Certain bacteria present on frog skin can prevent infection by the pathogenic fungus Batrachochytrium dendrobatidis (Bd), conferring disease resistance. Previous studies have used agar-based in vitro challenge assays to screen bacteria for Bd-inhibitory activity and to identify candidates for bacterial supplementation trials. However, agar-based assays can be difficult to set up and to replicate reliably. To overcome these difficulties, we developed a semi-quantitative spectrophotometric challenge assay technique. Cell-free supernatants were prepared from filtered bacterial cultures and added to 96-well plates in replicated wells containing Bd zoospores suspended in tryptone-gelatin hydrolysate-lactose (TGhL) broth medium. Plates were then read daily on a spectrophotometer until positive controls reached maximum growth in order to determine growth curves for Bd. We tested the technique by screening skin bacteria from the Australian green-eyed tree frog Litoria serrata. Of bacteria tested, 31% showed some degree of Bd inhibition, while some may have promoted Bd growth, a previously unknown effect. Our cell-free supernatant challenge assay technique is an effective in vitro method for screening bacterial isolates for strong Bd-inhibitory activity. It contributes to the expanding field of bioaugmentation research, which could play a significant role in mitigating the effects of chytridiomycosis on amphibians around the world. KEY WORDS: Chytridiomycosis · Challenge assay · Bacterial metabolite · Amphibian disease · BioaugmentationResale or republication not permitted without written consent of the publisher

Molecular detection ofNeisseria gonorrhoeae causes the second most prevalent bacterial sexually transmitted infection (STI) in men and women globally (42). As clinical signs of gonococcal infection may overlap with those of other STIs, laboratory testing is crucial for appropriate diagnosis and subsequent adequate treatment. The accurate diagnosis of gonorrhea relies on laboratory tests that are sensitive, specific, reproducible, and robust because of nonspecific clinical manifestations or lack of symptoms in women as well as men (43). Nucleic acid amplification tests (NAATs) have a number of recognized advantages over other diagnostic assays, which include increased sensitivity of detection and ease of sample collection (including use of self-collected samples) and transport, compared to culture-based methods (13,26,27,31,32). A number of commercial NAAT systems and assays developed "in-house" are currently in use for the detection of urogenital gonococcal infection, and their use has seen many of the anticipated benefits of NAATs. However, with increasing use of commercial and "in-house" NAAT systems over time and in different geographical settings, the need for both a considered approach to the application of NAATs and for an awareness of their limitations has arisen. These considerations include the sensitivity and specificity of the primary NAAT screening test, and where used, supplementary "confirmatory" tests as well as the prevalence of infection in various population groups (18). A number of assays have been shown to cross-react with other Neisseria species (10,20,23,29,36). The Centers for Disease Control and Prevention (CDC) (Atlanta, Georgia) and the Australian Public Health Laboratory Network have proposed a number of testing algorithms for confirmation of STIs by NAATs, which require the use of additional or supplementary assays (12,28,43). There is increasing evidence for the rise in infection in extragenital sites, which include the rectum and oropharynx, particularly in population groups of men who have sex with men (38). Commercial NAAT systems currently on the market have not been cleared by the FDA for diagnosis of specimens from the rectum, pharynx, or conjunctiva; however, as detection of N. gonorrhoeae by NAAT is more sensitive than culture (43), laboratories continue to offer these tests to diagnose extragenital gonorrhea (4). Extragenital sites carry a number of commensal Neisseria species and commonly Neisseria meningitidis, which due to having a high nucleic acid homology to N. gonorrhoeae may cross-react in the NAAT assay utilized (9). As crossreactions occur in screening assays, steps to include at least one

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