APPLIED AND ENVIRONMENTAL MICROBIOLOGY, July 1976, p. 190-191 Copyright © 1976 American Society for Microbiology
Vol. 32, No. 1 Printed in U.S.A.
Phage Typing System for Salmonella enteritidis M. GERSHMAN Departments of Microbiology and Animal and Veterinary Sciences, University of Maine, Orono, Maine 04473
Received for publication 13 February 1976
A system is described for the phage typing of Salmonella enteritidis. The system was developed using a number of bacteriophages that were isolated from sewage. tivity was too extensive to permit single-plaque isolations, the assaying procedure was repeated using a series of diluted filtrates. Recovered phage was purified by repeated plating and harvesting and brought to titer by the procedure described by Swanstrom and Adams (7). Phages of sufficiently high titer were then diluted and tested against all the S. enteritidis cultures collected for this investigation. The phages used in this study were used at a routine test dilution of not less than 10-3. Phage isolates were selected and maintained for regular use if they were stable and potentially suitable for type differentiations. Cultures to be typed were lightly inoculated (1). Salmonella enteritidis is frequently impli- into 3 ml of nutrient broth and incubated at cated in disease outbreaks. It is consistently 37°C for 1.5 h or until turbidity was barely reported as one of the 10 most recovered sero- perceivable. A small quantity of the broth cultypes of human origin in the United States (2). ture was then flooded onto a nutrient agar Given the frequency of isolations, a phage set plate, in the manner described above, and spothas been developed in our laboratory to charac- ted with standardized drops of phage using septerize these microorganisms. Aside from relat- arate 1-ml syringes fitted with 26-gauge ing an isolate to an outbreak, phage typing can needles. The plates were incubated overnight be used for surveillance, assessing strain distri- at 37°C and read the next day. The cultures bution, and ascertaining the effectiveness of were examined with the aid of xlO aplanat therapeutic measures. hand lens and viewed through the bottom of the The 183 cultures used in this project were plate. Susceptibility to a phage was demonobtained from our own diagnostic service and strated by areas of clearing that ranged from the National Animal Disease Laboratory, isolated plaques to confluent lysis. Phage activAmes, Iowa. ity was recorded on the basis of the reactions Untreated sewage samples were secured lo- described in Table 1. cally from a number of treatment plants. Each In all, 26 phages were isolates; however, only 100-ml sample of raw sewage was enriched with eight were kept for service as they constituted a a 1.5-h nutrient broth culture of one of 56 ran- practical minimum for producing maximum domly chosen S. enteritidis cultures used pri- patterns of delineation. The lytic patterns of marily for detecting phage. The samples were these eight phages are described in Table 1. incubated for 18 h at 37°C and passed through a Using these isolates we were able to type all of 0.45-,um membrane filter (Millipore Corp.). the 183 cultures and classify them into 17 disEach filtrate was then assayed for phage by tinct groups. It is conceivable that more types spotting the surface of a nutrient agar plate exist and will be revealed as new cultures are that had been previously flooded with a 1.5-h examined. The characteristic patterns of the nutrient broth culture of the inoculum initially phage types observed were reproducible, reused for enrichment and dried for 15 min at flecting the stability and practicality of the sysroom temperature. After 18 to 24 h of incuba- tem. Some of the S. enteritidis cultures used in tion at 37°C the plates were eximined for the this study were isolated from a common outpresence of plaques. In cases where phage ac- break. The phage patterns of these isolates
Sonnenschein, in the 1920's, isolated specific phages for Salmonella paratyphi B and Salmonella typhi, and suggested that they be used for the rapid identification of these organisms (5, 6). In 1938 Craigie and Yen (3, 4) introduced a phage typing scheme for S. typhi which proved to be a valuable and sensitive tool in the control of typhoid fever. The ability of phage to distinguish varieties among apparently identical serotypes led to the development and acceptance of phage typing as a significant epidemiological procedure. Indeed, a number of Salmonella serotypes are now routinely typed in various public health laboratories throughout the world
190
191
NOTES
VOL. 32, 1976
TABLE 1. Typing pattern of Salmonella enteritidis with typing phages at routine test dilutionsa Strains of S. enteritidis
Typing phages
Type strains 1
2
3
4
5
6
7
8
No.
%
2.7 5 OL OL
Vol. 32, No. 1 Printed in U.S.A.
Phage Typing System for Salmonella enteritidis M. GERSHMAN Departments of Microbiology and Animal and Veterinary Sciences, University of Maine, Orono, Maine 04473
Received for publication 13 February 1976
A system is described for the phage typing of Salmonella enteritidis. The system was developed using a number of bacteriophages that were isolated from sewage. tivity was too extensive to permit single-plaque isolations, the assaying procedure was repeated using a series of diluted filtrates. Recovered phage was purified by repeated plating and harvesting and brought to titer by the procedure described by Swanstrom and Adams (7). Phages of sufficiently high titer were then diluted and tested against all the S. enteritidis cultures collected for this investigation. The phages used in this study were used at a routine test dilution of not less than 10-3. Phage isolates were selected and maintained for regular use if they were stable and potentially suitable for type differentiations. Cultures to be typed were lightly inoculated (1). Salmonella enteritidis is frequently impli- into 3 ml of nutrient broth and incubated at cated in disease outbreaks. It is consistently 37°C for 1.5 h or until turbidity was barely reported as one of the 10 most recovered sero- perceivable. A small quantity of the broth cultypes of human origin in the United States (2). ture was then flooded onto a nutrient agar Given the frequency of isolations, a phage set plate, in the manner described above, and spothas been developed in our laboratory to charac- ted with standardized drops of phage using septerize these microorganisms. Aside from relat- arate 1-ml syringes fitted with 26-gauge ing an isolate to an outbreak, phage typing can needles. The plates were incubated overnight be used for surveillance, assessing strain distri- at 37°C and read the next day. The cultures bution, and ascertaining the effectiveness of were examined with the aid of xlO aplanat therapeutic measures. hand lens and viewed through the bottom of the The 183 cultures used in this project were plate. Susceptibility to a phage was demonobtained from our own diagnostic service and strated by areas of clearing that ranged from the National Animal Disease Laboratory, isolated plaques to confluent lysis. Phage activAmes, Iowa. ity was recorded on the basis of the reactions Untreated sewage samples were secured lo- described in Table 1. cally from a number of treatment plants. Each In all, 26 phages were isolates; however, only 100-ml sample of raw sewage was enriched with eight were kept for service as they constituted a a 1.5-h nutrient broth culture of one of 56 ran- practical minimum for producing maximum domly chosen S. enteritidis cultures used pri- patterns of delineation. The lytic patterns of marily for detecting phage. The samples were these eight phages are described in Table 1. incubated for 18 h at 37°C and passed through a Using these isolates we were able to type all of 0.45-,um membrane filter (Millipore Corp.). the 183 cultures and classify them into 17 disEach filtrate was then assayed for phage by tinct groups. It is conceivable that more types spotting the surface of a nutrient agar plate exist and will be revealed as new cultures are that had been previously flooded with a 1.5-h examined. The characteristic patterns of the nutrient broth culture of the inoculum initially phage types observed were reproducible, reused for enrichment and dried for 15 min at flecting the stability and practicality of the sysroom temperature. After 18 to 24 h of incuba- tem. Some of the S. enteritidis cultures used in tion at 37°C the plates were eximined for the this study were isolated from a common outpresence of plaques. In cases where phage ac- break. The phage patterns of these isolates
Sonnenschein, in the 1920's, isolated specific phages for Salmonella paratyphi B and Salmonella typhi, and suggested that they be used for the rapid identification of these organisms (5, 6). In 1938 Craigie and Yen (3, 4) introduced a phage typing scheme for S. typhi which proved to be a valuable and sensitive tool in the control of typhoid fever. The ability of phage to distinguish varieties among apparently identical serotypes led to the development and acceptance of phage typing as a significant epidemiological procedure. Indeed, a number of Salmonella serotypes are now routinely typed in various public health laboratories throughout the world
190
191
NOTES
VOL. 32, 1976
TABLE 1. Typing pattern of Salmonella enteritidis with typing phages at routine test dilutionsa Strains of S. enteritidis
Typing phages
Type strains 1
2
3
4
5
6
7
8
No.
%
2.7 5 OL OL
