Oryza sativa) is produced and consumed by nearly 90% of the population in Asia with less than 9% produced outside Asia. Hence, reports on large scale grain losses were alarming and resulted in a heightened awareness on the importance of rice plants’ health and increased interest against phytopathogens in rice. To serve this interest, this review will provide a summary on bacterial rice pathogens, which can potentially be controlled by plant growth-promoting bacteria (PGPB). Additionally, this review highlights PGPB-mediated functional traits, including biocontrol of bacterial rice pathogens and enhancement of rice plant’s growth. Currently, a plethora of recent studies address the use of PGPB to combat bacterial rice pathogens in an attempt to replace existing methods of chemical fertilizers and pesticides that often lead to environmental pollutions. As a tool to combat bacterial rice pathogens, PGPB presented itself as a promising alternative in improving rice plants’ health and simultaneously controlling bacterial rice pathogens in vitro and in the field/greenhouse studies. PGPB, such as Bacillus, Pseudomonas, Enterobacter, Streptomyces, are now very well-known. Applications of PGPB as bioformulations are found to be effective in improving rice productivity and provide an eco-friendly alternative to agroecosystems.As a major food crop, rice ( Oryza sativa) is a staple food in Asia and parts of African countries. Over 90% of the world’s rice is produced in the Asian region and consumed as the main food source. With an ever-increasing human population, it is challenging to ensure food security for the general population. To counter the demand, rice production needs to be increased to fulfill this need [Xanthomonas [Rice  are indigenous. PGPB display beneficial effects on their host plant and play a major role in the biocontrol of phytopathogens . With riin vitro . Charactin vitro . Direct in vitro . Thus, tBacillus [Pseudomonas [Enterobacter [Streptomyces [Pseudomonas fluorescens [Applications of PGPB on rice have shown notable successes. A plethora of PGPB from the genera Bacillus , Pseudomudomonas , Enterobrobacter , and Strptomyces  were reporescens ,16, and orescens  have shoorescens ,19,20. Corescens ,22,23. HXanthomonas oryzae pv. oryzae, Xanthomonas oryzae pv. oryzicola, Burkholderia glumae, and Burkholderia gladioli, are spread rapidly and sporadically under favorable conditions and cause tremendous obstacles to rice production [Rice diseases caused by bacteria are a major bottleneck towards sustainable productivity of rice, especially in Asia and parts of the African countries. In severe epidemics, reduction in rice has reached more than 60% and millions of hectares of rice are infected annually ,25,26. Boduction ,28. Bactoduction .Xanthomonas, Burkholderia, Pseudomonas, Pantoea, Erwinia, Acidovorax, Dickeya, and Enterobacter. Bacterial species belonging to the genus Xanthomonas, including X. oryzae pv. oryzae  and X. oryzae pv. oryzicola , are well-known bacterial diseases of rice. It is noteworthy that studies in recent years have indicated numerous bacterial species from the genus Burkholderia and Pantoea as the next major pathogens of rice [Bacterial pathogens infect the rice plant at all parts including the seed, foliar, leaf sheath, grain, culm, and root . Numerou of rice ,29,30. With regard to bacterial pathogenicity, plant pathology and genomic studies have revealed the detection of virulence factors including degradative enzymes, extracellular polysaccharides, and components of quorum sensing signaling molecules, which are in-volved in the communication between the host and pathogen that contribute to rice dis-eases ,32,33. THowever, the effectiveness of these approaches is somehow inefficient due to the polymorphisms and chemical resistance developed in virulent strains . InteresRhizosphere is the soil surrounding plant roots that are rich in nutrients and a potent habitat for microbes that thrive on root exudates known as rhizodeposits . RhizodePGPB’s ability as a biocontrol agent is linked to how they improve plant growth and suppress phytopathogens either by direct and or by indirect mechanisms . The chaIndirect mechanisms require the involvement of the plants’ defensive metabolic processes that respond to the signal sent from the PGPB. The mechanisms include: (i) induced systemic resistance (ISR) to plant phytopathogens (biotic stress), and (ii) protection against environmental stress (abiotic stress) . In thisBacillus, Pseudomonas, Enterobacter, and Streptomyces were all extensively studied as biocontrol agents as soon as their antagonistic activity against rice pathogens was recognized. Apart from these four groups, other genera are highlighted in the section below.Biocontrol is a promising strategy to control phytopathogens, which could be an alternative for chemical fertilizers and pesticides. The implementation of PGPB as a biocontrol agent to inhibit the growth of phytopathogens has become widespread due to environmental concerns. This strategy has received great attention as it provides a safe, inexpensive, long-lasting, and environmentally friendly alternative . BacteriBacillus spp. are Gram-positive bacteria belonging to the phyla Firmicutes, class Bacilli, and family Bacillaceae. It can be characterized as rod-shaped and endospore-forming bacteria. The ability to produce endospores when facing harsh conditions enabled this bacterial species to survive in various habitats including animal feces [Bacillus spp. as PGPB have been proven to confer numerous advantages in the agricultural sector [Bacillus spp. in rice: (i) increase yield; (ii) improve tolerance to abiotic stresses; (iii) decrease in disease occurrence. al feces , bee proal feces , soil [1al feces , food [1al feces , and aqual feces . Bacillul sector . There aBacillus spp. on crop roots caused an increase in crop yields [Bacillus was found to improve growth, yield, and zinc (Zn) translocation of Basmati rice. Basmati-385 and Super Basmati rice yields were improved by more than 22% and 18%, respectively, upon inoculation with Zn-solubilizing strains that were identified as Bacillus spp. by 16S rRNA gene analysis [Bacillus subtilis, Pseudomonas fluorescens, and Azospirillum brasilens. The integration of inorganic fertilizers with a consortium of PGPB positively affected rice yields and contributed to reducing chemical nitrogen fertilizers by 25% [Bacillus pumilus strain TUAT-1 and N fertilizer reportedly led to higher tiller numbers of rice at the maximum tillering stage [The colonization of p yields . Evidentanalysis . Growth s by 25% . Moreoveng stage . Bacillus amyloliquefaciens strain NBRI-SN13 isolated from the alkaline soil of Banthara, Lucknow, was found to possess PGPB activities and improve stress tolerance in rice [B. amyloliquefaciens strain NBRI-SN13 positively modulated stress-responsive gene expressions, such as dehydrin (DHN) and late embryogenesis abundant (LEA), under various abiotic stresses  and phytohormone (abscisic acid) treatments [Bacillus tequilensis strain UPMRB9 and Bacillus aryabhattai strain UPMRE6 on rice plants were shown to have beneficial effects on photosynthesis, transpiration, and stomatal conductance [B. tequilensis strain UPMRB9 on the MR297 rice variety improved total chlorophyll content by 28% and reduced electrolyte leakage by 92% [B. tequilensis strain UPMRB9 and B. aryabhattai strain UPMRE6 on rice plants. The results suggested a synergistic effect between PGPB and rice plants on the mechanisms of the plant’s salt tolerance, suggesting the application of PGPB for salinity mitigation practice for coastal rice cultivation. Moreover, the potential application of Bacillus to mitigate drought stress in rice has also been demonstrated [Bacillus altitudinis strain FD48 found an increased relative water content, chlorophyll stability index, and membrane stability index in rice. Abiotic stresses, such as salinity and drought, pose major threats to rice growth and yield. Interestingly,  in rice ,113. Tiweatments . The resductance . Shultane by 92% . Incremenstrated . InoculaBacillus controls the proliferation of phytopathogens by suppressing plant immunity [Bacillus can inhibit proliferation of phytopathogens as well as enhance plant immunity directly  and indirectly (by producing antioxidant enzymes). As confirmed through genomic analysis, the Bacillus genome is composed of antimicrobial metabolite gene clusters  and gene-encoding proteins  that function to suppress plant immunity [Bacillus. Further studies should be conducted on the Bacillus antimicrobial compounds and antioxidant enzymes beyond in silico genome analysis to understand its contribution in mediated rice plant ISR.In plant disease management, immunity ,118. Supimmunity . The mecimmunity ,119. It immunity ,120. TheBacillus spp. are known to activate ISR. This has been verified in vitro that rice seeds treated with Bacillus spp. showed an elevation of ISR in rice against X. oryzae pv. oryzae [Bacillus-treated seeds exhibited an increased synthesis of defense-related enzymes including PAL, PO, and PPO. On another related study, the induction of systemic resistance against a fungal pathogen, namely, Rhizoctonia solani  was detected through an increased level of PAL and PO in rice treated with B. subtilis [B. subtilis on rice leaves under greenhouse conditions triggered the accumulation of pathogenesis-related proteins (thaumatin and β-1-3-glucanases) that play important roles for the induction of resistance in rice plant.. oryzae . Bacillusubtilis . The treBacillus spp. as PGPB is linked to their ability to suppress phytopathogens by secretion of antimicrobial metabolites [Bacillus spp. trigger the pathways of ISR, which contributes to the suppressive effect of plant immunity [Bacillus strains, NH-100 and NH-217, were effective against rice bakanae disease [Bacillus velezensis strain HN-2 displayed antibacterial activities against X. oryzae pv. oryzae and effectively inhibited its infection on rice [B. amyloliquefaciens in bean plants was determined to enhance the plant’s ISR against a fungal pathogen, Botrytis cinerea, infection [B. velezensis strain FZB42 (previously B. amyloliquefaciens strain FZB42) to suppress rice diseases caused by Xanthomonas [Xanthomonas virulence, cell division, protein synthesis, and cell wall synthesis.It is well known that the activity of abolites . The secimmunity ,125. Antimmunity . Sarwar  on rice . It is wnfection . Similarnfection . Furtherthomonas . The resPseudomonas spp. are Gram-negative, polar-flagellated, and rod-shaped bacteria. This bacterial genus belongs to the phyla Proteobacteria, class Gammaproteobacteria, and family Pseudomonadaceae. Species of Pseudomonas and their products have been used in large-scale for biotechnological applications [Pseudomonas spp. are ubiquitous in agricultural soils and have many plant growth-promoting traits. Moreover, Pseudomonas is a notable bacterial genus because some species are known as clinically important opportunistic human pathogen, plant pathogen, and biocontrol agent. Notable examples include the human pathogen, Pseudomonas aeruginosa [Pseudomonas syringae [Pseudomonas putida and Pseudomonas fluorescens [ications . Pseudomruginosa , the plasyringae , and theorescens ,134. P. aeruginosa strains have been intensively studied as an opportunistic human pathogen, especially in an immunocompromised host (a host with a weakened immune system). Interestingly in biocontrol applications, the use of P. aeruginosa strains as PGPB has been demonstrated. Strains of P. aeruginosa have been reported to be used in seed treatments of rice [P. aeruginosa strains showed plant growth-promoting activity and ISR in rice. Pathogenesis-related peroxidases that are involved in ISR in rice plants were detected in all the P. aeruginosa strains and showed antifungal activity against phytopathogenic fungi . In 2017, P. aeruginosa strain BRp3 (identified using 16S rRNA gene sequencing) isolated from rice rhizosphere was found able to solubilize phosphorus (97 μg/mL) and produced IAA (30 μg/mL) and siderophores (15 mg/L) in vitro [X. oryzae pv. oryzae and its potential to promote the growth and yield of Super Basmati rice. of rice . All P. in vitro . MS analP. syringae are noted for their diverse and host-specific interactions with a plethora of plant species including rice [P. syringae colonizes plant tissues by entering plant leaves through the stomata, multiplies in the intercellular space (apoplast), and eventually produces necrotic lesions that are often surrounded by chlorotic halos [P. syringae pv. syringae strain 260-02 was found exhibiting non-pathogenic behavior [P. syringae pv. tomato strain DC3000, against the B. cinerea fungus and the Cymbidium ringspot virus. Controversially, the pathogenic status of P. syringae has been rising. Considering the genome plasticity of strain 260-02 that could switch to pathogenic behavior through horizontal gene transfer mechanisms, the introduction of this strain into an ecosystem as a biocontrol agent is extremely dangerous.Strains of ing rice ,137. As ic halos . Howeverbehavior . Strain Psuedomonas as PGPB on rice plant is well reported [P. putida strain RRF3 have been demonstrated through transcriptomic analysis of rice plant roots [P. putida (bacterium) and Chlorella vulgaris  has proven to ameliorate arsenic toxicity in rice [−1 dw) and shoot (51 mg kg−1 dw) in a consortium of inoculated seedlings as compared to arsenic exposed plants . These results suggested that this consortium might alleviate arsenic stress and improve growth of rice seedlings along with a reduction in arsenic levels. Moreover, in vitro characterization of plant growth-promoting activities of Pseudomonas pseudoalcaligenes showed that this bacterium has a higher phosphate solubilization and productions of ACC deaminase, siderophores, IAA, and gibberellins [P. pseudoalcaligenes suppressed Magnaporthe grisea  infection by producing lytic enzymes, namely, chitinase and β-1, 3-glucanase. In greenhouse studies, inoculation of P. pseudoalcaligenes resulted in the improvement of dry weight, plant height, and root length of rice plants compared to inoculation of B. pumilus. The implementation of various species of reported ,141. Fornt roots . Overall in rice . The poserellins . In addiPseudomonas such as P. fluorescens are commonly isolated from rice rhizosphere [P. fluorescens isolated from rice rhizosphere soils in Karnataka, India possesses antibacterial activities against X. oryzae pv. oryzae [β-1, 3-glucanase, cellulase, and salicylic acid. One P. fluorescens strain, namely, Pf9, was found to effectively control the causal agent of Bacteria Leaf Blight (BLB) disease. In untreated controls, BLB disease incidence was 80% and this was reduced to 20% in plants raised from strain Pf9 treated seeds. Moreover, biological control of X. oryzae pv. oryzae by plant-associated P. fluorescens producing antimicrobial compound, DAPG, also has been reported [X. oryzae pv. oryzae in laboratory assays and reduced BLB disease in net-house (59%) and field (64%) experiments. In 2016, the application of P. fluorescens (PGPRPf) in Giza 179 rice cultivar has been tested in the nursery and field [Pf exhibited an increase in seed germination, seedling vigor, and yield. In addition, the application of inorganic nitrogen in combination with PGPRPf  in the field improved rice yields. PGPB strains belonging to fluorescent zosphere . For ins. oryzae . All 10 reported . DAPG wand field . InoculaEnterobacter spp. are Gram-negative, rod-shaped, and non-spore-forming bacteria belonging to the phyla Proteobacteria, class Gammaproteobacteria, and family Enterobacteriaceae. The potential of Enterobacter to contribute to the development of sustainable agricultural systems as PGPB has been reviewed previously [Enterobacter functions in three different ways: (i) synthesizing particular compounds for the plants, (ii) facilitating the uptake of certain nutrients from the soil, and (iii) lessening or preventing the plants from diseases. These can be characterized by the determination of antagonistic and plant growth-promoting activities [eviously . Howevertivities .Enterobacter cloacae strain B8 [X. oryzae pv. oryzae. In 2020, two bacterial strains (BSB1 and BCB11) isolated from the field showed antagonistic activities towards B. glumae were identified belonging to the genus Enterobacter [Enterobacter tabaci as analyzed based on 16S rRNA gene sequences and phylogenetic analyses. Strain BSB1 proved to be the best inorganic phosphorus solubilizer with a solubilization index (SI) of 4.5. Noteworthy, the EtOAc extract of strain BCB11 was found to inhibit the growth of B. glumae strains by 85–95%. Metabolomic analysis of EtOAc extract based on GC–MS showed that the main compound present is 3-phenylpropanoic acid (46.7%). This compound showed antibacterial activity with a minimum inhibitory concentration (MIC) of 1000 mg/L against five strains of B. glumae. The results suggested that Enterobacter is a promising strain as a PGPB and as a source of compound that could inhibit the growth of B. glumae.train B8  and stratrain B8  have beerobacter . The strEnterobacter have exhibited positive effects on rice plant growth. In 2018, multi-heavy-metal-resistant-PGPB isolated from metal-contaminated rice rhizosphere were identified as Enterobacter sp. strain K2 using phenotypic characterization and MALDI-TOF MS [Enterobacter that conferred cadmium-tolerance in rice seedlings and are potentially to be applied as PGPB in contaminated fields. Mitra et al. reported that Enterobacter sp. strain S2 showed multiple heavy metal resistance on cadmium (3500 µg/mL), lead (2500 µg/mL), and arsenic (1050 µg/mL) [α-keto-butyrate/mg protein/h), and IAA (726 µg/mL). Inoculation of strain S2 with rice seedlings significantly enhanced various morphological and biochemical characters of seedling growth compared to un-inoculated seedlings under cadmium stress. Up until now, multi-heavy-metal-resistant strains of I-TOF MS . Strain 0 µg/mL) . Strain Enterobacter aerogenes strain K6  was isolated from rice rhizosphere contaminated with a variety of heavy metals/metalloid near an industrial area [Enterobacter has also been reported to be resistant to chromium (Cr(VI)) and possesses plant growth-promoting activities. For example, Enterobacter cloacae strain CTWI-06 was shown to be resistant to 3500 ppm of Cr(VI) [R. solani ITCC 2060 and Phytium debaryanum ITCC 5488) were recorded, as well as improved productivity of Mahalakshmi rice in pot culture. The results suggested the potential application of Cr(VI)-reducing strain, CTWI-06, as a bioremediation agent of Cr(VI) in chromium-contaminated soil.Moreover, a multi-heavy-metal-resistant-PGPB strain identified as ial area . Strain f Cr(VI) . Under oEnterobacter spp. are not only focusing on the determination of heavy-metal-resistant-PGPB. Several studies also reported the potential of Enterobacter to enhance tolerance of rice plants to salt in soil. For instance, inoculation of Enterobacter sp. strain SE-5 resulted in an increment of mature rice plant biomass under salt and cadmium stresses [gfp). In addition, strain SE-5 was found to survive in different soil layers for more than 90 days. Overall, the results suggested that strain SE-5 is potentially proliferating-transmitting in mature rice plants and rhizosphere soil during plant growth. Moreover, salt-tolerant PGPB isolated from rice fields, namely, Enterobacter sp. strain P23, were also shown to promote rice seedling growth under salt stress [Enterobacter was also found exhibiting ACC deaminase activity [Enterobacter sp. E5) and engineered  strains promoted the growth of sprouts. The promoting effects were more profound with engineered strain than with wild strain. The engineered strain improved saline resistance of sprouts under salt concentrations from 10 to 25 gL−1 and promoted longer roots and shoots than the wild strain. These results suggested that bacterial ACC deaminases play a role in plant-produced ACC degradation and thus inhibit plant ethylene production.Current explorations of stresses . Strain t stress . This efactivity . InoculaStreptomyces spp. are the most attractive bacterial genus within the scientific community due to their capability to produce various bioactive compounds, which are consequently invaluable in the medical and agricultural fields. Streptomyces spp. received a worldwide attention due to their potential as producers of extracellular enzymes [Streptomyces spp. are complex filamentous Gram-positive bacteria belonging to the phyla Actinobacteria, class Actinomycetes, and family Streptomycetaceae.  enzymes  and as i enzymes . StreptoStreptomyces are complexly reproduced. Unlike most bacteria that divide by binary fission, Streptomyces spp. grow as a mycelium of branching hyphal filaments and reproduce in a mold-like manner by sending up aerial branches that turn into chains of spores [Streptomyces spp. can also be observed through their large genome size (more than 8 Mbp with a high G + C content), which is often associated with their ability to survive in various environments [Streptomyces strains as a biocontrol agent [Bacteria from the genus f spores . The comronments ,160. Intol agent . Streptomyces spp. were reported to aid in plant development as PGPB [Streptomyces spp. as PGPB has provided information on their beneficial traits related to the antagonistic and plant growth-promoting activities [Streptomyces clade isolated from the Colombian Caribbean Sea showed antibacterial activities against B. glumae, B. gladioli, and B. plantarii [Streptomyces spp. have also been reported to exhibit antagonistic activities against X. oryzae [X. oryzae pv. oryzicola [X. oryzae pv. oryzae [B. glumae and B. gladioli [P. fuscovaginae [E. chrysanthemi [D. zeae [ as PGPB ,164,165.tivities . Bacterilantarii . Moreove. oryzae ,169, X. ryzicola ,171, X. . oryzae ,173,174,gladioli , P. fuscovaginae , E. chrysanthemi , and D. [D. zeae . Streptomyces strains [Streptomyces spp. are reportedly able to produce antimicrobial metabolites, including blasticidin-S [Streptomyces spp. also produce a large number of other bioactive metabolites, including VOCs that stimulate plant growth both directly and indirectly [Streptomyces spp., particularly with regard to the antagonistic activity against bacterial rice pathogens as well as the ISR of rice plants.Genomically, the key pathways relating to plant growth-promoting activity, such as siderophores, IAA, HCN, chitinase, and cellulase, have also been decoded in the genome of  strains . It is nicidin-S , kasugamicidin-S , polyoxiicidin-S , and oliicidin-S , that weicidin-S  and enhaicidin-S . Other tdirectly ,184. MucAcidovorax [Rhizobium [Burkholderia [Serratia [Azotobacter [Klebsiella [Alcaligenes [Ochrobactrum [Pseudacidovorax [Azospirillum [Herbaspirillum [Acinetobacter and Pantoea) and Firmicutes  have been reported as PGPB and showed antagonistic effects against X. oryzae pv. oryzae [Stenotrophomonas maltophila UKA-72 and Rhizobium radiobacter UKA-24) and Firmicutes (B. pumilus UKA-27) exhibited antimicrobial activities against fungal  and bacterial  pathogens.Bacterial genera belonging to the phyla Proteobacteria, including idovorax , Rhizobihizobium ,186, Burholderia ,188, SerSerratia , Azotobatobacter , Klebsiealigenes , Ochrobaobactrum , Pseudacidovorax , Azospirpirillum ,193, andpirillum ,193, hav. oryzae . Moreove. oryzae . BacteriSo far, omics technologies have led to the exploration of crop rhizobiome through the metagenomics approach to understanding plant–microbe interactions . Such inBioformulations are defined as any biologically active substances derived from microbial biomass or products containing microbes and microbial metabolites that are used in plant growth promotion, nutrient acquisition, and disease control in an eco-friendly manner . The appP. aeruginosa strain BRp3 has been suggested as a bio-inoculant for Super Basmati rice and showed antagonistic activities against X. oryzae pv. oryzae [−1) significantly enhanced grain and straw yield with an increase of 51% and 55%, respectively. Colonization studies under field conditions using viable count detected the colonization of strain BRp3 on rice roots and shoots up to 60 days, suggesting its application as rhizobacteria-inoculants. Many reports suggested that bioformulations are easy to deliver, able to enhance plant growth and stress resistance, and able to increase plant biomass and yield. Additionally, any bioformulation with an increased shelf life with simultaneous actions of biocontrol and biofertilizer activities under field conditions could open the way for technological exploitation and marketing ,207. Wit. oryzae . The inoP. fluorescens, as a bioformulation in rice has been reported. Seed treatment with bioformulation of P. fluorescens strain RRb 11 increased the plant growth-promoting parameters of Pusa Basmati 1 rice [X. oryzae pv. oryzae) but enhances germination, increases height, dry matter, and yield. In addition, Jambhulkar and Sharma showed antagonistic potential of P. fluorescens strain RRb-11 against X. oryzae pv. oryzae [P. fluorescens strain SP007s, has also been applied in Thailand [X. oryzae pv. oryzae) and fungal  pathogens.In another related study, the efficacy of PGPB, i 1 rice . The tal. oryzae . The resThailand . The appAzospirillum spp.; BioGroe®  containing P. fluorescens, B. subtilis, B. amyloliquefaciens, and Candida tropicalis; Dimargon®  containing Azotobacter chroococcum; EMAS  containing Azospirillum lipoferum, Acinetobacter beijerinckii, Aeromonas punctate, and Aspergillus niger; BioPower  containing multiple strains of nitrogen-fixing bacteria.The use of PGPB in the form of bioformulation is an eco-friendly and inexpensive alternative to chemical fertilizers and pesticides ,214. It To enhance yield and protect crops against pests and pathogens, the use of chemical fertilizers and pesticides have been crucial in ensuring food security to feed the ever-increasing human population . RepeateThe introduction of the research results for industrial exploitation is not necessarily easy. Several PGPB bioformulations published worldwide demonstrated outstanding biocontrol activities in vitro ,227. OftXanthomonas, Burkholderia, and Pantoea. Interestingly, PGPB could be a possible alternative in controlling bacterial rice pathogens, which involves the application of eco-friendly microbes that control pathogens and improve plant growth. With the awareness on the usage of PGPB in the agricultural sector, their isolation and characterization are highly demanded, especially for use in rice plant protection. Varieties of bacterial genera, including Bacillus, Pseudomonas, Enterobacter, and Streptomyces, are promising as rice plant co-inoculants. Their application in the form of bioformulations could potentially improve the sustainable production of rice. The use of a bacterium or a consortium of PGPB in correct bioformulations provides a remarkable solution for a more sustainable agricultural future. In conclusion, the studies mentioned in this review support the progress in characterizing PGPB and designing bioformulations for use in rice plant protection to manage bacterial rice pathogens. In addition, this review highlighted the importance of continuing research on the applicability of PGPB, which, up to now, are scarcely used as rice plant co-inoculants to enhance rice production and ensure food security.A plethora of studies have addressed the ability of PGPB to promote plant growth on rice plants and its biocontrol activity against bacterial rice pathogens. As this review has made clear, bacterial rice pathogens can be caused by a variety of bacterial genus, including


Candida albicans, Candida glabrata, Candida krusei, Cryptococcus neoformans, and unicellular yeast-like Saccharomyces cerevisiae. Candida albicans alone accounts for about 70% of all global fungal infections including periodontal disease. This review intends to discuss the scope of Hstn 5 as a novel recourse for the control of fungal infections.Fungi are reported to cause a range of superficial to invasive human infections. These often result in high morbidity and at times mortality. Conventional antifungal agents though effective invariably exhibit drug interactions, treatment-related toxicity, and fail to elicit significant effect, thus indicating a need to look for suitable alternatives. Fungi thrive in humid, nutrient-enriched areas. Such an environment is well-supported by the oral cavity. Despite this, there is a relatively low incidence of severe oral and periodontal fungal infections, attributed to the presence of antimicrobial peptides hosted by saliva, viz. histatin 5 (Hstn 5). It displays fungicidal activity against a variety of fungi including  Candida albicans is documented as the prime agent for mucosal and systemic infections. It is responsible for about 70% of all fungal infections [Fungal infections impact the quality of life in the inflicted patients . Furtherfections  includinThe mainstay of treatment lies with azoles, polyenes, and echinocandins. Azoles such as fluconazole and itraconazole inhibit ergosterol biosynthesis in the fungal cell membrane . PolyeneSaliva is an extracellular fluid synthesized and secreted by salivary glands inside the oral cavity. It is viscoelastic in nature and vital for managing oral health and homeostasis. It is composed of water (99.5%), ions, proteins, and enzymes . AntimicHis1 and His2 located on chromosome 4, respectively. Hstn 1 undergoes proteolysis to produce Hstn 2, while proteolysis of Hstn 3 leads to the production of all other subtypes [Histatins (Hstns) are histidine-rich, low molecular weight, cationic, antimicrobial peptides present in human saliva and the oral cavity. Hstns exhibit a broad spectrum of antifungal activities and reportedly play a significant role in controlling periodontal and oral fungal infections ,15. Manysubtypes .C. albicans, the most prevalent fungi causing periodontal disease, skin, and other superficial infections [Out of all Hstns, Hstn 5 shows strong antifungal potential against fections ,17,18. Hfections .Hstn 5 is a naturally occurring peptide in humans, with no reported cross-reactivity in any human cells or tissues. Overall Hstn 5 showcases significant potential as a novel and highly effective antifungal agent . A signiA plethora of bioactivity displayed by Hstn 5 stimulates the compilation of this review which describes the structure and source of this salivary peptide and its potential as a novel antifungal therapeutic recourse, its purported mechanism of action, and plausible applications in diverse fungal infections including oral and periodontal infections.Peptides and their structural modification to produce products with high bioavailability and commercial applicability has been a subject of continuous interest for the scientific community.The structure of Hstn 5 is Asp-Ser-His-Ala-Lys-Arg-His-His- Gly-Tyr-Lys-Arg-Lys-Phe-His-Glu-Lys-His-His-Ser-His-Arg-Gly-Tyr. It is represented as DSHAKRHHGYKRKFHEKHHSHRGY. Hstn 5 though comprising of only 24 amino acids yet shows the highest candidacidal activity among all Hstns . The strHstns, being cationic amphipathic molecules with alpha-helical conformation target the anionic microbial surface . Hstn 5 Three mechanisms are proposed for the uptake of Hstn 5 which potentially work in tandem. In addition to the uptake after a direct interaction with the plasma membrane, transport-mediated and receptor-mediated endocytosis are the other two proposed uptake pathways . The conC. albicans and are involved in traversing Hstn 5 across the plasma membrane. Binding occurs in an energy-independent fashion and is required for localized retention before energy-mediated uptake. The fungal polyamine transporters Dur3 and Dur31 eventually actively transport Hstn 5 into the cells [Lower extracellular concentration directs the operation of receptor-mediated endocytosis thus internalizing Hstn 5in the vacuoles. Higher physiological concentrations lead to Hstn 5 accumulation in the cytoplasm through transport mediated uptake. However, at intermediate concentrations, the accumulation of this peptide inside the cell is not site-specific ,35. The he cells .+) [Hstn 5 causes exudation of intracellular potassium ions (K+)  leading +) . Trk 1 t+) .C. albicans. This released ATP mediates cell killing by binding to the purinergic receptors of the cell membrane, P2X [C. albicans. Cellular respiration blocked by sodium azide [Release of intracellular ATP after incorporation of Hstn 5 is also considered to be a cause of cell death in ane, P2X . Thus, iane, P2X . The mitum azide  or otherum azide . Studiesum azide . Thus, ium azide .C. albicans cells showed phosphorylation of Hog1. This induces the production of glycerol resulting in osmotic stress [C. albicans degrades Hstn 5 by employing aspartyl proteases, thus hampering its fungicidal activity [The mitogen-activated protein kinases (MAPK) signaling pathwayis stimulated by many antifungal drugs . Hstn 5 c stress . These oc stress . Cek1 isc stress . Phosphoc stress . Consequactivity .A goal of modern therapeutics is to develop non-invasive antifungal agents to prevent fungal growth. In this regard, Hstn 5, has attracted considerable interest in many therapeutic areas.C. albicans in the oral cavity. It is a common fungal disease observed in immunocompromised patients [C. albicans [C. albicans contamination [Oral candidiasis is a fungal infection affecting the oral mucosa caused by the overgrowth of patients . Howeveralbicans . Hstn 5 albicans . P113, aalbicans . Likewismination . Thus, imination .C. albicans is reported to be one of the prime causative organisms for periodontal disease. The carriage of this pathogen is directly related to the severity of the condition [C. albicans in synergy with bacteria invade the dental tissues resulting in the proliferation and exudation of enzymes that eventually cause tissue degradation [Periodontal disease constitutes a series of inflammatory conditions related to the tooth and its supporting tissues. Approximately 10% of the global population is affected by severe periodontitis ,58. In tondition . C. albiradation . Biofilmradation . These bradation ,64. Howeradation . Hstn 5 radation . Hstn 5 radation . All theC. albicans is involved in nearly 76–89% of all the infection cases being reported [C. albicans strains which indicate the possible role of drug-resistant strains in recurrent episodes of disease [Vulvovaginal candidiasis is an inflammatory disease, affecting the female genital tract. A retrospective study, including over 950,000 women who followed gynecological practices showed that approximately 70–75% of the women population experience vaginal candidiasis at least once in their lifetime, and up to 50% of them suffer from recurrent candidiasis. Vulvovaginal candidiasis is caused by the uncontrolled growth of Candida yeast species . Althougreported . Use of reported . Fluconareported . A study disease . In addi disease . Hence t disease . The ant disease .Peptides are naturally occurring macromolecules and closely mimic the intrinsic signaling entities which make them highly contemporary therapeutics. Further, a lot of work has been undertaken to design novel peptides and suitable delivery systems and penetration enhancers to ensure half-life augmentation and better permeation of these agents. All this has collectively broadened the scope of the application of peptides.Hstn 5, a cationic histidine-rich salivary peptide of human and higher primates has shown a promising potential to reduce mycological morbidity among immunocompromised patients. Different mechanisms are reported to support its antifungal activity. Further, the physiological nature of Hstn 5 ensures the absence of toxicity. While there is substantial evidence on the use of Hstn 5, however, a lot is desired on developing a suitable delivery system for the same to maintain its stability without compromising efficacy. The future thus foresees a bright perspective for the inclusion of novel drug delivery system which can facilitate the promise of this peptide.


Oxytropis glabra DC. is a perennial poisonous plant to livestock belonging to the genus Oxytropis, Leguminosae, mainly distributed in Northwestern China. As a poisonous grass, this species protects plant diversity in degraded grasslands by sheltering adjacent plants. In this study, the complete chloroplast genome with a total size of 122,094 bp was reported. Our annotations showed that the chloroplast genome contains 109 genes, including 76 protein-coding genes, 29 tRNA genes, and four rRNA genes. This work presents complete chloroplast genome information, which will be valuable for studying the evolution and genetic diversity of O. glabra. Oxytropis glabra DC. (Leguminosae) is a perennial poisonous plant to livestock belonging to Leguminosae, mainly distributed in Northwestern China. As a common poisonous grass, O. glabra protects plant diversity in degraded grasslands by sheltering adjacent plants. In addition, some species of Oxytropis were reported to have high flavonoids, such as Oxytropis falcata Bunge. The whole herb of Oxytropis falcata has a variety of pharmacological activities, including anti-inflammatory and antioxidant drugs . The voucher specimen  was stored in the herbarium of Tarim University. The total genomic DNA from leaves was extracted using CTAB method (Doyle and Doyle http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). The whole chloroplast genome was assembled using GetOrganelle v1.7.3    and Bayesian inference (BI)  . The ML nce (BI)  based onnce (BI) . This re


Purpose: To broaden the mutation and phenotype spectrum of the GJA8 and CHMP4B genes and to reveal genotype-phenotype correlations in a cohort of Chinese patients with congenital cataracts (CCs).Methods: Six Chinese Han families with CCs inherited in an autosomal dominant (AD) pattern were recruited for this study. All patients underwent full ocular examinations. Genomic DNA was extracted from the leukocytes of peripheral blood collected from all available patients and their unaffected family members. Whole-exome sequencing (WES) was performed on all probands and at least one of their parents. Candidate variants were further confirmed by Sanger sequencing. Bioinformatic analysis with several computational predictive programs was performed to assess the impacts of the candidate variants on the structure and function of the proteins.Results: Four heterozygous candidate variants in three different genes  were identified in affected individuals from the six families, including two novel missense variants , one missense mutation (CRYBB2: c.562C > T/p. R188C), and one small deletion (GJA8: c.426_440delGCTGGAGGGGACCCT/p.143_147delLEGTL). The three missense mutations were predicted as deleterious in all four computational prediction programs. In the homologous model, the GJA8: p.143_147delLEGTL mutation showed a sequence deletion of five amino acids at the cytoplasmic loop of the Cx50 protein, close to the third transmembrane domain. Patients carrying mutations in the same gene showed similar cataract phenotypes at a young age, including total cataracts, Y-sutural with fetal nuclear cataracts, and subcapsular cataracts.Conclusion: This study further expands the mutation spectrum and genotype-phenotype correlation of CRYBB2, GJA8, and CHMP4B underlying CCs. This study sheds light on the importance of comparing congenital cataract phenotypes in patients at the same age stage. It offers clues for the pathogenesis of CCs and allows for an early prenatal diagnosis for families carrying these genetic variants. Similar cataract patterns were observed in patients carrying the same mutations or different mutations in the same genes. Although CCs are a phenotypically heterogeneous disease, genotype-phenotype correlation analysis of cataract morphology may allow for more efficient genetic and prenatal diagnosis of families carrying these genetic alterations. It could also reveal the cataract genesis mechanisms of candidate genes in the different stages of lens development.Congenital cataracts (CCs) are defined as an opacity of the lens with onset from birth. It is one of the most common avoidable causes of visual impairment and blindness in children worldwide . It accotl.edu/) . With thtl.edu/) . In our tl.edu/) . Furtherhttps://www.wma.net/what-we-do/medical-ethics/declaration-of-helsinki/), revised in 2013. Informed consent was provided by all of the participants or their guardians. Detailed histories of birth conditions and ophthalmology were collected. A full ophthalmic examination was performed on all available subjects. It included a best-corrected visual acuity assessment, slit-lamp biomicroscopy, fundus photography, B-ultrasound scan, retinoscopy with cycloplegia, anterior segment biometry measurements performed with a Pentacam rotating Scheimpflug camera , and ocular biometry measurements performed with an A-ultrasound scan or an IOL Master 500 .A total of 25 participants (18 patients and seven unaffected relatives) in six Chinese families  were recGenomic DNA was extracted from leukocytes obtained from peripheral blood samples, as previously described . Whole-ehttp://grch37.ensembl.org/Homo_sapiens/Info/Index, Sep. 2015 data release), the dbSNP database , and the gnomAD database . All alleles with minor allele frequencies (MAFs) larger than 0.0002 were removed. The frequency was based on the incidence of CCs  (http://genetics.bwh.harvard.edu/pph2/index.shtml) (http://provean.jcvi.org/index.php) (http://www.mutationtaster.org/) (https://www.fruitfly.org/seq_tools/splice.html) (https://genome.ucsc.edu/index.html). The filtered results were analyzed in the Human Gene Mutation Database  and the Cat-Map database  to confirm whether the candidate variants we detected had been reported before. All candidate variants were further filtered by the phenotypes of the patients carrying these mutations and the cosegregation analysis. The ProtScale program  was used to predict protein hydrophobicity. In addition, homologous structures of wild-type and mutant human βB2-crystalline protein, connexin 50 (Cx50), and chromatin-modifying protein 4b were modeled by the SWISS-MODEL program based on the template of the resolved structure of No. 1blb, 7jjp, and AF-Q9H444-F1 (https://beta.swissmodel.expasy.org/)  , SIFT andex.php) , and Muter.org/) . The patce.html) . The evaCRYBB2, GJA8, and CHMP4B) were identified in affected individuals from six Chinese Han families. Two variants were novel: GJA8 (OMIM: 600897): c.64G > C (p. G22R), and CHMP4B (OMIM: 610897): c.587C > G (p.S196C). The other mutants included a missense mutation: CRYBB2 (OMIM: 123620): c.562C > T (p. R188C), and a small deletion: GJA8 (OMIM: 600897): c.426_440delGCTGGAGGGGACCCT (p.143_147delLEGTL), which has been previously reported in other Chinese families  to a positively charged hydrophilic residue (arginine). It is located in the first transmembrane region of the Cx50 protein  of the Cx50 protein, close to the third transmembrane domain  with CCs and seven unaffected family members  from six Chinese Han families. All patients had bilateral cataracts and a family history of autosomal dominant (AD) inheritance patterns. None of them reported cataract-causing situations during pregnancy, including virus infection , histories of medication use, toxin, or X-ray exposure. None of the unaffected family members carried the candidate variants. A summary of the general information and ocular phenotypes of all available patients in the six families is shown in CRYBB2: c.562C > T (p.R188C). All probands in the three families had total cataracts in both eyes before 5 months of age. The affected mother of CC1 underwent cataract surgery 23 years ago. The affected mother of CC2 and affected father of CC3 had cataract surgery more than 30 years ago. Therefore, there were no detailed cataract phenotype records. The cataract phenotypes of the probands in CC2 and CC3 are shown in All patients in families CC1, CC2, and CC3 carried the same missense mutation, C > T p.R8C. All pGJA8 gene were separately detected in families CC4 and CC5. The candidate variant of family CC4 is at exon 2 of GJA8. It is a base substitution change: c.64G > C (p.G22R). The proband of family CC4 (III:1) was a 4-month-old boy. He had bilateral fetal nuclear opacities with fuzzy Y-sutural opacity . According to previous reports, patients carrying the R188H mutant had anterior axial embryonal nuclear cataracts in a German family, and anterior axial embryonal nuclear cataracts (a 4-year-old patient) and coralliform cataracts (a 29-year-old patient) in a Chinese family  family. It is one of the components of the endosomal sorting complex required for transport III (ESCRT-III), a complex involved in the degradation of surface receptor proteins and the formation of endocytic multivesicular bodies  were found to be associated with posterior subcapsular or posterior polar cataracts  had common anterior segment characteristics , and minor(s)' legal guardian/next of kin, for the publication of any potentially identifiable images or data included in this article.XW, DW, WC, and HL designed this study. XW, DW, and QW analyzed and interpreted the data for the work. QW, WHua, MD, XZ, DL, ZL, JL, WHu, XLi, XLin, and QZ collected and measured data. XW and DW drafted the work. QW and DL revised it critically for important intellectual content. All authors discussed the results and commented on the manuscript.This research was funded by the Science and Technology Planning Projects of Guangdong Province, Grant No. 2019B030316012 and the National Natural Science Foundation of China, Grant Nos. 81822010, 81770967, and 82000946. The APC was funded by the Science and Technology Planning Projects of Guangdong Province, Grant No. 2019B030316012.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.


Enterobacterales are a major contributor. This study aimed to gain a deeper insight into the AMR burden of wild animals. In total, 1595 fecal samples were collected by two systematic searches in Mecklenburg-Western Pomerania, north-east Germany. Samples were screened for ESBL-carrying Escherichia (E.) coli and isolates found were further analyzed using antimicrobial susceptibility testing and whole-genome sequencing. We found an estimated prevalence of 1.2% ESBL-producing E. coli in wild boar and 1.1% in wild ruminants. CTX-M-1 was the most abundant CTX-M type. We also examined fecal samples from wild boar and wild ruminants using shotgun metagenomics to gain insight into the resistome in wild animals. The latter revealed significantly lower normalized counts for AMR genes in wildlife samples compared to farm animals. The AMR gene levels were lower in wild ruminants than in wild boar. In conclusion, our study revealed a low prevalence of ESBL-producing E. coli and a low overall AMR gene burden in wild boar and wild ruminants, probably due to the secluded location of the search area.Antimicrobial resistance (AMR) is a serious global health threat and extended-spectrum beta-lactamase (ESBL)-producing  The treatment of bacterial infections depends heavily on the availability of effective antimicrobial agents. Worldwide, the emergence and spread of antimicrobial resistance (AMR) is considered a serious public health threat . In addiAntibiotic resistance is a natural phenomenon ; howeverEnterobacterales (ESBL-PE) are among the most critical antibiotic-resistant bacteria [According to the WHO’s “Global Priority List of Antibiotic-Resistant Bacteria for Research, Discovery and Development of New Antibiotics”, extended-spectrum β-lactamase (ESBL)-producing bacteria . The enzbacteria . There abacteria  and ESBLbacteria .Acinetobacter baumannii, Pseudomonas aeruginosa and carbapenemase-producing Enterobacteriaceae infections in humans [Of particular concern is the emergence of Gram-negative bacteria that are resistant to three of the most important antimicrobial classes . 3MRGN occur in human healthcare settings , as welln humans . In recon humans . E. coli among humans, livestock and wildlife, reinforcing the role of these bacteria in transmission events, not only in veterinary settings, but also in public health, and vice versa [E. coli [In general, wild animals do not come into direct contact with antimicrobials. Nevertheless, they may be exposed to AMRB or antimicrobial residues by foraging and drinking in environments contaminated with anthropogenic sources ,16,21. Sce versa ,23. Howece versa . PublishE. coli in wild boar have recently received, only a few whole-genome sequencing (WGS) data sets are available on these resistant bacteria. With the present study, we aimed to fill this knowledge gap.Despite the increasing attention that ESBL-carrying E. coli represent only one example of antibiotic-resistant bacteria. In addition to the aforementioned studies, which are based on analyses of individual isolates-, resistance genes can be detected using sequence-based methods. Metagenome analyses offer the possibility to characterize the bacterial resistome and provide insight into the abundance and diversity of antimicrobial resistance genes (AMRG). For urban wastewater, there is a strong correlation with socioeconomic, health, and environmental factors [Of course, ESBL-bearing  factors ,26. Rece factors . A recen factors .E. coli isolates and the resistomes collected from wildlife in a rather limitedly investigated area in the northeast of Germany. We particularly addressed (i) the geno- and phenotypic resistance profiles of ESBL-producing E. coli, (ii) the phylogenetic (sequence types) and virulence characteristics of these bacteria to place them into a broader context and assess, whether they belong to clonal lineages, associated with a high-risk at the international level, and (iii) the microbiome of selected fecal samples to reveal their respective resistome. We hypothesized that the resistance burden in wildlife is generally lower in wildlife than in farm animals in the same area. Therefore and in accordance with the list of “critically important antimicrobials for human medicine” published by the WHO  as well E. coli. Fecal samples from wild boar and wild ruminants were collected during two periods in February 2020 and 2021. The fresh samples were subsequently analyzed in the laboratory for the presence of ESBL-producing E. coli were isolated from 820 fecal samples. Of these, 13 isolates originated from feces of wild boar (n = 693) and three from wild ruminants (n = 127). In 2021, only three isolates were obtained, and these were all isolated from wild boar fecal samples (n = 623). In 152 samples of wild ruminant feces, we did not find any ESBL-producing E. coli.In 2020, a total of 16 putative ESBL-E. coli in wild boar and 1.1%  in wild ruminants was calculated. For the individual collection periods, this results in 1.9% (13 of 693) positive fecal samples for wild boar in 2020 and 0.5% (3 of 623) in 2021. For wild ruminants, 2.4% (3 of 127) of samples were positive in 2020 and none (0 of 152) in 2021.Thus, an estimated prevalence of 1.2%  0.7–2.0%; 16 of 1316) of ESBL-producing All 19 isolates were resistant to the beta-lactam antibiotics amoxicillin/clavulanic acid, ampicillin, cefotaxime, ceftazidime, and cefepim. Six isolates were resistant to tetracycline (32%). In addition, two of the three isolates from 2021 (numbers 1031 and 1114) exhibited resistance to gentamicin and tobramycin, whereas the third isolate from 2021 (number 1115) was resistant to ciprofloxacin and trimethoprim/sulfamethoxazole. All isolates were sensitive to imipenem, meropenem, colistin, and amikacin. Thus, six of the 19 isolates phenotypically fulfilled the definition of multidrug-resistant (resistant to at least three classes of antibiotics). Details are given in E. coli [n = 9), followed by ST398 (n = 4). ST543 appeared three times and ST2768 two times, ST6448 was found only once. Interestingly, ST398 appeared in both sampling periods, but only in wild boar samples. In contrast, ST216 and ST543 were identified in different species (wild boar and wild ruminants), but only during the first sampling round. By analyzing WGS data obtained for the 19 suspected ESBL isolates, we identified three different phylotypes , all of which are mainly associated with commensal E. coli . MLST  antibiotics were detected in three isolates  by sequence analysis. These three isolates were obtained from wild boar samples collected in 2021. However, phenotypically, resistance to tobramycin and gentamicin was present only in isolates 1031 and 1114. Resistance genes conferring tetracycline resistance  were present in three isolates , while phenotypic resistance was found in three additional isolates . Isolate 1115 was resistant to trimethoprim/sulfonamide both phenotypically and genotypically , whereas isolates 1031 and 1114 carried resistance genes to sulfonamides only  . Resistance to phenicol  antibiotics was detected genotypically in the three isolates . Beta-lactamases of the CTX-M type were present in all isolates (CTX-M1 and 55), other class A enzymes were genotypically absent as were plasmid-encoded beta-lactamases of classes B-D. By contrast, the chromosomally encoded beta-lactamases (AmpC and AmpH) were present in all isolates. Genes conferring resistance to aminoglycoside , and heme receptors (chu). Furthermore, we found genes that confer resistance to heavy metals . In addition, we detected a few genes typical for extra-intestinal pathogenic E. coli (ExPEC). Details can be found in To gain insight into the virulence of the isolates, we searched for virulence genes associated with pathogenic bacteria. The main focus was on iron acquisition and resistance to heavy metals. We found a number of genes that play a role in iron uptake, such as enterobactin  positive samples from wild boar (2016) and 2.3% (13 of 573) from roe deer (2017) [E. coli samples (19/375) (either CTX-M-1bla  or SHV-12bla ). However, this match could only be confirmed for the samples from 2020. In the framework of the German National Zoonoses Monitoring Program, samples from hunted wild boar and roe deer were collected nationwide in 2016 and 2017, respectively. Selective isolation of ESBL-/AmpC-producing r (2017) . Only for (2017) . They con = 7 and 2013–2014, n = 39). The hunts took place in 37 forests distributed across the territory of Poland (wild boar (n = 332), red (n = 225), roe (n = 76), and fallow (n = 24) deer. Of 553 E. coli isolated, 11 were cefotaxime resistant. Nine of them were retrieved from wild boar , fallow deer or red deer (a single isolate each) [A study conducted in Poland included a total of 660 fecal samples collected during two winter seasons from game animals shot during 46 hunts  .E. coli was determined in a study from southern Europe in a total of 1504 fecal samples from wild boar. This study was performed in northern Italy and consisted of four hunting areas comprising approximately 1.800 km2. Samples were collected in three consecutive hunting years between 2018 and 2020 [A significantly higher estimated prevalence of 15.96% of ESBL/AmpC-producing and 2020 .E. coli in wildlife would help to better assess the AMR burden in wildlife. Due to the convergence of habitats, the contact of wildlife with other animals  and humans becomes more and more common, which subsequently leads to enhanced exchange of antibiotic resistance both in the context of actual bacterial isolates but also mobile genetic elements. As previously mentioned, it appears that wild animals that live in close proximity to human populations carry higher levels of AMRB and AMRG [For the future, it would be useful to compare the estimated prevalences of the individual studies in a systematic review. Such a meta-analysis of ESBL-and AMRG ,38. Thisand AMRG . A recenand AMRG .E. coli are summarized below.Analysis of the whole-genome sequence data revealed broad agreement of phenotypic and genotypic results. For the observed phenotypic resistances, genotypic resistance determinants were always found as well . DetailsIn our study, we detected an ESBL-genotype for all isolates  phenotypically resistant against cefotaxime. Even if only ESBL-bearing isolates from the above-mentioned studies are taken into account, the detections in our study are still significantly lower than those of other studies. The sampling areas are located in Mecklenburg-Western Pomerania in the northeast of Germany. This region has the lowest human population density in Germany. The sampling areas thus represent a rather secluded part of nature. The associated low contact intensity between humans and agriculture could be an explanation for the low detection frequencies. Unexpectedly, the majority of ESBL-positive isolates was found in 2020 (16/19) and only a small number in 2021 (3/19). In February 2021, it was very cold (partly −15 °C) and there were heavy snowfalls (average temperature: 0.18 °C). In contrast, February 2020 was rather mild (average temperature: 5.50 °C) . It is pE. coli from human fecal samples, ElBaradei et al. found high abundances of ESBL carriers or even multidrug resistance. This is alarming, since these strains are associated with an increased risk of infection [E. coli clones belonging to the commensal phylogroup A were increasingly reported in association with ESBL production [E. coli strains in humans, domestic animals, and environmental sources, making them a substantial public health threat [It is of course hardly possible to draw conclusions about the health status of the animals based solely on the nature of the fecal sample. However, it can at least be stated that no intestinal problem, such as diarrhea, was present at the time of defecation, since the consistency of the fecal sample was recorded. Consequently, the sequence analysis of the isolates revealed that they all belong to commensal phylogroups . In a recent study of commensal nfection . Similaroduction . Over thh threat .E. coli isolates revealed five different MLST sequence (ST) types . ST216 was detected most frequently, with nine of the 19 isolates belonging to this ST. E. coli ST216 is known to carry resistance genes to a variety of antibiotics and has been detected several times in nosocomial infections [E. coli seems to be particularly capable of capturing and transmitting genes encoding beta-lactamases and carbapenemases [E. coli belonged to these STs (ST131 and ST648) [Sequence analyses of the 19 ESBL-bearing fections ,46. In pfections  and animenemases ,50,51. Uenemases . In theiE. coli carrying this plasmid. CTX-M-1 is frequently associated with IncN, which occurs throughout Europe and is mainly isolated from E. coli from animal sources [E. coli from the fecal microbiota of pigs [n = 9) or FIB (n = 11) replicon. FII replicons (n = 2) were less common. In general, IncF is the most abundant plasmid and predominantly detected in E. coli. The resistance genes most frequently described in combination with IncF plasmids are ESBL genes [In our study, IncN was the most abundant inc/rep type, with 16 of the 19 isolated ESBL- sources . In part of pigs . Other fBL genes . In our BL genes .Bacteroidaceae, Oscillospiraceae, and Lachnospiraceae)  . Interes animals . When co animals . Only a few publications are available that allow a comparison of data on the resistome of wild boar. Skarzynska et al. applied a metagenomic approach to investigate the resistome of different wildlife species. They found a distribution of each antibiotic group in wild boar that was largely consistent with the distribution in our study : Aminoglycosides: 12.8% vs. 8.3–10.6%; beta-Lactams 9.0% vs. 5.8–6.9%; Glycopeptides 3.8% vs. 0.7–1.2%; Macrolide 9.0% vs. 17.5–17.9% (part of MLS Class); Nitroimidazole 2.6% vs. not present; Quinolone 1.3% vs. 1.3–1.4%; Sulfonamides 6.4 vs. 0.5%; Tetracyclines 56.4% vs. 28.1–29.2%) , but wild ruminants were not included. Therefore, our resistome data for wild ruminants, as well as the microbiome data, have to remain without comparison with data from other studies, at least for the present moment. However, the high similarity between the wild pig and wild ruminant resistomes in our study is striking . NeverthE. coli and an overall low AMR gene burden in wildlife. Shotgun metagenomics revealed significantly lower normalized AMR genes levels in wildlife samples compared to farm animals. In this regard, this confirms our research hypothesis. However, due to its secluded location, the sampling site is a well-known hunting area and therefore an attractive source for acquiring wild game meat. In addition, the forest provides an excellent recreation area for humans [In addition to the limited comparability of the prevalences found in the individual studies, another limitation of our study is the selection of antibiotics tested in the AST. It is only a selection of the available and used substances. In conclusion, our study revealed a detectable but low prevalence of ESBL-producing r humans . The intr humans . Especiar humans . Thus, tTherefore, our data provide a first but also concerning glimpse into a so-far under-investigated part of our environment, that definitely deserves more attention in studies of AMR spread. The collections of fecal samples were carried out in four hunting grounds in Mecklenburg-Western Pomerania. During two collection periods in February 2020 and 2021, the hunting grounds were systematically searched for feces from wild boar and wild ruminants . The searches were performed along transects covering the entire study area. Only fecal samples clearly belonging to the target species were included in the study. In addition, samples had to be fresh, i.e., no more than 24 h old. Age was determined by shape and surface moisture. Samples that did not meet these criteria were not included in the study. During the sample collections in February 2020, a total of 820 fresh fecal samples were collected. Of these, 127 were from wild ruminants  and 693 from wild boar. During the February 2021 sample collections, a total of 775 fresh fecal samples were collected. Of these, 152 were from wild ruminants and 623 were from wild boar.E. coli colonies were identified based on colony morphology (red-purple colonies) and subcultured until pure cultures were achieved. The pure cultures were used for further verification and characterization.Fecal samples were streaked on CHROMID ESBL chromogenic medium  using swabs and subsequently incubated at 37 °C overnight. Putative antibiotic-resistant E. coli isolate to be tested, which was adjusted to a McFarland standard of 0.5 in 0.9% NaCl. The inoculum (50 µL) was then diluted in 11.5 mL Mueller-Hinton II broth . Thereafter, 100 µL of this dilution were transferred into Merlin MICRONAUT S 96-well AST plates for livestock (“E1-301-100: Zoonosis Monitoring”) . The results were visually assessed after incubation for 18–24 h at 37 °C. The decision on turbidity and microbial growth was made by trained scientists. Growth was considered to have occurred when turbidity appeared at the bottom of the well. Tests were considered valid only, if growth had also been observed in the internal growth control.To determine the minimum inhibitory concentrations (MIC) of important antimicrobial compounds, e.g., colistin, AST was carried out in a 96-well plate microdilution procedure. To this end, we prepared an inoculum of the http://www.eucast.org, last accessed 1 October 2021) and European Commission Implementing Decision (EU) 2020/1729 were used for the evaluation.The European Committee for Antimicrobial Susceptibility Testing (EUCAST) Breakpoint Tables for the Interpretation of MICs and Zone Diameters . DNA extraction was performed using the MasterPure™ DNA Puri-fication Kit for Blood, Version II . DNA extraction was performed using the MasterPure™ DNA Purification Kit for Blood, Version II  and subsequently quantified using QuBit fluorometer . DNA samples were then shipped to StarSEQ. Library preparation was performed by StarSEQ as published elsewhere [WGS was performed for the overall 19 ESBL/AmpC suspect lsewhere , followehttp://sourceforge.net/projects/bbmap/, last accessed 1 October 2021), the following steps were performed with raw sequencing reads: adapter-trimming , filtering for contaminants , and quality-trimming . Quality controlling of all reads (trimmed reads and raw reads) was performed using FastQC v. 0.11.9 . Using the shovill v. 1.1.0 assembly pipeline  in combination with SPAdes v. 3.15.0 [https://github.com/tseemann/mlst, last accessed 1 October 2021) and ABRicate v. 1.0.0 , respectively. Third-party databases ) were used for the analyses of both tools.Using BBDuk from BBTools v. 38.89  and roe deer (n = 2) were subjected to metagenomic sequencing. For DNA extraction the QIAamp Fast DNA Stool Mini Kit  was used applying a modified protocol developed by Knudsen et al. [Fecal samples from wild boar . LGC the Allegro Targeted Genotyping Kit (TECAN) and the plexWell™ 384 Library Preparation Kit (seqWell™). Firstly, Allegro Targeted Genotyping Kit was used for (i) fragmentation, (ii) end-Repair and Adaptor Ligation, and (iii) purification and secondly, plexWell™ 384 Library Preparation Kit was used for plate-specific barcoding. Libraries were amplified in an emulsion PCR for 14 cycles using standard Illumina primers and afterwards libraries were size selected by a size selection on an LMP-Agarose gel, removing fragments smaller than 300 bp and those larger than 800 bp. A final library purification step and quality control of libraries were performed via Fragmentanalyzer  and Qubit. Sequencing was done on an Illumina NovaSeq 6000 SP FC- 2 × 250 bp paired-end read length  following the manufactures’ instructions. Metagenome sequencing data supplied by LGC was then analyzed using AMR++ and the MEGARes v2.0 database ,70 usingn represents each individual ARG in the MEGARes database, N(AMRseq) indicates the number of reads identified in the analysis using the AMR++ pipeline, and N(16Sseq) is the number of reads found to map to bacterial 16S rRNA gene reads in the analysis using METAXA2. L(reads) corresponds to the length of the sequencing reads , L(AMRrefseq) is the length of the AMR reference gene in the MEGARes database, and L(16Srefseq) corresponds to length of the 16S rRNA gene sequence .For normalization of the number of AMR reads, we analyzed the raw data (fastq datasets) using METAXA2 v. 2.2.3  to enumeThe calculation was done for each sample separately.https://researchdata.edu.au/indexed-reference-databases-kma-ccmetagen/1371207, accessed 15 July 2021) . Second, a quality filtering step was performed in the pipeline through CCMetagen (default settings were used).The CCMetagen pipeline was used for accurate taxonomic assignments of the quality-controlled read sets. The CCMetagen pipeline links the two tools KMA (version 1.3.0) and CCMetagen (version 1.2.2) ,73. In t


Plasmodium lactate dehydrogenase (pLDH) is a common target in malaria rapid diagnostic tests (RDTs). These commercial antibody capture assays target either Plasmodium falciparum–specific pLDH (PfLDH), P. vivax–specific pLDH (PvLDH), or a conserved epitope in all human malaria pLDH (PanLDH). However, there are no assays specifically targeting P. ovale, P. malariae or zoonotic parasites such as P. knowlesi and P. cynomolgi. A malaria multiplex array, carrying the specific antibody spots for PfLDH, PvLDH, and PanLDH has been previously developed. This study aimed to assess potential cross-reactivity between pLDH from various Plasmodium species and this array. We tested recombinant pLDH proteins, clinical samples for P. vivax, P. falciparum, P. ovale curtisi, and P. malariae; and in vitro cultured P. knowlesi and P. cynomolgi. P. ovale-specific pLDH (PoLDH) and P. malariae-specific pLDH (PmLDH) cross-reacted with the PfLDH and PanLDH spots. Plasmodium Knowlesi-specific pLDH (PkLDH) and P. cynomolgi-specific pLDH (PcLDH) cross-reacted with the PvLDH spot, but only PkLDH was recognized by the PanLDH spot. Plasmodium ovale and P. malariae can be differentiated from P. falciparum by the concentration ratios of PanLDH/PfLDH, which had mean (range) values of 4.56 (4.07–5.16) and 4.56 (3.43–6.54), respectively, whereas P. falciparum had a lower ratio of 1.12 (0.56–2.61). Plasmodium knowlesi had a similar PanLDH/PvLDH ratio value, with P. vivax having a mean value of 2.24 (1.37–2.79). The cross-reactivity pattern of pLDH can be a useful predictor to differentiate certain Plasmodium species. Cross-reactivity of the pLDH bands in RDTs requires further investigation. Plasmodium falciparum in sub-Saharan Africa and to a lesser extent P. vivax in Asia and South America.Plasmodium species—such as P. falciparum, P. vivax, P. malariae, and P. ovale spp. —that cause natural human malaria via parasite speciation and treatment of infection.–P. malariae and P. ovale spp. are prevalent in Africa and other malaria-endemic regions but receive little attention because of clinically mild symptoms.,Global malaria programs have been focused largely on P. knowlesi and P. cynomolgi. P. knowlesi, commonly found in natural hosts including long-tailed macaques (Macaca fascicularis) and pig-tailed macaques (M. nemestrina) in Southeast Asian countries, has been documented in humans as a result of increased human–macaque contacts as a result of habitat disruption and destruction by extensive population growth and deforestation in the areas where natural hosts and the anopheline mosquito vectors carrying P. knowlesi exist.,P. cynomolgi, often detected with other simian parasites such as P. inui, P. coatneyi, or P. fieldi in the macaque population in Southeast Asia, is a close relative of P. vivax.,P. cynomolgi has been reported in Southeast Asia.–In addition, malaria can be caused in humans by simian parasites such as P. knowlesi infection.–P. knowlesi has a relatively short asexual life cycle of approximately 24 hours, compared with approximately 48 hours for P. falciparum, P. vivax, and P. ovale, and 72 hours for P. malariae.P. knowlesi infection can rapidly replicate to reach a high parasite density and progress to severe malaria similar to that from P. falciparum if there is no prompt medical treatment.,P. knowlesi  may have been prevalent at low levels for many decades, a relative surge has arisen as a result of a dramatic reduction in prevalence of P. vivax and P. falciparum over the past decade.,P. knowlesi by microscopy is unreliable largely because of its similar morphological features with P. falciparum at the early trophozoite stage and to P. malariae at the other stages, notably trophozoite band forms.,Severe malarial symptoms and even death have been described with human P. knowlesi, there are no rapid diagnostic tests (RDTs) specifically designed for P. knowlesi identification. Currently available malaria RDTs use several key biomarkers to differentiate between species: P. falciparum-specific histidine-rich protein 2 (HRP2) or/and lactate dehydrogenase (PfLDH) for P. falciparum, and P. vivax-specific lactate dehydrogenase (PvLDH) for P. vivax. Some malaria RDTs detect pan-malarial biomarkers such as the pan-Plasmodium lactate dehydrogenase (pLDH) or Plasmodium aldolase (pAldo) that can, in principle, detect but not differentiate all Plasmodium species.® , Entebe Malaria Cassette, BinaxNOWTM Malaria , and Paramax-3TM ) between parasites of two major Plasmodium species and P. knowlesi parasites, and poor sensitivity in cases of low parasitemia with P. knowlesi infection, revealing the need for caution in data interpretation if confirmatory testing is not conducted.,P. knowlesi, several groups reported the stronger performance of molecular techniques, such as nested polymerase chain reaction (PCR) assay, loop-mediated isothermal amplification (LAMP), and real-time PCR assays.–P. ovale- and P. malariae-specific RDTs, also leading to the underreporting of these more widely spread forms of human malaria.,Plasmodium species.,P. cynomolgi (PcLDH) and P. vivax (PvLDH) indicated the close similarity of pLDH isoforms, whereas the absence of reactivity of the P. falciparum-specific antibodies with other simian parasites except P. knowlesi suggested the possible use of these antibodies to distinguish P. knowlesi from the other simian parasites.In addition to the limitations of microscopy to accurately identify P. vivax diagnosis with RDTs.hrp2/hrp3 gene deletions in P. falciparum infections leading to false negative RDT results, pLDH is increasingly a key biomarker for RDT-based diagnosis of malaria.,Plasmodium species to the different pLDH assays used in RDTs—namely P. falciparum pLDH, P. vivax pLDH, and PanLDH. In previous studies, we described the development and validation of a laboratory-based multiplex platform, the Human Malaria Array , that can quantify HRP2 and pLDH from P. falciparum and P. vivax.,Plasmodium species, may increase our understanding of the performance of current RDTs in these species.The pLDH is the primary target analyte for P. malariae and P. ovale and by using culture-adapted strains of both P. cynomolgi and P. knowlesi.,P. falciparum and P. vivax parasites, and the time-course expression of P. knowlesi-specific pLDH (PkLDH) protein in culture by a multiplex reference assay.Here, analysis of cross-reactivity of this reference assay was investigated with clinical samples of P. falciparum, 62 P. vivax, 17 P. malariae, and 8 P. ovale) from febrile patients with malaria mono-infection were obtained from the Foundation for Innovative New Diagnostics (FIND) Specimen Bank. Nested PCR with microscopy was used to identify the species of human malaria parasites in the samples. Parasite density in clinical samples was confirmed by either microscopy or quantitative PCR (qPCR) methods.A total of 116 clinical blood samples  gradient solution 4 hours apart, retaining the upper brown portion in the first centrifugation and the lower RBC portion for the second centrifugation, to achieve the highest synchronization rate. After synchronization, synchrony was confirmed, and parasite density in the culture was determined by staining thin-film smear using a Hemacolor® staining kit  and counting parasites in a minimum of 2,000 RBCs using a microscope. Following the second synchronization, the culture was incubated and harvested at different time points. For kinetics of PkLDH, infected RBC (iRBC) cultures that were incubated in the culture tubes in three replicates were harvested at 0, 1, 2, 6, 19, 21, 25, 27, 28, 29, and 30 hours. After centrifugation, the cell culture supernatant was collected, and the remaining iRBC pellets were washed with phosphate-buffered saline (PBS) and resuspended in PBS at the original culture volume. The total number of iRBCs was counted using an Accuri™ C6‐UV flow cytometer The Plasmodium cynomolgi parasites were cultured in complete RPMI media supplemented with GlutaMAXTM  and 200 µM hypoxanthine  as described previously.2, 5% CO2, and 5% O2. Three lots of in vitro culture as both iRBC pellet and culture supernatant were prepared on the second day after synchronization and frozen prior to shipment to PATH  for the antigen quantification.Plasmodium species-specific pLDH proteins and human LDH protein for use as reference proteins. Based on the DNA sequence information available from GenBank, the full-length ldh genes specific for P. falciparum, P. vivax, P. ovale curtisi, and P. malariae and the human full-length ldh gene were synthesized and cloned into the prokaryotic expression vector pOPIN-NHis3C to express in RosettaTM (DE3)pLysS competent cells . Bacterial culture was performed in 1 L TB media (Novagen) containing antibiotics at 30°C for around 24 hours. Culture was harvested by centrifugation at 5,000 × g for 30 minutes at 4°C and resuspended in lysis buffer. After lysing using sonication, the lysate was centrifuged at 20,000 × g for 30 minutes at 4°C, and the supernatant representing the soluble fraction was isolated. The soluble fraction was subjected to an immobilized metal affinity chromatography using a HisTrapTM Fast Flow column  followed by size-exclusion chromatography (SEC)® 26/600 Superdex® 200 column . The SEC elution fractions were analyzed by resolving on 12% SDS-PAGE. The SEC elution fractions of interest were pooled and concentrated using an Amicon® centrifuge filter . The sample was filtered through a 0.22 µm filter and stored at −80°C. The amount and purity of His-tag-containing recombinant pLDH and human LDH proteins were estimated by absorbance at 280 nm using NanoDropTM  spectrophotometer and SDS-PAGE, respectively. All recombinant pLDH and human LDH proteins showed more than 95% purity  and were kept at −80°C for long-term storage until future use.PATH collaborated with the University of Queensland Protein Expression Facility  to develop PfLDH, PvLDH, and PanLDH in iRBC pellets, culture supernatant, clinical samples, and samples spiked with recombinant pLDH proteins were tested using the Q-PlexTM Human Malaria Array  to target five different analytes—HRP2, PfLDH, PvLDH, PanLDH, and C-reactive protein (CRP)—according to the manufacturer’s instructions.P. vivax and P. falciparum parasites were individually spotted for the capture of PvLDH and PfLDH, respectively, with a third spot representing a capture antibody to Plasmodium LDH as a PanLDH determinant. A broadly reactive PanLDH antibody was used as the detector antibody for all three assays . For comparison of reactivity pattern, iRBC pellets of synchronized P. cynomolgi and P. knowlesi cultures and P. vivax clinical samples were diluted at 200 and 40 p/µL of parasite density and tested on the 5-Plex. For the samples at high concentration, which falls outside the standard range, the samples were subsequently diluted 1:20 and 1:400 with assay diluent, and the dilution factors were applied for the final concentration of antigens.The presence and quantification of /PfLDH and PanLDH/PvLDH are presented as mean ± SD. Student’s t-test was used to determine significant differences between ratio values of PanLDH and species-specific pLDH of recombinant and native proteins, estimated by reference assay. In addition, a two-way analysis of variance (ANOVA) with Sidak multiple comparison test was used to establish statistical significance between groups (P < 0.05).All data were analyzed using GraphPad Prism . Pearson’s correlation coefficient was calculated for the PanLDH level based on the 5-Plex and for parasite density based on microscopy or qPCR. The results relative to ratios of PanLDHP. falciparum, P. vivax, P. malariae, and P. ovale curtisi, and clinical whole blood samples infected with each of four Plasmodium species were tested on the 5-Plex to examine the presence and level of reactivity to the assays for PanLDH, PfLDH, and PvLDH. Analysis showed that good specificity on PfLDH and PvLDH spots was found with recombinant PfLDH and PvLDH proteins, respectively . Interestingly, recombinant P. ovale-specific pLDH (PoLDH) and P. malariae-specific pLDH (PmLDH) proteins showed reactivity with both PanLDH and PfLDH spots of the 5-Plex. However, a notable difference was observed with the relative signal from the PanLDH spot, in which both PoLDH and PmLDH recombinant proteins showed relatively less signal, resulting in lower measured concentration as compared with recombinant PfLDH or PvLDH proteins for a given concentration . Furthermore, the relation between level of PanLDH and parasite density was assessed with clinical samples infected by each Plasmodium species , P. vivax  and P. malariae  but relatively poor correlation with parasite density for P. ovale .For the reactivity to native pLDH from four  species . The corPlasmodium species . The PanLDH concentration measurement in the 5-Plex is calibrated to recombinant PfLDH and so the ratio of close to 1 for PanLDH/PfLDH is to be expected.The ratio of PanLDH to species-specific pLDH was similar for both native pLDH from clinical samples and the same recombinant proteins across the four  species . SpecifiP. knowlesi and P. cynomolgi on each assay for PanLDH, PfLDH, and PvLDH, a series of dilutions (200 and 40 p/µL) of whole blood sample spiked with synchronized P. cynomolgi and P. knowlesi iRBC pellets were tested with the 5-Plex. The results were comparable to that of a clinical P. vivax blood sample. The results showed the similar reactivity between PkLDH and PvLDH proteins for binding to the PanLDH and PvLDH spots, and with comparable PanLDH/PvLDH ratio values between these two Plasmodium species at 2.7 for PkLDH and 2.8 for PvLDH at 200 p/µL of parasite density , similar to the previously published finding of 27 hours.PkLDH was found in iRBC pellets rather than in the supernatant, both pellets and supernatant showed progressively increased concentration of PkLDH (∼8 times more after 19 hours compared with 1 hour for both pellets and supernatant) during ring and trophozoite stages . The maximal amount of PkLDH is reached in the pellet during the schizont stage (21 to 27 hours), but a continuous accumulation of PkLDH released from the schizont rupture in the supernatant was observed over this period of time. Interestingly, during the first life cycle, parasite density seems to be decreased during the schizont stage, and the number of parasites in the second life cycle is increased after the released cells infect the fresh cells.For the time-course study,  results  demonstrPfLDH, and PvLDH and a qualitative measure for all malaria species (PanLDH) as well as measure the human biomarker CRP.PfLDH and PvLDH,hrp2/hrp3 deletions both in whole blood pelletsP. falciparum and P. vivax, using the described biomarkers.,Plasmodium species such as P. malariae and P. ovale, and two simian Plasmodium species, P. knowlesi and P. cynomolgi, which have been recognized to cause human infection, has not been fully explored. As the 5-Plex includes a PanLDH spot detecting all Plasmodium species, in this study, we first evaluated the potential cross-reactivity of pLDH proteins from these parasites against pan-malaria and species-specific spots using both recombinant and native proteins.The human malaria array (5-Plex) was developed to provide quantitative measurement of the malaria antigens HRP2, P. falciparum, P. malariae, P. ovale curtisi, and P. vivax offer great diagnostic value for the appropriate evaluation of the assay performance when there are limited clinical blood samples. Our study demonstrated that these recombinant pLDH proteins behave similarly to native pLDH proteins from corresponding Plasmodium species as demonstrated by PanLDH/species-specific pLDH ratio while being distinct from the equivalent host LDH protein .The recombinant pLDH proteins of  protein . HoweverPlasmodium species, but obtaining native proteins from live parasites is a major challenge, especially with species of P. vivax, P. ovale, and P. malariae, for which continuous culture systems have not been established unlike P. falciparum strains.–P. falciparum parasites and their respective native proteins.The significant variation of PanLDH values in the estimated concentration of recombinant pLDH proteins suggest two possibilities: 1) some minor variance in the amino acid sequence of the conserved region or that of the surrounding area that enhances or reduces the antigen–antibody reaction, and 2) inefficient protein folding during protein expression/purification might have occurred when using a prokaryotic expression system. The second possibility may be addressed by evaluating the native pLDH proteins purified from different Supplemental Figure 3). The results suggest that this could be an artifact derived from the protein purification processes.The pLDH amount measured as PanLDH correlates to parasite density equally across species, as shown by the normalized binding reactivity of native pLDH proteins with respect to the given parasite number . It will therefore be of interest to investigate the extent of the level of cross-reactivity, which may have a significant influence on interpreting diagnostic test results. The PanLDH/species-specific pLDH ratio values for P. malariae, P. ovale, and P. vivax were significantly different from that for P. falciparum  and found a conserved region (the region forming the loop structure) as the potential site recognized by pan-malaria antibodies and several species-specific regions as described previously .–PcLDH protein as compared with three other parasite species including P. falciparum, P. vivax, and P. knowlesi that would explain our observation. It would be interesting to explore binding characteristics of commercially available pan-malaria antibodies to the laboratory-adopted P. cynomolgi strain or recombinant PcLDH reagents and investigate the extent of sequence conservation within the conserved region of PcLDH protein from the cultured P. cynomolgi in a future study.The reactivity patterns of pLDH from LDH spot . UnfortuPkLDH exhibited significant cross-reactivity to both the PanLDH and PvLDH spots, but no or little cross-reactivity with the PfLDH spot on the 5-Plex. A previous study looking at cross-reactivity between pLDH proteins from different Plasmodium species and different antibodies to pLDH highlighted that PkLDH can react to antibodies specific to both PfLDH and PvLDH.PkLDH and the PvLDH antibody pair was observed. Additionally, the PanLDH/PvLDH ratio for P. knowlesi was similar to that seen when measuring PvLDH from the P. vivax clinical samples. Although the PanLDH and PvLDH spots on the 5-Plex can detect the presence of the PkLDH antigen, they were unable to distinguish P. knowlesi from P. vivax infection. To differentiate P. knowlesi from other Plasmodium species, development of an expanded array that hosts a P. knowlesi-specific antigen assay is required.PkLDH in a culture system. The results exhibited accumulation of PkLDH over time in culture, except with a slight decrease in PkLDH concentration seen in samples from the late schizont stage at around 25–28 hours. The pellet fraction contains the largest total amount of PkLDH until 29 hours after synchronization. At an inflection point where more PkLDH is contained in the supernatant, the erythrocytic cycle is completed through the rupturing of the schizonts to create new ring-stage parasites. The overall decrease in PkLDH seen starting at hour 25 may be explained by the lability of PkLDH and consequently an overall drop in PkLDH concentration until the parasites mature again. It should also be noted that the reactivity pattern of PkLDH proteins as a function of PanLDH/PvLDH ratio was maintained at 2.11 ± 0.24 and 2.35 ± 0.24 in both cell pellet and supernatant compartments, respectively, throughout all development stages .This is the first study to demonstrate the dynamic kinetic expression of Plasmodium species investigated and observed in this study across the 5-Plex PfLDH, PvLDH, and PanLDH assays echoes results from previous studies investigating both antibody reactivity to pLDH and RDT reactivity to pLDH.,,,P. ovale, P. malariae, and P. knowlesi, and also to the less-common species known to infect humans, including P. cynomolgi and P. simium. This will be particularly relevant to countries approaching elimination and/or that have a significant relative burden of P. knowlesi.,hrp2/hrp3 deletions in P. falciparum infections and pLDH as the most commonly used target analyte for non-P. falciparum malaria RDTs, understanding the dynamics and cross-reactivity of pLDH between the different human Plasmodium species is essential. Additionally, given the known cross-reactivities between different Plasmodium species, new diagnostic markers for better differentiating Plasmodium species should be explored.The differential binding of pLDH from the different Supplemental materials