A tentative national reference procedure for isolation and enumeration of Escherichia coli from bivalve molluscan shellfish by most probable number method

In the UK several quantitative methods exist for the examination of bivalve molluscan shellfish for sewage contamination. These methods include roll tubes, pour plates and most probable number (MPN) techniques, but there is no national standard method. A comparative study was made of the most commonly used methods for detection of Escherichia coli in bivalve shellfish. Schemes employing solid media, such as the roll tube and pour plate methods, underestimated faecal contamination in shellfish tissue compared with a liquid MPN multiple test-tube method using minerals-modified-glutamate broth (MMGB) as primary enrichment medium. The composition of MMGB apparently permits repair of sublethally injured cells of E. coli. Incorporation of resuscitation stages into the pour plate technique did not yield higher counts. A standardized MPN technique for examination of bivalve molluscan shellfish for E. coli content is proposed as a possible national reference procedure pending further collaborative assessment.     

Rapid estimation of Escherichia coli in live marine bivalve shellfish using automated conductance measurement

Conductance measurement for quantitative estimations of Escherichia coli in live bivalve shellfish was evaluated as an alternative to the conventional most probable number (MPN) method used in France. The sensitivity of the two techniques was comparable. A single regression line ( r =-0·968, P < 10^-6) between log₁₀ MPN and detection time (DT) was used to estimate E. coli concentrations for all shellfish examined. Estimation accuracy was ± 0·92 log unit. Repeatability was better for DT than the log₁₀ of MPN estimations (average coefficients of variation 2·7 and 9·3%, respectively). The conductance signal was attributable to E. coli in 96% of cases, and only 0·7% of E. coli cultures failed to exhibit a signal within 20 h. The conductance method reduces analysis handling time and is much easier to use than the MPN method. Moreover, results can be obtained within 5–9 h compared to 3 d for the MPN method.     

Calibration of the impedance method for rapid quantitative estimation of Escherichia coli in live marine bivalve molluscs

AIM: Calibration of impedance measurement was performed vs the Association Fran?oise de Normalisation (AFNOR) MPN method with a view to rapid enumeration of Escherichia coli in live marine bivalve molluscs. METHODS AND RESULTS: Linear regression models between log10 MPN and detection time (DT) were adjusted for several shellfish types, growth media, and impedance instruments (BacTrac and Malthus systems). Escherichia coli concentrations could be estimated from DT using a single regression line for BacTrac 4100 with M1 medium (R2 = 87.8%) and Malthus with M2 medium (R2 = 86.7%), and two regression lines for BacTrac 4110 with M2 medium (R2 = 86.4 and 88.2%). The uncertainty of the predicted bacterial concentration was around +/-0.43 log unit for duplicate sample analysis. The impedance signal was attributable to E. coli in 99% of cases. All cultures containing E. coli produced an impedance signal with BacTrac 4100 and BacTrac 4110, whereas 5.6% did not exhibit a signal with Malthus. CONCLUSIONS: Impedance measurement is a possible alternative to the MPN method for rapid quantitative estimation of E. coli in live bivalve shellfish. SIGNIFICANCE AND IMPACT OF THE STUDY: The impedance method reduces analysis handling time considerably and is much easier to use than the MPN method. Moreover, results can be obtained within 5-10 h, allowing rapid intervention to ensure public health protection in case of shellfish contamination.     

Levels of male-specific RNA bacteriophage and Escherichia coli in molluscan bivalve shellfish from commercial harvesting areas

AIMS: Current measures for controlling the public health risks associated with bivalve molluscan shellfish consumption rely on the use of Escherichia coli to indicate the sanitary quality of shellfish harvesting areas. However, it has been demonstrated that E. coli is an inadequate indicator of the viral risk associated with shellfish. An alternative indicator organism, male-specific RNA (FRNA) bacteriophage has been proposed for this role. This study compared the distribution of E. coli and FRNA bacteriophage in shellfish harvesting areas. METHODS AND RESULTS: A total of 608 shellfish samples from 49 shellfish harvesting areas were analysed for E. coli and FRNA bacteriophage using standard published methods. The geometric mean concentration of FRNA bacteriophage in all samples was over three times greater than that of E. coli (1800 and 538 counts/100 g for FRNA bacteriophage and E. coli, respectively). In contrast to E. coli, FRNA bacteriophage concentrations were strongly influenced by season with a geometric mean count of 4503 PFU/100 g in the winter (October-March) compared with 910 PFU/100 g in the summer (April-September). CONCLUSIONS: FRNA bacteriophage were present in shellfish at higher concentrations than E. coli. Elevated levels of FRNA bacteriophage observed in the winter concur with the known increased viral risk associated with shellfish harvested at that time of year in the UK. Levels of FRNA bacteriophage found in many shellfish from category B harvesting areas would not be eliminated by conventional treatment processes. SIGNIFICANCE AND IMPACT OF THE STUDY: Data from this study will inform future proposals to introduce FRNA bacteriophage as an indicator of the viral risk associated with shellfish.     

Recovery of Escherichia coli and Coliforms from Macerated Shellfish

A comparison of manual and mechanical maceration techniques for the bacteriological examination of molluscan shellfish suggests that mechanical maceration leads to an improved recovery of Escherichia coli . Extensive use of such a technique in the routine examination of shellfish has introduced a standard considerably more rigorous than that proposed originally.     

Detection of bacteriophage tracers in bivalve molluscan shellfish

Procedures to enumerate commonly used bacteriophage tracers in bivalve molluscan shellfish were evaluated. Bacteriophages specific to Serratia marcescens, Escherichia coli and Enterobacter cloacae can be recovered from shellfish flesh by chloroform treatment of homogenates and subsequent clarification of samples by centrifugation. Bacteriophages were enumerated using a soft agar overlay technique. Hard clams appeared to release toxic compounds during homogenization which dramatically reduced counts of Ent. clocae bacteriophage.     

Estimation of Escherichia coli in raw ground beef

This study was undertaken to establish and evaluate more rapid methods of estimating Escherichia coli in ground beef than the standard most probable number (MPN) technique. Direct inoculation of and modifications to EC medium gave unreliable estimates of the presumptive E. coli count. Solid media incubated at an elevated temperature were compared to the MPN technique. Anderson and Baird-Parker's tryptone bile agar (TBA) method and prepoured plates of Endo, Levine eosin methylene blue (EMB), and violet red bile (VRBA) agars incubated at 44 degree C gave equivalent counts to the standard MPN method. Anderson and Baird-Parker TBA was the most selective solid medium for E. coli estimation, but all selective media incubated at elevated temperature reduced apparent E. coli counts by as much as 50%. Indole-producing and lactose-fermenting Enterobacteriaceae, capable of growth at elevated temperature, were tested for their growth on TBA, EMB, and VRBA at elevated temperature. TBA was selective for E. coli biotype I compared to other Enterobacteriaceae that predominate in meats. VRBA and EMB incubated at elevated temperature were not as selective as TBA, but differences in colonies could be observed between typical E. coli colonies and other Enterobacteriaceae on these media. Therefore, VRBA incubated at elevated temperature is proposed as a quality assurance screening test for presumptive E. coli in ground meat. Resuscitation techniques and prepoured plates with VRBA increased recovery levels of presumptive E. coli, but, under the conditions of this study, not to levels that represented a significant practical difference.     

Maximizing recovery and detection of Cryptosporidium parvum oocysts from spiked eastern oyster (Crassostrea virginica) tissue samples

Numerous studies have documented the presence of Cryptosporidium parvum, an anthropozoonotic enteric parasite, in molluscan shellfish harvested for commercial purposes. Getting accurate estimates of Cryptosporidium contamination levels in molluscan shellfish is difficult because recovery efficiencies are dependent on the isolation method used. Such estimates are important for determining the human health risks posed by consumption of contaminated shellfish. In the present study, oocyst recovery was compared for multiple methods used to isolate Cryptosporidium parvum oocysts from oysters (Crassostrea virginica) after exposure to contaminated water for 24 h. The immunomagnetic separation (IMS) and immunofluorescent antibody procedures from Environmental Protection Agency method 1623 were adapted for these purposes. Recovery efficiencies for the different methods were also determined using oyster tissue homogenate and hemolymph spiked with oocysts. There were significant differences in recovery efficiency among the different treatment groups (P < 0.05). We observed the highest recovery efficiency (i.e., 51%) from spiked samples when hemolymph was kept separate during the homogenization of the whole oyster meat but was then added to the pellet following diethyl ether extraction of the homogenate, prior to IMS. Using this processing method, as few as 10 oocysts could be detected in a spiked homogenate sample by nested PCR. In the absence of water quality indicators that correlate with Cryptosporidium contamination levels, assessment of shellfish safety may rely on accurate quantification of oocyst loads, necessitating the use of processing methods that maximize oocyst recovery. The results from this study have important implications for regulatory agencies charged with determining the safety of molluscan shellfish for human consumption.     

Evaluation of the MicroFoss system for enumeration of total viable count, Escherichia coli and coliforms in ground beef

This study was conducted to evaluate the performance of the MicroFoss system (Biosys, Ann Arbor, MI) for enumeration of total viable organisms, Escherichia coli and coliforms in ground beef. The system performance was compared to that of the USDA Bacteriological Analytical Method (BAM) reference culture methods. The correlation coefficients for the regression lines comparing the MicroFoss system detection times to the results of plate count methods for the total viable counts, coliform counts and the most probable number (MPN) method for E. coli were -0.95, -0.96 and -0.97, respectively. Tests comparing the reproducibility of data generated independently by two technicians on the same batch of samples showed no significant differences (P>0.05) in the MicroFoss detection times and culture results. The plate count methods for the total viable counts and coliform counts, and the MPN method for E. coli required 10, 11 and 22 times, respectively, the amount of time to complete tests compared to the length of time required to perform these tests using the MicroFoss system. The MicroFoss system produced reproducible data and provided a rapid and cost-efficient alternative method for enumeration of TVC, coliforms and E. coli in ground beef.     

Estimation of Escherichia coli in raw ground beef.

This study was undertaken to establish and evaluate more rapid methods of estimating Escherichia coli in ground beef than the standard most probable number (MPN) technique. Direct inoculation of and modifications to EC medium gave unreliable estimates of the presumptive E. coli count. Solid media incubated at an elevated temperature were compared to the MPN technique. Anderson and Baird-Parker's tryptone bile agar (TBA) method and prepoured plates of Endo, Levine eosin methylene blue (EMB), and violet red bile (VRBA) agars incubated at 44 degree C gave equivalent counts to the standard MPN method. Anderson and Baird-Parker TBA was the most selective solid medium for E. coli estimation, but all selective media incubated at elevated temperature reduced apparent E. coli counts by as much as 50%. Indole-producing and lactose-fermenting Enterobacteriaceae, capable of growth at elevated temperature, were tested for their growth on TBA, EMB, and VRBA at elevated temperature. TBA was selective for E. coli biotype I compared to other Enterobacteriaceae that predominate in meats. VRBA and EMB incubated at elevated temperature were not as selective as TBA, but differences in colonies could be observed between typical E. coli colonies and other Enterobacteriaceae on these media. Therefore, VRBA incubated at elevated temperature is proposed as a quality assurance screening test for presumptive E. coli in ground meat. Resuscitation techniques and prepoured plates with VRBA increased recovery levels of presumptive E. coli, but, under the conditions of this study, not to levels that represented a significant practical difference.     

Most probable number methodology for quantifying dilute concentrations and fluxes of Escherichia coli O157:H7 in surface waters

Aims:  To better understand the transport and enumeration of dilute densities of Escherichia coli O157:H7 in agricultural watersheds, we developed a culture-based, five tube-multiple dilution most probable number (MPN) method.
Methods and Results:  The MPN method combined a filtration technique for large volumes of surface water with standard selective media, biochemical and immunological tests, and a TaqMan confirmation step. This method determined E. coli O157:H7 concentrations as low as 0·1 MPN per litre, with a 95% confidence level of 0·01–0·7 MPN per litre. Escherichia coli O157:H7 densities ranged from not detectable to 9 MPN per litre for pond inflow, from not detectable to 0·9 MPN per litre for pond outflow and from not detectable to 8·3 MPN per litre for within pond. The MPN methodology was extended to mass flux determinations. Fluxes of E. coli O157:H7 ranged from <27 to >10⁴ MPN per hour.
Conclusion:  This culture-based method can detect small numbers of viable/culturable E. coli O157:H7 in surface waters of watersheds containing animal agriculture and wildlife.
Significance and Impact of the Study:  This MPN method will improve our understanding of the transport and fate of E. coli O157:H7 in agricultural watersheds, and can be the basis of collections of environmental E. coli O157:H7.     

Maximizing Recovery and Detection of Cryptosporidium parvum Oocysts from Spiked Eastern Oyster (Crassostrea virginica) Tissue Samples

Numerous studies have documented the presence of Cryptosporidium parvum, an anthropozoonotic enteric parasite, in molluscan shellfish harvested for commercial purposes. Getting accurate estimates of Cryptosporidium contamination levels in molluscan shellfish is difficult because recovery efficiencies are dependent on the isolation method used. Such estimates are important for determining the human health risks posed by consumption of contaminated shellfish. In the present study, oocyst recovery was compared for multiple methods used to isolate Cryptosporidium parvum oocysts from oysters (Crassostrea virginica) after exposure to contaminated water for 24 h. The immunomagnetic separation (IMS) and immunofluorescent antibody procedures from Environmental Protection Agency method 1623 were adapted for these purposes. Recovery efficiencies for the different methods were also determined using oyster tissue homogenate and hemolymph spiked with oocysts. There were significant differences in recovery efficiency among the different treatment groups (P < 0.05). We observed the highest recovery efficiency (i.e., 51%) from spiked samples when hemolymph was kept separate during the homogenization of the whole oyster meat but was then added to the pellet following diethyl ether extraction of the homogenate, prior to IMS. Using this processing method, as few as 10 oocysts could be detected in a spiked homogenate sample by nested PCR. In the absence of water quality indicators that correlate with Cryptosporidium contamination levels, assessment of shellfish safety may rely on accurate quantification of oocyst loads, necessitating the use of processing methods that maximize oocyst recovery. The results from this study have important implications for regulatory agencies charged with determining the safety of molluscan shellfish for human consumption.     

Evaluation of recovery methods to detect faecal streptococci in polluted waters

AIMS: This paper compares the faecal streptococci count on 25 samples of polluted waters obtained with three techniques: most probable number (MPN), membrane filtration (MF) and pour plate (PP) methods. Although the PP method is a simple technique, familiar to water bacteriologists, it is not recommended in the international methods. METHODS AND RESULTS: For the MPN method, azide dextrose broth and ethyl violet azide broth were employed. For the MF technique, Millipore filters were placed onto azide maltose agar (KF agar), while for the PP method, 1 ml of a decimal water dilution was added to (Kennel Faecal) KF medium. Regression analysis and Friedman's ANOVA were performed to determine the relationship between faecal streptococci counts obtained with the three techniques. Statistical analysis of the results showed that the MPN, MF and PP techniques were equally valid with respect to faecal streptococci enumeration in polluted waters. CONCLUSION: Since the PP method was found to be as good as the other techniques, it may be preferred in polluted waters. It is more economical in terms of both time and materials than the MPN count, and it is as accurate as the MF count. SIGNIFICANCE AND IMPACT OF THE STUDY: This study indicates that the PP method, although not recommended internationally, is a reliable alternative to MF and MPN.     

水体中大肠杆菌的生物检测法—斑贝检测法

大肠杆菌是水体污染程度的重要指示菌 ,因而对其要求快速、准确的检测。常规检测法的优点是简便、快速 ,但缺点是当菌数过多会影响准确计数 ,而菌体过少则会出现漏检的情况。综述了一种新的大肠杆菌生物检测法 ,该法应用斑贝作为预测水体中菌密度升高的一种指示生物 ,因其具备快速积累并保留水体中大肠杆菌的能力 ,可有效地避免低菌数时的漏检情况 ,能使水域管理者降低正常监测大肠杆菌临界量所需的取样频率 ,因而提供了一个更高效的细菌污染的河水监测方案。     

Estimation of Low Numbers of Escherichia coli Bacteriophage by Use of the Most Probable Number Method

The estimation of low numbers of the Escherichia coli bacteriophage was made possible by use of the most probable number (MPN) method. This method is similar to the technique used for counting coliform bacteria. The statistical results were computed by referring to tables. The method makes it possible to record values as low as two particles per 100 ml of sample. The direct plate count and MPN method were found to be in good correlation for T2 bacteriophage and bulk T bacteriophage in samples obtained from a sewage treatment plant and from contaminated seawater.     

Enumeration of Escherichia coli O157 in cattle faeces using most probable number technique and automated immunomagnetic separation

AIMS: To determine the numbers of Escherichia coli O157 present in the faeces of naturally infected cattle. METHODS AND RESULTS: A combination of the most probable number (MPN) technique and automated immunomagnetic separation (AIMS) was used to enumerate E. coli O157 in cattle faeces from both pasture-fed and grain-fed animals. A total of 22 E. coli O157 positive faecal samples were enumerated for E. coli O157 (10 from pasture-fed and 12 from grain-fed animals). The numbers of E. coli O157 in cattle faeces varied from undetectable (<3 MPN g-1 of faeces) to 2.4 x 104 MPN g-1. There was no significant difference (P = 0.06) between the numbers of E. coli O157 in pasture-fed or grain-fed cattle faeces, although the geometric mean (antilog of the mean of log10 transformed MPN values) was higher in grain-fed (130 MPN g-1) than in pasture-fed (13 MPN g-1). CONCLUSIONS: Although the number of samples tested is small, the results indicate that E. coli O157 make up a small proportion of the total E. coli population present in cattle faeces. SIGNIFICANCE AND IMPACT OF THE STUDY: Information on the numbers of E. coli O157 present in cattle will assist in developing more robust quantitative risk assessments and formulating intervention strategies.     

Comparison of culture-based methods to enumerate Escherichia coli in tropical and temperate freshwaters

Aims: The specificity of a method for the enumeration of Escherichia coli (chromocult agar, CC) was tested using freshwater samples from a tropical area (Cuba Island) by isolating colonies and identifying them with API (Appareillage et procédé d’identification) strips. Enumerations of E. coli by the most probable number (MPN) microplate method were compared with counts on chromogenic and fluorogenic agar media [CC, rapid E. coli (REC), fluorocult] in tropical and temperate freshwater samples. Methods and Results: A high percentage of specificity (95·7%) for the CC agar enumeration of E. coli was observed. High regression coefficients (log‐log linear regressions) were found between E. coli counts on agar media and by the MPN method. In the tropical environment, counts with REC medium were significantly different from those obtained with the other methods. MPN counts were found to be significantly higher than those obtained using the plate counts methods in the temperate environment. Conclusions: Escherichia coli enumeration methods based on glucuronidase activity appear to be suitable for the evaluation of microbiological quality in the tropical environment featured in this study. Significance and Impact of the Study: The methods for the enumeration of E. coli tested in this study should help improve the evaluation of microbiological contamination of Cuban freshwaters.