Relationship among fecal coliforms, Escherichia coli, and Salmonella spp. in shellfish

The relationship of fecal coliforms, Escherichia coli, and Salmonella spp. was examined in freshly harvested and stored shellfish. In 16 of 40 freshly collected oyster samples, fecal coliform levels were above the recommended wholesale level suggested by the National Shellfish Sanitation Program (less than or equal to 230/100 g), and Salmonella spp. were present in three of these samples. Salmonella spp. were not, however, present in any sample containing less than 230 fecal coliforms per 100 g. Analysis of the data suggests that low fecal coliform levels in both fresh and stored oysters are good indicators of the absence of Salmonella spp., but that high levels of fecal coliforms are somewhat limited in predicting the presence of Salmonella spp. E. coli levels correlated very strongly with fecal coliform levels in both fresh and stored oysters and clams, suggesting that there is no advantage in replacing fecal coliforms with E. coli as an indicator of shellfish quality.     

Effects of storage on microbial loads of two commercially important shellfish species, Crassostrea virginica and Mercenaria campechiensis.

The effects of storage on the microbial load in two commercially important species of shellfish were examined. Oysters (Crassostrea virginica) were stored as shellstock, shucked meats, and fully processed meats at four temperatures for up to 21 days, and clams (Mercenaria campechiensis) were stored only as shellstock. The concentrations of most microbiological groups of organisms increased with the duration and temperature of storage in both shellfish species, although the increases were significantly lower in claims. Concentrations of Vibrio cholerae rose by approximately 1 log in oysters stored as shellstock after 7 days at 2 degrees C, and Lac+ vibrios increased 2 logs at 8 degrees C. Total counts of bacteria, fungi, coliforms, fecal streptococci, Aeromonas hydrophila, and clostridia were significantly higher in shucked oysters than in those stored as shellstock. Fecal coliforms were statistically the same, but V. cholerae, Vibrio parahaemolyticus, and the Lac+ vibrios were higher in oysters stored as shellstock. The concentrations of all microbial groups were higher in fully processed oysters than in shucked meats, with the exception of the vibrios, which showed no significant difference among the treatments. The results showed that although traditional methods of storing shellfish resulted in an overall increase in the microbial load, vibrio levels increased only in oysters stored as shellstock. Although fecal coliform and total bacterial counts did not correlate with those for vibrios in fresh oysters, strong correlations were observed in oysters stored for 7 days, suggesting that these indicators may be useful in monitoring oyster quality when meats are stored for a limited time as shellstock.     

Effects of storage on microbial loads of two commercially important shellfish species, Crassostrea virginica and Mercenaria campechiensis

The effects of storage on the microbial load in two commercially important species of shellfish were examined. Oysters (Crassostrea virginica) were stored as shellstock, shucked meats, and fully processed meats at four temperatures for up to 21 days, and clams (Mercenaria campechiensis) were stored only as shellstock. The concentrations of most microbiological groups of organisms increased with the duration and temperature of storage in both shellfish species, although the increases were significantly lower in claims. Concentrations of Vibrio cholerae rose by approximately 1 log in oysters stored as shellstock after 7 days at 2 degrees C, and Lac+ vibrios increased 2 logs at 8 degrees C. Total counts of bacteria, fungi, coliforms, fecal streptococci, Aeromonas hydrophila, and clostridia were significantly higher in shucked oysters than in those stored as shellstock. Fecal coliforms were statistically the same, but V. cholerae, Vibrio parahaemolyticus, and the Lac+ vibrios were higher in oysters stored as shellstock. The concentrations of all microbial groups were higher in fully processed oysters than in shucked meats, with the exception of the vibrios, which showed no significant difference among the treatments. The results showed that although traditional methods of storing shellfish resulted in an overall increase in the microbial load, vibrio levels increased only in oysters stored as shellstock. Although fecal coliform and total bacterial counts did not correlate with those for vibrios in fresh oysters, strong correlations were observed in oysters stored for 7 days, suggesting that these indicators may be useful in monitoring oyster quality when meats are stored for a limited time as shellstock.     

Human enteroviruses in oysters and their overlying waters.

The presence of enteroviruses in oysters and oyster-harvesting waters of the Texas Gulf coast was monitored over a period of 10 months. Viruses were detected in water and oyster samples obtained from areas both open and closed to shellfish harvesting. Viruses were detected periodically in waters that met current bacteriological standards for shellfish harvesting. No significant statistical relationship was demonstrated between virus concentration in oysters and the bacteriological and physiochemical quality of water and shellfish. Viruses in water were, however, moderately correlated with total coliforms in water and oysters and with fecal coliforms in oysters. Total coliforms in water were realted to total coliforms in sediment were related only to total coliforms in sediment. Among the physiochemical characteristics of water, turbidity was related statistically to the organic matter content of water and to fecal coliforms in water. There was a marked effect of rainfall on the bacteriological quality of water. Of a total of 44 water samples, 26 yielded virus in concentrations from 4 to 167 plaque-forming units per 100-gallon (ca. 378.5-liter) sample. Of a total of 40 pools of 10 to 12 oysters each, virus was found in 14 pools at a concentration of 6 to 224 plaque-forming units per 100 g of oyster meat. On five occasions, virus was found in water samples when no virus could be detected in oysters harvested from the same sites. This study indicates that current bacteriological standards for determining the safety of shellfish and shellfish-growing waters do no reflect the occurrence of enteroviruses.     

Human enteroviruses in oysters and their overlying waters.

The presence of enteroviruses in oysters and oyster-harvesting waters of the Texas Gulf coast was monitored over a period of 10 months. Viruses were detected in water and oyster samples obtained from areas both open and closed to shellfish harvesting. Viruses were detected periodically in waters that met current bacteriological standards for shellfish harvesting. No significant statistical relationship was demonstrated between virus concentration in oysters and the bacteriological and physiochemical quality of water and shellfish. Viruses in water were, however, moderately correlated with total coliforms in water and oysters and with fecal coliforms in oysters. Total coliforms in water were realted to total coliforms in sediment were related only to total coliforms in sediment. Among the physiochemical characteristics of water, turbidity was related statistically to the organic matter content of water and to fecal coliforms in water. There was a marked effect of rainfall on the bacteriological quality of water. Of a total of 44 water samples, 26 yielded virus in concentrations from 4 to 167 plaque-forming units per 100-gallon (ca. 378.5-liter) sample. Of a total of 40 pools of 10 to 12 oysters each, virus was found in 14 pools at a concentration of 6 to 224 plaque-forming units per 100 g of oyster meat. On five occasions, virus was found in water samples when no virus could be detected in oysters harvested from the same sites. This study indicates that current bacteriological standards for determining the safety of shellfish and shellfish-growing waters do no reflect the occurrence of enteroviruses.     

Prevalence of Salmonella spp. in oysters in the United States

Food-borne diseases such as salmonellosis can be attributed, in part, to the consumption of raw oysters. To determine the prevalence of Salmonella spp. in oysters, oysters harvested from 36 U.S. bays (12 each from the West, East, and Gulf coasts in the summer of 2002, and 12 bays, four per coast, in the winter of 2002-2003) were tested. Salmonella was isolated from oysters from each coast of the United States, and 7.4% of all oysters tested contained Salmonella. Isolation tended to be bay specific, with some bays having a high prevalence of Salmonella, while other bays had none. Differences in the percentage of oysters from which Salmonella was isolated were observed between the summer and winter months, with winter numbers much lower probably due to a variety of weather-related events. The vast majority (78/101) of Salmonella isolates from oysters were Salmonella enterica serovar Newport, a major human pathogen, confirming the human health hazard of raw oyster consumption. Contrary to previous findings, no relationship was found between the isolation of fecal coliforms and Salmonella from oysters, indicating a necessity for specific monitoring for Salmonella and other pathogens rather than the current reliance on fecal coliform testing.     

Isolation of Klebsielleae from within living wood.

Previous studies from this laboratory have documented the presence of coliform bacteria emanating from wooden reservoirs containing finished drinking water. Coliforms were identified as Klebsiella pneumoniae and Enterobacter spp. In the present report, evidence is presented which suggests that the origin of these coliforms is from the wood used to construct the reservoirs. In liquid expressed from freshly cut redwood, total bacterial counts in the range of 10(5) to 10(6)/ml were commonly observed. When present, coliform counts were over 10(3)/ml of expressed liquid. E. agglomerans was the most prevalent coliform present, but Klebsiella was isolated from freshly cut logs. Citrobacter freundii was also occasionally isolated. No fecal coliform-positive Klebsiella were obtained from any of the samples. Highest total bacteria and coliform counts were observed in sapwood specimens. Coliforms were present throughout sapwood as evidenced by contact plating serial sections of freshly cut wood. Scanning electron micrographs illustrate the presence of bacterial colonies within sapwood tracheids. Other wood species also contained coliform bacteria but in numbers lower than found in redwood.     

Isolation of Klebsielleae from within living wood.

Previous studies from this laboratory have documented the presence of coliform bacteria emanating from wooden reservoirs containing finished drinking water. Coliforms were identified as Klebsiella pneumoniae and Enterobacter spp. In the present report, evidence is presented which suggests that the origin of these coliforms is from the wood used to construct the reservoirs. In liquid expressed from freshly cut redwood, total bacterial counts in the range of 10(5) to 10(6)/ml were commonly observed. When present, coliform counts were over 10(3)/ml of expressed liquid. E. agglomerans was the most prevalent coliform present, but Klebsiella was isolated from freshly cut logs. Citrobacter freundii was also occasionally isolated. No fecal coliform-positive Klebsiella were obtained from any of the samples. Highest total bacteria and coliform counts were observed in sapwood specimens. Coliforms were present throughout sapwood as evidenced by contact plating serial sections of freshly cut wood. Scanning electron micrographs illustrate the presence of bacterial colonies within sapwood tracheids. Other wood species also contained coliform bacteria but in numbers lower than found in redwood.     

Bacteriological water quality effects of hydraulically dredging contaminated upper Mississippi River bottom sediment.

Bacteriological effects of hydraulically dredging polluted bottom sediment in the navigation channel of the Upper Mississippi River (river mile 827.5 [about 1,332 km] to 828.1 [about 1,333 km]) were investigated. Bottom sediment in the dredging site contained high total coliform densities (about 6,800 most-probable-number total coliform index per g [dry weight] and 3,800 membrane filter total coliforms per g [dry weight]), and fecal coliforms comprised an average 32% of each total coliform count. Total coliform and fecal coliform densities in water samples taken immediately below the dredge discharge pipe were each approximately four times corresponding upstream values; fecal streptococcus densities were approximately 50 times corresponding upstream values. Correlation analysis indicated that mean turbidity values downstream to the dredging operation were directly and significantly (r greater than 0.94) related to corresponding total coliform, fecal coliform, and fecal streptococcus densities. Salmonellae and shigellae were not recovered from either upstream or downstream water samples. Turbidity and indicator bacteria levels had returned to predredge values within less than 2 km below the dredge spoil discharge area at the prevailing current velocity (about 0.15 m/s).     

Detection of coliform bacteria in water by polymerase chain reaction and gene probes.

Polymerase chain reaction (PCR) amplification and gene probe detection of regions of two genes, lacZ and lamB, were tested for their abilities to detect coliform bacteria. Amplification of a segment of the coding region of Escherichia coli lacZ by using a PCR primer annealing temperature of 50 degrees C detected E. coli and other coliform bacteria (including Shigella spp.) but not Salmonella spp. and noncoliform bacteria. Amplification of a region of E. coli lamB by using a primer annealing temperature of 50 degrees C selectively detected E. coli and Salmonella and Shigella spp. PCR amplification and radiolabeled gene probes detected as little as 1 to 10 fg of genomic E. coli DNA and as a few as 1 to 5 viable E. coli cells in 100 ml of water. PCR amplification of lacZ and lamB provides a basis for a method to detect indicators of fecal contamination of water, and amplification of lamB in particular permits detection of E. coli and enteric pathogens (Salmonella and Shigella spp.) with the necessary specificity and sensitivity for monitoring the bacteriological quality of water so as to ensure the safety of water supplies.     

Concentration of Escherichia coli in sediments as an indicator of the sanitary status of oyster (Crassostrea virginica) aquaculture sites

The objective of the study was to determine whether there are differences in Escherichia coli counts in relation to seasonal and/or spatial distribution patterns in a conditional shellfish‐growing zone located in the Richibucto estuary, New Brunswick, Canada. E. coli concentrations in surface water, sediments, suspended and bottom cultured American oysters (Crassostrea virginica) were determined. Contamination levels in water were significantly lower than in sediments, bottom and suspended cultured oysters (P < 0.01; P < 0.05, P < 0.01, n = 303, respectively). No significant difference in fecal contamination was observed between suspended and bottom cultured oysters (P > 0.05, n = 303). Seasonal variations (temporal) had a significant influence on fecal coliform contamination of all components (P < 0.01, n = 303, linear model). E. coli concentration levels were as much as ten times higher in sediments than in water. Furthermore, results suggest that E. coli concentration levels in sediments are a more reliable indicator of the sanitary status of a grow‐out operation than E. coli levels in water. Considering only sampling dates when contamination levels in oysters exceeded the established threshold of 230 MPN, less than 50% of the cases were predicted by water contamination while as many as 89.9% of the samples were predicted by sediment contamination using the proposed threshold of 75 MPN per 100 g. This paper emphasizes the importance of acknowledging the significant role sediments play in the dynamics of contamination by E. coli in areas that are currently exploited or are being considered for shellfish aquaculture. The presence of E. coli in sediments should not be overlooked as an integral factor in assessing the environmental sanitary status.     

Microbial impact of Canada geese (Branta canadensis) and whistling swans (Cygnus columbianus columbianus) on aquatic ecosystems.

Quantitative and qualitative analyses of the intestinal bacterial flora of Canada geese and whistling swans were carried out with the finding that wild birds harbor significantly more fecal coliforms than fecal streptococci. The reverse was typical of captive and fasting birds. Neither Salmonella spp. nor Shigella spp. were isolated from 44 migratory waterfowl that were wintering in the Chesapeake Bay region. Enteropathogenic Escherichia coli were detected in seven birds. Geese eliminated 10(7) and swans 10(9) fecal coliforms per day. Results of in situ studies showed that large flocks of waterfowl can cause elevated fecal coliform densities in the water column. From the data obtained in this study, it is possible to predict the microbial impact of migratory waterfowl upon aquatic roosting sites.     

Microbial impact of Canada geese (Branta canadensis) and whistling swans (Cygnus columbianus columbianus) on aquatic ecosystems.

Quantitative and qualitative analyses of the intestinal bacterial flora of Canada geese and whistling swans were carried out with the finding that wild birds harbor significantly more fecal coliforms than fecal streptococci. The reverse was typical of captive and fasting birds. Neither Salmonella spp. nor Shigella spp. were isolated from 44 migratory waterfowl that were wintering in the Chesapeake Bay region. Enteropathogenic Escherichia coli were detected in seven birds. Geese eliminated 10(7) and swans 10(9) fecal coliforms per day. Results of in situ studies showed that large flocks of waterfowl can cause elevated fecal coliform densities in the water column. From the data obtained in this study, it is possible to predict the microbial impact of migratory waterfowl upon aquatic roosting sites.     

Detection of coliform bacteria in water by polymerase chain reaction and gene probes

Polymerase chain reaction (PCR) amplification and gene probe detection of regions of two genes, lacZ and lamB, were tested for their abilities to detect coliform bacteria. Amplification of a segment of the coding region of Escherichia coli lacZ by using a PCR primer annealing temperature of 50 degrees C detected E. coli and other coliform bacteria (including Shigella spp.) but not Salmonella spp. and noncoliform bacteria. Amplification of a region of E. coli lamB by using a primer annealing temperature of 50 degrees C selectively detected E. coli and Salmonella and Shigella spp. PCR amplification and radiolabeled gene probes detected as little as 1 to 10 fg of genomic E. coli DNA and as a few as 1 to 5 viable E. coli cells in 100 ml of water. PCR amplification of lacZ and lamB provides a basis for a method to detect indicators of fecal contamination of water, and amplification of lamB in particular permits detection of E. coli and enteric pathogens (Salmonella and Shigella spp.) with the necessary specificity and sensitivity for monitoring the bacteriological quality of water so as to ensure the safety of water supplies.     

LENTICULE discs provide a homogenous format for external quality assessment samples: a comparison with freeze-dried samples for shellfish microbiology

AIMS: The aim was to compare the variability in Escherichia coli enumeration data and detection of Salmonella spp. between four samples of LENTICULE discs and freeze-dried samples for the Health Protection Agency's External Quality Assessment (EQA) scheme for shellfish microbiology. METHODS AND RESULTS: Four samples of known but undisclosed microbiological content were dispatched in both freeze-dried and LENTICULE disc formats to 57 participating laboratories in 20 countries. Participants examined samples using their routine methods for the most probable number (MPN) of E. coli per 100 g and the presence/absence of Salmonella spp. There was no significant difference between the Food and Environmental Proficiency Testing Unit and participating laboratories for E. coli and Salmonella spp. results. There were significantly less outlying results using the LENTICULE discs than freeze-dried sample format and equivalent or less variance for the former for E. coli MPN. There was no significant difference between LENTICULE discs and freeze-dried samples for the presence/absence of Salmonella spp. CONCLUSIONS: Overall the results indicated that there was equivalent or less variance in results for the LENTICULE discs than for freeze-dried samples, therefore LENTICULE discs are a homogenous and stable matrix for EQA samples. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides validation data for the replacement of freeze-dried samples by LENTICULE discs for the Health Protection Agency Shellfish EQA Scheme.     

Incidence of R factors in coliform, fecal coliform, and Salmonella populations of the Red River in Canada.

Coliforms, fecal coliforms, and Salmonella were isolated from the Red River, Manitoba, Canada, and identified. These organisms were then examined for resistance to 12 antibiotics. Some fecal coliforms were resistant to all 12 antibiotics, and 18% of the Salmonella isolates were resistant to one or more antibiotics. A total of 52.9% of the fecal coliforms resistant to three or more antibiotics were able to transfer single or multiple resistance (R) determinants to the Salmonella recipient, and 40.7% could transfer R determinants to the Escherichia coli recipient. Of the resistant Salmonella, 57% transferred one or two determinants to the Salmonella recipient, and 39% transferred one or two determinants to the E. coli recipient. It was calculated that populations of fecal coliforms containing R factors were as high as 1,400 per 100 ml and that an accidental intake of a few milliliters of water could lead to transient or permanent colonization of the digestive tract. Consideration of data on bacteria with R factors should be made in future water quality deliberations and in discharge regulations.     

Ecology, serology, and enterotoxin production of Vibrio cholerae in Chesapeake Bay.

A total of 65 isolates of Vibrio cholerae, serotypes other than O--1, have been recovered from water, sediment, and shellfish samples from the Chesapeake Bay. Isolations were not random, but followed a distinct pattern in which salinity appeared to be a controlling factor in V. cholerae distribution. Water salinity at stations yielding V. cholerae (13 out of 21 stations) was 4 to 17 0/00, whereas the salinity of water at stations from which V. cholerae organisms were not isolated was less than 4 or greater than 17 0/00. From results of statistical analyses, no correlation between incidence of fecal coliforms and V. cholerae could be detected, whereas incidence of Salmonella species, measured concurrently, was clearly correlated with fecal coliforms, with Salmonella isolated only in areas of high fecal coliform levels. A seasonal cycle could not be determined since strains of V. cholerae were detectable at low levels (ca. 1 to 10 cells/liter) throughout the year. Although none of the Chesapeake Bay isolates was agglutinable in V. cholerae O group 1 antiserum, the majority for Y-1 adrenal cells. Furthermore, rabbit ileal loop and mouse lethality tests were also positive for the Chesapeake Bay isolates, with average fluid accumulation in positive ileal loops ranging from 0.21 to 2.11 ml/cm. Serotypes of the strains of V. cholerae recovered from Chesapeake Bay were those of wide geographic distribution. It is concluded from the data assembled to date, that V. cholerae is an autochthonous estuarine bacterial species resident in Chesapeake Bay.     

Effectiveness of standard UV depuration at inactivating Cryptosporidium parvum recovered from spiked Pacific oysters (Crassostrea gigas)

When filter-feeding shellfish are consumed raw, because of their ability to concentrate and store waterborne pathogens, they are being increasingly associated with human gastroenteritis and have become recognized as important pathogen vectors. In the shellfish industry, UV depuration procedures are mandatory to reduce pathogen levels prior to human consumption. However, these guidelines are based around more susceptible fecal coliforms and Salmonella spp. and do not consider Cryptosporidium spp., which have significant resistance to environmental stresses. Thus, there is an urgent need to evaluate the efficiency of standard UV depuration against the survival of Cryptosporidium recovered from shellfish. Our study found that in industrial-scale shellfish depuration treatment tanks, standard UV treatment resulted in a 13-fold inactivation of recovered, viable C. parvum oocysts from spiked (1 x 10(6) oocysts liter (-1)) Pacific oysters. Depuration at half power also significantly reduced (P < 0.05; ninefold) the number of viable oocysts recovered from oysters. While UV treatment resulted in significant reductions of recovered viable oocysts, low numbers of viable oocysts were still recovered from oysters after depuration, making their consumption when raw a public health risk. Our study highlights the need for increased periodic monitoring programs for shellfish harvesting sites, improved depuration procedures, and revised microbial quality control parameters, including Cryptosporidium assessment, to minimize the risk of cryptosporidiosis.     

Comparison of four membrane filter methods for fecal coliform enumeration in tropical waters.

Four membrane filter methods for the enumeration of fecal coliforms were compared for accuracy, specificity, and recovery. Water samples were taken several times from 13 marine, 1 estuarine, and 4 freshwater sites around Puerto Rico, from pristine waters and waters receiving treated and untreated sewage and effluent from a tuna cannery and a rum distillery. Differences of 1 to 3 orders of magnitude in the levels of fecal coliforms were observed in some samples by different recovery techniques. Marine water samples gave poorer results, in terms of specificity, selectivity, and comparability, than freshwater samples for all four fecal coliform methods used. The method using Difco m-FC agar with a resuscitation step gave the best overall results; however, even this method gave higher false-positive error, higher undetected-target error, lower selectivity, and higher recovery of nontarget organisms than the method using MacConkey membrane broth, the worst method for temperate waters. All methods tested were unacceptable for the enumeration of fecal coliforms in tropical fresh and marine waters. Thus, considering the high densities of fecal coliforms observed at most sites in Puerto Rico by all these methods, it would seem that these density estimates are, in many cases, grossly overestimating the degree of recent fecal contamination. Since Escherichia coli appears to be a normal inhabitant of tropical waters, fecal contamination may be indicated when none is present. Using fecal coliforms as an indicator is grossly inadequate for the detection of recent human fecal contamination and associated pathogens in both marine and fresh tropical waters.