8% with initial 10 mg/L. The removal mechanism of BPA was further explored in terms of microbial degradation, EPS adsorption, and intermediate degradation products.

This study was designed to evaluate the abilities of phage P22 to lyse, eradiate, and disperse the biofilm cells of Salmonella enterica serovar Typhimurium ATCC 19585 (STWT), ciprofloxacin-induced Typhimurium ATCC 19585 (STCIP), S. Typhimurium KCCM 40253 (STKCCM), and multidrug-resistant S. Typhimurium CCARM 8009 (STCCARM) in association with hydrophobicity, auto-aggregation, motility, protein content, extracellular DNA, and depolymerase activity. The affinity to hexadecane was significantly increased in STWT, STKCCM, and STCCARM cells after P22 infection. All strains tested showed relatively higher auto-aggregation abilities in the presence of P22 than the absence of P22. STKCCM showed the greatest auto-aggregative ability (23%) in the presence of P22, while STWT showed the least auto-aggregative ability (9%) in the absence of P22. The bacterial swimming motility affected the bacterial attachment at the early stage of biofilm formation. The red, dry and rough morphotype was observed for all strains tested. The numbers of STWT, STCIP, and STKCCM planktonic cells were considerably reduced by 7.2, 5.0, and 5.0 log CFU/ml, respectively, and STWT, STCIP, and STKCCM biofilm-forming cells were reduced by 5.8, 4.5, and 4.9 log, respectively, after 24 h of phage infection. The depolymerase produced by phages were confirmed by the presence of outer rim of plaques. Phages could be considered as promising alternatives for the control of biofilms due to their advantages including enzymatic degradation of extracellular biofilm matrix. The study would provide useful information for understanding the dynamic interactions between phages and biofilms and also designing the effective phage-based control system as an alterative strategy against biofilms.

Auto-aggregation is a desired property for probiotic strains because it is suggested to promote colonization of the human intestine, to prevent pathogen infections and to modulate the colonic mucosa. We recently reported the generation of adapted mutants of Lactiplantibacillus plantarum NZ3400, a derivative of the model strain WCFS1, for colonization under adult colonic conditions of PolyFermS continuous intestinal fermentation models. Here we describe and characterize the emerge of an auto-aggregating phenotype in L. plantarum NZ3400 derivatives recovered from the modelled gut microbiota.
L. plantarum isolates were recovered from reactor effluent of four different adult microbiota and from spontaneously formed reactor biofilms. Auto-aggregation was observed in L. plantarum recovered from all microbiota and at higher percentage when recovered from biofilm than from effluent. Further, auto-aggregation percentage increased over time of cultivation in the microbiota. Starvation of the gut microbiota by interrupting the inflow of nutritive medium enhanced auto-aggregation, suggesting a link to nutrient availability. Auto-aggregation was lost under standard cultivation conditions for lactobacilli in MRS medium. However, it was reestablished during growth on sucrose and maltose and in a medium that simulates the abiotic gut environment. Remarkably, none of these conditions resulted in an auto-aggregation phenotype in the wild type strain NZ3400 nor other non-aggregating L. plantarum, indicating that auto-aggregation depends on the strain history. Whole genome sequencing analysis did not reveal any mutation responsible for the auto-aggregation phenotype. Transcriptome analysis showed highly significant upregulation of LP_RS05225 (msa) at 4.1-4.4 log2-fold-change and LP_RS05230 (marR) at 4.5-5.4 log2-fold-change in all auto-aggregating strains compared to non-aggregating. These co-expressed genes encode a mannose-specific adhesin protein and transcriptional regulator, respectively. Mapping of the RNA-sequence reads to the promoter region of the msa-marR operon reveled a DNA inversion in this region that is predominant in auto-aggregating but not in non-aggregating strains. This strongly suggests a role of this inversion in the auto-aggregation phenotype.
L. plantarum NZ3400 adapts to the in vitro colonic environment by developing an auto-aggregation phenotype. Similar aggregation phenotypes may promote gut colonization and efficacy of other probiotics and should be further investigated by using validated continuous models of gut fermentation such as PolyFermS.