The role of N-acetylcysteine on adhesion and biofilm formation of Candida parapsilosis isolated from catheter-related candidemia.

Objectives. Anti-fungal agents are increasingly becoming less effective due to the development of resistance. In addition, it is difficult to treat Candida organisms that form biofilms due to a lack of ability of drugs to penetrate the biofilms. We are attempting to assess the effect of a new therapeutic agent, N-acetylcysteine (NAC), on adhesion and biofilm formation in Candida parapsilosis clinical strains. Meanwhile, to detect the transcription level changes of adhesion and biofilm formation-associated genes (CpALS6, CpALS7, CpEFG1 and CpBCR1) when administrated with NAC in C. parapsilosis strains, furthermore, to explore the mechanism of drug interference on biofilms.Hypothesis/Gap statement. N-acetylcysteine (NAC) exhibits certain inhibitory effects on adhesion and biofilm formation in C. parapsilosis clinical strains from CRBSIs through: (1) down-regulating the expression of the CpEFG1 gene, making it a highly potential candidate for the treatment of C. parapsilosis catheter-related bloodstream infections (CRBSIs), (2) regulating the metabolism and biofilm -forming factors of cell structure.Methods. To determine whether non-antifungal agents can exhibit inhibitory effects on adhesion, amounts of total biofilm formation and metabolic activities of C. parapsilosis isolates from candidemia patients, NAC was added to the yeast suspensions at different concentrations, respectively. Reverse transcription was used to detect the transcriptional levels of adhesion-related genes (CpALS6 and CpALS7) and biofilm formation-related factors (CpEFG1 and CpBCR1) in the BCR1 knockout strain, CP7 and CP5 clinical strains in the presence of NAC. To further explore the mechanism of NAC on the biofilms of C. parapsilosis, RNA sequencing was used to calculate gene expression, comparing the differences among samples. Gene Ontology (GO) enrichment analysis helps to illustrate the difference between two particular samples on functional levels.Results. A high concentration of NAC reduces the total amount of biofilm formation in C. parapsilosis. Following co-incubation with NAC, the expression of CpEFG1 in both CP7 and CP5 clinical strains decreased, while there were no significant changes in the transcriptional levels of CpBCR1 compared with the untreated strain. GO enrichment analysis showed that the metabolism and biofilm-forming factors of cell structure were all regulated after NAC intervention.Conclusions. The non-antifungal agent NAC exhibits certain inhibitory effects on clinical isolate biofilm formation by down-regulating the expression of the CpEFG1 gene, making it a highly potential candidate for the treatment of C. parapsilosis catheter-related bloodstream infections.

Enhancing small-scale acetification processes using adsorbed Acetobacter pasteurianus UMCC 2951 on κ-carrageenan-coated luffa sponge.

Background: This study explored the utilization of luffa sponge (LS) in enhancing acetification processes. LS is known for having high porosity and specific surface area, and can provide a novel means of supporting the growth of acetic acid bacteria (AAB) to improve biomass yield and acetification rate, and thereby promote more efficient and sustainable vinegar production. Moreover, the promising potential of LS and luffa sponge coated with κ-carrageenan (LSK) means they may represent effective alternatives for the co-production of industrially valuable bioproducts, for example bacterial cellulose (BC) and acetic acid. Methods: LS and LSK were employed as adsorbents for Acetobacter pasteurianus UMCC 2951 in a submerged semi-continuous acetification process. Experiments were conducted under reciprocal shaking at 1 Hz and a temperature of 32 °C. The performance of the two systems (LS-AAB and LSK-AAB respectively) was evaluated based on cell dry weight (CDW), acetification rate, and BC biofilm formation. Results: The use of LS significantly increased the biomass yield during acetification, achieving a CDW of 3.34 mg/L versus the 0.91 mg/L obtained with planktonic cells. Coating LS with κ-carrageenan further enhanced yield, with a CDW of 4.45 mg/L. Acetification rates were also higher in the LSK-AAB system, reaching 3.33 ± 0.05 g/L d as opposed to 2.45 ± 0.05 g/L d for LS-AAB and 1.13 ± 0.05 g/L d for planktonic cells. Additionally, BC biofilm formation during the second operational cycle was more pronounced in the LSK-AAB system (37.0 ± 3.0 mg/L, as opposed to 25.0 ± 2.0 mg/L in LS-AAB). Conclusions: This study demonstrates that LS significantly improves the efficiency of the acetification process, particularly when enhanced with κ-carrageenan. The increased biomass yield, accelerated acetification, and enhanced BC biofilm formation highlight the potential of the LS-AAB system, and especially the LSK-AAB variant, in sustainable and effective vinegar production. These systems offer a promising approach for small-scale, semi-continuous acetification processes that aligns with eco-friendly practices and caters to specialized market needs. Finally, this innovative method facilitates the dual production of acetic acid and bacterial cellulose, with potential applications in biotechnological fields.

Temperatures above 37°C increase virulence of a convergent Klebsiella pneumoniae sequence type 307 strain.

Background: Convergence of Klebsiella pneumoniae (KP) pathotypes has been increasingly reported in recent years. These pathogens combine features of both multidrug-resistant and hypervirulent KP. However, clinically used indicators for hypervirulent KP identification, such as hypermucoviscosity, appear to be differentially expressed in convergent KP, potential outbreak clones are difficult to identify. We aimed to fill such knowledge gaps by investigating the temperature dependence of hypermucoviscosity and virulence in a convergent KP strain isolated during a clonal outbreak and belonging to the high-risk sequence type (ST)307. Methods: Hypermucoviscosity, biofilm formation, and mortality rates in Galleria mellonella larvae were examined at different temperatures (room temperature, 28°C, 37°C, 40°C and 42°C) and with various phenotypic experiments including electron microscopy. The underlying mechanisms of the phenotypic changes were explored via qPCR analysis to evaluate plasmid copy numbers, and transcriptomics. Results: Our results show a temperature-dependent switch above 37°C towards a hypermucoviscous phenotype, consistent with increased biofilm formation and in vivo mortality, possibly reflecting a bacterial response to fever-like conditions. Furthermore, we observed an increase in plasmid copy number for a hybrid plasmid harboring carbapenemase and rmpA genes. However, transcriptomic analysis revealed no changes in rmpA expression at higher temperatures, suggesting alternative regulatory pathways. Conclusion: This study not only elucidates the impact of elevated temperatures on hypermucoviscosity and virulence in convergent KP but also sheds light on previously unrecognized aspects of its adaptive behavior, underscoring its resilience to changing environments.

Molecular characterization of Pseudomonas aeruginosa from diabetic foot infections in Tunisia.

Background. Pseudomonas aeruginosa is an invasive organism that frequently causes severe tissue damage in diabetic foot ulcers.Gap statement. The characterisation of P. aeruginosa strains isolated from diabetic foot infections has not been carried out in Tunisia.Purpose. The aim was to determine the prevalence of P. aeruginosa isolated from patients with diabetic foot infections (DFIs) in Tunisia and to characterize their resistance, virulence and molecular typing.Methods. Patients with DFIs admitted to the diabetes department of the International Hospital Centre of Tunisia, from September 2019 to April 2021, were included in this prospective study. P. aeruginosa were obtained from the wound swabs, aspiration and soft tissue biopsies during routine clinical care and were confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Antimicrobial susceptibility testing, serotyping, integron and OprD characterization, virulence, biofilm production, pigment quantification, elastase activity and molecular typing were analysed in all recovered P. aeruginosa isolates by phenotypic tests, specific PCRs, sequencing, pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing.Results. Sixteen P. aeruginosa isolates (16.3 %) were recovered from 98 samples of 78 diabetic patients and were classified into 6 serotypes (O:11 the most frequent), 11 different PFGE patterns and 10 sequence types (three of them new ones). The high-risk clone ST235 was found in two isolates. The highest resistance percentages were observed to netilmicin (69 %) and cefepime (43.8 %). Four multidrug-resistant (MDR) isolates (25 %) were detected, three of them being carbapenem-resistant. The ST235-MDR strain harboured the In51 class 1 integron (intI1 +aadA6+orfD+qacED1-sul1). According to the detection of 14 genes involved in virulence or quorum sensing, 5 virulotypes were observed, including 5 exoU-positive, 9 exoS-positive and 2 exoU/exoS-positive strains. The lasR gene was truncated by ISPpu21 insertion sequence in one isolate, and a deletion of 64 bp in the rhlR gene was detected in the ST235-MDR strain. Low biofilm, pyoverdine and elastase production were detected in all P. aeruginosa; however, the lasR-truncated strain showed a chronic infection phenotype characterized by loss of serotype-specific antigenicity, high production of phenazines and high biofilm formation.Conclusions. Our study demonstrated for the first time the prevalence and the molecular characterization of P. aeruginosa strains from DFIs in Tunisia, showing a high genetic diversity, moderate antimicrobial resistance, but a high number of virulence-related traits, highlighting their pathological importance.

Biogenic amine tryptamine in human vaginal probiotic isolates mediates matrix inhibition and thwarts uropathogenic E. coli biofilm.

Probiotics offer a promising prophylactic approach against various pathogens and represent an alternative strategy to combat biofilm-related infections. In this study, we isolated vaginal commensal microbiota from 54 healthy Indian women to investigate their probiotic traits. We primarily explored the ability of cell-free supernatant (CFS) from Lactobacilli to prevent Uropathogenic Escherichia coli (UPEC) colonization and biofilm formation. Our findings revealed that CFS effectively reduced UPEC's swimming and swarming motility, decreased cell surface hydrophobicity, and hindered matrix production by downregulating specific genes (fimA, fimH, papG, and csgA). Subsequent GC-MS analysis identified Tryptamine, a monoamine compound, as the potent bioactive substance from Lactobacilli CFS, inhibiting UPEC biofilms with an MBIC of 4 µg/ml and an MBEC of 8 µg/ml. Tryptamine induced significant changes in E. coli colony biofilm morphology, transitioning from the Red, Dry, and Rough (RDAR) to the Smooth and White phenotype, indicating reduced extracellular matrix production. Biofilm time-kill assays demonstrated a four-log reduction in UPEC viability when treated with Tryptamine, highlighting its potent antibacterial properties, comparable to CFS treatment. Biofilm ROS assays indicated a significant elevation in ROS generation within UPEC biofilms, suggesting a potential antibacterial mechanism. Gene expression studies with Tryptamine-treated samples showed a reduction in expression of curli gene (csgA), consistent with CFS treatment. This study underscores the potential of Tryptamine from probiotic Lactobacilli CFS as a promising antibiofilm agent against UPEC biofilms.

Three-Dimensional Rotary Culture to Model Mycobacterial Biofilms in Low-Shear Detergent-Free Liquid Suspension.

In vitro biofilm models have allowed researchers to investigate the role biofilms play in the pathogenesis, virulence, and antimicrobial drug susceptibility of a wide range of bacterial pathogens. Rotary cell culture systems create three-dimensional cellular structures, primarily applied to eukaryotic organoids, that better capture characteristics of the cells in vivo. Here, we describe how to apply a low-shear, detergent-free rotary cell culture system to generate biofilms of Mycobacterium bovis BCG. The three-dimensional biofilm model forms mycobacterial cell aggregates in suspension as surface-detached biomass, without severe nutrient starvation or environmental stress, that can be harvested for downstream experiments. Mycobacterium bovis BCG derived from cell clusters display antimicrobial drug tolerance, presence of an extracellular matrix, and evidence of cell wall remodeling, all features of biofilm-associated bacteria that may be relevant to the treatment of tuberculosis.

Coexistence of ß-lactamase genes and biofilm forming potential among carbapenem-resistant Acinetobacter baumannii in Lahore, Pakistan.

INTRODUCTION: Our goal was to investigate the antimicrobial resistance due to beta-lactamase genes and virulent determinants (biofilm-forming ability) expressed by Acinetobacter collected from health settings in Pakistan. A cross-sectional study was conducted for the molecular characterization of carbapenemases and biofilm-producing strains of Acinetobacter spp. METHODOLOGY: Two twenty-three imipenem-resistant Acinetobacter isolates were analyzed from 2020 to 2023.The combination disk test and modified hodge test were performed. Biofilm forming ability was determined by polystyrene tube assay. Multiplex polymerase chain reaction (PCR) for virulent and biofilm-forming genes, and 16S rRNA sequencing were performed. RESULTS: 118 (52.9%) carbapenem-resistant Acinetobacter (CR-AB) were isolated from wounds and pus, 121 (54.2%) from males, and 92 (41.2%) from 26-50-years-olds. More than 80% of strains produced ß-lactamases and carbapenemases. Based on the PCR amplification of the ITS gene, 174 (78.0%) CR-AB strains were identified from CR-Acinetobacter non-baumannii (ANB). Most CR-AB were strong and moderate biofilm producers. Genetic analysis revealed the blaOXA-23, blaTEM, blaCTX-M blaNDM-1 and blaVIM were prevalent in CR-AB with frequencies 91 (94.8%), 68 (70.8%), 19 (19.7%), 53 (55.2%), 2 (2.0%) respectively. Among virulence genes, OmpA was dominant in CR-AB isolates from wound (83, 86.4%), csuE 63 (80.7%) from non-wound specimens and significantly correlated with blaNDM and blaOXA genes. Phylogenetic analysis revealed three different clades for strains based on specimens. CONCLUSIONS: CR-AB was highly prevalent in Pakistan and associated with wound infections. The genes, blaOXA-23, blaTEM, blaCTX-M, and blaNDM-1 were detected in CR-AB. Most CR-AB were strong biofilm producers with virulent genes OmpA and csuE.

Methanogenesis in the presence of oxygenic photosynthetic bacteria may contribute to global methane cycle.

Accumulating evidences are challenging the paradigm that methane in surface water primarily stems from the anaerobic transformation of organic matters. Yet, the contribution of oxygenic photosynthetic bacteria, a dominant species in surface water, to methane production remains unclear. Here we show methanogenesis triggered by the interaction between oxygenic photosynthetic bacteria and anaerobic methanogenic archaea. By introducing cyanobacterium Synechocystis PCC6803 and methanogenic archaea Methanosarcina barkeri with the redox cycling of iron, CH4 production was induced in coculture biofilms through both syntrophic methanogenesis (under anoxic conditions in darkness) and abiotic methanogenesis (under oxic conditions in illumination) during the periodic dark-light cycles. We have further demonstrated CH4 production by other model oxygenic photosynthetic bacteria from various phyla, in conjunction with different anaerobic methanogenic archaea exhibiting diverse energy conservation modes, as well as various common Fe-species. These findings have revealed an unexpected link between oxygenic photosynthesis and methanogenesis and would advance our understanding of photosynthetic bacteria's ecological role in the global CH4 cycle. Such light-driven methanogenesis may be widely present in nature.

Transcriptome Analysis of Human Dermal Cells Infected with Candida auris Identified Unique Pathogenesis/Defensive Mechanisms Particularly Ferroptosis.

Candida auris is an emerging multi-drug resistant yeast that can cause life-threatening infections. A recent report clarified the ability of C. auris to form a biofilm with enhanced drug resistance properties in the host skin's deep layers. The formed biofilm may initiate further bloodstream spread and immune escape. Therefore, we propose that secreted chemicals from the biofilm may facilitate fungal pathogenesis. In response to this interaction, the host skin may develop potential defensive mechanisms. Comparative transcriptomics was performed on the host dermal cells in response to indirect interaction with C. auris biofilm through Transwell inserts compared to planktonic cells. Furthermore, the effect of antifungals including caspofungin and fluconazole was studied. The obtained data showed that the dermal cells exhibited different transcriptional responses. Kyoto Encyclopedia of Genes and Genomes and Reactome analyses identified potential defensive responses employed by the dermal cells and potential toxicity induced by C. auris. Additionally, our data indicated that the dominating toxic effect was mediated by ferroptosis; which was validated by qRT-PCR, cytotoxicity assay, and flow cytometry. On the other hand, the viability of C. auris biofilm was enhanced and accompanied by upregulation of MDR1, and KRE6 upon interaction with dermal cells; both genes play significant roles in drug resistance and biofilm maturation, respectively. This study for the first-time shed light on the dominating defensive responses of human dermal cells, microbe colonization site, to C. auris biofilm and its toxic effects. Further, it demonstrates how C. auris biofilm responds to the defensive mechanisms developed by the human dermal cells.

Marine biofilm formation on flax fibre reinforced biocomposites.

Artificial reefs represent useful tools to revitalize coastal and ocean ecosystems. Their formulation determines the biofilm formation which is the prerequisite for the colonization process by marine micro- and macroorganisms. In comparison with concrete, biobased polymers offer improved characteristics, including architecture, formulation, rugosity and recycling. This article aims to explore a new scale of artificial reef made of biocomposites reinforced with a high flax fibre (Linum utilatissimum) content (30%). Cellular adhesion and resulting biofilm formation were assessed using two marine microorganisms: Pseudoalteromonas sp. 3J6 and Cylindrotheca closterium. The influence of flax fibre leachates and plastic monomers on the growth of those marine microorganisms were also evaluated. Results indicated that the introduction of flax fibres inside the polymer matrix modified its physicochemical properties thus modulating adhesion and biofilm formation depending on the microorganism. This study gives insights for further developments of novel functionalized artificial reefs made of biocomposites.

KKL-35 inhibits growth of Staphylococcus aureus by systematically changing bacterial phenotypes.

KKL-35 is a new oxadiazole compound with potent broad-spectrum antibacterial activity against a number of gram-positive and gram-negative bacteria. However, its influences on bacterial growth are unclear. This study is to investigate phenotypic changes of Staphylococcus aureus (SA) caused by KKL-35 and evaluate antibacterial activity of combinations of KKL-35 with 7 class of antibiotics available in medical facilities. KKL-35-treated SA showed significantly lower survival under stresses of NaCl and H2O2 than DMSO (21.03 ± 2.60% vs. 68.21 ± 5.31% for NaCl, 4.91 ± 3.14% vs. 74.78 ± 2.88% for H2O2). UV exposure significantly decreased survival of SA treated with KKL-35 than DMSO-treated ones (23.91 ± 0.71% vs. 55.45 ± 4.70% for 4.2 J/m2, 12.80 ± 1.03% vs. 31.99 ± 5.99% for 7.0 J/m2, 1.52 ± 0.63% vs. 6.49 ± 0.51% for 14.0 J/m2). KKL-35 significantly decreased biofilm formation (0.47 ± 0.12 vs. 1.45 ± 0.21) and bacterial survival in the serum resistance assay (42.27 ± 2.77% vs. 78.31 ± 5.64%) than DMSO. KKL-35 significantly decreased ethidium bromide uptake and efflux, as well as the cell membrane integrity. KKL-35 had low cytotoxicity and low propensity for resistance. KKL-35 inhibited SA growth in concentration-independent and time-dependent manners, and showed additivity when combined with the majority class of available antibiotics. Antibiotic combinations of KKL-35 with ciprofloxacin, rifampicin, or linezolid significantly decreased bacterial loads than the most active antibiotic in the corresponding combination. Thus, KKL-35 inhibits growth of SA by decreasing bacterial environmental adaptations, biofilm formation, membrane uptake and efflux, as well as increasing antibiotic sensitivity. Its potent antibacterial activity, low cytotoxicity, low propensity for resistance, and wide choices in antibiotic combinations make KKL-35 a promising leading compound to design new antibiotics in monotherapies and combination therapies to treat bacterial infections.

Effect of L-HSL on biofilm and motility of Pseudomonas aeruginosa and its mechanism.

Pseudomonas aeruginosa (P. aeruginosa) biofilm formation is a crucial cause of enhanced antibiotic resistance. Quorum sensing (QS) is involved in regulating biofilm formation; QS inhibitors block the QS signaling pathway as a new strategy to address bacterial resistance. This study investigated the potential and mechanism of L-HSL (N-(3-cyclic butyrolactone)-4-trifluorophenylacetamide) as a QS inhibitor for P. aeruginosa. The results showed that L-HSL effectively inhibited the biofilm formation and dispersed the pre-formed biofilm of P. aeruginosa. The production of extracellular polysaccharides and the motility ability of P. aeruginosa were suppressed by L-HSL. C. elegans infection experiment showed that L-HSL was non-toxic and provided protection to C. elegans against P. aeruginosa infection. Transcriptomic analysis revealed that L-HSL downregulated genes related to QS pathways and biofilm formation. L-HSL exhibits a promising potential as a therapeutic drug for P. aeruginosa infection. KEY POINTS: • Chemical synthesis of N-(3-cyclic butyrolactone)-4-trifluorophenylacetamide, named L-HSL. • L-HSL does not generate survival pressure on the growth of P. aeruginosa and can inhibit the QS system. • KEGG enrichment analysis found that after L-HSL treatment, QS-related genes were downregulated.

Bacterial persisters: molecular mechanisms and therapeutic development.

Persisters refer to genetically drug susceptible quiescent (non-growing or slow growing) bacteria that survive in stress environments such as antibiotic exposure, acidic and starvation conditions. These cells can regrow after stress removal and remain susceptible to the same stress. Persisters are underlying the problems of treating chronic and persistent infections and relapse infections after treatment, drug resistance development, and biofilm infections, and pose significant challenges for effective treatments. Understanding the characteristics and the exact mechanisms of persister formation, especially the key molecules that affect the formation and survival of the persisters is critical to more effective treatment of chronic and persistent infections. Currently, genes related to persister formation and survival are being discovered and confirmed, but the mechanisms by which bacteria form persisters are very complex, and there are still many unanswered questions. This article comprehensively summarizes the historical background of bacterial persisters, details their complex characteristics and their relationship with antibiotic tolerant and resistant bacteria, systematically elucidates the interplay between various bacterial biological processes and the formation of persister cells, as well as consolidates the diverse anti-persister compounds and treatments. We hope to provide theoretical background for in-depth research on mechanisms of persisters and suggest new ideas for choosing strategies for more effective treatment of persistent infections.

[Advances in mechanisms of biofilm formation and drug resistance of Staphylococcus aureus].

Staphylococcus aureus is a common pathogenic bacterium. However, due to the abuse of antibiotics, multiple drug-resistant S. aureus (DR S. aureus) has emerged in a large number, which seriously threatens human health. DR S. aureus usually forms biofilms by attaching on contact surfaces and secreting macromolecules including polysaccharides, proteins, and lipids, thus encasing themselves in a self-generated polymeric matrix. A biofilm provides an efficacious barrier that protects bacteria from detrimental environmental factors. Simultaneously, it protects DR S. aureus from the host immune system and attenuates the penetration and killing effects of drugs, serving as a key structure for the development of drug resistance. Therefore, gaining an in-depth understanding of the DR S. aureus biofilm is crucial for treating related infectious diseases. In this paper, we summarize recent research progress in the biofilm formation mechanism, drug resistance mechanism, and measures for inhibition and clearance of DR S. aureus and provide an outlook on the future research directions.

[Investigation of Biofilm Formation, Anti-Quorum Sensing Activity and Antimicrobial Resistance in Corynebacterium Species Isolated from Clinical Samples]. / Klinik Örneklerden Izole Edilen Corynebacterium Türlerinde Biyofilm Olusumunun, Anti-Quorum Sensing Aktivitesinin ve Antimikrobiyal Direnç Durumunun Arastirilmasi.

An increasing number of different clinical infections caused by Corynebacteria have been reported in the last decade. The aim of this study was to evaluate the antibiotic resistance rates, biofilm formation capacities and to investigate the ''anti-quorum-sensing (anti-QS)'' activities of corynebacteria, which were divided into three groups according to the type of growth in culture (pure, with another pathogenic bacterium and polymicrobial growth). In total 240 Corynebacterium spp. isolates from different clinical specimens sent to the medical microbiology laboratories of Düzce University Faculty of Medicine Hospital and Basaksehir Çam and Sakura City Hospital between June 2021 and June 2022 were classified into three groups: pure, isolated with another pathogen and polymicrobial, according to their growth patterns in culture. Bacteria were identified by matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) Biotyper (Bruker, Germany) at an external centre. Antibiotic susceptibilities were determined by disc diffusion method and for vancomycin broth microdilution method was used. Results were interpreted according to EUCAST recommendations. The biofilm-forming properties of the isolates were determined quantitatively. Bioactive components of 17 isolates with strong biofilm formation were extracted and anti-QS activity was determined by agar diffusion method using Chromobacterium violaceum ATCC 12472 strain and then violacein pigment production was measured quantitatively. Of the 240 Corynebacterium spp. isolates, 138 (58%) were pure, 52 (22%) were isolated with another pathogen and 50 (20%) were part of a polymicrobial infection. Of the isolates, 140 were identified as C.striatum, 34 as C.amycolatum and 24 as Corynebacterium afermentans. When the antibiotic resistance rates of the Corynebacterium isolates were analysed according to the groups, the resistance rates to rifampicin and tetracycline antibiotics were found to be statistically significantly lower in the polymicrobial group than in the other groups. The resistance rates to penicillin, clindamycin, ciprofloxacin, moxifloxacin, rifampicin, tetracycline and linezolid were 96.7%, 88.3%, 86.3%, 73.8%, 62.5%, 59.2% and 0.8%, respectively. While all isolates were susceptible to vancomycin, linezolid resistance was detected in two C.afermentans isolates. When the biofilm formation ability was analysed, it was observed that 87 (36.3%) isolates formed biofilm. The biofilm formation rate of the isolates in the polymicrobial growth group was lower than the other two groups. The anti-QS activity of 17 isolates with strong biofilm formation was investigated and none of the Corynebacterium extracts tested were found to have anti-QS activity (inhibition of violacein pigment production without inhibiting bacterial growth) in the QS study with C.violaceum, whereas five isolate extracts had antibacterial activity (inhibition of bacterial growth). Four of the bacterial extracts with antimicrobial activity belonged to C.amycolatum and one to C.afermentans. In conclusion, when both antibiotic resistance rates and biofilm formation rates were analysed, the corynebacteria growing in culture with another pathogen showed similar characteristics to the corynebacteria growing as a pure culture. Therefore, it was thought that corynebacteria growing with another pathogen should not be ignored. In addition, the antimicrobial effects of some corynebacterial extracts suggested that more QS studies should be carried out with microbiota bacteria.

[Effect of Quorum Sensing Systems on Biofilm Formation and Virulence in Salmonella]. / Salmonella'da Çogunlugu Algilama Sistemlerinin Biyofilm Olusumu ve Virülans Üzerine Etkisi.

In recent years, as the paradigm of communication between cells has been clarified, the ability of bacteria to change their gene expression patterns in response to various extracellular signals has attracted great interest. In particular, intracellular and intercellular communication between bacterial populations, called quorum sensing (QS), is essential for coordinating physiological and genetic activities. QS studies are critical, particularly in elucidating the regulatory mechanisms of infectious processes in food-borne pathogens. Elucidating the QS mechanisms in Salmonella is effective in silencing the virulence factors in the fight against this bacterium. The aims of this study were; to create luxS gene mutants that play a vital role in the QS activity of Salmonella and to determine the effect of this mutation on the expression of virulence genes in the bacteria and to determine the impact of synthetic N-hexanoyl-homoserine lactone (C6HSL) on biofilm formation and AI-2 signaling pathway of Salmonella wild strain and luxS gene mutants. luxS gene mutants were constructed by recombining the gene region with the chloramphenicol gene cassette based on homologous region recombination. In the luxS mutants obtained in this way, the expression of eight different virulence genes (hilA, invA, inv, glgC, fimF, fliF, lpfA, gyrA), which have essential roles in Salmonella pathogenicity, was determined by quantitative real-time reverse transcriptase polymerase chain reaction (rRT-qPCR) method and compared with natural strains. As a result of these studies, it was determined that the expression of each gene examined was significantly reduced in luxS mutant strains. The relative AI-2 activities of Salmonella strains were analyzed depending on time. It was determined that the highest activity occurred at the fourth hour and the AI-2 activities of luxS mutants were reduced compared to the wild strain. Finally, it was determined that C6HSL increased the biofilm activity of Salmonella Typhimurium DMC4, SL1344 wild strains, and mutants, mainly at the 72nd hour. In conclusion, our results proved that C6HSL stimulated QS communication in all strains and increased biofilm of Salmonella formation and autoinducer activity. This situation determines that Salmonella responds to external signals by using QS systems. In addition, this research contributed to provide additional information on interspecies communication mechanisms to develop strategies to prevent biofilm formation of this pathogen.

Fluid flow drives phenotypic heterogeneity in bacterial growth and adhesion on surfaces.

Bacteria often thrive in surface-attached communities, where they can form biofilms affording them multiple advantages. In this sessile form, fluid flow is a key component of their environments, renewing nutrients and transporting metabolic products and signaling molecules. It also controls colonization patterns and growth rates on surfaces, through bacteria transport, attachment and detachment. However, the current understanding of bacterial growth on surfaces neglects the possibility that bacteria may modulate their division behavior as a response to flow. Here, we employed single-cell imaging in microfluidic experiments to demonstrate that attached Escherichia coli cells can enter a growth arrest state while simultaneously enhancing their adhesion underflow. Despite utilizing clonal populations, we observed a non-uniform response characterized by bistable dynamics, with co-existing subpopulations of non-dividing and actively dividing bacteria. As the proportion of non-dividing bacteria increased with the applied flow rate, it resulted in a reduction in the average growth rate of bacterial populations on flow-exposed surfaces. Dividing bacteria exhibited asymmetric attachment, whereas non-dividing counterparts adhered to the surface via both cell poles. Hence, this phenotypic diversity allows bacterial colonies to combine enhanced attachment with sustained growth, although at a reduced rate, which may be a significant advantage in fluctuating flow conditions.

Legionella colonization and 3D spatial location within a Pseudomonas biofilm.

Biofilms are known to be critical for Legionella settlement in engineered water systems and are often associated with Legionnaire's Disease events. One of the key features of biofilms is their heterogeneous three-dimensional structure which supports the establishment of microbial interactions and confers protection to microorganisms. This work addresses the impact of Legionella pneumophila colonization of a Pseudomonas fluorescens biofilm, as information about the interactions between Legionella and biofilm structures is scarce. It combines a set of meso- and microscale biofilm analyses (Optical Coherence Tomography, Episcopic Differential Interference Contrast coupled with Epifluorescence Microscopy and Confocal Laser Scanning Microscopy) with PNA-FISH labelled L. pneumophila to tackle the following questions: (a) does the biofilm structure change upon L. pneumophila biofilm colonization?; (b) what happens to L. pneumophila within the biofilm over time and (c) where is L. pneumophila preferentially located within the biofilm? Results showed that P. fluorescens structure did not significantly change upon L. pneumophila colonization, indicating the competitive advantage of the first colonizer. Imaging of PNA-labelled L. pneumophila showed that compared to standard culture recovery it colonized to a greater extent the 3-day-old P. fluorescens biofilms, presumably entering in VBNC state by the end of the experiment. L. pneumophila was mostly located in the bottom regions of the biofilm, which is consistent with the physiological requirements of both bacteria and confers enhanced Legionella protection against external aggressions. The present study provides an expedited methodological approach to address specific systematic laboratory studies concerning the interactions between L. pneumophila and biofilm structure that can provide, in the future, insights for public health Legionella management of water systems.

Beyond antibiotics: CRISPR/Cas9 triumph over biofilm-associated antibiotic resistance infections.

A complex structure known as a biofilm is formed when a variety of bacterial colonies or a single type of cell in a group sticks to a surface. The extracellular polymeric compounds that encase these cells, often consisting of proteins, eDNA, and polysaccharides, exhibit strong antibiotic resistance. Concerns about biofilm in the pharmaceutical industry, public health, and medical fields have sparked a lot of interest, as antibiotic resistance is a unique capacity exhibited by these biofilm-producing bacteria, which increases morbidity and death. Biofilm formation is a complicated process that is controlled by several variables. Insights into the processes to target for the therapy have been gained from multiple attempts to dissect the biofilm formation process. Targeting pathogens within a biofilm is profitable because the bacterial pathogens become considerably more resistant to drugs in the biofilm state. Although biofilm-mediated infections can be lessened using the currently available medications, there has been a lot of focus on the development of new approaches, such as bioinformatics tools, for both treating and preventing the production of biofilms. Technologies such as transcriptomics, metabolomics, nanotherapeutics and proteomics are also used to develop novel anti-biofilm agents. These techniques help to identify small compounds that can be used to inhibit important biofilm regulators. The field of appropriate control strategies to avoid biofilm formation is expanding quickly because of this spurred study. As a result, the current article addresses our current knowledge of how biofilms form, the mechanisms by which bacteria in biofilms resist antibiotics, and cutting-edge treatment approaches for infections caused by biofilms. Furthermore, we have showcased current ongoing research utilizing the CRISPR/Cas9 gene editing system to combat bacterial biofilm infections, particularly those brought on by lethal drug-resistant pathogens, concluded the article with a novel hypothesis and aspirations, and acknowledged certain limitations.