JOURNAL OF CLINICAL MICROBIOLOGY, Dec. 1991, p. 2817-2823 0095-1137/91/122817-07$02.00/0 Copyright © 1991, American Society for Microbiology
Vol. 29, No. 12
Phage Typing of Salmonella enteritidis in the United States F. W. HICKMAN-BRENNER,l* A. D. STUBBS,2 AND J. J. FARMER I1l' Enteric Bacteriology Section, Enteric Diseases Branch, Division of Bacterial and Mycotic Diseases, National Center for Infectious Diseases, Centers for Disease Control, Atlanta, Georgia 30333,1 and Southeast Regional Laboratory, Food and Drug Administration, Atlanta, Georgia 303092 Received 17 May 1991/Accepted 18 September 1991
The number of reported isolates of Salmonella enteritidis has increased dramatically in the last 10 years. For phage typing has been a useful epidemiologic tool for studying outbreaks of S. typhi and S. typhimurium. In 1987, Ward et al. (L. R. Ward, J. De Sa, and B. Rowe, Epidemiol. Infect. 99:291-294, 1987) described a phage typing scheme for S. enteritidis. This system differentiated 27 phage types by use of 10 typing phages. With these phages, we typed 573 strains of S. enteritidis from humans (42 outbreaks), animals, food, and the environment. Ninety-six percent of the strains were typeable. The most common phage types were 8 (48.2%), 13a (20.1%), 13 (7.8%), and 14b (7.8%). Most of the strains were specifically collected from egg-related outbreaks in the northeastern United States in 1988 and 1989, probably accounting for the distribution of the four most common types in this sample. This system was particularly useful for differentiating a group of animal strains that had a number of diverse phage types. For 49 animal strains typed, 16 different patterns were obtained. Phage type 8 represented 32% of these strains, but no other phage type represented more than 8% of these strains. One-half of the 16 animal strains that were phage type 8 were from poultry. This phage typing system will be useful for comparing phage types found in the United States with those types encountered worldwide and for determining whether virulent strains of phage type 4 are entering the United States. Additional phage typing systems as well as molecular techniques are being studied to determine whether they can differentiate strains of phage types 8 and 13a. many years
The rate of isolation of Salmonella serotype Enteritidis has been increasing dramatically in the last 10 years (particularly in the northeastern United States), and this serotype has been the second most common one reported in that country (S. typhimurium has been the most common one). In 1989, 8,340 isolates of S. enteritidis were reported (4). Detailed information concerning reported outbreaks of S. enteritidis was collected at the Centers for Disease Control from January 1985 through May 1987. For 65 outbreaks reported, a food vehicle was established for 35. For 27 of the 35, or 77%, the food vehicle was grade A shell eggs. Eggs were traced to 13 different farms or distributors in seven states. In nursing homes, 10 of 130 infected persons died, a fatality rate of 8%, compared with an overall fatality rate of 0.5% (18). Since phage typing has been a useful laboratory tool for investigating outbreaks of S. typhi and S. typhimurium, the phage typing scheme for S. enteritidis, described in 1987 by Ward et al. (21), was obtained from the World Health Organization (WHO) International Center for Enteric Phage Typing, London, United Kingdom. Since the increase in S. enteritidis appears to be worldwide (17), we also wanted to compare the phage types found internationally with those found in the United States. This system was used to type 573 strains of S. enteritidis isolated in the United States. The data obtained and our experience with this system are presented here. This system is also being used at the U.S. Department of Agriculture (USDA), the Food and Drug Administration, and many national centers throughout the world. In the future there will be reports on the national and worldwide distributions of phage types. We hope that our experience with this phage typing system will benefit those reading such reports.
*
Corresponding author.
MATERIALS AND METHODS
Media. The media used for phage typing have been previously described (2, 7, 12). Modified phage agar contained 1.3% agar instead of the 2% previously used. Blood agar consisting of Trypticase soy agar and 5% sheep blood (BBL Microbiology Systems, Cockeysville, Md.) was used as the plating medium. Isolates were stored on semisolid Trypticase soy agar containing 15 g of Trypticase peptone (BBL), 5 g of Phytone peptone, 5 g of sodium chloride, 4 g of agar, and 1,000 ml of distilled water. Processing of diagnostic strains. Isolates of S. enteritidis with accompanying epidemiologic data were sent by state health departments and other laboratories for phage typing. Isolated colonies were tested. Incubation was done at 36 1°C unless otherwise noted. All stocks were stored at room temperature. For this study we made no distinction between the terms "isolate" and "strain." Preparation of phage agar plates for phage typing. Plates were prepared as previously described (7, 12). Modified phage agar plates (15 by 100 mm) contained 30 ml of medium. Preparation of broth cultures. Strains to be phage typed were inoculated from 24-h cultures on blood agar into 3 ml of phage broth (2) in screw-cap tubes (16 by 125 mm) to yield a heavy suspension (turbidity equal to a no. 1 MacFarland standard [11]). The cultures were grown for 2 h on a roller drum (20 rpm) located in a walk-in incubator. After 2 h, the broth was very turbid (equal to a no. 5 or 6 MacFarland standard). Preparation of lawns. Each broth, prepared as described above, was poured directly onto a dry modified phage agar plate. After the excess fluid was removed, the plates were ready for phage application. Application of phages. A phage applicator was used to deliver the 10 phages in the system (see Table 1) to the lawns 2817
2818
J. CLIN. MICROBIOL.
HICKMAN-BRENNER ET AL.
TABLE 1. Titration of the S. enteritidis phages of Ward et al. (21) with the phage type 1 strain as the host Result at dilution: Phage
1 2 3 4 5 6 7 8 9 10
10-1
1V-2
1O-3
CL OL OL OL OL SOL OL OL OL OL
CL SCL OL
SOL 250 SOL
OL OL 4+ SOL SOL OL SOL
RTD
10-4
io-0 iooa _b 2 x 10-3 25 10-2
50 200 20 SOL 100 distinct plaques, too numerous to count, but not confluent; SCL, semiconfluent lysis; SOL, semiopaque lysis; CL, confluent lysis; OL, opaque lysis; and
Vol. 29, No. 12
Phage Typing of Salmonella enteritidis in the United States F. W. HICKMAN-BRENNER,l* A. D. STUBBS,2 AND J. J. FARMER I1l' Enteric Bacteriology Section, Enteric Diseases Branch, Division of Bacterial and Mycotic Diseases, National Center for Infectious Diseases, Centers for Disease Control, Atlanta, Georgia 30333,1 and Southeast Regional Laboratory, Food and Drug Administration, Atlanta, Georgia 303092 Received 17 May 1991/Accepted 18 September 1991
The number of reported isolates of Salmonella enteritidis has increased dramatically in the last 10 years. For phage typing has been a useful epidemiologic tool for studying outbreaks of S. typhi and S. typhimurium. In 1987, Ward et al. (L. R. Ward, J. De Sa, and B. Rowe, Epidemiol. Infect. 99:291-294, 1987) described a phage typing scheme for S. enteritidis. This system differentiated 27 phage types by use of 10 typing phages. With these phages, we typed 573 strains of S. enteritidis from humans (42 outbreaks), animals, food, and the environment. Ninety-six percent of the strains were typeable. The most common phage types were 8 (48.2%), 13a (20.1%), 13 (7.8%), and 14b (7.8%). Most of the strains were specifically collected from egg-related outbreaks in the northeastern United States in 1988 and 1989, probably accounting for the distribution of the four most common types in this sample. This system was particularly useful for differentiating a group of animal strains that had a number of diverse phage types. For 49 animal strains typed, 16 different patterns were obtained. Phage type 8 represented 32% of these strains, but no other phage type represented more than 8% of these strains. One-half of the 16 animal strains that were phage type 8 were from poultry. This phage typing system will be useful for comparing phage types found in the United States with those types encountered worldwide and for determining whether virulent strains of phage type 4 are entering the United States. Additional phage typing systems as well as molecular techniques are being studied to determine whether they can differentiate strains of phage types 8 and 13a. many years
The rate of isolation of Salmonella serotype Enteritidis has been increasing dramatically in the last 10 years (particularly in the northeastern United States), and this serotype has been the second most common one reported in that country (S. typhimurium has been the most common one). In 1989, 8,340 isolates of S. enteritidis were reported (4). Detailed information concerning reported outbreaks of S. enteritidis was collected at the Centers for Disease Control from January 1985 through May 1987. For 65 outbreaks reported, a food vehicle was established for 35. For 27 of the 35, or 77%, the food vehicle was grade A shell eggs. Eggs were traced to 13 different farms or distributors in seven states. In nursing homes, 10 of 130 infected persons died, a fatality rate of 8%, compared with an overall fatality rate of 0.5% (18). Since phage typing has been a useful laboratory tool for investigating outbreaks of S. typhi and S. typhimurium, the phage typing scheme for S. enteritidis, described in 1987 by Ward et al. (21), was obtained from the World Health Organization (WHO) International Center for Enteric Phage Typing, London, United Kingdom. Since the increase in S. enteritidis appears to be worldwide (17), we also wanted to compare the phage types found internationally with those found in the United States. This system was used to type 573 strains of S. enteritidis isolated in the United States. The data obtained and our experience with this system are presented here. This system is also being used at the U.S. Department of Agriculture (USDA), the Food and Drug Administration, and many national centers throughout the world. In the future there will be reports on the national and worldwide distributions of phage types. We hope that our experience with this phage typing system will benefit those reading such reports.
*
Corresponding author.
MATERIALS AND METHODS
Media. The media used for phage typing have been previously described (2, 7, 12). Modified phage agar contained 1.3% agar instead of the 2% previously used. Blood agar consisting of Trypticase soy agar and 5% sheep blood (BBL Microbiology Systems, Cockeysville, Md.) was used as the plating medium. Isolates were stored on semisolid Trypticase soy agar containing 15 g of Trypticase peptone (BBL), 5 g of Phytone peptone, 5 g of sodium chloride, 4 g of agar, and 1,000 ml of distilled water. Processing of diagnostic strains. Isolates of S. enteritidis with accompanying epidemiologic data were sent by state health departments and other laboratories for phage typing. Isolated colonies were tested. Incubation was done at 36 1°C unless otherwise noted. All stocks were stored at room temperature. For this study we made no distinction between the terms "isolate" and "strain." Preparation of phage agar plates for phage typing. Plates were prepared as previously described (7, 12). Modified phage agar plates (15 by 100 mm) contained 30 ml of medium. Preparation of broth cultures. Strains to be phage typed were inoculated from 24-h cultures on blood agar into 3 ml of phage broth (2) in screw-cap tubes (16 by 125 mm) to yield a heavy suspension (turbidity equal to a no. 1 MacFarland standard [11]). The cultures were grown for 2 h on a roller drum (20 rpm) located in a walk-in incubator. After 2 h, the broth was very turbid (equal to a no. 5 or 6 MacFarland standard). Preparation of lawns. Each broth, prepared as described above, was poured directly onto a dry modified phage agar plate. After the excess fluid was removed, the plates were ready for phage application. Application of phages. A phage applicator was used to deliver the 10 phages in the system (see Table 1) to the lawns 2817
2818
J. CLIN. MICROBIOL.
HICKMAN-BRENNER ET AL.
TABLE 1. Titration of the S. enteritidis phages of Ward et al. (21) with the phage type 1 strain as the host Result at dilution: Phage
1 2 3 4 5 6 7 8 9 10
10-1
1V-2
1O-3
CL OL OL OL OL SOL OL OL OL OL
CL SCL OL
SOL 250 SOL
OL OL 4+ SOL SOL OL SOL
RTD
10-4
io-0 iooa _b 2 x 10-3 25 10-2
50 200 20 SOL 100 distinct plaques, too numerous to count, but not confluent; SCL, semiconfluent lysis; SOL, semiopaque lysis; CL, confluent lysis; OL, opaque lysis; and
