DRUG RESISTANCK OF FURUNCULOSIS IN TROUT 



559 



Tift 



Figure 1. — Sensitivity of Aeromonas salmonicida in vitro 

 to various antibiotics as determined with three media: 

 Trypticase Soy agar (TSA), Mueller Hinton agar (MH), 

 and standard furunculosis agar (#4). The antibiotic 

 and quantity used on Multidisks, reading clockwise, 

 were as follows: Upper: Tetracycline (TE), 10/ig.; 

 erythromycin (E), 10 /ig.; terramycin (T), 10 /ig. ; car- 

 boniycin (CA), 5 /ig.; bacitracin (B), 5 units; aureomycin 

 (A),10Atg. Lower: Viomycin (V), 10 /jg. ; Chloromycetin 

 (C), 10 ^g. ; polymyxin B, (PB), 10 units; penicillin (V), 

 1.5 units; streptomycin (dihydro) (S), 10 /ig- i neomycin 

 (X), 10 ^g. The presence of a clear zone indicates 

 sensitivity. 



as proteose peptone #3, vitamin-free casamino 

 aciils, nutrient brotli, and N-Z-Case were tested. 

 Surprisingly, the most promising results were 

 obtained witli nutrient broth, and this medium was 

 used in all subsequent experiments unless other- 

 wise in<licate(l. Since the sodium salts of sulfon- 



amides give alkaline solutions and their solubility 

 and ionization decreases with decreasing alkalinity 

 of the medium, nutrient broth was buffered by 

 adding 0.5 percent of secon<larv sodium phospliate 

 and the pH of the medium adjusted as required. 

 In the unbuffered nutrient broth (original pH, 6.8) 

 sodium sulfamerazine precipitated if added in 

 concentration up to 1 :2^Q. In such a medium the 

 sulfa-sensitive and sulfa-resistant strains of A. 

 Kalmonicida grew e(|uaily well. 



Since Griffin et al. (1953) found that A. salmoni- 

 cida has a wide pH tolerance, the buffered nutrient 

 brotli was adjusted to ])H 7.0, 7.5, 8.0, 8.5, and 9.0. 

 The more alkaline the medium, the more striking 

 were the differences noted between the sulfa- 

 sensitive and sulfa-resistant strains. The all- 

 round best restdts were obtained at pH 8.0 to 

 8.5: therefore, all std)sequent tests were run within 

 this pH range unless otherwise specified. 



According to Northey (1948), "Frequently it 

 is found that once an organism has become resist- 

 ant to one of the potent sulfonamide drugs it is 

 resistant to all tlie commonly used sulfa drugs." 

 Therefore it sliould be advantageous to select for 

 a routine in-vitro test with A. salmonicida a 

 sulfonamide, or sulfonamides, which would be the 

 most convenient to work with. With this in mind, 

 several commoidy useil sulfonamides were tested 

 with the two resistant and two sensitive strains 

 of ^4. salnuinicida. Sodiiun salts prepared in the 

 laboratory were serially diluted in the buffered 

 nutrient broth adjusted to pH 8.0 to 8.5. The 

 results are presented in table 6. Results for sulfa- 

 guanidine are omitted from the table because a 

 soluble sodium salt could not be prepared within 

 the desired pH range. 



On the basis of these results, all available 

 strains (84) of A. salmonicida were tested with 

 sulfadiazine, though sulfamerazine or sulfisoxazole 

 couki be used equally well. Results are presented 

 in table 4. It is evident that the largest number 

 of resistant cultures were isolated at Leetown in 



1955 from treated or control trout during a period 

 when treatments with sulfonamides were being 

 caiTied out. Cultures isolated at Leetown in 



1956 before any treatments started were pre- 

 dominantly sensitive. There were some inter- 

 mediate strains which produced a scanty growth 

 in the medium with sodium sulfadiazine. 



The highest dilutions of sidfonamides arresting 

 growth of .1. salmonicida in a buffered nutrient 



