Combat phytopathogenic bacteria employing Argirium-SUNCs: limits and perspectives.

Bacterial plant diseases are difficult to control as the durability of deployed control measures is thwarted by continuous and rapid changing of bacterial populations. Although application of copper compounds to plants is the most widespread and inexpensive control measure, it is often partially efficacious for the frequent appearance of copper-resistant bacterial strains and it is raising concerns for the harmful effects of copper on environment and human health. Consequently, European Community included copper compounds in the list of substances candidates for substitution. Nanotechnologies and the application of nanoparticles seem to respond to the need to find new very effective and durable measures. We believe that Argirium-SUNCs®, silver ultra nanoclusters with an average size of 1.79 nm and characterized by rare oxidative states (Ag2+/3+), represent a valid candidate as a nano-bactericide in the control of plant bacterial diseases. Respect to the many silver nanoparticles described in the literature, Argirium-SUNCs have many strengths due to the reproducibility of the synthesis method, the purity and the stability of the preparation, the very strong (less than 1 ppm) antimicrobial, and anti-biofilm activities. In this mini-review, we provide information on this nanomaterial and on the possible application in agriculture. KEY POINTS: • Argirium-SUNCs have strong antimicrobial activities against phytopathogenic bacteria. • Argirium-SUNCs are a possible plant protection product. • Argirium-SUNCs protect tomato plants against bacterial speck disease.

Characterization of Pseudomonas spp. contamination and in situ spoilage potential in pasteurized milk production process.

To investigate the prevalence of Pseudomonas in the pasteurized milk production process and its effect on milk quality, 106 strains of Pseudomonas were isolated from the pasteurized milk production process of a milk production plant in Shaanxi Province, China. The protease, lipase and biofilm-producing capacities of the 106 Pseudomonas strains were evaluated, and the spoilage enzyme activities of their metabolites were assessed by simulating temperature incubation in the refrigerated (7 °C) and transport environment (25 °C) segments and thermal treatments of pasteurization (75 °C, 5 min) and ultra-high temperature sterilization (121 °C, 15 s). A phylogenetic tree was drawn based on 16S rDNA gene sequencing and the top 5 strains were selected as representative strains to identify their in situ spoilage potential by examining their growth potential and ability to hydrolyze proteins and lipids in milk using growth curves, pH, whiteness, Zeta-potential, lipid oxidation, SDS-PAGE and volatile flavor compounds. The results showed that half and more of the isolated Pseudomonas had spoilage enzyme production and biofilm capacity, and the spoilage enzyme activity of metabolites was affected by the culture temperature and sterilization method, but ultra-high temperature sterilization could not completely eliminate the enzyme activity. The growth of Pseudomonas lundensis and Pseudomonas qingdaonensis was less affected by temperature and time, and the hydrolytic capacity of extracellular protease and lipase secreted by Pseudomonas lurida was the strongest, which had the greatest effect on milk quality. Therefore, it is crucial to identify the key contamination links of Pseudomonas, the main bacteria responsible for milk spoilage, and the influence of environmental factors on its deterioration.

Vitamin D and vitamin K1 as novel inhibitors of biofilm in Gram-negative bacteria.

BACKGROUND: The persistent surge in antimicrobial resistance represents a global disaster. The initial attachment and maturation of microbial biofilms are intimately related to antimicrobial resistance, which in turn exacerbates the challenge of eradicating bacterial infections. Consequently, there is a pressing need for novel therapies to be employed either independently or as adjuvants to diminish bacterial virulence and pathogenicity. In this context, we propose a novel approach focusing on vitamin D and vitamin K1 as potential antibiofilm agents that target Gram-negative bacteria which are hazardous to human health. RESULTS: Out of 130 Gram-negative bacterial isolates, 117 were confirmed to be A. baumannii (21 isolates, 17.9%), K. pneumoniae (40 isolates, 34.2%) and P. aeruginosa (56 isolates, 47.9%). The majority of the isolates were obtained from blood and wound specimens (27.4% each). Most of the isolates exhibited high resistance rates to ß-lactams (60.7-100%), ciprofloxacin (62.5-100%), amikacin (53.6-76.2%) and gentamicin (65-71.4%). Approximately 93.2% of the isolates were biofilm producers, with 6.8% categorized as weak, 42.7% as moderate, and 50.4% as strong biofilm producers. The minimum inhibitory concentrations (MICs) of vitamin D and vitamin K1 were 625-1250 µg mL-1 and 2500-5000 µg mL-1, respectively, against A. baumannii (A5, A20 and A21), K. pneumoniae (K25, K27 and K28), and P. aeruginosa (P8, P16, P24 and P27) clinical isolates and standard strains A. baumannii (ATCC 19606 and ATCC 17978), K. pneumoniae (ATCC 51503) and P. aeruginosa PAO1 and PAO14. Both vitamins significantly decreased bacterial attachment and significantly eradicated mature biofilms developed by the selected standard and clinical Gram-negative isolates. The anti-biofilm effects of both supplements were confirmed by a notable decrease in the relative expression of the biofilm-encoding genes cusD, bssS and pelA in A. baumannii A5, K. pneumoniae K28 and P. aeruginosa P16, respectively. CONCLUSION: This study highlights the anti-biofilm activity of vitamins D and K1 against the tested Gram-negative strains, which emphasizes the potential of these vitamins for use as adjuvant therapies to increase the efficacy of treatment for infections caused by multidrug-resistant (MDR) strains and biofilm-forming phenotypes. However, further validation through in vivo studies is needed to confirm these promising results.

Host-Microbiome Crosstalk in Chronic Wound Healing.

The pathogenesis of chronic wounds (CW) involves a multifaceted interplay of biochemical, immunological, hematological, and microbiological interactions. Biofilm development is a significant virulence trait which enhances microbial survival and pathogenicity and has various implications on the development and management of CW. Biofilms induce a prolonged suboptimal inflammation in the wound microenvironment, associated with delayed healing. The composition of wound fluid (WF) adds more complexity to the subject, with proven pro-inflammatory properties and an intricate crosstalk among cytokines, chemokines, microRNAs, proteases, growth factors, and ECM components. One approach to achieve information on the mechanisms of disease progression and therapeutic response is the use of multiple high-throughput 'OMIC' modalities (genomic, proteomic, lipidomic, metabolomic assays), facilitating the discovery of potential biomarkers for wound healing, which may represent a breakthrough in this field and a major help in addressing delayed wound healing. In this review article, we aim to summarize the current progress achieved in host-microbiome crosstalk in the spectrum of CW healing and highlight future innovative strategies to boost the host immune response against infections, focusing on the interaction between pathogens and their hosts (for instance, by harnessing microorganisms like probiotics), which may serve as the prospective advancement of vaccines and treatments against infections.

Escherichia coli isolated from pyometra and cystitis in the same animal exhibit a wide phenotypic similarity.

AIMS: Pyometra and cystitis caused by Escherichia coli are common diseases identified in canine or feline females. The origin of pyometra infection remains uncertain, and effective prevention strategies for this disease are still unknown. This study aimed to provide a phenotypic characterization, including antimicrobial resistance and virulence profiles, of endometrial pathogenic (EnPEC) and uropathogenic (UPEC) E. coli strains isolated simultaneously from the same animal. METHODS AND RESULTS: Sixteen E. coli strains, from eight different animals, were analyzed in this study. The antimicrobial susceptibility profile of EnPEC and UPEC strains was determined using the disc diffusion method, which showed a similar susceptibility profile among strains (EnPEC and UPEC) from the same animal. The virulence profile of the strains was assessed through biofilm formation, as well as serum resistance abilities. EnPEC and UPEC strains from the same animal exhibited slight variations in their virulence and antimicrobial resistance capabilities. Overall, most of the strain pairs showed a high similarity in their ability to establish biofilms and survive in serum complement activity. CONCLUSIONS: Overall, strains of E. coli isolated from both pyometra and cystitis in the same animal, despite presenting distinct clinical diseases, exhibit a wide phenotypic similarity, suggesting a common origin for the strains.

Deletion of codY impairs Staphylococcus epidermidis biofilm formation, generation of viable but non-culturable cells and stimulates cytokine production in human macrophages.

Introduction. Staphylococcus epidermidis biofilms are one of the major causes of bloodstream infections related to the use of medical devices. The diagnosis of these infections is challenging, delaying their treatment and resulting in increased morbidity and mortality rates. As such, it is urgent to characterize the mechanisms employed by this bacterium to endure antibiotic treatments and the response of the host immune system, to develop more effective therapeutic strategies. In several bacterial species, the gene codY was shown to encode a protein that regulates the expression of genes involved in biofilm formation and immune evasion. Additionally, in a previous study, our group generated evidence indicating that codY is involved in the emergence of viable but non-culturable (VBNC) cells in S. epidermidis.Gap statement/Hypothesis. As such, we hypothesized that the gene codY has have an important role in this bacterium virulence.Aim. This study aimed to assess, for the first time, the impact of the deletion of the gene codY in S. epidermidis virulence, namely, in antibiotic susceptibility, biofilm formation, VBNC state emergence and in vitro host immune system response.Methodology. Using an allelic replacement strategy, we constructed and then characterized an S. epidermidis strain lacking codY, in regards to biofilm and VBNC cell formation, susceptibility to antibiotics as well as their role in the interaction with human blood and plasma. Additionally, we investigate whether the codY gene can impact the activation of innate immune cells by evaluating the production of both pro- and anti-inflammatory cytokines by THP-1 macrophages.Results. We demonstrated that the deletion of the gene codY resulted in biofilms with less c.f.u. counts and fewer VBNC cells. Furthermore, we show that although WT and mutant cells were similarly internalized in vitro by human macrophages, a stronger cytokine response was elicited by the mutant in a toll-like receptor 4-dependent manner.Conclusion. Our results indicate that codY contributes to S. epidermidis virulence, which in turn may have an impact on our ability to manage the biofilm-associated infections caused by this bacterium.

Exploring the dynamics of mixed-species biofilms involving Candida spp. and bacteria in cystic fibrosis.

Cystic fibrosis (CF) is an inherited disease that results from mutations in the gene responsible for the cystic fibrosis transmembrane conductance regulator (CFTR). The airways become clogged with thick, viscous mucus that traps microbes in respiratory tracts, facilitating colonization, inflammation and infection. CF is recognized as a biofilm-associated disease, it is commonly polymicrobial and can develop in biofilms. This review discusses Candida spp. and both Gram-positive and Gram-negative bacterial biofilms that affect the airways and cause pulmonary infections in the CF context, with a particular focus on mixed-species biofilms. In addition, the review explores the intricate interactions between fungal and bacterial species within these biofilms and elucidates the underlying molecular mechanisms that govern their dynamics. Moreover, the review addresses the multifaceted issue of antimicrobial resistance in the context of CF-associated biofilms. By synthesizing current knowledge and research findings, this review aims to provide insights into the pathogenesis of CF-related infections and identify potential therapeutic approaches to manage and combat these complex biofilm-mediated infections.

Comprehensive strategies for controlling Listeria monocytogenes biofilms on food-contact surfaces.

Listeria monocytogenes biofilms formed on food-contact surfaces within food-processing facilities pose a significant challenge, serving as persistent sources of cross-contamination. In this review, we examined documented cases of foodborne outbreaks and recalls linked to L. monocytogenes contamination on equipment surfaces and in the food production environment, provided an overview of the prevalence and persistence of L. monocytogenes in different food-processing facilities, and discussed environmental factors influencing its biofilm formation. We further delved into antimicrobial interventions, such as chemical sanitizers, thermal treatments, biological control, physical treatment, and other approaches for controlling L. monocytogenes biofilms on food-contact surfaces. This review provides valuable insights into the persistent challenge of L. monocytogenes biofilms in food processing, offering a foundation for future research and practical strategies to enhance food safety.

Biofilms and core pathogens shape the tumor microenvironment and immune phenotype in colorectal cancer.

Extensive research has explored the role of gut microbiota in colorectal cancer (CRC). Nonetheless, metatranscriptomic studies investigating the in situ functional implications of host-microbe interactions in CRC are scarce. Therefore, we characterized the influence of CRC core pathogens and biofilms on the tumor microenvironment (TME) in 40 CRC, paired normal, and healthy tissue biopsies using fluorescence in situ hybridization (FISH) and dual-RNA sequencing. FISH revealed that Fusobacterium spp. was associated with increased bacterial biomass and inflammatory response in CRC samples. Dual-RNA sequencing demonstrated increased expression of pro-inflammatory cytokines, defensins, matrix-metalloproteases, and immunomodulatory factors in CRC samples with high bacterial activity. In addition, bacterial activity correlated with the infiltration of several immune cell subtypes, including M2 macrophages and regulatory T-cells in CRC samples. Specifically, Bacteroides fragilis and Fusobacterium nucleatum correlated with the infiltration of neutrophils and CD4+ T-cells, respectively. The collective bacterial activity/biomass appeared to exert a more significant influence on the TME than core pathogens, underscoring the intricate interplay between gut microbiota and CRC. These results emphasize how biofilms and core pathogens shape the immune phenotype and TME in CRC while highlighting the need to extend the bacterial scope beyond CRC pathogens to advance our understanding and identify treatment targets.

Antimicrobial efficacy of direct air gas soft jet plasma for the in vitro reduction of oral bacterial biofilms.

The aim of this study was to evaluate the antimicrobial efficacy of an air gas soft jet CAP for its potential use in removing oral biofilms, given that plasma-based technologies have emerged as promising methods in periodontology. Two types of biofilms were developed, one by Streptococcus mutans UA 159 bacterial strain and the other by a complex mixture of saliva microorganisms isolated from a patient with periodontitis. This latter biofilm was characterized via Next Generation Sequencing to determine the main bacterial phyla. The CAP source was applied at a distance of 6 mm for different time points. A statistically significant reduction of both CFU count and XTT was already detected after 60 s of CAP treatment. CLSM analysis supported CAP effectiveness in killing the microorganisms inside the biofilm and in reducing the thickness of the biofilm matrix. Cytotoxicity tests demonstrated the possible use of CAP without important side effects towards human gingival fibroblasts cell line. The current study showed that CAP treatment was able to significantly reduce preformed biofilms developed by both S. mutans and microorganisms isolated by a saliva sample. Further studies should be conducted on biofilms developed by additional saliva donors to support the potential of this innovative strategy to counteract oral pathogens responsible for periodontal diseases.

Characterization and therapeutic potential of MRABP9, a novel lytic bacteriophage infecting multidrug-resistant Acinetobacter baumannii clinical strains.

Acinetobacter baumannii is one of the most important pathogens of healthcare-associated infections. The rising prevalence of multidrug-resistant A. baumannii (MRAB) strains and biofilm formation impact the outcome of conventional treatment. Phage-related therapy is a promising strategy to tame troublesome multidrug-resistant bacteria. Here, we isolated and evaluated a highly efficient lytic phage called MRABP9 from hospital sewage. The phage was a novel species within the genus Friunavirus and exhibited lytic activity against 2 other identified MRAB strains. Genomic analysis revealed it was a safe virulent phage and a pectate lyase domain was identified within its tail spike protein. MRABP9 showed potent bactericidal and anti-biofilm activity against MRAB, significantly delaying the time point of bacterial regrowth in vitro. Phage administration could rescue the mice from acute lethal MRAB infection. Considering its features, MRABP9 has the potential as an efficient candidate for prophylactic and therapeutic use against acute infections caused by MRAB strains.

Genetic, virulence, and antimicrobial resistance characteristics associated with distinct morphotypes in ST11 carbapenem-resistant Klebsiella pneumoniae.

ST11 is the most common lineage among carbapenem-resistant Klebsiella pneumoniae (CRKP) infections in Asia. Diverse morphotypes resulting from genetic mutations are associated with significant differences in microbial characteristics among K. pneumoniae isolates. Here, we investigated the genetic determinants and critical characteristics associated with distinct morphotypes of ST11 CRKP. An ST11-KL47 CRKP isolate carrying a pLVPK-like virulence plasmid was isolated from a patient with a bloodstream infection; the isolate had the "mcsw" morphotype. Two distinct morphotypes ("ntrd" and "msdw") were derived from this strain during in vitro passage. Whole genome sequencing was used to identify mutations that cause the distinct morphotypes of ST11 CRKP. Transmission electron microscopy, antimicrobial susceptibility tests, growth assays, biofilm formation, virulence assays, membrane permeability assays, and RNA-seq analysis were used to investigate the specific characteristics associated with different morphotypes of ST11 CRKP. Compared with the parental mcsw morphotype, the ntrd morphotype resulted from mutation of genes involved in capsular polysaccharide biosynthesis (wza, wzc, and wbaP), a result validated by gene knockout experiments. This morphotype showed capsule deficiency and lower virulence potential, but higher biofilm production. By contrast, the msdw morphotype displayed competition deficiency and increased susceptibility to chlorhexidine and polymyxin B. Further analyses indicated that these characteristics were caused by interruption of the sigma factor gene rpoN by insertion mutations and deletion of the rpoN gene, which attenuated membrane integrity presumably by downregulating the phage shock protein operon. These data expand current understanding of genetic, virulence, and antimicrobial resistance characteristics associated with distinct morphotypes in ST11 CRKP.

GlmS plays a key role in the virulence factor expression and biofilm formation ability of Staphylococcus aureus promoted by advanced glycation end products.

Staphylococcus aureus (S. aureus) is well known for its biofilm formation ability and is responsible for serious, chronic refractory infections worldwide. We previously demonstrated that advanced glycation end products (AGEs), a hallmark of chronic hyperglycaemia in diabetic tissues, enhanced biofilm formation by promoting eDNA release via sigB upregulation in S. aureus, contributing to the high morbidity and mortality of patients presenting a diabetic foot ulcer infection. However, the exact regulatory network has not been completely described. Here, we used pull-down assay and LC-MS/MS to identify the GlmS as a candidate regulator of sigB in S. aureus stimulated by AGEs. Dual-luciferase assays and electrophoretic mobility shift assays (EMSAs) revealed that GlmS directly upregulated the transcriptional activity of sigB. We constructed NCTC 8325 ∆glmS for further validation. qRT-PCR analysis revealed that AGEs promoted both glmS and sigB expression in the NCTC 8325 strain but had no effect on NCTC 8325 ∆glmS. NCTC 8325 ∆glmS showed a significant attenuation in biofilm formation and virulence factor expression, accompanied by a decrease in sigB expression, even under AGE stimulation. All of the changes, including pigment deficiency, decreased haemolysis ability, downregulation of hla and hld expression, and less and sparser biofilms, indicated that sigB and biofilm formation ability no longer responded to AGEs in NCTC 8325 ∆glmS. Our data extend the understanding of GlmS in the global regulatory network of S. aureus and demonstrate a new mechanism by which AGEs can upregulate GlmS, which directly regulates sigB and plays a significant role in mediating biofilm formation and virulence factor expression.

Cultivable epiphytic bacteria of the Chlorophyta Ulva sp.: diversity, antibacterial, and biofilm-modulating activities.

AIMS: Macroalgae harbor a rich epiphytic microbiota that plays a crucial role in algal morphogenesis and defense mechanisms. This study aims to isolate epiphytic cultivable microbiota from Ulva sp. surfaces. Various culture media were employed to evaluate a wide range of cultivable microbiota. Our objective was to assess the antibacterial and biofilm-modulating activities of supernatants from isolated bacteria. METHODS AND RESULTS: Sixty-nine bacterial isolates from Ulva sp. were identified based on 16S rRNA gene sequencing. Their antibacterial activity and biofilm modulation potential were screened against three target marine bacteria: 45%, mostly affiliated with Gammaproteobacteria and mainly grown on diluted R2A medium (R2Ad), showed strong antibacterial activity, while 18% had a significant impact on biofilm modulation. Molecular network analysis was carried out on four bioactive bacterial supernatants, revealing new molecules potentially responsible for their activities. CONCLUSION: R2Ad offered the greatest diversity and proportion of active isolates. The molecular network approach holds promise for both identifying bacterial isolates based on their molecular production and characterizing antibacterial and biofilm-modulating activities.

Antimicrobial and anti-biofilm properties of oleuropein against Escherichia coli and fluconazole-resistant isolates of Candida albicans and Candida glabrata.

BACKGROUND: Side effects associated with antimicrobial drugs, as well as their high cost, have prompted a search for low-cost herbal medicinal substances with fewer side effects. These substances can be used as supplements to medicine or to strengthen their effects. The current study investigated the effect of oleuropein on the inhibition of fungal and bacterial biofilm in-vitro and at the molecular level. MATERIALS AND METHODS: In this experimental study, antimicrobial properties were evaluated using microbroth dilution method. The effect of oleuropein on the formation and eradication of biofilm was assessed on 96-well flat bottom microtiter plates and their effects were observed through scanning electron microscopy (SEM). Its effect on key genes (Hwp1, Als3, Epa1, Epa6, LuxS, Pfs) involved in biofilm formation was investigated using the quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) method. RESULTS: The minimum inhibitory concentration (MIC) and minimum fungicidal/bactericidal concentration (MFC/MBC) for oleuropein were found to be 65 mg/ml and 130 mg/ml, respectively. Oleuropein significantly inhibited biofilm formation at MIC/2 (32.5 mg/ml), MIC/4 (16.25 mg/ml), MIC/8 (8.125 mg/ml) and MIC/16 (4.062 mg/ml) (p < 0.0001). The anti-biofilm effect of oleuropein was confirmed by SEM. RT-qPCR indicated significant down regulation of expression genes involved in biofilm formation in Candida albicans (Hwp1, Als3) and Candida glabrata (Epa1, Epa6) as well as Escherichia coli (LuxS, Pfs) genes after culture with a MIC/2 of oleuropein (p < 0.0001). CONCLUSIONS: The results indicate that oleuropein has antifungal and antibacterial properties that enable it to inhibit or destroy the formation of fungal and bacterial biofilm.

Antibacterial activities of Miang extracts against selected pathogens and the potential of the tannin-free extracts in the growth inhibition of Streptococcus mutans.

Bacterial pathogens have remained a major public health concern for several decades. This study investigated the antibacterial activities of Miang extracts (at non-neutral and neutral pH) against Bacillus cereus TISTR 747, Escherichia coli ATCC 22595, Salmonella enterica serovar Typhimurium TISTR 292 and Streptococcus mutans DMST 18777. The potential of Polyvinylpolypyrrolidone (PVPP)-precipitated tannin-free Miang extracts in growth-inhibition of the cariogenic Streptococcus mutans DMST 18777 and its biofilms was also evaluated. The tannin-rich fermented extracts had the best bacterial growth inhibition against S. mutans DMST 18777 with an MIC of 0.29 and 0.72 mg/mL for nonfilamentous fungi (NFP) Miang and filamentous-fungi-processed (FFP) Miang respectively. This observed anti-streptococcal activity still remained after PVPP-mediated precipitation of bioactive tannins especially, in NFP and FFP Miang. Characterization of the PVPP-treated extracts using High performance liquid chromatography quadrupole-time of flight-mass spectrometry (HPLC-QToF-MS) analysis, also offered an insight into probable compound classes responsible for the activities. In addition, Crystal violet-staining also showed better IC50 values for NFP Miang (4.30 ± 0.66 mg/mL) and FFP Miang (12.73 ± 0.11 mg/mL) against S. mutans DMST 18777 biofilms in vitro. Homology modeling and molecular docking analysis using HPLC-MS identified ligands in tannin-free Miang supernatants, was performed against modelled S. mutans DMST 18777 sortase A enzyme. The in silico analysis suggested that the inhibition by NFP and FFP Miang might be attributed to the presence of ellagic acid, flavonoid aglycones, and glycosides. Thus, these Miang extracts could be optimized and explored as natural active pharmaceutical ingredients (NAPIs) for applications in oral hygienic products.

Antibacterial activity of Hungarian varietal honeys against respiratory pathogens as a function of storage time.

Today, antibiotic therapies that previously worked well against certain bacteria due to their natural sensitivity, are becoming less effective. Honey has been proven to inhibit the biofilm formation of some respiratory bacteria, however few data are available on how the storage time affects the antibacterial effect. The activity of black locust, goldenrod, linden and sunflower honeys from three consecutive years (2020, 2021, 2022) was analyzed in 2022 against Gram-negative (Haemophilus influenzae, H. parainfluenzae, Pseudomonas aeruginosa) and Gram-positive (Streptococcus pneumoniae) bacteria using in vitro microbiological methods. After determining the physicochemical parameters of honey, broth microdilution was applied to determine the minimum inhibitory concentration of each honey type against each bacterium, and crystal violet assay was used to test their antibiofilm effect. The possible mechanism of action was explored with membrane degradation test, while structural changes were illustrated with scanning electron microscopy. Honeys stored for one or two years were darker than fresh honeys, while older honeys had significantly lower antibacterial activity. The most remarkable inhibitory effect was exerted by linden and sunflower honeys, and P. aeruginosa proved to be the most resistant bacterium. Based on our results, honey intended for medicinal purposes should be used as fresh as possible during a treatment.

A genetically encoded biosensor to monitor dynamic changes of c-di-GMP with high temporal resolution.

Monitoring changes of signaling molecules and metabolites with high temporal resolution is key to understanding dynamic biological systems. Here, we use directed evolution to develop a genetically encoded ratiometric biosensor for c-di-GMP, a ubiquitous bacterial second messenger regulating important biological processes like motility, surface attachment, virulence and persistence. The resulting biosensor, cdGreen2, faithfully tracks c-di-GMP in single cells and with high temporal resolution over extended imaging times, making it possible to resolve regulatory networks driving bimodal developmental programs in different bacterial model organisms. We further adopt cdGreen2 as a simple tool for in vitro studies, facilitating high-throughput screens for compounds interfering with c-di-GMP signaling and biofilm formation. The sensitivity and versatility of cdGreen2 could help reveal c-di-GMP dynamics in a broad range of microorganisms with high temporal resolution. Its design principles could also serve as a blueprint for the development of similar, orthogonal biosensors for other signaling molecules, metabolites and antibiotics.

Growing a Cystic Fibrosis-Relevant Polymicrobial Biofilm to Probe Community Phenotypes.

Most in vitro models lack the capacity to fully probe bacterial phenotypes emerging from the complex interactions observed in real-life environments. This is particularly true in the context of hard-to-treat, chronic, and polymicrobial biofilm-based infections detected in the airways of individuals living with cystic fibrosis (CF), a multiorgan genetic disease. While multiple microbiome studies have defined the microbial compositions detected in the airway of people with CF (pwCF), no in vitro models thus far have fully integrated critical CF-relevant lung features. Therefore, a significant knowledge gap exists in the capacity to investigate the mechanisms driving the pathogenesis of mixed species CF lung infections. Here, we describe a recently developed four-species microbial community model, including Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus sanguinis, and Prevotella melaninogenica grown in CF-like conditions. Through the utilization of this system, clinically relevant phenotypes such as antimicrobial recalcitrance of several pathogens were observed and explored at the molecular level. The usefulness of this in vitro model resides in its standardized workflow that can facilitate the study of interspecies interactions in the context of chronic CF lung infections.

Wastewater treatment bacteria show differential preference for colonizing natural biopolymers.

Most reduced organic matter entering activated sludge systems is particulate (1-100-µm diameter) or colloidal (0.001-1-µm diameter), yet little is known about colonization of particulate organic matter by activated sludge bacteria. In this study, colonization of biopolymers (chitin, keratin, lignocellulose, lignin, and cellulose) by activated sludge bacteria was compared with colonization of glass beads in the presence and absence of regular nutrient amendment (acetate and ammonia). Scanning electron microscopy and quantitative PCR revealed chitin and cellulose were most readily colonized followed by lignin and lignocellulose, while keratin and glass beads were relatively resistant to colonization. Bacterial community profiles on particles compared to sludge confirmed that specific bacterial phylotypes preferentially colonize different biopolymers. Nitrifying bacteria proved adept at colonizing particles, achieving higher relative abundance on particles compared to bulk sludge. Denitrifying bacteria showed similar or lower relative abundance on particles compared to sludge. KEY POINTS: • Some activated sludge bacteria colonize natural biopolymers more readily than others. • Nitrifying bacteria are overrepresented in natural biopolymer biofilm communities. • Biopolymers in wastewater likely influence activated sludge community composition.