APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Apr. 1977, Copyright C 1977 American Society for Microbiology
p.
829-835
Vol. 33, No. 4 Printed in U.S.A.
Ambient-Temperature Primary Nonselective Enrichment for Isolation of Salmonella spp. from an Estuarine Environment J. B. KAPER, G. S. SAYLER,' M. M. BALDINI,2 AND R. R. COLWELL* Department of Microbiology, University of Maryland, College Park, Maryland 20742
Received for publication 21 September 1976
A primary, nonselective, ambient-temperature enrichment procedure for isolation of Salmonella spp. is described. The procedure was superior to elevatedtemperature selective enrichment for Salmonella when estuarine water samples were examined. Five Chesapeake Bay stations were monitored, over an 8-month period, for the presence of salmonellae. Of 72 water and sediment samples collected, 17 (23.6%) yielded Salmonella spp. Seven serotypes were identified among the isolates. A seasonal pattern was noted for the incidence of the salmonellae. A most probable number procedure, performed by membrane filtration and nonselective enrichment, yielded Salmonella most probable number indices as high as 110 per 100 g of sediment. The results suggest that new methods, such as the one described in this report, are required for isolation of human intestinal pathogens from estuaries and coastal waters.
Isolation of Salmonella spp. from water and sediment samples collected from Chesapeake Bay and its subestuaries was difficult when recommended procedures (4, 17) for isolation of Salmonella from food and water samples were employed. Salmonellae are usually isolated by employing highly selective enrichment and plating media incubated at an elevated temperature (41.5°C) after inoculation (1). Most investigators use this approach for detection of human pathogens in all types and classes of water, although, in fact, elevated temperatures and highly selective media are aimed at detection of Salmonella in water heavily contaminated with sewage (9, 19). Interestingly, Harvey and Price (9) observed that a lower incubation temperature (37°C) may be required for samples containing smaller populations of Salmonella or organisms in need of "resuscitation." The importance of resuscitating stressed Enterobacteriaceae before transfer to a selective medium was reported by Mossel et al. (13). In addition, Bissonnette et al. (3) demonstrated that stressed Escherichia coli can be revived by inoculation into a nonselective medium. Physical and chemical characteristics of the environment, as well as the residence time of the microorganisms, can influence recovery. For example, heat tolerance of Salmonella in samples collected from areas of in situ tempera-
tures less than 350C is significantly less than that of Salmonella recovered from samples collected where warmer in situ temperatures are found (2, 14). Baird-Parker et al. (2) showed heat tolerance to be inversely proportional to salinity, and Ng et al. (14) reported that carbon-limited cells may be more heat resistant than nitrogen-limited cells. Thus, environmental factors appear to exert a significant effect on recovery of Salmonella from extraintestinal sources. Salmonella spp. may be poor competitors in the aquatic environment (10), and recovery of these organisms may depend on factors not usually considered for clinical or food samples. In a previous study (18), an ambient-temperature primary nonselective method was used in parallel with selenite cystine and tetrathionate media for the isolation of Salmonella spp. in Chesapeake Bay. Thirty-four water and sediment samples were examined, and four (11.8%) were found to contain Salmonella spp. Serologically confirmed Salmonella spp. were recovered only when the nonselective enrichment method was employed. It is the objective of this report, therefore, to describe in detail the primary nonselective enrichment method for recovery of Salmonella spp. from estuarine samples and its application in a study of the Upper Chesapeake Bay.
' Present address: Graduate Program in Ecology, University of Tennessee, Knoxville, TN 37916. 2 Present address: U.S. Environmental Protection Agency, Marine Field Station, Health Effects Research Laboratory-Cin., West Kingston, RI 02892.
MATERIALS AND METHODS Sampling. Seventy-two Chesapeake Bay water and sediment samples were collected between 2 October 1975 and 27 May 1976. The samples were col829
830
APPL. ENVIRON. MICROBIOL.
KAPER ET AL.
lected at five sampling stations located in the midregion of Chesapeak Bay (Fig. 1). The stations included three sites in Baltimore Harbor and two relatively unpolluted areas in Chesapeake Bay, Chesapeake Beach and Eastern Bay. Water samples were collected with a 2-liter sterile Niskin bag sampler (General Oceanics, Miami, Fla.). Top water was sampled at ca. 1 m below the surface, and bottom water was sampled 1 to 2 m above the sediment-water interface. The upper 10 cm of the sediment was sampled by using a nonaseptic Petite Ponar grab, and the sediment samples were aseptically subsampled for microbiological examination. At each station, only one sample of each type, i.e., top water, bottom water, and sediment, was collected and examined on each cruise. Methods employed for measurement of temperature, dissolved oxygen, salinity, and transparency have been reported elsewhere (17). All samples were processed aboard ship immediately after collection. The inoculated media were incubated at 350C aboard ship and were transported to the laboratory upon docking. Approximately 1 h elapsed between docking and arrival at the laboratory. Subsequent elevated-temperature incubation (42.5 and 44.5°C) and bacteriological analyses were performed in the laboratory. Bacteriological examination. Total viable bacterial counts were determined by using Upper Bay yeast extract agar, the formula for which has been reported elsewhere (17).
Total coliforms were estimated by using a threedilution, three-tube replication of lactose broth (Difco) in a most probable number (MPN) series. Sample volumes of 10.0, 1.0, and 0.1 ml of top and bottom water or of a 1:10 dilution of sediment were inoculated into the appropriate tubes. In the spring and fall, additional dilutions were made at the Baltimore Harbor stations so that 0.1-ml amounts of 1:10 and 1:100 dilutions were also inoculated. Lactose MPN tubes were incubated at 35°C for 48 h, after which they were examined for growth and gas. Tubes showing growth and gas production were recorded as presumptive for total coliforms. Positive lactose broth tubes were used to inoculate tubes of EC broth (Difco) which were incubated at 44.5 + 0.5°C. Tubes showing growth and gas after 24 h were recorded as confirmed for the presence of fecal coliforms. The MPN index for both total and fecal coliforms was computed by using published tables (1). The procedure used to isolate Salmonella was a primary, nonselective, ambient-temperature enrichment method, devised because of the failure of selective enrichment to yield confirmed Salmonella (18). Duplicate tubes of double-strength lactose, dulcitol, and inositol broths were each inoculated with 10 ml of water sample or sediment dilution. The latter two media were prepared by the same formula as lactose broth except for the substitution of dulcitol or inositol for lactose. Top- and bottom-water samples were filtered through a 0.45-,um membrane
FIG. 1. Chesapeake Bay stations sampled in this study.
VOL. 33, 1977
NONSELECTIVE ENRICHMENT OF SALMONELLA
by using 100-ml volumes (later increased to 250-ml volumes for non-Baltimore Harbor stations). The membrane filters were rolled up after filtration and placed in single-strength lactose, dulcitol, and inositol broths. In addition to the sediment dilution, 50and 100-g sediment samples were placed into 100and 150-ml volumes, respectively, of enrichment broths. The inoculated enrichment media were allowed to incubate at ambient temperature, ca. 25°C, for approximately 4 h, followed by incubation at 35°C for a combined incubation of approximately 24 h. After primary nonselective (i.e., ambient-temperature) enrichment, 1 ml was transferred to 10 ml of selenite cystine broth (Difco) and incubated for 24 h at 42.5 + 0.5°C for selective enrichment. After selective enrichment, the selenite cystine broth cultures were streaked onto a variety of isolation media, including brilliant green agar (Bioquest), bismuth sulfite agar (Difco), salmonella-shigella agar (Difco), and XLD agar (Difco). The inoculated plates were then incubated at 35°C for 24 to 48 h. Characterization of isolates. Isolated colonies were screened on Kliger iron agar (Difco), and pected Salmonella cultures were biochemically characterized by using the API 20 system (Analytab Products Inc., Plainview, N.Y.). Identification to the species level was achieved by using the API computerized profile index. Confirmation of Salmonella by serological technique was performed at the Center for Disease Control, Atlanta, Ga. Salmonella MPN. On the May 1976 cruise, an MPN procedure was utilized to enumerate Salmonella in the Baltimore Harbor. Two sets of a threedilution, three-tube replication series were used for each sample. One set included dulcitol broth and the other, inositol broth. Sample volumes of 100, 10, and sus-
831
1 ml for water and 100, 10, and 1 g for sediment were inoculated into the respective tubes and flasks by the procedures outlined above. Cultures were incubated and transferred as described above, and presumptive Salmonella identification was based on biochemical results from the API 20 system and was duplicated by conventional tube tests.
RESULTS AND DISCUSSION Recovery of Salmonella. Of 72 samples of Chesapeake Bay water and sediment examined, 17 (23.6%) were found to contain Salmonella spp. All of the strains, isolated by using the nonselective enrichment method, were identified as S. enteritidis. The strains characterized to date comprise seven serotypes, including S. enteritidis serotypes derby, saintpaul, enteritidis, typhimurium, agona, amsterdam, and london (Table 1). The first 4 serotypes are listed among the 10 most frequently reported from human specimens in the United States during the period 1963-1974 (16). The other serotypes are not as common, with the exception of S. enteritidis serotype agona, the frequency of which has been increasing since 1972 (16). During the years 1972-1974, agona, thought to have been introduced into the United States by Peruvian fish meal, has been among the 10 serotypes most frequently isolated from humans (16). The fact that the majority of these serotypes are associated with human infections emphasizes the importance of water as a reservoir in transmission of salmonellosis. This fact, together with the possibility of fish as vectors
TABLE 1. Recovery of Salmonella enteriditis from samples of water and sediment by use of the primary, ambient-temperature, nonselective enrichment procedure Station Data Enrichment Sample typea Serotypes isolated Jones Falls BW October 1975 Inositol derby S Dulcitol derby, london S Inositol agona, derby
Fort McHenry
November 1975
TW
Dulcitol
typhimurium
April 1976
TW S S
Dulcitol Dulcitol Inositol
enteriditis amsterdam agona
October 1975
TW BW BW BW
Lactose
Dulcitol Inositol Lactose
derby
TW BW BW
Dulcitol Dulcitol Inositol
April 1976
derby agona derby, saint-paul
derby derby derby
TW Lactose October 1975 Colgate Creek derby BW, Bottom water collected 1 m above the sediment-water interface; S, sediment grab sample collected from the upper 10 cm of the sediment; TW, top water collected from the surface. a
832
KAPER ET AL.
APPL. ENVIRON. MICROBIOL.
(12), indicates a need to study further the environmental parameters of disease caused by Salmonella. The S. enteritidis strains were isolated from samples collected at three stations in Baltimore Harbor. At Jones Falls (station CBB09, Fig. 1), eight samples (44.4%) yielded Salmonella (Table 2). Fifty percent of the top-water and sediment samples were positive for Satmonella spp., and 33.3% of bottom water samples vwere also positive. Jones Falls is a shallow, lowsalinity site located in the northwest branch of Baltimore Harbor (Table 2), at the confluence of Jones Falls, a stream carrying sewage treatment effluent, and Baltimore Harbor. It is distinguished from the other stations by its extremely high fecal coliform MPN, which is rarely less than 2,400 per 100 ml. Seven of the samples (38.9%) collected at the Fort McHenry station (station CBB08, Fig. 1) were positive for Salmonella spp. Of the samples taken, 50% of both top and bottom water were positive, whereas only one sediment sample yielded Salmonella. Fort McHenry is about twice as deep as Jones Falls; the water is slightly more brackish, with a lower fecal coliform MPN (Table 3), and well mixed. The Fort McHenry station is not adjacent to any point source of pollution and, of the three harbor
stations, best reflects the condition of the inner harbor.
Colgate Creek (station CBB07, Fig. 1) is the deepest of the three stations examined in this study (Table 4), comparable to Fort McHenry in salinity but generally an order of magnitude lower in fecal coliform MPN. However, it is also exposed continually to petroleum and heavymetal pollution. Salmonellae were isolated from two ofthe samples (11.1%), top and bottom water, but not from sediment. Chesapeake Beach (station 8410A, Fig. 1) and Eastern Bay (station 8540S, Fig. 1) were found to be free from detectable salmonellae. These stations are more brackish (8 to 10%) and have far lower fecal coliform MPN indices, usually 2,400 >24,000 2,400 >2,400 >2,400 >24,000
>2,400 >24,000 2,400 >2,400 >2,400 >24,000
0 1 0 0 1 NDd
0.7 0.5 0.7 0.7 1.0 0.8
20.8 16.9 10.1 9.2 16.0 20.8
0.38 0.24 0.09 0.95 0.07 0.78
>2,400 >24,000 2,400 >2,400 >2,400 >24,000
>2,400 >24,000 2,400 >2,400 210 >24,000
1 0 0 0 0 ND
>24,000 >240,000 >240,000 >24,000 >24,000 >240,000
>24,000 >240,000 >240,000 11,000 11,000 110,000
Temp
37.0 8.9 3.5 3.9 120.0
120.0
Dissolved oxygen. b Total viable count per 1 ml of water or 1 g of sediment. c Most probable number per 100 ml of water or 100 g of sediment. d Serotypes for Salmonella isolated in May have not yet been determined. a
No. of
(0C)
par-
Coliform
MPNc
Fecal coliform MPN
Salmo-
nella serotypes
3 0 0 0 2
ND
833
NONSELECTIVE ENRICHMENT OF SALMONELLA
VOL. 33, 1977
TABLE 3. Physical, chemical, and bacteriological parameters measured at the Fort McHenry station Sample type and date
Top water October 1975 ... November 1975 December 1975 March 1976 .... April 1976 ..... May 1976 ...... Bottom water October 1975. .. November 1975 December 1975 March 1976 .... April 1976 ..... May 1976
......
Depth
TVC
Trans-
Salin-
DOg
(i/y)
ter)
ency
0 0 0 0 0 0
3.5 5.0 4.1 3.8 4.7 4.2
8.2 8.8
0.7 1.4 1.5 1.3 1.0 1.0
20.3 16.4 7.2 8.9 16.0 19.3
1.7 0.025 3.6 9.2 0.18 3.0
4.0 11.8 8.9 8.0 6.7
3.3 6.5 5.3 4.3 6.0
20.3 17.0 7.9
10.0 9.6
0.7 1.4 1.5 1.3 1.0
13.9
0.79 0.11 0.01 7.2 0.24
8.7
4.7
6.4
1.0
18.8
3.8
(m)
10.7 9.1 9.3
7.6 9.5
(
(OC)
(x 105)
Coliform MPNc
Fecal coliform MPN
No. of Salmonella rotypes
)
8.6
Sediment October 1975 ... November 1975 December 1975
2.0 85.0 3.3
March 1976
13.0
April 1976 May 1976
25.0 20.0
>2,400
1,100
1
4,600 240
0 0
>2,400 >2,400 >24,000
2,400 93 >2,400 >2,400 4,600
>2,400 2,100 460 >2,400 >2,400 11,000
460 1,200 21 150 >2,400 11,000
3 0 0 0 1 ND
>24,000 >240,000 460,000 >24,000 >24,000 110,000
11,000 2,400 230
p.
829-835
Vol. 33, No. 4 Printed in U.S.A.
Ambient-Temperature Primary Nonselective Enrichment for Isolation of Salmonella spp. from an Estuarine Environment J. B. KAPER, G. S. SAYLER,' M. M. BALDINI,2 AND R. R. COLWELL* Department of Microbiology, University of Maryland, College Park, Maryland 20742
Received for publication 21 September 1976
A primary, nonselective, ambient-temperature enrichment procedure for isolation of Salmonella spp. is described. The procedure was superior to elevatedtemperature selective enrichment for Salmonella when estuarine water samples were examined. Five Chesapeake Bay stations were monitored, over an 8-month period, for the presence of salmonellae. Of 72 water and sediment samples collected, 17 (23.6%) yielded Salmonella spp. Seven serotypes were identified among the isolates. A seasonal pattern was noted for the incidence of the salmonellae. A most probable number procedure, performed by membrane filtration and nonselective enrichment, yielded Salmonella most probable number indices as high as 110 per 100 g of sediment. The results suggest that new methods, such as the one described in this report, are required for isolation of human intestinal pathogens from estuaries and coastal waters.
Isolation of Salmonella spp. from water and sediment samples collected from Chesapeake Bay and its subestuaries was difficult when recommended procedures (4, 17) for isolation of Salmonella from food and water samples were employed. Salmonellae are usually isolated by employing highly selective enrichment and plating media incubated at an elevated temperature (41.5°C) after inoculation (1). Most investigators use this approach for detection of human pathogens in all types and classes of water, although, in fact, elevated temperatures and highly selective media are aimed at detection of Salmonella in water heavily contaminated with sewage (9, 19). Interestingly, Harvey and Price (9) observed that a lower incubation temperature (37°C) may be required for samples containing smaller populations of Salmonella or organisms in need of "resuscitation." The importance of resuscitating stressed Enterobacteriaceae before transfer to a selective medium was reported by Mossel et al. (13). In addition, Bissonnette et al. (3) demonstrated that stressed Escherichia coli can be revived by inoculation into a nonselective medium. Physical and chemical characteristics of the environment, as well as the residence time of the microorganisms, can influence recovery. For example, heat tolerance of Salmonella in samples collected from areas of in situ tempera-
tures less than 350C is significantly less than that of Salmonella recovered from samples collected where warmer in situ temperatures are found (2, 14). Baird-Parker et al. (2) showed heat tolerance to be inversely proportional to salinity, and Ng et al. (14) reported that carbon-limited cells may be more heat resistant than nitrogen-limited cells. Thus, environmental factors appear to exert a significant effect on recovery of Salmonella from extraintestinal sources. Salmonella spp. may be poor competitors in the aquatic environment (10), and recovery of these organisms may depend on factors not usually considered for clinical or food samples. In a previous study (18), an ambient-temperature primary nonselective method was used in parallel with selenite cystine and tetrathionate media for the isolation of Salmonella spp. in Chesapeake Bay. Thirty-four water and sediment samples were examined, and four (11.8%) were found to contain Salmonella spp. Serologically confirmed Salmonella spp. were recovered only when the nonselective enrichment method was employed. It is the objective of this report, therefore, to describe in detail the primary nonselective enrichment method for recovery of Salmonella spp. from estuarine samples and its application in a study of the Upper Chesapeake Bay.
' Present address: Graduate Program in Ecology, University of Tennessee, Knoxville, TN 37916. 2 Present address: U.S. Environmental Protection Agency, Marine Field Station, Health Effects Research Laboratory-Cin., West Kingston, RI 02892.
MATERIALS AND METHODS Sampling. Seventy-two Chesapeake Bay water and sediment samples were collected between 2 October 1975 and 27 May 1976. The samples were col829
830
APPL. ENVIRON. MICROBIOL.
KAPER ET AL.
lected at five sampling stations located in the midregion of Chesapeak Bay (Fig. 1). The stations included three sites in Baltimore Harbor and two relatively unpolluted areas in Chesapeake Bay, Chesapeake Beach and Eastern Bay. Water samples were collected with a 2-liter sterile Niskin bag sampler (General Oceanics, Miami, Fla.). Top water was sampled at ca. 1 m below the surface, and bottom water was sampled 1 to 2 m above the sediment-water interface. The upper 10 cm of the sediment was sampled by using a nonaseptic Petite Ponar grab, and the sediment samples were aseptically subsampled for microbiological examination. At each station, only one sample of each type, i.e., top water, bottom water, and sediment, was collected and examined on each cruise. Methods employed for measurement of temperature, dissolved oxygen, salinity, and transparency have been reported elsewhere (17). All samples were processed aboard ship immediately after collection. The inoculated media were incubated at 350C aboard ship and were transported to the laboratory upon docking. Approximately 1 h elapsed between docking and arrival at the laboratory. Subsequent elevated-temperature incubation (42.5 and 44.5°C) and bacteriological analyses were performed in the laboratory. Bacteriological examination. Total viable bacterial counts were determined by using Upper Bay yeast extract agar, the formula for which has been reported elsewhere (17).
Total coliforms were estimated by using a threedilution, three-tube replication of lactose broth (Difco) in a most probable number (MPN) series. Sample volumes of 10.0, 1.0, and 0.1 ml of top and bottom water or of a 1:10 dilution of sediment were inoculated into the appropriate tubes. In the spring and fall, additional dilutions were made at the Baltimore Harbor stations so that 0.1-ml amounts of 1:10 and 1:100 dilutions were also inoculated. Lactose MPN tubes were incubated at 35°C for 48 h, after which they were examined for growth and gas. Tubes showing growth and gas production were recorded as presumptive for total coliforms. Positive lactose broth tubes were used to inoculate tubes of EC broth (Difco) which were incubated at 44.5 + 0.5°C. Tubes showing growth and gas after 24 h were recorded as confirmed for the presence of fecal coliforms. The MPN index for both total and fecal coliforms was computed by using published tables (1). The procedure used to isolate Salmonella was a primary, nonselective, ambient-temperature enrichment method, devised because of the failure of selective enrichment to yield confirmed Salmonella (18). Duplicate tubes of double-strength lactose, dulcitol, and inositol broths were each inoculated with 10 ml of water sample or sediment dilution. The latter two media were prepared by the same formula as lactose broth except for the substitution of dulcitol or inositol for lactose. Top- and bottom-water samples were filtered through a 0.45-,um membrane
FIG. 1. Chesapeake Bay stations sampled in this study.
VOL. 33, 1977
NONSELECTIVE ENRICHMENT OF SALMONELLA
by using 100-ml volumes (later increased to 250-ml volumes for non-Baltimore Harbor stations). The membrane filters were rolled up after filtration and placed in single-strength lactose, dulcitol, and inositol broths. In addition to the sediment dilution, 50and 100-g sediment samples were placed into 100and 150-ml volumes, respectively, of enrichment broths. The inoculated enrichment media were allowed to incubate at ambient temperature, ca. 25°C, for approximately 4 h, followed by incubation at 35°C for a combined incubation of approximately 24 h. After primary nonselective (i.e., ambient-temperature) enrichment, 1 ml was transferred to 10 ml of selenite cystine broth (Difco) and incubated for 24 h at 42.5 + 0.5°C for selective enrichment. After selective enrichment, the selenite cystine broth cultures were streaked onto a variety of isolation media, including brilliant green agar (Bioquest), bismuth sulfite agar (Difco), salmonella-shigella agar (Difco), and XLD agar (Difco). The inoculated plates were then incubated at 35°C for 24 to 48 h. Characterization of isolates. Isolated colonies were screened on Kliger iron agar (Difco), and pected Salmonella cultures were biochemically characterized by using the API 20 system (Analytab Products Inc., Plainview, N.Y.). Identification to the species level was achieved by using the API computerized profile index. Confirmation of Salmonella by serological technique was performed at the Center for Disease Control, Atlanta, Ga. Salmonella MPN. On the May 1976 cruise, an MPN procedure was utilized to enumerate Salmonella in the Baltimore Harbor. Two sets of a threedilution, three-tube replication series were used for each sample. One set included dulcitol broth and the other, inositol broth. Sample volumes of 100, 10, and sus-
831
1 ml for water and 100, 10, and 1 g for sediment were inoculated into the respective tubes and flasks by the procedures outlined above. Cultures were incubated and transferred as described above, and presumptive Salmonella identification was based on biochemical results from the API 20 system and was duplicated by conventional tube tests.
RESULTS AND DISCUSSION Recovery of Salmonella. Of 72 samples of Chesapeake Bay water and sediment examined, 17 (23.6%) were found to contain Salmonella spp. All of the strains, isolated by using the nonselective enrichment method, were identified as S. enteritidis. The strains characterized to date comprise seven serotypes, including S. enteritidis serotypes derby, saintpaul, enteritidis, typhimurium, agona, amsterdam, and london (Table 1). The first 4 serotypes are listed among the 10 most frequently reported from human specimens in the United States during the period 1963-1974 (16). The other serotypes are not as common, with the exception of S. enteritidis serotype agona, the frequency of which has been increasing since 1972 (16). During the years 1972-1974, agona, thought to have been introduced into the United States by Peruvian fish meal, has been among the 10 serotypes most frequently isolated from humans (16). The fact that the majority of these serotypes are associated with human infections emphasizes the importance of water as a reservoir in transmission of salmonellosis. This fact, together with the possibility of fish as vectors
TABLE 1. Recovery of Salmonella enteriditis from samples of water and sediment by use of the primary, ambient-temperature, nonselective enrichment procedure Station Data Enrichment Sample typea Serotypes isolated Jones Falls BW October 1975 Inositol derby S Dulcitol derby, london S Inositol agona, derby
Fort McHenry
November 1975
TW
Dulcitol
typhimurium
April 1976
TW S S
Dulcitol Dulcitol Inositol
enteriditis amsterdam agona
October 1975
TW BW BW BW
Lactose
Dulcitol Inositol Lactose
derby
TW BW BW
Dulcitol Dulcitol Inositol
April 1976
derby agona derby, saint-paul
derby derby derby
TW Lactose October 1975 Colgate Creek derby BW, Bottom water collected 1 m above the sediment-water interface; S, sediment grab sample collected from the upper 10 cm of the sediment; TW, top water collected from the surface. a
832
KAPER ET AL.
APPL. ENVIRON. MICROBIOL.
(12), indicates a need to study further the environmental parameters of disease caused by Salmonella. The S. enteritidis strains were isolated from samples collected at three stations in Baltimore Harbor. At Jones Falls (station CBB09, Fig. 1), eight samples (44.4%) yielded Salmonella (Table 2). Fifty percent of the top-water and sediment samples were positive for Satmonella spp., and 33.3% of bottom water samples vwere also positive. Jones Falls is a shallow, lowsalinity site located in the northwest branch of Baltimore Harbor (Table 2), at the confluence of Jones Falls, a stream carrying sewage treatment effluent, and Baltimore Harbor. It is distinguished from the other stations by its extremely high fecal coliform MPN, which is rarely less than 2,400 per 100 ml. Seven of the samples (38.9%) collected at the Fort McHenry station (station CBB08, Fig. 1) were positive for Salmonella spp. Of the samples taken, 50% of both top and bottom water were positive, whereas only one sediment sample yielded Salmonella. Fort McHenry is about twice as deep as Jones Falls; the water is slightly more brackish, with a lower fecal coliform MPN (Table 3), and well mixed. The Fort McHenry station is not adjacent to any point source of pollution and, of the three harbor
stations, best reflects the condition of the inner harbor.
Colgate Creek (station CBB07, Fig. 1) is the deepest of the three stations examined in this study (Table 4), comparable to Fort McHenry in salinity but generally an order of magnitude lower in fecal coliform MPN. However, it is also exposed continually to petroleum and heavymetal pollution. Salmonellae were isolated from two ofthe samples (11.1%), top and bottom water, but not from sediment. Chesapeake Beach (station 8410A, Fig. 1) and Eastern Bay (station 8540S, Fig. 1) were found to be free from detectable salmonellae. These stations are more brackish (8 to 10%) and have far lower fecal coliform MPN indices, usually 2,400 >24,000 2,400 >2,400 >2,400 >24,000
>2,400 >24,000 2,400 >2,400 >2,400 >24,000
0 1 0 0 1 NDd
0.7 0.5 0.7 0.7 1.0 0.8
20.8 16.9 10.1 9.2 16.0 20.8
0.38 0.24 0.09 0.95 0.07 0.78
>2,400 >24,000 2,400 >2,400 >2,400 >24,000
>2,400 >24,000 2,400 >2,400 210 >24,000
1 0 0 0 0 ND
>24,000 >240,000 >240,000 >24,000 >24,000 >240,000
>24,000 >240,000 >240,000 11,000 11,000 110,000
Temp
37.0 8.9 3.5 3.9 120.0
120.0
Dissolved oxygen. b Total viable count per 1 ml of water or 1 g of sediment. c Most probable number per 100 ml of water or 100 g of sediment. d Serotypes for Salmonella isolated in May have not yet been determined. a
No. of
(0C)
par-
Coliform
MPNc
Fecal coliform MPN
Salmo-
nella serotypes
3 0 0 0 2
ND
833
NONSELECTIVE ENRICHMENT OF SALMONELLA
VOL. 33, 1977
TABLE 3. Physical, chemical, and bacteriological parameters measured at the Fort McHenry station Sample type and date
Top water October 1975 ... November 1975 December 1975 March 1976 .... April 1976 ..... May 1976 ...... Bottom water October 1975. .. November 1975 December 1975 March 1976 .... April 1976 ..... May 1976
......
Depth
TVC
Trans-
Salin-
DOg
(i/y)
ter)
ency
0 0 0 0 0 0
3.5 5.0 4.1 3.8 4.7 4.2
8.2 8.8
0.7 1.4 1.5 1.3 1.0 1.0
20.3 16.4 7.2 8.9 16.0 19.3
1.7 0.025 3.6 9.2 0.18 3.0
4.0 11.8 8.9 8.0 6.7
3.3 6.5 5.3 4.3 6.0
20.3 17.0 7.9
10.0 9.6
0.7 1.4 1.5 1.3 1.0
13.9
0.79 0.11 0.01 7.2 0.24
8.7
4.7
6.4
1.0
18.8
3.8
(m)
10.7 9.1 9.3
7.6 9.5
(
(OC)
(x 105)
Coliform MPNc
Fecal coliform MPN
No. of Salmonella rotypes
)
8.6
Sediment October 1975 ... November 1975 December 1975
2.0 85.0 3.3
March 1976
13.0
April 1976 May 1976
25.0 20.0
>2,400
1,100
1
4,600 240
0 0
>2,400 >2,400 >24,000
2,400 93 >2,400 >2,400 4,600
>2,400 2,100 460 >2,400 >2,400 11,000
460 1,200 21 150 >2,400 11,000
3 0 0 0 1 ND
>24,000 >240,000 460,000 >24,000 >24,000 110,000
11,000 2,400 230
