Interaction of constituents of MDT regimen for leprosy with Mycobacterium leprae HSP18: impact on its structure and function.

Mycobacterium leprae, the causative organism of leprosy, harbors many antigenic proteins, and one such protein is the 18-kDa antigen. This protein belongs to the small heat shock protein family and is commonly known as HSP18. Its chaperone function plays an important role in the growth and survival of M. leprae inside infected hosts. HSP18/18-kDa antigen is often used as a diagnostic marker for determining the efficacy of multidrug therapy (MDT) in leprosy. However, whether MDT drugs (dapsone, clofazimine, and rifampicin) do interact with HSP18 and how these interactions affect its structure and chaperone function is still unclear. Here, we report evidence of HSP18-dapsone/clofazimine/rifampicin interaction and its impact on the structure and chaperone function of HSP18. These three drugs interact efficiently with HSP18 (having submicromolar binding affinity) with 1 : 1 stoichiometry. Binding of these MDT drugs to the 'α-crystallin domain' of HSP18 alters its secondary structure and tryptophan micro-environment. Furthermore, surface hydrophobicity, oligomeric size, and thermostability of the protein are reduced upon interaction with these three drugs. Eventually, all these structural alterations synergistically decrease the chaperone function of HSP18. Interestingly, the effect of rifampicin on the structure, stability, and chaperone function of this mycobacterial small heat shock protein is more pronounced than the other two MDT drugs. This reduction in the chaperone function of HSP18 may additionally abate M. leprae survivability during multidrug treatment. Altogether, this study provides a possible foundation for rational designing and development of suitable HSP18 inhibitors in the context of effective treatment of leprosy.

Autophagy-Associated IL-15 Production Is Involved in the Pathogenesis of Leprosy Type 1 Reaction.

Leprosy reactional episodes are acute inflammatory events that may occur during the clinical course of the disease. Type 1 reaction (T1R) is associated with an increase in neural damage, and the understanding of the molecular pathways related to T1R onset is pivotal for the development of strategies that may effectively control the reaction. Interferon-gamma (IFN-γ) is a key cytokine associated with T1R onset and is also associated with autophagy induction. Here, we evaluated the modulation of the autophagy pathway in Mycobacterium leprae-stimulated cells in the presence or absence of IFN-γ. We observed that IFN-γ treatment promoted autophagy activation and increased the expression of genes related to the formation of phagosomes, autophagy regulation and function, or lysosomal pathways in M. leprae-stimulated cells. IFN-γ increased interleukin (IL)-15 secretion in M. leprae-stimulated THP-1 cells in a process associated with autophagy activation. We also observed higher IL15 gene expression in multibacillary (MB) patients who later developed T1R during clinical follow-up when compared to MB patients who did not develop the episode. By overlapping gene expression patterns, we observed 13 common elements shared between T1R skin lesion cells and THP-1 cells stimulated with both M. leprae and IFN-γ. Among these genes, the autophagy regulator Translocated Promoter Region, Nuclear Basket Protein (TPR) was significantly increased in T1R cells when compared with non-reactional MB cells. Overall, our results indicate that IFN-γ may induce a TPR-mediated autophagy transcriptional program in M. leprae-stimulated cells similar to that observed in skin cells during T1R by a pathway that involves IL-15 production, suggesting the involvement of this cytokine in the pathogenesis of T1R.

Identification of sensitive indicators in immune response for leprosy affected patients: An observational clinical study of safety and immunogenicity of influenza vaccine.

ABSTRACT: Cured leprosy patients have special physical conditions, which could pose challenges for safety and immunogenicity after immunization. We performed an observational clinical study aimed to identify the safety and immunogenicity of influenza vaccine in cured leprosy patients. A total of 65 participants from a leprosarium were recruited into leprosy cured group or control group, and received a 0.5 ml dose of the inactivated split-virion trivalent influenza vaccine and a follow-up 28 days proactive observation of any adverse events. Hemagglutination and hemagglutination inhibition test was performed to evaluate serum antibody titer, flow cytometry was conducted to screen of cytokines level. The total rate of reactogenicity was 0.0% [0/41] in leprosy cured group and 37.5% [9/24] in control group. The seroconversion rate for H1N1 was difference between leprosy cured group and control group (41.83% vs 79.17%, P = .0082), but not for H3N2 (34.25% vs 50.00%, P = .4468). At day 0, leprosy cured group have relatively high concentration of interleukin-6, interleukin-10, tumor necrosis factor, interferon-γ, and interleukin-17 compared to control group. The interleukin-2 concentration increased 2 weeks after vaccination compared to pre-vaccination in leprosy cured group, but declined in control group (0.92 pg/ml vs -0.02 pg/ml, P = .0147). Leprosy cured group showed a more rapid down-regulation of interleukin-6 when influenza virus was challenged compared to control group (-144.38 pg/ml vs -11.52 pg/ml, P < .0001). Subgroup analysis revealed that the immunization administration declined interleukin-17 concentration in Tuberculoid type subgroup, but not in Lepromatous type subgroup or control group. Clinically cured leprosy patients are relatively safe for influenza vaccine. Leprosy cured patient have immune deficit in producing antibody. Interleukin-6 and interleukin-17 were 2 sensitive indicators in immune response for leprosy affected patients. The identification of indicators might be help management of leprosy and used as predictive markers in leprosy early symptom monitoring.

The cellular architecture of the antimicrobial response network in human leprosy granulomas.

Granulomas are complex cellular structures composed predominantly of macrophages and lymphocytes that function to contain and kill invading pathogens. Here, we investigated the single-cell phenotypes associated with antimicrobial responses in human leprosy granulomas by applying single-cell and spatial sequencing to leprosy biopsy specimens. We focused on reversal reactions (RRs), a dynamic process whereby some patients with disseminated lepromatous leprosy (L-lep) transition toward self-limiting tuberculoid leprosy (T-lep), mounting effective antimicrobial responses. We identified a set of genes encoding proteins involved in antimicrobial responses that are differentially expressed in RR versus L-lep lesions and regulated by interferon-γ and interleukin-1ß. By integrating the spatial coordinates of the key cell types and antimicrobial gene expression in RR and T-lep lesions, we constructed a map revealing the organized architecture of granulomas depicting compositional and functional layers by which macrophages, T cells, keratinocytes and fibroblasts can each contribute to the antimicrobial response.

Reverse vaccinology and subtractive genomics approaches for identifying common therapeutics against Mycobacterium leprae and Mycobacterium lepromatosis

Mycobacterium leprae and Mycobacterium lepromatosis are gram-positive bacterial pathogens and the causative agents of leprosy in humans across the world. The elimination of leprosy cannot be achieved by multidrug therapy alone, and highlights the need for new tools and drugs to prevent the emergence of new resistant strains. Methods In this study, our contribution includes the prediction of vaccine targets and new putative drugs against leprosy, using reverse vaccinology and subtractive genomics. Six strains of Mycobacterium leprae and Mycobacterium lepromatosis (4 and 2 strains, respectively) were used for comparison taking Mycobacterium leprae strain TN as the reference genome. Briefly, we used a combined reverse vaccinology and subtractive genomics approach. Results As a result, we identified 12 common putative antigenic proteins as vaccine targets and three common drug targets against Mycobacterium leprae and Mycobacterium lepromatosis. Furthermore, the docking analysis using 28 natural compounds with three drug targets was done. Conclusions The bis-naphthoquinone compound Diospyrin (CID 308140) obtained from indigenous plant Diospyros spp. showed the most favored binding affinity against predicted drug targets, which can be a candidate therapeutic target in the future against leprosy.(AU)

Experimental Modeling of Leprosy in BALB/c, BALB/c Nude, CBA, and C57BL/6ТNF-/- Mice.

Leprosy was modeled in an experiment on BALB/c, BALB/cNude, CBA, and C57BL/6ТNF-/- mice using three Mycobacterium leprae strains obtained from patients with a diagnosis of A30 according to ICD-10 from different regions of the Russian Federation. Proliferation of M. leprae of the used strains showed a temporal-quantitative dependence on the used mouse line. CBA and BALB/cNude mice were optimal for strain R and BALB/c and BALB/cNude lines were optimal for strain I. BALB/cNude mice infected with strain I had low lifespan. M. leprae strain M showed low proliferation activity in BALB/cNude and C57BL/6ТNF-/- mice.

Leprosy piRnome: exploring new possibilities for an old disease.

Leprosy, which is caused by the human pathogen Mycobacterium leprae, causes nerve damage, deformity and disability in over 200,000 people every year. Because of the long doubling time of M. leprae (13 days) and the delayed onset of detectable symptoms, which is estimated to be approximately 3-7 years after infection, there is always a large percentage of subclinically infected individuals in the population who will eventually develop the disease, mainly in endemic countries. piRNAs comprise the largest group of small noncoding RNAs found in humans, and they are distinct from microRNAs (miRNAs) and small interfering RNAs (siRNAs). piRNAs function in transposon silencing, epigenetic regulation, and germline development. The functional role of piRNAs and their associated PIWI proteins have started to emerge in the development of human cancers and viral infections, but their relevance to bacterial diseases has not been investigated. The present study reports the piRNome of human skin, revealing that all but one of the piRNAs examined are downregulated in leprosy skin lesions. Considering that one of the best characterized functions of piRNAs in humans is posttranscriptional mRNA silencing, their functions are similar to what we have described for miRNAs, including acting on apoptosis, M. leprae recognition and engulfment, Schwann cell (SC) demyelination, epithelial-mesenchymal transition (EMT), loss of sensation and neuropathic pain. In addition to new findings on leprosy physiopathology, the discovery of relevant piRNAs involved in disease processes in human skin may provide new clues for therapeutic targets, specifically to control nerve damage, a prominent feature of leprosy that has no currently available pharmaceutical treatment.

Clinicobacteriological evaluation of leprosy patients with 1-5 skin lesions.

Background: Mycobacterium leprae is a noncultivable mycobacteria, and diagnosis of the disease is based on its clinical and histopathological characteristics and finding the bacteria in skin scrapings and in biopsies taken from the patients. The aim of this study was to shed light on the clinical classification (based on the number of skin lesions) used extensively in the field where patients classified as paucibacillary (PB) were positive on skin smears and histopathology leading to treatment failure and drug resistance. Methods: In this study, we enrolled untreated 62 leprosy patients with 1-5 skin lesions and did a detailed bacterio-histopathological analysis by slit-skin smears (SSSs) and histopathology. Results: Of 62 patients analyzed, 15 patients came out to be multibacillary (MB) and 47 were PB by SSS and histopathology. Conclusion: The findings of the present study showed that the WHO classification of leprosy based on the number of lesions seems to be inappropriate as it considers a number of MB lesions as PB only, thus misleading the treatment strategies. Hence, it is essential that a comprehensive clinicobacteriological assessment of leprosy cases should be done to ensure the appropriate bacillary status and guiding the appropriate treatment strategy.

Metabolism and interactions of antileprosy drugs.

Leprosy is a chronic infectious disease caused my Mycobacterium leprae that primarily affects peripheral nervous system and extremities and is prevalent in tropical countries. Treatment for leprosy with multidrug regimens is very effective compared to monotherapy especially in multibacillary cases. The three major antileprosy drugs currently in use are 4, 4'-diaminodiphenyl sulfone (DDS, dapsone), rifampicin, and clofazimine. During multidrug therapy, the potent antibiotic rifampicin induces the metabolism of dapsone, which results in decreased plasma half-life of dapsone and its metabolites. Furthermore, rifampicin induces its own metabolism and decreases its half-life during monotherapy. Rifampicin upregulates several hepatic microsomal drug-metabolizing enzymes, especially cytochrome P450 (CYP) family that in turn induce the metabolism of dapsone. Clofazimine lacks significant induction of any drug-metabolizing enzyme including CYP family and does not interact with dapsone metabolism. Rifampicin does not induce clofazimine metabolism during combination treatment. Administration of dapsone in the acetylated form (acedapsone) can release the drug slowly into circulation up to 75 days and could be useful for the effective treatment of paucibacillary cases along with rifampicin. This review summarizes the major aspects of antileprosy drug metabolism and drug interactions and the role of cytochrome P450 family of drug metabolizing enzymes, especially CYP3A4 during multidrug regimens for the treatment of leprosy.

Myelin breakdown favours Mycobacterium leprae survival in Schwann cells.

Leprosy neuropathy is a chronic degenerative infectious disorder of the peripheral nerve caused by the intracellular obligate pathogen Mycobacterium leprae (M. leprae). Among all nonneuronal cells that constitute the nerve, Schwann cells are remarkable in supporting M. leprae persistence intracellularly. Notably, the success of leprosy infection has been attributed to its ability in inducing the demyelination phenotype after contacting myelinated fibres. However, the exact role M. leprae plays during the ongoing process of myelin breakdown is entirely unknown. Here, we provided evidence showing an unexpected predilection of leprosy pathogen for degenerating myelin ovoids inside Schwann cells. In addition, M. leprae infection accelerated the rate of myelin breakdown and clearance leading to increased formation of lipid droplets, by modulating a set of regulatory genes involved in myelin maintenance, autophagy, and lipid storage. Remarkably, the blockage of myelin breakdown significantly reduced M. leprae content, demonstrating a new unpredictable role of myelin dismantling favouring M. leprae physiology. Collectively, our study provides novel evidence that may explain the demyelination phenotype as an evolutionarily conserved mechanism used by leprosy pathogen to persist longer in the peripheral nerve.

Epitope imprinting of Mycobacterium leprae bacteria via molecularly imprinted nanoparticles using multiple monomers approach.

Mycobacterium leprae causes endemic disease leprosy which becomes chronic if not treated timely. To expedite this 'timely diagnosis', and that also at an early stage, here an attempt is made to fabricate an epitope-imprinted sensor. A molecularly imprinted polymer nanoparticles modified electrochemical quartz crystal microbalance sensor was developed for sensing of Mycobacterium leprae bacteria through its epitope sequence. Multiple monomers, 3-sulphopropyl methacrylate potassium salt, benzyl methacrylate and 4-aminothiophenol were utilized to imprint this bacterial epitope. Imprinted nanoparticles were electropolymerized on gold coated quartz electrode. The sensor was able to show specific binding towards the blood samples of infected patients, even in the presence of 'matrix' and other plasma proteins such as albumin and globulin. Even other peptide sequences, similar to epitope sequences only with two amino acid mismatches were also unable to show any binding. Sensor withstood analytical tests viz. selectivity, specificity, matrix effect, detection limit (0.161 nM), quantification limit (and 0.536 nM), reproducibility (RSD 2.01%). Hence a diagnostic tool for bacterium causing leprosy is successfully fabricated in a facile manner which will broaden the clinical access and efficient population screening can be made feasible.

Immunodiagnostic of leprosy exploiting a photoelectrochemical platform based on a recombinant peptide mimetic of a Mycobacterium leprae antigen.

The first serum diagnosis of leprosy based on the detection of antibodies of patients using a recombinant mimetic peptide (PGL1M3R) as recognition element and exploiting a photoelectrochemical sensor is presented in this work. The photoeletrochemical platform consists of cadmium sulphide and nickel hydroxide electrodeposited on fluorine-doped tin oxide coated glass slide (CdS/Ni(OH)2/FTO). The optical band gap and flat band potential of the photoelectroactive materials were evaluated by UV-Vis spectroscopy and electrochemical impedance spectroscopy. The spatial photoelectrochemical response of the platform was evaluated by Scanning Electrochemical Microscopy and the morphology of the films was investigated by Scanning Electron Microscopy (SEM). The photoelectrochemical response of the CdS/Ni(OH)2/FTO platform was optimized by evaluating the effects of the kind, concentration, and pH of the buffer. Furthermore, the applied potential to the CdS/Ni(OH)2/FTO platform was also investigated. The CdS/Ni(OH)2/FTO photoelectrochemical platform was modified with a synthetic peptide by using glutaraldehyde as cross-linking reagent and chitosan (CS) for the covalent coupling of the peptide to the photoelectrochemical platform (PGL1M3R/CdS/Ni(OH)2/FTO). The photoelectrochemical immunosensor is able to distinguishing between positive and negative leprosy human sera samples diluted from 1:640 up to 1:10240. Furthermore, to test the specificity of the sensor, samples from tuberculosis and leishmaniasis patients were analyzed using the proposed photoelectrochemical immunosensor.

Neutrophil extracellular traps contribute to the pathogenesis of leprosy type 2 reactions.

Up to 50% of patients with the multibacillary form of leprosy are expected to develop acute systemic inflammatory episodes known as type 2 reactions (T2R), thus aggravating their clinical status. Thalidomide rapidly improves T2R symptoms. But, due to its restricted use worldwide, novel alternative therapies are urgently needed. The T2R triggering mechanisms and immune-inflammatory pathways involved in its pathology remain ill defined. In a recent report, we defined the recognition of nucleic acids by TLR9 as a major innate immunity pathway that is activated during T2R. DNA recognition has been described as a major inflammatory pathway in several autoimmune diseases, and neutrophil DNA extracellular traps (NETs) have been shown to be a prime source of endogenous DNA. Considering that neutrophil abundance is a marked characteristic of T2R lesions, the objective of this study was to investigate NETs production in T2R patients based on the hypothesis that the excessive NETs formation would play a major role in T2R pathogenesis. Abundant NETs were found in T2R skin lesions, and increased spontaneous NETs formation was observed in T2R peripheral neutrophils. Both the M. leprae whole-cell sonicate and the CpG-Hlp complex, mimicking a mycobacterial TLR9 ligand, were able to induce NETs production in vitro. Moreover, TLR9 expression was shown to be higher in T2R neutrophils, suggesting that DNA recognition via TLR9 may be one of the pathways triggering this process during T2R. Finally, treatment of T2R patients with thalidomide for 7 consecutive days resulted in a decrease in all of the evaluated in vivo and ex vivo NETosis parameters. Altogether, our findings shed light on the pathogenesis of T2R, which, it is hoped, will contribute to the emergence of novel alternative therapies and the identification of prognostic reactional markers in the near future.

Development of a combined RLEP/16S rRNA (RT) qPCR assay for the detection of viable M. leprae from nasal swab samples.

BACKGROUND: Leprosy continues to be a health problem in endemic areas. More than 200,000 new cases of leprosy per year suggest that transmission of the disease is still ongoing, presumably as airborne infection through nasal droplets. Late diagnosis supports continued transmission and increases the individual risk for functional disabilities. Laboratory tools are considered beneficial to facilitate early detection and clinical assessment of cases. The aim of this study was to validate molecular tools allowing detection, quantification and assessment of viability of M. leprae from nasal swab samples which are easy to obtain without the need of any invasive procedures. METHODS: Validation of two real-time PCRs detecting M. leprae DNA (RLEP qPCR) and RNA (16S rRNA RT qPCR) was conducted on "must not detect"/"must detect" samples and 160 pre-treatment nasal swab samples from 20 clinically diagnosed multibacillary (MB) leprosy patients from Togo. RESULTS: Both assays were 100% M. leprae specific and showed analytical sensitivities of three templates each. Out of 20 clinically diagnosed MB leprosy patients, 15 (75.0%) had a positive RLEP qPCR result from nasal swab samples. The 16S rRNA RT qPCR detected viable bacilli in nasal swab samples of ten out of these 15 RLEP positive patients (66.7%). CONCLUSION: The combined RLEP/16S rRNA (RT) qPCR assay provides a sensitive and specific tool to determine the bacterial load and viability of M. leprae from nasal swab samples and is applicable for early diagnosis, monitoring treatment response and investigating the role of nasal carriage of M. leprae in human-to-human transmission through aerosol infection.

Invasion of human microvascular endothelial cells by Mycobacterium leprae through Mce1A protein.

In patients with lepromatous leprosy, Mycobacterium leprae is often observed inside the human microvascular endothelial cells (HMVEC) surrounding Schwann cells (SC) at the site of lesions in the peripheral nerves. Based on this observation, it is considered that the nasal mucous may be the invasion pathway for M. leprae and HMVEC serve as an important reservoir for the bacteria before they invade SC. In light of previous research which revealed that Mce1A protein mediates bacterial invasion into nasal epithelial cells and HMVEC, we conducted a study to determine whether the invasion of M. leprae into HMVEC can be suppressed by blocking the Mce1A protein. In this study, we analyzed bacterial invasive activity by adding recombinant Escherichia coli, which express the active region (InvX:72 a.a.) of Mce1A protein on their external membrane, into cultured HMVEC, using the adhesin involved in the diffuse adherence mechanism. The number of bacteria that invaded into the cells was then measured by a colony counting method. The active region of Mce1A was divided into four sections, and hyperimmune antisera was prepared for each section for analyzing the inhibitory effect against invasion. The invasive activity was suppressed by antibodies against InvX regions 1-24 a.a., 25-46 a.a. and 58-72 a.a. This suggests that the InvX regions 1-24 a.a., 25-46 a.a. and 58-72 a.a. of Mce1A protein play an important role in the invasion of M. leprae into HMVEC and that it may be possible to suppress entry of M. leprae in HMVEC with antibodies against these regions.

Archival, paleopathological and aDNA-based techniques in leprosy research and the case of Father Petrus Donders at the Leprosarium 'Batavia', Suriname.

OBJECTIVE: We assessed whether Petrus Donders (died 1887), a Dutch priest who for 27 years cared for people with leprosy in the leprosarium Batavia, Suriname, had evidence of Mycobacterium (M.) leprae infection. A positive finding of M. leprae ancient (a)DNA would contribute to the origin of leprosy in Suriname. MATERIALS: Skeletal remains of Father Petrus Donders; two additional skeletons excavated from the Batavia cemetery were used as controls. METHODS: Archival research, paleopathological evaluation and aDNA-based testing of skeletal remains. RESULTS: Neither archives nor inspection of Donders skeletal remains revealed evidence of leprosy, and aDNA-based testing for M. leprae was negative. We detected M. leprae aDNA by RLEP PCR in one control skeleton, which also displayed pathological lesions compatible with leprosy. The M. leprae aDNA was genotyped by Sanger sequencing as SNP type 4; the skeleton displayed mitochondrial haplogroup L3. CONCLUSION: We found no evidence that Donders contracted leprosy despite years of intense leprosy contact, but we successfully isolated an archaeological M. leprae aDNA sample from a control skeleton from South America. SIGNIFICANCE: We successfully genotyped recovered aDNA to a M. leprae strain that likely originated in West Africa. The detected human mitochondrial haplogroup L3 is also associated with this geographical region. This suggests that slave trade contributed to leprosy in Suriname. LIMITATIONS: A limited number of skeletons was examined. SUGGESTIONS FOR FURTHER RESEARCH: Broader review of skeletal collections is advised to expand on diversity of the M. leprae aDNA database.

Delayed Diagnosis of Leprosy in a Micronesian Soldier - Case Report.

Lepromatous leprosy represents a cutaneous infection by the bacterium Mycobacterium leprae. Once considered a common, fatal disease, leprosy has become increasingly rare with modern, inexpensive antibiotics. Most healthcare workers will never encounter a case of leprosy due to the low prevalence of the disease. However, military physicians, through deployments and contact with foreign-born servicemembers, are one of the first lines of defense against this disease. With an unknown method of transmission and an insidiously slow replication, it can take years for the disease to fully manifest. There are multiple cutaneous manifestations associated with the infection that can mimic other infectious etiologies, stalling appropriate diagnosis and treatment. To determine which treatment course is recommended requires evaluation of disease dissemination and the level of host immune response. As the incidence of reported leprosy cases continues to decline, disease education on diagnosis and treatment is imperative to enhance early detection and intervention. Understanding the populations at risk for leprosy and its insidious presentation will aid the practitioner in minimizing disease burden for both U.S. servicemembers and our foreign partners.

Looking Backward To Move Forward: the Utility of Sequencing Historical Bacterial Genomes.

Many pathogens that caused devastating disease throughout human history, such as Yersinia pestis, Mycobacterium tuberculosis, and Mycobacterium leprae, remain problematic today. Historical bacterial genomes represent a unique source of genetic information and advancements in sequencing technologies have allowed unprecedented insights from this previously understudied resource. This minireview brings together example studies which have utilized ancient DNA, individual historical isolates (both extant and dead) and collections of historical isolates. The studies span human history and highlight the contribution that sequencing and analysis of historical bacterial genomes have made to a wide variety of fields. From providing retrospective diagnosis, to uncovering epidemiological pathways and characterizing genetic diversity, there is clear evidence for the utility of historical isolate studies in understanding disease today. Studies utilizing historical isolate collections, such as those from the National Collection of Type Cultures, the American Type Culture Collection, and the Institut Pasteur, offer enhanced insight since they typically span a wide time period encompassing important historical events and are useful for the investigating the phylodynamics of pathogens. Furthermore, historical sequencing studies are particularly useful for looking into the evolution of antimicrobial resistance, a major public health concern. In summary, although there are limitations to working with historical bacterial isolates, especially when utilizing ancient DNA, continued improvement in molecular and sequencing technologies and the resourcefulness of investigators mean this area of study will continue to expand and contribute to the understanding of pathogens.