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Transmission of M. ulcerans, the etiological agent of Buruli ulcer, from the environment to humans remains an enigma despite decades of research. Major transmission hypotheses propose 1) that M. ulcerans is acquired through an insect bite or 2) that bacteria enter an existing wound through exposure to a contaminated environment. In studies reported here, a guinea pig infection model was developed to determine whether Buruli ulcer could be produced through passive inoculation of M. ulcerans onto a superficial abrasion. The choice of an abrasion model was based on the fact that most bacterial pathogens infecting the skin are able to infect an open lesion, and that abrasions are extremely common in children. Our studies show that after a 90d infection period, an ulcer was present at intra-dermal injection sites of all seven animals infected, whereas topical application of M. ulcerans failed to establish an infection. Mycobacterium ulcerans was cultured from all injection sites whereas infected abrasion sites healed and were culture negative. A 14d experiment was conducted to determine how long organisms persisted after inoculation. Mycobacterium ulcerans was isolated from abrasions at one hour and 24 hours post infection, but cultures from later time points were negative. Abrasion sites were qPCR positive up to seven days post infection, but negative at later timepoints. In contrast, M. ulcerans DNA was detected at intra-dermal injection sites throughout the study. M. ulcerans was cultured from injection sites at each time point. These results suggest that injection of M. ulcerans into the skin greatly facilitates infection and lends support for the role of an invertebrate vector or other route of entry such as a puncture wound or deep laceration where bacteria would be contained within the lesion. Infection through passive inoculation into an existing abrasion appears a less likely route of entry. Author Summary: Buruli ulcer, a severe skin disease in West and Central Africa results in significant disability. The causative bacterium, M. ulcerans has been detected in many aquatic sources, but how bacteria enter the skin is an enigma. Two major hypotheses for infection are 1) that bacteria are injected into the skin through the bite of an aquatic insect, or 2) that bacteria enter open wounds on a person's body. In this study, we use a guinea pig infection model to evaluate whether application of M. ulcerans to an open abrasion produces Buruli ulcer. Our results show that despite topical application of very large numbers of M. ulcerans, we are unable to produce infection in open abrasions. These results are extremely surprising because most bacteria such as Staphylococcus or Streptococcus can readily infect abrasions. In contrast, intra-dermal injection of M. ulcerans into the skin of guinea pigs consistently produced an ulcer. Our studies are the first to explore the route of infection of M. ulcerans in an experimental model. These results suggest that Buruli ulcer is not likely to be due to passive entry of bacteria into pre-existing abrasions and supports the role of biting invertebrates, puncture wounds, or lacerations as a requirement for infection. [ABSTRACT FROM AUTHOR]