Increased Biofilm Formation Research Articles

Subinhibitory Concentrations of Biogenic Silver Nanoparticles Affect Motility and Biofilm Formation in Pseudomonas aeruginosa.

Biogenic silver nanoparticles (bio-AgNPs) are increasingly recognized as an antibiofilm and antivirulence strategy against P. aeruginosa, a bacterium that causes chronic infections in immunocompromised and cystic fibrosis patients. This study aimed to investigate the effects of subinhibitory concentrations of bio-AgNPs on motility and biofilm formation in P. aeruginosa. Bio-AgNPs were synthesized via reduction of ionic silver catalyzed by cell-free culture filtrate from Fusarium oxysporum. A total of 17 P. aeruginosa isolates and strains were evaluated for swarming, swimming, and twitching motility in the presence and absence (control) of bio-AgNPs, including 10 clinical isolates from patients with and without cystic fibrosis, 5 environmental isolates obtained from the public water supply system, and 2 reference strains (PAO1 and PA14). Isolates were identified by biochemical and molecular methods. Minimum inhibitory concentrations (MICs) were determined by the broth microdilution method. Swarming, swimming, and twitching motility assays were performed in Petri dishes. Biofilm formation capacity was assessed quantitatively by the crystal violet method. MIC values ranged from 15.62 to 62.50 µM. The results showed that subinhibitory concentrations of bio-AgNPs (½ MIC, 7.81–31.25 µM) significantly increased (p < 0.05) swarming, swimming, and twitching motility in 40.0, 40.0, and 46.7% of isolates, respectively. Subinhibitory bio-AgNP treatment enhanced (p < 0.05) biofilm formation capacity in PA14 and a cystic fibrosis isolate (P11). It is concluded that subinhibitory concentrations of bio-AgNPs increased biofilm formation and swarming, swimming, and twitching motility in PA14 and some P. aeruginosa isolates. These virulence factors are directly involved with quorum-sensing systems. Further research should investigate the effects of AgNPs on P. aeruginosa quorum sensing to help elucidate their mechanism of action at subinhibitory concentrations.

Bacterial surface, biofilm and virulence properties of Listeriamonocytogenes strains isolated from smoked salmon and fish food contact surfaces

Biofilm formation is one of the defense mechanisms of bacteria against disinfectants and antimicrobials. The aim of this study was to determine biofilm-forming L.monocytogenes from fish processing and salmon surfaces. Biofilm formation at 15, 25, 37, and 40 °C from 1 to 6-days period, adhesion to glass, polypropylene and stainless-steel surfaces, bacterial surface charge and hydrophobicity was determined. Adhesion behavior of the strains was evaluated using Surface Plasmon Resonance (SPR) technique. Totally 32 L.monocytogenes strains belonging to serogroups IIa (n:17), IIc(n:14) and IVb(n:1) were detected from 1320 swabs and 16 smoked salmons. Biofilm formation tests revealed that 21 strains form biofilm on microplate by increasing time and temperature. Although all strains strongly formed biofilm on glass surfaces, two strains slightly adhered polypropylene surfaces. High surface roughness of stainless-steel FeCrNi alloy (Ra = 4.15 nm) and CoCrMo alloy (Ra = 10.75 nm) increased biofilm formation of L.monocytogenes on stainless-steel surfaces. Zeta potential results showed that non-biofilm formers were more negatively charged after 6-days and hydrophobicity couldn't give a distinct distribution among biofilm formers and non-formers. SPR analysis method was evaluated to distinguish biofilm formers to adhere SPR gold chip surfaces. PCR results revealed that all strains were positive for hylA, iap, actA, plcA, plcB, fri, flaA, inlA, inlB, inlC, inlJ, and lmo1386 genes. Additionally, all strains were susceptible to penicillin, ampicillin, meropenem, erythromycin and trimethoprim-sulfamethoxazole. Biofilm-forming, virulence properties of L.monocytogenes strains isolated from fish processing surfaces and smoked salmons were evaluated and SPR was used to differentiate biofilm formers as a sensitive technique for biofilm studies.

Roles of ompA of Citrobacter werkmanii in bacterial growth, biocide resistance, biofilm formation and swimming motility

The genus Citrobacter is commonly found in environmental and industrial settings, some members of which have been used for bioremediation of heavy metals owing to the absorption ability of their biofilms. Although our previous studies have found that the outer membrane protein A (OmpA) contributes to the process of Citrobacter werkmanii biofilm formation, the underlying mechanisms remain elusive. Therefore, we deleted ompA from the genome of C. werkmanii and investigated its phenotypes in comparison to the wild type strain (WT) and the complementary strain using biochemical and molecular techniques including RNA-Seq. Our results demonstrated that the deletion of ompA led to an increase in biofilm formation on both polystyrene and glass surfaces due to upregulation of some biofilm formation related genes. Meanwhile, swimming ability, which is mediated by activation of flagellar assembly genes, was increased on semi-solid plates in the ∆ompA strain when compared with WT. Additionally, inactivation of ompA also caused increased 1,2-benzisothiazolin-3-one (BIT) resistance, differential responses to Ca2+ stress, curli protein expression and cellulose production. Finally, ∆ompA caused differential expression of a total of 1470 genes when compared with WT, of which 146 were upregulated and 1324 were downregulated. These genes were classified into different Gene Ontology (GO) and KEGG pathways. In summary, ompA in C. werkmanii contributes to a variety of biological functions and may act as a target site to modulate biofilm formation. KEY POINTS: • ompA is a negative regulator for biofilm formation by C. werkmanii. • ompA inhibits swimming motility of C. werkmanii. • ompA deletion causes different expression profiles in C. werkmanii.

Parallel Evolution of Enhanced Biofilm Formation and Phage-Resistance in Pseudomonas aeruginosa during Adaptation Process in Spatially Heterogeneous Environments

An opportunistic pathogen Pseudomonas aeruginosa has a versatile phenotype and high evolutionary potential to adapt to various natural habitats. As the organism normally lives in spatially heterogeneous and polymicrobial environments from open fields to the inside of hosts, adaptation to abiotic (spatial heterogeneity) and biotic factors (interspecies interactions) is a key process to proliferate. However, our knowledge about the adaptation process of P. aeruginosa in spatially heterogeneous environments associated with other species is limited. We show herein that the evolutionary dynamics of P. aeruginosa PAO1 in spatially heterogeneous environments with Staphylococcus aureus known to coexist in vivo is dictated by two distinct core evolutionary trajectories: (i) the increase of biofilm formation and (ii) the resistance to infection by a filamentous phage which is retained in the PAO1 genome. Hyperbiofilm and/or pili-deficient phage-resistant variants were frequently selected in the laboratory evolution experiment, indicating that these are key adaptive traits under spatially structured conditions. On the other hand, the presence of S. aureus had only a marginal effect on the emergence and maintenance of these variants. These results show key adaptive traits of P. aeruginosa and indicate the strong selection pressure conferred by spatial heterogeneity, which might overwhelm the effect of interspecies interactions.

Evaluation of Antagonism Activity and Control of Vibrio alginolyticus in Artemia Culture Using Mixed Probiotic

Supplementation with mixed probiotic in aquaculture has been proven to benefit the hosts as disease resistance tool. In this study, a mixed probiotic which consisted of three isolated strains (Lysinibacillus fusiformis strain SPS11, A2, and Bacillus megaterium strain I24) was formulated for the in vitro assays against Vibrio alginolyticus and in vivo preliminary study towards Artemia nauplii. These strains showed antagonism activities against V. alginolyticus in in vitro assay. An increase in biofilm formation of this mixed probiotic was observed which indicated that the strains could work synergistically with each other to confer benefits to the hosts. Enrichment of Artemia nauplii with the formulated mixed probiotic was done to investigate its role in enhancing resistance against the V. alginolyticus. Artemia nauplii were cultured in two different concentrations of mixed probiotic (106 and 108 CFU mL-1) and challenged via immersion method. The mixed probiotic at both concentrations resulted in significantly higher survival of Artemia compared to the challenged group with no probiont added (106 CFU mL-1, 65.00 ± 0.00 % and 108 CFU mL-1, 77.50 ± 3.53 %). Significant reduction of Vibrio loads was observed in Artemia and its culture water supplemented with mixed probiotic at 108 CFU mL-1 whereas there was no reduction of Vibrio at 106 CFU mL-1. This study suggests that the usage of formulated mixed probiotic at high concentration (108 CFU mL-1) as opposed to single-strain probiotic can confer protection against V. alginolyticus infection towards Artemia.

Differential expression of bio-active metabolites produced by chitosan polymers-based Bacillus amyloliquefaciens fermentation

Bacillus amyloliquefaciens strain PPL shows a potential for the control of phytopathogenic fungi. In the present study, upon growing the strain PPL on various forms of chitosan (0.5 % powder, 0.1 % soluble, and 0.15 % colloidal) as the carbon source, the antifungal activity on tomato Fusarium wilt correlated with the activity of chitosanase and β-1,3-glucanase. The colloidal substrate-based strain PPL fermentation displayed the highest degree of spore germination inhibition (79.5 %) and biocontrol efficiency (76.0 %) in tomato by increased biofilm formation. The colloidal culture upregulated the expression of chitosanase gene (5.9-fold), and the powder attributed to the expression of cyclic lipopeptides-genes (2.5–5.7 fold). Moreover, the three chitosan cultures induced the morphological changes of Fusarium oxysporum. These results suggest that the choice of growth substrate synergistically affects the production of secondary metabolites by PPL strain, and consequently its antifungal activity.

Influence of UVA radiation on growth, biofilm formation and bioleaching capacity of Leptospirillum ferrooxidans

Ultraviolet-A (UVA) radiation is one of the most stressful oxidative agents that bacteria have to face in natural environments. The effect of this radiation on growth, survival, biofilm formation, and bioleaching activity of two Leptospirillum ferrooxidans strains was studied, comparing the collection strain DSM2705 with FAM-RA2, a strain recently isolated from Famatina (La Rioja, Argentina), at 3869 m above sea level (ma.s.l.). We demonstrated that the isolate FAM-RA2 was more UVA resistant than the collection strain DSM2705 in kinetic growth and biofilm formation assays. However, both strains presented a similar behavior in their bioleaching capacity. Higher levels of intracellular reactive oxygen species (ROS) after UVA exposure were detected in DSM2705 strain, suggesting the presence of stronger defensive mechanisms against oxidative stress in FAM-RA2 strain. Assays of biofilm formation showed that UVA radiation induced cell adhesion and extracellular polymeric substance (EPS) production in the collection strain DSM2705, but not in the FAM-RA2 strain. The ability of UVA radiation to increase biofilm formation in DSM2705 led us to suppose that this agent could promote its bioleaching activity; however, under these studied conditions, this stress agent did not enhance metal sulfide dissolution. This study provides first insights into the effects of UVA radiation on the physiological features of acidophilic microorganisms.

D-mannose-sensitive pilus of Acinetobacter baumannii is linked to biofilm formation and adherence onto respiratory tract epithelial cells.

Acinetobacter baumannii is an important nosocomial pathogen. To better understand the role of CsuA/BABCDE pilus of A. baumannii in virulence, bacterial biofilm formation, adherence and carbohydrate-mediated inhibition were conducted. CsuA/BABCDE pilus-producing (abbreviated Csu pilus) operon of A. baumannii ATCC17978 was cloned for analysis of biofilm formation on an abiotic plastic plate, bacterial adherence to respiratory epithelial human A549cells and carbohydrate-mediated inhibition. The carbohydrates used for inhibition of biofilm formation and adherence to A549cells included monosaccharides, pyranosides, and mannose-polymers. The Csu pilus of A. baumannii ATCC17978 was cloned and expressed into a non-pilus-producing Escherichia coli JM109, and was knocked out as well. The recombinant Csu (rCsu) pilus on E. coli JM109/rCsu pilus-producing clone observed by both electro-microscopy and atomic force microscopy showed abundant, while Csu-knockout A. baumannii ATCC17978 mutant appeared less or no pilus production. The E. coli JM109/rCsu pilus-producing clone significantly increased biofilm formation and adherence to A549cells; however, the Csu-knockout mutant dramatically lost biofilm-making ability but, in contrast, increased adherence. Moreover, both of biofilm formation and adherence could be significantly inhibited by d-mannose and methyl-α-d-mannopyranoside in Csu pilus-producing E. coli JM109, whereas in A. baumannii ATCC17978, high concentration of carbohydrates was required for the inhibition, suggesting that Csu pilus is sensitive to d-mannose. This is the first study confirming that Csu pilus of A. baumannii belongs to mannose-sensitive type 1 pilus family and contributes to biofilm formation and bacterial adherence to human epithelial cells.

Distinct clonal lineages and within-host diversification shape invasive Staphylococcus epidermidis populations.

S. epidermidis is a substantial component of the human skin microbiota, but also one of the major causes of nosocomial infection in the context of implanted medical devices. We here aimed to advance the understanding of S. epidermidis genotypes and phenotypes conducive to infection establishment. Furthermore, we investigate the adaptation of individual clonal lines to the infection lifestyle based on the detailed analysis of individual S. epidermidis populations of 23 patients suffering from prosthetic joint infection. Analysis of invasive and colonizing S. epidermidis provided evidence that invasive S. epidermidis are characterized by infection-supporting phenotypes (e.g. increased biofilm formation, growth in nutrient poor media and antibiotic resistance), as well as specific genetic traits. The discriminating gene loci were almost exclusively assigned to the mobilome. Here, in addition to IS256 and SCCmec, chromosomally integrated phages was identified for the first time. These phenotypic and genotypic features were more likely present in isolates belonging to sequence type (ST) 2. By comparing seven patient-matched nasal and invasive S. epidermidis isolates belonging to identical genetic lineages, infection-associated phenotypic and genotypic changes were documented. Besides increased biofilm production, the invasive isolates were characterized by better growth in nutrient-poor media and reduced hemolysis. By examining several colonies grown in parallel from each infection, evidence for genetic within-host population heterogeneity was obtained. Importantly, subpopulations carrying IS insertions in agrC, mutations in the acetate kinase (AckA) and deletions in the SCCmec element emerged in several infections. In summary, these results shed light on the multifactorial processes of infection adaptation and demonstrate how S. epidermidis is able to flexibly repurpose and edit factors important for colonization to facilitate survival in hostile infection environments.

The three NADH dehydrogenases of Pseudomonas aeruginosa: Their roles in energy metabolism and links to virulence.

Pseudomonas aeruginosa is a ubiquitous opportunistic pathogen which relies on a highly adaptable metabolism to achieve broad pathogenesis. In one example of this flexibility, to catalyze the NADH:quinone oxidoreductase step of the respiratory chain, P. aeruginosa has three different enzymes: NUO, NQR and NDH2, all of which carry out the same redox function but have different energy conservation and ion transport properties. In order to better understand the roles of these enzymes, we constructed two series of mutants: (i) three single deletion mutants, each of which lacks one NADH dehydrogenase and (ii) three double deletion mutants, each of which retains only one of the three enzymes. All of the mutants grew approximately as well as wild type, when tested in rich and minimal medium and in a range of pH and [Na+] conditions, except that the strain with only NUO (ΔnqrFΔndh) has an extended lag phase. During exponential phase, the NADH dehydrogenases contribute to total wild-type activity in the following order: NQR > NDH2 > NUO. Some mutants, including the strain without NQR (ΔnqrF) had increased biofilm formation, pyocyanin production, and killed more efficiently in both macrophage and mouse infection models. Consistent with this, ΔnqrF showed increased transcription of genes involved in pyocyanin production.

Effect of antimicrobial peptides on planktonic growth, biofilm formation and biofilm-derived bacterial viability of Streptococcus pneumoniae

Streptococcus pneumoniae is a leading cause of pneumonia mortality globally. Pneumococcal disease is often associated with prolonged colonisation of hosts and this process is facilitated by biofilm formation that is largely resistant to conventional antibiotics. We investigated the effects of antimicrobial peptides (AMPs) lysozyme, lactoferrin, LL37 and a combination of all three on planktonic growth, biofilm formation and biofilm-derived bacterial viability by S. pneumoniae, serotype 23F. Planktonic growth and biofilm-derived bacterial viability were determined using standard colony-forming techniques, while biofilm formation was measured using a crystal violet based spectrophotometric method. Relative to controls, lysozyme significantly reduced biofilm formation (0.08 OD vs. 0.10 OD at 570 nm, p = 0.01), while LL37 and the AMP combination increased biofilm formation (0.14 OD vs. 0.10 OD at 570 nm, p = 0.01). The combination of AMPs significantly decreased planktonic growth (1.10 × 108 colony-forming units per millilitres [CFU/mL] vs. 2.13 × 108 CFU/mL, p = 0.02). Biofilm-derived bacterial viability was greatly reduced by exposure to a combination of AMPs (1.05 × 105 CFU/mL vs. 1.12 × 106 CFU/mL, p = 3.60 × 10−8). Streptococcus pneumoniae displays marked resistance to the individual AMPs. A combination of lysozyme, lactoferrin and LL37 effectively inhibited planktonic growth and biofilm-derived bacterial viability; however, persister cell growth was still evident after exposure.

Transcriptomic Basis of Serum Resistance and Virulence Related Traits in XDR P. aeruginosa Evolved Under Antibiotic Pressure in a Morbidostat Device.

Colistin is a last resort antibiotic against the critical status pathogen Pseudomonas aeruginosa. Virulence and related traits such as biofilm formation and serum resistance after exposure to sub-inhibitory levels of colistin have been underexplored. We cultivated P. aeruginosa in a semi-automated morbidostat device with colistin, metronidazole and a combination of the two antibiotics for 21 days, and completed RNA-Seq to uncover the transcriptional changes over time. Strains became resistant to colistin within this time period. Colistin-resistant strains show significantly increased biofilm formation: the cell density in biofilm increases under exposure to colistin, while the addition of metronidazole can remove this effect. After 7 days of colistin exposure, strains develop an ability to grow in serum, suggesting that colistin drives bacterial modifications conferring a protective effect from serum complement factors. Of note, strains exposed to colistin showed a decrease in virulence, when measured using the Galleria mellonella infection model. These phenotypic changes were characterized by a series of differential gene expression changes, particularly those related to LPS modifications, spermidine synthesis (via speH and speE) and the major stress response regulator rpoS. Our results suggest a clinically important bacterial evolution under sub-lethal antibiotic concentration leading to potential for significant changes in the clinical course of infection.

Candida parapsilosis Colony Morphotype Forecasts Biofilm Formation of Clinical Isolates.

Candida parapsilosis is a frequent cause of fungal bloodstream infections, especially in critically ill neonates or immunocompromised patients. Due to the formation of biofilms, the use of indwelling catheters and other medical devices increases the risk of infection and complicates treatment, as cells embedded in biofilms display reduced drug susceptibility. Therefore, biofilm formation may be a significant clinical parameter, guiding downstream therapeutic choices. Here, we phenotypically characterized 120 selected isolates out of a prospective collection of 215 clinical C. parapsilosis isolates, determining biofilm formation, major emerging colony morphotype, and antifungal drug susceptibility of the isolates and their biofilms. In our isolate set, increased biofilm formation capacity was independent of body site of isolation and not predictable using standard or modified European Committee on Antimicrobial Susceptibility Testing (EUCAST) drug susceptibility testing protocols. In contrast, biofilm formation was strongly correlated with the appearance of non-smooth colony morphotypes and invasiveness into agar plates. Our data suggest that the observation of non-smooth colony morphotypes in cultures of C. parapsilosis may help as an indicator to consider the initiation of anti-biofilm-active therapy, such as the switch from azole- to echinocandin- or polyene-based strategies, especially in case of infections by potent biofilm-forming strains.

Effects of ClpP protease on biofilm formation of Enterococcus faecalis.

Enterococcus faecalis (E. faecalis), one of the main pathogens responsible for refractory periapical periodontitis and nosocomial infections, exhibits markedly higher pathogenicity in biofilms. Objectives Studies have shown that caseinolytic protease P (ClpP) is involved in biofilm formation. However, to date, few studies have investigated the role of ClpP in the survival of E. faecalis, and in enhancing biofilm formation. Therefore, we investigated the role of ClpP in the formation of E. faecalis biofilms.Methodology In our study, we used homologous recombination to construct clpP deleted and clpP complement strains of E. faecalis ATCC 29212. A viable colony counting method was used to analyze the growth patterns of E. faecalis. Crystal violet staining (CV) and confocal scanning laser microscopy (CLSM) were used to characterize biofilm mass formation and scanning electron microscopy (SEM) was used to observe the biofilm microstructure. Data was statistically analyzed via Student’s t-test or one-way analysis of variance (ANOVA).Results The results exhibited altered growth patterns for the clpP deletion strains and depleted polysaccharide matrix, resulting in reduced biofilm formation capacity compared to the standard strains. Moreover, ClpP was observed to increase biofilm formation in E. faecalis.Conclusion Our study shows that ClpP can increase biofilm formation in E. faecalis and emphasizes the importance of ClpP as a potential target against E. faecalis.

Combined effects of soft drinks and nicotine on Streptococcus mutans metabolic activity and biofilm formation.

The purpose of this study was to explore the effects of nicotine on the activity of Streptococcus mutans (S. mutans) in soft drinks. Regular soft drinks contain large proportions of high-fructose corn syrup (HFCS), which increases the activity of S. mutans resulting in high-caries risk compared with sugar-free soft drinks. Nicotine use exhibits a strong correlation with increased S. mutans biofilm formation. The soft drinks chosen were (Coca-Cola Classic, Diet Coke, Coca-Cola Zero Sugar, Caffeine-Free Coca-Cola, Caffeine-Free Diet Coke, Caffeine-Free Coca-Cola Zero Sugar). S. mutans was grown overnight in tryptic soy broth; nicotine was diluted in tryptic soy broth supplemented with 1.0% sucrose followed by soft drinks in dilution of 1:3. Total growth absorbance and biofilm growth were determined by spectrophotometry, absorbance measured to determine biofilm formation, and metabolic activity quantified. One-way ANOVA showed a considerable effect for HFCS and caffeine in the presence of nicotine and their interaction in all measures. Results showed sugar-free caffeinated colas demonstrated significant effect in inhibiting S. mutans biofilm formation and metabolic activity with nicotine. Nicotine-induced S. mutans increased biofilm formation and metabolic activity in the presence of HFCS and caffeine in soft drinks. In conclusion, smokers should consider sugar-free caffeinated versions to minimize the chance of developing dental caries dut to the reduction of biofilm formation.

Biofilm Formation on Breast Implant Surfaces by Major Gram-Positive Bacterial Pathogens.

Bacterial biofilm on surfaces of mammary implants is a predisposing factor for several outcomes. Because Gram-positive bacteria are potential agents of biomaterial-associated infections (BAIs), their abilities to form biofilm on breast implants should be elucidated. The aim of this study was to evaluate biofilm formation on different mammary prosthesis surfaces by major Gram-positive bacterial pathogens involved in BAIs. We initially evaluated biofilm formation on polystyrene plates with and without fibrinogen or collagen for 1 reference strain and 1 clinical isolate of Enterococcus faecalis, Staphylococcus aureus, Staphylococcus epidermidis, and Streptococcus pyogenes. We also tested the ability of clinical isolates to form biofilm on 4 different implant surfaces: polyurethane foam and smooth, microtextured, and standard textured silicone. Biofilm structure and cell viability were observed by scanning electron microscopy and confocal laser scanning microscopy. All strains showed strong biofilm formation on polystyrene. After fibrinogen or collagen treatment, biofilm formation varied. With fibrinogen, reference strains of S. aureus and S. pyogenes increased biofilm formation (P < 0.05). Reference strains of all species and the clinical isolate of S. pyogenes increased biofilm formation after collagen treatment (P < 0.05). In general, S. aureus showed higher capacity to produce biofilm. Scanning electron microscopy showed that biofilm attached to all surfaces tested, with the presence of extracellular polymeric substances and voids. Viable cells were more frequent for E. faecalis and S. pyogenes. All species produced biofilm on all prosthesis surfaces and under different conditions. Micrographies indicated thicker bacterial biofilm formation on microtextured and/or standard textured silicone by all species, except E. faecalis.

Impact of cigarette smoke on respiratory pathogens in biofilm production

Biofilm formation by respiratory pathogen favour their colonization, persistence and virulence. Cigarette smoke have the ability to cause induction and increase in biofilm formation. This quantitative in vitro study is aimed at understanding the dose-dependent change in the biofilm formation, which can be correlated to the degree of virulence of the respiratory pathogens. Materials and Methods:In the present study, 77 respiratory isolates of non-smokers were included. They were processed and exposed to varying concentrations of Cigarette Smoke Extract (CSE).Quantification of biofilm production was done according to the Microtitre dish biofilm formation assay. Fischer’s exact test was used to compare the biofilm production status. Results:We observed that 42[80.77%] isolates out of 52 biofilm producers and 12[48%] isolates out of 25 non-biofilm producers were found to show an increase in Biofilm index (BFI) with exposure to CSE. Among the total of 77 respiratory isolates, 32 isolates accounting for 41.57% [95% CI; 30.6% to 52.6%] showed a dose-related augmentation of biofilm formation on exposure to CSE. Interpretation and Conclusions:Progressive increase of biofilm with increase in concentration of CSE was the major finding. This emphasizes the need to initiate appropriate empirical therapy for smokers at the earliest. Keywords: Biofilm producer, Cigarette smoke extract, CSE, Dose related augmentation, Respiratory pathogens, Staphylococcus aureusa

Through quorum sensing, Pseudomonas aeruginosa resists noble metal-based nanomaterials toxicity.

Noble metal-based nanomaterials (NMNs), such as platinum nanoparticles (Pt@NPs) and palladium nanoparticles (Pd@NPs), are increasingly being used as antibacterial agents. However, little information is available on bacterial resistance to NMNs. In this study, owing to their oxidase-like and peroxidase-like properties, both Pt@NPs and Pd@NPs induce reactive oxygen species (ROS) and manifest antibacterial activities: 6.25μg/mL of either Pt@NPs or Pd@NPs killed >50% of Staphylococcus aureus strain ATCC29213. However, Pseudomonas aeruginosa strain PAO1 completely resisted 12.5μg/mL of Pt@NPs and 6.25μg/mL of Pd@NPs. Compared to the non-NMN groups, these NMNs promoted 2-3-fold upregulation of the quorum sensing (QS) gene lasR in strain PAO1. In fact, the lasR gene upregulation induced a 1.5-fold reduction in ROS production and increased biofilm formation by 11% (Pt@NPs) and 27% (Pd@NPs) in strain PAO1. The ΔlasR mutants (lasR gene knock out in strain PAO1), became sensitive to NMNs. The survival rates of ΔlasR mutants at 12.5μg/mL Pt@NPs and Pd@NPs treatments were only 77% and 58%, respectively. This is the first report indicating that bacteria can resist NMNs through QS. Based on these results, evaluation of the ecological risks of using NMNs as antibacterial agents is necessary.

Antifungal and Antibiofilm Activities of Selective Serotonin Reuptake Inhibitors Alone and in Combination with Fluconazole.

Candida spp. are clinically important pathogens that cause difficulties for treatment by biofilm formation. Considering antifungal resistance rates and the limitations in the discovery of new antifungals, the antifungal and antibiofilm effects of various drugs used for different therapeutic purposes are becoming more important. The goal of our study was to determine the antifungal and antibiofilm effects of the selective serotonin reuptake inhibitors (SSRIs), namely sertraline (SRT), paroxetine (PRX), and fluoxetine (FLX) alone and in combination with fluconazole (FLC) against Candida spp. Twenty Candida spp. strains isolated from clinical samples from Ege University Hospital were identified by the Dalmau method and matrix-assisted laser desorption ionization time of flight mass spectrometry. The minimum inhibitory concentrations (MICs) of the SSRIs and FLC were detected by broth microdilution method. Synergistic interactions between the SSRIs and FLC were investigated by checkerboard assay. The antibiofilm effects of the SSRIs were determined by spectrophotometric microplate method. Among the isolates, five different Candida spp. (C. albicans, C. glabrata, C. krusei, C. tropicalis, and C.parapsilosis) were identified. The MICs of the SSRIs ranged between 16-512 μg/mL. While SRT showed the highest antifungal effect, the antibiofilm efficacy of FLX was higher than that of the other agents. Moreover, FLX and PRX showed a synergistic effect with FLC in 13 and 19 isolates, respectively. Four isolates were strong biofilm producers while nine isolates were moderate biofilm producers. C. parapsilosis strains showed higher biofilm production than the other species. At MIC/2 concentration, FLX and SRT alone inhibited mature biofilms in six and five isolates, respectively, while PRX caused increases biofilm formation in seven isolates. This study revealed that MIC/2 concentrations of SSRIs could have antifungal and antibiofilm effects. SRT and FLX alone or in combination with antifungals may possibly have therapeutic potential for combating fungal infections.

Chemical Composition of Natural Hydrolates and Their Antimicrobial Activity on Arcobacter-Like Cells in Comparison with Other Microorganisms.

Hydrolates obtained via the hydrodistillation and steam distillation of Lavandula angustifolia Mill., Syzygium aromaticum L., Foeniculum vulgare Mill., and Laurus nobilis L. were analyzed by gas chromatography with flame ionization detector (GC-FID) and gas chromatography coupled to mass spectrometry (GC-MS). Additionally, the hydrolates were evaluated for antimicrobial activity (disk-diffusion and microdilution method), influence on biofilm formation (Christensen method) and cytotoxicity of concentrated hydrolates against human cell lines (A549) by xCELLigence system. Using chemical analysis, 48, 9, 13 and 33 different components were detected in lavender, clove, fennel and laurel hydrolates, respectively. Lavender hydrolate contained the largest proportion of 1,8-cineol, linalool furanoxide, and linalool. The main components of laurel hydrolate were 1,8-cineol, 4-terpineol and α-terpineol. Fenchone and estragole were the most abundant in fennel hydrolate, and eugenol and eugenyl acetate in clove hydrolate. Concentrated hydrolates showed significant antimicrobial activity. Clove hydrolate was among the most antimicrobially active agents, most preferably against C. albicans, with an inhibition zone up to 23.5 mm. Moreover, concentrated hydrolates did not show any cytotoxic effect again8 st human A549 cells. In the presence of the non-concentrated hydrolates, significantly reduced biofilm formation was observed; however, with concentrated clove hydrolate, there was an increase in biofilm formation, e.g., of A. thereius, A. lanthieri, and A. butzleri. Research shows new findings about hydrolates that may be important in natural medicine or for preservation purposes.