Mycobacterium ulcerans low infectious dose and mechanical transmission support insect bites and puncturing injuries in the spread of Buruli ulcer

Addressing the transmission enigma of the neglected disease Buruli ulcer (BU) is a World Health Organization priority. In Australia, we have observed an association between mosquitoes harboring the causative agent, Mycobacterium ulcerans, and BU. Here we tested a contaminated skin model of BU transmission by dipping the tails from healthy mice in cultures of the causative agent, Mycobacterium ulcerans. Tails were exposed to mosquito (Aedes notoscriptus and Aedes aegypti) blood feeding or punctured with sterile needles. Two of 12 of mice with M. ulcerans contaminated tails exposed to feeding A. notoscriptus mosquitoes developed BU. There were no mice exposed to A. aegypti that developed BU. Eighty-eight percent of mice (21/24) subjected to contaminated tail needle puncture developed BU. Mouse tails coated only in bacteria did not develop disease. A median incubation time of 12 weeks, consistent with data from human infections, was noted. We then specifically tested the M. ulcerans infectious dose-50 (ID50) in this contaminated skin surface infection model with needle puncture and observed an ID50 of 2.6 colony-forming units. We have uncovered a biologically plausible mechanical transmission mode of BU via natural or anthropogenic skin punctures.


Introduction
Among the 17 neglected tropical diseases the World Health Organization (WHO) has targeted for control and elimination, only Leprosy and Buruli ulcer (BU) have unknown modes of transmission [1]. The search to understand how humans contract BU spans more than 70 years since the causative agent, Mycobacterium ulcerans, was first identified [2]. There are persistent and emerging foci of BU cases across the world, in particular Africa and Australia [3]. BU is characterized by necrotizing skin lesions, caused by localized proliferation of M. ulcerans in subcutaneous tissue. BU is rarely fatal, but untreated infections leave patients with significant disfigurement and disability, with damaging personal and economic consequences [4,5]. Researchers have long been struck by the characteristic epidemiology of BU, with cases occurring in highly geographically circumscribed regions (sometimes less than a few square kilometres) and risk factors for infection that include gardening, insect bites and proximity to (but not necessarily contact with) lacustrine/riverine regions [6][7][8][9][10][11][12][13][14]. Human-to-human spread is considered unlikely [14]. Disease transmission is thought to occur by contact with an environment contaminated with Mycobacterium ulcerans but exactly where the pathogen resides and why it appears so geographically restricted have yet to be determined. [15].
M. ulcerans is very slow growing (doubling time >48 hrs) and this poses a problem for source tracking efforts as it is difficult to isolate the bacteria in pure culture from complex environmental specimens [16]. M. ulcerans has only once been isolated from a non-clinical source, an aquatic water bug (Gerridae) from Benin [16]. Quantitative PCR targeting M. ulcerans-specific DNA is the most frequently used technique in surveys of environmental specimens. A comprehensive review of the many field and lab studies that have examined transmission of BU has highlighted the range of organisms from aquatic plants, snails, insects, fish, amphibia, and in Australia certain native marsupials that can serve as potential reservoirs for M. ulcerans [15,[17][18][19][20]. Since the first observation that biting aquatic insects can harbour M. ulcerans [21], studies of BU transmission have largely focused on the potential for insects to biologically vector M. ulcerans implying that M. ulcerans undergoes a propagative or reproductive mode of development in an insect [22][23][24][25][26]. Several case-control studies, including from both Australia and Africa have suggested insects may play a role in transmission [10,11]. For example, in southeastern Australia, we noted Buruli lesions on exposed areas likely to attract biting insects, some patients with every brief exposure times to endemic areas [27,28] and in 2004 we began a study that identified M. ulcerans DNA associated with mosquitoes captured in endemic areas [22]. However, there is no compelling experimental evidence for single-mode biological transmission of M. ulcerans via insect vectors.

Ethics statement
The animal ethics committee (AEC) of the University of Melbourne approved all animal experiments under approval number AEC: 1312775.2, in accordance with the National Health and Medical Research Council Australian code for the care and use of animals for scientific purposes 8th edition (2013).

Bacterial isolates and culture conditions
M. ulcerans strain JKD8049 and bioluminescent M. ulcerans JKD8049 (harbouring plasmid pMV306 hsp:luxG13) [29,30] were cultured in 7H9 broth or Middlebrook 7H10 agar, containing 10% oleic-albumin-dextrose-catalase growth supplement (Middlebrook, Becton Dickinson, Sparks, MD, USA) and 0.5% glycerol (v/v) at 30˚C. Colony counts from bacterial cultures or tissue specimens were performed using spot plating. Five x 3μl volumes of serial 10-fold dilutions (10 −1 to 10 −5 ) of a culture or tissue preparation were spotted onto 7H10 agar plates with a 5x5 grid marked. The spots were allowed to dry, the plates loosely wrapped in plastic bags and then incubated as above for 10 weeks before counting colonies. Data analysis was performed using GraphPad Prism v 7.0a. All culture extracts were screened by LC-MS for the presence of mycolactones as previously described to ensure bacteria used in transmission experiments remained fully virulent [31].

Experimental animals
BALB/c mice were purchased from ARC (Canning Vale, Australia) and housed in individual ventilated cages. Upon arrival, animals were acclimatizing for 5 days. Food and water were given ad libitum.

Aedes notoscriptus and Aedes aegypti rearing
Wild caught mosquitoes were sourced from around Cairns, Queensland, Australia. A. notoscriptus and A. aegypti colonies were reared in a Physical Containment Level 2 (PC2) laboratory environment at 26˚C using previously described methods, with the addition of brown paper used as the oviposition substrate for A. notoscriptus [32]. Females were aged for at least one week prior to blood feeding ensure maturity, during which they were mated (seminal accessory fluid is required to stimulate searching and feeding behavior in females). The adults were provided with access to a 10% sucrose solution, which was withdrawn 24h prior to a blood feeding experiment.

Mosquito-mouse transmission experiments
Six-week old female BALB/c mice were anaesthetized and their tails coated in a thin film of M. ulcerans wild type strain JKD8049 by dipping the tails in a Petri dish containing 20mL of bacterial culture (concentration~10 6 CFU/mL) ( Table 1). Tails were allowed to air-dry for 5 minutes after dipping. The tail only was then exposed to a 200mm x 200mm x 200mm cage of 20 adult female mosquitoes for a period of 15 minutes. Twenty mosquitoes were used per feeding bout to minimize stress on the mice. The number of insects biting each mouse was recorded over the exposure period by continuous observation. Tails were not wiped or disinfected postbiting. Mice were then observed weekly for up to six months for signs of tail lesions. Sterile needle stick (25G or 30G needle) and no-trauma were used as controls. An additional control consisted of tails dipped in sterile culture broth only and subjected to mosquito biting or sterile needle stick (See Table 1 for experiment design). The concentration of bacteria used was calculated by dilution plating as described above. Contingency tables and Fisher's exact test were used to test if instances of mosquito-associated or needle stick-associated disease transmission were significantly different to no trauma (GraphPad Prism v 7.0a).

Infectious dose calculations and infectious dose 50 experiment
To estimate the infectious dose, we first measured the surface area of five dissected naïve mouse-tails to obtain an average surface area (493.3 ± 41.1 mm 2 ). Using ten naïve mouse-tails and a precision balance, we then calculated the average volume of M. ulcerans 7H9 Middlebrook culture adhering to the tail surface (32.4 ± 4.2 μL), the concentration of bacteria in the cultures used, and the surface area of the tips of 25G and 30G needles used to deliver the puncture wounds (0.207 mm 2 and 0.056 mm 2 respectively). These parameters were then used to calculate the infectious dose, assuming the bacteria were evenly distributed over the tail surface ( Fig 1B). A standard protocol to calculate an ID50 was followed. Four-week old female BALB/ c mice were anaesthetized and their tails coated as described above for the transmission experiments with 10-fold dilutions of bioluminescent M. ulcerans JKD8049 from 10 6 to 10 CFU/mL in each Petri dish, using five mice per dilution, followed by a single needle stick puncture with a 25G needle. The mice were monitored using a Lumina XRMS Series III In Vitro Imaging System (IVIS) (Perkin Elmer). Images were captured with the following settings: binning of 4 (medium), Field of View (FOV) of 24, Relative aperture at f1.2 and exposure time of 180s. Bioluminescence was calculated using Living Image software V4.1. The number of mice per dilution developing BU up to six-months was recorded. Mice were observed up to six-months. The data were plotted and a standard curve was fitted using least squares non-linear regression. The ID50 and 95% confidence intervals were interpolated with reference to the standard curve using GraphPad Prism v 7.0a.

Real time quantitative PCR
For each mosquito that blood-fed DNA was individually extracted from the dissected head, abdomen and legs of each insect using the MoBio Powersoil DNA extraction kit following manufacturer's instructions (MoBio Laboratories Inc., Carlsbad CA USA). The insects were held by the wings and each body portion (legs, abdomen, head) individually and separately removed with sterile, fine forceps to avoid cross contamination during dissections. Body parts were stored individually (legs were pooled per insect) in sterile 1.5mL tubes at -20˚C until DNA extraction. DNA was similarly extracted from mouse tissue. Procedural extraction control blanks (sterile water) were included at a frequency of 10% to monitor potential PCR contamination, in addition to no-template negative controls. IS2404 quantitative PCR (qPCR) was performed as described

Preparation of mouse tissue for analysis
At the end of the experimental period or when a clinical end-point was reached mice were humanely killed. The region of a mouse-tail spanning a likely lesion was cut into three equal sections for histology, qPCR and CFU counts. Individual tail pieces for CFU counts were weighed and placed into sterile 2ml screw capped tube containing 0.5g of large glass beads and 600μl of sterile 1x PBS. Tissues were homogenized using four rounds of 2x 30second pulses in a high-speed tissue-disruptor at 6500 rpm, with tubes placed on ice for 5 minutes between each round. A 300μl volume of this homogenate was decontaminated with 300μl of 2% NaOH (v/v) and incubated at room temperature for 15 minutes. The preparation was neutralized drop-wise with a 10% solution of orthophosphoric acid (v/v) with added bromophenol blue until the solution changed from blue to clear. The mixtures were diluted in PBS and CFUs determined by spot plating as described above.

Histology
Sections of mouse-tails were fixed in 10% (w/v) neutral-buffered-formalin and imbedded in paraffin. Each mouse-tail was sectioned transversely (4μm thick) and subjected to Ziehl-Neelson and hematoxylin/eosin staining. The fixed and stained tissue sections were examined by light microscopy.

M. ulcerans is efficiently transmitted to a mammalian host by mechanical means
In experiment one, we established a murine model of M. ulcerans transmission that represented a skin surface contaminated with the bacteria and then subjected to a minor penetrating trauma, via either a mosquito bite or needle stick puncture. For this experiment, only Aedes notoscriptus mosquitoes were used, a local species previously associated with BU in south east Australia [22]. We first coated the tails of 12 mice with M. ulcerans then exposed only the tails to A. notoscriptus. Six of the 12 mice exposed to mosquitoes were bitten once each, and of these six mice, two developed BU lesions (Table 1, Fig 1B, Fig 2A). Histology of these lesions confirmed a subcutaneous focus of AFB, within a zone of necrotic tissue. There was also characteristic epithelial hyperplasia adjacent to the site of infection (Fig 2B and 2C). Material extracted from the lesions was IS2404 qPCR-positive and culture positive for M. ulcerans (S1 Table). Mice bitten by mosquitoes but with tails coated only with sterile culture media did not develop lesions (Table 1). In the same experiment, we also subjected five mice to a single needle stick puncture. Each mouse had their tail coated with M. ulcerans as for the mosquito biting. Four of these five mice developed M. ulcerans positive lesions (Table 1, Fig 2D), with subcutaneous foci of infection and viable bacteria (Fig 2F). The histology of these lesions was the same as the mice subjected to mosquito blood feeding, however bacterial burden was higher following needle stick puncture (Fig 2C compared with Fig 2F). Six mice with their tails coated with M. ulcerans but not subjected to a puncturing injury did not develop lesions and remained healthy until the completion of the experiment at six months. This experiment suggested that minor penetrating skin trauma (defined here as a puncture <0.5mm diameter and <2mm deep) to a skin surface contaminated with M. ulcerans is sufficient to cause infection. It also revealed a means by which mosquitoes could act as mechanical vectors of M. ulcerans.
In experiment two, using approximately the same dose of bacteria to coat the mouse-tails, we repeated experiment-1 but with Aedes aegypti, because of the close association of this mosquito to humans world-wide and their potential to vector pathogens. Despite multiple mosquito bites per mouse in the second experiment compared to the first, none of the five Aedes aegypti-exposed mice developed lesions (Table 1). However, as for experiment 1, four of five mice subjected to single, needle stick puncture developed M. ulcerans positive tail lesions (Table 1). A third needle stick puncture experiment was then conducted, this time using a smaller diameter, 30-gauge needle, to assess the impact of a smaller injury. There were 13/14 mice that developed BU when subjected to a single needle stick puncture through a contaminated skin surface, while eight mice with contaminated skin but no injury did not progress to disease (Table 1). Thus, across the three experiments there were 21/24 mice (88%) with needle stick puncture that developed BU, suggesting that this is an efficient mode of disease transmission (Table 1). Either A. notoscriptus mosquito bite or needle stick trauma significantly increased the risk of developing BU in our mouse skin surface contamination model (Tables 2 & 3).

Higher M. ulcerans burden on mosquitoes is associated with transmission
We assessed the likely burden of M. ulcerans by individual IS2404 qPCR of the head, abdomen and legs for each mosquito that blood fed (Fig 3). A summary of these results is shown in Fig  3A. We noted that the bacterial load (expressed as genome equivalents [GE]) was significantly higher in the mosquito heads associated with mice that developed lesions (p<0.05) (Fig 3B, S1  Table). These data point to a threshold, above which some mosquitoes may become competent mechanical vectors for M. ulcerans transmission.

Estimation of incubation period and infectious dose of transmission model-1
Based on the time until a tail lesion was first observed, and when using the highest concentration of bacteria (dose range 9-55 CFU, Table 1) we estimated a median incubation period (IP) of 12 weeks (Fig 4A). This result overlaps with the IP in humans for BU, estimated in different epidemiological studies from 4-10 weeks in Uganda during the 1960s [14] and 4-37 weeks in south east Australia [28]. We then estimated the infectious dose-50 (ID50). We used six different concentrations of M. ulcerans to coat the tails of mice (n = 5 mice/dilution), subjected each mouse-tail to a single needle stick puncture, and then observed the number of mice for each dilution that developed Buruli ulcer, allowing an ID 50 estimate of 2.6 CFU (95% CI 1.6-3.6 CFU) (Fig 4B). To our knowledge this is the first estimate of an M. ulcerans infectious dose and indicates that like Mycobacterium tuberculosis and Mycobacterium leprae, a small quantity of this slow growing mycobacterium is sufficient to cause disease.

Discussion
Pathogen transmission by arthropods is generally characterized by either biological transmission, such as malaria [34] or mechanical transmission, where replication or biological transformation of the pathogen within the vector is not necessary for disease spread [35,36]. Here, we show for the first time an efficient mechanical mode of transmission of Mycobacterium ulcerans to a mammalian host that implicates both puncturing injuries and arthropods. In our study, the uninfected host is externally contaminated with M. ulcerans that is certainly plausible in many areas of the world. We propose that a micro-puncture wound of any sort whether it is by natural means e.g., a thorn, arthropod bite or artificially induced via a human-mediated puncture has the potential to inject M. ulcerans and generate an ulcer. This research was designed around established frameworks for implicating vectors in disease transmission and provides the necessary causational evidence to help resolve the 80-year mystery on how M. ulcerans is spread to people [15,37]. The efficient establishment of BU we have shown here via minor penetrating trauma such as a needle puncture through a contaminated skin surface helps fulfil one of four Barnett Criteria [37]. In vector ecology, mechanical transmission is defined as a non-circulative process involving accidental transport of the pathogen [36]. That is, the pathogen, in some fashion, nonspecifically associates or contaminates the mouthparts (stylet) of an arthropod vector. Insect mechanical transmission of BU implies that if M. ulcerans were ingested and then egested via regurgitation or salivation, the mechanism would act more like a syringe than a needle [38]. Such a mode of M. ulcerans disease transmission is supported by previous laboratory studies in which Naucoris and Belostmatid water bugs were contaminated via feeding on maggot prey that had been injected with M. ulcerans or fed naturally on dietary contaminated larval mosquito prey [26,39].
Our demonstration in the current study of mechanical transmission suggests there are potentially multiple or parallel pathways of M. ulcerans infection [37]. Examples of bacterial diseases with multiple transmission modes include tularemia, plague and trachoma [40,41]. Support for our mechanical transmission model also comes from the many field reports over the decades of M. ulcerans infection following trauma to the skin. Case reports have noted BU following a suite of penetrating injuries ranging from insect bites (ants, scorpions), snake bite, human bite, splinters, gunshot, hypodermic injections of medication and vaccinations [42][43][44]. Epidemiologists in Uganda during the 1960s and 70s suggested sharp-edged grasses might introduce the bacteria [45]. However, a recent laboratory study established that abrasions of the skin in Guinea pig models and subsequent application of M. ulcerans was not enough to cause an ulcer, however, this same study established that a subcutaneous injection would cause an ulcer [46]. As a sequel to this study in Guinea pigs, we raised the question of how likely it was that mammalian skin could be sufficiently coated in M. ulcerans that an injury from natural or anthropogenic sources could lead to infection. Other explanations for the transmission of M. ulcerans include linkages with human behavior that increase direct contact with human skin and contaminated water [15]. A recent study from Cameroon recorded the persistence of M. ulcerans over a 24-month period in a waterhole used by villagers (including BU patients) for bathing [47]. A similar study in Ghana documented a 90% positivity rate for MU for water bodies frequented by community members for bathing and washing purposes [48]. Hence, it is reasonable to envisage a scenario where a villager's skin surface becomes contaminated after bathing in such a water body and is primed for infection if (i) the concentration of bacteria is sufficiently high, and (ii) an inoculating event occurs. Whereas, in Australia, earlier studies have shown that M. ulcerans contamination of possum feces in and around the gardens of BU patients might present a similar skin surface contamination model in this region [17,49]. Future experiments will address the possibility that insect vectors may be able to move M. ulcerans from one source and inject it into an animal or human. Our focus on mechanical mosquito transmission arose from previous surveys in southeastern Australia where a strong association between M. ulcerans positive mosquitoes and human cases of BU has shown that M. ulcerans has not only been found on adult mosquitoes from both lab and field studies but also a biological gradient, where maximum likelihood estimates (MLE) of the proportion of M. ulcerans-positive mosquitoes increased as the number of cases of BU increased [22,39,[50][51][52][53]. However, a recent study in Benin, West Africa found no evidence of M. ulcerans in association with adult mosquitoes [54]. The authors concluded that the mode of transmission might differ between southeastern Australia and Africa. Although, laboratory and fieldwork in West Africa suggest that aquatic insects, including mosquito larvae, play a role as reservoirs in nature for M. ulcerans that may be indirectly tied to transmission by serving as dispersal mechanisms [21,26,55]. Epidemiological studies have shown that direct contact with water is not a universal risk factor for BU [8,11]. Prior exposure to insect bites and gardening are also independent risk factors for developing BU, while use of insect repellent is protective [11,56]. Null hypothesis (no difference in means) was rejected (p<0.05)* (Kruskal-Wallis test). Horizontal bar indicates the mean bacterial load per mosquito and error bars depict standard deviation. The y-axis is GE and x-axis is experiment. The qPCR data for individual insects is contained in S1 Table. https://doi.org/10.1371/journal.pntd.0005553.g003 Laboratory support to show mosquitoes can be competent vectors to spread BU is important additional evidence required to satisfy accepted vector ecology criteria for implicating insects in disease transmission [15,37]. We found that infection was established following very minor penetrating trauma. Mosquitoes, in general, feed by insertion of a stylet, sheathed within the proboscis, beneath the skin of a host. The stylet has an approximate diameter 10 μM tapering to 1 μM at its tip and extending 1-2 mm below the skin surface. We estimated the density of M. ulcerans on the mouse-tails surface was 100-200 CFU/mm 2 . Thus, the number of bacteria potentially injected during mosquito feeding through this contaminated surface is likely to be low, but this is consistent with our infectious dose estimates from needle-stick punctures, indicating an ID50 of only 2.6 CFU (Fig 4B). Aedes notoscriptus mosquitoes are approximately twice as large as Aedes aegypti. Larger size may imply a longer stylet length, longer blood feeding time with a deeper penetration to a depth that may initiate M. ulcerans infection more frequently than that from Aedes aegypti mosquitoes that are smaller and do not blood feed as long. Such subtle differences in mosquito morphology and behavior may indicate why mechanical transmission in any form may be uncommon. There are strong parallels here with Mycobacterium leprae, the agent of leprosy. Like BU, the mode of transmission of the leprosy bacillus is unclear, but the infective dose is known to be very low (10 bacteria) and epidemiological evidence suggests multiple transmission pathways, including entry of the bacteria after skin trauma [57,58]. Our infective dose estimate for M. ulcerans is consistent with observations that pathogens producing locally acting molecules to cause disease (e.g. the polyketide toxin mycolactone of M. ulcerans) have lower infective doses [59].
In summary, we have uncovered a highly efficient (88% rate) for needle stick skin punctures and a lower rate for mosquito punctures, suggesting a plausible mechanical transmission mode of M. ulcerans infection via anthropogenic or natural skin-puncturing microtrauma. We conclude from these experiments that reduction of exposure to insect bites, access to clean water for bathing, and prompt treatment of wounds and existing BU are concrete measures likely to interrupt BU transmission.