Association of exopolysaccharide genes in biofilm developing antibiotic-resistant Pseudomonas aeruginosa from hospital wastewater

The study aimed to examine the relationship between antibiotic resistance, biofilm formation and genes responsible for biofilm formation. Sixty-six Pseudomonas aeruginosa isolates were obtained from hospital wastewater and analyzed for their antibiotic resistance. Biofilm production among the isolates was tested by quantitative method crystal violet assay. Biofilm-associated genes among these isolates psl, alg, and pel were also checked. The maximum resistance was observed for ampicillins (88.24%) followed by nalidixic (83.82%), and nitrofurantoin (64.71%), respectively. Biofilm phenotypes are distributed in the following categories: high 39.39% (n = 26); moderate 57.57% (n = 38), and weak 3.0% (n = 2). Among the total isolates, biofilm-associated genes were detected in 84.84% (n = 56) of isolates and the remaining isolates 15.15% (n = 10) did not harbor any genes. In this study, pslB was the most predominant gene observed (71.21%, n = 47) followed by pslA (57.57%, n = 38), pelA (45.45%, n = 30), algD (43.93%, n = 29), and pelD (27.27%, n = 18), respectively. The present study reveals that the majority of the isolates are multidrug resistant being moderate and high biofilm formers. The study implies that biofilm acts as a machinery for bacteria to survive in the hospital effluent which is an antibiotic stress environment.

Active rheumatoid arthritis in a mouse model is not an independent risk factor for periprosthetic joint infection

Introduction Periprosthetic joint infection (PJI) represents a devastating complication of total joint arthroplasty associated with significant morbidity and mortality. Literature suggests a possible higher incidence of periprosthetic joint infection (PJI) in patients with rheumatoid arthritis (RA). There is, however, no consensus on this purported risk nor a well-defined mechanism. This study investigates how collagen-induced arthritis (CIA), a validated animal model of RA, impacts infectious burden in a well-established model of PJI. Methods Control mice were compared against CIA mice. Whole blood samples were collected to quantify systemic IgG levels via ELISA. Ex vivo respiratory burst function was measured via dihydrorhodamine assay. Ex vivo Staphylococcus aureus Xen36 burden was measured directly via colony forming unit (CFU) counts and crystal violet assay to assess biofilm formation. In vivo, surgical placement of a titanium implant through the knee joint and inoculation with S. aureus Xen36 was performed. Bacterial burden was then quantified by longitudinal bioluminescent imaging. Results Mice with CIA demonstrated significantly higher levels of systemic IgG compared with control mice (p = 0.003). Ex vivo, there was no significant difference in respiratory burst function (p = 0.89) or S. aureus bacterial burden as measured by CFU counts (p = 0.91) and crystal violet assay (p = 0.96). In vivo, no significant difference in bacterial bioluminescence between groups was found at all postoperative time points. CFU counts of both the implant and the peri-implant tissue were not significantly different between groups (p = 0.82 and 0.80, respectively). Conclusion This study demonstrated no significant difference in S. aureus infectious burden between mice with CIA and control mice. These results suggest that untreated, active RA may not represent a significant intrinsic risk factor for PJI, however further mechanistic translational and clinical studies are warranted.

Evaluation of biofilm-forming abilities of Listeria monocytogenes (ATCC 19115) and efficacy of different washing methods for removal of biofilm on apple

Fresh produce can be contaminated at any stage along the food supply chain. In this study, apple was chosen to determine the time course of biofilm formation by Listeria monocytogenes (ATCC 19115), as well as to compare the efficacy of different household washing methods such as scrubbing with hands under running tap water, soaking with and without commercial vegetable wash with different treatment times in removing the biofilm formation by L. monocytogenes on apple surface. The biofilm formation was quantified using crystal violet assay and the result showed that L. monocytogenes took 18 hrs to form matured biofilm on apple surface. Besides, scrubbing apples with hands under running tap water for 30 s and 60 s were the most effective method which significantly removed (P<0.05) biofilm formed on the apple surface with approximately 5.93 log reduction. Soaking apples with vegetable wash for 5 mins and 10 mins were also found to be significantly effective (P<0.05) in reducing L. monocytogenes biofilm. Since L. monocytogenes can form matured biofilm on fresh produce, therefore efficient washing step is important before consuming fresh produce to lower the risk of foodborne illness.

Physicochemical Properties, Cytocompatibility and Antibiofilm Activity of a New Calcium Silicate Sealer

The aim of this study was to evaluate the physicochemical properties, cytocompatibility and antibiofilm activity of a new calcium silicate-based endodontic sealer, Sealer Plus BC (MK Life, Brazil), in comparison with TotalFill BC Sealer (FKG Dentaire SA, Switzerland) and AH Plus (Dentsply, Germany). Setting time and flow were evaluated based on ISO 6876 standard. The pH was evaluated after different periods, and radiopacity by radiographic analysis (mmAl). Solubility (% mass loss) and volumetric change (by micro-CT) were assessed after 30 days of immersion in distilled water. Cytocompatibility was assessed by methyltetrazolium (MTT) and neutral red (NR) assays, after exposure of Saos-2 cells to the sealer extract for 24 h. An additional analysis was performed by using MTT assay after 1, 3 and 7 days of exposure of Saos-2 to the sealers 1:8 dilution extracts. Antibiofilm activity against Enterococcus faecalis and/or Candida albicans was evaluated by crystal violet assay and modified direct contact test. The physicochemical properties were analyzed using ANOVA/Tukey tests; MTT and NR data were analyzed by ANOVA and Bonferroni tests; the antimicrobial tests were analyzed by Kruskal-Wallis and Dunn tests (α=0.05). Sealer Plus BC had proper setting time, radiopacity, flow and alkalization capacity. Sealer Plus BC was significantly more soluble than AH Plus (p<0.05) and presented volumetric change similar to AH Plus and TotalFill BC (p>0.05). Sealer Plus BC presented antibiofilm activity and no cytotoxic effect. In conclusion, although Sealer Plus BC had higher solubility, this sealer showed proper physicochemical properties, cytocompatibility, and antibiofilm activity.

New Evidence for Artemisia absinthium L. Application in Gastrointestinal Ailments: Ethnopharmacology, Antimicrobial Capacity, Cytotoxicity, and Phenolic Profile

Artemisia absinthium L. (Asteraceae) is traditionally used for gastrointestinal ailments and disorders linked to numerous risk factors including microbial infections. We aimed to provide contemporary evidence for its ethnopharmacological use and determine its antimicrobial capacity and mode of action, cytotoxicity, and phenolic constituents. Ethnopharmacological survey was conducted using semistructured interviews. Antimicrobial and antibiofilm capacities were determined by microdilution/crystal violet assay, respectively. Modes of action tested include estimation of exopolysaccharide production (congo red binding assay) and interference with membrane integrity (crystal violet uptake and nucleotide leakage assay). Cytotoxicity was determined using crystal violet assay. Polyphenolic profiling was done by advanced liquid chromatography/mass spectrometry (UHPLC-LTQ OrbiTrap MS). Artemisia absinthium in Serbia is traditionally used for gastrointestinal disorders, among others. Further study revealed high antifungal capacity of herb ethanolic extract towards range of Candida species (MIC 0.5–1 mg/mL) along with promising antibacterial activities (MIC 0.25–4 mg/mL). Interference with membrane integrity could be observed as a possible antimicrobial mechanism. Antibiofilm potential can be considered as high (towards C. krusei) to limited (towards P. aeruginosa) and moderate based on reduction in exopolysaccharide content. In concentrations up to 400 µg/mL, no cytotoxicity was observed towards HaCaT and HGF-1 cell lines. Polyphenolic analysis revealed twenty-one different constituents. A. absinthium usage as a gastrointestinal ailment remedy has been confirmed in vitro by its antimicrobial capacity towards microorganisms whose presence is linked to the diseases and associated complications and noncytotoxic nature of the natural product. The observed activities could be attributed to the present phenolic compounds.

Pyrogallol Induces Antimicrobial Effect and Cell Membrane Disruption on Methicillin Resistant Staphylococcus aureus (MRSA)

Background: Pyrogallol is present naturally in numerous plants and is also an important functional group in many polyphenol compounds. Objectives: The antibacterial activity, efficacy and mechanism of pyrogallol towards MRSA strains were evaluated in this study. Methods: Microbroth dilution method was used to determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). Time-kill kinetic assay was adopted to determine the killing pattern of pyrogallol towards MRSA. The antibacterial mechanism was determined using scanning electron microscopy (SEM), Fourier Transform-Infrared (FT-IR) spectroscopy and crystal violet assay. Results: Pyrogallol exhibited strong antibacterial activity against MRSA with MIC and MBC 15.6 µg/mL. Pyrogallol could inhibit the exponential growth of MRSA and kill the bacterial cells at higher concentrations. Pyrogallol was found targeting the cell membrane fatty acids, proteins/peptides, polysaccharides/carbohydrates and peptidoglycan of cell walls in the antibacterial mechanism. This has been confirmed through SEM, FT-IR spectroscopy and crystal violet assay. Conclusion : Overall, the findings suggest that pyrogallol has the potential to be used as antibiotics which are used to treat multidrug-resistant bacteria.

Coexistence of Virulence Factors and Efflux Pump Genes in Clinical Isolates of Pseudomonas aeruginosa: Analysis of Biofilm-Forming Strains from Iran

Background. Biofilm formation and efflux pumps (EPs) correlation play a critical role in the pathogenicity and antibiotic resistance of Pseudomonas aeruginosa. In this study, biofilm formation and EP’s collaborative role in clinical isolates of P. aeruginosa infection were investigated. Methods. Eighty-six (86) P. aeruginosa isolates were collected from different clinical specimens and were confirmed using different biochemical tests. The formation of biofilm was investigated by using a crystal violet assay. Also, EP genes were identified by the PCR method. Results. Based on the results, gentamicin-resistant (n = 50, 66.29%) and ciprofloxacin-resistant (n = 61, 69.66%) strains were the most frequent and colistin (n = 1, 1.12%) and ceftazidime (n = 12, 7.86%) resistant strains were the least prevalent. Furthermore, 22 isolates (31.42%) were MDR, and 11 isolates (12.35%) were XDR strains. Also, 19 isolates (22.47%) were classified as strong biofilm, 29 isolates (21.34%) as moderate biofilm, and 3 (11.23%) isolates as weak biofilm producers. The distribution of the EP genes was as follows: mexA (n = 44, 34.83%), mexB (n = 33, 32.58%), oprM (n = 59, 29.21%), oprD (n = 61, 30.33%), tetA (n = 22, 25.58%), tetR (n = 19, 22.09%), and emrE (n = 21, 24.41%). However, there was a strong significant association between biofilm formation and EPs in P. aeruginosa. Conclusions. In this study, we suggested that the presence of a multidrug resistance efflux pump, MexEF-OprN, significantly reduced P. aeruginosa pathogenicity. In contrast, the presence of the MexAB-OprM and MexCD-OprJ pumps did not affect virulence.

Active Rheumatoid Arthritis in a Mouse Model is not an Independent Risk Factor for Periprosthetic Joint Infection

Introduction Periprosthetic joint infection (PJI) represents a devastating complication of total joint arthroplasty associated with significant morbidity and mortality. Literature suggests a possible higher incidence of periprosthetic joint infection (PJI) in patients with rheumatoid arthritis (RA). There is, however, no consensus on this relative risk nor a well-defined mechanism of this purported risk. This study investigates how collagen-induced arthritis (CIA), a validated animal model of RA, impacts infectious burden in a well-established model of PJI. Methods Control mice were compared against CIA mice. Whole blood samples were collected to quantify systemic IgG levels via ELISA. Ex vivo respiratory burst function was measured via dihydrorhodamine assay. Ex vivo Staphylococcus aureus Xen36 burden was measured directly via colony forming unit (CFU) counts and crystal violet assay to assess biofilm formation. In vivo , surgical placement of a titanium implant through the knee joint and inoculation with S. aureus Xen36 was performed. Bacterial burden was then quantified by longitudinal bioluminescent imaging. Results Mice with CIA demonstrated significantly higher levels of systemic IgG compared with control mice (p = 0.003). Ex vivo, there was no significant difference in respiratory burst function (p=0.89) or S. aureus bacterial burden as measured by CFU counts (p=0.91) and crystal violet assay (p=0.96). In vivo , no significant difference in bacterial bioluminescence between groups was found at all postoperative time points. CFU counts of both the implant and the peri-implant tissue were not significantly different between groups (p=0.82 and 0.80, respectively). Conclusion This study demonstrated no significant difference in S. aureus infectious burden between mice with CIA and control mice. These results suggest that untreated, active RA may not represent a significant intrinsic risk factor for PJI. Further mechanistic translational and clinical studies are, however, warranted to thoroughly investigate the infectious risk of RA.

The Inhibitory Effects of Ficin on Streptococcus mutans Biofilm Formation

To investigate the effects of ficin on biofilm formation of conditionally cariogenic Streptococcus mutans (S. mutans). Biomass and metabolic activity of biofilm were assessed using crystal violet assay, colony-forming unit (CFU) counting, and MTT assay. Extracellular polysaccharide (EPS) synthesis was displayed by SEM imaging, bacteria/EPS staining, and anthrone method while acid production was revealed by lactic acid assay. Growth curve and live/dead bacterial staining were conducted to monitor bacterial growth state in both planktonic and biofilm form. Total protein and extracellular proteins of S. mutans biofilm were analyzed by protein/bacterial staining and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), severally. qRT-PCR was conducted to detect acid production, acid tolerance, and biofilm formation associated genes. Crystal violet assay, CFU counting, and MTT assay showed that the suppression effect of ficin on S. mutans biofilm formation was concentration dependent. 4 mg/mL ficin had significant inhibitory effect on S. mutans biofilm formation including biomass, metabolic activity, EPS synthesis, and lactic acid production ( p < 0.05 ). The growth curves from 0 mg/mL to 4 mg/mL ficin were aligned with each other. There was no significant difference among different ficin groups in terms of live/dead bacterial staining result ( p > 0.05 ). Protein/bacterial staining outcome indicated that ficin inhibit both total protein and biofilm formation during the biofilm development. There were more relatively small molecular weight protein bands in extracellular proteins of 4 mg/mL ficin group when compared with the control. Generally, ficin could inhibit biofilm formation and reduce cariogenic virulence of S. mutans effectively in vitro; thus, it could be a potential anticaries agent.

Biofilm formation, icaABCD genes and agr genotyping of Staphylococcus aureus from fish and ground beef

A total of 46 Staphylococcus aureus isolates from fish and ground beef were tested for the agr types, icaABCD genes, and biofilm formation at 12, 25 and 37 °C by the microtiter plate and the MTT assays. All isolates were positive for the icaABD genes, while 97.8% were positive for the icaC. All isolates produced biofilms at 37 and 25 °C, but 93.5% of them were also biofilm producers at 12 °C. There was no significant difference in biofilm formation between 25 and 37 °C using the crystal violet assay (P > 0.05). However, statistically significant differences were detected between 12 and 25 °C as well as 12 and 37 °C (P < 0.05). All isolates were significantly different in biofilm production by the MTT assay at all tested temperatures. Furthermore, a relationship between the presence of the icaABCD genes and biofilm formation was observed. The agr type I was the most prevalent (54.4%) among the isolates, followed by agr type II (41.3%) and agr type III (9.6%). In this study, the S. aureus isolates exhibited biofilm formation ability responsible for persistence of bacteria in foods, which may lead to food spoilage and human health problems.