Biofilm Formation and Phospholipase and Proteinase Production in Cryptococcus neoformans Clinical Isolates and Susceptibility towards Some Bioactive Natural Products.

Background: Cryptococcosis is one of the most common fungal infections in immunocompromised patients, which is caused by Cryptococcus neoformans. However, relatively little is known about the virulence factors of C. neoformans and the incidence of antifungal drug resistance in C. neoformans is rapidly increasing. This study was undertaken to investigate the virulence factors in C. neoformans, thymol, curcumin, piperine, gallic acid, eugenol, and plumbagin for their potential antimicrobial activity against C. neoformans. Methods: The production of phospholipase and proteinase was detected using standard methods. Biofilm formation was determined using the microtiter plate method. The broth microdilution method was used to determine the antifungal activity. The antibiofilm activity was assessed using the safranin staining method. Results: All isolates of C. neoformans produced biofilms with optical density values ranging from 0.16 to 0.89. A majority of C. neoformans isolates that were tested exhibited strong phospholipase (7/8) and proteinase (5/8) production. Plumbagin (with minimum inhibitory concentration values ranging from 4 to 16 µg/mL) showed the highest antifungal activity followed by thymol (with minimum biofilm inhibitory concentration values ranging from 8 to 64 µg/mL). In addition, plumbagin showed the highest antibiofilm activity with minimum biofilm inhibitory concentration and minimum biofilm eradication concentration values ranging from 4 to 16 µg/mL and 32 to 256 µg/mL, respectively. Conclusion: Plumbagin, compared to other natural products studied, was the most efficient in terms of antifungal and antibiofilm activities. Hence, plumbagin could be used in combination with antifungals for the development of new anticryptococcal drugs.

Biofilm Formation, Pyocyanin Production, and Antibiotic Resistance Profile of Pseudomonas aeruginosa Isolates from Wounds.

Pseudomonas aeruginosa is one of the most frequently resistant and dangerous bacteria isolated from infected wounds of patients. This study aimed to determine the prevalence of P. aeruginosa from infected wounds of patients in the Dschang District Hospital to evaluate their antibiotic susceptibility profiles and their ability to swarm and swim and correlate pyocyanin production with biofilm formation. Wound swab samples were collected and the identification of P. aeruginosa was performed using microbiological and biochemical tests. Their antimicrobial susceptibility was determined by the broth microdilution method. Swarming and swimming were determined by measuring the diameters of motility in semisolid/low-viscosity media. Furthermore, pyocyanin production and biofilm formation were evaluated spectrophotometrically using a microtiter plate. The prevalence of P. aeruginosa from infected wounds in our study population was 26%. All P. aeruginosa isolates were resistant to streptomycin and paromomycin, and the frequency of multidrug resistance (MDR) was 65.8%. All P. aeruginosa isolates showed the ability to produce biofilm and pyocyanin. Out of the 37 isolates screened, 19 including the reference strains (51.4%) were strong biofilm producers. A significant positive correlation was observed among biofilm formation, pyocyanin production, and the antibiotic resistance profile of the isolates. Findings from this study suggest that infected wounds could act as a reservoir for MDR and virulent P. aeruginosa. The presence of strong biofilm producers of P. aeruginosa in infected wounds is a serious public health concern. Therefore, surveillance programs to monitor and control MDR P. aeruginosa in these patients are required to prevent their dissemination in hospital settings.

Antibiofilm Activity of Curcumin and Piperine and Their Synergistic Effects with Antifungals against Candida albicans Clinical Isolates.

Background: Candidiasis is the common name for diseases caused by yeast of the genus Candida. Candida albicans is one of the most implicated species in superficial and invasive candidiasis. Antifungals, polyenes, and azoles have been used to treat candidiasis. However, due to the development of antifungal resistance, research of natural substances with potential antifungal effects at low concentrations or combined is also a possibility. Methods: The broth microdilution method was used to evaluate the antifungal activity. The biofilm formation was assessed using the microtiter plate method. The antibiofilm activities were assessed using micro plaque tetrazolium salt assay (MTT). The combination effect of antifungal with natural substances was made using the checkerboard method. Results: Among our isolates, clotrimazole was the most resistant, but amphotericin B was the most effective antifungal. The biofilm was formed by all isolates of C. albicans. Curcumin and piperine displayed antibiofilm activity with minimum biofilm inhibitory concentration (MBIC) and minimum eradicating concentration (MBEC) ranging from 64 to 1024 µg/mL and 256 to 2048 µg/mL. In combination, piperine presented double synergistic effects compared to curcumin with all antifungals tested. Curcumin shows more synergistic effect when combined with polyenes than with azoles. However, piperine shows a more synergistic effect when combined with azoles compared to polyenes. Conclusion: C. albicans was susceptible to curcumin and piperine both on planktonic cells and biofilm. The combination of curcumin and piperine with antifungals has shown synergistic effects against multiresistant clinical isolates of Candida albicans representing an alternative drug research for the treatment of clinical candidiasis.

Antibiotic Resistance Profile, Biofilm Formation Ability, and Virulence Factors Analysis of Three Staphylococcus spp. Isolates From Urine.

Background Staphylococcus spp. is one of the most causative agents of urinary tract infections (UTIs). This study aimed to investigate the antibiotic resistance profile and the virulence factors, including the biofilm formation ability of Staphylococcus spp. isolates from urine. Methodology The agar disk diffusion method was used to test the susceptibility of Staphylococcus isolates to ten antibiotics. The biofilm formation ability was determined using the safranin microplate-based method, and the phospholipase, esterase, and hemolysin activities were assessed by the agar plate method. Results During the study period, a prevalence of 18.12% of urinary tract infections caused by the identified Staphylococci was obtained. All the isolated Staphylococcus aureus and S. epidermidis were resistant to cefazolin. Multi-drug resistance (MDR) was recorded in 80.01%, 81.49%, and 76.20% of S. aureus, S. epidermidis, and S. saprophyticus isolates, respectively. Most of the isolates were moderate biofilm formers, while 44.44%, 31.75%, and 30.16% were positive for phospholipase, esterase, and hemolysin activities, respectively. No relevant correlations were observed between the ability of biofilm formation and the resistance to antibiotics or the expression of virulence factors investigated. Conclusion This study shows that Staphylococcus spp. isolates from patients with clinical manifestations of UTIs expressed a high degree of virulence factors, including the ability of biofilm formation, and exhibited multi-drug resistance to the majority of antimicrobials commonly used for the treatment of Staphylococcal infections.

Antibiotic-Potentiating Effect of Some Bioactive Natural Products against Planktonic Cells, Biofilms, and Virulence Factors of Pseudomonas aeruginosa.

Background: Pseudomonas aeruginosa is an opportunistic human pathogen that causes infections that are mediated by both virulence factor production and biofilm formation. In addition, many antibiotics are increasingly losing their efficacy due to the development of resistance. The screening of potentially bioactive natural compounds that have both antivirulence and antibiofilm activities to enhance antibiotic efficacy and reverse antibiotic resistance is a good strategy to overcome these issues. In this study, the antibacterial, antibiofilm, and antivirulence factor activities of some bioactive natural products in combination with conventional antibiotics were evaluated against clinical isolates of P. aeruginosa. Methods: The broth microdilution method was used to determine the antibacterial and antibiofilm activities. The checkerboard method was used to evaluate the combination interactions. Spectrophotometric and agar plate techniques were used to assess the effect of the combination on the pyocyanin production and the motility in P. aeruginosa ATCC 27853 strain. Results: Out of the eighteen combinations tested, ten exhibited synergistic effects against planktonic cells, seven against biofilm inhibition, and five against the eradication of mature biofilm of P. aeruginosa biofilm. The best synergistic effect was the association of amikacin and sinapic acid against planktonic cells (FICI = 0.08) with a 70-fold reduction in the MIC value of amikacin. The same combination showed significant synergistic inhibition of biofilm formation (FICI = 0.1) and biofilm eradication (FICI = 0.15) reducing the MBIC and MBEC of amikacin by 32-fold. Some selected synergistic combinations showed statistically significant differences (p < 0.01 or p < 0.001) in the inhibition of virulence factors compared to the antimicrobials alone. Conclusion: In summary, this study revealed sinapic acid as an antibiotic adjuvant and antivirulence compound to overcome P. aeruginosa infections. This finding indicates that the combinations of amikacin plus sinapic acid, ceftazidime plus thymol, and norfloxacin plus curcumin could be considered promising candidates for the development of combination therapies targeting virulence factors against P. aeruginosa infections.

Phytochemical Analysis and Antifungal Potentiating Activity of Extracts from Loquat (Eriobotrya japonica) against Cryptococcus neoformans Clinical Isolates.

Eriobotrya japonica (loquat) has been used in African traditional medicine with numerous beneficial health effects. The extracts from loquat contain several bioactive compounds with a plethora of pharmacological properties. However, a scientific study on the activity against the aetiological agent of cryptococcosis has not yet been reported. Therefore, this study aimed to investigate the antifungal potential of various extracts from Eriobotrya japonica against clinical isolates of Cryptococcus neoformans. Quantitative and qualitative phytochemical analyses of extracts were made by following standard procedures. The broth microdilution method and the checkerboard methods were used to determine the antifungal activity and the combination of extracts with antifungals drugs. The methanol extract of seeds and the hexane extract of leaves exhibited the best significant antifungal activity with MIC values of 32 µg/mL. Furthermore, the combination of both extracts with nystatin and clotrimazole showed synergistic interactions with a 32-fold reduction in the MIC values of nystatin. Our findings indicate that Eriobotrya japonica extracts are a potential source of new antifungals that could be developed for use in the treatment of cryptococcosis. The anticryptococcal and antifungal activities potentiating activity of the studied extracts indicate their potential in the management of cryptococcosis. Further study should be considered to identify the bioactive principles against Cryptococcus neoformans.