Polystyrene Microtiter Plates Research Articles

Evaluation of Antimicrobial Activity of Selected Plant Extracts against Staphylococcus Strains Isolated from Wound Infections.

Wound infections are a significant medical problem, with Staphylococcus species being one of the most common etiological factors. Treatment is complex due to bacterial antibiotic resistance and the ability to form a biofilm. The aim of this study was to determine the drug susceptibility of the clinical isolates of Staphylococcus species obtained from wound infections, assess their ability to form a biofilm in vitro, evaluate the level of extracellular slime synthesis, and test the antistaphylococcal properties of selected plant extracts against plate-cultured bacteria and activity against mature biofilms. A total of 20 Staphylococcus strains were evaluated. The antibiotic susceptibility and sensitivity of the strains to the tested extracts were determined using the disc diffusion method. The production of extracellular bacterial slime was assessed using Congo Red agar plates. The biofilm formation and the effect of plant extracts on the biofilm layer were examined using the MTT method on polystyrene microtiter plates. Diverse drug susceptibility profiles, slime production ability, and in vitro biofilm formation were observed among the tested strains. It was found that aqueous extracts from the Serpylli herba, Arctii lappae folium, Taraxaci folium, and Galii aparini herba showed antibacterial activity against some of the tested strains. In contrast, their antibiofilm activity was not confirmed. Among all tested preparations, the most promising antimicrobial extracts in both planktonic cultures and biofilm were Thymus serpyllum herb and Taraxacum officinale leaf extracts. These results allowed us to conclude their potential application in medicine to support the treatment of challenging wound conditions.

Comparative Adhesion of Pseudomonas aeruginosa to Human Oral Mucosal Epithelial Cells and Polystyrene Surfaces

Background: The adhesion of bacteria to biotic and abiotic surfaces reflects their ability to cause infectious diseases. The distinction between the ability of Pseudomonas aeruginosa bacteria to adhere to biotic and abiotic surfaces is not clear in the literature. Objectives: The current study aims to shed light on the extent of similarities and differences between P. aeruginosa in terms of their ability to adhere to different biotic and abiotic surfaces. Materials and Methods: ten isolates of P. aeruginosa were isolated from 100 wound and burn swabs. The isolates were identified using biochemical and phenotypic tests in addition to VITIK-II technology. The susceptibility to antibiotics was estimated using the disc diffusion method. The microtiter-spectrophotometric was used to measure the biofilm formation on polystyrene microtiter plates. The Human oral mucosal epithelial (OMECs) were used to evaluate the adhesion of P. aeruginosa isolates. Plate count and direct bacterial count were used to count the adhered bacteria to human OMECs in vitro. Results: Norfloxacin showed the highest antibacterial effect while the lowest antibacterial effect was seen in the case of using when Amoxicillin and Cefixime (all isolates were resistant). All isolates from, Pa2 form the highest biofilm followed by Pa6, while the lowest biofilm formation was seen in the case of Pa4. While Pa 6 showed the highest ability to attach to human OMECs followed by Pa2. Conclusion: It can be concluded that P. aeruginosa isolates are resistant to most antibiotics and that their ability to adhere and biofilm formation depends on the type of surface.

Effect of Piper Cubeba Extract on Biofim Producing Pseudomonas Aeruginosa Isolated from Clinical Samples

Background: The species of Pseudomonas aeruginosa is a nosocomial bacterium that increases the risk of multidrug-resistant infections, especially in vulnerable patients. The plant Piper cubeba, commonly referred to as cubeb or tailed pepper, it is specifically cultivated for its fruit and essential oil. The plant belongs to the genus Piper. The study aims at assessing the effectiveness of Piper cubeba extract against different P. aeruginosa strains that could produce biofilm and carry blaIMP gene. Method: Isolates from 85 samples (17 sputum, 40 urine and 28 diarrhea samples). Collected from Al Nasiriyah Teaching Hospital during the period of December 2021 to March 2022. Samples were cultivated using routine cultivation methods, catalase test, oxidase reaction, and the Api20E system were performed to identify P. aeruginosa. Results: Out of a total of 17 isolated samples of Pseudomonas aeruginosa, 3 (17.64%) were from sputum, 6(35.29%) from urine, and 8 (47.05%) diarrhea samples. Out of 17 isolates 6 were sensitive in stool isolates 4 isolates were moderate sensitive and 7 isolates were resistance. Conclusion at concentrations of 25, 50, 75, and 100 mg/mL for Piper cubeba, respectively, the greatest MBC of plant extracts was noted. Also, all the isolates were capable of producing a biofilm by using presterilized 96-well polystyrene microtiter plates. Out of 17 isolates, 9 had a potent biofilm ability and the remaining 7 were moderate biofilm producers. Additionally, 7 of the isolates (41.17%) carried the blaIMP gene. Conclusion: Piper cubeba extract has potential as an antimicrobial agent against both sensitive and moderately resistant strains of Pseudomonas aeruginosa. This antimicrobial effect becomes more pronounced with an increase in the concentration of the extract. Additionally, Pseudomonas aeruginosa that produce biofilms tend to consistently produce more Metallo-beta-lactamase (MBL), which can further contribute to antibiotic resistance.

Capacidade de formação de biofilme de Salmonella enterica subsp. enterica proveniente de fábricas de rações para suínos

ABSTRACT: Animal feed has been considered an important vehicle for introducing Salmonella enterica subsp. enterica in pig farms. Salmonella survival and persistence in feed mill environments have been associated with biofilm-forming ability. This study evaluated 54 Salmonella isolates from swine-feed mills for: i. phenotypic expression of curli fimbriae and cellulose; ii. pellicle formation at the air-liquid interface; iii. adhesion on polystyrene microtiter plates; and iv. the presence of the main genes associated with biofilm formation. Regarding phenotypic cell morphology assays, all Salmonella isolates presented morphotype RDAR at 28 °C and SAW at 37 °C. Rigid pellicle formation at the air-liquid interface was observed in 51.85% (28/54), while fragile pellicle was seen in 18.52% (10/54), and 29.62% (16/54) were not able to produce pellicle. Biofilm quantification on polystyrene microtiter plates showed that 98.15% (53/54) of Salmonella isolates were able to form biofilms at 28 °C, while 83.33% (45/54) of the isolates were classified as non-adherent at 37 °C. The genes csgD, fimA, adrA, and bapA were found in all isolates evaluated. These results indicated that Salmonella serovars from swine-feed mills have the biofilm-forming ability.

New Amino-Anthracene-9, 10-Dione Derivatives: Synthesis and Pharmacological Evaluation as Powerful Neuroprotective and Antidepressant Agents

The monoamine oxidase (MAO) enzyme resides in the outer mitochondrial membranes of all body cells, including the ones found in the brain, liver, and intestinal mucosa. Dopamine, serotonin, norepinephrine, tyramine, and tryptamine are extrinsic and indigenous amines that MAO oxidatively deaminates. MAO-A has been correlated with depression and other mental health issues, while MAO-B has been associated with the conditions Alzheimer’s and Parkinson’s disease. In a targeted assessment of naturally occurring anthraquinones, two isoforms of recombinant human MAOs were utilized. The inhibitory effects of purpurin and alizarin on MAO-A were observed, with calculated IC50 values of 2.50 and 30.1 M, respectively. This research on anthraquinones, purpurin, and alizarin offers promising new information that might eventually be used as a lead molecule in the discovery of the unique synthetic anthraquinones, anthracene 9, 10-dione compounds 1 to 9. On 96-well black polystyrene microtiter plates, both MAOs inhibiting action of 9 distinct synthetic anthracene 9,10-dione compounds has been evaluated. Compared to clorgyline, compounds 1, 2, 5, 8, and 9 inhibit MAO-A significantly, while compounds 1, 3, 5, 8, and 9 considerably inhibit MAO-B. Significant findings indicate that these compounds may be beneficial When employed to treat depression owing to their intense selective MAO-B activity and antidepressant effects as a result of their selective MAO-inhibition.

Identification, proteolytic activity quantification and biofilm-forming characterization of Gram-positive, proteolytic, psychrotrophic bacteria isolated from cold raw milk.

Psychrotrophic bacteria of raw milk face the dairy industry with significant spoilage and technological problems due to their ability to produce heat-resistant enzymes and biofilms. Despite extensive information about Gram-negative psychrotrophic bacteria in milk, little is known about Gram-positive psychrotrophic bacteria in milk, and their proteolytic activity and biofilm-forming characteristics. In the present study, Gram-positive, proteolytic, psychrotrophic bacteria of cold raw milk were identified, and their proteolytic activity and biofilm-forming capacity were quantified. In total, 12 genera and 22 species were represented among the bacterial isolates, however 50% belonged to three genera, namely Staphylococcus (19.4%), Bacillus (16.7%), and Enterococcus (13.9%). Different levels of proteolytic activity were detected in the identified isolates, even among the strains belonging to the same species. In addition, proteolytic activity was significantly higher at 25°C than at 7°C for all isolates. The crystal violet staining assay in polystyrene microtitre plates revealed a high level of variation in the biofilm-forming capacity at 7°C. After 72 hours of incubation, 11.1% of the strains did not produce a biofilm, while 27.8%, 52.8%, and 8.3% produced low, moderate, and high amounts of biofilm on polystyrene, respectively. The psychrotrophic bacteria were also able to produce biofilms on the surface of stainless steel coupons in ultra-high temperature milk after 72 h of incubation at 7°C; the number of attached cells ranged from 1.34 to 5.11 log cfu/cm2. These results expand the knowledge related to the proteolytic activity and biofilm-forming capacity of Gram-positive psychrotrophic milk bacteria.

Bioactive Compounds from P. pertomentellum That Regulate QS, Biofilm Formation and Virulence Factor Production of P. aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen responsible for many nosocomial infections. This bacterium uses Quorum Sensing (QS) to generate antimicrobial resistance (AMR) so its disruption is considered a novel approach. The current study describes the antibiofilm and QS inhibitory potential of extract and chemical components from Piper pertomentellum. The methodo- logy included the phytochemical study on the aerial part of the species, the determination of QS inhibition efficacy on Chromobacterium violaceum and the evaluation of the effect on biofilm formation and virulence factors on P. aeruginosa. The phytochemical study led to the isolation and identification of a new piperamide (ethyltembamide 1), together with four known amides (tembamide acetate 2, cepharadione B 3, benzamide 4 and tembamide 5). The results indicated that the ethanolic extract and some fractions reduced violacein production in C. violaceum, however, only the ethanolic extract caused inhibition of biofilm formation of P. aeruginosa on polystyrene microtiter plates. Finally, the investigation determined that molecules (1-5) inhibited the formation of biofilms (50% approximately), while compounds 2-4 can inhibit pyocyanin and elastase production (30-50% approximately). In this way, the study contributes to the determination of the potential of extract and chemical constituents from P pertomentellum to regulate the QS system in P. aeruginosa.

Effect of subinhibitory doses of rifaximin on in vitro Pseudomonas aeruginosa adherence and biofilm formation to biotic and abiotic surface models.

The adhesion of Pseudomonas aeruginosa to biotic and abiotic surfaces is responsible for the persistence and development of bacterial infection. To fill the gap in the knowledge regarding the relationship between rifaximin susceptibility and biofilm formation, and to investigate the effect of subinhibitory doses of rifaximin on the adhesion and biofilm formation. A total of 10 isolates of P. aeruginosa were obtained from 110 urine samples of urinary tract infection (UTI) patients. Biofilm formation on polystyrene microtiter plates, minimum inhibitory concentrations (MICs) of rifaximin against the 10 isolates of P. aeruginosa (Pa1-Pa10), the effect of sub-MICs of rifaximin (0.5 × MIC, 0.25 × MIC, 0.125 × MIC, and 0.06 × MIC) on biofilm formation by the Pa4 isolate to polystyrene microtiter plates, and the adhesion to human epithelial cells (HECs) in vitro were evaluated. The MICs of rifaximin against 10 isolates ranged from 62.5 μg/mL to 1000 μg/mL. The Pa4 isolate produced the highest level of biofilm formation, while the MIC of Pa4 was 125 μg/mL. There was no correlation between bacterial susceptibility to rifaximin and biofilm formation (r: -0.016; p > 0.05). Sub-MIC doses of rifaximin significantly reduced the biofilm formation on abiotic surfaces, while only 0.5 × MIC, 0.25 × MIC and 0.12 × MIC of rifaximin reduced the adhesion to HECs significantly (p < 0.05) in a dose-dependent manner. This pioneering study demonstrated the negative effect of sub-MIC doses of rifaximin on biofilm formation and adhesion to abiotic and biotic surfaces in vitro.

DETECTION AND QUANTIFICATION OF BIOFILM FORMED BY CANDIDA SPECIES ISOLATED FROM CLINICAL SAMPLES AT TERTIARY CARE HOSPITAL.

Candida speciesis a part of commensalin healthy humans but they can cause opportunistic infections, especially in immunocompromised individuals, patient admitted in ICUs(Intensive care units) and HIV patients. biofilm act as protective shield of microorganism. Candida species form most common fungal biofilm, which is extremely difficult to treat. Purpose: The biofilm forming candida is difficult to eradicate with usual antifungal drug and often cause chronic infections. Understanding of biofilm process is very important for effective control strategies of biofilm associated infections and improvement in patient management. Method:The observational cross sectional prospective study was conducted on forty patients samples including urine, BAL/sputum, blood culture, body fluids, pus, swab, indwelling devicesand tissue showing candida species growth. Biofilm was detected using pre-sterilized 96 well polystyrene microtitre plate method. Results: Among 40 isolates, 22 isolates were detected positive, whereas 18 isolates were negative for biofilm formation. C. tropicalis had formed maximum strong biofilms among all species isolated. These biofilm acts resisting antifungal treatment and withstanding the competitive pressure from other organisms, these are difficult to treat.Changing trend with shift toward non albicans. Conclusion:Candida tropicalis as the predominant pathogen causing candidia infectionsand it had highest capacity to form biofilm.

Biofilm formation by selected microbial strains isolated from wastewater and their consortia: mercury resistance and removal potential

Wastewater often contains an increased amount of mercury and, at the same time, resistant microorganisms. During wastewater treatment, a biofilm of indigenous microorganisms is often unavoidable. Therefore, the objective of this research is to isolate and identify microorganisms from wastewater and investigate their ability to form biofilms for possible application in mercury removal processes.The resistance of planktonic cells and their biofilms to the effects of mercury was investigated using Minimum Biofilm Eradication Concentration-High Throughput Plates. The formation of biofilms and the degree of resistance to mercury were confirmed in polystyrene microtiter plates with 96 wells. Biofilm on AMB Media carriers (Assisting Moving Bad Media) was quantified using the Bradford protein assay. The removal of mercury ions by biofilms formed on AMB Media carriers of selected isolates and their consortia was determined by a removal test in Erlenmeyer flasks simulating MBBR.All isolates in planktonic form showed some degree of resistance to mercury. The most resistant microorganisms (Enterobacter cloacae, Klebsiella oxytoca, Serratia odorifera, and Saccharomyces cerevisiae) were tested for their ability to form biofilms in the presence and absence of mercury, both in polystyrene plates and on ABM carriers. The results showed that among planktonic forms, K. oxytoca was the most resistant. A biofilm of the same microorganisms was more than 10-fold resistant. Most consortia biofilms had MBEC values > 100,000 μg/mL. Among individual biofilms, E. cloacae showed the highest mercury removal efficiency (97.81% for 10 days). Biofilm consortia composed of three species showed the best ability to remove mercury (96.64%–99.03% for 10 days).This study points to the importance of consortia of different types of wastewater microorganisms in the form of biofilms and suggests that they can be used to remove mercury in wastewater treatment bioreactors.

Antibiofilm activity of LAE (ethyl lauroyl arginate) against food-borne fungi and its application in polystyrene surface coating

Several filamentous fungi species as Fusarium oxysporum or Cladosporium sp. can form biofilms by themselves or by participating in polymicrobial biofilms with bacteria. However, despite the high impact of biofilm on the food industry and the high efforts done to control biofilm produced by bacteria in the food area, there has been little study of strategies to control fungal biofilm in this area. In this study, the antibiofilm activity of the safe antimicrobial compound ethyl lauroyl arginate (LAE) was investigated against food spoilage fungi (Cladosporium cladosporioides, Aspergillus ochraceus, Penicillium italicum, Botrytis cynerea and Fusarium oxyspoum). Finally, the efficacy of a varnish-based coating incorporating LAE and coated onto polystyrene microtiter plates has been evaluated as a strategy to reduce fungal biofilm formation. The results of the 2,3-bis-(2-metoxi-4-nitro-5-sulfofenil)-2H-tetrazoilo-5-carboxanilida (XTT) assay, which measure the biofilm metabolic activity of moulds, demonstrated that LAE reduced significantly the formation of fungal biofilm at concentrations from 6 to 25 mg/L. This reduction was confirmed by the micrographs obtained by scanning electronic microscopy (SEM). In addition, LAE also showed antifungal activity against established biofilms. Particularly, it reduced their metabolic activity and viability at concentrations from 6 to 25 mg/L according to results obtained in the XTT assay and observations made by confocal laser scanning microscopy (CLSM). Finally, active coating incorporating from 2% of LAE proved to reduce significantly the biofilm formation in C. cladosporioides, B. cynerea and F. oxyspoum according to the results obtained in the XTT assay. However, the released studies indicated that the retention of LAE in the coating should be improved to prolong their activity.

PREVALENCE, PHENOTYPIC-GENOTYPIC RESISTANCE AND BIOFILM FORMATION OF Staphylococcus aureus IN CHICKEN MEAT WITH REFERENCE TO ITS PUBLIC HEALTH HAZARD

The purpose of this study was to determine the prevalence, antimicrobial resistance in S. aureus and presence of enterotoxigenic as well as biofilm-forming genes in S. aureus bacteria isolated from broiler meat from traditional shops and supermarkets in Sharkia, Egypt. For this determination, two hundred fresh raw chicken meat cotton swabs were collected from the breast and thighs (100 each). S. aureus was highlighted through a coagulase test, and then phenotypic and genotypic characterizations were studied. Uniplex PCR was used to identify the occurrence of enterotoxin genes in the selected isolates. Finally, they were subjected to a biofilm formation test using 96-well flat bottom polystyrene microtiter plates; besides, biofilm-forming genes were investigated. Nineteen isolates out of the 200 samples tested positive for S. aureus (9.5 percent), No Vancomycin resistance strains were obtained, nor did any ciprofloxacin isolates for S. aureus while, all isolates were 100 % resistant for streptomycin, amoxicillin- clavulanic acid and sulfamethoxazole-trimethoprim S. aureus isolates were found resistant to at least one antibiotic. The multiple antibiotic resistance (MAR) index of S. aureus isolates was ranged from 0.3 to 0.7 with an average of 0.4. In The mecicillin resistant S.aureus (MRSA) strains carried mecA gene with a percentage of 5%, while the blaZ gene was distributed with a percentage of 5.5%, 11/200). The obtained isolates gave 60% strong biofilm formation and 40% were non-formers, with nil results for moderate or weak production isolates. The icaA gene was 100%, in comparison to icaD which was zero. It should be noted that strong biofilm former strains were only 100% positive for sea and seb. This study pointed out the higher prevalence of MDR isolates of S. aureus isolates in chicken meat due to inadequate handling and insufficient sanitary equipment and post microbial contamination. This finding highlighted the importance of broiler meat as a reservoir for antimicrobial-resistant strains of S. aureus and biofilm-forming of Staphylococcus aureus.

WOUND HEALING AND ANTIFUNGAL EVALUATIONS OF SOME SURVEYED PLANTS OF GWADABAWA/ILLELA COMMUNITIES OF SOKOTO STATE-NIGERIA

Objective: This study aimed to evaluate the wound healing and antifungal properties of five earlier surveyed plants of Gwadabawa and Illela communities of Sokoto State-Nigeria cited for their acclaimed therapeutic purpose. Methods: Air-dried plant parts species for each plant were extracted by maceration using methanol. These were concentrated in vacuo to obtain crude methanolic extract (CME). The CME was partly defatted and partitioned with n-butanol to solubilize bioactive compounds in the organic phase, leaving sugars, amino acids, and salty compounds in the aqueous phase. The resulting concentrated n-butanol extracts were then screened by subjecting each extract to in vitro antifungal assay for the determination of the minimum inhibitory concentration (MIC) on Aspergillus niger in a 96-well flat bottom polystyrene microtiter plate using the broth microdilution method as outlined in the 2021 Clinical and Laboratory Standard Institute guideline; while the CME above for each plant, was subjected to wound healing assay using the wound excision model. Results: Our findings showed all five plant extracts were active with variable antifungal properties of MIC values ranging from 250 to 3.9 (mg/ml). The lowest activity was recorded for Waltheria indica, (with a MIC of 125 mg/ml), while the highest activity was indicated for Faidherbia albida (with a MIC of 3.90625 mg/ml). The other three extracts exhibited moderate activity at test concentration with a MIC of 7.8125 mg/ml. Similarly, the CME showed a comparable wound healing effect for all the plant extracts concerning the control groups (5 mg/ml povidone Iodine, PI as positive control and distilled water, DW as negative control). Increased tissue contraction of lesions on the excised skin of rats was observed to significantly differ accordingly based on the applied treatment with the graded doses of the test sample concentrations used (1.25, 2, and 5) mg/ml. Epithelial closure in all the rats occurred after 14 d, and more so, on the 16th 16th-day wounding, the wounds were almost scarless, while those of blank control (DW) had obvious scars. Conclusion: The wound healing and antifungal potentials of the crude extracts of the selected plants were confirmed. Thus, the five screened plant extracts may possibly be further investigated and developed into drugs for topical treatment of fungal-infected wounds, in line with their earlier folklore documentation.

High Virulence and Multidrug Resistance of Escherichia coli Isolated in Periodontal Disease.

Periodontal disease is caused by different gram-negative anaerobic bacteria; however, Escherichia coli has also been isolated from periodontitis and its role in periodontitis is less known. This study aimed to determine the variability in virulence genotype, antibiotic resistance phenotype, biofilm formation, phylogroups, and serotypes in different emerging periodontal strains of Escherichia coli, isolated from patients with periodontal disease and healthy controls. E. coli, virulence genes, and phylogroups, were identified by PCR, antibiotic susceptibility by the Kirby-Bauer method, biofilm formation was quantified using polystyrene microtiter plates, and serotypes were determined by serotyping. Although E. coli was not detected in the controls (n = 70), it was isolated in 14.7% (100/678) of the patients. Most of the strains (n = 81/100) were multidrug-resistance. The most frequent adhesion genes among the strains were fimH and iha, toxin genes were usp and hlyA, iron-acquisition genes were fyuA and irp2, and protectin genes were ompT, and KpsMT. Phylogroup B2 and serotype O25:H4 were the most predominant among the strains. These findings suggest that E. coli may be involved in periodontal disease due to its high virulence, multidrug-resistance, and a wide distribution of phylogroups and serotypes.

Salmonella Derby from pig production chain over a 10-year period: antimicrobial resistance, biofilm formation, and genetic relatedness.

The aim of this study was to evaluate 140 Salmonella Derby isolates collected over a 10-year period from porcine origins (environment, pig carcass, lymph nodes, intestinal content, and pork) for their phenotypic and genotypic antimicrobial resistance, their ability to produce biofilm, and their genetic relatedness. The minimum inhibitory concentration (MIC) was determined using microdilution broth method and antimicrobial resistance genes were investigated by PCR. The quantification of biofilm formation was performed in sterile polystyrene microtiter plates. Genetic relatedness was determined by Xba-I macrorestriction analysis. The highest frequencies of non-wildtype (nWT) populations were observed against tetracycline (75.7%), streptomycin (70%), and colistin (11.4%), whereas wildtype populations were observed against ciprofloxacin, ceftazidime, and gentamicin. The resistance genes found were blaTEM (ampicillin), aadA variant (streptomycin/spectinomycin), tetA (tetracycline), and floR (florfenicol). On 96-well polystyrene microtiter plate, 68.6% of the isolates proved to be biofilm producers. Among 36 S. Derby isolates selected to PFGE analysis, 22 were clustered with 83.6% of similarity. Additionally, 27 isolates were clustered in 11 pulsotypes, which presented more than one strain with 100% of similarity. Most of S. Derby isolates were able to form biofilm and were classified as nWT or resistant to tetracycline, streptomycin, and colistin. PFGE allowed the identification of closely related S. Derby isolates that circulated in pig slaughterhouses and pork derived products along a decade.

Hypervirulence and Multiresistance to Antibiotics in Klebsiella pneumoniae Strains Isolated from Patients with Hospital- and Community-Acquired Infections in a Mexican Medical Center.

Klebsiella pneumoniae is a pathogenic bacterium associated with different infectious diseases. This study aimed to establish the different association profiles of virulence genes related to the hypermucoviscous phenotype (HM), capsular serotypes, biofilm formation, and multidrug resistance in K. pneumoniae strains from patients with hospital- and community-acquired infections. K. pneumoniae virulence genes and capsular serotypes were identified by PCR, antibiotic susceptibility by the Kirby–Bauer method, HM by the string test, and biofilm formation by measurement in polystyrene microtiter plates. Of a total of 150 strains from patients with hospital- (n = 25) and community-acquired infections (n = 125), 53.3% (80/150) were HM-positive and 46.7% (70/150) were HM-negative. HM-positive (68/80) and HM-negative (67/70) strains were biofilm-forming. Moreover, 58.7% (47/80) HM-positive and 57.1% (40/70) HM-negative strains were multidrug-resistant. Among HM-positive, HM-negative, and serotypes K1 (25/150), K2 (48/150), and non-K1/K2 strains, (77/150) the frequently detected adhesion genes were fimH, mrkD, ycfM, and kpn; entB, irp2, irp1, and ybtS, for iron acquisition; and rmpA for protectins. The gene association pattern fimH/kpn/mrkD/ycfM/entB/irp1/irp2/ybtS/fyuA (18/150) was frequent among the strains. K. pneumoniae strains from patients with hospital- and community-acquired infections demonstrated a wide diversity of virulence gene profiles related to phenotype (hypermucoviscosity, multidrug resistance, and biofilm formation) and serotypes.

P103 Role of biofilm production in recalcitrant tinea

Poster session 1, September 21, 2022, 12:30 PM - 1:30 PMObjectiveTo determine the role of biofilm production in dermatophytic isolates from tinea infections of recalcitrant skin lesions of study patients.MethodsAn observational study conducted in UCMS and GTB Hospital Delhi, in forty clinically diagnosed and mycologically confirmed cases of recalcitrant tinea infection of glabrous skin to analyze the role of biofilm production in dermatophytes.After taking written informed consent from the study population sample collection (skin scraping) was done.The scraping was then mounted in 10% potassium hydroxide (KOH) for direct microscopic examination followed by culture on Sabouraud Dextrose Agar (SDA) media with antibiotics (Chloramphenicol, Gentamicin, Cycloheximide).The fungal growth was then subjected to LPCB mount (Lactophenol cotton blue).The isolates were allowed to form in-vitro biofilms on polystyrene microtiter plates.Quantification of biofilm biomass was done using crystal violet staining and measuring the optical density (OD) at 570 nm and classified as non-adherent/non-producer, weak moderate, and strong biofilm producers.ResultsTinea corporis and cruris were the most common clinical types of dermatophytosis. T. mentegrophytes-complex was the most common dermatophyte isolated from the clinical specimens.Majority (86.84%) of isolates formed strong (OD >4 ODc) biofilms.ConclusionThere has been an increase in the incidents of chronic and recalcitrant dermatophytosis of skin.The predominance of T. mentegrophytes-complex as observed in our study highlights the importance of the pathogen in causation of current and chronic and recalcitrant dermatophytosis in India.High rate of in-vitro strong biofilm formation by the isolates indicates that these organisms might be forming biofilms in-vivo leading to chronicity and poor response to therapy.

Synthesis, optimization, and physicochemical characterization of selenium nanoparticles from polysaccharide of mangrove Rhizophora mucronata with potential bioactivities

BackgroundSelenium (Se) is considered an important micronutrient for human and animal health having potential biological functions and it is required for the prevention and treatment of diseases. The present study was designed to optimize the synthesis conditions of selenium nanoparticles by the Taguchi design method using sodium selenite as a precursor, ascorbic acid as a reductant, and Rhizophora mucronata leaves polysaccharide (RMLP) as a stabilizing agent. MethodsIn the present studies, RMLP-SeNPs were synthesized by using a polysaccharide (RMLP) that was previously isolated from mangrove Rhizophora mucronata leaves. The synthesized RMLP-SeNPs were characterized by UV-Vis, HR-TEM, DLS, FTIR, and TGA-DSC analysis. In addition to this, in vitro antibacterial activity of RMLP-SeNPs was determined by using the disk diffusion method according to the standard protocols. Antibiofilm activity of RMLP-SeNPs was determined by 96-wells polystyrene microtiter plate methods. Moreover, in vitro, antioxidant capacity such as ABTS, DPPH, and reducing power of RMLP-SeNPs was evaluated. ResultsThe result demonstrates that the optimal condition for the synthesis of RMLP-SeNPs was 2 h of reaction time, 0.04 M of Vc concentration, 1 mg/mL of RMLP, and pH 7.5. The particle size of synthesized RMLP-SeNPs was around 54.85 nm under optimal conditions by DLS measurement. HR-TEM results showed that RMLP-SeNPs were a uniform spherical shape with an average size of 31.82 nm. UV-vis analysis showed the synthesis of RMLP-SeNPs with a characteristic peak at 265 nm which was further confirmed by FTIR and EDX spectrum. Further, the RMLP-SeNPs showed excellent scavenging capacity on DPPH, ABTS, and reducing power as compared to other forms of selenium. Moreover, the RMLP-SeNPs show good antimicrobial and antibiofilm activity against tested strains such as Aeromonas hydrophila, Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa and exhibited a lesser toxic effect in brine shrimp lethality assay. ConclusionThese findings provide an opportunity to use selenium nanoparticles in food industries, biomedical, cosmetics, and pharmaceutical applications, especially to develop novel antioxidant and antimicrobial compounds.

Enterococcus faecalis Shields Porphyromonas gingivalis in Dual-Species Biofilm in Oxic Condition.

Aim: To develop a reproducible biofilm model consisting of Enterococcus faecalis (E. faecalis) and Porphyromonas gingivalis (P. gingivalis) and to evaluate the interaction between the two bacterial species. Methodology: E. faecalis and P. gingivalis were grown in mono-culture, sequential, and co-culture models for 96 h in a 96-well polystyrene microtiter plate under both aerobic and anaerobic conditions separately. The viability of the two bacterial species in the biofilms was quantified by polymerase chain reaction (qPCR). Biofilm thickness and protein contents were measured using confocal laser scanning microscopy (CLSM). Two-way analysis of variance (ANOVA) was performed to analyze cell viability and biofilm thickness among different culture models cultivated under either aerobic or anaerobic conditions. The level of significance was set at p < 0.05. Results: Different culture models tested did not show any significant difference between the viable cell counts of both E. faecalis and P. gingivalis cultivated under aerobic and anaerobic conditions (p > 0.05). Biofilm was significantly thicker (p < 0.05) in the co-culture models compared to the mono-culture and sequential models. Protein contents in the biofilms were more pronounced when both bacterial species were co-cultured under aerobic conditions. Conclusions: E. faecalis appeared to shield P. gingivalis and support its continued growth in oxic (aerobic) conditions. The co-culture model of E. faecalis and P. gingivalis produced a significantly thicker biofilm irrespective of the presence or absence of oxygen, while increased protein contents were only observed in the presence of oxygen.

Inhibitory Effect of Bacillus licheniformis Strains Isolated from Canine Oral Cavity.

Bacillus licheniformis is used in a broad spectrum of areas, including some probiotic preparations for human and veterinary health. Moreover, B. licheniformis strains are known producers of various bioactive substances with antimicrobial and antibiofilm effects. In searching for new potentially beneficial bacteria for oral health, the inhibitory effect of B. licheniformis strains isolated from canine dental biofilm against pathogenic oral bacteria was evaluated. The antimicrobial effect of neutralized cell-free supernatants (nCFS) was assessed in vitro on polystyrene microtiter plates. Furthermore, molecular and morphological analyses were executed to evaluate the production of bioactive substances. To determine the nature of antimicrobial substance present in nCFS of B. licheniformis A-1-5B-AP, nCFS was exposed to the activity of various enzymes. The nCFS of B. licheniformis A-1-5B-AP significantly (p < 0.0001) reduced the growth of Porphyromonas gulae 3/H, Prevotella intermedia 1/P and Streptococcus mutans ATCC 35668. On the other hand, B. licheniformis A-2-11B-AP only significantly (p < 0.0001) inhibited the growth of P. intermedia 1/P and S. mutans ATCC 35668. However, enzyme-treated nCFS of B. licheniformis A-1-5B-AP did not lose its antimicrobial effect and significantly (p < 0.0001) inhibited the growth of Micrococcus luteus DSM 1790. Further studies are needed for the identification of antimicrobial substances.