Microbial biofilm is an aggregation of microbial species that are either attached to surfaces or organized into an extracellular matrix. Microbes in the form of biofilms are highly resistant to several antimicrobials compared to planktonic microbial cells. Their resistance developing ability is one of the major root causes of antibiotic resistance in health sectors. Therefore, effective antibiofilm compounds are required to treat biofilm-associated health issues. The awareness of biofilm properties, formation, and resistance mechanisms facilitate researchers to design and develop combating strategies. This review highlights biofilm formation, composition, major stability parameters, resistance mechanisms, pathogenicity, combating strategies, and effective biofilm-controlling compounds. The naturally derived products, particularly plants, have demonstrated significant medicinal properties, producing them a practical approach for controlling biofilm-producing microbes. Despite providing effective antibiofilm activities, the plant-derived antimicrobial compounds may face the limitations of less bioavailability and low concentration of bioactive molecules. The microbes-derived and the phytonanotechnology-based antibiofilm compounds are emerging as an effective approach to inhibit and eliminate the biofilm-producing microbes. A biofilm is a complex of micro-organisms that sustains a structured and organized pathway for their growth, proliferation, and survival on any surface ,2. A sinThe development of microbial resistance to antibiotics reduces or inhibits the efficacy of antibiotics. Consequently, it has been demonstrated that the improper use of antibiotics leads to the emergence of antibiotic resistance . Many anThis study explores the new combat approaches that avoid existing resistance mechanisms. Using different natural products, such as plant-derived components, bee products, marine-derived components, and plant-based nanomaterials, is gaining great attention from researchers to design novel antibiofilm compounds to avoid AMR. In the current review, the emerging global concerns regarding the development of biofilm resistance and their control by describing some potential therapeutic compounds have been recapitulated.Biofilm can be defined as the complex aggregation of micro-organisms, firmly adhered to a surface and micro-colonized into extracellular polymeric substances (EPS) matrix. EPS is comprised of exopolysaccharides, nucleic acids, lipids, and proteins . The emeIn addition, the EPS also improves biofilm tolerance toward different antimicrobial agents and immune cells. The biofilm maturation needs several developmental phases with particular features, resulting in biofilm development on the surfaces. Additionally, understanding every developmental phase is essential for designing and applying proper antimicrobial agents against microbial biofilms. Each developmental phase is briefly elaborated on in Biofilm development is initiated with the attachment of microbial cells to any surface. Several sensing pathways  facilitate the microbes’ identification of the surface . After iSurface adhesion is essentially the required parameter for biofilm development. The adhesion initiation and the biofilm dispersal start with the adherence capacity of a specific bacterial species concerning the host surface. The surface attachment, along with biofilm development, is the survival strategy of the microbes that typically adhere to themselves in a particular way to meet their environmental and nutritional requirements. The surface adhesion consists of two phases: the primary/reversible phase and the secondary/irreversible attachment phase . Both ofThe formation of mature biofilm is followed by early biofilm development when the microbes begin to multiply and divide, creating micro-colonies incorporated with the EPS matrix . The EPSEscherichia coli produces N-acetyl-heparosan lyase and Pseudomonas aeruginosa, P. fluorescens produces alginate lyase, and Streptococcus equisimilis produces hyaluronidase. The development of flagella originates with the upregulation of protein expression by microbial cells. It allows the microbes to move to a new place for colonization and supports them in spreading biofilm-associated diseases. The remodeling of the EPS matrix and the dispersal pathways activation may induce dispersion of biofilm that can assist in combating biofilms [During the dispersal phase, the micro-organisms of sessile biofilm begin to disperse and change into motile form. On the other hand, the microbes that do not produce extracellular polysaccharides directly scatter into the environment by applying a mechanical force. The dispersed microbial communities produce saccharolytic enzymes, which release surface microbes towards a new place for colonization. The biofilms .Biofilm is a complex of variability and heterogeneity consisting of 10–25% microbial cells and a 75–90% self-developed EPS matrix . MoreoveP. aeruginosa biofilms [Polysaccharides developed by microbes can be categorized into two categories: hetero-polysaccharides and homo-polysaccharides. Most polysaccharides are heterogeneous in nature, while only a few are homogenous in nature, such as glucans, sucrose-based fructans, and cellulose . Severalbiofilms . Some poP. aeruginosa were observed in outer-membrane vesicles as membrane proteins. Some of them were secreted and lysed by cell-derived proteins [The biofilm complex can have a substantial number of extracellular proteins . Their iproteins .Several extracellular enzymes have also been found in microbial biofilms; some of them are involved in biopolymer degradation. The extracellular enzyme substrates contain water-insoluble components , water-soluble compounds , and the biofilm entraps organic particles . AdditioStaphylococcus aureus biofilm, while in S. epidermidis biofilms, it is produced as a minor constituent [P. aeruginosa and facilitates intercellular interactions [eDNA is one of the prime components of the EPS matrix, which is essential for the accumulation of microbes within the biofilm. The amount of eDNA production may vary even among closely linked microbial species. eDNA is the primary structural constituent in the matrix of a stituent . It has stituent ,43. eDNAractions .P. aeruginosa biofilm formation, while in Bacillus cereus the eDNA acts similar to adhesins [Listeria monocytogenes [P. aeruginosa biofilms against different antibiotics [Moreover, the eDNA was observed to inhibit the adhesins ,46. Okshadhesins . eDNA haytogenes . Wilton ibiotics .Rhodococcus spp. generate hydrophobic EPS, and can attach to Teflon and may colonize the waxen surfaces with the help of hydrophobic EPS [The extracellular polysaccharides, proteins, and eDNA are quite hydrated (hydrophilic) molecules, while the other components of EPS exhibit hydrophobic properties. Some bacterial species, such as obic EPS . In the obic EPS . An impoobic EPS .Water is recognized as the largest constituent (accounts for up to 97%) of the EPS matrix of most of the biofilms, and it retains the biofilm hydrated and protects against desiccation . The watThe antimicrobial resistance and the other functional characteristics of microbial biofilms are linked with the structure of the biofilm, matrix shape, and the 3D organization of microbes . The locP. aeruginosa, an EPS known as alginate facilitates biofilm formation and its architectural stability [The cell-cell communication, environmental influences, and the secondary messengers, such as c-di-GMP and cAMP, structure the biofilms by providing microbes with better environmental adaptability . Severaltability .Vibrio cholera and E. coli facilitates biofilm development in a three-dimensional configuration [Bacillus subtilis biofilm, and maintaining the biofilm matrix’s integrity [The EPS in guration ,61. An Entegrity . The arcntegrity .Aspergillus fumigatus biofilm formation. MAPKs reduce the A. fumigatus adhesion in biofilm formation. The ΔpphA strain was found to be more susceptible to cell wall destroying antimicrobials, had less chitin, and enhanced β--glucan, resulting in reduced adherence and biofilm formation [Manifoili et al. determined the impact of three different mitogen-activated protein kinases (MAPKs)  and protein phosphatases (PhpA) on ormation .S. aureus (strain LAC) by promoting bacterial adhesion and accumulation [Cronobacter sakazakii survival and biofilm formation under NaCl-stressed conditions [Streptococcus parasanguinis [The bacterial cell wall-linked fibronectin-binding proteins (FnBPs) (FnBPB and FnBPA) mediate the biofilm development of methicillin-resistant mulation . The outnditions . A surfaanguinis .bhp- and ica-dependent modes. AI-2 regulates the QS in S. epidermidis through increased transcription levels of bhp (biofilm-linked protein containing icaR) and ica operon [Intracellular cyclic dinucleotide and extracellular quorum sensing (QS) signaling cascades are crucial in biofilm development. It has been reported that these two signaling pathways may coincide or link up and synergistically mediate biofilm formation . QS is ta operon .P. aeruginosa include integrated QS (IQS), PQS, rhl, and las. These QS systems activate each other by regulating QS-associated genes [P. aeruginosa biofilm formation [QS signaling cascades observed in ed genes . The smaormation . SeveralSeveral antimicrobial resistance mechanisms have been identified, and biofilm development is one of the major factors of resistance emergence. Mechanisms allowing microbial biofilms to resist or tolerate the antimicrobials’ actions are discussed here.2O2 (hydrogen peroxide), to microbial cells inside the biofilm. It has been observed that P. aeruginosa in the planktonic state is more susceptible to H2O2, while in the form of a biofilm, it can survive even at a very high concentration of H2O2 [As EPS is crucial for a biofilm’s architectural stability, it also acts as a physical barrier, protecting or shielding the embedded microbes against antimicrobials, ultraviolet light, etc. ,86. The  of H2O2 .Pantoea agglomerans YS19, expresses SPM43.1 protein (acid-resistant) at high levels to resist harsh environmental conditions [The heterogeneity inside the developed biofilms averts the entire eradication of all involved microbial cells by antimicrobials. There are oxygen and nutrient gradients from the top-bottom of microbial biofilms. From the top-bottom of the biofilm matrix, the oxygen and nutrient reduction leads to a reduced growth rate and metabolic activity . The pronditions ,91.V. harveyi, (c) autoinducing peptide 1 (AIP-1) by S. aureus, (d) N-(3-oxoacyl)-l-homoserine lactone (3-oxo-AHL), (e) diffusible signaling factor (DSF), (f) N-(3-hydroxyacyl) homoserine lactone (3-hydroxy-AHL), (g) 2-heptyl-3-hydroxy-4(1H)-quinolone by P. aeruginosa, and (h) hydroxy-palmitic acid methyl ester (PAME). They are presented in Quorum sensing (QS) in microbes is a cell-cell communication mediated by activating specific signaling molecules, facilitating environmental adaptation to microbes . QS is aEnvironmental factors, such as nutrient deficiency, pH, antimicrobials, and salt concentrations, regulate QS-mediated activity in bacterial biofilms . The feeThe efflux pumps (proteinaceous) entrenched in the cytoplasmic membranes act as active transporters. The multidrug and toxic compound extrusion family (MATE), the ATP-binding cassette family (ABC), the small multidrug resistance family (SMR), the resistance-nodulation-division family (RND), and the major facilitator superfamily (MF) are commonly reported efflux pump classes in different bacteria ,101.P. aeruginosa pathogen [Burkholderia cepacia against tobramycin [Several molecular studies have revealed that increased efflux pumps are a popular and criticizing resistance mechanism in microbial biofilms . This mepathogen . The PA1pathogen . Numeroubramycin .Numerous factors are known to contribute to pathogenicity in biofilm-producing microbes. Microbial biofilms release different extracellular substances, altering the gene regulation of several microbial virulence factors. Moreover, the biofilm-producing microbes strengthen the maturation rate of biofilms to escape from host defenses, enhance the activity of β-lactamase, and for plasmid-mediated gene transfer resulting in intense virulence and antimicrobial resistance with enhanced mutation rate and efflux pump. The properties of the extracellular matrix contribute to the biofilm’s pathogenicity, offering a defensive barrier with less antimicrobial and immune cell penetration .The MIC of antibiotics is significant against planktonic microbes but not effective against biofilm microbes . MicrobiP. aeruginosa and S. aureus, can cause chronic biofilm infections, and hospitalized individuals (approximately 8–10%) are more vulnerable to carrying infections.The pathogenic activity of microbes in the form of biofilms is significantly higher, and they can escape from host defense cells and antibiotics. In numerous infections, biofilm bacteria are concerned with the pathogenesis and clinical symptoms . OpportuE. coli, L. monocytogenes), food intoxication , and gastroenteritis  can be caused by biofilm-producing pathogens [Biofilm-associated infections pose a threat to human health. Over the last few decades, innovative methods have been discovered to control microbial infections. Biofilm formation in the era of the food industry poses a serious threat to human health. Biofilms may contain only one type of bacteria, different bacterial species, or fungal species that may be pathogenic and may only target immunocompromised patients . Systematic diseases , hydrophobicity, exopolysaccharides (EPS) production by microbes, and the bacterial appendages , contribute to a crucial role in biofilm formation . Some st E. coli ,125. BioThe global rise in antibiotic resistance has led to the failure of antibiotics. The ABR has become a major threat to human health. Therefore, alternative therapies have been reported to eliminate or inhibit biofilm formation and their associated infections. Different points can be targeted for biofilm inhibition and eradication at different stages of biofilm formation . These cMany natural compounds can act as biofilm-controlling compounds by interfering with QS, possessing antiadhesive properties, and inhibiting biofilm formation .P. aeruginosa by polymorphonuclear leukocytes (PMNs) within the immune response of a mouse infection model [Chamaemelum nobile is a naturally occurring, well-known plant for its antimicrobial, anti-inflammatory, antiseptic, spasmolytic, anticatarrhal, sedative, and carminative properties. It can inhibit P. aeruginosa biofilm by disrupting QS [E. coli to uroepithelial cells [Helicobacter pylori to the gastric mucous of humans [Streptococci spp. biofilm formation [Fritillaria verticillata, Rhus verniciflua, Cocculus trilobus, and Liriope platyphylla, have been analyzed for their antibiofilm activity. Comparatively, Cocculus trilobus (ethyl acetate fraction) has shown the highest antibiofilm activity against Gram-positive bacteria by providing effective antiadhesive activity [Herba patriniae, with medicinal properties, has averted the gene expression of six genes linked with biofilm development and EPS production in P. aeruginosa [Ginkgo biloba, exhibited antitumor, antimicrobial, and neuroprotective and is active against S. aureus strains and E. coli biofilm formation [Several naturally occurring compounds can be used as antibiofilm molecules to eradicate or inhibit biofilm development. The garlic extract can significantly block QS and may promote rapid virulence attenuation  of on model . Perssonon model . Chamaempting QS . Proanthal cells . Cranberf humans , and it ormation ,152. Eigactivity . A Chineruginosa . The Ginormation ,156.Dioon spinulosum plant extract against the clinical isolates of P. aeruginosa. The in vitro analysis of antibiofilm activity showed a 77.1–34.3% reduction in biofilm formation at 250–500 μg/mL concentrations. Both in vitro and in vivo investigations revealed a significant reduction in P. aeruginosa biofilm formation. However, preclinical studies leading to clinical studies are recommended to allow its practical application in treating P. aeruginosa infections [Apium graveolens, Plantago ovata, Vitis vinifera, Viscus album, Senna acutifolia, and Melissa officinalis, against Aggregatibacter actinomycetemcomitans. The A. actinomycetemcomitans was collected from patients with dental caries. The obtained results indicated that the A. actinomycetemcomitans was more sensitive to S. acutifolia and M. officinalis, with zone inhibitions of 33 and 35 mm, respectively [Encephalartos laurentianus (methanol extract) against C. albicans. The in vitro antibiofilm analysis revealed a 62.5–25% reduction in C. albicans cell percentage. The in vivo evaluations of E. laurentianus performed on C. albicans infected rats resulted in an increased survival rate with a protective effect against renal damage caused by C. albicans [Elekhnawy et al. determined the antiquorum sensing and biofilm inhibitory potentials of fections . Obaid eectively . Negam ealbicans .Acalypha wilkesiana, Alchornea laxiflora, Ficus exasperata, Jatropha gossypiifolia, Morinda lucida, and Ocimum gratissimum plant extracts against poultry pathogens . All plant extracts showed effective biofilm inhibition of approximately >50% against the tested micro-organisms [Malva sylvestris methanolic extract against some human pathogens, such as E. coli, S. aureus, P. aeruginosa, E. faecalis, and K. pneumoniae. The highest biofilm inhibition was examined against S. aureus (89.19%), K. pneumoniae (95.46%), and E. faecalis (98.79%) with 40 μg/mL MIC [Olawuwo et al. investigated the in vitro antibiofilm activity of rganisms . Fathi eg/mL MIC .Paederia foetida against E. coli, Mycobacterium smegmatis with 30–50% inhibition, respectively [Cinnamomum buramanii against periodontal pathogens, such as Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis. The outcomes revealed that all C. buramanii concentrations showed effective antibiofilm activity against both periodontal pathogens [Priyanto et al. studied the antibiofilm potential of leaf extract of ectively . Panjaitathogens .Artemisia arborescens plant extracts against S. aureus (ATCC 25923), E. coli (ATCC 25922), P. aeruginosa (ATCC 15442), E. faecalis (29212), and C. albicans (10231). The hot methanol extract showed the highest antibiofilm activity against S. aureus, E. coli, and C. albicans with 58–67% inhibition [Cymbopogon spp. (Cymbopogon proximus and Cymbopogon citratus) against Malassezia furfur and Candida spp. The EOs of C. proximus and C. citratus showed significant biofilm inhibition ranging from 27.65 ± 11.7 to 96.39 ± 2.8 against all the tested organisms. Based on the reported results, the EOs of both Cymbopogon spp. can be used for the prevention of Malassezia and Candida infections [Plescia et al. determined the antibiofilm potential of hibition . Rhimi efections .Allium sativum  against nosocomial and food pathogens S. aureus, E. coli, L. monocytogenes, and Acinetobacter baumannii. The EOs from the bulbs and aerial parts of Bianco del Veneto showed significant inhibitory activity against all tested bacteria, more prominently against L. monocytogenes 64.29–60.55%, respectively. The EOs from the aerial parts of Staravec exhibited effective inhibition more effectively against Acinetobacter baumannii (45.61%), while EOs from the bulbs of Staravec showed no inhibition. The outcomes revealed their potential application as potential antibiofilm agents in the food industry and health sector as well [Nazzaro et al. determined the antibiofilm activity of EOs of aerial parts and bulbs of two different cultivars of  as well .Jatropha flowering plant. The EOs were obtained from J. intigrimma, J. gossypiifolia, and J. roseae. The 7.81, 15.63, 31.25, 62.5, 125, 250, 500, and 1000 μg/mL concentrations were used to evaluate the antibiofilm activity. J. intigrimma EO exhibited 100% biofilm inhibitory activity at 31.25 μg/mL. J. roseae EO showed 100% inhibition at 250 μg/mL, while J. gossypiifolia EO revealed less effective biofilm inhibition even at 1000 μg/mL. However, it can be suggested that J. intigrimma and J. roseae EOs can be used as promising antibiofilms and furthering in vivo investigations is also highly recommended [Thymus algeriensis, Eucalyptus globulus, and Origanum glandulosum. The EOs from all three plants showed significant antibiofilm activity with low minimum inhibitory concentrations (MICs) ranging between 0.078–1.25 μg/mL against C. albicans, E. coli, E. faecalis, L. monocytogenes, S. aureus, and S. Typhimurium. The order of biofilm inhibition against the tested bacteria was revealed as T. algeriensis > O. glandulosum > E. globulus EOs. The T. algeriensis EO showed the highest inhibition against L. monocytogenes (80.95%), and E. coli (77.83%) at MICs [Gamal El-Din et al. investigated the in vitro antibiofilm potential of EOs of three species of ommended . Djebill at MICs . Some otBee products are effectively being studied for their wide range of antibacterial, antioxidant, antiviral, antifungal, and anticancerous activities. Honey and its bioactive components are well recognized for their potential antibacterial effects against a wide range of bacteria and even against several antibiotic-resistant bacteria ,186,187.P. aeruginosa, methicillin-resistant S. aureus (MRSA), and methicillin-sensitive S. aureus (MSSA) biofilms. Manuka and Sidr honey have notably more effective antibiofilm activities against P. aeruginosa, MSSA, and MRSA, ranging from 91%, 63–82%, and 63–73%, respectively [K. pneumoniae [E. coli [P. aeruginosa [Ureaplasma urealyticum, and Ureaplasma parvum [Bouchelaghem et al. collected propolis from six Hungarian regions and evaluated the in vitro antibiofilm activity by using ethanolic extract (EEP) alone and in combination with vancomycin against MSSA and MRSA. The EEP significantly prevented planktonic growth. The EEP in combination with vancomycin synergistically showed effective inhibition and degradation against biofilm formation and maturation, respectively. The EEP at a concentration of 200 μg/mL against MSSA and MRSA showed 47–87% biofilm degradation, respectively . Alandejectively . Manuka eumoniae ,191,192,[E. coli , multidra parvum .S. aureus, vancomycin-resistant S. aureus (VRSA), P. aeruginosa, Enterobacter cloacae, and S. haemolyticus. Bee venom showed a considerable reduction in biofilm formation, ranging between 63.8–92% [Fadl et al. reported the antibiofilm potential of bee venom against biofilm-forming MDR bacteria, such as 63.8–92% . Bouchel63.8–92% .˂100 nm) with a large surface area and may provide several biological, chemical, and biomedical applications [Nanotechnology-based nanomaterials (NMs) are very small in size . Three types of Ag NPs were prepared to investigate the antibiofilm activity, including cinnamon Ag NPs synthesized by biofilms . Muthulaytogenes . Salem eectively . Some plS. aureus biofilms [P. aeruginosa biofilm [Liposomes can significantly enhance the interaction with bacterial membranes and mediate penetration into mature biofilms due to their high biocompatibility. The fusogenic liposome can be significantly employed to encapsulate drugs or antimicrobial agents with optimized release to increase the antibiofilm efficacy . Vancomybiofilms . Meropen biofilm . The druS. aureus biofilms. CA-SLNs showed a two-fold higher anti-biofilm activity against S. aureus biofilm. Ninety-seven percent of the free CA was released in 2 h, while it takes 12 h to reach 96% release when it is in the encapsulated form [Solid lipid NPs (SLNs) containing surfactants stabilized lipid cores are spherical colloidal NMs (with 10–1000 nm diameters) . Cefuroxted form .P. aeruginosa than a free QSI [P. aeruginosa biofilms. Se-NPs and Te-NPs disrupted QS signaling and significantly reduced the bacterial biovolume, resulting in biofilm formation inhibition of 70–80% [Numerous QS inhibitor (QSI) NMs have been developed to inhibit or eradicate biofilm formation . These Qfree QSI . Tellurif 70–80% . HoweverE. coli [E. coli. Miconazole encapsulated CDs attached to polypropylene and polyethylene showed 87–96% inhibition against the biofilm formation of C. albicans [C. albicans biofilm. Thymol and anidulafungin encapsulated cyclodextrins showed significant antibiofilm activity against the biofilms of C. albicans with 75–64% inhibition, respectively [Nguyen et al. investigated the anti-biofilm activity of a ubiquitous flavanone, naringenin (NAR), encapsulated β-CD and chitosan against the biofilm of E. coli . NAR nanalbicans . CDs coaectively .P. aeruginosa, S. aureus, Acinetobacter baumannii, etc. Hydrogels can be used as promising biomaterials to treat and control multidrug-resistant bacterial infections. Some other NMs have also been reported for combating bacterial biofilms  showed potential anti-biofilm activity against MRSA due to its 2,3-dihydroxybenzoic acid-mediated iron chelating ability [Streptomyces sp. (isolated from Iraqi marine sediment) exhibited significant potential for designing and developing new anti-biotics for treating urinary tract infections. The extract showed biofilm inhibition against P. mirabilis (uropathogenic bacteria) even at sub-MIC by interrupting the QS signals [Many micro-organisms produce several types of bioactive molecules with anti-microbial properties to benefit from other micro-organisms. Several investigations have determined the various secondary metabolites with potential anti-biofilm properties extracted from different bacterial and fungal species. Streptomyces species are known as the most promising sources of biofilm-controlling compounds. Methanolic compounds extracted from  ability . Ethyl-a signals .V. cholera biofilms at 60.1 µM (IC50). Additionally, the inhibitory effect of auromomycin was significantly enhanced by adding some antibiotics  at their sub-inhibitory concentrations [Dilsea pulchra, has been reported to have effective anti-biofilm properties [Marine-derived bacteria are well categorized as a prime microbial group found in the marine environment. Peach et al. discovered and characterized the auromomycin chromophore as a potential inhibitor against trations . A red aoperties . Some syoperties ,266,267.1–6, S. mutans, S. epidermidis, S. aureus, and P. aeruginosa biofilms [1, γ-hydroxy-γ-lactam, and the compound-2, (E)- γ-alkylidene- γ-lactam were found to be most effective against S. epidermidis with IC50 values of 13.3 and 12.2 µg/mL, respectively. The compound-3 and 4, and (Z)-γ-alkylidene-γ-lactam were observed to be most significant against P. aeruginosa with IC50 values of 0.7 and 0.6 µg/mL, respectively. The compound-5 was most effective against S. aureus with 53.1% biofilm inhibition at 44 µg/mL. The compound-2, 4, and 6 inhibited the biofilm formation of S. mutans [P. aeruginosa, V. harveyi, S. aureus, and Chromobacterium violaceum [Pereira et al. determined the biofilm inhibitory effect of brominated alkylidene lactams  inhibited the biofilm formation of P. aeruginosa PAO1 by interfering with rhl and the las system [bacteria . Actinobs system .Oceanobacillus sp. (XC22919) and analyzed their antibiofilm activity. All three compounds exhibited significant biofilm inhibitory activity against P. aeruginosa biofilm in a dose-dependent manner by inhibiting QS activity [Chen et al. isolated three bioactive compounds -butyramide, and 2-methyl-N-(2’-phenylethyl)-butyramide) from a marine bacterium activity .A bibliographic search was conducted to screen relevant scientific publications before August 2022, on bacterial biofilm formation, resistance development mechanisms, and biofilm control strategies. The online search engines of Google scholar, PubMed, ScienceDirect, and Web of Science Core Collection databases were used. The search strategy consisted of separate or simultaneous use, under different combination forms, of several particular keywords including “biofilm formation”, “microbial biofilm composition”, antimicrobial resistance”, “plant-derived anti-biofilm compounds”, “bee products”, “marine derived compounds”, and “phyto-nanotechnology”. Firstly, all titles and abstracts of the search results were individually examined to evaluate whether the articles met inclusion criteria, meaning reporting results in evaluating the antibiofilm activity of plant products, bee products, and nanomaterials against biofilm-forming bacteria with significant outcomes. All the duplicated papers and those published in a language other than English or irrelevant publications that did not contribute to retrieving meaningful results in the goal of assessing natural strategies as potential weapons against bacterial biofilms were excluded. Furthermore, the selected articles were entirely read to obtain significant material based on their experimental outcomes.Over the last three decades, biofilm formation has become a potential threat in the food and health sector. Many biofilm-forming microbial species can develop resistance to harsh environmental conditions. Several antibiotics and different disinfectants are used in hospitals and the food industry. Several biofilm-forming foodborne pathogens have been found to cause outbreaks. Several chronic infections are associated with biofilm-forming microbes. Therefore, several strategies have been designed and analyzed to inhibit microbial growth. However, the emergence of antimicrobial and multidrug resistance forced researchers to study all growth features of microbes and their resistance mechanisms for developing effective combating strategies and significant biofilm-controlling compounds. Many researchers are discovering and developing effective anti-biofilm compounds using natural products. In this review, recent studies were reviewed, focusing on biofilm-controlling compounds, such as natural plants, bee products, nanomaterials, microbes, and marine-derived anti-biofilm compounds to reduce or eradicate microbial biofilms and their associated infections as well. These antibiofilm compounds possess a significant potential to overcome the antimicrobial resistance linked to biofilm formation. On the basis of their potential antibiofilm properties with less to no toxicity and increased bioavailability, they can be suggested to treat biofilm-associated infections.


Parkinson’s disease (PD) is the second most common neurodegenerative disease with a fast-growing prevalence. Developing disease-modifying therapies for PD remains an enormous challenge. Current drug treatment will lose efficacy and bring about severe side effects as the disease progresses. Extracts from Ginkgo biloba folium (GBE) have been shown neuroprotective in PD models. However, the complex GBE extracts intertwingled with complicated PD targets hinder further drug development. In this study, we have pioneered using single-nuclei RNA sequencing data in network pharmacology analysis. Furthermore, high-throughput screening for potent drug-target interaction (DTI) was conducted with a deep learning algorithm, DeepPurpose. The strongest DTIs between ginkgolides and MAPK14 were further validated by molecular docking. This work should help advance the network pharmacology analysis procedure to tackle the limitation of conventional research. Meanwhile, these results should contribute to a better understanding of the complicated mechanisms of GBE in treating PD and lay the theoretical ground for future drug development in PD. Parkinson’s disease (PD) is mainly manifested by progressive motor impairment , leadingThe development of novel PD therapeutics is in urgent demand. Without available disease-modifying therapy, current treatments for PD are only symptomatic . Long-tein vivo and in vitro PD models, including toxin-induced PD models on rats , have gradually come to attention as novel therapies for PD. Herbal medicines generally share the advantages of multilevel functions with fewer adverse effects . Among t on rats , toxin-i on rats , transge on rats , and in l models . Subsequl models , and thel models . The varHerein, advances in analytical pharmacy and bioinformatics would help to detangle the complex molecular mechanisms underlying the therapeutic efficacy of GBE in PD. The single-nuclei RNA sequencing (snRNA-seq) has emerged as a powerful tool for identifying and characterizing cell types, states, and lineages . RecentlThe deep learning approach can be another handy tool to guide pharmacological studies, including drug-target prediction, drug repurposing, and novel drug discovery . ExperimIn this study, we tended to identify active components in GBE for PD along with its cell-type-specific targets. Network pharmacology analysis was conducted, integrating data from snRNA-seq and existing drug datasets. A cell-type-specific compound-target-pathway network was established, and DTI was subsequently investigated with a deep learning algorithm. Then, we validated the results by molecular docking. This research will contribute to a better understanding of the molecular mechanisms of treating PD with GBE.https://old.tcmsp-e.com/index.php, version 2.3), TCMID  and SymMap  databases rendered 307, 94 and 319 ingredients of GBE, respectively. All data were collected on 18 May 2022.Firstly, components of GBE were collected via searching the terms: “ginkgo folium,” “folium ginkgo,” and “Yinxingye” in databases. TCMSP  (https:/), TCMID  (http://d SymMap  (http://https://pubchem.ncbi.nlm.nih.gov/) to identify each component.Secondly, PubChem CID was retrieved from PubChem  AND (components OR ingredients OR metabolite)" and added ginkgolide K, which was not timely updated in databases .http://www.swissadme.ch) (Po/w), number of H-bond acceptors, number of H-bond acceptors, number of rotatable bonds, and topological polar surface area (TPSA). ADMETlab 2.0 (https://admetmesh.scbdd.com/) was employed to evaluate compound pharmacokinetics and toxicity. To assess components’ oral bioactivity, HobPre (www.icdrug.com/ICDrug/ADMET) (http://structure.bioc.cam.ac.uk/pkcsm).Thirdly, chemical properties and pharmacokinetic profiles of components were retrieved. The chemical properties of components were annotated via SwissADME (adme.ch) , which pTlab 2.0  (https:/g/ADMET) , a classg/ADMET)   database, and 272 potential molecular targets were retrieved. SymMap database integrates target information from HIT (http://lifecenter.sgst.cn/hit/), TCMSP, HPO (https://hpo.jax.org/app/), DrugBank (https://go.drugbank.com/), NCBI(https://www.ncbi.nlm.nih.gov/) and HERB (http://herb.ac.cn/) databases. Additional pharmacoproteomic and pharmaco-transcriptomic data were obtained manually. Additional ginkgolide J, ginkgolide M, and ginkgolide K targets data, which is not included in the above databases, was retrieved from the Comparative Toxicogenomics Database (CTD) (http://ctdbase.org/). All data were collected on 18 May 2022. After removing duplicates, 283 genes were identified as putative GBE targets for PD.Every selected component has been searched in SymMap    (URL: htGene expression profile of different cell types from the idiopathic Parkinson’s disease patient’s brain snRNA-seq  GSE1577. The cutProtein-protein interaction (PPI) network of all targets was constructed using Cytoscape software (version 3.9.1) with data from STRING   and enrichplot (version 1.14.2). All mentioned analysis was conducted on R version 4.1.2.R package topGO (version 2.46.0) and cluster profile (version 4.2.2) was employed to conduct Gene Ontology (GO) and KEGG pathway analysis. Reference gene data were retrieved using R package, org. Hs.eg.db (version 3.14.0). The https://github.com/kexinhuang12345/DeepPurpose) was used to calculate the binding score between selected targets and their proven ligands. In this pre-trained model, Convolutional Neural Network (CNN) was chosen to encode SMILES of components and the amino acid sequence. The Binding Database (BindingDB), a public drug-target binding benchmark dataset, was employed to provide measured binding affinities. DeepPurpose generates predictions via a Multi-Layer Perceptron (MLP), one of the most common artificial neural networks. All amino acid sequences of the selected targets were collected from UniProt (https://www.uniprot.org/). The SMILES of each component were obtained from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/).Pre-trained model CNN_CNN_BindingDB provided by DeepPurpose  (https:/ UniProt  (https:/http://www.swissdock.ch/). 3D structure of MAPK14 protein was obtained from RCSB PDB (https://www.rcsb.org/) with PDB ID: 1WBS. The chemical structure of ginkgolide J and ginkgolide A was obtained from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/). The DockPrep plugin of Chimera  was employed to prepare the structures before docking. Docking results were analyzed and visualized using UCSF Chimera  and LigPlot  between all 25 active compounds in GBE and all 47 PD targets in three cell types. Detailed results are listed in Parkinson’s disease (PD) is a neurodegenerative disorder due to selective loss of dopaminergic neurons in the substantia nigra and Lewy body formation . PD patiDeveloping effective therapeutics to slow or halt the progression of PD remains a top priority for researchers. So far, no agents have been proven with sufficient evidence for disease-modifying effects in PD . CurrentG. biloba, a medicinal plant belonging to the Ginkgoaceae family, is considered the oldest tree alive in the world   have shed light on drug development for PD. Herbal products have gradually gained acceptance in treating neurodegenerative diseases for their multi-functional characteristic with relatively fewer adverse effects . Herbal he world . GBE hashe world . Commercase (AD) . In manyase (AD) . Variousase (AD) . Existinase (AD)  and oxidase (AD)  in PD moase (AD) .However, GBE composition may vary across studies, largely dependent on the complicated extraction procedure .83, protocatechuic acid, and chrysin  technique was recently developed and applied to study the midbrain transcriptome of PD patients. This cell-specific data allowed the chance to delineate GBE effects in different cell types. Moreover, advances in deep learning algorithms conferred the potential to conduct high-throughput screening for potent drug-target interaction (DTI) between GBE components and related PD targets.A total of 25 potentially active components were obtained after selection by Lipinski’s Rule of Five. As mentioned above, part of the ingredients has already been reported for efficacy in PD. Components information was manually curated and supplemented from published reports. Certain ingredients like ginkgolide K were not documented in existing compounds databases since they were not identified as components of GBE until recently . With adMainly, selected GBE compounds largely constitute flavonoids and flavone derivates, such as luteolin, kaempferol, and apigenin. As the main ingredients of GBE, flavonoids are among the most studied herbal products in medical applications . Also, oIn order to identify GBE targets in a cell-type-specific manner, we analyzed snRNA-seq data on PD midbrain samples . Since pThe component-target-pathway network showed that potential mechanisms were complex interactions between multiple components, targets, and pathways in GBE therapy for PD. Nearly all potentially active components were linked with more than one target for PD. Similarly, most potential targets were regulated by multiple components in GBE.Herein, we exploited recently developed deep learning technology to help detangle complex drug-target interactions. DTI between all potential compounds and targets was predicted with DeepPurpose, a deep learning method-based approach for drug discovery. Our results suggested relatively strong interactions between ginkgolides and several PD targets participating in core biological pathways. Notably, ginkgolide A, B, J, and K showed similar binding patterns to targets, possibly due to their similar chemical structures.In vivo or/and in vitro laboratory work is still required to establish concrete interaction between predicted DTI in this study.Published experimental reports supported the reliability of our bioinformatic methods. For instance, a recent study on lipopolysaccharide (LPS) induced inflammation models has shown ginkgolide A as a modulator for MAPK . AccordiTaken together, through the integration of data from snRNA-seq and employing a deep learning algorithm, a cell-type-specific targets and compound network was established. This work took advantage of recently advanced bioinformatics approaches. Herein, an unprecedented procedure of conducting network pharmacology analyses in a cell-type-specific manner was established. This work will better facilitate our understanding of GBE mechanisms in treating PD. Moreover, identified interaction of drugs and targets would lay a theoretical foundation for the development PD drugs.


Diagnosing and treating many infectious diseases depends on correctly identifying the causative pathogen. Characterization of pathogen-specific nucleic acid sequences by PCR is the most sensitive and specific method available for this purpose, although it is restricted to laboratories that have the necessary infrastructure and finance. Microscopy, rapid immunochromatographic tests for antigens, and immunoassays for detecting pathogen-specific antibodies are alternative and useful diagnostic methods with different advantages and disadvantages. Detection of ribosomal RNA molecules in the cytoplasm of bacterial and protozoan pathogens by fluorescence in-situ hybridization (FISH) using sequence-specific fluorescently labelled DNA probes, is cheaper than PCR and requires minimal equipment and infrastructure. A LED light source attached to most laboratory light microscopes can be used in place of a fluorescence microscope with a UV lamp for FISH. A FISH test hybridization can be completed in 30 min at 37 °C and the whole test in less than two hours. FISH tests can therefore be rapidly performed in both well-equipped and poorly-resourced laboratories. Highly sensitive and specific FISH tests for identifying many bacterial and protozoan pathogens that cause disease in humans, livestock and pets are reviewed, with particular reference to parasites causing malaria and babesiosis, and mycobacteria responsible for tuberculosis. Pneumocystis carinii strains in sputum and tissue from patients [The in-situ hybridization (ISH) technique for examining the formation and detection of RNA-DNA or DNA-DNA nucleotide complementary hybrids in cells utilizing radioactively labelled oligonucleotides as probes was first described in 1969 . ISH perpatients . FISH aspatients ,18,19,20The application of FISH assays for detecting and identifying microbial pathogens has advanced considerably since the turn of the century . FISH teRibosomal RNA molecules possess genus, species and strain-specific regions. Hence, 16S rRNA sequences have been used to establish phylogenetic relationships among bacteria . The binMycobacterium tuberculosis (MTB), which is primarily responsible for human tuberculosis, caused an estimated 1.4 million deaths worldwide in 2020 [The World Health Organization estimated 241 million cases of malaria and 627,000 resulting deaths worldwide in 2020 . Infecti in 2020 . Malaria in 2020 . While t in 2020 ,29. Plasmodium falciparum is responsible for most of the annual 241 million global malaria infections, together with an estimated 4.5 million cases of Plasmodium vivax and fewer cases of Plasmodium malariae and Plasmodium ovale [Plasmodium knowlesi, which normally infects macaque and leaf monkeys, causes a significant number of dead-end human infections in Southeast Asian countries [Plasmodium knowlesi is difficult to differentiate from human malaria parasites in Giemsa-stained blood smears that are commonly used for diagnosing malaria in endemic areas [Plasmodium cynomolgi and Plasmodium inui have also been reported in Southeast Asia [Plasmodium brasilianum and Plasmodium simium infect platyrrhine monkeys in South and Central America, are genetically almost identical to the human malaria parasites P. malariae and P. vivax respectively, and likely to have been derived from the human parasites by anthroponosis [P. simium and P. brasilianum can also infect humans by zoonosis but their differentiation from P. vivax and P. malariae respectively in stained blood smears is not possible [um ovale . Plasmodountries . Plasmodic areas . Occasioast Asia . Plasmodoponosis . P. simipossible . Plasmodium falciparum accounted for 70.5%, P. vivax 10.0%, P. ovale 5.5%, and P. malariae 2.6% of the infections, all of which had been acquired outside the US [Plasmodium species were responsible for 1.0% of infections [There were 2171 US cases of malaria reported to the US Centers for Disease Control and Prevention (CDC) in 2017 according to the latest available CDC report . Plasmode the US . Infectifections . The ideP. falciparum (PfHRP2) and pan Plasmodium-specific lactate dehydrogenase and aldolase, have more recently proved helpful in resource-limited locations [PfHRP2 gene deletions in P. falciparum in malaria-endemic areas of Africa has lately increased the false negativity rates for P. falciparum [Plasmodium at the species level, but the procedure is not suitable for resource-poor settings and field use [Giemsa-stained thick and thin blood smear microscopy has been the most widely used technique globally for diagnosing malaria. It however requires time and an experienced microscopist for optimal sensitivity of detection and for identifying the infecting species of parasite. A sensitivity of >150 parasites per µL is typically achieved during routine microscopy ,55. Rapiield use . The Looield use . Flow cyield use ,59. Plasmodium genus-specific FISH test utilizing a DNA probe hybridizing to 18S rRNA [Simple, rapid and specific FISH tests for malaria, that can be easily performed in resource-constrained diagnostic laboratories, have many advantages ,42. Thes18S rRNA  are reprPlasmodium genus-specific FISH test identified all common species of human malaria parasites with 100% specificity when compared with several other common human blood-borne pathogens [The athogens . Plasmodium genus-specific FISH test, and designed to specifically identify P. falciparum, utilized a mixture of P. falciparum 18S rRNA-specific probes labeled with Alexa 488 green and the Plasmodium genus-specific probe labeled with a Texas Red in a multiplex format. Plasmodium falciparum fluoresced green  understanding its epidemiology, and (ii) its pathogenicity and drug treatment options can differ from human malaria caused by P. falciparum, P. malariae, P. ovale and P. vivax that also occur in Southeast Asia. Reliable RDTs for specifically detecting P. knowlesi are not yet available [P. knowlesi 18S rRNA (termed the PK-FISH test) specifically identified P. knowlesi in blood smears [Zoonotic fections ,63,64,65vailable . Howeverd smears , as showPlasmodium genus-specific test and the PF-FISH and PV-FISH tests [P. knowlesi [P. knowlesi at the low limit of 16 P. knowlesi parasites per µL, even in the concomitant presence of P. falciparum at approximately 500 parasites per µL [P. knowlesi and other human malaria parasite species are common [P. knowlesi zoonosis is prevalent [The PK-FISH test, like the analogous SH tests , identifknowlesi . The PK-s per µL , which is per µL ,55. Thise common ,63. The revalent ,65,66.Plasmodium infections  usefully complement Giemsa-stained blood smear microscopy and RDTs for routine diagnosis and screening for malaria, and (ii) identify the species of infecting Plasmodium (including in mixed infections), in both endemic and non-endemic countries.A large number of malaria tests are performed for diagnostic and screening purposes in malaria-endemic countries. Tests in malaria-free countries are utilized for (i) screening passengers arriving from malaria-endemic countries to prevent the reintroduction of malaria if mosquito vectors are present in the country of arrival, and (ii) confirming malaria in arriving travelers who have malaria-like symptoms. FISH tests are more costly and complex to perform than Giemsa-stained blood smear microscopy and RDTs, but significantly less so than NAA-dependent PCR and LAMP, for detecting table in  below. FBabesia are apicomplexan protozoan parasites, like Plasmodium, that infect and replicate within red blood cells to cause babesiosis in humans, livestock and pets [Babesia from animal reservoirs function as vectors to transmit infections to humans [Babesia microti, B. duncani and B. divergens are largely responsible for human infections in the US [Babesia microti, B. divergens, B. venatorum and B. crassa are responsible for human babesiosis in Eurasia [Babesia infect cattle , horses , dogs , cats (B. felis), deer , mice  and other animals [Ixodes scapularis, I. ricinus, I. persulcatus and Dermacentor albipictus are vectors that transmit Babesia to humans [and pets ,67,68,69o humans ,67,68,69o humans . The CDCo humans . Babesian the US . Babesia Eurasia ,68,69,70 animals ,69. The o humans . Babesia by immunoassays, and (iii) Babesia-specific nucleic acid sequences by PCR [Borrelia bacteria), share many clinical manifestations, and occur as coinfections [Babesia species are not readily distinguished from the ring and trophozoite stages of P. falciparum by microscopy in areas where babesiosis and malaria are co-endemic [Babesia infections [Laboratory tests commonly used for diagnosing babesiosis involve the detection of (i) parasites in stained blood smears by microscopy, (ii) serum antibodies to s by PCR ,70. Babefections ,74,75,76fections ,77,78, u-endemic . Furtherfections ,68,69,70fections ,80,81 anfections . HoweverBabesia parasites, with many advantages for use in resource-limited laboratories, has been developed [Babesia genus FISH test, it is based on DNA probes that specifically hybridize to the multiple copies of Babesia 18S rRNA present in the parasite cytoplasm. Like other rRNA-directed FISH tests, the Babesia genus FISH test does not require NAA—a process that is sensitive to NAA inhibitors sometimes present in blood [A FISH test that identifies all common species of eveloped ,44. Termin blood .Babesia genus-specific FISH test detects B. microti, B. duncani and B. divergens, as well as the two important parasites causing bovine babesiosis, B. bovis and B. bigemina [The bigemina , as illuBabesia genus-specific FISH test, in conjunction with an IFA test for detecting serum antibodies to B. duncani and B. microti, on clinical samples originating from USA, Australia, Europe and elsewhere, showed that the global prevalence of B. duncani infections had hitherto been under-estimated [Babesia genus-specific FISH test was highly specific and did not detect other pertinent pathogens found in human blood [Borrelia and Plasmodium [Bartonella that infect humans and domestic pets [The stimated . Furtheran blood , includiasmodium , as welltic pets ,84. Babesia infections in patients and animals. FISH tests can be used in mixed infections. FISH is also useful for histopathological investigations to identify Babesia parasites sequestered in tissues [Babesia FISH tests, that are presently being developed, can address more precise diagnostic requirements in babesiosis. The prevalence of human babesiosis has probably been underestimated throughout the world . Babesio tissues . SpeciesMycobacterium tuberculosis (MTB) and the closely related species Mycobacterium bovis, both of which belong to the Mycobacterium tuberculosis complex (MTBC) [Mycobacterium avium complex (MAC), M. kansasii, M. fortuitum, M. xenopi, M. abscessus, and M. simiae also occur worldwide, making their differential diagnosis important for clinical purposes [Norcadia species widely distributed in the environment, also needs to be differentiated from MTB during lung infections [Pulmonary mycobacterial infections in humans are caused mostly by x (MTBC) . Infectipurposes ,85,86,87purposes . Norcardfections .® MTB/RIF system or Xpert , is a PCR-based nucleic acid amplification (NAA) technique that detects specific DNA sequences of MTB [Microscopic examination for acid-fast staining (AFS) bacilli, e.g., with the Ziehl-Neelsen stain, in sputum or tissue plays an important role in the diagnosis of tuberculosis . AFS does of MTB ,92,93. Xs of MTB ,92,93. Is of MTB . ImmunocMycobacterium tuberculosis complex (MTBC) and a green fluorescent probe specific for the Mycobacterium and Nocardia genera (MN Genus) 16S rRNA to detect and distinguish MTBC from other mycobacteria and Nocardia species. A complementary MTBC-MAC FISH test used green and orange fluorescent probes for 23S rRNA that respectively differentiate MTBC and MAC [A simple and rapid test for directly identifying MTB and NTM in sputum and tissues that can be used by resource-limited laboratories in endemic countries is therefore expected to greatly aid tuberculosis control worldwide ,93. Two  and MAC ,29,30.Mycobacterium species from reference cultures, except M. wolinskyi, reacted positively with the MN Genus-specific probe and only the M. tuberculosis complex species reacted positively with the MTBC- specific probe in the MN Genus—MTBC FISH test. Only the M. tuberculosis complex species reacted positively with the MTBC-specific probe and only the M. avium complex species reacted positively with the MAC–specific probe in the MTBC-MAC FISH test [Nocardia reacted positively with the MN Genus probe but not with the MTBC- and MAC-specific probes in the MN Genus-MTBC and the MTBC-MAC tests [4 bacteria per ml [M. tuberculosis, M. avium and M. kansasii [All ISH test . NocardiAC tests . The esta per ml . Resultskansasii  are reprM. abscessus, an NTM, that reacted only with the MN Genus-specific probe.The FISH tests used for culture identification can also be used for directly detecting mycobacteria in sputum . Figure rpoB gene coding for the β subunit of RNA polymerase has been described [rpoB FISH test however required enzyme digestion of sputum, concentration of mycobacteria by centrifugation, hybridization overnight and a UV fluorescence microscope for visualizing results [Another FISH test specific for MTBC in sputum targeting the escribed . The rpo results . Other P results ,14,15,16 results . They ar results . All FIS results ,97.FISH tests for tuberculosis meet internationally-expressed needs for diagnosing tuberculosis in respiratory samples ,93. TheyTests that depend on amplifying specific nucleic acid sequences of pathogens and the subsequent detection and/or sequencing of the amplified nucleic acids are widely regarded as the gold standard for diagnostic tests because of their high sensitivity and specificity. Two NAA techniques that can be used for RNA and DNA, are based on PCR  and LAMPThe simplicity, cost, modest infrastructure/equipment/reagent requirements, reagent stability, good diagnostic parameters, and the ability to identify pathogens at the species level, suggest that FISH tests can be used in advanced as well as resource-constrained diagnostic laboratories throughout the world. FISH tests, therefore, can complement existing diagnostic tests in both disease-endemic and non-endemic countries. FISH tests are particularly advantageous for identifying pathogens at the species level. Flow cytometry combined with FISH has been shown able to rapidly identify potentially pathogenic bacteria present in food, water, air and biofilms formed on various abiotic surfaces . The use


Gardnerella species might increase susceptibility. Not all lactobacilli are equally protective. Recent research suggests that D(−) isomer lactic acid producing lactobacilli  may protect against infection with Chlamydia trachomatis, an important cause of PID. Lactobacillus iners, which produces L(+) isomer lactic acid, may be less protective. We investigated the microbiome in stored vaginal samples from participants who did or did not develop PID during the prevention of pelvic infection (POPI) chlamydia screening trial.A lactobacilli-dominated vaginal microbiome may protect against pelvic inflammatory disease (PID), but one dominated by Long-read 16S rRNA gene nanopore sequencing was used on baseline vaginal samples (one per participant) from all 37 women who subsequently developed clinically diagnosed PID during 12-month follow-up, and 111 frequency matched controls who did not, matched on four possible risk factors for PID: age <20 versus ≥20, black ethnicity versus other ethnicity, chlamydia positive versus negative at baseline and ≥2 sexual partners in the previous year versus 0–1 partners.L. iners, L. crispatus and Gardnerella vaginalis. There was no association between a more diverse, G. vaginalis dominated microbiome and subsequent PID, although increased Shannon diversity was associated with black ethnicity (p=0.002) and bacterial vaginosis (diagnosed by Gram stain p<0.0001). Women who developed PID had similar relative abundance of protective D(−) isomer lactic acid producing lactobacilli to women without PID, but numbers of PID cases were small.Samples from 106 women  were suitable for analysis. Three main taxonomic clusters were identified dominated by In the first-ever community-based prospective study of PID, there was no clear association between the vaginal microbiome and subsequent development of PID. Future studies using serial samples may identify vaginal microbial communities that may predispose to PID. Chlamydia trachomatis and Neisseria gonorrhoeae cause up to 30% of PID, but in many cases, the aetiology is polymicrobial or unclear.C. trachomatis infection develop upper tract infection. A healthy, low diversity, vaginal microbiome dominated by lactobacilli may protect against disease.Gardnerella vaginosis and/or Atopobium vaginae) may increase susceptibility to STIs by causing cervicomucosal barrier disruption and epithelial portals of entry for ascending infection.Pelvic inflammatory disease (PID) is an infection-induced inflammation of the female upper reproductive tract that can lead to infertility and ectopic pregnancy. Lactobacillus crispatus, L. jensenii and L. gasseri) may be associated with long-term protection against C. trachomatis infection, possibly by reducing epithelial cell recycling.L. iners, which mainly produces L(+) isomer lactic acid, may be associated with increased susceptibility to infection.Not all lactobacilli are equal.A recent systematic review called for longitudinal studies of vaginal microbiota and infections to lay the foundation for possible prevention and treatment strategies.Conventional short-read 16S rRNA gene sequencing is constrained in its ability to comprehensively speciate pathogens within a sample because of the limited 16S variable regions that are usually sequenced. Long-read sequencing platforms such as PacBio and Oxford Nanopore Technologies’ MinION allow for full sequencing of the 16S rRNA gene for microbiome studies.C. trachomatis,C. trachomatis infection or bacterial vaginosis (BV).We used long-read 16S rRNA gene nanopore sequencing to investigate bacterial communities in 37 women who developed clinical PID within the following year, and 111 frequency matched control women who did not. Since D(−) isomer lactic acid producing lactobacilli may protect against One hundred and forty-eight frozen, baseline vaginal samples (one sample per participant) were retrieved from all 37 women who subsequently developed clinically diagnosed PID in the next year and 111 controls with no PID, as part of the POPI trialDNA was isolated from vaginal samples using QIAamp DNA Mini Kit (Qiagen), following the manufacturer’s protocol for Gram-positive and difficult-to-lyse bacteria, and quantified using Qubit 3.0 Fluorometer. 16S rRNA genes in each sample were amplified with the primers, B27F (TTTCTGTTGGTGCTGATATTGCAGRGTTYGATYMTGGCTCAG) and B1492R (ACTTGCCTGTCGCTCTATCTTCRGYTACCTTGTTACGACTT), which were modified from S-D-bact-0008-c-S20 and S-*-Univ-1492-a-A-19.The sequence analysis protocol was based on previously published methods.L. crispatus, L. jensenii and L. gasseri), for three clinical diagnoses: pelvic inflammatory disease, bacterial vaginosis or C. trachomatis. Finally, we investigated beta diversity using non-metric multidimensional scaling (NMDS) of Bray-Curtis distances.The relative abundance of the dominant species was explored (including a combination of D(−) isomer lactic acid producing lactobacilli Gardnerella vaginalisG. vaginalis as a surrogate for bacterial vaginosis.)The study was constrained by the number of women diagnosed with PID. We estimated that a sample size of 148 women (37 who developed PID and 111 who did not develop PID) would detect a significant difference  if vaginal samples from 35% of women with PID had Burkholderia gladioli. Although a rare cause of opportunistic infection in humans,B. gladioli is unusual, and the Burkholderia genus has been associated with contamination in other microbiome studies.B. gladioli were collected within a specific time period at the end of the study . In light of this, all 42 samples containing more than 1% B. gladioli were removed from further analysis.  Inclusion or exclusion of these samples had little effect on the conclusions of this study.An initial heatmap representation of the relative abundances  showed fhe study , suggest10.1136/sextrans-2021-055260.supp1Supplementary dataC. trachomatis, 21% (22) had bacterial vaginosis , 4% (4) had Mycoplasma genitalium and 2% (2) Neisseria gonorrhoeae (diagnosed by Gen-Probe PCR). The estimated mean time from baseline sample to diagnosis of PID, where data were available (in 22 of 23 women), was 35 weeks (range 13–52 weeks).Samples from 106 participants (23 with PID) had adequate sequencing data and were included in the analysis. The median age of the women in the cohort was 19 years (range 16–27), 40% (42) described their ethnic group as black and 54% (57) reported ≥two sexual partners in the previous year. At baseline, 22% of women (23) had L. iners, L. crispatus and G. vaginalis, with a smaller number of samples dominated by L. jensenii and L. gasseri. The lactobacillus dominated clusters showed lower Shannon diversity than the G. vaginalis cluster.C. trachomatis, BV, age, ethnicity and multiple partners) showed no association between a more diverse microbiome and subsequent PID  or concurrent C. trachomatis infection (p=0.38). However, there was a significantly higher diversity in samples from participants aged ≥20 years (p=0.048), participants of black ethnic group (p=0.002) and those with a diagnosis of bacterial vaginosis .Comparing Shannon alpha diversity  across sLactobacillus iners, Lactobacillus crispatus, Gardnerella vaginalis, Lactobacillus jensenii and Lactobacillus gasseri (including a combination of D(−) isomer lactic acid producing lactobacilli L. crispatus, L. jensenii and L. gasseri), related to the three main clinical diagnoses:BV at baseline.PID within the next 12 months.C. trachomatis at baseline.C. trachomatis, was associated with lower abundance of D(−) isomer lactic acid producing lactobacilli species . This shows that unlike BV, neither PID nor C. trachomatis. However for BV, three clusters correlated with the dominant species shown in L. iners, L. crispatus and G. vaginalis), with dimensions one and two distinguishing BV versus no BV  isomer lactic acid producing lactobacilli, but this did not apply to women who developed PID.G. vaginalis taxonomic cluster was associated with black ethnicity, an association that lends internal validity to this study.This study is unique as it is the only community-based prospective study of the vaginal microbiome and PID, and the first to explore the possible association of PID with low levels of vaginal D(−) isomer lactic acid producing lactobacilli. Previous studies were based in hospitals or clinics,L. iners, one by L. crispatus and a more diverse cluster dominated by anaerobes. Further strengths include our pilot work confirming the integrity of stored samplesTo our knowledge, this is also the first study to use long-read sequencing on self-taken vaginal swabs. Combined with evidence that unsupervised self-collected vaginal swabs give similar microbiota results to clinician-collected swabs,B. gladioli was a contaminant. Removal of affected samples did not seem to influence the results, although it did reduce the power of the study. PID is a clinical diagnosis of low specificity, with around a third of diagnoses not confirmed on laparoscopy. This could also weaken the power of the study. However, diagnosis was confirmed by three experienced sexual health physicians.The main limitation is that this was an exploratory study with a small number of PID cases. However, it is similar in size to earlier studiesC. trachomatis or N. gonorrhoeae.C. trachomatis, N. gonorrhoeae and M. genitalium by PCR.Gardnerella species,Gardnerella genus, and they are not yet included in NCBI and other 16S rRNA databases. In addition, the taxon for bacterial vaginosis associated bacteria such as BVAB-1 (Candidatus Lachnocurva vaginae) may be misclassified as Shuttleworthia using the NCBI database. Finally, we analysed by the dominant species within a community state typeAnother weakness is that the sensitivity of samples stored for 16 years may be reduced. Nonetheless, other sequencing studies using stored samples have shown reliable results.C. trachomatis infection. Most recently, Trent and colleagues analysed samples from 26 women aged 13–25 years with PID who were enrolled in the Technology Enhanced Community Health Nursing study.There is a dearth of studies of vaginal microbiota in women with and without PID,L. crispatus was progressively replaced by L. iners in the shift from a healthy to an infected microbiome. As in our study, this was mainly characterised by anaerobic genera such as Gardnerella, Prevotella, Megasphaera, Roseburia and Atopobium. We did not find an association between C. trachomatis and L. iners dominated vaginal microbiota,C. trachomatis infection were small. Finally, our finding of lower rates of protective D(−) isomer lactic acid producing lactobacilli in women with (vs without) concurrent bacterial vaginosis confirms other studies.Other studies of the vaginal microbiome in healthy ethnically diverse women have found similar main clusters/community state types to those in our study, two dominated by lactobacilli and one with higher proportions of anaerobic organisms.This study supports the feasibility of larger scale community-based projects, the use of self-collected vaginal swabs and the integrity of stored samples. The significantly higher abundance of D(−) isomer lactic acid producing lactobacilli in women without bacterial vaginosis is in line with other studies suggesting a protective role for these lactobacilli.There are few data on vaginal microbiota and subsequent development of pelvic inflammatory disease (PID).Gardnerella species might increase susceptibility.A lactobacilli-dominated vaginal microbiome may protect against pelvic inflammatory disease, but one dominated by In this first ever community-based prospective study of PID, there was no clear association between vaginal microbiota and development of PID in the next 12 months.In line with previous studies, findings highlight the need for large serial sampling studies to identify vaginal microbiota that might predispose to PID.