Globally, leishmaniasis is one of the most neglected tropical and subtropical diseases, in which hosts are diseased after receiving the infectious bite of Phlebotominae sand flies infected with protozoan parasites of Leishmania genus (Torres-Guerrero et al., 2017). It is estimated that 0.7-1 million new cases of leishmaniasis occur annually (WHO, 2022). The disease is clinically characterized by a diverse spectrum of manifestations, varying from self-limiting cutaneous leishmaniasis (CL) to progressive and potentially fatal visceral leishmaniasis (VL) (Murray et al., 2005). Cutaneous leishmaniasis is the most common form of leishmaniasis worldwide, and virtually 95% of the cases occur in the Americas, the Mediterranean Basin, the Middle East, and Central Asia (Hepburn, 2003; WHO, 2022). As the most frequent presentation of the disease, CL causes skin lesions that evolve from nodules to painless ulcers, which can leave long-lasting scars and serious disability or stigma (Bilgic-Temel et al., 2019; WHO, 2022).

Clinical features of leishmaniasis is extremely intricate and depend on multiple factors, especially infecting parasite species (Oshaghi et al., 2009; Thakur et al., 2018; Kato et al., 2021) and their virulence factors such as lipophosphoglycan, metalloprotease GP63, and elongation factor 1 alpha (Gupta et al., 2022), as well as genetic background of the vertebrate (Sakthianandeswaren et al., 2009; Krayem and Lipoldová, 2021), immune homeostasis of the host, and vector-derived product (Serafim et al., 2021). However, balance between type 1 and type 2 immune responses, along with other regulatory mechanisms, plays an essential role in determining the outcome of leishmaniasis (Costa-da-Silva et al., 2022). Studies have signified that a set of cytokines are respectively involved in the leishmaniasis progression, and host protection entails cytokines, including TGF-β and interleukins (IL)-4, IL-5, IL-6, IL-9, IL-10, IL-27, and IL-33 and also inducible nitric oxide synthase (iNOS), interferon (IFN)-γ, tumor necrosis factor-α, IL-2, IL-7, IL-8, IL-12p40, IL-15, IL-22, and IL-23. However, the cytokines IL-1, IL-3, IL-4, IL-13, IL-17, and IL-18 have a dual role not only in the disease progression but also in host resistance (Dayakar et al., 2019). In addition, IL-4 and IL-10 have been reported to be associated with the visceralization of cutaneous L. major infection (Heinzel et al., 1989; Heinzel et al., 1991).

During the last few years, research has exhibited that vector-derived factors of sand fly (e.g. saliva and gut microbiota) and Leishmania (e.g. the promastigote secretory gel and exosomes) origin are actively involved in vector transmission and facilitate parasite survival and its establishment in the host (Serafim et al., 2021). It is well known that leishmaniasis transmission occurs in complex biological systems, including the human host, parasite, sand fly vector, and in some cases, one or more animal reservoirs (WHO, 2010). Efforts made so far toward the development of an efficient and a safe drug or vaccine against leishmaniasis have been unsuccessful or partially successful (Altamura et al., 2022; de Vries and Schallig, 2022; Ghafari and Parvizi, 2022). Hence, to constrain and finally eliminate leishmaniasis, comprehensive research programs focusing on the role of all components of the disease and their interaction are needed.

It has fully been accepted that microbiota, bacteria in particular, are inextricably linked to leishmaniasis, an issue neglected in the early theory of infectious disease causation. Interaction of microbiota with the components of a disease cycle, starting from the vector gut and continuing to the vertebrate host, can be unilateral, bilateral, or multilateral and can have adverse, neutral or beneficial effects for the partners (Maleki-Ravasan et al., 2015; Gimblet et al., 2017; Dey et al., 2018; Karimian et al., 2018; Borbon et al., 2019; Karimian et al., 2019; Campolina et al., 2020).

Once Leishmania parasites reach the insect’s alimentary canal, they encounter a commensal bacterial community, where multiple microbiota-parasite-host interactions determine the ultimate fate of the parasite journey (Telleria et al., 2018). Microbiota play an important role in the physiology of nutrition, digestion, and maturation of the vector’s innate immune system (Dillon and Dillon, 2004; Weiss and Aksoy, 2011). It has been shown that eliminating or changing the microbiota can alter the development of the Leishmania parasite in the vector’s gut (Kelly et al., 2017; Louradour et al., 2017). Gut microbiota can also affect the parasite infection by activating the innate immune pathways of the vector (Diaz-Albiter et al., 2012). Likewise, Leishmania infection may also lead to a prompt loss of bacterial diversity throughout the course of infection (Kelly et al., 2017).

A large number of bacteria including the members of Enterobacter cloacae and Bacillus subtilis complexes are commensal bacteria in the gut of sand flies that transmit causative agents of CL and VL in the Old and New Worlds (Oliveira et al., 2000; Hurwitz et al., 2011; Akhoundi et al., 2012; Maleki-Ravasan et al., 2015; Fraihi et al., 2017; Gunathilaka et al., 2020; Karimian et al., 2022). Both bacteria have the ability to regulate insect immune responses (Eappen et al., 2013; Heerman et al., 2015; Zhang et al., 2021) and produce secondary metabolites that show activity against insects and their harboring microorganisms (Eappen et al., 2013; Caulier et al., 2019; Zhang et al., 2021). Therefore, they probably play an essential role in sand fly vector competence for Leishmania parasites (Louradour et al., 2017). Consequently, E. cloacae and B. subtilis can be considered as a shuttle system to deliver, express and spread foreign inserts to be used as promising candidates for the paratransgenic approach (Hurwitz et al., 2011; Dehghan et al., 2017; Dehghan et al., 2022).

Altogether, bacterial components of the sand fly microbiota can interfere with the development of Leishmania parasite inside the midgut of the sand fly vector (Telleria et al., 2018) or outside the midgut, in the skin of the vertebrate (Dey et al., 2018). Regarding the first part, as mentioned above, relatively suitable studies are available, but for the second part, one survey has suggested that eliminating the vector gut microbiota or blocking the vertebrate host’s IL-1β before parasite transmission abrogates neutrophils recruited to the site of sand fly bite and declines Leishmania dissemination (Dey et al., 2018).

Given the above arguments, it is apparent that the bacteria in the sand fly’s gut are a perpetual companion of Leishmania parasites, and the consequences of concomitance of these bacteria and the mentioned parasites have not been investigated in the initiation, continuation, and termination of the infection process. Hence, a laboratory model of Leishmania infection was developed in BALB/c mice through needle injection of parasites with or without sand fly gut bacteria to determine the outcomes of accompanying bacteria, in terms of type and number, in the Leishmania wound formation and local immune responses. The results of the present study disclosed that bacterial co-infection has a profound effect on the balance of pro- and anti-inflammatory cytokine expression, thus the severity of L. major lesions.

The findings of the present study demonstrated that E. cloacae and B. subtilis bacteria significantly influence the infection process caused by L. major parasite, and the results can be used in the interpretation of the natural infection transmission through sand fly bites. Bacterial co-infection had a profound impact on the expression balance of pro- and anti-inflammatory cytokines at the beginning, middle, and end of infection course. These effects varied based on the type and dose of bacteria inoculum. From the microscopic and macroscopic point of view, the worst type of wound was observed in the group receiving low-dose E. cloacae and B. subtilis bacteria plus L. major parasite, leading to a decrease in the expression of iNOS and an increase in the expression of other cytokines together with raising inflammatory cells. However, the same treatment with increasing dose of bacteria showed different results in terms of wound morphology and the expression of cytokines and inflammatory cells.

There is sufficient evidence suggesting that studies of the evolutionary course of leishmaniasis, as an ancient disease, are consistent with the development of infectious disease modeling theories. At the beginning of the 20^th century, early studies had focused on the identification of Leishmania parasites as an etiological agent and also sand flies as the transmission vectors of leishmaniasis (Steverding, 2017). Later, factors other than the main partners of the disease were identified, which described the other mechanisms involved in the transmission of the disease in more detail (Serafim et al., 2021). Recently, a new multiscale model of infectious disease systems, namely the replication-transmission relativity theory, has been developed. This theory denotes that at each level of organization of an infectious disease system, pathogens must succeed at both the microscale (where pathogen replication often occurs) and the macroscale (where pathogen transmission often occurs) if they are going to spread and persist at a special level of organization of an infectious disease system (Garira, 2019). However, due to the neglect of the dynamics of the disease and the interaction between micro and macro scales, leishmaniasis still remains a major health problem in many countries, including Iran. Hence, the present study was designed and carried out in line with the multiscale replication-transmission relativity theory to show that the microbiota of the sand fly gut may affect the survival, reproduction, pathogenesis, and spread of the Leishmania parasite.

Lately, it has been suggested that the relationship between Leishmania and the microbiota may extend beyond the vector midgut. During sand fly feeding, bacteria from the insect’s gut or in the skin of the vertebrate host can enter the bite site. It has been proven that the gut microbiota of sand flies are transferred while sugar feeding (Maleki-Ravasan, in press) or are co-egested with the Leishmania parasite (Dey et al., 2018). Using a murine VL model of vector-transmitted L. donovani parasite, Dey and colleagues demonstrated that the gut microbes of the Lutzomyia longipalpis are entered the host skin, where they induce inflammation and IL-1β production by neutrophils. IL-1β then acts as a primary autocrine signal to attract neutrophils to the bite site. The same authors have also displayed that the microbe-induced immune response controls the downstream events governing L. donovani dissemination (Dey et al., 2018). Regarding the simultaneous infection of the microbiota of vertebrate skin with the Leishmania parasite, it has been found that this type of inoculation aggravates the disease both by promoting more inflammation and neutrophil recruitment and by increasing neutrophil apoptosis and delaying the resolution of the inflammatory response (Borbon et al., 2019).

The present research is the first study investigating the role of bacteria isolated from sand fly gut in the wound formation and local immune responses in BALB/c mice infected with L. major. While this study was a laboratory model, without inclusion of saliva or unculturable bacteria found in microbiota from the sand fly, its results can be used to deduce the key role of bacteria in the infectious bite of sand flies. The most important issue in our investigation was the type and the number of bacteria transmitted during the sand fly bite. Our previous study uncovered that culturable bacteria constitute a small portion of microbiota transmitted during bites (Maleki-Ravasan, in press), which should be taken into account in future studies. Therefore, tactically, the consequences of the association of a Gram-positive bacterium with a Gram-negative bacterium alongside the Leishmania parasite were investigated in the present study. On the one hand, the average number of parasites and bacteria egested by a sand fly is estimated to be about 1,000 and 45,000 (Maleki-Ravasan, in press; Rogers et al., 2004), respectively, and on the other hand, the number of 10²–10⁷ Leishmania parasites has been indicated to cause ulcers in an animal model (Kimblin et al., 2008; Loeuillet et al., 2016). Considering these data, the number of parasites was selected as 1.5×10⁶ and that of bacteria as 1.5×10³ and 1.5×10⁶ in low and high doses, respectively ( Table 1 ).

The gut of insects, compared to mammals’ gut, harbors relatively fewer microbial species, but the gut of most of them contains specialized bacteria (Engel and Moran, 2013). Insects such as sand flies have a symbiotic relationship with their gut microbiota, which has become a necessary evolutionary consequence of their survival in extreme environmental conditions (Gupta and Nair, 2020). Various evolutionary processes in the insect body have led to different types of symbiotic relationships, from free living to an obligate or facultative symbiosis (Gupta and Nair, 2020). There is insufficient information on the type of symbiosis between B. subtilis and E. cloacae together and with the host insect, but evidence of their co-occurrence in the gut of eight sand fly species (Maleki-Ravasan et al., 2015; Karimian et al., 2019; Karimian et al., 2022) and their transmission while biting (Maleki-Ravasan, in press) have been provided. While these two bacteria were pre-coexisted in sand fly gut, in the present study, they first were isolated, then sub-cultured separately and finally combined while injecting into the mice, which resulted in relatively severe symptoms of L. major, though it may not be the case in natural settings. All these issues require more detailed and accurate investigations.

In the current study, the thickness of the ear pinna lesion in BALB/c mice was found to be bacteria dose-dependent. Thus, the highest thickness in low- and high-dose groups was related to the groups receiving the parasite plus two bacteria and the parasite alone, respectively ( Figure 1 ). The results of ear lesion thickness and the abundance of tissue chronic inflammatory cells ( Figure 6 ) are consistent with the findings of a recent study (Dey et al., 2018). However, the contradictory performance of high doses of bacteria in wound formation is probably due to the interaction of the Gram-positive with Gram-negative bacteria, which requires more in-depth studies.

Measurement of the parasite load during the experimental infection period showed that the number of live parasites was much higher at the end than in the middle of the infection period. In addition, there was no significant difference between the parasite loads of the groups with or without bacteria, indicating that microbiota more likely have an impact only on the pathogenesis of the parasite not on its number ( Figure 3 ). The results of both ear lesion thickness and parasite load examined herein were in agreement with the Borbon et al.’s findings (Borbon et al., 2019).

In the immunology of leishmaniasis, balance between Th1 and Th 2 along with regulatory mechanisms determines the outcome of leishmaniasis. In general, Th1-type response mediates host resistance, and Th2-type response associates with disease progression in experimental infection with L. major in a mouse model (Dayakar et al., 2019). The results of our study showed that the local expression of pro- and anti-inflammatory cytokines in mice receiving different inoculations of bacteria changed and caused the disease to worsen. By comparing the low-dose group with high-dose group, we found that the simultaneous injection of two bacteria together with parasites causes a 35- and 970-time increase in the expression of IL-4 and IL-1β in the low-dose group. In both groups, the lowest iNOS expression was observed in the group receiving two bacteria plus parasites at 1 dpi. A significant increased expression of IFN-γ was also observed on the first day of all treatments in the low-dose group, while in the high-dose group, the elevated expression of the cytokine was detected only in the treatments receiving Bs+EC (P<0.0001). Coinfection of two bacteria with the parasite led to 260-fold increase in the expression of IL-12p40 in the low-dose group compared to the high-dose group. Moreover, the simultaneous infection of the same bacteria without parasite resulted in 328-fold increase in the expression of IL-12p40 in the high-dose group compared to the low-dose group ( Figures 4 , 5 ). Also, by comparing the mean expression of cytokines in mice receiving two bacteria along with parasites compared to the control group, it was found that the expression of IL-10 is completely dose-dependent ( Figures S1 , S2 ). Therefore, as the results imply, the bacteria of the sand fly’s gut act as mice immunomodulators in adjusting the outcome of leishmaniasis.

As stated above, leishmaniasis has a complex epidemiology, and apart from main partners of the disease, various factors are responsible for the severity of the parasite’s pathogenicity. Thus, it is necessary to deeply analyze the role of neglected factors, such as microbiota, in modulating Leishmania pathogenesis, in order to achieve a comprehensive view of the complicated interaction of Leishmania parasite with its hosts. Current leishmaniasis prevention and control measures and access to valid diagnostic methods and effective treatments are insufficient. Therefore, these deficiencies could have significant implications for the disease, including increase in the incidence of leishmaniasis in the endemic foci and its neighboring localities, the spread of Leishmania species into new areas going unnoticed, increase in treatment failure, and the development of resistance to treatments (Berriatua et al., 2021).

The results of this study suggest that the co-infection of sand fly gut bacteria with L. major aggravates the pathological responses of BALB/c mice. This finding gives new insight into using the capability of the vector-derived microbiota in modulating the vertebrate immune system for the benefit of the host or using appropriate antibiotics together with antileishmanial drugs. The design of the present study and the proposed model included features that were easily controlled, but there are many factors in patients - including host-specific variability - that cannot be managed and should await future studies. The data represented in the present study can be a small step to initiate a new series of studies, though it faces some limitations, such as failing to consider the systemic immune responses of mice and the role of sand fly saliva in the pathogenesis of Leishmania parasite. This pioneering study can be expanded to other levels of organization of leishmaniasis via applying advanced OMICS technologies, with the contribution of all partners of leishmaniasis. Perhaps, it is better to describe the process of wound formation in leishmaniasis from the time of the sand fly bite to the formation of nodules and wounds and even its recovery considering the role of microbiota in more detail.

The original contributions presented in the study are included in the article/ Supplementary Material, further inquiries can be directed to the corresponding author/s.

The animal study was reviewed and approved by Ethics Commission of IPI and IUMS (ethical codes: IR.PII.REC.1399.027 and IR.IUMS.FMD.REC.1400.385).

NM-R conceived and coordinated the study. NM-R, MN-R and FA designed and performed the majority of the experiments. FA, MN-R, and NM-R conducted animal studies. FA, MN-R, AB, and AM contributed to microbial culture and Leishmania maintenance. FK performed statistical analyses. FA, MN-R, RH, and PP contributed to real-time PCR assays and parasite burden. RH, and FA contributed to the histopathological investigations. NM-R, PP, and RH analyzed all the data. NM-R and FA wrote the paper with input from all of the authors. All authors contributed to the article and approved the submitted version.