Due to the ever-increasing number of multidrug-resistant bacteria, research concerning plant-derived compounds with antimicrobial mechanisms of action has been conducted. Pentacyclic triterpenes, which have a broad spectrum of medicinal properties, are one of such groups. Asiatic acid (AA) and ursolic acid (UA), which belong to this group, exhibit diverse biological activities that include antioxidant, anti-inflammatory, diuretic, and immunostimulatory. Some of these articles usually contain only a short section describing the antibacterial effects of AA or UA. Therefore, our review article aims to provide the reader with a broader understanding of the activity of these acids against pathogenic bacteria. The bacteria in the human body can live in the planktonic form and create a biofilm structure. Therefore, we found it valuable to present the action of AA and UA on both planktonic and biofilm cultures. The article also presents mechanisms of the biological activity of these substances against microorganisms. In recent years, there has been a growing interest in natural sources of medicinal substances. In the era of the growing resistance of bacteria to antibiotics, an intensive search is being made for effective and safe plant-derived compounds that could become a useful tool both in terms of prevention and treatment of diseases of bacterial aetiology. It is believed that plant products used as part of phytotherapy may support standard pharmacotherapy. Their potential use in terms of controlling chronic and/or recurrent inflammation caused by bacterial biofilm formation is of particular interest.A bacterial biofilm is a community of intercommunicating microorganisms settled on a specific surface, adhering to each other, and surrounded by a layer of organic and inorganic substances produced by these microorganisms. The biofilm consists of aggregates of bacterial cells embedded within an extracellular polymeric substance (EPS), covering biotic and abiotic surfaces . BiofilmProblems with the treatment of chronic and/or recurrent infections caused by bacteria living in biofilm result not only from the difficulty of penetration of drugs through its structure, but they may also be caused by changes in the expression of genes encoding virulence factors, genes that are important in the process of formation and development of the biofilm structure, and genes encoding drug transporters and regulatory proteins ,5,6.Bacterial cells forming a biofilm structure have an altered metabolism compared to forms living in the suspended matter, that is, planktonic forms. In deeper layers of the biofilm, there are super-resistant cells, “persisters”, that have low metabolic activity, which weakens the effect of antimicrobial agents, so that these cells can survive in the presence of lethal concentrations of antibiotics and re-establish the biofilm after the end of the therapeutic cycle ,7.gft genes’ expression [The structure of the biofilm and the low metabolic activity of the bacteria living in it mean that the minimum drug concentrations necessary to inhibit their growth are even 100–1000 times higher than the concentrations of the same drugs used to eliminate bacteria that grow as planktonic forms. Such high doses of antibiotics cannot be used in the pharmacotherapy of human infections due to their toxicity ,5,8. Bacpression  and in cCentella asiatica and Arctostaphylos uva-ursi, are frequently used prophylactically in the form of dietary supplements and nutraceuticals, as well as they are used as a support for the treatment of many diseases, especially those with inflammatory causes [For the reasons discussed above, plant-derived compounds are being sought; they may be applied as an alternative or supportive strategy to antibiotic therapy, as well as they may prevent biofilm formation or facilitate biofilm eradication. In recent years, there has been an increasing interest in pentacyclic triterpenes (TPs). They are polycyclic organic compounds of plant origin, used in traditional phytotherapy due to their wide spectrum of valuable medicinal properties. Plants rich in TPs, for example, y causes ,16,17.30H48O5, the molecular weight of AA is 488.70 g/mol [C. asiatica, known as Gotu kola or kodavan. The total fraction of TPs of C. asiatica contains 30% AA, 40% asiaticoside and 30% madecassic acid [C. asiatica has been one of the main medicinal ingredients used in traditional African, Ayurvedic and Chinese medicine as a panacea and it has been recommended primarily for neuropsychiatric disorders and for the treatment of wounds, leprosy, and syphilis [C. asiatica is an ingredient in ointments, cosmetics, and toothpaste [Asiatic acid  is a monocarboxylic acid derived from the hydride of ursane. In the structural formula, ursane is substituted with a carboxyl group at C-28 and hydroxyl groups at C-2, C-3, and C-23  A. The mo70 g/mol ,19. AA, 70 g/mol . Particusic acid . This plsic acid . For ovesyphilis ,22. Nowaothpaste ,22.30H48O3, the molecular weight of UA is 456.70 g/mol [Ursolic acid  less commonly known as urson, is a hydroxy monocarboxylic acid derived from the hydride of ursane. In the structural formula, ursane is substituted with a carboxyl group at C-28 and a 3β-hydroxyl group at C-3 B. The mo70 g/mol ,24,25,26A.uva-ursi, also known as bearberry. The leaf of A. uva-ursi is a pharmacopoeial raw material and it contains approx. 0.75% UA and several other pharmacologically active compounds: 6–12% arbutin and its metabolites , approx. 20% tannins, 1.2–1.5% flavonoids , uvaol, polyphenolic acids  [UA is most found in the free form or as an aglycone in the case of triterpenoid saponins. UA is present in many plant species belonging to angiosperms, ferns, and bryophytes ,28,29,30ic acid) ,32.A. uva-ursi can be found in the arctic and temperate zones of the northern hemisphere—in Europe, Asia, and North America. In Poland, it can be found mainly in the northern part of the lowlands, in dry pine forests and heathlands, rarely in the mountains. In 2014, A. uva-ursi was included in the list of species under strict protection (Red List) [A. uva-ursi have been used for centuries in European traditional medicine as a diuretic, astringent, and antiseptic to treat urinary tract infections (UTIs) and relieve pain concerning nephrolithiasis. In contrast, Native Americans used leaves of A. uva-ursi to treat headaches, prevent and treat scurvy and prevent miscarriages. In modern phytotherapy, the leaf of A. uva-ursi is used alone in the form of decoctions and as an ingredient of urological herbal mixtures. Moreover, the A. uva-ursi leaf extract is an ingredient of pharmaceutical compounded preparations—liquids and pills with antibacterial, anti-inflammatory, and diuretic properties, which are recommended for the treatment of UTIs [Sphagnum magellanicum, Sphagnum tenellum, Sphagnum rubellum, Polytrichum juniperum, Sphagnum fallax, and Aulacomnium palustre. However, only the extract from S. magellanicum showed antimicrobial activity [ed List) . Leaves  of UTIs . UA is aactivity .Many studies have proved several molecular and pharmacological properties that are common to AA and UA: antioxidant, anti-inflammatory, anti-free radical, cytoprotective, apoptosis-regulating, and receptor- and enzyme-modulating activities. In various in vitro and in vivo studies, AA and UA have also been found to affect growth factors, transcription factors, and cell signalling. The above-mentioned properties result in the following therapeutic actions that are common to AA and UA: anticancer, hypotensive, cardioprotective, anti-infarction, anti-stroke, antihyperlipidemic, antidiabetic, hepatoprotective, gastroprotective, nephroprotective, diuretic, immunostimulatory, neuroprotective, nootropic, anti-Parkinson’s, anti-Alzheimer’s, anti-osteoporosis, antiprotozoal , antifungal, antimycobacterial, antiviral  and radioprotective (anti-aging) actions. Furthermore, AA supports the treatment of burns and non-healing diabetic wounds, and it also exhibits spermicidal activity. UA, in turn, can accelerate wound healing, stimulate muscle growth and reduce fat gain, as well as it is an ingredient of dermatological preparations, cosmetics, and nutritional supplements for athletes ,42,43,44Antibacterial activity is one of the many properties of pentacyclic triterpenes. Their activity is related to changes in the structure and functioning of the bacterial cell structures , cell morphology, gene expression, and processes such as adhesion and biofilm formation  10,13,1,45,46,47In studies devoted to the antibiofilm effects of AA and UA, there was a more effective effect of AA, probably resulting from the slightly different molecular structure of this compound compared to UA. This is because AA has an additional hydroxyl group at C-23, which gives a more hydrophilic nature to the whole molecule, making it easier for this acid to penetrate inside the EPS of the biofilm and thus reach bacterial cells ,49.The anti-adhesion activity of AA and UA, contributing to the reduced ability of bacteria to adhere to epithelial cells , is one of the mechanisms of their antimicrobial activity . ImpaireAnother known mechanism for the antibacterial activity of AA and UA is the impairment of the integrity and irreversible damage to the bacterial cell membrane. It has been determined that AA impairs potassium cation (K+) homeostasis, which is regulated by Kdp membrane transporters. K+ is involved in many aspects of bacterial physiology, for example, growth, survival, and virulence. AA increases the release of K+ from the cytoplasm and excessive loss of K+ leads to bacterial cell death. Moreover, the treatment of bacteria with AA causes an unfavourable release of DNA and RNA nucleotides, which may also cause cell death. Although AA can damage the lipid membrane, it may not be able to penetrate the EPS barrier that is a component of the biofilm ,50.The molecular mechanism of UA activity also involves the inclusion of its molecules into the inner membrane of the bacterial cell and interference with the formation of hydrogen bonds between phosphatidylethanolamine (PE) and phosphatidylglycerol (PG), which are major components of this membrane. UA molecules can also disorganise the packing of hydrophobic chains of phospholipid membranes. It is also possible that UA not only disrupts the PE-PG packing but also locally alters their mutual ratios, making bacteria more susceptible to antibiotics .Bacillus subtilis, Staphylococcus aureus) and Gram-negative  strains. Most of the synthesised UA derivatives showed inhibitory activities against both Gram-positive and Gram-negative bacteria, adopting minimum inhibitory concentration (MIC) values of 3.9–15.6 mg/L, which were similar to the MIC values of amikacin—the positive control in that study. These results proved that the presence of the carbazole moiety alters the physicochemical properties of UA, including improvement of its water solubility, which leads to improved bioavailability and biological activity compared to the parent UA molecule.After a thorough understanding of the mechanism of action of AA and UA, the next issue to be addressed is to improve their low in vivo bioavailability that limits the clinical application of TPs in question. Therefore, structural modifications of UA have been conducted in recent years to improve its biological activity and bioavailability. The aim of one of the studies by Gu et al.  was to dB. subtilis ATCC 6051, S. aureus ATCC 25923, and Streptomyces scabies), with negligible or weak activity against Gram-negative bacteria  compared to UA. The weak activity of UA and its derivatives is due to the presence of an outer membrane in Gram-negative bacteria, which is a selective barrier against the penetration of these compounds into the cell. The structure-activity relationship analysis of UA derivatives showed that the introduction of two hydroxyl groups into UA at C-1 and C-2 along with a short alkyl ester group at C-17 strongly enhances the growth-inhibitory activity of Gram-positive bacteria. The authors showed that UA derivatives alter the expression levels (up- or down-regulation) of genes related to peptidoglycan metabolism and cellular respiration metabolism, as well as genes involved in bacterial virulence.Huang et al.  synthesiAcinetobacter baumannii ATCC 19606 and its clinical colistin-resistant strains. It is interesting that despite the lack of antibacterial properties of pure UA, the authors showed very good antibiofilm properties of one of its derivatives -3-hydroxyurs-12-en28-amide) against all tested A. baumannii strains. The authors also found that this amide derivative down-regulates the expression of biofilm development (bap) and quorum sensing (abaR) genes of A. baumannii suggesting this compound might hinder quorum sensing leading to stop biofilm formation.Usmani et al.  investigS. aureus were 64 and 32 mg/L, respectively. The authors demonstrated that UA-Ch-NPs significantly decreased the expression of icaA and icaD genes, which are engaged in biofilm formation, indicating that UA-Ch-NPs could be a promising material for antibacterial and antibiofilm applications.Ghasemzadeh et al.  investigBacillus cereus and B. subtilis strains. Unfortunately, contrary to expectations, “encapsulation” reduced the antimicrobial activity of UA. Therefore, further studies are necessary to improve its antimicrobial activity.The weaker activity of UA compared to AA is due to its poor solubility in water. To improve the bioavailability of UA, Oprean et al.  encapsulHowever, there are relatively few literature reports that include studies describing the antimicrobial effects of AA and UA. Experiments conducted in recent years have proved that these acids, alone and combined with antibiotics, have significant antimicrobial activity. Existing studies focus primarily on bacteria living in the suspended matter, that is, planktonic forms. MIC and minimum bactericidal concentration (MBC) values of both acids were determined and their effects on bacterial survival, cell morphology, and bacterial membrane structure were described. The effects of AA and UA on the cell surface hydrophobicity, motility, and synthesis of fimbriae, which are virulence factors of significant importance for biofilm formation, were also shown in those studiesPlanktonic forms of bacteria are free-living single cells that move freely in body fluids. They are more susceptible to used antibiotics compared to their biofilm forms, and they are more easily identified and eliminated by the host immune system . In manyThe studies presented below used AA isolated by researchers from various plants rich in pentacyclic triterpenes  or purchSome articles are limited to identifying only MIC values  or zonesThe MIC value is the lowest concentration of the antibacterial substance that inhibits bacterial growth. Determination of the MIC is the most basic laboratory measurement of the activity of an antimicrobial agent against microorganisms. A low MIC value indicates that less antibacterial agent concentration is needed to inhibit bacterial growth. Knowing the MIC values of antibiotics is very important as it allows clinicians to select the appropriate antibiotic for a patient with a specific infection and determine the effective dose of the antibiotic.Bacteria exposed to an insufficient concentration of a particular drug can evolve resistance to this drug. Therefore, the knowledge of MIC values is helpful in the treatment of patients and prevents the development of drug-resistant strains.The antimicrobial activity of pure compound isolated from crude extract can be considered significant (very good) when its MIC is below 10 mg/L, moderate (good) when between 10 and 100 mg/L, and low (weak) when more than 100 mg/L .S. guineense leaves showed significant activity against Gram-positive B. subtilis (MIC = 0.75 mg/L) and Gram-negative E. coli (5 mg/L) [E. faecalis [S. aureus [E. coli, P. aeruginosa, and E. cloacae as well as Gram-positive S. aureus showed the lowest sensitivity to AA (MIC >128 mg/L) [E. coli, for which the AA MIC ranged from 512 mg/L to >1024 mg/L [The data in (5 mg/L) . AA show(5 mg/L) ,60,61,62faecalis ,63 and S28 mg/L) ,63,64. A024 mg/L ,10,47.S. guineense leaves. That acid revealed significant antibacterial activity against B. subtilis, E. coli, and S. sonnei strains and its MIC values were 0.75, 5.0, and 30.0 mg/L, respectively. Taemchuay et al. [S. aureus and the reference strain S. aureus ATCC 25923, were in the range of 20–160 mg/L for AA isolated from C. asiatica leaves. Acebey-Castellon et al. [S. lancifolia leaves exhibited stronger antibacterial activity against E. faecalis ATCC 29212 (MIC = 128 mg/L) and weaker activity against S. aureus ATCC 25923, E. coli ATCC 25922, and P. aeruginosa ATCC 27853 (MICs > 128 mg/L).Djoukeng et al.  studied y et al.  found thn et al.  determinE. coli strains isolated from the urine of patients with pyelonephritis was also analysed by Wojnicz et al. [E. coli to uroepithelial cells, significantly reducing the number of bacilli adhering to the epithelium. AA at a concentration of 50 mg/L also induced morphological changes in bacterial cells. An increased percentage of short filaments (5–15 μm) and the presence of long filaments (>15 μm) were observed in cultures treated with AA [The effect of AA on clinical z et al. ,47. The z et al. . At a co with AA .E. coli O157:H7, S. typhimurium DT104, P. aeruginosa, as well as Gram-positive bacteria: L. monocytogenes, S. aureus, E. faecalis, B. cereus. The MIC values of that acid against those bacterial strains were in the range of 20–40 mg/L, while the MBC values were in the range of 32–52 mg/L. AA reduced the survival of all bacterial strains as early as in 6-h cultures. AA at 1 × MIC concentration impaired cell membrane integrity in 40–56% bacterial cells, while at 2 × MIC concentration it caused loss of K+ ions and nucleotides in 71–89% of bacterial cells.The study by Liu et al.  revealedE. faecalis, the bacteria associated with serious nosocomial UTIs, particularly among catheterised patients. Those strains were resistant to, for example, gentamicin, nitrofurantoin, ampicillin, and trimethoprim/sulphamethoxazole. The MIC values of AA against the analysed strains were 64–128 mg/L. The anti-growth effect of sub-inhibitory concentrations (0.75 × MIC) of AA was observed after 2-, 4-, 6- and 24-h incubation. AA showed the highest activity after 24 h, where bacterial survival was reduced by 250-fold compared to the control sample. It was also observed that E. faecalis cells were larger and formed aggregates. These changes may be caused by AA impairing cell division, resulting in changes in cell morphology such as increased cell diameter or the occurrence of aggregates. Furthermore, the authors showed that AA significantly reduced or completely inhibited the ability of E. faecalis to synthesise enzymes and toxins that damage host tissues such as lipase, lecithinase, gelatinase, and haemolysins.Wojnicz et al.  studied C. difficile. The MIC values of AA were 10–20 mg/L. AA inhibited bacterial motility, induced membrane damage and morphological changes in cells, thus causing leakage of intracellular substances (proteins and nucleic acids) as early as 30 min of exposure. AA at concentrations of 10 and 100 mg/L damaged the spore surface, impairing the sporulation and germination of spores. According to the study by Han et al. [E. coli ATCC 8739 and S. aureus ATCC 29213 as standard dressings soaked in penicillin at a concentration of 25 mg/L. In contrast, AA at a concentration of 15 µM (30%) showed a stronger inhibitory effect against both tested strains than penicillin.Harnvoravongchai et al.  found thn et al. , AA soluS. robusta resin exhibited good antimicrobial activity against E. coli ATCC 25938 and P. aeruginosa with the MIC value of 64 mg/L.In the study conducted by Bharitkar et al. , AA isolSome researchers define the antimicrobial activity of AA by determining the zone of bacterial growth inhibition around a disc soaked with a specific concentration of it . The sizK. pneumoniae (13–28 mm) using AA in concentrations of 25–100 mg/L [B. subtilis using concentrations of 75 and 100 mg/L and S. aureus by treating it with AA at a concentration of 100 mg/L [B. cereus, only a very high concentration of AA (2000 mg/L) isolated from M. malabathricum leaves showed antibacterial activity [P. aeruginosa strain exposed to AA at the concentrations of 75 and 100 mg/L [B. cereus [B. subtilis [S. aureus [S. pneumoniae [E. coli [H. pylori [K. pneumoniae [P. aeruginosa [100 mg/L . The hig100 mg/L . In the activity . On the 100 mg/L . The dia. cereus , B. subtsubtilis , S. aure. aureus ,67,68, a[E. coli ,68, H. peumoniae , and P. ruginosa .C. asiatica. At concentrations of 500–1000 mg/L, AA had antimicrobial activity against H. pylori ATCC 45903, E. coli ATCC 29952, S. pneumoniae, and S. aureus. The disk diffusion method was used for the assessment of antibacterial activity; the zones of inhibition were 12 mm (1000 mg/L) and 8 mm (500 mg/L) for H. pylori, 8 mm (1000 mg/L) for S. aureus, and 7 mm (1000 mg/L) for E. coli and S. pneumoniae. On the other hand, asiaticoside did not show antimicrobial activity against any of the above-mentioned strains.Norzaharaini et al.  investigM. malabathricum leaves exhibited antibacterial activity against B. cereus ATCC 10876, S. aureus ATCC 25923, and K. pneumoniae. For AA at a concentration of 2000 mg/L, the zones of inhibition of the analysed strains were 13.5, 11.0, and 9.0 mm, respectively, while for AA at a concentration of 1000 mg/L they were 12.0, 10.5, and 8.0 mm, respectively. AA at concentrations of 500 and 250 mg/L inhibited the growth of B. cereus  and S. aureus (10.0 and 9.0 mm), however, it was not active against K. pneumoniae. None of the used AA concentrations had antibacterial activity against B. subtilis, E. coli ATCC 25922, P. aeruginosa ATCC 17853, and S. typhi.Wong et al. , using tK. pneumoniae, P. aeruginosa, B. subtilis, and S. aureus by identifying zones of bacterial growth inhibition at different acid concentrations . K. pneumoniae proved to be the most susceptible to AA, as the zone of inhibition of growth of these bacteria appeared already at the lowest AA concentration (25 mg/L) and it was 13 mm. The P. aeruginosa strain was the most resistant to AA, as a zone of growth inhibition of 6 mm appeared only after using the concentration of 100 mg/L.Ashella and Fleming  conducteS. robusta resin showed antimicrobial activity against E. coli ATCC 25938 and P. aeruginosa. The zones of inhibition for AA at the same concentration of 64 mg/L were 7 and 8 mm, respectively.In the study by Bharitkar et al. , AA isolMany articles describe the activity of UA against planktonic forms of bacteria. UA used in these experiments was plant-derived as shown in These articles include a very diverse range of research, for example, determination of MIC values , the sizA. scholaris showed significant activity against E. faecalis (MIC = 1 mg/L) and L. monocytogenes (MIC = 2 mg/L) [S. officinalis also showed very good activity (MIC = 4 mg/L) against E. faecalis and E. faecium [S. mutans, S. sobrinus and S. gordonii [E. faecalis (MIC = 4 mg/L) [S. aureus (4 ≤ MIC ≤ 8 mg/L) [S. epidermidis (MIC = 4 mg/L) [L. monocytogenes (6.5 ≤ MIC ≤ 8 mg/L) [A. scholaris [S. officinalis [V. paradoxa [B. cereus [S. aureus [S. pneumoniae [B. cereus [L. monocytogenes [S. aureus [S. mutans [S. pneumoniae [A. caveae [E. coli [K. pneumoniae [S. choleraesuis [V. cholerae [The data presented in  2 mg/L) . UA isol faecium . On the gordonii ,71, E. f 4 mg/L) , S. aure 8 mg/L) ,73,74 an 4 mg/L) , as well 8 mg/L) ,76,77. Tcholaris , S. offiicinalis  and V. pparadoxa  was dete. cereus , S. aure. aureus ,78 and Seumoniae . UA showeumoniae ,85,86,87eumoniae ,79,82. Oeumoniae ,89,90,91eumoniae ,88,92,93. mutans , S. pneueumoniae , and 5 s[E. coli ,47,82,95eumoniae , S. choleraesuis , V. cholcholerae ,82.S. aureus (21 mm) and Gram-negative S. boydii (19 mm) treated with UA (100 mg/L) isolated from H. corymbosa, which proves its high activity antibacterial action against these bacteria [M. malabathricum [B. subtilis (8.0–10.5 mm) and S. aureus (7.5–10.5 mm) and Gram-negative S. typhi (9.5–11.0 mm). The higher the acid concentration, the larger the zone of inhibition of growth. Interestingly, this relationship was not observed in the case of B. cereus, where the zone of growth inhibition was 7.0 mm regardless of the concentration of UA (250–2000 mg/L) [bacteria . On the athricum  and used00 mg/L) .When analysing the data contained in O-fatty acids (C12–C18) against various bacterial species. UA was isolated from the leaves of D. melanoxylon. The MIC values of UA were 25 mg/L against B. subtilis ATCC 6051 and P. syringae ATCC 13457, while 50 mg/L against B. sphaericus ATCC 14577, S. aureus ATCC 9144, E. coli ATCC 25922, and S. typhi ATCC 23564. It should be noted that the antibacterial activity of UA derivatives against the analysed strains varied and it was dependent on the bacterial species, however, only in a few cases, it was better than that of pure UA.Mallavadhani et al.  investigM. fallax and its semi-synthetic derivatives compared to the strains involved in plaque and caries formation: E. faecalis ATCC 4082, S. salivarius ATCC 25975, S. mitis ATCC 49456, S. mutans ATCC 25275, S. sobrinus ATCC 33478 and S. sanguinis ATCC 10556. The MIC values of UA against all the aforementioned strains were in the range of 50–80 mg/L. In contrast, the MIC values of UA derivatives were generally higher. The stronger antibacterial activity of UA compared to its derivatives suggests that the free hydroxyl group at C-3 and the carboxyl group at C-17 are primarily responsible for this activity.Scalon Cunha et al.  evaluateS. aureus (MRSA), penicillin-resistant S. pneumoniae (PRSP), and vancomycin-resistant enterococci (VRE): E. faecalis and E. faecium. The MIC values were 8, 8, and 4 mg/L, respectively. At a concentration of 2 × MIC, UA showed the bactericidal activity against VRE for at least 48 h. UA showed a stronger bactericidal activity against E. faecium than ampicillin. UA showed only a bacteriostatic activity against E. faecalis. However, UA was not active against Gram-negative bacteria: P. aeruginosa, E. coli, S. marcescens, or against mutants of E. coli and P. aeruginosa, which are hypersensitive to drugs due to the absence of multidrug efflux pumps (MDR pumps). The MIC value against those strains was >128 mg/L.According to the study by Horiuchi et al. , UA exhiM. elegans against B. subtilis, S. aureus, P. aeruginosa, S. typhi, E. coli, and S. flexneri. UA revealed the antibacterial activity against B. subtilis, S. aureus, P. aeruginosa, S. typhi for which growth inhibition zones were 12, 15, 16, and 18 mm, respectively. UA did not inhibit the growth of the other two strains. The study by Ahmad et al.  determinE. coli ATCC 25922, S. aureus ATCC 25923 and ATCC 29213, E. faecalis ATCC 29212 and P. aeruginosa ATCC 27853, as well as five antibiotic-resistant clinical isolates: E. coli, S. aureus, E. faecium, E. faecalis, and P. aeruginosa. UA was found to be highly effective against Gram-positive bacteria: E. faecalis ATCC 29212 (MIC = 4 mg/L) and S. aureus ATCC 25923, S. aureus ATCC 29213 (MIC = 8 mg/L). UA had no antibacterial activity against E. coli ATCC 25922, P. aeruginosa ATCC 27853, and all clinical strains (MIC > 256 mg/L).Fontanay et al.  determinB. dracunculifolia leaves had antimicrobial activity against S. aureus ATCC 43300 (MRSA). The growth inhibitory concentration for 50% was IC50 = 5 mg/L and the MIC value was 10 mg/L. Da Silva Filho et al.  showed tV. macrocarpon cranberry fruit. The MIC value of the extract determined against strains of uropathogenic E. coli ATCC 700336 and ATCC 25922 was >256 mg/L. The extract fraction at 10 mg/L also inhibited COX-2 activity and the activity of nuclear transcription factor, NF-κβ.Huang et al.  identifiM. ligustroides and its derivatives against B. cereus ATCC 14579, V. cholerae ATCC 9458, S. choleraesuis ATCC 10708, K. pneumoniae ATCC 10031, and S. pneumoniae ATCC 6305. UA was active only against B. cereus, showing the MIC value of 20 mg/L; the MIC values for other strains were 1000 mg/L or higher. UA derivatives were effective only against S. pneumoniae; their MIC values were 50 mg/L.Cunha et al.  evaluateL. monocytogenes. After 24-h incubation in the presence of UA at a concentration of 0.7 × MIC (4.5 mg/L), bacterial cell length was reduced by 20% compared to the control and it did not exceed 2 μm.According to Kurek et al. , UA imprH. corymbosa against 4 strains of Gram-positive bacteria: B. subtilis, B. cereus, S. lutea, and S. aureus, as well as 10 Gram-negative strains: S. sonnei, S. dysenteriae, S. boydii, S. paratyphi A, E. coli, P. aeruginosa, S. typhi, S. flexneri, V. cholereae, and K. pneumonia. UA at 100 mg/L showed significant activity against S. aureus (21 mm diameter zone of inhibition) and S. boydii (19 mm), whereas a moderate activity against P. aeruginosa (10 mm). Unfortunately, a weak activity of UA against other microorganisms (zones of inhibition of 6–10 mm) was reported.Sultana et al.  studied S. lancifolia leaves showed a stronger antibacterial activity against Gram-positive S. aureus ATCC 25923 and E. faecalis ATCC 29212, and a weaker activity against Gram-negative E. coli ATCC 25922 and P. aeruginosa ATCC 27853. The MIC values ranged from 16 to >128 mg/L and they were lowest against E. faecalis. Acebey-Castellon et al.  found thS. aureus ATCC 29213, S. aureus ATCC 43300 (MRSA), and 42 clinical isolates of S. aureus. The MIC and MBC values of UA against S. aureus ATCC 29213 were 7.81 mg/L and 124.96 mg/L, respectively. MRSA strain was more resistant to UA, the MIC and MBC values were 15.62 mg/L and 249.92 mg/L, respectively. The synergistic effect of UA and the antibiotic was proved by the combination of 0.25 × MIC of norfloxacin with 1 × MIC of UA, which inhibited the growth of 21 of 42 clinical strains, while the combination of 0.5 × MIC of norfloxacin with 2 × MIC of UA inhibited as many as 90% of the strains. Against S. aureus strain ATCC 29213, UA at a concentration of 1 × MIC showed no postantibiotic effect (PAE), while at a concentration of 2 × MIC it exerted a very short PAE (2 h). Administration of UA combined with norfloxacin resulted in the prolongation of PAE up to 14.5 h (at 1 × MIC of UA) and 21 h (at 2 × MIC of UA). There was a significant prolongation (25.3–29.1 h) of the postantibiotic sub-MIC effect (PASME) through preincubation of S. aureus ATCC 29213 cells in sub-inhibitory concentrations of norfloxacin. The synergistic effect of UA and norfloxacin may be due to initial damage to the bacterial membrane caused by lipophilic UA, which increases its permeability to norfloxacin. Enhanced intracellular accumulation of norfloxacin results in increased bacterial DNA damage and thus longer PAE and PASME.Filocamo et al.  analysedS. mutans ATCC 25175, S. sobrinus ATCC 33478, 40 clinical strains of S. mutans, and 15 clinical strains of S. sobrinus, which are involved in dental caries. The MIC value of UA against both reference strains was 2 mg/L while the MBC values were >4 mg/L for S. mutans ATCC 25175 and 8 mg/L for S. sobrinus ATCC 33478. The MIC value of UA was 2 mg/L for most clinical strains. The exceptions were 1 strain of S. mutans and three strains of S. sobrinus (MIC = 4 mg/L). Based on the obtained results, the authors suggested that UA at concentrations >8 mg/L could be used as an ingredient in oral hygiene products to prevent dental caries.Kim et al.  determinCarissa macrocarpa in their study. The acid revealed a moderate antibacterial activity against three Gram-negative bacterial strains: K. pneumoniae ATCC 700603, P. aeruginosa ATCC 35032, E. coli ATCC 25922, as well as four Gram-positive strains: S. saprophyticus ATCC 35552, S. aureus ATCC 25923, S. aureus ATCC 43300, and E. faecium ATCC 19434. The MIC values of that acid were 250–500 mg/L. Moodley et al., in their study, also identified the effect of UA at concentrations of 0.5 × MIC, 1 × MIC, and 2 × MIC on the adhesion of all the above-listed strains to polystyrene surfaces. The adhesion of E. coli and S. aureus cells was reduced under the influence of all acid concentrations used. In the case of P. aeruginosa, there was reduced adhesion only with exposures to UA at 0.5 × MIC and 1 × MIC concentrations while there was enhanced adhesion during the exposure to 2 × MIC. On the other hand, the adhesion of S. saprophyticus was enhanced during exposure to 0.5 × MIC and it was reduced under the influence of 1 × MIC and 2 × MIC. It should be noted that the adhesion of K. pneumoniae to polystyrene surfaces enhanced after their exposure to all UA concentrations under analysis.Moodley et al.  used UA Kim et al.  identifiP. aeruginosa, L. monocytogenes, S. aureus ATCC 29213, and S. epidermidis ATCC 12228. MIC values ranged from 7.5 mg/L for S. epidermidis to 22.5 mg/L for P. aeruginosa. Furthermore, the authors demonstrated the synergism of action of UA combined with β-lactam antibiotics (ampicillin and oxacillin). It should be noted that bacteria that were cultured in the presence of both components (UA + oxacillin or UA + ampicillin) became more susceptible to each of them. The most significant effect was observed for S. aureus and UA + ampicillin combination. The MIC value for ampicillin was reduced as much as 16-fold, that is, from 4 mg/L to 0.25 mg/L. These results indicate that UA when administered in combination with β-lactams, may be useful in the treatment of bacterial infections.According to the study by Kurek et al. , UA reveM. malabathricum leaves exhibited antimicrobial activity against S. aureus ATCC 25923, B. subtilis, B. cereus ATCC 10876, and S. typhi. For UA at 2000 mg/L, the zones of inhibition of the tested strains were 10.5, 10.5, 7.0, and 11.0 mm; for UA at 1000 mg/L, they were 9.5, 9.5, 7.0, and 10.0 mm; for UA at 500 mg/L, they were 8.5, 9.0, 7.0, and 10.0 mm; and for UA at 250 mg/L, they were 7.5, 8.0, 7.0 and 9.5 mm, respectively. UA was not active against E. coli ATCC 25922, P. aeruginosa ATCC 17853, and K. pneumoniae at any of the analysed concentrations.Wong et al.  proved uE. coli bacilli adhering to uroepithelial cells. That acid, at a concentration of 50 mg/L, also induced morphological changes in bacterial cells. In UA-treated cultures, there were increased numbers of short filaments (5–15 μm). There were also long filaments (>15 μm), thickened cells with swollen filaments, and cells partially devoid of a cell wall (“ghost cells”).Wojnicz et al. , in addiS. australis against 12 bacterial strains. UA revealed the strongest activity against S. aureus ATCC 6538 (MIC = 32 mg/L). UA was also effective against E. coli ATCC 25922, K. pneumoniae ATCC 10031, and S. flexneri ATCC 12022 (MIC = 64 mg/L). In the case of E. coli ATCC 27 and P. aeruginosa ATCC 15442, the MIC value was identical (512 mg/L). The six other strains, that is, S. aureus ATCC 12692, S. aureus ATCC 12624, B. cereus ATCC 33018, A. caveae ATCC 15468, V. cholera ATCC 15748, and L. monocytogenes ATCC 19117 were insusceptible to UA (MIC ≥1024 mg/L). The researchers also found the synergism of action of UA combined with aminoglycosides . MIC values of those antibiotics were reduced in the presence of UA against almost all microorganisms, excluding gentamicin and amikacin against E. coli ATCC 25922 and kanamycin against P. aeruginosa ATCC 15442 and K. pneumoniae ATCC 10031.Do Nascimento et al.  determinE. tereticornis leaves and its lipophilic derivatives. Three strains were used in a study by Dwivedi et al.: nalidixic acid-sensitive E. coli CA8000, nalidixic acid-resistant E. coli DH5α, and the MDREC-KG4 clinical isolate of E. coli that is resistant to multiple antibiotics, including tetracycline. The MIC value of UA against all E. coli strains was 1000 mg/L. Similarly, the MIC values of lipophilic derivatives against bacilli were very high (500–1000 mg/L), indicating their lack of antibacterial activity. However, it should be emphasised that the authors found the synergistic effect of both UA and its derivatives combined with nalidixic acid against E. coli CA8000 and E. coli DH5α, as well as with tetracycline against E. coli MDREC-KG4. When applied at a concentration of 10 mg/L, UA and its derivatives combined with nalidixic acid reduced the MIC values of this antibiotic against E. coli CA8000 and E. coli DH5α by 2-fold (UA) and 4–8-fold (UA derivatives). On the other hand, UA and its derivatives at a concentration of 50 mg/L and combined with tetracycline, reduced the MIC values of this antibiotic against E. coli MDREC-KG4 strain by 2-fold and 8-fold, respectively.Dwivedi et al.  investigS. mutans UA159. UA inhibited transcription of all genes involved in the first step  and second step of peptidoglycan biosynthesis , and of most genes involved in the third step of murein synthesis . These studies clearly indicate that inhibition of gene expression is one of the mechanisms of the antimicrobial activity of UA.Park et al.  determinA. scholaris leaves, against Gram-positive strains of E. faecalis ATCC 29212 (MIC = 1 mg/L), L. monocytogenes ATCC 7644 (MIC = 2 mg/L), B. cereus ATCC 9139 (MIC = 8 mg/L), methicillin-susceptible S. aureus (MSSA) ATCC 29213 (MIC = 16 mg/L) and S. aureus ATCC 43300 (MRSA) (MIC = 64 mg/L), while a weaker antibacterial activity (MIC >128 mg/L) was against Gram-negative E. coli ATCC 35150, P. aeruginosa ATCC 27853 and S. enterica ATCC 13311. Synergism was also found in the joint action of 0.5 × MIC of UA and 0.5 × MIC of ampicillin or tetracycline against B. cereus strains, MSSA and MRSA. The reduction in viable cell counts in 24-h cultures was >4 log10. A study by Wang et al.  revealedWang et al.  investigV. paradoxa leaves revealed good antimicrobial activity against reference S. aureus ATCC 33591 and clinical MRSA strains [UA isolated from  strains . The MICE. coli ATCC 25922, P. aeruginosa ATCC 27853, and S. aureus ATCC 29213 strains. The MIC values against these bacteria ranged from 64–256 mg/L, while the MBC values ranged from 256–512 mg/L. According to further studies, UA can generate the production of reactive oxygen species (ROS), especially superoxide anion radical and hydroxyl radical, in bacterial cells. They cause enhanced electron transport chain activity, resulting in oxidative stress, lipid peroxidation, and oxidation of 2-deoxyribose in DNA, leading to bacterial cell death. Increased amounts of ROS are associated with dysfunctions in the conversion reactions of glutathione, a natural free radical scavenger. The authors proved that UA decreased the level of reduced glutathione in bacterial cells while increasing the number of its oxidised molecules, which contributed to a significant decrease in bacterial survival due to an increase in oxidative stress indices.Oloyede et al.  found thE. faecalis strains, also analysed the antibacterial effect of UA against cocci that are a common cause of UTIs, especially in catheterised patients. The bacteria were resistant to gentamicin, nitrofurantoin, ampicillin, and trimethoprim/sulphamethoxazole. The MIC values of UA against E. faecalis strains were 32–512 mg/L, which corresponds to other available literature data. It was noted that the growth of planktonic forms of E. faecalis was inhibited under the influence of 0.75 × MIC of UA as early as in the 2-h culture, while in the 6-h culture, the greatest 780-fold reduction was observed in the number of CFU/mL of viable cells compared to the control sample. It was also found that in the presence of UA, E. faecalis cells were larger and they formed aggregates instead of characteristic chains. It is likely that UA impairs cell division processes, which may cause phenotypic changes in cell morphology, such as increased cell diameter and the presence of irregular aggregates. Moreover, it was found that UA significantly reduced, and in some cases completely inhibited, the ability of E. faecalis strains to synthesise DNase and extracellular enzymes  and haemolysin that damage host tissues.Wojnicz et al. , in addiO-α-L-arabinopyranoside (URS), isolated from A. henryi leaves, against S. aureus strains. The MIC values were found to be 3.125 mg/L against MSSA strain and 6.25 mg/L against MRSA strains. The authors also found the existence of synergism between URS and oxacillin against MRSA strains. The addition of 0.5 × MIC of URS reduced the MIC values of oxacillin by 2–32-fold according to strain. The study also determined the effect of URS on the morphology of MRSA strain cells. URS at a concentration of 0.5 × MIC damaged the bacterial cell membrane and caused surface roughness. The authors also noted a slight decrease in membrane protein PBP2A levels under the influence of URS. A significant decrease in PBP2A levels, cell membrane disintegration, and bacterial cell lysis were only observed in the assay containing the combination of URS with oxacillin.Zhou et al.  investigS. typhi and Shigella spp., as well as Gram-positive S. aureus and S. pneumoniae. The MIC value of hydrazide of UA against the above-mentioned strains was >256 mg/L. Furthermore, it was found that the antibacterial activity of the hydrazide of UA might be enhanced by producing complexes with various metals , then the MIC values were 4–32 mg/L. The complex with triphenyltin proved to be the most potent antimicrobial agent, with the MIC values of 4 mg/L against S. pneumoniae and 8 mg/L against other strains.Jabeen et al.  investigE. coli (MIC = 256 mg/L) and K. pneumoniae (MIC = 512 mg/L). The researchers found that UA exhibited the synergism of action with colistin, causing a significant 16-fold reduction in colistin MIC for E. coli and a 4-fold for K. pneumoniae. Moreover, UA enhanced the permeability of the bacterial outer membrane, and that facilitated the transport of colistin into the bacterial cell. Furthermore, UA inhibited the activity of efflux pumps, which in turn impeded the removal of the antibiotic from the bacterial cell.Sundaramoorthy et al.  determinWhen analysing the antibacterial activity of AA or UA, it can be noticed that their antimicrobial activity (MIC/inhibition zone) may be different for the same bacterial species. However, there are several different aspects to consider. First, there are many different bacterial strains within a species, all from different origins . Secondly, the acids used in separate studies often have different origins, that is, they are isolated from different plant species. Moreover, the methods of extracting these metabolites often vary.It is currently known that more than 99% of both commensal and pathogenic bacteria in the human body occur in a biofilm form. Biofilms are formed on the surface of dead cells (skin microflora), viable cells (mucous membrane microflora), and abiotic surfaces found in the human body . Consortia formed from pathogenic bacteria are difficult to destroy with antibiotics, so it is useful to learn about the effects of AA and UA on biofilm structure and their interaction with drugs used in the treatment of bacterial infections.A negligible number of articles have been devoted to the effect of AA on biofilm formation and eradication. Therefore, we decided to analyse available research in the current review in which the antibiofilm properties of AA combined with antibiotics were also highlighted.P. aeruginosa biofilm. A rotating disk reactor (RDR) was used in that study as a model device to study the susceptibility of biofilms to antibiotics and other compounds. The MIC value of AA for P. aeruginosa was >128 mg/L. AA at concentrations of 10, 50, and 100 mg/L was used in that study. Only a small degree of biofilm reduction was observed under the influence of AA at concentrations of 50 and 100 mg/L. However, it should be noted that the lowest concentration of AA (10 mg/L) enhanced the susceptibility of biofilm-living P. aeruginosa to both tobramycin (100 mg/L) and ciprofloxacin (10 mg/L), which previously showed no antibiofilm effect.Garo et al.  investigE. coli strains , with virulence traits relevant to biofilm formation, were used in that study. AA at a concentration of 50 mg/L weakly inhibited the biofilm formation by E. coli strains. A significantly better effect was obtained when AA was used in combination with ciprofloxacin. Similarly, during the eradication of biofilm from urological catheters, statistically significant results were obtained only when AA was used in combination with ciprofloxacin. The number of viable bacteria was reduced to 12%. In another study, Wojnicz et al. [E. faecalis strains. The MIC values of AA against the analysed strains were 64–128 mg/L. The complete inhibition of biofilm synthesis by tested cocci was demonstrated throughout the duration of the experiment (1–10 days). The survival rate of bacteria in the biofilm mass was significantly reduced under the influence of AA compared to control samples. Wojnicz et al.  conductez et al.  investigC. difficile R20291.Surprising results were obtained by Harnvoravongchai et al. . The resE. coli CFT073, E. cloacae ATCC-BAA 2468, P. aeruginosa ATCC 25000 strains. AA also changed the morphology of these bacteria.The recent study of Sycz et al.  revealedMuch more attention was paid to the antibiofilm properties of UA compared to AA. The following articles describe the effects of UA on biofilm mass formation and the expression of genes encoding virulence factors associated with biofilm synthesis. Few articles highlighted the synergistic effect of UA and antibiotics on biofilm formation.Diospyros dendo leaves. UA at a concentration of 10 mg/L already showed significant antimicrobial activity against 24-h single-species biofilms formed by E. coli (including ATCC 25404), P. aeruginosa, and V. harveyi strains, reducing the amount of biofilm mass they produced by 72%, 87%, and 57%, respectively. Interestingly, UA at concentrations of 10 and 30 mg/L did not inhibit the growth of the aforementioned strains growing in planktonic forms. It was also found that UA did not affect quorum sensing. In contrast, UA at concentrations of 10 and 30 mg/L induced the expression of genes encoding proteins related to chemotaxis , heat shock , and membrane transport . It should be noted that overexpression of the motAB gene makes cells too motile to remain stable within the biofilm environment, resulting in reduced biofilm formation. Conversely, low cell motility caused by loss of the motAB gene promotes bacterial conjugation, which promotes biofilm development. The authors found that UA (at concentrations of 10 and 30 mg/L) inhibited the operon cysDJK regulated by CysB. The CysB protein is a transcriptional regulator of LysR that controls the expression of genes involved in cysteine biosynthesis and sulphur metabolism. The CysB pathway is an interesting potential target pathway for UA and other TPs. According to that study, UA can modulate cysB gene expression in E. coli. The cysB mutant enhanced biofilm formation by 2–10-fold compared to the isogenic cysB strain.Ren et al.  analysedP. aeruginosa, L. monocytogenes, S. aureus ATCC 29213 and S. epidermidis ATCC 12228 strains had approx. 4-fold higher resistance to UA, compared to planktonic cells. Administration of UA along with β-lactam antibiotics (ampicillin and oxacillin) reduced MBIC (Minimum Biofilm Inhibitory Concentration) of both UA and antibiotics against the aforementioned bacterial strains.According to Kurek et al. , single-S. mutans UA159 cocci that play a key role in the pathogenesis of dental caries. UA in concentrations of 0.1, 0.2, and 0.5 % (w/w) was used for saturating the composite resin disks in that study. The disks were soaked in a culture of S. mutans, which was incubated for 24 h and then plated on nutrient agar to measure the number of CFU/mL. It was found that the number of bacteria reduced with an increasing concentration of UA. However, it should be noted that a better antibacterial effect of UA was obtained in cultures where glucose was the carbohydrate source than in those containing sucrose. Kim et al.  evaluateS. mutans UA159 strain, the researchers used three other strains from the genus Streptococcus , as well as two strains from the genus Actinomyces . The MIC values of UA were significantly lower against Actinomyces spp. (16 mg/L and 32 mg/L) than its MIC values against Streptococcus spp. (64–256 mg/L). The researchers found that UA at the sub-inhibitory concentration (0.25 × MIC) inhibited biofilm formation by S. mutans and S. gordonii on titer plates as well as S. mutans and A. viscosus on the tooth surface. The study also attempted to eradicate mature biofilms formed by S. mutans and A. viscosus from the tooth surface using supra-inhibitory concentrations of UA. A much better effect was obtained against A. viscosus, which is largely related to the less compact structure of the biofilm formed by that strain compared to the biofilm formed by S. mutans. The exopolysaccharide of S. mutans, due to the presence of extracellular matrix glucans, is much denser and much more compact, which presumably made it much more difficult for UA to penetrate the inside of the biofilm formed by these cocci and for the bacteria to survive in central parts of the biofilm.Zhou et al.  also invL. monocytogenes—haemolytic activity and biofilm synthesis. After the MIC (8 mg/L) and MBIC (24 mg/L) values of UA had been measured, the researchers determined the effect of sub-inhibitory concentrations of UA on the production of listeriolysin O by L. monocytogenes, biofilm formation ability, and survival of bacteria living in the biofilm mass. It was reported that UA at a concentration of 0.75 × MIC inhibited the activity of listeriolysin O almost by 3-fold. A concentration of 0.5 × MBIC of UA attenuated biofilm formation by more than 60% and it reduced the survival of L. monocytogenes cells in biofilms by 56%.Kurek et al.  investigS. aureus strains that are a common cause of infective endocarditis. Titre plates with wells coated with fibrinogen, fibronectin, and collagen were used in that study. UA at a concentration of 0.75 × MIC (187.5 mg/L) significantly reduced the bacterial adhesion to surfaces coated with matrix proteins: collagen by 73.2%, fibronectin by 58.8%, and fibrinogen by 65.9%. The impairment of adhesion activities of staphylococci under the influence of UA contributed to significant inhibition of biofilm formation by these bacteria on analysed surfaces (70–86%).The aim of a study by Micota et al.  was to dS. aureus. According to the researchers, the presence of UA resulted in reduced expression of genes encoding adhesins  and some genes related to metabolism , which are considered important for biofilm survival.Qin et al.  proved tStreptococcus bacteria that are the main aetiological agent of dental caries in humans. Reference strains of S. sobrinus ATCC 33478 and S. mutans UA159 as well as 2 clinical strains of S. mutans (KCOM 1207 and KCOM 1128) were used in that study. The MIC values of UA were 128–256 mg/L and the MBC values ranged from 256–512 mg/L, according to the analysed strain. Interestingly, both the MIC and MBC values of xylitol were identical regardless of the analysed strain. The most UA-susceptible strains were found to be S. mutans KCOM 1207 and S. sobrinus ATCC 33478. The synergism of action of UA and xylitol was investigated by using combinations of these two components at different concentrations. Combinations of 20% xylitol with sub-inhibitory concentrations of UA (16 or 32 mg/L) significantly reduced biofilm formation by analysed streptococci.Zou et al.  determinLepidozia chordulifera against reference strains of P. aeruginosa ATCC 27853 and S. aureus ATTC 6538P. UA at a concentration of 50 mg/L did not decrease the amount of biofilm mass produced by P. aeruginosa, but it resulted in a 33% increase in biofilm production by S. aureus compared to the control. Moreover, UA had a stimulating effect on the growth of both P. aeruginosa and S. aureus, increasing the number of these bacteria by 41% and 12%, respectively. It should be noted that despite the lack of antibiofilm properties, UA reduced the activity of elastase (LasB), produced by P. aeruginosa, by 96%. It is believed that this enzyme affects biofilm architecture and functionality [Gilabert et al.  investigionality , while iionality .Arctium lappa leaves, on the ability of biofilm synthesis by P. aeruginosa ATCC 9027. UA, along with rutin, caffeic acid, coumaric acid, and quercetin, was found to be one of the five best-performing antibiofilm substances present in A. lappa leaves. The lowest UA concentration that completely inhibited biofilm formation by P. aeruginosa was 500 mg/L.Lou et al.  conducteS. aureus ATCC 2592 (MSSA), which can form a vancomycin-resistant biofilm, was used in that study. The identified MIC and MBC values for UA were 60 mg/L and >200 mg/L, respectively. It was found that UA inhibited biofilm mass growth by 46.5%. The expression of six genes  involved in biofilm formation in UA-treated bacteria was also investigated. Based on the analysis of obtained results, the mechanism of biofilm formation of the MSSA strain was different from that of the MRSA strain analysed by Qin et al. [agr) in the MSSA strain. These findings also suggest that biofilms of the MSSA strain may be more resistant to antibiotics than biofilms of the MRSA strain that has a fully functional agr.Tan et al.  extendedn et al. . The difE. coli strain CFT073 (ATCC 700928) and 10 uropathogenic clinical E. coli strains with genes encoding proteins that are important in biofilm formation were used in those studies. On titre plates, both ursolic acid and its combination with ciprofloxacin showed anti-biofilm activity, especially in older biofilms. There was a decrease in both the amount of produced biofilm mass and the number of viable bacteria. Unfortunately, UA used alone had a weak effect on the eradication of biofilm from urological catheters. Statistically significant eradication of the biofilm mass was obtained only after treatment with a mixture of UA and ciprofloxacin.Studies determining the effect of UA and UA combined with ciprofloxacin on the process of biofilm formation and eradication were also conducted by Wojnicz et al. . The resP. acnes, an opportunistic strain of Gram-positive bacteria that is resistant to many tetracyclines and macrolide antibiotics. P. acnes grows as a biofilm on biomedical materials (implants) and in hair follicles of the skin, causing acne vulgaris. Myrtus communis leaf extract, in which UA content was quantified by HPLC and it was 20%, showed significant antibacterial activity against P. acnes strains that are insusceptible to erythromycin and clindamycin, growing both in suspended matter and forming a biofilm. The analysed extract, at concentrations of 10–1000 mg/L, inhibited biofilm formation and reduced the structured 48-h biofilm of P. acnes. The authors also determined the antibacterial properties of the extract (10 mg/L) combined with erythromycin (1000 mg/L) or clindamycin (500 mg/L). In those combinations, the analysed extract restored susceptibility of P. acnes strains to both erythromycin and clindamycin.The subject of a study by Feuillolay et al.  was P. aS. mutans UA159 on the tooth surface. According to Chung et al. , when adS. marcescens. These bacilli may be the cause of catheter-related UTIs. UA was found to inhibit biofilm formation by the analysed bacterial strain.Ray et al.  analysedE. faecalis strains. Although UA at a concentration of 0.75 × MIC did not exhibit any significant inhibitory effect on biofilm mass synthesis, it significantly reduced the survival of cocci at all stages of 10-day biofilm development.Other Wojnicz et al. studies  investigS. aureus strains with icaD adhesion gene involved in biofilm production. Inhibition of biofilm formation was observed in 40 isolates of the analysed strains, and it was 48.6% for UA applied at a concentration of 30 mg/L and 71.5% when the UA concentration was 60 mg/L, respectively.Jyothi et al.  investigMalus domestica) against Gram-positive bacteria: E. faecalis ATCC 29212, S. aureus ATCC 25904, and S. epidermidis ATCC 35984. Only UA at a concentration of 100 μM showed antimicrobial activity against planktonic cells and it inhibited biofilm synthesis by all bacterial strains under analysis.Silva et al.  investigS. mutans and the structural integrity of its biofilms by interacting with the catalytic centre of glucosyltransferases, the key enzymes required in EPS synthesis. Lyu et al. [S. mutans, S. sanguinis, and S. gordonii) by inhibiting the expression of gft genes and in consequence leading to inhibition of EPS formation.As has been demonstrated by Liu et al. , UA decru et al.  found thThe studies published so far show that antibacterial activity of AA and UA is related to changes in the structure and functioning of the bacterial cell structures , cell morphology, expression of genes encoding virulence factors such as P fimbriae, curli fimbriae, and hydrophobicity. Both pentacyclic triterpenes can affect the adhesion of bacteria to host cells and the process of biofilm formation, but the exact molecular mechanisms of this activity are still not fully explained. Therefore, our article also presents and summarises suggested by researchers’ mechanisms of the biological action of these substances against microorganisms.The article describes how to improve the poor availability of acids in vivo that limits their clinical application. Structural modifications of these substances have been conducted in recent years to improve their biological activity and bioavailability, such as designing and synthesising novel derivatives, improvement of its water solubility, encapsulation in carries .In our article, we also intend to highlight that in the perspective of further research, the existence of synergistic effects of AA and UA with antibiotics  should be taken into account. It is even more necessary to define the rules for antimicrobial activity of both acids validation and its conversion of in vitro potency into in vivo therapeutic activity. Then, AA and UA could serve as supplements to standard pharmacotherapy.


Drosophila UC model induced by sodium dodecyl sulfate (SDS), we investigated the defensive effect of UA on intestinal damage. The results showed that UA could significantly protect Drosophila from the damage caused by SDS exposure. Further, UA alleviated the accumulation of reactive oxygen species (ROS) and malondialdehyde (MDA) induced by SDS and upregulated the activities of total superoxide dismutase (T-SOD) and catalase (CAT). Moreover, the proliferation and differentiation of intestine stem cells (ISCs) as well as the excessive activation of the c-Jun N-terminal kinase (JNK)-dependent JAK/STAT signaling pathway induced by SDS were restored by UA. In conclusion, UA prevents intestine injury from toxic compounds by reducing the JNK/JAK/STAT signaling pathway. UA may provide a theoretical basis for functional food or natural medicine development.Ursolic acid (UA) is a bioactive molecule widely distributed in various fruits and vegetables, which was reported to play a therapeutic role in ulcerative colitis (UC) induced by toxic chemicals. However, the underlying mechanism has not been well clarified in vivo. Here, using a  Ulcerative colitis (UC) is a chronic and recurrent inflammatory bowel disease that is listed as one of the modern refractory diseases by the World Health Organization (WHO) . UC is cUrsolic acid (UA) is an anti-inflammatory natural triterpenoid found in large quantities in different fruits and vegetables, including apple, basil, cranberry, and peppermint ,9. SeverDrosophila melanogaster (hereinafter Drosophila) represents one of the most important model organisms and has made fundamental contributions to different areas of biology [Drosophila contains the foregut, proventriculus, midgut and hindgut, and the intestinal function of Drosophila is similar to that of mammals [Drosophila midgut harbors multipotent adult stem cells that are essential to renew the gut in homeostatic conditions and upon stress-induced regeneration, and the Drosophila midgut has emerged in recent years as a model system to decipher regulatory mechanisms of stem cell biology [Drosophila [Drosophila to human beings [Drosophila has become a powerful model to study intestinal physiology and pathology [Drosophila has been used to screen plant extracts with potential applications in many chemical-induced intestinal disorders [ biology . The dig mammals ,28. The  biology ,30. Composophila . In addin beings ,33. Therathology . For exaisorders ,35.Drosophila was employed to investigate the effect and regulatory mechanism of UA on the intestine disruption induced by SDS. The results indicated that the decreased survival rate and mobility of adult flies caused by SDS could be restored by UA. In addition, the increased reactive oxygen species (ROS) and malondialdehyde (MDA) levels caused by SDS could be reduced by UA. Further studies indicated that the activated JNK and JAK/STAT pathways induced by ROS in UC models could be rescued by UA. These results elucidate the protective effect of UA on UC caused by environmental stresses. This study is of great significance for UA application and may improve the medicinal and nutritional value of UA in the future.In this study, 2DCFDA) was purchased from Thermo Fisher Scientific Co., Ltd. . All other reagents used were of analytical grade and commercially available.The UA (purity ≥ 98%) was purchased from Aladdin Co., Ltd. . The SDS (#L4509) and Brilliant Blue FCF (#80717) were purchased from Shanghai Macklin Biochemical Co., Ltd. . The phosphorylated JNK  antibody and Triton X-100 (#T8787) were purchased from Sigma-Aldrich Trading Co., Ltd. . The Cy3-conjugated goat anti-rat IgG (#A0521), 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride , phosphate buffered saline , total superoxide dismutase (T-SOD) assay kit (#S0101), catalase (CAT) assay kit (#S0051) and lipid peroxidation (MDA) assay kit (#S0131) were purchased from Beyotime Biotechnology . The 2′,7′-dichlorodihydrofluorescein diacetate . esg-Gal4; UAS-GFP; tub-Gal80ts (esg-GAL4ts) and Myo1A-Gal4; UAS-GFP; tub-Gal80ts (Myo1A-GAL4ts) flies were kindly gifted from Dr. Lihua Jin  [esg-GAL4ts and Myo1A-GAL4ts flies were collected within 48 h after eclosing and aged for 5 days before shifting to 29 °C  to induce GFP expression. Animals of the other genotypes were cultured under 25 °C and 60% humidity with a 12 h light:12 h dark cycle, unless otherwise noted. All flies were cultured on corn-yeast standard food as the previous report [The following flies were used: w1118 (V#60000) was obtained from the Vienna , China) . 10×STAT, China) . The esgs report . The conFor survival experiments, groups of ~3 days newly emerged flies  reared on normal food (NF) or UA-containing food (100 μM) were starved for 2 h before being transferred into vials containing three layers of filter paper soaked with 200 μL of 5% sucrose (control group) or 5% sucrose solution containing 0.5% SDS . To summarize, the control group flies were emergenced on normal food for 7 days and then fed with 5% sucrose (NF-SUC). The UC model group flies were emergenced on normal food for 7 days and then fed with 5% sucrose supplemented with 0.5% SDS (NF-SDS). The experimental group flies were emergenced on 100 μM UA food for 7 days and then fed with 5% sucrose supplemented with 0.5% SDS (UA-SDS). The filter papers and solution were changed once every 24 h. The surviving flies were counted and recorded every day. The experiment was repeated three times.w/v) for 24 h (as stated in Climbing ability assay was performed as described previously . Ten neww/v) for 24 h (as stated in Climbing ability assay was performed as described previously . Ten neww/v) for 48 h (as stated in Twelve newly eclosed (~3 day old) female flies were maintained on UA-supplemented (100 μM) or control diet for 7 days and then exposed to sucrose or SDS  or 5% sucrose as negative control (w/v). For the assay, the flies were starved for 2 h every other day and then treated for 6 h with the SDS/sucrose solution soaked on a Whatman filter paper before they were relocated to Brilliant Blue FCF-dyed food. After feeding for 7 days, the flies were frozen to death, photographed and then homogenized with 1× PBS and centrifuged. The intestine integrity was determined by the absorbance of the supernatant at 625 nm as the previous reports [The Smurf assay was performed as previously described with minor modifications . Briefly reports ,40.w/v) and incubated at 25 °C for 48 h. A total of 10~15 intestines were dissected in cold 1× PBS and incubated in H2DCFDA (5 µM in PBS) for 10 min, then washed 3 times in cold 1× PBS for 5 min, and immediately observed under a Nikon ECLIPES Ti2-U microscope . The data presented are from three independent experiments.Adult females were exposed to SDS .Data were analyzed by using Student’s t-test to compare between groups and one-way analysis of variance (ANOVA) for multiple groups. The statistical results were presented as mean ± SEM. Asterisks indicate the critical levels of significance (* Drosophila UC model induced by the inflammatory reagent SDS (NF-SDS). The UC model group flies (NF-SDS) showed shorter life spans and decreased mobility compared with the control group (NF-SUC) (p < 0.001) and from 2.5% to 30% (p < 0.001) in the UA-SDS group, respectively, compared with the NF-SDS group  A–D. As sDS group A,B. In aDS group C,D. Thesp < 0.001). In addition, the intestinal epithelium is susceptible to damage caused by the chemical compound SDS [Drosophila.The male flies and female flies upon SDS exposure exhibited largely similar phenotypes, except that the phenotypes of the female flies were more severe than those of the male flies. In addition, the protection of UA on female flies against SDS was better than the males. Therefore, only the results of female flies are shown hereafter. As shown in ound SDS . SDS-indound SDS ,43. The ound SDS ,45. To iesg-Gal4ts  [+ cells were clustered and showed aberrant morphology  . After 4rphology A. A statrphology B. UA suprphology A,B.Myo1A is specifically expressed in enterocytes (ECs) [Myo1A-GAL4ts to analyze the numbers of ECs. As shown in Myo1A GFP+ cells, compared with the control group). As expected, the increased ECs in the NF-SDS group could be significantly attenuated by UA; however, the number of ECs in the UA-SDS group remained larger than the control group (p < 0.05). These results indicated that the ISCs proliferation and differentiation induced by SDS could be rescued by UA.es (ECs) . Subsequ2DCFDA, a cell-permeable probe, was used to detect the intracellular ROS levels. Adult flies were exposed to SDS for 48 h and a robust fluorescence signal was observed in NF-SDS compared with control. The enhanced fluorescence signal in the posterior midgut of the NF-SDS flies was markedly reduced in the UA-SDS group flies (p < 0.05) compared with the UC model group  and catalase (CAT), help organisms resist oxidative damage . To furtDrosophila intestine from SDS-induced damage through the JNK signaling pathway.Many studies demonstrated that ROS can activate the JNK signaling pathway, and the ROS/JNK signaling plays a vital role in environmental stress responses . MoreoveDrosophila intestine homeostasis from toxic chemical-induced disruption.JNK acts synergistically with JAK/STAT in response to tissue damage in fly tissues . IntestiDrosophila intestine homeostasis is mediated by the reduction of the JNK/JAK/STAT signaling pathway. These results provide in vivo evidence for the protective effect of UA on SDS-induced intestinal dysfunction. Therefore, our findings may provide a theoretical basis for functional food or natural medicine development of UA.The intestinal epithelium is a prominent defense barrier against environmental stresses , and theROS plays an important role in maintaining intestinal homeostasis and improving stress tolerance in organisms . ExcessiDrosophila to environmental challenges. It is unknown whether there are other pathways that participate in this process. Additional protective molecular pathways stimulated as response to UA may be further explored by using high-throughput transcriptome-wide RNA sequencing in the future. Even though Drosophila has many organs that function like mammalian organs [Drosophila genome is homologous to that of humans [Several signaling pathways have been implicated in maintaining intestinal homeostasis, such as JNK, JAK/STAT, Notch and Hippo pathways ,64,65. Fn organs , there af humans , and wheDrosophila adults from SDS-induced intestinal damage. The behavioral defects, morphological defects and the ROS production caused by SDS could be rescued by UA. Further studies indicated that UA could remarkably attenuate the SDS-induced excessive ISC proliferation and differentiation. Moreover, the protective effect of UA on UC is mediated by the JNK/JAK/STAT signaling pathway. Therefore, our data provided evidence for UA to be potentially developed as promising alternative functional foods and medicines for UC.In summary, UA could protect


Objective: The key element in the pathogenesis of systemic sclerosis (SSc) is microcirculatory changes in several vascular beds. Uric acid is associated with endothelial dysfunction and therefore, microvascular damage. The aim of this study was to examine the association between uric acid (UA) and peripheral microvascular involvement in patients with SSc.Methods: We included consecutive, consenting patients with SSc. Serum UA, urea and creatinine were measured, and glomerular filtration rate (GFR) was calculated with CKD-EPI. All participants underwent nailfold video-capillaroscopy (NVC) to evaluate the microcirculation.Results: A total of 64 patients (95.3% women) were included in the study. UA levels were significantly associated with the number of avascular areas , whereas no correlation was shown for the GFR . A significant trend of UA in the three capillaroscopic patterns was shown . Multivariate analysis showed that male gender  and UA  were independently associated with the number of avascular areas.Conclusion: These data suggest that UA levels are significantly associated with the capillaroscopic patterns, reflecting a progressive microvasculopathy. In systemic sclerosis (SSc), inflammation and microvascular dysfunction appear to be the main events that progressively stimulate fibrotic process. The precise etiology of fibrotic changes remains partially understood and may include impaired communication between endothelial cells, epithelial cells and fibroblasts, lymphocyte activation, autoantibody production, inflammation, and connective tissue fibrosis . AlteratUric acid (UA) is the final oxidation product of purine metabolism. Elevated serum UA levels have been associated with endothelial dysfunction, possibly by decreasing nitric oxide availability  and stimIn this context, the aim of this study was to investigate the potential relationship between UA levels and microvascular alterations assessed by NVC in a large well-characterized cohort of SSc patients.The study included consecutive patients with SSc attending the Scleroderma Clinic of the Fourth Department of Internal Medicine, Hippokration General Hospital, Thessaloniki, Greece, between March 2018 and September 2020, who were screened for the study. All patients satisfied the revised EULAR/ACR criteria for the diagnosis of SSc . The excAll participants underwent a thorough physical examination and demographic data were collected by a questionnaire. Complete medical history was also recorded which included the duration of the disease, existence of pulmonary hypertension, pulmonary fibrosis, or esophageal motility disorders, as documented by imaging or endoscopic examination, respectively, as well as medication and cardiovascular risk factors .Various hematological and biochemical laboratory parameters such as routine biochemistry and hematology, lipid and bone profile tests, inflammatory markers such as erythrocyte sedimentation rate (ESR) and Creactive protein (CRP), immunological markers such as antinuclear antibodies (ANA), anti-centromeric antibodies (ACA), and anti-topoisomerase IIa (anti-scl-70) antibodies were tested. Serum UA and creatinine levels were measured with photometric measurement of the solution and GRF was calculated using the CKD-EPI equation . NVC assStudy participants underwent NVC using an Optilia Digital Capillaroscope and a 200 × contact lens and the photos collected were analyzed with OptiPix Capillaroscopy  software system. Prior to performing the test, patients were placed in a quiet environment at a temperature between 20 and 25°C. A drop of cedar oil was placed on each fingernail prior to observation for better image analysis. The findings were classified in one of the following qualitative patterns: early, active, and late NVC pattern . The “ean, %). Comparisons for continuous variables were performed with the student's t-test or the Mann-Whitney U test, according to the normality of the distribution. Multiple comparison analysis was performed with the ANOVA or the Kruskal-Wallis tests, according to normality. Chi-square or Fishers exact test was used for comparisons of categorical variables. Uni- and multivariable linear regression analysis was performed to explore the parameters possibly associated with the number of avascular areas. We first tested for univariate associations and included in the multivariable model only variables with associations of p < 0.2 in univariate analysis. We report β-coefficients with 95% confidence intervals (CI). P < 0.05 (two-tailed) were considered statistically significant for all comparisons.Statistical analysis was performed with Statistical Package for Social Sciences 22 . Continuous variables were expressed as mean values ± standard deviation (SD) or median [interquartile range] according to the normality of distribution tested with the Kolmogorov-Smirnov or the Shapiro-Wilk test. Categorical variables were presented as absolute frequencies and percentages , whereas no correlation was shown for the eGFR . All the other NVC parameters measured showed no correlation with the levels of UA. There was no significant correlation with CSURI .The correlation of UA, urea, creatinine, and GFR with the parameters of NVC is presented in p = 0.028) (p = 0.019 and p = 0.052), while the degree of NVC changes was not associated with creatinine or eGFR levels.Within-groups comparisons revealed a significant trend of UA levels in the capillaroscopy patterns reflective of progressive micro-vasculopathy  . SimilarIn order to identify possible factors associated with the avascular areas in NVC, we have performed a uni- and multivariable linear regression analysis with a number of avascular areas being the dependent variable and several demographic, anthropometric, medical history, and laboratory parameters as the independent variables. As shown in The main finding of our study is the positive correlation between NVC structural alterations and serum UA concentrations in patients with SSc. In particular advancing stages of SSc-related microangiopathy as determined by both NVC “scleroderma patterns” and the number of avascular areas are associated with higher UA levels, indicating UA as a potential biomarker of peripheral vascular damage in SSc.This possibility may reflect biologically relevant metabolic procedures involved in the pathogenesis and progression of SSc-related microvasculopathy. For example degradation of UA by the oxidative action of the enzyme xanthine oxidoreductase leads to the production of reactive oxygen species and the initiation of several detrimental procedures such as increased cytokine production, inflammation, and endothelial activation all of which contribute to vascular injury . Taking Thus, it is not surprising that previous reports have demonstrated significant associations between high UA levels and various aspects of SSc vasculopathy. In fact elevated UA levels do not only confer a diagnostic value in the identification of SSc individuals at early, asymptomatic stages of PAH as indicated by DETECT study , but theOur findings are in line with a previous small study  and confOn the other hand, the relationship between high UA and various markers of microvascular dysfunction in different vascular beds namely retinal arteriolar narrowing , microalThe main limitation of our study is the single-center, cross-sectional design which precludes any causal relationships between UA levels and vasculopathy as discussed above. However, NVC was performed in all fingers except thumbs and the acquisition of two adjacent images from each finger according to the updated European League against Rheumatism (EULAR) recommendations . We tookIn conclusion, serum levels of UA are significantly associated with progressive micro-vasculopathy based on the qualitative NVC pattern. These results provide evidence that UA may be a significant mediator of the microvascular damage in these patients.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Ethics Committee of School of Medicine, Aristotle University Thessaloniki, Greece. The patients/participants provided their written informed consent to participate in this study.EP collected the data and drafted the paper with support from TD. SS contributed to the perception of the study and the editing of the paper. ET collected the data. AM verified the analytical methods and performed the analysis. GK and AG were involved in planning and supervised the work and critically reviewed the paper. TD supervised the project and contributed to drafting the paper. All authors discussed the results and contributed to the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Background: Despite the rising prevalence of infertility, studies have indicated that in the United States fertility awareness remains low. No published study to date, however, has investigated the impact of any racial or ethnic disparities in fertility awareness.Materials and Methods: We conducted a cross-sectional survey of people self-identifying as female, aged 18–45 years, via Amazon Mechanical Turk in August 2020. The study was approved by the institutional review board at the University of Colorado. The survey consisted of demographic questions and a validated questionnaire, the Fertility and Infertility Treatment Knowledge Score (FIT-KS). Participants were classified as non-Hispanic White (NHW) or “Minority” race/ethnicity.Results: A total of 476 women completed the survey, 405 of which were included in analysis. Of those, 54.6% self-identified as NHW and 45.4% were in the Minority group. The median FIT-KS was 51.7% (16 items answered correctly). The Minority group scored significantly lower than the NHW participants overall  and in all three subscales (p < 0.05). The Minority group was significantly more likely to underestimate the rate of miscarriage  and had a lower awareness of risk factors that can impact fertility including smoking , obesity , and/or a history of gonorrhea/chlamydia infection .Conclusions: Minority women appear to have a lower fertility awareness than their NHW counterparts. Addressing these disparities and improving fertility education in diverse communities may lead to a reduction in clinically significant infertility disparities. In the United States, these disparities often persist throughout one's reproductive lifetime, from birth to menopause.3 As women in the United States are continuing to delay childbearing for various reasons, reports have shown that there has been increasing cases of infertility and smaller than desired family sizes.5 In fact, data from the National Survey of Family Growth highlight that from 2011–2015 to 2015–2017 there has been a rise in the prevalence of impaired fecundity and an increasing percentage of women seeking infertility services.7 Furthermore, studies have also shown that not only do racial and ethnic minorities including Black and Hispanic women experience infertility significantly more frequently than their Caucasian counterparts, but they are also significantly less likely to receive infertility treatment.9A key example of a racial and ethnic disparity that exists in reproductive medicine is the prevalence, diagnosis, and treatment of infertility.10–13 Using the Fertility and Infertility Treatment Knowledge Score (FIT-KS), Kudesia et al.11 highlighted that gaps in fertility knowledge exist in the general reproductive-age U.S. female population, and even among medical trainees. In this seminal study, female medical trainees from two large urban academic medical centers including medical students and obstetric and gynecology residents were found to have substantial gaps in their fertility knowledge.11Despite the rising prevalence of infertility and subsequent smaller than desired family sizes, fertility awareness defined by the International Glossary on Infertility and Fertility Care as “the understanding of one's reproduction, fecundity, fecundability, and related risk factors” remains low.14Notably, meta-analyses have suggested a slight increase in fertility awareness among more educated women and among those who have had difficulties in conceiving; yet to the best of our knowledge, no study to date has investigated the presence of any racial or ethnic disparities in fertility awareness.It is imperative to identify and address any meaningful differences in the level of fertility understanding among women from various diverse backgrounds. Increased fertility understanding is likely to help women to make informed decisions regarding planning a desired pregnancy delay, preventing pregnancy when not desired, or actively trying to become pregnant. The overriding objective of our study is to identify any patterns in fertility knowledge gaps among a diverse patient population. Enhanced knowledge of any relevant disparities will serve to enrich individualized reproductive education and counseling regarding fertility-based planning.XM  and disseminated to participants living in the United States via the Web-based platform Amazon Mechanical Turk (MTurk) from August 6 to 9, 2020. MTurk is an online crowdsourcing forum where requesters can publish and request the completion of various human intelligence tasks, including surveys, to willing participants in the general population. The survey was made public only to registered MTurk users living in the United States who self-identified as female. After completion of the survey in its entirety, the participants were compensated with $1.00. The study was approved by the Colorado Multiple Institutional Review Board.This was a cross-sectional study using a previously validated questionnaire, the FIT-KS. The study instrument was uploaded to Qualtrics Survey SoftwareThe survey was made available in both English and Spanish and consisted of 29 questions surrounding topics related to fertility and infertility risk factors as well as 10 demographic questions. The survey was translated into Spanish by a certified Medical Spanish translator.The questions were separated into three sections, the first of which covered topics related to natural fertility such as the average rate of fecundability in a given menstrual cycle, the average rate of spontaneous abortion, the fertility window in a given menstrual cycle, and male versus female infertility and contribution to fertilization, among others (20 items). The second group of questions explored topics related to infertility risk factors in a “true” or “false” format including factors, such as tobacco use, obesity, malnutrition, alcohol consumption, caffeine intake, history of sexually transmitted infections, history of pregnancy termination, history of contraception use, and use of certain sexual lubricants (1 item). Finally, the last section explored infertility treatment options as well as their corresponding success rates and cost using the most recent Society for Reproductive Technology data (8 items). The specific questions are available in .11 after it underwent extensive review by experts in the field and face validation by the general population living in the United States. First, the questions were ensured to be of appropriate complexity and accuracy by a panel of 15 reproductive endocrinologists. Next, the survey was distributed to 10 participants unaffiliated with the medical field to ensure clarity and was analyzed for item difficulty, instrument reliability, internal consistency, item consistency, and item discriminability. Finally, the survey underwent both discriminative and convergent validity by correlating the total test scores with the level of the participant's gynecologic training as well as by comparing responses to a previously validated survey with several overlapping questions published by Lampic et al.15The questionnaire was designed and published in 2017 by Kudesia et al.In addition, demographic information including race, ethnicity, preferred language, marital status, gravidity, parity, insurance type, and education level was also collected. The classification of race and ethnicity was selected by the participant, and the various options were defined by the investigator, using a similar format to that seen on the U.S. Census Bureau including White, Black or African American, Asian, American Indian, Alaska Native, Native American or Other Pacific Islander, Hispanic or Latinx, or not Hispanic or Latinx. The participant was also given the opportunity to self-describe their racial background if it was not included in the available options. Due to the number of subjects self-identifying in each racial and ethnic minority group, all participants who identified as a race other than non-Hispanic White (NHW) were combined into the Minority group. However, 16 As some of these topics are explored in the questionnaire, the decision was made to exclude women younger than 18 years to avoid any potential confounding variable such as accessibility.Additional inclusion criteria included women between the ages of 18 and 45 years and able to read and write in English or Spanish. The age range of 18–45 years was chosen as this is the age-range that the Centers for Disease Control and Prevention defines as Women of reproductive age. Additionally, the lower limit of 18 years old was also used as according to the latest reports from the Guttmacher Institute, there are still several states in the United States that require parental consent or notification for certain reproductive health services in this population including abortions and contraception.t-tests or nonparametric equivalents for continuous variables and chi-squared or Fisher's exact test (for variables with cell sizes <5) for categorical or dichotomous variables. Variables were significant (p < 0.1) in bivariate comparisons, and the Minority group was entered into a linear regression model to predict percent correct on the FIT-KS scale. IBM® SPSS Version 27 was used for all analyses.Responses to items from the FIT-KS were scored as correct or incorrect. These responses were summed, and a percent correct variable was calculated. We computed descriptive statistics including tests of normality for continuous variables. To compare responses of Minority participants with NHW respondents, we used Student's The survey was published on the MTurk site, and 476 women completed the survey. Of these, 85% were included in the final analysis (405). The main reason for not being included in the analysis was not meeting the age inclusion criteria (57 respondents were aged 46 years or older and 1 participant aged 17 years or younger). Additionally, 13 participants did not answer the question about their age and were therefore excluded from analysis.The remaining responses included in analysis were from a diverse group of reproductive-aged women who were predominantly English-speaking, had a college degree or higher, and were privately insured . The majp ≤ 0.001). Additionally, the Minority group had a significantly higher number of participants with a master's degree or higher , but they were significantly less likely to have private insurance (57.6% vs. 65.6%). There was no significant difference between the two groups in terms of language preference (p = 0.33). Most participants in both groups spoke English as a preferred language , whereas only 4 total participants (1.0%) preferred to take the questionnaire in Spanish. Other preferred languages included Tamil (3), Korean (1), Hindi (1), Portuguese (5), French (1), and Italian (1).While there was a fairly even distribution of responses from women of all age categories in both groups, the Minority group was significantly more likely to be <26 years old , Minority participants were less likely to be nulligravid  and nulliparous . The Minority group was also more likely to desire pregnancy in the next year   .p < 0.001). In logistic regression analysis, having private insurance  and nulliparity  was positively associated with percent correct, and desiring pregnancy was negatively associated . The Minority group was an independent predictor of percent correct .Among all respondents, of a maximum possible score of 29, the median score was 51.7% (16 answered correctly), with a range from 24.1% to 86.2% (7–25 answered correctly). Overall, there was a significant difference in the median scores between the NHW and Minority groups, with the Minority group scoring significantly lower than the NHW group  , lifestyle risk factors impacting fertility (question 13), and infertility treatment options (questions 14–29), the Minority group scored significantly lower in all three categories  . These d< 0.001) .p = 0.003; 44.3% correct vs. 27.9%, p = 0.001). Additionally, while the majority of both groups were aware that both a man and woman can contribute to a couple's infertility, the Minority group was significantly less likely to answer this question correctly .In regard to natural fertility knowledge, on average, women from both groups tended to underestimate the rate of miscarriage in a reproductive-aged woman  and overestimate the age that fertility declines most precipitously . However, the Minority group was even more likely to overestimate and answer these questions incorrectly , obesity , and/or a history of gonorrhea/chlamydia infection . Approximately half of participants overall believed that safe pregnancy termination can negatively impact a woman's fertility, yet the Minority group was significantly more likely to incorrectly answer that this statement is true .In terms of lifestyle factors that can impact fertility, the Minority group scored significantly lower on questions related to risk factors that can decrease a woman's chance of fertility, including smoking  and egg cryopreservation . In general, respondents tended to overestimate the success of ART and underestimate the chance of twins. Furthermore, the NHW group was significantly more likely to overestimate the average price of one in vitro fertilization (IVF) cycle in the United States .With regard to infertility treatment options, respondents were generally aware of the artificial reproductive technology (ART) options and definitions; however, the Minority group was less familiar with intrauterine insemination  and are less familiar with the average cost of an IVF cycle than the Minority women. NHW women are more likely to be privately insured than the Minority women, and findings from our study suggest that private insurance may portend a higher fertility awareness.25 While MTurk participants are compensated and rated by their thorough completion of published tasks, the workers are completing such tasks in an unsupervised environment with little verification surrounding the accuracy of such responses. Nonetheless, many studies have shown MTurk to be a trustworthy and reliable source of information that may even be superior to that of other samples and has now become the largest online crowdsourcing platform in the world.26 Furthermore, while the FIT-KS questionnaire was first reviewed by a panel of reproductive endocrinologists for appropriate depth and then administered to laypeople for face validation in the original study, the questions are designed to be quite difficult and therefore may not accurately reflect a clinically relevant fertility understanding among the general population. Finally, although in our supplementary analysis we found that the differences in fertility awareness among the Minority group persisted when divided into NHB, Hispanic, and NHO, the study was not adequately powered to address any specific differences among the individual racial or ethnic Minority groups. Despite these limitations, we believe that the findings from this study highlight the need for culturally competent and individualized counseling in reproductive and fertility-based education. More research is needed to investigate possible nuances in the various racial and ethnic Minority groups and to determine the most effective interventions to improve fertility awareness among an ever-increasing diverse population.While this was a novel study highlighting racial and ethnic disparities in fertility-related knowledge, there certainly were several limitations. To start, the data collection platform MTurk used to obtain the survey responses may recruit participants who are not representative of the U.S. population and may therefore hinder the generalizability and external validity of such findings. To complete a survey published on MTurk, one must have reliable access to internet and a functional computer, tablet, or smartphone. MTurk participants, therefore, on average tend to be more highly educated and younger than the national population.Fertility awareness overall remains quite low among reproductive-aged women in the United States, and no improvements in these knowledge gaps have been made over the past several of years. Importantly, these knowledge gaps are even more significant among racial and ethnic Minority women. Addressing these disparities and improving fertility education in underserved communities may lead to a reduction in clinically significant infertility disparities. Not only can improved fertility awareness help those who are actively trying to become pregnant achieve their goal of parenthood, but it may also provide information to those avoiding or delaying pregnancy as more women are relying on natural family planning to prevent an unwanted pregnancy. Further work is needed regarding the development and implementation of culturally sensitive and individualized reproductive education and counseling regarding fertility-based planning.