A comparison of ten USEPA approved total coliform/E. coli tests

Since 2002, the United States Environmental Protection Agency (USEPA) has approved ten enzyme-based total coliform and E. coli detection tests for examination of drinking water. These tests include: Colilert^®, Colilert-18^®, Colisure^®, m-Coli Blue 24^®, Readycult^® Coliforms 100, Chromocult^®, Coliscan^®, E*Colite^®, Colitag™ and MI Agar. The utility of the enzyme based test systems is based on both the ability of the test to detect the target organisms at low levels and the ability of the test system to suppress the growth of non-target organisms that might result in false positive results. Differences in the ability of some of these methods to detect total coliform and E. coli, as well as suppress Aeromonas spp., a common cause of “false positive” results, have been observed. As a result, this study was undertaken to elucidate the strengths and weaknesses of each method. Water samples were collected from three geographically and chemically diverse groundwaters in Wisconsin. One-hundred milliliter aliquots were individually spiked with both low concentrations (one to ten organisms) and high concentrations (fifty to one-hundred) of each of five different total coliform organisms (Serratia, Citrobacter, Enterobacter, E. coli, & Klebsiella). These spiked samples were used to test the capability of ten enzyme-based test systems to both detect and enumerate the spiked organisms. In addition, 100 ml samples were independently spiked with two different strains of Aeromonas spp. at six different levels, to assess the ability of each enzyme-based test to suppress Aeromonas spp. Analysis of the data indicated that wide variability exists among USEPA approved tests to detect and quantify total coliforms, as well as suppress Aeromonas spp.     

A comparison between minerals-modified glutamate medium and lauryl tryptose lactose broth for the enumeration of Escherichia coli and coliform organisms in water by the multiple tube method.

In a multi-laboratory trial, minerals-modified glutamate medium (MMGM) was compared with lauryl tryptose lactose broth (LTLB) in the multiple tube method for the enumeration of coliform organisms, including Escherichia coli, in water. Samples of raw and chlorinated waters yielded a total of 2313 positive tube-reactions with MMGM and 2174 with LTLB. These were interpreted either as E. coli; other coliform organisms; or as false positive reactions. The results at first reading (18 or 24 h) and at 48 h have been analysed statistically in terms of (i) most probable numbers of coliform organisms; (ii) positive reactions and their interpretation; and (iii) whether or not the sample yielded any E. coli or other coliform organisms. All three analyses indicated the same trends. For the detection of E. coli in raw waters LTLB was better than MMGM at 18-24 h, but MMGM was better at 48 h with waters containing small numbers of coliform organisms; for raw waters with greater numbers of organisms, both media performed equally well. Analysis of a subset of samples read at both 18 and 24 h indicated that the superiority of LTLB over MMGM with raw waters disappeared by 24 h. For chlorinated waters, LTLB yielded more positive gas reactions at 18-24 h, but fewer of these were E. coli than with MMGM; at 48 h MMGM was clearly better than LTLB for total coliform organisms including E. coli--especially if the numbers were small. MMGM therefore remains the medium of choice for the detection of E. coli as an indicator of faecal contamination of chlorinated drinking water supplies. It is also better for the detection of small numbers of E. coli in other waters.     

Effect of the implementation of HACCP on the microbiological quality of meals at a university restaurant

A study was conducted to evaluate the microbiological quality, including total mesophilic counts and markers of bacteriological hygiene, as indicator of food safety of three categories of the most consumed meals in a university restaurant, before and after implementation of the HACCP system and personnel training. Cold gastronomy products, cooked warm-served products, and cooked cold-served products were tested for bacterial contamination. Throughout the experiment, 894 samples were examined for total counts of aerobic bacteria, counts of indicator organisms (coliform organisms and Escherichia coli) and pathogens (Staphylococcus aureus, Bacillus cereus, Salmonella spp., and Listeria monocytogenes). Implementation of the HACCP system, together with training in personnel hygiene, good manufacturing practices, and cleaning and sanitation procedures, resulted in lower aerobic plate counts and a lower incidence of S. aureus, coliform organisms, E. coli, and B. cereus, whereas Salmonella spp. and L. monocytogenes were not found in all samples studied. The microbial results of this study demonstrate that personnel training together with HACCP application contributed to improve the food safety of meals served in the restaurant studied.     

Occurrence of drug-resistant bacteria in communal well water around Port Harcourt, Nigeria

A total of 108 raw water samples was collected from 36 wells at nine shanty settlements around Port Harcourt, Nigeria, over a period of 7 months. Samples were analysed for their bacteriological quality. Selected bacterial strains isolated from the samples were tested for their susceptibility to ten commonly used antibiotics. The organisms isolated include Pseudomonas spp., Klebsiella spp., Staphylococcus spp., Proteus spp., Enterococcus faecalis, Aeromonas spp., Escherichia coli, Chromobacterium spp., Flavobacterium spp., and Serratia spp. Out of 300 strains tested, 23 (6.9%) were susceptible to all the antibiotics, 277 (92.3%) were resistant to at least one antibiotic and 232 (77.3%) were resistant to two or more antibiotics. The epidemiological significance of these results is discussed.     

An evaluation of the hydrogen sulphide water screening test and coliform counts for water quality assessment in rural Malaysia.

The H2S water screening test and the membrane filtration faecal coliform count were compared with Escherichia coli counts for water samples collected from household water sources and domestic drinking water in rural Malaysia. Water samples were taken from 151 wells, 44 taps supplying water from the treated municipal supply and 192 domestic stored water supplies. E. coli were detected in 20% of the samples (42% of wells, 7% of tap water and 6% of drinking water). Excellent correlation (Spearman's rank correlation rs = 0.93) was found between the faecal coliform and E. coli counts for all sample types. The H2S method was poorly correlated whether read at 18 or 30 h. False positive rates were highest for well water, and false negative rates were highest for both well and drinking water samples, with low E. coli counts. The faecal coliform test was an excellent predictor of the presence of E. coli in these water samples, while the H2S test was very inadequate.     

Molecular identification of coliform bacteria isolated from drinking water reservoirs with traditional methods and the Colilert-18 system

The accuracy of a traditional method (lactose utilization with acid and gas production) for the detection of coliform bacteria and E. coli was tested in comparison with method ISO 9308-1 (based on acid formation from lactose) and the Colilert-18 system (detection of beta-galactosidase). A total of 345 isolates were identified after isolation from water samples using API 20E strips. The Colilert-18 led to the highest number of positive findings (95% of the isolates were assigned to coliforms), whereas the ISO-9308-1 method resulted only in 29% coliform findings. With the traditional method only 15% were rated positive. Most of the isolates were identified by the API 20E system as Enterobacter spp. (species of the Enterobacter cloacae complex), Serratia spp., Citrobacter spp.and Klebsiella spp.; but species identification remained vague in several cases. A more detailed identification of 126 pure cultures by using 16S rRNA gene sequence analysis and analysis of the hsp60 gene resulted in the identification of Enterobacter nimipressuralis, E. amnigenus, E. asburiae, E. hormaechei, and Serratia fonticola as predominat coliforms. These species are beta-galactosidase positive, but show acid formation from lactose often after a prolonged incubation time. They are often not of fecal origin and may interfere with the ability to accurately detect coliforms of fecal origin.     

Membrane filtration media for the enumeration of coliform organisms and Escherichia coli in water: comparison of Tergitol 7 and lauryl sulphate with Teepol 610.

In a multi-laboratory trial with the membrane filtration technique, three surfactants--Teepol 610 (T610), Tergitol 7 (T7) and sodium lauryl sulphate (LS)--were compared in media for the enumeration of coliform organisms and Escherichia coli in water. A total of 179 samples of water (87 raw and 92 marginally chlorinated) were examined for colony counts of coliform organisms, and 185 water samples (94 raw and 91 marginally chlorinated) for E. coli. Slight differences in the confirmed colony counts between the three media were noted, but few of these were observed consistently in every laboratory. In most laboratories, T7 gave slightly higher counts of E. coli than LS with chlorinated waters; a higher incidence of false-positive results for E. coli at 44 degrees C was also noted with T7. As there were no outstanding differences in the trial, sodium lauryl sulphate, which is chemically defined, cheap and readily available, is therefore recommended for use at a concentration of 0 . 1% instead of Teepol 610 in the standard medium for the enumeration of coliform organisms and E. coli in water by the membrane filtration technique.     

A microbiological study of bottled mineral water marketed in Ludhiana

The microbiological quality of bottled mineral water marketed in Ludhiana was examined, Twenty three brands were analyzed for presumptive coliform count by multiple tube tests, and E. coli count was confirmed by Eijkman test. Bacterial and fungal loads were tested by membrane filtration test. Out of 23 only one sample (4.4%) showed the presumptive coliform count to be 460 most probable number (MPN)l 1 00ml,and 1 was found to be positive when tested by Eijkman test for Ecoli. In the membrane filtration test three samples (13%) showed more than two types of bacteria. Different types of bacteria isolated included Bacillus sp (19/23). Pseudomonas spp (13123), Ecoli, Klebsiella sp and S.albus one each Fungi was isolated from five of twenty three. (22%) samples. Only one brand of mineral water was unfit for human consumption. The rest of the samples were contaminated with non pathogenic flora.     

大肠杆菌O157胶体金免疫层析快速筛查方法的建立

目的建立一种大肠杆菌O157的胶体金免疫层析快速筛查方法。方法利用胶体金免疫层析技术,采用双抗体夹心法检测大肠杆菌O157,对该法进行敏感性、特异性和食品样品的适用性分析。结果该法能在15分钟内完成检测,检测不同的肠杆菌科细菌(非O157型大肠杆菌、沙门菌、志贺菌、变形杆菌、柠檬酸杆菌、肠杆菌、沙雷菌、耶尔森菌)、葡萄球菌、李斯特菌、气单胞菌、弧菌等共24种30株常见细菌,未发现有交叉反应,显示该法特异性良好。检测大肠杆菌O157的最低检出浓度为1×105cfu/ml。方法适用于奶粉、面粉、淀粉、咖啡粉、饼干、蛋糕、果冻、燕窝和果汁等食品样品的检测。结论新建立的大肠杆菌O157胶体金免疫层析试验简便、快速,特异性和灵敏度较好,适用于现场样品的快速筛查。     

DNA-based real-time detection and quantification of aeromonads from fresh water beaches on Lake Ontario

The present study was designed to develop a novel, rapid, direct DNA-based protocol to enumerate aeromonads in recreational waters. An Aeromonas genus-specific real-time quantitative polymerase chain reaction (Q-PCR) protocol was developed and optimized using newly designed genus-specific oligonucleotide primers derived from the gyrase B subunit (GyrB) gene. A standard curve was developed based on the PCR protocol with a minimum quantification limit of 10 cell equivalents ml^-1 achieved using an autoclaved water sample from recreational water spiked with known quantities of an Aeromonas ATCC strain. The Q-PCR protocol was validated and applied to detect and quantify the total number of aeromonads in water samples collected from two fresh water beaches on Lake Ontario. The Q-PCR protocol revealed significantly higher numbers of aeromonads in all water samples than a culture-based assay at both beaches. Foreshore sand was found to serve as a reservoir of high concentrations of Aeromonas similar to this phenomenon noted for enteric bacteria like Eschershia coli. The new real-time Q-PCR protocol facilitated the rapid quantification of total numbers of Aeromonas cells present in recreational water samples in <3 hours without culturing.     

Drinking water test methods in crisis-afflicted areas: comparison of methods under field conditions

To simplify the testing of drinking water in crisis-afflicted areas (as in Kosovo in 2007), rapid test methods were compared with the standard test. For Escherichia coli and coliform pathogens, rapid tests were made available: Colilert(?)-18, P/A test with 4-methylumbelliferyl-β-D-glucoronid, and m-Endo Broth. Biochemical differentiation was carried out by Enterotube? II. Enterococci were determined following the standard ISO test and by means of Enterolert?. Four hundred ninety-nine water samples were tested for E. coli and coliforms using four methods. Following the standard method, 20.8% (n=104) of the samples contained E. coli, whereas the rapid tests detected between 19.6% (m-Endo Broth, 92.0% concordance) and 20.0% (concordance: 93.6% Colilert-18 and 94.8% P/A-test) positive samples. Regarding coliforms, the percentage of concordant results ranged from 98.4% (P/A-test) to 99.0% (Colilert-18). Colilert-18 and m-Endo Broth detected even more positive samples than the standard method did. Enterococci were detected in 93 of 573 samples by the standard method, but in 92 samples by Enterolert (concordance: 99.5%). Considering the high-quality equipment and time requirements of the standard method, the use of rapid tests in crisis-afflicted areas is sufficiently reliable.     

Pathogen and indicator variability in a heavily impacted watershed

Water samples were collected from 36 locations within the Grand River Watershed, in Southwestern Ontario, Canada from July 2002 to December 2003 and were analyzed for total coliforms, fecal coliforms, Escherichia coli, Escherichia coli O157:H7, and thermophilic Campylobacter spp. A subset of samples was also analyzed for Cryptosporidium spp., Giardia spp., culturable human enteric viruses, and Clostridium perfringens. Storm and snowmelt events were sampled at two locations including a drinking water intake. For the majority of the events, the Spearman rank correlation test showed a positive correlation between E. coli levels and turbidity. Peaks in pathogen numbers frequently preceded the peaks in numbers of indicator organisms and turbidity. Pathogen levels sometimes decreased to undetectable levels during an event. As pathogen peaks did not correspond to turbidity and indicator peaks, the correlations were weak. Weak correlations may be the result of differences in the sources of the pathogens, rather than differences in pathogen movement through the environment. Results from this investigation have implications for planning monitoring programs for water quality and for the development of pathogen fate and transport models to be used for source water risk assessment.     

Pathogen and indicator variability in a heavily impacted watershed

Water samples were collected from 36 locations within the Grand River Watershed, in Southwestern Ontario, Canada from July 2002 to December 2003 and were analyzed for total coliforms, fecal coliforms, Escherichia coli, Escherichia coli O157:H7, and thermophilic Campylobacter spp. A subset of samples was also analyzed for Cryptosporidium spp., Giardia spp., culturable human enteric viruses, and Clostridium perfringens. Storm and snowmelt events were sampled at two locations including a drinking water intake. For the majority of the events, the Spearman rank correlation test showed a positive correlation between E. coli levels and turbidity. Peaks in pathogen numbers frequently preceded the peaks in numbers of indicator organisms and turbidity. Pathogen levels sometimes decreased to undetectable levels during an event. As pathogen peaks did not correspond to turbidity and indicator peaks, the correlations were weak. Weak correlations may be the result of differences in the sources of the pathogens, rather than differences in pathogen movement through the environment. Results from this investigation have implications for planning monitoring programs for water quality and for the development of pathogen fate and transport models to be used for source water risk assessment.     

Faecal contamination indicators, Salmonella, Vibrio and Aeromonas in water used for the irrigation of agricultural products

The faecal contamination indicators (total coliforms, faecal coliforms, Escherichia coli, enterococci) and the genera Salmonella, Vibrio, Aeromonas were investigated in water samples used for irrigation. During 4 months, 52 samples were taken. The methods used were: multiple tube fermentation method for faecal contamination indicators and membrane filtration techniques for salmonella, aeromonas and vibrio. Two samples were positive for Salmonella spp., fourteen for Aeromonas spp. and no samples for Vibrio spp. No correlation was found between aeromonas and the indicators of faecal contamination. Regarding Aeromonas spp., 21.6% of the strains were adhesive and 12.6% cytotoxic: this confirms the possible role of aeromonas in human pathologies. These results are important to determine the quality of irrigation water in relation to human health. In fact, the spray or sprinkler irrigation produces bioaerosol, which can contaminate the crops that are likely to be eaten uncooked. In addition, the flood or furrow irrigation represents a risk to field workers.     

Microbial flora and food borne pathogens on minced meat and their susceptibility to antimicrobial agents

Background

Food-borne pathogens are the leading cause of illness and death in developing countries. Changes in eating habits, mass catering, unsafe food storage conditions and poor hygiene practices are major contributing factors to food associated illnesses. In Ethiopia, the widespread habit of raw beef consumption is potential cause for food borne illnesses. The present study aimed at investigating the microbial quality of meat available in common retail shops, restaurants and abattoir of Jimma City and determining susceptibility pattern of bacterial isolates.

Method

A total of 165 samples from food establishments, butcher shops and a slaughter houses were processed and analyzed for the presence of indicator bacterial and potential food pathogens using standards methods. Antimicrobial susceptibility test was performed for Salmonella, Shigella and Staphylococcus aureus isolates using Kirby-Bauer disk diffusion method.

Results

A total of 165 samples were collected from twenty four hotels and five butchers and an abattoir. Various food borne pathogens were isolated in 13 (43.3%) and indicator organisms in 29 (96.7%) out of the thirty food establishments (hotels, butchery and abattoir). Overall, ten different bacterial species were isolated which included, proteus spp 89 (53.9%), E. coli 44 (26.6%), Providencia spp 23 (13.9%) Citrobacter spp 15(9%), Pseudomonas spp 9 (5.5%), Klebsiella spp 2 (1.2%), Enterobacter spp 2 (1.2%), Salmonella spp 2(1.2%), and Shigella species 1 (0.6%). Out of the 44 E. coli isolates 37 (84%) were thermo tolerant E. coli and out of the gram positive organisms identified 20 (12.1%) were Staphylococcus aureus isolates. From the two Salmonella isolates one was susceptible against all 12 tested antimicrobials, while the other to all the 11 except cephalexin. Shigella dysentery was resistant only to co-trimoxazole and tetracycline. Out of the 20 S. aureus isolates, 90% showed resistance to oxacillin, 85% to ampicillin, 65% to erythromycin, 60% to amoxicillin, 35% to streptomycin, and 20% to vancomycin and all isolates were sensitive to co-trimoxazole (100%). In this study, 90% (18/20) of the S. aureus isolates were Methicillin Resistant Staphylococcus aureus.

Conclusion

In this study high percentage of indicator organisms as well as food borne pathogens were identified, which shows unhygienic condition of handling and processing in the food establishments. Our data also confirmed the presence of resistant food pathogens; particularly Staphylococcus aureus isolates which are Methicillin Resistant Staphylococcus aureus and multidrug resistant that emphasizes close follow up in the utilization of antibiotics. Therefore, meat handlers and sellers should be educated on the adverse effect of lack of proper personal, environmental hygiene and sanitation. In addition, consumers should be made aware of the risk of consuming raw and inadequately cooked meat.     

食品中大肠菌群快速检测效果评价

摘要:目的　分析复合显色培养基在食品检验中快速测定大肠菌群的作用。方法　以大肠菌群标准菌株及其他食源性致病菌标准菌株、85份食品样品为研究对象对象,同时采取国标GB4789.3中对大肠菌群的检测方法以及复合显色培养法进行大肠菌群的检测,比较两者的检测时间﹑准确性等。综合评价该复合显色培养基的效果。结果　将标准菌株大肠杆菌、弗氏柠檬酸杆菌、阴沟肠杆菌、阪崎肠杆菌、产气肠杆菌和肺炎克雷伯菌接种在复合显色平板及VRBA平板,均见生长,呈蓝绿色。而福氏志贺菌、副溶血性弧菌和金黄色葡萄球菌非大肠菌群属细菌在复合显色平板上培养18 h后,菌落无生长。检测食品样本的阳性率与国标大肠菌群VRBA平板法差异无统计学意义（P＞0.05）。结论　复合显色培养基可以快速检测食物中大肠菌群并且准确度高,值得在食品检测中使用。     

Determination of total aerobic and indicator bacteria on some raw eaten vegetables from wholesalers in Ankara, Turkey

The aim of this study was to investigate the bacteriological quality of some raw eaten salad vegetables obtained from wholesalers in Ankara, Turkey. A total of 180 samples including lettuces, cos lettuce, iceberg lettuce, parsley, dill and carrots were analyzed for total aerobic bacteria, total coliform bacteria and E. coli between January 2004 and April 2004. Compact Dry media (Nissui Pharmaceutical Co. Ltd., Japan) were used for bacteriological analysis. Outer leaves of lettuce, cos lettuce and iceberg-lettuce samples had higher bacterial loads (3.3-7.4 log(10)CFU(-1)) than inner leaves. Neither total aerobic and coliform bacteria nor E .coli were isolated from seven inner leaves (two cos and five iceberg-lettuce samples). E. coli was significantly more often detected on parsley (21/30) and dill samples (12/30) (p<0.05). Consequently, we showed that fresh salad vegetables (especially parsley, dill and cos lettuce) might contain pathogenic microorganisms and represent a risk for consumers regarding foodborne disease. The importance of adequate measures throughout the farm-to-table food chain was emphasized.     

Microbiological evaluation of sugarcane juice sold at street stands and juice handling conditions in São Carlos, São Paulo, Brazil

Fresh sugarcane juice is sold by street vendors without any heat treatment in S?o Carlos, S?o Paulo, Brazil. Twenty-four samples of point-of-sale juice were tested by standard methods to determine heterotrophic bacteria, total and thermo-tolerant coliform counts, Salmonella, and parasites in the juice. 25% of samples showed poor sanitary conditions, with thermo-tolerant coliform levels higher than allowed by Brazilian standards. Salmonella spp. and parasites were absent in all samples. Thermo-tolerant coliforms were detected on the hands of 37% of juice handlers, and heterotrophic bacterial counts reached 2.0 x 10(3) cfu/per hand. Escherichia coli was detected in one hand sample, and no Salmonella spp. was detected. Screening questionnaires were used to interview the vendors, and 62% of interviewees were either unfamiliar with or failed to adopt adequate hygiene for food handling.     

Characterization of Escherichia coli and Salmonella enterica from cattle feed ingredients

The objectives of this study were (1) to evaluate the frequency with which feed ingredients or mixed feeds in cattle feedlots were contaminated with Escherichia coli and Salmonella spp. and (2) to evaluate the antimicrobial susceptibility patterns of non-type-specific Escherichia coli and Salmonella spp. recovered from feed ingredients or mixed feeds. Approximately 30 individual samples were collected from each of several feed commodities present on two cattle feedlots each month for 1 year. Half of the samples were cultured for Escherichia coli, and the other half were cultured for Salmonella spp. E. coli was recovered from 48.2% (516/1070) of the samples and from all feed ingredient types at least once. Salmonella spp. were recovered from 5.3% (57/1070) of samples. Overall, 40.3% (207/514) of E. coli isolates and 54.4% (31/57) of Salmonella spp. isolates were susceptible to all antimicrobials tested in the panel. Bacterial contamination of feed ingredients used at cattle feedlots with enteric bacteria is relatively common. In some cases, the enteric organisms are resistant to one or more antimicrobials. Feed ingredients may be a source of genetic elements associated with antimicrobial resistance for feedlot cattle. To be successful in minimizing foodborne pathogens and antimicrobial resistance in the cattle feedlot setting, it is important to consider the myriad of potential sources of these organisms or genetic elements.