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.     

Bacteriological quality of some dairy products (kariesh cheese and ice cream) in alexandria

The present study estimated the total viable bacterial density, total and faecal coliforms, and E. coli in Kariesh cheese and ice cream. The study included 160 ice cream and kariesh cheese samples (80 samples each). Ice cream samples were 47 packed (33 cup and 14 stick) and 33 open samples while kariesh cheese samples were 62 open, 18 packed samples (8 of known brand and 10 of unknown brand). Samples were collected from supermarkets, shops and street vendors. All samples were analyzed for enumeration of total viable heterotrophic bacteria using standard pour plate method, and for the determination of the total coliforms, fecal coliforms and E. coli using multiple tube dilution method. Ice cream samples, showed that the total bacterial count was >/=1.5x105 cfu/g in 26 (32.5%) samples, total coliforms were >/= 10 MPN/g in 36 (45.0%) samples, fecal coliforms were detected in 45 (56.3%) samples ,and E. coli was detected in 34 (42.5%). kariesh cheese samples, showed a total coliforms of >/= 10 MPN/g in 54 (67.5%) samples, while fecal coliforms were detected in 64 (80%) samples, and E. coli was detected in 60 (75%). It is recommended to use and implement immediate regulatory measures like good manufacturing practices as well as distribution and retail storage practices for ensuring microbiological safety of ice cream and kariesh cheese.     

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 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.     

Are the defined substrate-based methods adequate to determine the microbiological quality of natural recreational waters?

Monitoring the microbiological quality of water used for recreational activities is very important to human public health. Although the sanitary quality of recreational marine waters could be evaluated by standard methods, they are time-consuming and need confirmation. For these reasons, faster and more sensitive methods, such as the defined substrate-based technology, have been developed. In the present work, we have compared the standard method of membrane filtration using Tergitol-TTC agar for total coliforms and Escherichia coli, and Slanetz and Bartley agar for enterococci, and the IDEXX defined substrate technology for these faecal pollution indicators to determine the microbiological quality of natural recreational waters. ISO 17994:2004 standard was used to compare these methods. The IDEXX for total coliforms and E. coli, Colilert^®, showed higher values than those obtained by the standard method. Enterolert^® test, for the enumeration of enterococci, showed lower values when compared with the standard method. It may be concluded that more studies to evaluate the precision and accuracy of the rapid tests are required in order to apply them for routine monitoring of marine and freshwater recreational bathing areas. The main advantages of these methods are that they are more specific, feasible and simpler than the standard methodology.     

Use of the ISO 9308-1 procedure for the detection of E. coli in water utilizing two incubation temperatures and two confirmation procedures and comparison with defined substrate technology

Disinfected and non-disinfected samples have been used to determine the accuracy of the ISO procedure (ISO 9308-1) for detection of E. coli in drinking water. Samples were analysed using the ISO procedure at both 36 and 44°C and using the defined substrate technology method Colilert-18^®/Quanti-Tray^® (Colilert-18). Utilizing the confirmation procedure described in ISO 9308-1, large numbers of false positive E. coli results were obtained using the ISO primary isolation procedure at 36°C. However, when glucuronidase production was used as the confirmation procedure, the ‘confirmed’ count of E. coli was lowest with ISO 9308-1 performed at 36°C. When TTC medium was incubated at 36°C confirmation using production of indole at 44°C resulted in 29% more ‘E. coli’ being recovered than when confirmation was performed using production of glucuronidase. When 44°C was used as the primary isolation temperature the difference between the number of ‘confirmed’ E. coli identified using the two confirmation procedures was less than 1% and was not significant. Identification of isolates which ‘confirmed’ when using production of indole at 44°C as the test method but °which failed to produce b-D-glucuronidase, showed that the majority of these isolates were Klebsiella oxytoca.     

Microbial quality of water in rural communities of Trinidad.

A cross-sectional study was conducted in four rural communities of northeastern Trinidad to determine the microbial quality of water supply to households and that quality's relationship to source and storage device. Of the 167 household water samples tested, total coliforms were detected in 132 of the samples (79.0%), fecal coliforms in 102 (61.1%), and E. coli in 111 (66.5%). There were significant differences among the towns in the proportion of the samples contaminated with coliforms (P < 0.001) and E. coli (P < 0.001). Of 253 strains of E. coli studied, 4 (1.6%) were mucoid, 9 (3.6%) were hemolytic, and 37 (14.6%) were nonsorbitol fermenters. Of 69 isolates of E. coli tested, 10 (14.5%) were verocytotoxigenic. Twenty-eight (14.0%) of 200 E. coli isolates tested belonged to enteropathogenic serogroups. Standpipe, the most common water source, was utilized by 57 (34.1%) of the 167 households. Treated water (pipeborne in homes, standpipes, or truckborne) was supplied to 119 households (71.3%), while 48 households (28.7%) used water from untreated sources (rain, river/stream, or well) as their primary water supply. The type of household storage device was associated with coliform contamination. Water stored in drums, barrels, or buckets was more likely to harbor fecal coliforms (74.2% of samples) than was water stored in tanks (53.3% of samples), even after controlling for water source (P = 0.04). Compared with water from other sources, water piped into homes was significantly less likely to be contaminated with total coliforms (56.9% versus 88.8%, P < 0.001) and fecal coliforms (41.2% versus 69.8%, P < 0.01), even when the type of storage device was taken into account. However, fecal contamination was not associated with whether the water came from a treated or untreated source. We concluded that the drinking water in rural communities in Trinidad was grossly unfit for human consumption, due both to contamination of various water sources and during household water storage.     

大肠杆菌检验方法的探究与分析

目的观察不同方法下呋喃唑酮片对大肠杆菌的检出率,分析探讨科学有效的检验方法和检验价值。方法分别应用一次离心法、离心滤膜法、离心双重滤膜结合法进行大肠杆菌检验,观察对比大肠杆菌检出率情况。结果一次离心法检验中,胆盐乳糖增菌液呈现微浑,伊红美蓝琼脂平板分离没有菌落长出;离心滤膜法检验中,胆盐乳糖增菌液呈现澄清,伊红美蓝琼脂平板分离没有菌落长出;离心双重滤膜法检验中,胆盐乳糖增菌液呈现浑浊、产气,伊红美蓝琼脂平板分离有典型大肠杆菌菌落长出。应用离心双重滤膜法检验的检出效果,明显优于应用一次离心法检验、离心滤膜法检验的效果,组间差异显著(P<0.05)。应用人工污染大肠杆菌进行证实,呋喃唑酮片以离心双重滤膜结合法进行检验,阳性检出率为100%。结论应用离心双重滤膜结合法进行呋喃唑酮片大肠杆菌的检验,操作简单,检出率高,值得临床上应用推广。     

一种改良的化妆品微生物检测方法的探讨

目的:用改良法检测化妆品中的微生物,将检测结果与《化妆品卫生规范》(2007年版)方法进行比较。方法:分别用《化妆品卫生规范》(2007年版)方法和改良方法对1000件样品进行菌落总数、霉菌和酵母菌、粪大肠菌群、铜绿假单胞菌和金黄色葡萄球菌的检测。改良方法为:用SCDLP增菌液代替生理盐水稀释样品,进行菌落总数、霉菌和酵母菌检测;SCDLP增菌后,用卵磷脂吐温80营养琼脂培养,对粪大肠菌群、铜绿假单胞菌和金黄色葡萄球菌筛选检测,并结合生化酶反应进行鉴定。结果:1000件样品的检测结果为:3件样品菌落总数指标超标;4件样品菌落总数和霉菌和酵母菌两项指标超标;1件样品菌落总数、霉菌和酵母菌、粪大肠菌群、铜绿假单胞菌和金黄色葡萄球菌五项指标均超标,共8件不合格样品,样品合格率为99.2%。两种方法检测结果相同。结论:改良法操作简便快捷,适合化妆品日常微生物的快速检测。     

Microbiological quality of drinking water in Akure metropolis,Nigeria

The microbiological qualities of four main sources of drinking water in Akure metropolis were investigated. These include wells, streams, tap and surface tank reservoirs. Results from these investigations inferred that there were high coliform counts in well and stream waters and the presence of Escherichia coli (E. coli) in four successive weekly water samples indicating that they were constantly being polluted with either human excrement or animal droppings. Although coliforms were occasionally detected in tap and surface tank waters, none of them was positive for E. coli test. Results from ANOVA test of most probably number (MPN) for coliforms and E. coli counts rejected the Null hypothesis (at 95% level) that there was no pollution. It also indicated that pollution that occurred was not by chance event.     

Comparison of two methods for evaluating the quality of stored drinking water in Abidjan, Côte d'lvoire, and review of other comparisons in the literature

Membrane filtration, multiple tube fermentation (the standard methods) and Colilert are techniques available for assessing drinking water quality, but there are no published comparisons of Colilert to standard methods in a developing country laboratory. We reviewed the published literature on Colilert and standard methods and conducted a study to compare Colilert with membrane filtration for the detection and enumeration of total coliforms and fecal coliforms (Escherichia coli bacteria) using 35 stored drinking water samples from households in Abidjan, C?te d'lvoire. Our study results are consistent with previous published studies conducted in developed countries. Results from Colilert and membrane filtration correlated for both total coliforms (r2 = 0.81) and E. coli (r2 = 0.93). Colilert is an acceptable method to measure the presence and quantity of coliforms in water samples in a developing country setting.     

即食凉拌食品中微生物污染调查分析及大肠菌群限量探讨

目的了解即食凉拌食品中微生物污染状况,为制定即食凉拌食品微生物限量标准提供依据。方法采用国家食源性疾病监测网监测方案和《中华人民共和国国家标准食品卫生微生物学检验方法》（GB/T4789-2003）检测凉拌食品中的沙门氏菌、志贺氏菌、O157∶H7/NM、单核细胞增生李斯特氏菌和大肠菌群。结果29家零售加工单位即食凉拌食品受大肠菌群污染差异无统计学意义（χ2=5.60,P〉0.05）,其零售的76份即食凉拌食品样品中均未检出目的致病菌。大肠菌群是主要污染菌,阳性率为71.05%,其中中式凉拌菜阳性率为70.23%,高于西式沙拉的68.97%,差异无统计学意义（t=0.92,P〉0.05）,菌浓度差异无统计学意义（t=0.7,P〉0.05）。超市零售加工的中式凉拌菜大肠菌群阳性率高于普通饭店和中高级饭店,差异有统计学意义（χ2=7.00,P〈0.05）,平均菌浓度差异有统计学意义（F=11.06,P〈0.01）。结论大肠菌群是凉拌食品主要的污染菌,应尽快出台即食凉拌食品卫生标准,建议即食凉拌食品大肠菌群限量≤100MPN/g。     

Specificity of a defined substrate method used to monitor balneability of tropical coastal waters impacted by polluted stormwater

Defined substrate (DS) is an alternative technique to monitoring the water quality based on species-specific enzyme activity. Although more sensitive and more specific than traditional media, there is some controversy over use in the warmer waters of tropical and subtropical environments, rich in organic matter and microorganism groups capable of interfering with results. The aim of this study was to test the specificity of DS method (Colilert, IDEXX) for detection of coliforms and Escherichia coli in stormwater seawater samples from a coastal city (Fortaleza, Brazil) compared to findings obtained with the multiple tube fermentation (MTF) method. The samples were collected from stormsewers and adjacent seashore locations. The most probable number (MPN) of total coliforms (TC), thermotolerant coliforms (TtC) and E. coli was determined and the selectivity of the enzymatic substrate medium in the seawater samples was tested. The MTF method showed samples from sampling points 1, 2 and 3 to be 13.3, 13.3 and 46.7%, respectively, above the legal cut-off value for coastal balneability. With the DS method, the corresponding figures were 60, 53.3 and 80% for E. coli. Overall, coliform levels were higher with the DS medium. Vibrios and aeromonads were isolated from E. coli-positive DS tubes.     

Evaluation of Lepto Dri Dot as a rapid test for the diagnosis of leptospirosis

Lepto Dri Dot is a new card agglutination test developed by the Dutch Royal Tropical Institute for the rapid diagnosis of leptospirosis. We evaluated the test in field conditions in The Andaman Islands. Patients suspected of leptospirosis who attended three primary health centres were included in the study. The test results were compared with blood culture or microscopic agglutination tests on paired serum samples; 74 of 124 patients were diagnosed as having leptospirosis based on these criteria. Lepto Dri Dot had a sensitivity of 67.6% (50/74) and a specificity of 66.0% (33/50) during week 1. During weeks 2-4 the values increased to 85.5% (47/55) and 80% (40/50) respectively. An IgM ELISA was also performed on the serum samples for comparison and this was marginally less sensitive, but more specific, during the first week of illness. The positivity rates for the Dri Dot test during days 2-3, 4-5 and 6-7 were 53.1% (17/32), 75.0% (18/24) and 83.3% (15/18), respectively. The corresponding values for ELISA were 28.1% (9/32), 54% (13/24) and 77.8% (14/18). Both Dri Dot and ELISA showed good agreement with the standard diagnostic criteria after the first week of illness (kappa= 0.65 and 0.74, respectively). The overall concordance of the two tests was 89.5 % (kappa = 0.79). The test does not require special storage or sophisticated equipment and can be performed by relatively low skilled personnel.     

Microbial quality of oysters sold in Western Trinidad and potential health risk to consumers

The prevalence and characteristics of Escherichia coli and Salmonella spp. as well as counts of E. coli in raw oysters, condiments/spices, and raw oyster cocktails sampled from 72 vendors across Western Trinidad were determined. The microbial quality of the water used in the preparation of raw oysters was also investigated. Of 200 samples each of raw oysters, condiments/spices and oyster cocktails tested, 154 (77.0%), 89 (44.5%) and 154 (77.0%) respectively yielded E. coli. The differences were statistically significant (P = < 0.001; chi square = 62.91). The mean E. coli count per g in the ready-to-eat oyster cocktail ranged from 1.5 x 10(3) +/- 2.7 x 10(3) in Couva to 8.7x10(6) +/- 4.9x10(7) in San Fernando. One hundred and forty-six (73.0%) oyster cocktails contaminated with E. coli had counts that exceeded the recommended standard of 16 per g. Of a total of 590 E. coli isolates from various sources tested, 24 (4.1%), 20 (3.4%) and 69 (11.7%) were mucoid, haemolytic and non-sorbitol fermenters respectively. Twelve (2.0%) isolates of E. coli were O157 strains, while 92 (46.0%) of 200 E. coli isolates tested belonged to enteropathogenic serogroups. Ninety (45.0%) and 73 (36.5%) of 200 water samples contained total coliforms and faecal coliforms respectively, with counts that exceeded 2.2 coliforms per 100 ml. Salmonella spp. were isolated from 7 (3.5%), 1 (0.5%) and 2 (1.0%) of 200 samples each, of raw oysters, condiments/spices and oyster cocktails respectively. Oysters pose a health risk to consumers in Trinidad, particularly from colibacillosis and salmonellosis, and the need for increased public awareness of this hazard cannot be over-emphasized.     

Microbiological evaluation of South Australian rock lobster meat.

Samples of frozen precooked rock lobster meat from five South Australian fish-processing plants situated in the West Coast and south-east regions were tested over a period of six months during the 1974/5 lobster fishing season. The most probable number (MPN) of E. coli and coliforms, Staphylococcus aureus and Salmonella, as well as total plate count (TPC) were determined in 480 samples. Monthly geometric mean TPC ranged from 1600/g to 25,000/g. The highest geometric mean of the MPN of coliforms and E. coli were 4.9/g and 1.8/g respectively. The highest geometric mean number of staphylococci was 18.6/g. Salmonella was not detected in the 480 units tested. Only 0.4% of the samples had TPC exceeding 100,000/g. Coliforms and E. coli were not present in 76.1% and 92.7% respectively of the samples tested. Staphylococcus aureus was not detected in 67.7% of the samples. The numbers of organisms in 82% of the samples fall within the microbiological standards proposed by the National Health and Medical Research Council of Australia for frozen precooked foods. The results of this study demonstrate the microbial quality of precooked lobster meat attainable when good manufacturing practices are used.     

Drinking water microbiological survey of the Northwestern State of Sinaloa, Mexico

A potable water survey, in two important municipalities of the state of Sinaloa, Mexico was conducted. Culiacan, capital city of Sinaloa and its neighboring municipality, Navolato were selected to enumerate Aeromonas hydrophila, Escherichia coli, fecal and total coliforms, Pseudomonas aeruginosa, and Heterotrophic plate count bacteria from 100 households' taps. Manganese; residual chlorine; pH; temperature and turbidity were also examined. Overall, Aeromonas hydrophila was not detected in any of the samples, 3% contained Escherichia coli, 28% had fecal and 46 total coliforms, P. aeruginosa was present in 15% of the samples. HPC bacteria were found in all of the samples but 43% had numbers greater than 500 CFU per ml. The average numbers obtained for the physico-chemical parameters were 0.15 mg/L; 0.32 mg/L; 6.5; 28.7 degrees C and 2.92 NTU for manganese, residual chlorine, pH, temperature and turbidity, respectively. The findings of the current study demonstrate that potable water from both municipalities can harbor substantial numbers of indicator and opportunistic pathogens suggesting that additional treatment in the household may be needed.     

肠球菌属耐药基因检测及耐药性分析

目的 探讨临床分离肠球菌标本的来源分布与耐药性及耐药基因.方法 选取2009年感染患者的肠球菌属分离株,采用常规方法进行菌种鉴定及药物敏感试验,并对其耐药基因进行检测.结果 粪肠球菌检出20株,占57.1％,屎肠球菌共分离到15株,占42.9％;21株多药耐药肠球菌中,阳性基因aac(6′)/aph(2″)、aph(3′)-Ⅲ、ant(6)-Ⅰ、ermB、tetM分别检出19、11、11、19、10株,检出率分别为90.5％、52.4％、52.4％、90.5％、47.6％.结论 临床分离的肠球菌属多药耐药严重;携带抗菌药物相关耐药基因是导致菌株对抗菌药物产生耐药的重要原因.     

The microbiological quality of potable water on board ships docking in the UK and the Channel Islands: an association of Port Health Authorities and Health Protection Agency Study

Providing safe potable water onboard vessels presents particular challenges and contamination can occur directly from source waters as well as during loading, storage and distribution. Between May and October 2005, 950 potable water samples were collected from 342 ships docking at ports. Comparison with Guidelines found 9% of samples contained coliforms, Escherichia coli or enterococci and 2.8% had faecal indicators (E. coli or enterococci). Action levels of aerobic colony count (ACC) bacteria were detected in 20% (22°C) and 21.5% (37°C) of samples. ACC results from one-off sampling are not informative as this does not enable port health authorities to monitor ACC trends. They should be removed as a routine criterion for remedial action and vessels should adopt the WHO Water Safety Plan approach, whilst continuing to monitor water quality with public health-based indicators (e.g. chlorine residual, coliforms, E. coli and enterococci). Logistic regression analyses identified practices associated with water quality. Practices protective against coliforms, E. coli or enterococci in potable supplies were: good hose hygiene, processing water onboard, maintaining free chlorine residual at 0.2 mg/L. This emphasizes the importance of good hygiene during potable water loading and maintaining adequate disinfection of supplies onboard.     

Microbiological and metal contamination of watercress in the Wellington region, New Zealand--2000 survey

OBJECTIVES: To investigate potential microbiological and metal contamination of watercress and to assess the public health risks associated with harvesting and consumption of watercress. METHOD: During March and April 2000, samples were taken from 11 known or potential watercress collection sites in the Wellington region. Microbiological testing included bacterial counts for presumptive and faecal coliforms (watercress); total coliforms (growing water); and Escherichia coli (E. coli) and presence/absence tests for Campylobacter species (growing water and watercress). Watercress concentrations of a range of metals were also measured. RESULTS: All of the sites showed significant levels of E. coli in samples of both watercress and water. The E. coli levels in water were well above recommended freshwater recreational contact safety guidelines at most sites. Campylobacter was detected in the growing waters at all sites (80% of the samples) and in 11% of the watercress samples. Mean metal concentrations in watercress did not exceed the NZ Food Regulations (1984) levels at any of the sites. However, lead concentrations at the urban sites and one of the semi-urban sites would have exceeded the new Australia New Zealand Food Standards Code maximum levels (2003). CONCLUSIONS AND IMPLICATIONS: The consumption of raw watercress contaminated with enteric pathogens could potentially cause serious gastrointestinal illness (e.g. campylobacteriosis) and people gathering watercress could also be at risk of infection from contact with contaminated surface waters.