Metabolic by-products of mosquito midgut bacteria, Enterobacter cloacae and Serratia marcescens, exhibit potent anti-parasitic effects on Plasmodium falciparum gametocytes in vitro

Metabolic by-products of mosquito midgut bacteria, Enterobacter cloacae and Serratia marcescens, exhibit potent anti-parasitic effects on Plasmodium falciparum gametocytes in vitro | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Metabolic by-products of mosquito midgut bacteria, Enterobacter cloacae and Serratia marcescens, exhibit potent anti-parasitic effects on Plasmodium falciparum gametocytes in vitro Esinam Abla Akorli, Stephanie N.A.S. Addo, Grace Odoom, Emmanuel Osei-Frempong, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6176263/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Malaria remains a significant public health challenge, necessitating innovative strategies to break transmission, such as interrupting successful Plasmodium falciparum development in the vector using microbiota. While this transmission-blocking has been demonstrated experimentally as low ookinete or oocyst intensity in the mosquito, little is known about the effect of these microbes on gametocytes, which are the first stages of the parasite that the mosquito host encounters. We have tested the effect of cell-free spent media from Enterobacter cloacae and Serratia marcescens on gametocytes of Plasmodium falciparum in vitro. The two bacteria were isolated from female Anopheles gambiae collected in Ghana, put into Luria or blood broth (LB or BB) medium and the spent media was harvested at 6 time points during the growth of the bacteria. Results The spent media exhibited strong anti-parasitic effects, significantly reducing parasite intensity compared to Escherichia coli controls. Compared to non-treated gametocytes, there was overall cytotoxic effect from all three bacteria with significant variance influenced by the bacteria species and product time point. Blood broth (BB)-based products achieved 25.7% greater mean parasite suppression than Luria broth (LB)-based products ( p = 0.028) suggesting that the production of the anti-parasitic substances is dependent on the growth conditions of the bacteria. Heat and proteinase K treatments revealed that LB-based anti-parasitic activity was partially protein-dependent and heat-sensitive, whereas BB-based activity remained largely unaffected, suggesting the presence of heat-stable, non-proteinaceous bioactive compounds. Finally, we observed that heat and proteinase K treatment effects varied between E. cloacae and S. marcescens . Conclusions These findings underscore the potential of natural mosquito midgut bacteria and their metabolic by-products in malaria intervention strategies. By demonstrating parasite suppression in an in vitro system independent of mosquito immune responses, our study suggests that bacterial-derived metabolites could be harnessed to disrupt parasite development in non-colonized mosquito populations. Future research should focus on identifying the specific bioactive compounds responsible for parasite reduction and assessing their feasibility for field application in malaria control programs. mosquito midgut microbiota Enterobacter cloacae Serratia marcescens Anopheles gambiae disease transmission blocking gametocytes Figures Figure 1 Figure 2 Figure 3 Figure 4 Background The global disease case and mortality rates for malaria have shown unprecedented upsurges following years of decline, challenging the prospects of achieving the goals set for 2030 [ 1 , 2 ]. The effectiveness of available insecticides and chemotherapies are threatened by vector and parasite resistance, respectively [ 3 , 4 ]. New tools at various stages of development and implementation have been proposed to address resistance especially in the control of the vector [ 5 ]. Rising concerns about residual malaria transmission [ 6 – 8 ] and the shift to outdoor biting in some of the major Anopheline vectors [ 9 – 11 ] suggest more integrated approaches which target different angles of the transmission cycle are needed. Disease transmission blocking, a strategy that prevents mosquito-to-human transmission, would effectively target parasites that escape drug therapy and those that are ingested by insecticide-resistant mosquitoes. The various developmental stages presented in the mosquito stages of the parasite offers several target points for disrupting development and breaking transmission [ 12 – 15 ]. The mosquito midgut is a very crucial environment in the development and transmission intensity of Plasmodium parasites. Gametocytes are the first stages of the parasite to interact with the vector. Macro- and micro-gametocytes fuse to form gametes which traverse the mosquito midgut to become ookinetes and then to oocysts which rupture after several hours to release sporozoites[ 14 ]. Bacteria resident within the mosquito midgut are involved in the mosquito-parasite interaction, where they influence ookinete and/or oocyst development [ 16 – 18 ]. While this is indeed promising for blocking transmission, it is yet to be established at which parasite stage these detrimental effects are initiated. For example, it has been shown that the number of ookinetes were higher in aseptic An. gambiae than those with intact microbiota following infection with P . falciparum suggesting that the anti-parasitic of effect of bacteria occurs before this stage [ 16 ]. This implies that the anti-Plasmodium action could as well occur at any point when the gametocytes entered the midgut to when they became motile ookinetes. Gametocytes are comparatively short-lived (~ 15 mins) in the host lumen and become ookinetes after about18 hours after the mosquito has taken an infected blood meal [ 14 ]. Therefore, their assessment of the bacterial effects is difficult to evaluate and often ignored. However, Plasmodium in vitro cultures provide suitable ways to study firsthand effects of treatment on gametocytes while using SYBR I Green assay to assess their survival [ 19 – 21 ]. The Anopheles mosquito midgut is dominated by Gram-negative bacteria [ 22 ]. The exceptional increase in Enterobacteriaceae after blood meals [ 23 , 24 ] and the demonstration of their anti-parasitic effects [ 17 , 25 – 29 ] is of particular interest to many laboratories that study mosquito microbiota for disease control. Enterobacter and Serratia are two key members of bacterial family Enterobacteriaceae that produce anti-parasitic factors that result in reduced parasite intensity [ 17 , 25 , 27 – 29 ]. While the microbial mode of action has been shown to essentially involve the mosquito immune system [ 25 , 29 , 30 ], there is growing evidence that there is also a direct interaction between bacteria and the parasite through the production of metabolites which have anti-plasmodial activities [ 28 , 31 ] and the formation of epithelial barrier that prevents the traverse of ookinetes across the mosquito midgut [ 32 ]. E. cloacae is known to release reactive oxygen species that directly affect the parasites [ 17 ], while Serratia spp. Show variation in their antagonistic modes of action towards Plasmodium [ 25 , 28 , 33 ]. Therefore, while both belong to the same bacterial family and proliferate in the midgut after the host takes a blood meal, they are likely to be affecting the parasite through different molecular processes and pathways, and at distinctive stages of the parasite life cycle. In this study, we assessed the effect of E. cloacae and S. marcescens metabolic by-products on P. falciparum gametocytes using an in vitro treatment assay. This allowed the observation of direct, non-immune effect of the by-products on this stage of the parasite. Methods Preparation of bacterial metabolic by-products Pure isolates of E. cloacae and S. marcescens previously obtained from the midgut of field-derived Anopheles mosquitoes [ 34 ] were revived from ultra-low storage by plating on sterile LB agar. The bacteria cells were inoculated into Luria-Bertani broth (LB) or Tryptic Soy Broth supplemented with 5% sheep (simply referred to as a ‘blood-base’ (BB) broth) and incubated overnight at 37ºC. The OD 600nm of the overnight culture was estimated with a UV-vis spectrophotometer (Varioskan Lux, Thermo Scientific) and set at an initial (stock) OD of 0.5–0.55 for each bacterium. 49 mL of fresh sterile broth (LB or BB) was prepared into tubes labelled with time points 0.5, 1, 2, 4, 6 and 12 hours, and 1 mL of the stock bacterial suspension was transferred into each tube. A control tube was prepared with 50 mL of broth without any inoculum. All tubes were incubated at 37ºC. After each designated time point, the respective tubes were removed from the incubator and centrifuged to separate the cells from the supernatant. The supernatants were ultra-frozen overnight and lyophilised by freeze-drying. The dried products were aliquoted into 2 mL cryovials and stored at -20°C until used. By-products from two isolates each of E. cloacae and S. marcescens were prepared in LB and BB as described. Escherichia coli (Migula) Castellani and Chalmers (ATCC ref#: 25922) was used as the control bacterium for the LB-based by-products only. Gametocyte treatment assays Plasmodium falciparum 3D7 was cultured under standard conditions using established protocols [ 35 ]. Gametocytes were obtained by doubling the volume of the culture medium on the 5th day after the parasites had been put into culture, stressing the parasites to switch to the gametocyte stage [ 36 , 37 ]. Thin blood smears were prepared on days 14 and 17, stained with Giemsa and viewed under a light microscope to confirm the presence of gametocytes. Gametocytaemia for the assay ranged between 1.3–1.7% and 2% haematocrit. The parasites were transferred in triplicates of 50 µL into a 96-well cell culture plate. The lyophilised LB- and BB-base by-products for E. cloacae , S. marcescens and E. coli (LB-base only), and the medium control (LB and BB with no inoculum) were reconstituted at a stock concentration of 50 mg/mL. Working concentrations of 3 mg/mL were prepared by diluting with 1× Phosphate Buffered Saline (PBS) and filtered through 0.22µ PES filters. 50 µL was added to the parasites in the wells. The control well was set-up with infected RBCs (iRBCs) in complete parasite medium at 2% haematocrit, with no by-product added. The plate was gassed and incubated at 37°C for 48 hours in a modular incubator chamber (Billups-Rothenberg, Inc.). SYBR Green-1 fluorescent dye was used to estimate the survival of Plasmodium gametocytes following treatment with bacterial by-products. 100 µL of SYBR Green-1 fluorescent (MSF) buffer (5% v/v) was added to each well and mixed by pipetting up and down. The plates were then wrapped with aluminium foil and incubated in the dark at room temperature for 24 hours to ensure optimal binding of the dye [ 38 , 39 ]. The SYBR Green fluorescence intensities were measured with a Varioskan LUX multimode microplate reader (ThermoFisher Scientific, USA) at excitation and emission wavelengths of 485nm and 530nm, respectively. Assessing effects of heat and proteinase treatment on by-products We investigated whether heat or proteinase K treatment of the bacterial by-products affects the survival of the Plasmodium . The experiment focused on LB- and BB-based by-products for the mosquito midgut bacteria, E. cloacae and S. marcescens . 50 µL of reconstituted by-products at 50 mg/mL was aliquoted into two equal volumes. One part was heat-treated at 56°C overnight. The second part was proteinase K-treated by adding 5 µL of 20mg/mL of proteinase K (Qiagen), heated at 56°C overnight and, the enzyme reaction was stopped at 72°C. Untreated bacterial by-products were used as control. All treatments were filtered, prepared to a working concentration and used in the gametocyte assay as previously described. Data analyses Raw data was obtained as absorbances. The average absorbance from wells treated with bacterial products were blanked by subtracting the average background absorbance of the non-bacterial broth (LB or BB) medium. Percentage parasite suppression was then calculated from the ratio of the difference in estimated parasite remaining after the treatment (untreated iRBCs - normalized treated iRBCs) to the control (untreated iRBCs). The parasite, time point, and bacteria genus were fitted as interactive independent variables in generalized linear models to determine their influence on parasite suppression. Where appropriate, a linear mixed effect (LMM) model was fitted to test the bacterial strain as a random effect variable. In such cases, the best fit model was estimated by comparing models with the Akaike Information Criterion (AIC). Pairwise comparisons of means between the independent factors were estimated using estimated marginal means ( emmeans ), accepting as significant adjusted p -values < 0.05. All analyses and plots were generated with custom R scripts. Results Effect of LB and BB-based bacterial by-products on Plasmodium gametocytes By-products from all the bacteria used in this study exhibited cytotoxicity on P. falciparum gametocytes. Variance in parasite suppression after exposure to the bacterial products was significantly influenced by the bacteria from which the product was obtained (F (2, 12) = 192.22, p < 0.001) and the product time point (F (5, 12) = 47.36, p < 0.001). In addition, the interaction between bacteria and product time point (F (10, 12) = 64.44, p < 0.001) was also significant indicating that the effect of bacterial product on the parasite varied across different time points (Table 1). The overall mean parasite suppression was higher with products from E. cloacae (79.7%) and S. marcescens (82.6%) than E. coli (45.9%) ( p = 7.69e-10) (Fig. 1 , Additional file 1). The time point effects of the bacterial products remained similar between E. cloacae and S. marcescens , and the highest difference between these and E. coli occurred for the 6-hour by-products (Fig. 1 , Additional file 1). At some time points, the parasite suppression was above 100% (Fig. 1 ). This resulted from a higher absorbance recorded for the broth medium control than for the treated samples. Negative normalised absorbance resulted in higher calculated parasite suppression relative to the untreated iRBCs i.e ([untreated iRBCs - normalized treated iRBCs]/ [untreated iRBCs] is > 1). The mean parasite suppression resulting from treatment with blood broth (BB) based products was 21.07 units higher than with LB-based products (Additional file 2), corresponding to a 25.7% increase (t = 2.289, p = 0.028). In the model that was fit to compare the two products, the time point was a major determinant of parasite suppression (F (5,36) = 31.82, p < 0.0001) and demonstrated significant differences between the two products at 0.5, 1 and 12hrs (Additional file 2). Parasite suppression was higher for blood-based products compared to LB medium at the earlier time points but was lower at 12 hours (Fig. 2 ). Effect of heat and proteinase K treatment on bacterial by-products A linear mixed effect model (LMM) best estimated the factors associated with parasite suppression given the treatment of the bacterial by-products with heat or proteinase K (pk) before adding then to the gametocyte culture (Additional file 3). For both LB and BB-based products, the treatment (normal, heat-treated or proteinase K treated) and product time point significantly affected the parasite, and the treatment also varied over time (treatment * time point) ( p < 0.01) (Table 3 ). With heat-treated LB-based by-products, there was decreased parasite suppression compared to the normal products, while similarly treated BB-based by-products did not result in a significant change in parasite suppression compared to unheated (normal) products (Fig. 3 ). In both LB and BB-based products, proteinase K treatment did not affect the overall mean parasite r suppression. However, the effect of proteinase K was dependent on the product time point (Table 3 , Fig. 3 ) indicating that the product at specific time points had protein components that contributed to the deleterious effect on the parasites. Proteinase K treatment resulted in decreased activity of 4 and 6-hour LB-based products (Fig. 3 ). Table 3 Analysis of variance table for LB and BB evaluating the effects of treatment, time, bacteria (bac), and their interactions on the paraste suppression. Linear mixed model= [p_sup ~ ttment * time * bac + (1|strain)]. Significant factors and their P-values are in bold. Sum Sq NumDF DenDF F values P -value LB treatment 7839.3 2 40.693 15.926 7.79e-06 time point 4519.6 5 39.596 3.6728 0.0080 bac 901.7 1 2.858 3.6638 0.156 treatment*time 16282.6 10 39.596 6.6158 6.31e-06 treatment*bac 957.3 2 40.693 1.9449 0.156 time*bac 1551.6 5 39.596 1.2608 0.300 treatment*time*bac 799.2 10 39.596 0.3247 0.970 BB treatment 3733.5 2 36 12.5829 7.18e-05 time point 9904.6 5 36 13.3527 2.19e-07 bac 2.2 1 36 0.015 0.903 treatment*time 17135.7 10 36 11.5505 1.54e-08 treatment*bac 26.4 2 36 0.0891 0.915 time*bac 924.3 5 36 1.2461 0.308 treatment*time*bac 1344.1 10 36 0.906 0.538 Sum Sq (Sum of Squares): Measures the variation explained by each factor. NumDF (Numerator Degrees of Freedom): Number of levels in the factor. DenDF (Denominator Degrees of Freedom): Residual degrees of freedom. F-value = Ratio of explained to unexplained variance The two bacterial products were analysed to assess the impact of heat and proteinase K treatment on the effect they had along the time points. E. cloacae LB-based products were most affected by heat at earlier time points of 1 and 2 hours, demonstrating decreased effect on parasite survival. Proteinase K treatment of 1-hour LB-based products, however, led to increased parasite suppression compared to untreated products at the same time point (Fig. 4 A). In contrast, S. marcescens LB-based products were affected by heat at 4 and 12 hours, and by proteinase K at 12 hours (Fig. 4 B). Parasites treated with heated and proteinase-treated E. cloacae 12-hour BB-based products showed higher suppression, but S. marcescens BB-based products were affected only by heat at 0.5 and 1-hour timepoints. This is suggestive of varied heat-sensitive and protein-based bioactive components at different time points during the culturing of E. cloacae and S. marcescens bacteria in LB or BB. Discussion This study highlights the potential for using the by-products from midgut bacteria to reduce Plasmodium falciparum . Parasite numbers significantly decreased in the presence of the cell-free spent media from E. cloacae and S. marcescens , two members of Enterobacteriaceae which have shown promise for parasite transmission-blocking [ 40 , 41 ]. While parasite cultures treated with E. coli products also showed reduced parasite numbers, the effect from the two midgut bacteria were more than 100% higher, indicating a stronger detrimental potential. These results support the use of natural microbiota for malaria intervention and uniquely highlight the potential of bioactive products from midgut bacteria as a novel strategy to reduce Plasmodium falciparum as early as the gametocyte stage, a critical yet underexplored target stage for malaria transmission-blocking. Compared to other studies [ 27 , 28 , 33 ], our in vitro approach allowed the evaluation of the antagonistic effect of the bacteria by-product outside of the mosquito host immune system. This demonstrates that parasite-blocking could be achieved in mosquitoes that do not naturally harbour these two bacteria in the midguts. However, whether such a scenario would lead to the same outcome as we have previously shown [ 27 ], requires further investigation. Our current results show that the bioactive substances that are released during culture of E. cloacae , S. marcescens and E. coli in regular Luria broth medium at 37℃ are toxic towards P. falciparum in a manner that is reflective of their growth dynamics (Additional file 4). The negative effect of the product on the parasite viability increased with product time point from lag (0.5-1hr) through the exponential (2-6hrs) phase and reduced thereafter (12hrs). The production of bacterial metabolites is generally known to be minimal during the lag phase where bacteria adjust to their environment [ 42 , 43 ], typically lasting between 1–6 hours depending on the bacteria and growth conditions. Despite this, the LB-base products from our lowest measured time point (0.5 hours) for E. cloacae and S. marcescens resulted in > 70% parasite suppression compared to E. coli , which is nonetheless a strong significant effect. For the two midgut bacteria investigated in this study there was a slight decrease in cell numbers at 1hour compared to 0.5 hour which also resulted in a dip in parasite suppression. The exponential phase, which could start between 1–2 hours under optimum conditions results in increased bacterial replication and production of several biological substances including amino acids, enzymes and organic acids that the bacteria require to proliferate [ 43 ]. The increased parasite suppression matched this growth phase and peaked at 6hours. Growing bacteria numbers therefore appears to have resulted in increased production of anti-parasitic by-products. The parasite suppression profile changes with blood-based products demonstrating the influence of growth conditions on the production of bacterial by-products [ 44 ]. The parasite suppression was observed above 100% even at 0.5 hours for BB-based products. Enterobacteriaceae are well-known for their haemolytic and haemagglutination activities, but the combination of these activities varies by species and strains [ 45 ]. We observed firsthand the lysis of blood during the culture of S. marcescens in the blood-based broth (Additional file 5). The haemolytic substances may have persisted after harvesting and processing the spent media and continued to lyse the blood cells in the Plasmodium culture. Haemoglobin released from lysed red blood cells can absorb weakly at the wavelengths used [ 46 ], and contribute with to the final absorbance read. This would explain the > 100% suppression observed resulting from higher absorbance compared to medium control, when the parasites were treated with BB products. Some clinical isolates of E. cloacae are known to also produce ⍺-haemolysin [ 47 ]. The parasite suppression profile did not differ between the two bacteria genera and considering that the two bacteria were isolated from the mosquito midgut where they encounter blood, it is plausible that these E. cloacae strains also have haemolytic properties. The use of heat and proteinase K treatment allowed basic insights into the properties of the bioactive by-products. The effect of proteinase K on LB-based products was more associated with time than the heat treatment. Proteinase reduced the efficacy of product time point 4 and 6hours, suggesting that the bioactive products at these time points could involve proteins. Heating the by-products, however, appeared to have provided a more conducive medium for parasite survival, indicative of heat-sensitive bioactive compounds in the LB-base by-products. LB medium when autoclaved has increased protein and antioxidant levels which can resist high temperatures [ 48 ]. However, as bacteria use up these components, they release metabolic by-products such as acids and, secrete proteins (e.g. toxins) and extracellular molecules, depending on the bacteria species [ 48 , 49 ]. The BB-based products were much less affected by the treatments suggestive of anti-parasitic components that are heat-stable and less proteinaceous. The two bacteria showed striking patterns in the comparison of their heat and proteinase K-treated products to the non-treated, which suggested differential compositions. The 1- and 2-hours LB-based products of E. cloacae were more heat-labile, and removing proteins increased the antagonistic property suggesting that the presence of proteins here may have interrupted with some of the bioactive components. S. marcescens had these characteristics at later time points from 4 hours. The spent media used in this study warrant detailed investigations into their compositions, antagonistic properties and modes of action towards the parasite. Conclusions This study demonstrates the potential of bacterial by-products from E. cloacae and S. marcescens as promising agents for reducing Plasmodium falciparum at the gametocyte stage, a critical target for malaria transmission-blocking. Our findings reveal that the anti-parasitic effects of these midgut bacteria-derived spent media are time-dependent and strongly associated with bacterial growth phases, with peak parasite suppression occurring during the exponential phase. The influence of culture conditions, particularly the differences between Luria broth and blood-based media, underscores the variability in bioactive compound production. The haemolytic activity observed in the blood-based cultures suggests that these bacterial strains may produce haemolysins or other cytotoxic compounds that contribute to parasite suppression. Additionally, our heat and proteinase K treatments suggest that the active components include both heat-sensitive and heat-stable molecules, with differential protein involvement between the two bacterial species. Overall, our study supports the growing interest in microbiota-based malaria interventions and highlights the need for further investigations into the specific bioactive compounds responsible for the observed anti-parasitic effects. Future research should focus on characterizing these bacterial metabolites, their mechanisms of action, and their potential application in vector control strategies to disrupt malaria transmission. Declarations Ethics approval and consent to participate Not applicable Consent for publication Not applicable Clinical Trial Number Not applicable Availability of data and materials All data generated or analysed during this study are included in this published article and its supplementary information files. Funding This study was funded by the Wellcome Trust Intermediate Fellowship [Grant number: 220737/Z/20/Z] to JA. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Author’s contributions JA conceptualized the study. JA, EAA designed the experiments. EAA, SNASA, GO, EO-F, JKB performed the experiments, EAA, SNASA, JA analysed the data and drafted the manuscript. All the authors have read and approved the final manuscript. Acknowledgement We acknowledge the advisory role played by Dr. Lisa Ranford-Cartwright (University of Glasgow) in the execution of this study. References World Health Organization. World Malaria Report 2024. Geneva: World Health Organization; 2024. Health Organization W. Global Technical Strategy for Malaria 2016-2030. World Health Organization. 2015. 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Nat Commun. 2015;6: 5921. doi:10.1038/ncomms6921 Akorli EA, Ubiaru PC, Pradhan S, Akorli J, Ranford-Cartwright L. Bio-products from Serratia marcescens isolated from Ghanaian Anopheles gambiae reduce Plasmodium falciparum burden in vector mosquitoes. Frontiers in Tropical Diseases. 2022;3. doi:10.3389/fitd.2022.979615 Bando H, Okado K, Guelbeogo WM, Badolo A, Aonuma H, Nelson B, et al. Intra-specific diversity of Serratia marcescens in Anopheles mosquito midgut defines Plasmodium transmission capacity. Sci Rep. 2013;3: 1641. doi:10.1038/srep01641 Eappen AG, Smith RC, Jacobs-lorena M. Enterobacter-Activated Mosquito Immune Responses to Plasmodium Involve Activation of SRPN6 in Anopheles stephensi. PLoS One. 2013;8: 1–8. doi:10.1371/journal.pone.0062937 Cirimotich CM, Dong Y, Garver LS, Sim S, Dimopoulos G. Mosquito immune defenses against Plasmodium infection. Dev Comp Immunol. 2010;34: 387–395. doi:10.1016/j.dci.2009.12.005 Bahia AC, Dong Y, Blumberg BJ, Mlambo G, Tripathi A, BenMarzouk-Hidalgo OJ, et al. Exploring Anopheles gut bacteria for Plasmodium blocking activity. Environ Microbiol. 2014;16: 2980–2994. doi:10.1111/1462-2920.12381 Rodgers FH, Gendrin M, Wyer CAS, Christophides GK. Microbiota-induced peritrophic matrix regulates midgut homeostasis and prevents systemic infection of malaria vector mosquitoes. Dimopoulos G, editor. PLoS Pathog. 2017;13: e1006391. doi:10.1371/journal.ppat.1006391 Gao H, Bai L, Jiang Y, Huang W, Wang L, Li S, et al. A natural symbiotic bacterium drives mosquito refractoriness to Plasmodium infection via secretion of an antimalarial lipase. Nat Microbiol. 2021. doi:10.1038/s41564-021-00899-8 Ezemuoka LC, Akorli EA, Aboagye-antwi F, Akorli J. Mosquito midgut Enterobacter cloacae and Serratia marcescens affect the fitness of adult female Anopheles gambiae s . l . PLoS One. 2020;15: e0238931. doi:10.1371/journal.pone.0238931 Moll K, Ljungström I, Perlmann H, Scherf A, Wahlgren M. Methods in Malaria Research. Manassas, Virginia; 2013. Ranford-Cartwright LC, Sinha A, Humphreys GS, Mwangi JM. New synchronization method for Plasmodium falciparum. Malar J. 2010;9: 1–5. doi:10.1186/1475-2875-9-170/COMMENTS Omorou R, Bin Sa’id I, Delves M, Severini C, Kouakou YI, Bienvenu AL, et al. Protocols for Plasmodium gametocyte production in vitro: an integrative review and analysis. Parasit Vectors. 2022;15: 1–12. doi:10.1186/S13071-022-05566-3/FIGURES/2 Dery V, Duah NO, Ayanful-Torgby R, Matrevi SA, Anto F, Quashie NB. An improved SYBR Green-1-based fluorescence method for the routine monitoring of Plasmodium falciparum resistance to anti-malarial drugs. Malar J. 2015;14: 1–6. doi:10.1186/S12936-015-1011-X/FIGURES/3 Cheruiyot AC, Auschwitz JM, Lee PJ, Yeda RA, Okello CO, Leed SE, et al. Assessment of the Worldwide Antimalarial Resistance Network Standardized Procedure for In Vitro Malaria Drug Sensitivity Testing Using SYBR Green Assay for Field Samples with Various Initial Parasitemia Levels. Antimicrob Agents Chemother. 2016;60: 2417. doi:10.1128/AAC.00527-15 Wilke ABB, Marrelli MT. Paratransgenesis: a promising new strategy for mosquito vector control. Parasit Vectors. 2015;8: 342. doi:10.1186/s13071-015-0959-2 Gendrin M, Christophides GK. The Anopheles Mosquito Microbiota and Their Impact on Pathogen Transmission. In: Manguin S, editor. Anopheles mosquitoes - New insights into malaria vectors. InTech; 2013. pp. 525–548. doi:10.5772/3392 Madigan MT, Martinko JM, Bender KS, Buckley DH, Stahl DA. Brock Biology of Microorganisms. 14th ed. Pearson; 2014. Rolfe MD, Rice CJ, Lucchini S, Pin C, Thompson A, Cameron ADS, et al. Lag Phase Is a Distinct Growth Phase That Prepares Bacteria for Exponential Growth and Involves Transient Metal Accumulation. 2011 [cited 15 Feb 2025]. doi:10.1128/JB.06112-11 Gonzalez JM, Aranda B. Microbial Growth under Limiting Conditions-Future Perspectives. Microorganisms. 2023;11: 1641. doi:10.3390/MICROORGANISMS11071641 Roland FP. Interaction of Blood with Enterobacteriaceae Hemolysis, Hemagglutination, Fibrinolysis. Am J Clin Pathol. 1977;67: 260–263. Available: http://ajcp.oxfordjournals.org/ Townsend D, D’Aiuto F, Deanfield J. Journal of Medical and Biological Engineering. J Med Biol Eng. 2014;34: 172–177. doi:10.5405/jmbe.1643 Prada J, Beutin L. Detection of Escherichia coli α-haemolysin genes and their expression in a human faecal strain of Enterobacter cloacae. FEMS Microbiol Lett. 1991;79: 111–114. doi:10.1111/j.1574-6968.1991.tb04514.x Wang H, Guo J, Chen X, He H. The Metabolomics Changes in Luria–Bertani Broth Medium under Different Sterilization Methods and Their Effects on Bacillus Growth. Metabolites. 2023;13: 958. doi:10.3390/METABO13080958 Maffei B, Francetic O, Subtil A. Tracking proteins secreted by bacteria: What’s in the toolbox? Front Cell Infect Microbiol. 2017;7: 269489. doi:10.3389/FCIMB.2017.00221/PDF Tables Tables 1 and 2 are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Additionalfile1.xlsx Additional file 1: Results from multiple means comparison for linear regression to test the association of parasite suppression with bacteria and by-product time point. The fitted model had the formula [p_sup~ bacteria * timepoint] where p_sup is the calculated parasite suppression. Additionalfile2.xlsx Additional file 2: Results linear regression to test the association of parasite suppression with LB- and BB- base by-products and time point. The fitted model had the formula [p_sup~ treatment * timepoint] where p_sup is the calculated parasite suppression. Additionalfile3.xlsx Additional file 3: Model comparison for testing effects of heat and proteinase-K treatments on the cytotoxic properties of bacterial by-products on gametocytes. The models were either linear with the treatment, time point and strain as interaction terms, or linear mixed effect with all the terms in the linear model but with strain and or bacteria as random effect terms. The ICC indicates the proportion of variance explained by the random effect variable. Additionalfile4.pdf Additional file 4: Growth plot of bacteria in Luria-Bertani (LB) and blood-base broth. Table1and2.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6176263","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":427394766,"identity":"afa9e881-ffd8-4991-a679-222db73b9b3f","order_by":0,"name":"Esinam Abla Akorli","email":"","orcid":"","institution":"Noguchi Memorial Institute for Medical Research","correspondingAuthor":false,"prefix":"","firstName":"Esinam","middleName":"Abla","lastName":"Akorli","suffix":""},{"id":427394767,"identity":"03600b3e-a20f-41f6-bef9-b87c0d663fd8","order_by":1,"name":"Stephanie N.A.S. Addo","email":"","orcid":"","institution":"Noguchi Memorial Institute for Medical Research","correspondingAuthor":false,"prefix":"","firstName":"Stephanie","middleName":"N.A.S.","lastName":"Addo","suffix":""},{"id":427394768,"identity":"fd8343c4-330b-41e5-8577-45b8a9724d50","order_by":2,"name":"Grace Odoom","email":"","orcid":"","institution":"Noguchi Memorial Institute for Medical Research","correspondingAuthor":false,"prefix":"","firstName":"Grace","middleName":"","lastName":"Odoom","suffix":""},{"id":427394769,"identity":"0008f129-f3a2-4aa2-a5d6-d55bafffa819","order_by":3,"name":"Emmanuel Osei-Frempong","email":"","orcid":"","institution":"Noguchi Memorial Institute for Medical Research","correspondingAuthor":false,"prefix":"","firstName":"Emmanuel","middleName":"","lastName":"Osei-Frempong","suffix":""},{"id":427394770,"identity":"e6384502-ef48-4097-8867-866d3f4eb247","order_by":4,"name":"Jeffrey K. Boateng","email":"","orcid":"","institution":"Noguchi Memorial Institute for Medical Research","correspondingAuthor":false,"prefix":"","firstName":"Jeffrey","middleName":"K.","lastName":"Boateng","suffix":""},{"id":427394771,"identity":"b52516e8-5fce-47ee-a8ef-a48fa410d15c","order_by":5,"name":"Jewelna Akorli","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABDUlEQVRIiWNgGAWjYBACxgY2IFkgIQflSUCEeYDYAK8WAwlj4rUwMIC1MCQ2QLQwENbC3H4s8XGFgUX6dvb2axI/d1gwGJxfwPjgbRuD3XZcDutJO2x4xkAid2fPmTLJ3jMSDAY3HjAbzm1jSN7ZgENLQ3qbZANQy4YbOWnSjG0gLQfYpHmBWgwO4NDS/xysJd0ASQv7b7xaZqQdA2lJMLiRfgyi5XwDGzNQix1uLc+SDYFaDDecOcNs2dsmwSN5g7FZcs45oCE4tBj2pxk+bKiokzc43v7wxs+2Ojm+84cPfnhTZmOPUwsiWHjAEcHDIAGOIwiJDcgjmOwPIDQ/xHR7HDpGwSgYBaNg5AEA9otbqs69WhwAAAAASUVORK5CYII=","orcid":"","institution":"Noguchi Memorial Institute for Medical Research","correspondingAuthor":true,"prefix":"","firstName":"Jewelna","middleName":"","lastName":"Akorli","suffix":""}],"badges":[],"createdAt":"2025-03-07 08:08:33","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6176263/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6176263/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":78324700,"identity":"7a56e3ba-5eb6-4c7f-9b6c-5def8dddb0c9","added_by":"auto","created_at":"2025-03-12 06:06:37","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":42737,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eParasite suppression effect of Luria broth (LB)-based bacterial culture products. \u003c/strong\u003eThe estimated parasite suppression (mortality) was calculated from the ratio of the difference between absorbance values from untreated infected samples (experimental control) and normalized (with LB only medium control) by-product treated samples, to the experimental control.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6176263/v1/2c5c2d7757217217d9ed0337.jpg"},{"id":78324701,"identity":"4bc184a9-ded4-4591-aeae-676d00d35470","added_by":"auto","created_at":"2025-03-12 06:06:37","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":35938,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparison of the parasite suppression effect of LB- and BB-based by-products from midgut bacteria. \u003c/strong\u003eSince the \u003cem\u003eE. cloacae\u003c/em\u003e and \u003cem\u003eS. marcescens\u003c/em\u003e were similar (Fig 1), their calculated estimates were averaged and plotted for each medium\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6176263/v1/cc132e7a82750cd7877d4e01.jpg"},{"id":78324703,"identity":"cf521686-645d-4b5c-8f7f-8fdbb927f1e3","added_by":"auto","created_at":"2025-03-12 06:06:37","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":79358,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eModel estimates for the effect of heat and proteinase K treatments of LB- and BB-based by-products on gametocytes.\u003c/strong\u003e Each dot represents the estimated change in the parasite suppression relative to the reference factor, and error bars are upper and lower limit 95% confidence intervals. Significant factors are in red.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6176263/v1/b5c05be2c2b21abee302b693.jpg"},{"id":78325369,"identity":"d384add9-93e5-4abe-969f-40ff4c7966c6","added_by":"auto","created_at":"2025-03-12 06:14:38","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":141981,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparison of parasite suppression for heated and proteinase K treated \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eE. cloacae\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eS. marcescens\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e by-products. \u003c/strong\u003eMeans were compared with pairwise t-test and asterisks show significant \u003cem\u003eP\u003c/em\u003e-values. Error bars represent standard error of mean (SEM).\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6176263/v1/0a5cfc5322915e4f8c88ec1a.jpg"},{"id":95185867,"identity":"b37fbe77-a47d-49f1-8ec1-0f7524440f04","added_by":"auto","created_at":"2025-11-05 09:09:25","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1275033,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6176263/v1/285105b4-4383-4921-9cd0-3b4ffe68a6c7.pdf"},{"id":78325363,"identity":"b4a6fb0c-eadf-419e-87d8-63de2905a16f","added_by":"auto","created_at":"2025-03-12 06:14:37","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":10524,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file 1: Results from multiple means comparison for linear regression to test the association of parasite suppression with bacteria and by-product time point. \u003c/strong\u003eThe fitted model had the formula\u003cstrong\u003e \u003c/strong\u003e[p_sup~ bacteria * timepoint]\u003cstrong\u003e \u003c/strong\u003ewhere p_sup is the calculated parasite suppression.\u003c/p\u003e","description":"","filename":"Additionalfile1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6176263/v1/190288ac759267774f20abf6.xlsx"},{"id":78325718,"identity":"c4ee2348-4080-44db-b694-6a39c4ee0049","added_by":"auto","created_at":"2025-03-12 06:22:38","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":10543,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file 2: Results linear regression to test the association of parasite suppression with LB- and BB- base by-products and time point. \u003c/strong\u003eThe fitted model had the formula\u003cstrong\u003e \u003c/strong\u003e[p_sup~ treatment * timepoint]\u003cstrong\u003e \u003c/strong\u003ewhere p_sup is the calculated parasite suppression.\u003c/p\u003e","description":"","filename":"Additionalfile2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6176263/v1/bce00b5283b491560b03020a.xlsx"},{"id":78324705,"identity":"1fa95e31-db4f-44f8-94fa-cdd1b245e7e8","added_by":"auto","created_at":"2025-03-12 06:06:37","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":14331,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file 3: Model comparison for testing effects of heat and proteinase-K treatments on the cytotoxic properties of bacterial by-products on gametocytes. \u003c/strong\u003eThe models were either linear with the treatment, time point and strain as interaction terms, or linear mixed effect with all the terms in the linear model but with strain and or bacteria as random effect terms. The ICC indicates the proportion of variance explained by the random effect variable.\u003c/p\u003e","description":"","filename":"Additionalfile3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6176263/v1/e26f1b990e9cd38187039e08.xlsx"},{"id":78324724,"identity":"b4370548-0042-4996-9e38-346ca3313527","added_by":"auto","created_at":"2025-03-12 06:06:38","extension":"pdf","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":52108,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file 4: Growth plot of bacteria in Luria-Bertani (LB) and blood-base broth.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Additionalfile4.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6176263/v1/5feaf0ed2c1eb5cc60f671ee.pdf"},{"id":78325377,"identity":"cd88e0b5-e51a-4762-878a-a42da10c01ca","added_by":"auto","created_at":"2025-03-12 06:14:38","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":14451,"visible":true,"origin":"","legend":"","description":"","filename":"Table1and2.docx","url":"https://assets-eu.researchsquare.com/files/rs-6176263/v1/0e3d95da0a8c08ca64eb4d8e.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Metabolic by-products of mosquito midgut bacteria, Enterobacter cloacae and Serratia marcescens, exhibit potent anti-parasitic effects on Plasmodium falciparum gametocytes in vitro","fulltext":[{"header":"Background","content":"\u003cp\u003eThe global disease case and mortality rates for malaria have shown unprecedented upsurges following years of decline, challenging the prospects of achieving the goals set for 2030 [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The effectiveness of available insecticides and chemotherapies are threatened by vector and parasite resistance, respectively [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. New tools at various stages of development and implementation have been proposed to address resistance especially in the control of the vector [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Rising concerns about residual malaria transmission [\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] and the shift to outdoor biting in some of the major Anopheline vectors [\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] suggest more integrated approaches which target different angles of the transmission cycle are needed. Disease transmission blocking, a strategy that prevents mosquito-to-human transmission, would effectively target parasites that escape drug therapy and those that are ingested by insecticide-resistant mosquitoes. The various developmental stages presented in the mosquito stages of the parasite offers several target points for disrupting development and breaking transmission [\u003cspan additionalcitationids=\"CR13 CR14\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe mosquito midgut is a very crucial environment in the development and transmission intensity of \u003cem\u003ePlasmodium\u003c/em\u003e parasites. Gametocytes are the first stages of the parasite to interact with the vector. Macro- and micro-gametocytes fuse to form gametes which traverse the mosquito midgut to become ookinetes and then to oocysts which rupture after several hours to release sporozoites[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Bacteria resident within the mosquito midgut are involved in the mosquito-parasite interaction, where they influence ookinete and/or oocyst development [\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. While this is indeed promising for blocking transmission, it is yet to be established at which parasite stage these detrimental effects are initiated. For example, it has been shown that the number of ookinetes were higher in aseptic \u003cem\u003eAn. gambiae\u003c/em\u003e than those with intact microbiota following infection with \u003cem\u003eP\u003c/em\u003e. \u003cem\u003efalciparum\u003c/em\u003e suggesting that the anti-parasitic of effect of bacteria occurs before this stage [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. This implies that the anti-Plasmodium action could as well occur at any point when the gametocytes entered the midgut to when they became motile ookinetes. Gametocytes are comparatively short-lived (~\u0026thinsp;15 mins) in the host lumen and become ookinetes after about18 hours after the mosquito has taken an infected blood meal [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Therefore, their assessment of the bacterial effects is difficult to evaluate and often ignored. However, \u003cem\u003ePlasmodium\u003c/em\u003e in vitro cultures provide suitable ways to study firsthand effects of treatment on gametocytes while using SYBR I Green assay to assess their survival [\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eAnopheles\u003c/em\u003e mosquito midgut is dominated by Gram-negative bacteria [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The exceptional increase in Enterobacteriaceae after blood meals [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] and the demonstration of their anti-parasitic effects [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan additionalcitationids=\"CR26 CR27 CR28\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] is of particular interest to many laboratories that study mosquito microbiota for disease control. \u003cem\u003eEnterobacter\u003c/em\u003e and \u003cem\u003eSerratia\u003c/em\u003e are two key members of bacterial family Enterobacteriaceae that produce anti-parasitic factors that result in reduced parasite intensity [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. While the microbial mode of action has been shown to essentially involve the mosquito immune system [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], there is growing evidence that there is also a direct interaction between bacteria and the parasite through the production of metabolites which have anti-plasmodial activities [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] and the formation of epithelial barrier that prevents the traverse of ookinetes across the mosquito midgut [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. \u003cem\u003eE. cloacae\u003c/em\u003e is known to release reactive oxygen species that directly affect the parasites [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], while \u003cem\u003eSerratia\u003c/em\u003e spp. Show variation in their antagonistic modes of action towards \u003cem\u003ePlasmodium\u003c/em\u003e [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Therefore, while both belong to the same bacterial family and proliferate in the midgut after the host takes a blood meal, they are likely to be affecting the parasite through different molecular processes and pathways, and at distinctive stages of the parasite life cycle. In this study, we assessed the effect of \u003cem\u003eE. cloacae\u003c/em\u003e and \u003cem\u003eS. marcescens\u003c/em\u003e metabolic by-products on \u003cem\u003eP. falciparum\u003c/em\u003e gametocytes using an in vitro treatment assay. This allowed the observation of direct, non-immune effect of the by-products on this stage of the parasite.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of bacterial metabolic by-products\u003c/h2\u003e \u003cp\u003ePure isolates of \u003cem\u003eE. cloacae\u003c/em\u003e and \u003cem\u003eS. marcescens\u003c/em\u003e previously obtained from the midgut of field-derived \u003cem\u003eAnopheles\u003c/em\u003e mosquitoes [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] were revived from ultra-low storage by plating on sterile LB agar. The bacteria cells were inoculated into Luria-Bertani broth (LB) or Tryptic Soy Broth supplemented with 5% sheep (simply referred to as a \u0026lsquo;blood-base\u0026rsquo; (BB) broth) and incubated overnight at 37\u0026ordm;C. The OD\u003csub\u003e600nm\u003c/sub\u003e of the overnight culture was estimated with a UV-vis spectrophotometer (Varioskan Lux, Thermo Scientific) and set at an initial (stock) OD of 0.5\u0026ndash;0.55 for each bacterium. 49 mL of fresh sterile broth (LB or BB) was prepared into tubes labelled with time points 0.5, 1, 2, 4, 6 and 12 hours, and 1 mL of the stock bacterial suspension was transferred into each tube. A control tube was prepared with 50 mL of broth without any inoculum. All tubes were incubated at 37\u0026ordm;C. After each designated time point, the respective tubes were removed from the incubator and centrifuged to separate the cells from the supernatant. The supernatants were ultra-frozen overnight and lyophilised by freeze-drying. The dried products were aliquoted into 2 mL cryovials and stored at -20\u0026deg;C until used. By-products from two isolates each of \u003cem\u003eE. cloacae\u003c/em\u003e and \u003cem\u003eS. marcescens\u003c/em\u003e were prepared in LB and BB as described. \u003cem\u003eEscherichia coli\u003c/em\u003e (Migula) Castellani and Chalmers (ATCC ref#: 25922) was used as the control bacterium for the LB-based by-products only.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eGametocyte treatment assays\u003c/h3\u003e\n\u003cp\u003e \u003cem\u003ePlasmodium falciparum\u003c/em\u003e 3D7 was cultured under standard conditions using established protocols [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Gametocytes were obtained by doubling the volume of the culture medium on the 5th day after the parasites had been put into culture, stressing the parasites to switch to the gametocyte stage [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Thin blood smears were prepared on days 14 and 17, stained with Giemsa and viewed under a light microscope to confirm the presence of gametocytes. Gametocytaemia for the assay ranged between 1.3\u0026ndash;1.7% and 2% haematocrit. The parasites were transferred in triplicates of 50 \u0026micro;L into a 96-well cell culture plate.\u003c/p\u003e \u003cp\u003eThe lyophilised LB- and BB-base by-products for \u003cem\u003eE. cloacae\u003c/em\u003e, \u003cem\u003eS. marcescens\u003c/em\u003e and \u003cem\u003eE. coli\u003c/em\u003e (LB-base only), and the medium control (LB and BB with no inoculum) were reconstituted at a stock concentration of 50 mg/mL. Working concentrations of 3 mg/mL were prepared by diluting with 1\u0026times; Phosphate Buffered Saline (PBS) and filtered through 0.22\u0026micro; PES filters. 50 \u0026micro;L was added to the parasites in the wells. The control well was set-up with infected RBCs (iRBCs) in complete parasite medium at 2% haematocrit, with no by-product added. The plate was gassed and incubated at 37\u0026deg;C for 48 hours in a modular incubator chamber (Billups-Rothenberg, Inc.). SYBR Green-1 fluorescent dye was used to estimate the survival of \u003cem\u003ePlasmodium\u003c/em\u003e gametocytes following treatment with bacterial by-products. 100 \u0026micro;L of SYBR Green-1 fluorescent (MSF) buffer (5% v/v) was added to each well and mixed by pipetting up and down. The plates were then wrapped with aluminium foil and incubated in the dark at room temperature for 24 hours to ensure optimal binding of the dye [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. The SYBR Green fluorescence intensities were measured with a Varioskan LUX multimode microplate reader (ThermoFisher Scientific, USA) at excitation and emission wavelengths of 485nm and 530nm, respectively.\u003c/p\u003e\n\u003ch3\u003eAssessing effects of heat and proteinase treatment on by-products\u003c/h3\u003e\n\u003cp\u003eWe investigated whether heat or proteinase K treatment of the bacterial by-products affects the survival of the \u003cem\u003ePlasmodium\u003c/em\u003e. The experiment focused on LB- and BB-based by-products for the mosquito midgut bacteria, \u003cem\u003eE. cloacae\u003c/em\u003e and \u003cem\u003eS. marcescens\u003c/em\u003e. 50 \u0026micro;L of reconstituted by-products at 50 mg/mL was aliquoted into two equal volumes. One part was heat-treated at 56\u0026deg;C overnight. The second part was proteinase K-treated by adding 5 \u0026micro;L of 20mg/mL of proteinase K (Qiagen), heated at 56\u0026deg;C overnight and, the enzyme reaction was stopped at 72\u0026deg;C. Untreated bacterial by-products were used as control. All treatments were filtered, prepared to a working concentration and used in the gametocyte assay as previously described.\u003c/p\u003e\n\u003ch3\u003eData analyses\u003c/h3\u003e\n\u003cp\u003eRaw data was obtained as absorbances. The average absorbance from wells treated with bacterial products were blanked by subtracting the average background absorbance of the non-bacterial broth (LB or BB) medium. Percentage parasite suppression was then calculated from the ratio of the difference in estimated parasite remaining after the treatment (untreated iRBCs - normalized treated iRBCs) to the control (untreated iRBCs).\u003c/p\u003e \u003cp\u003eThe parasite, time point, and bacteria genus were fitted as interactive independent variables in generalized linear models to determine their influence on parasite suppression. Where appropriate, a linear mixed effect (LMM) model was fitted to test the bacterial strain as a random effect variable. In such cases, the best fit model was estimated by comparing models with the Akaike Information Criterion (AIC). Pairwise comparisons of means between the independent factors were estimated using estimated marginal means (\u003cem\u003eemmeans\u003c/em\u003e), accepting as significant adjusted \u003cem\u003ep\u003c/em\u003e-values\u0026thinsp;\u0026lt;\u0026thinsp;0.05. All analyses and plots were generated with custom R scripts.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eEffect of LB and BB-based bacterial by-products on Plasmodium gametocytes\u003c/h2\u003e \u003cp\u003eBy-products from all the bacteria used in this study exhibited cytotoxicity on \u003cem\u003eP. falciparum\u003c/em\u003e gametocytes. Variance in parasite suppression after exposure to the bacterial products was significantly influenced by the bacteria from which the product was obtained (F\u003csub\u003e(2, 12)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;192.22, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and the product time point (F\u003csub\u003e(5, 12)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;47.36, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). In addition, the interaction between bacteria and product time point (F\u003csub\u003e(10, 12)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;64.44, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) was also significant indicating that the effect of bacterial product on the parasite varied across different time points (Table\u0026nbsp;1). The overall mean parasite suppression was higher with products from \u003cem\u003eE. cloacae\u003c/em\u003e (79.7%) and \u003cem\u003eS. marcescens\u003c/em\u003e (82.6%) than \u003cem\u003eE. coli\u003c/em\u003e (45.9%) (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.69e-10) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Additional file 1). The time point effects of the bacterial products remained similar between \u003cem\u003eE. cloacae\u003c/em\u003e and \u003cem\u003eS. marcescens\u003c/em\u003e, and the highest difference between these and \u003cem\u003eE. coli\u003c/em\u003e occurred for the 6-hour by-products (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Additional file 1). At some time points, the parasite suppression was above 100% (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). This resulted from a higher absorbance recorded for the broth medium control than for the treated samples. Negative normalised absorbance resulted in higher calculated parasite suppression relative to the untreated iRBCs i.e ([untreated iRBCs - normalized treated iRBCs]/ [untreated iRBCs] is \u0026gt;\u0026thinsp;1).\u003c/p\u003e \u003cp\u003eThe mean parasite suppression resulting from treatment with blood broth (BB) based products was 21.07 units higher than with LB-based products (Additional file 2), corresponding to a 25.7% increase (t\u0026thinsp;=\u0026thinsp;2.289, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.028). In the model that was fit to compare the two products, the time point was a major determinant of parasite suppression (F\u003csub\u003e(5,36)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;31.82, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) and demonstrated significant differences between the two products at 0.5, 1 and 12hrs (Additional file 2). Parasite suppression was higher for blood-based products compared to LB medium at the earlier time points but was lower at 12 hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eEffect of heat and proteinase K treatment on bacterial by-products\u003c/h2\u003e \u003cp\u003eA linear mixed effect model (LMM) best estimated the factors associated with parasite suppression given the treatment of the bacterial by-products with heat or proteinase K (pk) before adding then to the gametocyte culture (Additional file 3). For both LB and BB-based products, the treatment (normal, heat-treated or proteinase K treated) and product time point significantly affected the parasite, and the treatment also varied over time (treatment * time point) (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e3\u003c/span\u003e). With heat-treated LB-based by-products, there was decreased parasite suppression compared to the normal products, while similarly treated BB-based by-products did not result in a significant change in parasite suppression compared to unheated (normal) products (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). In both LB and BB-based products, proteinase K treatment did not affect the overall mean parasite r suppression. However, the effect of proteinase K was dependent on the product time point (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e3\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) indicating that the product at specific time points had protein components that contributed to the deleterious effect on the parasites. Proteinase K treatment resulted in decreased activity of 4 and 6-hour LB-based products (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cb\u003eAnalysis of variance table for LB and BB evaluating the effects of treatment, time, bacteria (bac), and their interactions on the paraste suppression.\u003c/b\u003e Linear mixed model= [p_sup\u0026thinsp;~\u0026thinsp;ttment * time * bac + (1|strain)]. Significant factors and their P-values are in bold.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSum Sq\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNumDF\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eDenDF\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eF values\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e-value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003etreatment\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7839.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e 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\u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ebac\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e901.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.858\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3.6638\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.156\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003etreatment*time\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16282.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e39.596\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6.6158\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e6.31e-06\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003etreatment*bac\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e957.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e40.693\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.9449\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.156\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003etime*bac\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1551.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e39.596\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.2608\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.300\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003etreatment*time*bac\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e799.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e39.596\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.3247\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.970\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003etreatment\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3733.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e12.5829\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e7.18e-05\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003etime point\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9904.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e13.3527\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e2.19e-07\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ebac\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.015\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.903\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003etreatment*time\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e17135.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e11.5505\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e1.54e-08\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003etreatment*bac\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e26.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.0891\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.915\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003etime*bac\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e924.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.2461\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.308\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003etreatment*time*bac\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1344.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.906\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.538\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"6\" nameend=\"c7\" namest=\"c2\"\u003e \u003cp\u003eSum Sq (Sum of Squares): Measures the variation explained by each factor.\u003c/p\u003e \u003cp\u003eNumDF (Numerator Degrees of Freedom): Number of levels in the factor.\u003c/p\u003e \u003cp\u003eDenDF (Denominator Degrees of Freedom): Residual degrees of freedom.\u003c/p\u003e \u003cp\u003eF-value\u0026thinsp;=\u0026thinsp;Ratio of explained to unexplained variance\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe two bacterial products were analysed to assess the impact of heat and proteinase K treatment on the effect they had along the time points. \u003cem\u003eE. cloacae\u003c/em\u003e LB-based products were most affected by heat at earlier time points of 1 and 2 hours, demonstrating decreased effect on parasite survival. Proteinase K treatment of 1-hour LB-based products, however, led to increased parasite suppression compared to untreated products at the same time point (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). In contrast, \u003cem\u003eS. marcescens\u003c/em\u003e LB-based products were affected by heat at 4 and 12 hours, and by proteinase K at 12 hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Parasites treated with heated and proteinase-treated \u003cem\u003eE. cloacae\u003c/em\u003e 12-hour BB-based products showed higher suppression, but \u003cem\u003eS. marcescens\u003c/em\u003e BB-based products were affected only by heat at 0.5 and 1-hour timepoints. This is suggestive of varied heat-sensitive and protein-based bioactive components at different time points during the culturing of \u003cem\u003eE. cloacae\u003c/em\u003e and \u003cem\u003eS. marcescens\u003c/em\u003e bacteria in LB or BB.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study highlights the potential for using the by-products from midgut bacteria to reduce \u003cem\u003ePlasmodium falciparum\u003c/em\u003e. Parasite numbers significantly decreased in the presence of the cell-free spent media from \u003cem\u003eE. cloacae and S. marcescens\u003c/em\u003e, two members of Enterobacteriaceae which have shown promise for parasite transmission-blocking [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. While parasite cultures treated with \u003cem\u003eE. coli\u003c/em\u003e products also showed reduced parasite numbers, the effect from the two midgut bacteria were more than 100% higher, indicating a stronger detrimental potential. These results support the use of natural microbiota for malaria intervention and uniquely highlight the potential of bioactive products from midgut bacteria as a novel strategy to reduce \u003cem\u003ePlasmodium falciparum\u003c/em\u003e as early as the gametocyte stage, a critical yet underexplored target stage for malaria transmission-blocking. Compared to other studies [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], our in vitro approach allowed the evaluation of the antagonistic effect of the bacteria by-product outside of the mosquito host immune system. This demonstrates that parasite-blocking could be achieved in mosquitoes that do not naturally harbour these two bacteria in the midguts. However, whether such a scenario would lead to the same outcome as we have previously shown [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], requires further investigation.\u003c/p\u003e \u003cp\u003eOur current results show that the bioactive substances that are released during culture of \u003cem\u003eE. cloacae\u003c/em\u003e, \u003cem\u003eS. marcescens\u003c/em\u003e and \u003cem\u003eE. coli\u003c/em\u003e in regular Luria broth medium at 37℃ are toxic towards \u003cem\u003eP. falciparum\u003c/em\u003e in a manner that is reflective of their growth dynamics (Additional file 4). The negative effect of the product on the parasite viability increased with product time point from lag (0.5-1hr) through the exponential (2-6hrs) phase and reduced thereafter (12hrs). The production of bacterial metabolites is generally known to be minimal during the lag phase where bacteria adjust to their environment [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], typically lasting between 1\u0026ndash;6 hours depending on the bacteria and growth conditions. Despite this, the LB-base products from our lowest measured time point (0.5 hours) for \u003cem\u003eE. cloacae\u003c/em\u003e and \u003cem\u003eS. marcescens\u003c/em\u003e resulted in \u0026gt;\u0026thinsp;70% parasite suppression compared to \u003cem\u003eE. coli\u003c/em\u003e, which is nonetheless a strong significant effect. For the two midgut bacteria investigated in this study there was a slight decrease in cell numbers at 1hour compared to 0.5 hour which also resulted in a dip in parasite suppression. The exponential phase, which could start between 1\u0026ndash;2 hours under optimum conditions results in increased bacterial replication and production of several biological substances including amino acids, enzymes and organic acids that the bacteria require to proliferate [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. The increased parasite suppression matched this growth phase and peaked at 6hours. Growing bacteria numbers therefore appears to have resulted in increased production of anti-parasitic by-products.\u003c/p\u003e \u003cp\u003eThe parasite suppression profile changes with blood-based products demonstrating the influence of growth conditions on the production of bacterial by-products [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. The parasite suppression was observed above 100% even at 0.5 hours for BB-based products. \u003cem\u003eEnterobacteriaceae\u003c/em\u003e are well-known for their haemolytic and haemagglutination activities, but the combination of these activities varies by species and strains [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. We observed firsthand the lysis of blood during the culture of \u003cem\u003eS. marcescens\u003c/em\u003e in the blood-based broth (Additional file 5). The haemolytic substances may have persisted after harvesting and processing the spent media and continued to lyse the blood cells in the \u003cem\u003ePlasmodium\u003c/em\u003e culture. Haemoglobin released from lysed red blood cells can absorb weakly at the wavelengths used [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e], and contribute with to the final absorbance read. This would explain the \u0026gt;\u0026thinsp;100% suppression observed resulting from higher absorbance compared to medium control, when the parasites were treated with BB products. Some clinical isolates of \u003cem\u003eE. cloacae\u003c/em\u003e are known to also produce ⍺-haemolysin [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. The parasite suppression profile did not differ between the two bacteria genera and considering that the two bacteria were isolated from the mosquito midgut where they encounter blood, it is plausible that these \u003cem\u003eE. cloacae\u003c/em\u003e strains also have haemolytic properties.\u003c/p\u003e \u003cp\u003eThe use of heat and proteinase K treatment allowed basic insights into the properties of the bioactive by-products. The effect of proteinase K on LB-based products was more associated with time than the heat treatment. Proteinase reduced the efficacy of product time point 4 and 6hours, suggesting that the bioactive products at these time points could involve proteins. Heating the by-products, however, appeared to have provided a more conducive medium for parasite survival, indicative of heat-sensitive bioactive compounds in the LB-base by-products. LB medium when autoclaved has increased protein and antioxidant levels which can resist high temperatures [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. However, as bacteria use up these components, they release metabolic by-products such as acids and, secrete proteins (e.g. toxins) and extracellular molecules, depending on the bacteria species [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. The BB-based products were much less affected by the treatments suggestive of anti-parasitic components that are heat-stable and less proteinaceous. The two bacteria showed striking patterns in the comparison of their heat and proteinase K-treated products to the non-treated, which suggested differential compositions. The 1- and 2-hours LB-based products of \u003cem\u003eE. cloacae\u003c/em\u003e were more heat-labile, and removing proteins increased the antagonistic property suggesting that the presence of proteins here may have interrupted with some of the bioactive components. \u003cem\u003eS. marcescens\u003c/em\u003e had these characteristics at later time points from 4 hours. The spent media used in this study warrant detailed investigations into their compositions, antagonistic properties and modes of action towards the parasite.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study demonstrates the potential of bacterial by-products from \u003cem\u003eE. cloacae\u003c/em\u003e and \u003cem\u003eS. marcescens\u003c/em\u003e as promising agents for reducing \u003cem\u003ePlasmodium falciparum\u003c/em\u003e at the gametocyte stage, a critical target for malaria transmission-blocking. Our findings reveal that the anti-parasitic effects of these midgut bacteria-derived spent media are time-dependent and strongly associated with bacterial growth phases, with peak parasite suppression occurring during the exponential phase. The influence of culture conditions, particularly the differences between Luria broth and blood-based media, underscores the variability in bioactive compound production. The haemolytic activity observed in the blood-based cultures suggests that these bacterial strains may produce haemolysins or other cytotoxic compounds that contribute to parasite suppression. Additionally, our heat and proteinase K treatments suggest that the active components include both heat-sensitive and heat-stable molecules, with differential protein involvement between the two bacterial species.\u003c/p\u003e \u003cp\u003eOverall, our study supports the growing interest in microbiota-based malaria interventions and highlights the need for further investigations into the specific bioactive compounds responsible for the observed anti-parasitic effects. Future research should focus on characterizing these bacterial metabolites, their mechanisms of action, and their potential application in vector control strategies to disrupt malaria transmission.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cem\u003eEthics approval and consent to participate\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eConsent for publication\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eClinical Trial Number\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAvailability of data and materials\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analysed during this study are included in this published article and its supplementary information files.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eFunding\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThis study was funded by the Wellcome Trust Intermediate Fellowship [Grant number: 220737/Z/20/Z] to JA. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAuthor’s contributions\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eJA conceptualized the study. JA, EAA designed the experiments. EAA, SNASA, GO, EO-F, JKB performed the experiments, EAA, SNASA, JA analysed the data and drafted the manuscript. All the authors have read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAcknowledgement\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eWe acknowledge the advisory role played by Dr. Lisa Ranford-Cartwright (University of Glasgow) in the execution of this study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eWorld Health Organization. World Malaria Report 2024. Geneva: World Health Organization; 2024.\u003c/li\u003e\n\u003cli\u003eHealth Organization W. Global Technical Strategy for Malaria 2016-2030. 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Lag Phase Is a Distinct Growth Phase That Prepares Bacteria for Exponential Growth and Involves Transient Metal Accumulation. 2011 [cited 15 Feb 2025]. doi:10.1128/JB.06112-11\u003c/li\u003e\n\u003cli\u003eGonzalez JM, Aranda B. Microbial Growth under Limiting Conditions-Future Perspectives. Microorganisms. 2023;11: 1641. doi:10.3390/MICROORGANISMS11071641\u003c/li\u003e\n\u003cli\u003eRoland FP. Interaction of Blood with Enterobacteriaceae Hemolysis, Hemagglutination, Fibrinolysis. Am J Clin Pathol. 1977;67: 260\u0026ndash;263. Available: http://ajcp.oxfordjournals.org/\u003c/li\u003e\n\u003cli\u003eTownsend D, D\u0026rsquo;Aiuto F, Deanfield J. Journal of Medical and Biological Engineering. J Med Biol Eng. 2014;34: 172\u0026ndash;177. doi:10.5405/jmbe.1643\u003c/li\u003e\n\u003cli\u003ePrada J, Beutin L. Detection of Escherichia coli \u0026alpha;-haemolysin genes and their expression in a human faecal strain of Enterobacter cloacae. FEMS Microbiol Lett. 1991;79: 111\u0026ndash;114. doi:10.1111/j.1574-6968.1991.tb04514.x\u003c/li\u003e\n\u003cli\u003eWang H, Guo J, Chen X, He H. The Metabolomics Changes in Luria\u0026ndash;Bertani Broth Medium under Different Sterilization Methods and Their Effects on Bacillus Growth. Metabolites. 2023;13: 958. doi:10.3390/METABO13080958\u003c/li\u003e\n\u003cli\u003eMaffei B, Francetic O, Subtil A. Tracking proteins secreted by bacteria: What\u0026rsquo;s in the toolbox? Front Cell Infect Microbiol. 2017;7: 269489. doi:10.3389/FCIMB.2017.00221/PDF\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 and 2 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"mosquito midgut microbiota, Enterobacter cloacae, Serratia marcescens, Anopheles gambiae, disease transmission blocking, gametocytes","lastPublishedDoi":"10.21203/rs.3.rs-6176263/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6176263/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eMalaria remains a significant public health challenge, necessitating innovative strategies to break transmission, such as interrupting successful \u003cem\u003ePlasmodium falciparum\u003c/em\u003e development in the vector using microbiota. While this transmission-blocking has been demonstrated experimentally as low ookinete or oocyst intensity in the mosquito, little is known about the effect of these microbes on gametocytes, which are the first stages of the parasite that the mosquito host encounters. We have tested the effect of cell-free spent media from \u003cem\u003eEnterobacter cloacae\u003c/em\u003e and \u003cem\u003eSerratia marcescens\u003c/em\u003e on gametocytes of \u003cem\u003ePlasmodium falciparum\u003c/em\u003e in vitro. The two bacteria were isolated from female \u003cem\u003eAnopheles gambiae\u003c/em\u003e collected in Ghana, put into Luria or blood broth (LB or BB) medium and the spent media was harvested at 6 time points during the growth of the bacteria.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe spent media exhibited strong anti-parasitic effects, significantly reducing parasite intensity compared to \u003cem\u003eEscherichia coli\u003c/em\u003e controls. Compared to non-treated gametocytes, there was overall cytotoxic effect from all three bacteria with significant variance influenced by the bacteria species and product time point. Blood broth (BB)-based products achieved 25.7% greater mean parasite suppression than Luria broth (LB)-based products (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.028) suggesting that the production of the anti-parasitic substances is dependent on the growth conditions of the bacteria. Heat and proteinase K treatments revealed that LB-based anti-parasitic activity was partially protein-dependent and heat-sensitive, whereas BB-based activity remained largely unaffected, suggesting the presence of heat-stable, non-proteinaceous bioactive compounds. Finally, we observed that heat and proteinase K treatment effects varied between \u003cem\u003eE. cloacae\u003c/em\u003e and \u003cem\u003eS. marcescens\u003c/em\u003e.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThese findings underscore the potential of natural mosquito midgut bacteria and their metabolic by-products in malaria intervention strategies. By demonstrating parasite suppression in an in vitro system independent of mosquito immune responses, our study suggests that bacterial-derived metabolites could be harnessed to disrupt parasite development in non-colonized mosquito populations. Future research should focus on identifying the specific bioactive compounds responsible for parasite reduction and assessing their feasibility for field application in malaria control programs.\u003c/p\u003e","manuscriptTitle":"Metabolic by-products of mosquito midgut bacteria, Enterobacter cloacae and Serratia marcescens, exhibit potent anti-parasitic effects on Plasmodium falciparum gametocytes in vitro","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-12 06:06:33","doi":"10.21203/rs.3.rs-6176263/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ddca5e13-6e90-44b6-8143-c1505ba4d1a3","owner":[],"postedDate":"March 12th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-11-05T09:09:07+00:00","versionOfRecord":[],"versionCreatedAt":"2025-03-12 06:06:33","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6176263","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6176263","identity":"rs-6176263","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}