EXPERIMENTAL INFECTIONS OF RANA-ESCULENTA WITH AEROMONAS-HYDROPHILA -A MOLECULAR MECHANISM FOR THE CONTROL OF THE NORMAL FLORA

Frogs can be useful models for studying the mechanisms that may regula te their natural microbial flora. Their skin glands produce a secretio n containing 20-30 different peptides, some antimicrobial some neurotr ophic. As they often live in soil or silt that is rich in microbes, th ey can be expected to be able to prevent or eliminate infections in ve ry short peroids of time. The bacterium Aeromonas hydrophila is widely distributed in nature and is considered as part of the natural flora of frogs and many animals, including humans. From an alternative frog strain of A. hydrophila, Bo-3, we isolated a spontaneous and stable mu tant (Bo-3N), resistant to nalidixic acid, here used to follow the hos t-microbe interactions in experimental infection of mouth and skin of Rana esculenta. The skin peptides had been previously isolated, sequen ced and cloned. We showed that skin treatment with a glucocorticoid (G C) cream blocked ne novo synthesis of these peptides and, simultaneous ly, prepropeptide mRNAs disappeared while I kappa B alpha was up-regul ated. Experimental mouth infections with 20 million cells of A. hydrop hila Bo-3N showed that a normal wild frog can eliminate the bacteria f rom the mouth within 15 min, while a frog pretreated with GC cream for 1 h could not reduce Bo-3N below 3500 colony-forming units (CFU)/5 mu l 'saliva'. An in vitro comparison showed that frog blood or serum al lowed bacteria to grow, while the skin secretion killed the bacteria w ithin 10 min. Using different enzyme-linked immunosorbent assays (ELIS As) with rabbit anti-Bo-3 serum as a positive control, we were able to rule out immunoglobulin G (IgG) binding to A. hydrophila. An assay fo r immunoglobulin M (IgM) (or some other serum component) in frog serum showed binding to A. hydrophila only corresponding to a few per cent of the positive control. For skin infections we bathed the frogs for 1 0 min in an overnight culture of Bo-3N diluted to about 10(7) CFU/ml. Electrical stimulation after the bath showed, for the total secretion, a two to fourfold increase in the antibacterial activity, while a pre treatment with GC cream reduced the activity to about one-third of tha t of the non-bathed control frog. HPLC analysis of the peptide pattern confirmed these findings. The survival value of antimicrobial peptide s have earlier been demonstrated in vivo and in vitro only in Drosophi la. The present experiments are the first combined in vivo and in vitv o demonstrations of the function of peptide antibiotics in a vertebrat e. One such function is involved in the control of the natural flora.