Microbiology Research 2013; volume 4:e4

Antibacterial activities Introduction Correspondence: Mark Merchant, DEof Bioche - of serum from the Komodo mistry, McNeese State University Dragon (Varanus komodoensis) The Komodo dragon (Varanus komodoensis) Lake Charles, LA 70609, USA. Tel. +1.337.4755773 is an endangered species of monitor lizard E-mail: [email protected] Mark Merchant,1 Danyell Henry,1 indigenous to only five islands in the Lesser Key words: innate immunity, lizard, reptile, Rodolfo Falconi,1 Becky Muscher,2 Sunda region of southeast Indonesia.1 It is the serum complement, varanid. Judith Bryja3 largest lizard in the world, reaching lengths of 2 1Department of Chemistry, McNeese more than 3 meters. These reptiles primarily Acknowledgements: the authors wish to acknowl- State University, Lake Charles, LA; inhabit the tropical savannahs, and their edge the help of San Antonio Zoo employees: J. 2Department of Herpetology, San boundary woodlands, of these islands. They Stephen McCusker (Director), Alan Kardon 3 (Curator of Herpetology and Aquarium), and Rob Antonio Zoo, TX; 3Department of feed as both scavenger and predators, eating a broad spectrum of food items. Coke and Jenny Nollman (Veterinarians), and Herpetology, Houston Zoo, TX, USA Komodo dragons kill prey items with a lethal Houston Zoo employees Rick Barongi (Director), bite that is believed to inject toxic bacteria into Stan Mays (Director of Herpetology), and Maryanne Tocidlowski (Veterinarian), for per- the blood stream of these animals.4-5 However, mission to work with zoo animals and collection Abstract recent studies have shown that Komodo drag- of samples. In addition, we also thank techni- on saliva also contains a potent and complex cians Courtney Threadgill, Candice Robinson, venom, which causes anticoagulation and Komodo dragons (Varanus komodoensis) and Herpetology/Aquarium staff (San Antonio induction of systemic shock in prey animals.6 Zoo) for help in restraining animals during blood are able to feed on large prey items by inject- After days, or even weeks, depending on the collection. ing a dose of toxic bacteria with their bite that, size of the prey and the number of bites, the over time, kills the prey by systemic infection. prey succumbs to the lethal effects of systemic Contributions: MM, provided grant funding, Dragons also suffer bites from other members infection or shock. However, despite the fact designed the experiments for the study, conduct- of their own species during territorial disputes that Komodo dragons fight and inflict bites on ed some of the laboratory work, wrote the major- and feeding frenzies. However, they do not suf- ity ofonly the manuscript; DHRF conducted the major- each other,2 these lizards are not known to be fer the same fate as their prey, suggesting that ity of the laboratory work, wrote portions of the affected by septic infections. This would seem they have developed a strong immunity to bac- manuscript; BM, JB, conceived of the general to indicate that these animals have developed terial infections. This study was undertaken to idea of the study, coordinated the collection of some type of immune mechanism(s) that pro- determine the antibacterial activities of serum usesamples, made small contributions to the devel- tect against potential sepsis due to bites from from the Komodo dragon. Bacterial cultures opment of the manuscript. other Komodo dragons. were treated with different volumes serum Despite the endangered status of the Funding: this research was funded by a McNeese from Varanus komodoensis and the growth was Komodo dragon, practically nothing is known State University Alumni Association Faculty monitored by optical density at 430 nm. In about the immune systems of these threatened Development Award. In addition, support was pro- addition, the serum was treated with protease, reptilians. Knowledge of the immune system vided by a NSF Research Commercialization/ chelators of divalent metal ions, or with mild Educational Enhancement Plan grant adminis- will be of interest to both evolutionary biolo- heat to determine the mechanism of antibac- tered by the Louisiana Board of Regents. gists and conservationists of these reptilians. terial activities. Treatment of bacterial cul- The primary goal of this project was to charac- tures with serum from Komodo dragons Conflict of interests: the authors declare no terize the antibacterial properties of serum (Varanus komodoensis) resulted in a volume- potential conflict of interests. from the Komodo dragon. dependent decrease in bacterial growth. Cultures of Escherichia coli, Staphylococcus Received for publication: 30 August 2011. Revision received: 14 August 2012. aureus, and Klebsiella oxytoca exhibited mod- Accepted for publication: 29 August 2012. erate-strong growth inhibition by V. komodoen- Materials and Methods sis serum, while cultures of Streptococcus epi- This work is licensed under a Creative Commons dermitis, Salmonella typhimurium, Providen - Attribution NonCommercial 3.0 License (CC BY- cia stuartii, and Shigella flexneri wereNon-commercial nearly Chemicals and biochemicals NC 3.0). completely obliterated for 24 h by only 10% Nutrient broth and low EEO agarose were ©Copyright M. Merchant et al., 2013 (v/v) serum. The antibacterial activity of V. purchased from Intermountain Scientific Licensee PAGEPress, Italy komodensis serum occurred very rapidly, as (Kaysville, UT). Bacterial cultures (American Microbiology Research 2013; 4:e4 18% of E. coli growth was inhibited by a five Type Culture Collection) were purchased from doi:10.4081/mr.2013.e4 min exposure to serum. Furthermore, 10- and Remel (Lenexa, KS). The following species 20-min incubations of E. coli with serum from were used for representatives of Gram- nega- V. komodoensis resulted in 43 and 68% inhibi- tive species: Escherichia coliform (ATCC tion of bacterial growth, respectively. The bac- 25922), Shigella flexnerii (ATCC 700930), and tericidal capacity of the serum against E. coli Klebsiella oxytoca (ATCC 49131), Salmonella Collection of blood samples Blood samples were collected from Komodo was 2,075,000 bacteria/µL serum, and was typhimurium (ATCC 14028), Providencia stu- inhibited by mild heat treatment, pronase, artii (33672) and the following Gram- positive dragons at the Houston and San Antonio zoos. EDTA, and phosphate, indicating that the anti- species: Staphylococcus aureus (ATCC 6538), Blood was drawn from the tail vein, transferred bacterial action is most probably due to the and Streptococcus epidermitis (ATCC 19615). to Vaccutainer™ tubes, and allowed to clot for presence of a potent serum complement pro- Pronase from Streptomyces griseus was pur- at least five h at ambient temperature before tein system. chased from Sigma Chemical Company (St. the serum was collected. The amount of blood Louis, MO). collected from each individual depended on the size of the animal, and was at the discretion of

[page 16] [Microbiology Research 2013; 4:e4] Article the attending veterinarian. Blood was collected The data in Table 1 exhibits the results of a from three adults (20-81.5 kg) and five juve- Results mechanistic study, showing the susceptibility niles (1.5-6.2 kg), and the serum was pooled so of the antibacterial effects to mild heat treat- that average antibacterial values for this The results in Figure 1 show that serum ment and chelators of divalent metal ions. species could be generated. The samples were from V. komodoensis inhibits the growth of Treatment of E. coli cultures with serum from stored at -20oC until used for the antibacterial Gram negative bacteria. The serum inhibited the Komodo dragon results in 96.4% of the bac- assays. The collection of blood from these ani- growth of E. coli in a volume-dependent man- teria killed. However, mild heat treatment o mals was conducted in accordance with the ner (Figure 1A). At 24 hrs, 10% V. komodoensis (56 C, 30 min) of the serum resulted in only animal care institutional policies of the serum (v/v) produced a 44% decrease in E. coli 4.0% antibacterial activity. Likewise, treatment Houston and San Antonio Zoos. growth, while 25, 50, 75, and 100% serum of the serum with 5 mM EDTA or 5 mM phos- caused 58, 75, 93, and 98% decreases in phate resulted in only 6.3% or 8.7% antibacter- Serum volume-dependent growth, respectively. By way of comparison, ial activity, respectively. incubation of 10% V. komodoensis serum with antibacterial assays Klebsiella oxytoca cultures resulted in 34% Two hundred µL samples containing various growth inhibition at 24 hrs, while 25% serum volumes (20-200 µL) of Komodo dragon serum caused a 65% decrease in bacterial density Discussion in sterile microtiter plates were inoculated (Figure 1B). All serum volumes at, or above, with five µL of bacterial culture from log phase 50% completely inhibited the growth of K. oxy- Eukaryotic organisms must continuously cultures and incubated for 24 h at 37oC. The toca cultures at 24 hrs. Serum from V. komod- defend themselves against infiltration and col- optical density of each sample was measured oensis (10% volume) inhibited Shigella onization by microorganisms. Host defense at 0, 3, 6 12 and 24 h using a Biorad flexnerii growth by 95% at 24 hrs (Figure 1D), occurs via complex mechanisms and can be Benchmark Plus™ microtiter plate reader at and exhibited similar results for cultures of divided into two distinct, but interrelated, 430 nm. Salmonella typhimurium (Figure 1E). Cultures types of responses: acquired immunity and of Providencia stuarti growth were completely innate immunity. Acquired immunity requires Kinetic antibacterial assay obliterated (100%) inhibition by only 10% prior exposure to a specific antigen before a serum (Figure 1C). fullonly immunological assault can be established Five hundred L of Komodo dragon serum µ Figure 2 illustrates the effects of serum by the host organism. In addition, the acquired were inoculated with 20 L of an E. coli culture µ from V. komodoensis on the growth of Gram immune response is complex and can often in log phase growth. At various time points, 50 positive bacteria. Serum (10% volume) from V. take several days to become fully activated. The L of a dilution of each sample were spread µ komodoensis inhibited the growthuse of innate immune response comprises a signifi- onto the surface of nutrient broth agar plates Staphylococcus aureus by 64% at 24 hrs, while cant portion of the immune system and acts as to determine the CFUs for each sample. The 25, 50, 75, and 100% serum resulted in 68, 84, an initial defense mechanism against micro- samples were typically plated at five different 94, and 93% reduction in growth, respectively bial growth shortly after infection occurs. dilutions (1:100-1:106, in sterile isotonic (Figure 2A). In contrast, 10% serum from V. These innate defense responses are activated saline) to obtain plates with a quantity of komodoensis inhibited the growth of shortly after exposure and act to slow or stop colonies such to provide a reasonable estimate Streptococcus epidermitis cultures by 49% an infection in the initial stages so that the of bacterial density (50-400 CFUs per plate). (Figure 2B). acquired immune response can be initiated. To determine possible mechanisms of antibac- The bacterial-killing capacity of serum from Also, the innate immune system functions in terial action, pooled serum from Komodo drag- V. komodoensis for E. coli is displayed in Figure the activation of the acquired immune system ons was incubated with one unit/mL pronase 3. A 20 µL sample of positive control culture of by generating chemotactic factors and produc- (isolated from Streptomyces griseus), 5 mM E. coli in log phase growth was found to have ing cytokines that initiate the development EDTA (pH 8.0), or 5 mM phosphate for 30 min 16.4±0.4 million viable bacteria. In contrast, and maturation of specific T-cell and B-cell at ambient temperature. In addition, four the addition of 25% (v/v) of serum from V. populations. Anecdotal evidence suggests that aliquots of serum were incubated at 56oC for komodoensis to the bacterial growth medium Komodo dragons are resistant to microbial caused a significant decrease (P<0.05) to 30 min. These serum samples (500 µL) were 5.2±2.1 million CFU. Further increases in all incubated with 20 µL of an E. coliNon-commercialculture in log phase growth. The samples were incubated serum to 50% resulted in decreased bacterial for 15 min at ambient temperature, diluted viability (1.3±0.2 million CFU). Because only Table 1. Effects of heat treatment, protease serially (1:100 -1:106) in sterile saline, and 50 20 µL of the bacterial culture were used for treatment, or metal chelators on the antibac- L of each sample were plated on nutrient colony determination, we calculated that one terial activity of serum from V. komodoensis µ L of serum from V. komodoensis has the against E. coli. The E. coli survival was deter- agarose as described above. µ capacity to kill approximately 2.24 million E. mined by manual colony counts. The results represent the means of four independent coli bacteria. determinations. Statistics and controls Figure 4 shows the results of a kinetic analy- All assays were conducted in quadruplicate, sis of the ability of serum from V. komodoensis Treatment CFU Antibacterial and the results are expressed as the means ± to inhibit the growth of E. coli bacterial cul- activity standard deviations for four independent repli- tures. The antibacterial activity occurred None 12.7 106 - cates. The statistical comparisons between quickly, as 18% of the bacteria (2.95 million) × Komodo dragon serum 0.46 106 96.4% groups were conducted using analyses of vari- were killed within 5 min of exposure to serum × 6 ance and Duncan’s post-hoc comparisons and from V. komodoensis. Further incubation Serum, 56°C, 30 min 12.2×10 4.0% 6 P<0.05 was chosen as the standard probability resulted in 43% (7.05 million) of the bacteria Serum + pronase 12.4×10 2.4% 6 of statistical significance. killed at 10 min of exposure, and the antibac- Serum + 5 mM EDTA 11.9×10 6.3% terial action was maximal at 20 min as 68% 6 Serum + 5 mM phosphate 11.6×10 8.7% (11.2 million) of the E. coli were killed. CFU, colony-forming units.

[Microbiology Research 2013; 4:e4] [page 17] Article infections. These animals often sustain seri- to mice, particularly Pasteurella multocida, of the Komodo dragons,4 supporting the idea ous injuries, including open wounds, due to supporting the idea that bite wounds inflicted that their immune systems are equipped to interspecies fighting in territorial disputes.7 by Komodo dragons may induce systemic sep- deal with the heavy bacteria load present in Despite the propensity of dragons to resist ticemia in their prey. However, it would be rea- their saliva. Antibacterial activities of other microbial infection, the mechanisms of immu- sonable to expect that Komodo dragons would reptilian species have been assessed, particu- nity are not well-characterized. have developed innate immune mechanisms larly those of several crocodilian species.8,9 Montgomery et al.4 isolated many bacterial to combat possible infection by the bacteria in Merchant et al.8 showed that the bactericidal species from both captive and wild Komodo their own saliva. In addition, antibodies capacity of serum from the American alligator dragons, some of which were extremely toxic against P. multocida were found in the plasma (Alligator mississippiensis) toward E. coli was

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Figure 1. Effects of serum from V. komodoensis on growth of Gram negative bacteria. Culture tubes containing 2 mL of 0, 10, 25, 50, 75 or 100% serum from V. komodoensis. The cultures were incubated at 37°C and their optical densities were measured (430 nm) at 0, 3, 6, 12, and 24 h post-inoculation. The results are expressed as the means ± S.D. for four independent determinations. A) Eschericia coliform, B) Klebsiella oxytoca, C) Providencia stuartii, D) Shigella flexneri, E) Salmonella typhimurium.

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54,000 bacteria/µL. Other reptilians have been described in this study could be a result of the potentially be altered by chronic stress of cap- shown to exhibit potent antibacterial activi- evolution of these ancient vertebrates under tivity. However, chronic stress typically acts as ties, particularly crocodilian species. Merchant the conditions of territorial aggression, feed- an immunosuppressive factor in vertebrates,12-17 et al.10 showed that serum from the American ing disputes, challenges for mating rights, etc. and thus the antibacterial activity of the serum alligator (Alligator missippiensis), when chal- Bull et al.11 reported that 70% of the feeding from wild V. komodoensis could potentially be lenged with cultures of E. coli as low as 106/mL, activities of V. komodoensis on large prey items underestimated in this study. could not kill enough bacteria to make a statis- occurred with multiple adult animals in close The antibacterial effects of Komodo dragon tical difference in the colony counts, relative to proximity. The broad spectrum of oral bacteria serum is most likely due to an efficient serum untreated controls cultures. The capacity of isolated from this species and the high bacter- complement innate immune-defense. The fact alligator serum to kill E. coli was approximate- ial load of their saliva, 4,11 would certainly be an that mild heat treatment (30 min. at 56oC) ly 54,000 bacteria/µL. However, the results issue if bite injuries occurred during territori- almost completely obliterated the observed shown in Figure 3 show that serum from V. al battles. Therefore, it is not surprising that activities is an indication that serum comple- komodoensis has substantially higher activity these animals could have evolved strong ment activity is responsible for the antibacter- than that of Alligator mississippiensis. The innate immunity defenses to deal with the ial activities. It has been well-established that serum from V. komodoensis showed an approx- potential for serious infection due to their serum complement proteins are extremely vul- imate 40-fold increase in the capacity to kill E. aggressive lifestyles. It must be noted that the nerable to mild heat treatment, as some of the coli, relative to that of Alligator mississippien- animals used in this study were captive ani- key proteins components are thermally unsta- sis. The extreme antibacterial properties mals, and thus the immune system could ble.18 In addition, the antibacterial activity was sensitive to pronase, indicating that at least one of the components required for the observed activity is proteinaceous in nature. This idea is reinforced by the dependence of the antibacterial activities on divalent metal ions (Table 1). It has long been known that divalentonly metals (magnesium and calcium) are required for mammalian serum complement activity.19-20 More recent studies have con- firmed the requirement for calcium and mag- use nesium for reptilian serum complement func- tion.21 It is not surprising that V. komodoensis would have serum complement proteins, as most vertebrate, as well as many ancient inver- tebrates, exhibit complement activity.22 However, the strength with which these pro- teins kill bacteria is staggering. Before this study, crocodilians were considered to have Figure 2. Effects of serum from V. komodoensis on growth of Gram positive bacteria. among the most potent serum complement Culture tubes containing 2 mL of 0, 10, 25, 50, 75 or 100% serum from Komodo dragons. defense systems.10 However, the results in this The cultures were incubated at 37°C and their optical densities were measured (430 nm) at study show that the potency of Komodo dragon 0, 3, 6, 12, and 24 h post-inoculation. The results are expressed as the means ± S.D. for four serum complement is much higher than that independent determinations. A) Staphylococcus aureus, B) Streptococcus epidermitis. of crocodilians.23 This study represents the first report on innate immune activity of V. komodoensis. Non-commercial References

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