Introduction

Over 50 years ago, Van Valen proposed ‘Red Queen Hypothesis’ in which the queen said to Alice, in ‘Through the Looking Glass,’

“It takes all the running you can do, to keep in the same place” [1]

To maintain their own survival, one species can increase its fitness at the expense of the other species [2]. Charles Darwin published his book “The Origin of Species” in 1859; the fields of evolutionary biology have come a long way since then, this book emphasized that natural selection evolves the organism which is totally different from their ancestor. The natural selection lies in the center of shaping the adaptations (morphological, physiological, and behavioral) to find out the inside story of living organism across the generation [3]. Genetic material is immortal and transfer information from one generation to another generation. Mutation and genetic recombination are the key players of variation.

Natural selection is working on the concept of ‘survival of the fittest’ and that’s why an evolutionary arm race, between host and pathogen, predator, and prey, continuously working in this dynamic environment [4, 5]. To make the environment dynamic, living, non-living, and different species of living things exist in a symbiotic environment. Symbiosis is a broad term which refers to 2 species existing together in an environment. The inter-specific correlation among eukaryotic and prokaryotic species existed during the course of evolution [5]. In this hill climbing fight for survival, human and microbial world are in a persistent fight.

Human beings are shielded by their immune system in combating infections caused via virulent organisms, namely viruses, fungi, bacteria, etc. As the part of patrolling, immune cells target and kill microbes roaming around the circulatory system, still immunity alone was not sufficient to tackle the contagious organism. Several anti-viral, antibacterial, and anti-parasitic were invented and became crucial to counter the contagious agents [6]. When it comes to ‘Minute forms of life,’ understanding their adaptation strategies during evolutionary arm race, to get selected by nature and maintaining their population through generations, demands our special attention. For example, viruses, bacteria, fungi, and parasites started to develop resistance which became one of the dominant strategies for survival [7].

Pathogenic microbes may be categorized on their infection methods. First, the extracellular microbes do not enter inside the cells but prefer to grow in external environment (body fluids with different nutrients). Second, the intracellular bacterial pathogen adapted a different strategy to escape from getting caught by host immune system and replicate inside host cell to increase their number causing severity of disease [8]. After invasion, host cells provide a heavenly environment for pathogen replication. However, each host cell has a limited carrying capacity for intracellular bacteria. Therefore, after reaching the carrying capacity, they must leave the infected cell and infect other cells to replicate further [9]. To date, it is not clearly understood how these intracellular bacteria multiply and spread their infection without being caught by the host immune system.

Scrub typhus (ST) is a remerging neglected disease that represents an acute fever sickness caused by the bacteria Orientia tsutsugamushi and spread by the larvae (chiggers) of Leptotrombidium mites. This disease is very severe public health problem in Southeast, East Asia, and the Pacific Islands, called as the ‘Tsutsugamushi Triangle’ [10]. Now scrub typhus (ST) is not only reported from tsutsugamushi triangle but evidence indicated that scrub typhus started to expand outside the Triangle in eastern Africa, France, the Middle East, and South America [11]. It frightens one billion people worldwide and around one million people are affected each year [12]. Many Sero-epidemiological data in Asia have shown seroprevalence rates are 9.3 to 27.9% alongside a notable increase in disease incidence. The median fatality rate for untreated ST and treated ST varied as 6.0% versus 1.4%. Patient with central nervous system involvement experience a high fatality rate of 13.6%, while those with multi-organ dysfunction had a rate of 24.1%. Additionally, ST infection during pregnancy is associated with a high risk of miscarriage and poor neonatal outcomes [13].

For the treatment of O. tsutsugamushi, antibiotics are still a medical miracle. Chloramphenicol, tetracycline (doxycycline), macrolides (azithromycin), and rifampin are still the best option for treating the disease ‘Scrub typhus’ [14,15,16,17]. O. tsutsugamushi started to develop strategies to tolerate and replicate even in the presence of antibiotics. This adaptation strategy of O. tsutsugamushi started to alarm for the discovery of new drugs.

In view of unusual biology for host–pathogen interaction, less susceptibility of present antibiotics, and outbreak of the scrub typhus across the world, the present review is conceptualized to find out the adaptation strategy acquired by the deadly pathogen O. tsutsugamushi. This review is also conceptualized to focus on antibiotics, less susceptibility toward antibiotics, and alternatives of antibiotics.

History

The history of O. tsutsugamushi is too old, for the first time, it was reported in China by Hong Ge, in 313 AD, and this organism is still very less reported and falls in the category of neglected diseases to date [18]. It was strived to gather the historical evidence from 313 AD to till date (Fig. 1).

Fig.1
figure 1

History and discoveries in scrub typhus

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Taxonomy and Classification

The genus Orientia belongs to the family Rickettsiaceae, order Rickettsiales and phylum Proteobacteria [19]. There are different strains of the bacteria due to the presence of variable membrane protein which includes Shimokoshi, TA763, Gilliam, Kawasaki, TA716, Boryong, Saitama, Ikeda, Kato, Karp, TA686, and Kuroki. This gram-negative bacterium is rod in shape and shows pleomorphism [10]. O. tsutsugamushi is obligate intracellular rickettsia which is classified as a biosafety level-3 (BSL-3) pathogen [20]. It is the causative agent of an acute febrile illness named Scrub typhus. The most neglected disease, as claimed by World Health Organization (WHO), is scrub typhus which requires hospitalization.

Transmission

The name ‘tsutsugamushi’ was originated from the Japanese word ‘tsutsuga’ which means illness and ‘mushi’ means insect, thus tsutsugamushi refers an illness caused due to an arthropod [21]. O. tsutsugamushi is transmitted by an arthropod named trombiculid mites (Acarina: Trombiculidae) commonly called as chiggers, when their larval mites feeds on human [22]. The carrier and main reservoir of Orientia species is mites. The only stage which is ecto-parasitic and feeds on serum exudates is the larvae of trombiculid mites, while all other active stages are free-living [22]. O. tsutsugamushi were transmitted and maintained in environment via vertical transmission, that is, infected adults to their larvae (Fig. S1); a study showed that horizontal transmission is also possible when an uninfected larva co-feeds with naturally infected larvae. This may explain the occurrence of many strains in individual larvae [23].

Life Cycle

Mites remain infected by O. tsutsugamushi in their different stages of life cycle (larva, nymph, adult, and egg) [24]. Through transovarial transmission female pass O. tsutsugamushi to their offspring via eggs and through transstadial transmission, the pathogen in the vector passes from one life stage to the next, i.e., from mite larva to nymph and nymph to adult (Fig. S1) [25]. Larva mites are the only parasitic stage; humans are the accidental and are dead-end host [26]. The incubation period of O. tsutsugamushi is approximately 5 to 14 days.

Symptoms and Complications

Infected Leptotrombidium mites bite to human thereafter infection start to manifest. Initial symptoms of scrub typhus include development of eschar (dark dry scab) at the bite site followed by fever, headache, myalgia, cough, generalized lymphadenopathy (swollen lymph nodes), nausea, vomiting, and abdominal pain (Fig. S2) [24]. Maximum percentage of scrub typhus patients share a common symptoms of fever acute undifferentiated febrile illness (AUFI) and headache followed by several chronic complications such as multi-organ failure occur in some case which includes jaundice, acute renal failure, pneumonitis, acute respiratory distress syndrome (ARDS), myocarditis (inflammed heart), septic shock, meningoencephalitis (inflammed brain), coagulopathy, pericarditis (swollen pericardium), and disseminated intravascular coagulation (DIC) (Fig. S2) [12]

Prior to understanding the adaptation strategy of pathogen, it is crucial to understand the cell biology of O. tsutsugamushi.

Components of Bacteria and Its Unique Features

Cell Wall

The cell wall of this intracellular pathogen does not completely match with either gram-positive bacteria or gram-negative bacteria. A recent study done by Atwal et al. indicated that O. tsutsugamushi lack the set of genes which encode for Lipopolysaccharides (LPS), while this group was able to identify the set of genes which were expressing peptidoglycan-like structure (Fig. S3) [27]. This component is crucial for bacterial growth, host cell invasion, and cell integrity [28]. The cell wall of Orientia is dynamic and different from truly gram-positive bacteria which are sensitive to several antibiotics targeting the cell wall. The dynamism of cell wall also help the pathogen to escape and is helpful in adaptation as intracellular pathogen [28]. An unrelated group of bacteria named chlamydiae shows similarity in a conserved gene set (murA-G) responsible for peptidoglycan synthesis. Gene murA-G regulates the shape, elongation, division, and sporulation. Another protein Class B penicillin-binding proteins (PBPs) responsible for peptidoglycan transpeptidase activity are also conserved in both Orientia and Chlamydiae [9].

Membrane Proteins

As the part of dynamic adaptation and smart invasion inside the host cell is governed by membrane proteins. The outer and inner membrane have plenty of specific proteins like Type-specific antigen (TSA56, TSA22, TSA47), Surface cell antigen (ScaA-F), htrA, and secretion systems which modulate host cell immune system for successful establishment and spread of intracellular pathogen O. tsutsugamushi [29].

Genomic Feature

The whole-genome sequencing of O. tsutsugamushi has been completed in the past. The information of sequenced genome of different strains is given in Table 1. Even many strains of O. tsutsugamushi genome have been sequenced, but in this study we are taking Ikeda strain as a model system for biology and pathogenesis of O. tsutsugamushi [30]. The complete genome sequence of Ikeda strain was done by Nakayama et al. and major portion of the genome consists of Coding sequences (CDSs-1967) following Transposable elements (619), O. tsutsugamushi amplified genetic elements (OtAGEs-185), Short repeats (165), tRNA genes (34), r-RNA genes (3), and other RNA genes (1) (Fig. 2).

Table 1 Sequencing and genome information of O. tsutsugamushi strains
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Fig. 2
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Peculiar features of whole genome of Ikeda strain of O. tsutsugamushi

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Ikeda strain has a single circular chromosome of 2,008,987 bp and having the coding sequence with number 1967 [31]. In this strain, no plasmid/prophage was reported, the protein-coding genes (1967ssCDSs) are higher compared to other member of Rickettsiales, and the second peculiar feature of its genome is repeated genes of 85 families (OtRG 1–85). In O. tsutsugamushi, several reports indicated that gene loss was at a high level, but enormous amplification of mobile elements induced in-depth genome shuffling. This resulted in origin of large number of repeated genes, and Nakayama et al. also reported 18 types of repeated sequence in O. tsutsugamushi genome which have exclusively amplified and scattered all around the genome; all these events allowed this bacterium a unique genome evolution. Poor co-linearity of O. tsutsugamushi genome to any of the rickettsial genome is another peculiar feature. There are many genes reported which do not belong to any of the repeated gene families are referred as Singleton genes [30].

Secretion System

Many intracellular bacterial pathogens have a protein complex on cell membrane referred to as Secretion system. Bacteria translocate various micro- and macromolecules to designate sub-cellular locations via secretion system which modulate host cell processes; secretion systems are classified from Type I to Type VI in gram-negative bacteria, which deliver a particular group of proteins; as per available report, different strains of O. tsutsugamushi utilize Type I and Type IV secretion system frequently [29].

Type 1 Secretion System (T1SS)

Type I secretion systems (T1SS) reported to secrete various protein/enzymes (namely adhesins, proteins, lipases, proteases, or pore-forming toxins) in the unfolded state, T1SS translocate proteins in a single step due to its following components: (a) Plasma membrane harbors ABC transporter which recognizes and translocates substrate. (b) Membrane fusion protein (MFP) is the linker protein connecting outer membrane and inner membrane. (c) Outer membrane protein (OMP) forms a channel and opens after substrate recognition and transport from ABC transporter, a 33 residue ankyrin repeat is the substrate of T1SS, expressed during infection [29]. Ank repertoires containing protein (Anks) are reported as crucial virulent factor of O. tsutsugamushi [31]. Anks have binding diversity via two reasons: (i) variation of Ank repeats and (ii) high rate of degeneracy of amino acids. Most O. tsutsugamushi Anks also carry an F-box domain that is capable of interacting with SKP1 of the SCF1 ubiquitin ligase complex [32], which normally functions in eukaryotic cells to tag proteins with ubiquitin for degradation by the 26 s proteasome degradation pathway.

Ikeda strain of O. tsutsugamushi genome has 47 Ank genes (one of the highest number of O. tsutsugamushi strains) [33]. The Ank ORF is crucial for modulation of host cellular processes. To initiate the modulation, Anks translocated from pathogen to host cell.

Type 4 Secretion System (TFSS)

However, conjugation systems are rarely reported in intracellular bacteria, but O. tsutsugamushi genome is exceptional with 359 tra genes responsible for conjugative TFSS [34]. Conjugative TFSS mediate the transport of DNA among bacteria and transport several effector proteins into the host for successful initiation of infection [35]. TFSS tra genes are arranged into 24 fragmented repeat clusters. In Boryong strain of O. tsutsugamushi, there are reports of having 27 TPR (Tetratricopeptide repeats) and 50 ankyrin repeat proteins, among them several proteins reported to mediate DNA and protein interaction with host cell. Amid sequenced Rickettsiales, O. tsutsugamushi is the only sequenced species characterized by having full-length spotT/relA gene with both synthase and hydrolase catalytic residues [34].

Target Cells of O. tsutsugamushi

The target cells of O. tsutsugamushi can be classified as non-phagocytic and phagocytic cells, in which the former comprises endothelial and fibroblast cells and latter comprises phagocytic macrophages, polymorphonuclear leukocytes (PMNs), and dendritic cells in vitro and in vivo [36].

Adaptive Strategies of Host Cell by Generating Immune Response After ST Infection

After infection, Dendritic cells (DCs) play a key role for initializing antigen-specific immune response. DCs are one of the major antigen-presenting cells and play a crucial role to connect innate and adaptive immune response. Maturation of DCs is a multistep process, namely antigen uptake, migration, expression of co-stimulatory molecules on cell surface, and through the secretion of cytokine and chemokine [37].

Just after infection of O. tsutsugamushi, maturation of monocyte-derived DCs are induced which can be measured by enhanced expression of CD80, CD83, CD86, and MHC class I molecules; after induction, DCs show an increased level of IL-12p70, TNF-α, IL-6, and IL-8. Matured DCs interact with T cells and production of interferon (IFN γ) is stimulated. On one hand, the protective immunity against intracellular pathogen is provided by IFN γ [38]; on the other hand, IFN γ is also responsible to activate the production of reactive oxygen species in macrophage to kill the intracellular pathogen [39]. As the part of adaptive strategy of host to control the severity and antigen-specific immune response against O. tsutsugamushi several cytokines, for example, IL-6 and IL-8 activate lymphocytes and are responsible for neutrophil migration. IL-12 was reported to induce differentiation of TH cells [40]. After phagocytosed by DCs, intracellular pathogen (Shigella flexneri [41] and Listeria monocytogenes [42]) are reported to enter in a degradation pathway (phagolysis). Just after phagolysis, major histocompatibility complex (MHC) presentation is done by antigenic short peptide which in turn activate T cells [43]. However, O. tsutsugamushi is reported to overcome the phagolysis mechanism which is different from Shigella flexneri and Listeria monocytogenes [38].

Infection Mechanism in Target Cell by O. tsutsugamushi

Attachment of O. tsutsugamushi to Host Cell

To establish a successful infection and growth of O. tsutsugamushi inside the cell requires efficient invasion of host cell. Intracellular invasion mechanism is controlled by signal transduction events which are complex and have not been clearly described. However, this review tries to compile up the molecular mechanism of invasion based on available literature till date. Cho et al. demonstrated that O. tsutsugamushi utilizes host integrin signaling pathways to mediate actin cytoskeleton rearrangement for entry into non-phagocytic host cell [18].

To initiate the attachment to the host cell O. tsutsugamushi utilizes TSA56-Fibronectin complex to interact with host cell integrin α5β1 (Fig. 3) [18]. The formation of TSA56-Fibronectin and integrin complex induces activation of signaling molecules at the inner surface of cytoplasmic membrane [44]. The signaling molecule are non-receptor tyrosine kinase, namely FAK and Src family, which is responsible for the focal adhesion, FAK is a 125 kDa protein regarded as key player in integrin mediated signaling [45]. Just after FAK activation, RhoA GTPase becomes active and mediates rearrangement of actin cytoskeleton as well as promotes bacterial uptake [18]. O. tsutsugamushi invade into the host cell via a zipper-like mechanism which is mediated by Clathrin protein [9]

Fig. 3
figure 3

Molecular mechanism of attachment and internalization involving bacterial protein TSA56, ScaC, and host protein Fibronectin-integrin complex and Clathrin protein

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Evolutionary Strategy Adapted by O. tsutsugamushi to Actively Escape from Host Cell Autophagy

Autophagy, an evolutionarily conserved catabolic mechanism, is regulated intracellularly to degrade the cytosolic components, like misfolded protein aggregates and defaced organelles, in a lysosome-dependent manner [46]. ATGs, a highly conserved autophagy-related gene, regulate autophagy. Immune cells specifically adapted an autonomous effector mechanism of innate immunity for degradation of microorganisms invading intracellularly through autophagy. Several escape mechanisms were acquired by intracellular pathogens by blocking host autophagic defense mechanism or altering host autophagic response. This can be achieved by different mechanisms such as (a) antagonizing autophagy initiation or auto-phagosomal maturation, (b) escaping autophagic recognition, and (c) using host autophagy component for their own survival [47]. On the other way, host autophagy generates nutrient which is utilized by intracellular pathogen for survival and growth [47]. There are reports that endolysosomal pathway may be utilized as a protective intracellular niche by intracellular pathogen [29].

In the case of O. tsutsugamushi, several studies indicated that this bacterium activates cellular autophagy, but at the same time it evades cellular autophagic system without fusing the lysosome (Fig. 4) [48]. In O. tsutsugamushi-infected polymorphonuclear leukocyte (PMNs), more autophagosomes are found, within 1-h post-infection (hpi) [49]. Just after entering the host cell early endosome is formed. After some time, late endosome is formed and before fusion of the lysosome it escapes from autophagy mechanism of host cell (Fig. 4); this mechanism is not fully understood. When O. tsutsugamushi was cultured in L929 cells and a hemolysin gene, tlyC, encodes phospholipase D protein [50]. This protein may disrupt the phagosomal membrane and allow the pathogen to discharge into the host cell’s cytoplasm [51]. O. tsutsugamushi now moves toward perinuclear region via microtubule mediated trafficking [52]. At perinuclear region in polysaccharide matrix bacterial replication takes place [9].

Fig. 4
figure 4

Schematic overview of escaping and budding out mechanism of O. tsutsugamushi in host cell involves several steps. (1) Attachment and internalization, (2) Escape to cytosol, (3) Induction and escape from autophagy, (4) Intracellular transport, (5) Bacterial replication, (6) Intracellular transport toward the cell wall, (7) Exit from the host cell

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Adapted Strategies of O. tsutsugamushi Inside the Host Cell (Hijacking the Host Cell 26 s-Proteasomal Degradation Machinery)

O. tsutsugamushi, an obligate auxotroph, relies on the host cell for amino acid (histidine and aromatic amino acid) [53]. During initial stage of invasion (24–48 h) minimal growth of O. tsutsugamushi is reported, after that it leads to log phase [54]. Requirement of amino acid was found less in initial phase (24 h). The demand of amino acids gets increased when the pathogen is ready for optimal growth (48 h). The pathogen reported to modulate host cellular processes to support its exponential growth [55].

An unfolded protein response (UPR) is evoked due to the accumulation of misfolded protein-guided stress of endoplasmic reticulum (ER) [55]. The UPR is regarded as a protective cellular pathway and reported as evolutionarily conserved. It relieves the stress of ER via inhibition of translation, enhancing protein folding capacity of ER and facilitates endoplasmic reticulum associated degradation (ERAD) (Fig. 5) [56]. ER recognize and transport misfolded newly synthesized protein to the 26 s-proteasome for degradation [57]. With the help of 26 s-proteasome unfolded peptides are degraded into amino acids via aminopeptidase enzyme [58]. As per available reports, viruses and some intracellular bacteria (O. tsutsugamushi) either induce or inhibit the UPR as per their requirement [56]. Optimum expression of Ank4 was reported during the 24 h for UPR induction [55]. O. tsutsugamushi effector Ank4 protein is linked to induce the UPR and inhibit ERAD during 24–48 h of infection. After expression Ank4 binds with ERAD chaperon Bat3 to inhibit ERAD and accumulate the huge number of misfolded proteins until 72 h. After that, O. tsutsugamushi utilizes ERAD-derived amino acids to benefit its replication [55]

Fig. 5
figure 5

ERAD pathway for degradation of misfolded ER proteins through 26 s-proteasomal machinery via a translocation, b aggregation, and c degradation

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Camouflaging and Spreading of Infection

Every host cell has a carrying capacity of pathogens for its survival. After achieving the carrying capacity, every intracellular pathogen needs to exit from the infected cell. Intracellular pathogen has adapted several mechanisms to exit infected host cell as follows: (a) Lysis of host cell, e.g., Plasmodium falciparum and Chlamydia spp. (b) Efflux of vacuole (harboring bacteria) (Cryptococcus neoformans), and (c) Actin-mediated protuberance into adjacent cells (e.g., Shigella flexneri, Listeria monocytogenes, Rickettsia rickettsii) [52]. However, O. tsutsugamushi has adapted a unique mechanism to exit the host cell by utilizing an unusual budding out mechanism (bacteria encased via host cell membrane) [59]. This encasing enables infection of newly adjacent cells exempting extreme cellular environment and remain hidden from the host immune cells by camouflaging. The utilized host cell membrane for bacterial envelope are specialized membrane micro-domains referred as lipid raft [60]. Lipid rafts are rich in glycosylphosphatidylinositol (GPI)-linked molecules, cholesterol, and glycosphingolipids and contain a 22-kDa protein, caveolin-1 [61]. Several reports indicated that lipid raft bacteria interactions may be initial event for bacterial entry [62, 63], but in O. tsutsugamushi, disruption of lipid rafts has no significant effect on entry into host cells [60]. The encasing process depends on a bacterial surface antigen htrA, a 47-kDa protein, which binds some proteins of the lipid raft and this binding of htrA and lipid raft protein initiate the exit of bacterium via budding (Fig. 4) [60].

Antibiotics and Their Susceptibility for the Treatment of Scrub Typhus

The disease scrub typhus can vary from asymptomatic [64] to lethal but it mainly causes severe febrile illness. For the treatment of scrub typhus, the first effective treatment was introduced in the form of antibiotic chloramphenicol [65]. Later tetracycline group of antibiotics were introduced and found comparatively more effective than existing ones. Among tetracyclines, the antibiotic doxycycline was reported as drug of choice till date [66]. Antibiotics of class Tetracycline, principally doxycycline, is equally effective. Several other antibiotics were used for the treatment of scrub typhus. The list of antibiotics currently recommended to treat scrub typhus includes the following (Table 2).

Table 2 Antibiotics used for treatment of scrub typhus
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Antibiotics: Resistance and Challenges in Developing New Antibiotics

As earlier it was said that only “survival of the fittest” evolved & evolved generation will dominate [67]. After the invention of antibiotics, the diseases caused by bacteria were regarded as diseases of the past. But the same time, bacteria also develop many mechanism to withstand and multiply in the presence of antibiotics [68]. Only a few groups of antibiotics are effective against O. tsutsugamushi, but bacteria started to show less susceptibility toward the present treatment options. The resistance in fluoroquinolones and less susceptibility reports toward doxycycline and chloramphenicol presented a pressure on macrolides group of antibiotics (azithromycin). In the current scenario, it is crucial to understand the reason behind the less susceptibility of antibiotics. There may be many reasons reported till date, among them one of the latest report [69] suggests that the concentration required to kill the bacteria is not reaching to the pathogen. If the right drug will not be prescribed for the right time, then there is no life for the existing antibiotic and in this race, it is evident that bacteria adapted smartly than the human being.

From the golden age of antibiotics till date, several drug-resistant bacteria (Methicillin-resistant Staphylococcus aureus (MRSA), Multidrug-resistant Mycobacterium tuberculosis (MDRTB), Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species (ESKAPE), and DIRTY DOZEN) have been developed [70]. Initially, MRSA and MDRTB directed the discovery and development of antibiotics [70]. After development of resistance in ESKAPE pathogens, the direction of discovery of antibiotics retracted toward these pathogens [71]. In 2017, World health Organization (WHO) again emphasized the need of new antibiotics against dirty dozen [71].

In this time duration, the neglected pathogens O. tsutsugamushi started to develop resistance toward the available antibiotics [72]. This bacteria is not only neglected in terms of scientific discovery but also in the field of drug discovery [69]. Doxycycline, chloramphenicol, fluoroquinolone, and rifampicin were the drugs which became choice of treatment in due time [17]. The first less susceptibility was reported in 1995 against Doxycycline [16]. From 1995 to till date, O. tsutsugamushi acquired resistance/less susceptibility toward other available drugs except azithromycin (Table 3) [73].

Table 3 Antibiotics showing less susceptibility or resistance
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As per report [74], there are unique and highly variable antibiotic resistance loci present in O. tsutsugamushi genomes in comparison with other rickettsial species. As earlier said, the genome of O. tsutsugamushi is highly repetitive. It possesses 2179 potential protein-coding loci. Among these, the number of presumed antibiotic-resistant loci is very low. Bacteria encode CRISPR-like elements that is more than 400 transposes, 60 phage integrases, and 70 reverse transcriptases in the major part of their protein-coding loci, explaining their capability to modify their genome under selection [34]. The O. tsutsugamushi was known to have high antigenic diversity. In India, for instance, Kato-like strains predominate (61.5%), followed by Karp-like strains (27.7%) and Gilliam and Ikeda strains [75]. In O. tsutsugamushi, genetic recombination occurs among diverse genotypes. The significant diversity and heterogeneity of putative antibiotic-resistant loci help to acquire antibiotic resistance under selection pressure and act as a challenge in developing new antibiotics.

Drug resistance in O. tsutsugamushi developed via horizontal gene transfer. O. tsutsugamushi genomes carry 359 tra genes which code integral components of conjugative type IV secretion system through which resistance genes are transferred horizontally. abaF, an efflux pump gene gets mutated due to antibiotic resistance and expressed actively. Another gene gyrA responsible for DNA gyrase enzyme gets mutated and becomes resistant to quinolones. [74]

New Approach for Effective Treatment and Drug in Pipeline

For treatment of scrub typhus, combination of antibiotics now became an attractive strategy to overcome the resistance. Combination therapy presents the best alternative to monotherapy due to the effectiveness and delay in development of resistance. A recent study by Varghese et al. reported the combination of doxycycline and azithromycin showed better results than giving alone [83]. The mode of actions of both the antibiotics are different, azithromycin binds the 23SrRNA of the 50S ribosomal subunit at the polypeptide exit tunnel, and doxycycline prevents aminoacyl-tRNA binding to the 30S ribosomal subunit, which results in complete blockage of protein synthesis with a consequently greater effect against O. tsutsugamushi [83].

A natural antibiotic Corallopyronin A isolated from an environmental soil bacteria named as Corallococcus coralloides is a new, highly effective agent for treating scrub typhus. Corallopyronin A (CorA) is a myxobacterial α-pyrone antibiotic which includes two side chains. Corallopyronin A was isolated from environmental bacteria and targets a bacterial enzyme called RNA polymerase. In preclinical trials, even low doses of the new antibiotic have proven to be very effective against Orientia tsutsugamushi [84].

Alternatives of Antibiotics

At present, antibiotics are the major weapon to treat the Scrub typhus, but development of less susceptibility/resistance against available antibiotics put our generation in dark [85]. This compelled us to think for the drugs beyond antibiotics and natural products became need of the hour for treating this neglected disease [86]. Nature and natural products always become the major supporter and guide for the humans being in the drug discovery [87]. Secondary metabolites of plants became the wonder drug antibiotics and became the major life-saving agent [14]. Plants secondary metabolites became one of the major source of new drugs or their molecules/compounds became the blueprint for the development of novel drugs [86]. As per report of WHO, maximum population depends on the natural products for their primary treatment [88]. Historically Indian and Chinese medicines are based on natural products. Several anti-parasitic and anti-cancerous drugs were developed from plants [89]. Several plants secondary metabolites like alkaloids, organosulfur compounds, terpenoids, and flavonoids reported to have antimicrobial activities for several bacterial pathogens [86]. In the era of drug resistance, these plants’ secondary metabolites are being observed as promising alternatives of antibiotics (Table 4).

Table 4 Antibacterial agents from plants
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Saponins, allicin, apigenin, eugenol, curcumin, piperine, gallic acid, kaempferol, etc. are the plant secondary metabolites which are used in clinical trials for the discovery and development as anti-infectives, but these compounds need to be repurposed as antibacterials for the treatment of Scrub typhus [86, 91,92,93,94,95,96,97]. The potential challenges for drug development of these compounds are as follows:

  1. 1

    In vitro antibacterial potential is found good but in preclinical and clinical stage maximum compounds do not qualify due to less efficacy and toxicity.

  2. 2

    The high cost and long time for the drug development.

  3. 3

    Secondary metabolite concentration varies from place to place and its industrial production affects secondary metabolite concentration.

Multiple secondary plant metabolites have been reported to be used for the treatment of MDR pathogens. But these researches are in infancy stage; similarly, Ayurveda recommends different plants (Veratrum, Belladonna, Gelsemium, Hyoscyamus, Dostemia contrayerba, Melissa officinalis) for the treatment of Scrub typhus [98]. These plants are reported to cure high-grade fever, sepsis, & complications of respiratory & nervous system.

Vaccine Development and Challenges Associated with it

Over the past 80 years, despite of many attempts to develop vaccine against O. tsutsugamushi, none of the attempts resulted in effective vaccine. To develop vaccines, many approaches were used, namely (1) cotton rat’s lungs infected with O. tsutsugamushi that were formalin-fixed homogenized, (2) formalin-killed O. tsutsugamushi, (3) live strain of O. tsutsugamushi with low virulence and inoculation of live virulent strain followed by antibiotic treatment, and (4) live irradiated O. tsutsugamushi and its recombinant fusion of 56-kDa and 47-kDa proteins [99]

The above vaccines have their own short comings either it may provide strong protection against homologous challenge but weak protection against heterologous challenge or it may provide active immunity by sustaining antibodies for only a short duration which diminishes through time [100]. In humans, homologous immunity lasts up to 3.5 years but heterologous immunity lasts only a few months and humans can get infected by multiple strains simultaneously [99]. The lack of knowledge for a common antigen for most of O. tsutsugamushi strains which can stimulate both cell-mediated and humoral immunity is the main issue in developing an effective clinical vaccine.

Future

In view of the novel cell biology of O. tsutsugamushi, there is need of further studies for deeper understanding of role of intracellular events, such as autophagy in scrub typhus pathogenesis. Further, investigations are needed to understand the role of biphasic metabolic differences between intracellular and extracellular bacterial stages in Orientia infection. O. tsutsugamushi has been reported to cause chronic latent infection, but the mechanism needs to be investigated in detail. However, there is limited knowledge of immune invasion strategies utilized by Orientia. Hence, a combined approach will be needed to understand the molecular, cellular, host–cell interaction, pathogenesis, genomic diversity, etc. Moreover, there are mixed reports of antibiotic susceptibility/resistance to available antibiotics, this needs to address precisely. There are limited antibiotics available to treat scrub typhus and there are also reports of antibiotic susceptibility/resistance to available antibiotics in Orientia. Thus, this is the need of hour to identify new antibiotics, repurposing of available drugs, and identification of novel drug targets to treat scrub typhus.

Summary

The earliest life forms (prokaryotes) are omnipresent and flourish in any type of environment, while evolutionarily evolved eukaryotes would die. This indicates that during the time of evolution robust environmental conditions make prokaryotes more resilient. O. tsutsugamushi adapted several mechanisms to become a model intracellular organism. Its unique cellular components make it a vibrant pathogen to evade the host cell machinery for chronic infection. At present, O. tsutsugamushi is not limited to the tsutsugamushi triangle but started to emerge different parts of the world. Less susceptibility toward available antibiotics makes it important to bring O. tsutsugamushi outside the neglected disease category. The discovery and development of novel multiplex subunit vaccines and broad-spectrum antibiotics will be the future research priority to overcome the acute and chronic infections caused by O. tsutsugamushi.