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.     

MALDiI Botyper与API20E对克罗诺杆菌(阪崎肠杆菌)的鉴定结果比较

目的:评价MALDI-TOF MS Biotyper鉴定克罗诺杆菌的效果。方法:应用MALDI-TOF MS Biotyper与API20E分别对32株克罗诺杆菌(28株分离株,4株参考菌株)与相近菌株阴沟肠杆菌、产气肠杆菌进行鉴定,并对鉴定结果分析比较。结果:MALDI-TOF MS Biotyper2.0将32株克罗诺杆菌鉴定到种、属水平分别为56.2%和37.5%;API20E为75%、21.9%。3株菌未获得鉴定结果,其余29株菌的鉴定结果相符。结论:MALDI-TOF MS Biotyper作为一种新的细菌鉴定手段,可用于克罗诺杆菌的鉴定。     

Bacterial pathogens recovered from vegetables irrigated by wastewater in Morocco

The authors obtained 50 vegetable samples from various regions in Morocco and examined them to determine the microbiological quality of these products. Aerobic count, coliform, enterococci, and Staphylococcus aureus were evaluated. This analysis revealed high levels of enterococci, fecal coliforms, and total coliforms. No coagulase-positive Staphylococcus aureus was detected in any of the samples analyzed. Biochemical identification of Enterobacteriaceae showed the presence of Citrobacter freundii (28 percent), Enterobacter cloacae (27 percent), Escherichia coli (16 percent), Enterobacter sakazakii (12 percent), Klebsiella pneumoniae (17 percent), Serratia liquefaciens (11 percent), and Salmonella arizonae (0.7 percent). The results clearly demonstrate that vegetables irrigated with untreated wastewater have a high level of microbiological contamination. Consequently, these vegetables may be a threat for the Moroccan consumer and may be considered a serious risk to Moroccan public health.     

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.     

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.     

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.     

The effect of oxidase positive bacteria on total coliform density estimates.

The most probable number method for enumerating coliforms was shown to give a false estimate of the coliform density. The inflated estimates were due to lactose positive, non-coliform bacteria whose presence was detected by an oxidase test. Thirty-six percent of all the samples examined contained oxidase positive bacteria that were able to produce gas from lactose. Their presence resulted in coliform density overestimates which ranged from 2 to 33 times the true value. The frequency of occurrence of inaccurate estimates was shown to be affected by water temperature but unrelated to environmental source.     

Diversity and antifungal susceptibility of yeasts isolated by multiple-tube fermentation from three freshwater lakes in Brazil

The diversity and antifungal resistance of yeasts able to grow at 37°C and the occurrence of bacterial indicators of water quality were studied in three lakes in Southeastern Brazil. The densities of yeasts, Escherichia coli, Enterococcus spp. and Pseudomonas aeruginosa were determined by the multiple-tube fermentation technique, and counts of heterotrophic bacteria were determined using the pour plate method. The yeasts were identified using physiological and molecular techniques and their resistance to amphotericin B, itraconazole and fluconazole was tested. Yeast occurrence was significantly correlated only with the density of fecal coliforms. Candida krusei, C. guilliermondii and C. tropicalis, the most frequently isolated yeast species, are associated with fecal contamination of water by warm-blooded animals. Yeast isolates were most resistant to amphotericin B (21.7%), followed by itraconazole (20%) and then fluconazole (2.8%). In addition to tests for the fecal coliform group, the density of yeasts grown at 37°C could be used as a complementary microbial indicator that aquatic environments contain organic matter of human origin. The incidence of yeast species resistant to three antifungal drugs shows that these microorganisms could pose a health risk to the people who use these lakes for recreation.     

Epidemiological role of arthropods detectable in health facilities.

A total of 161 arthropod specimens were collected from 55 sites in a health care facility during July and September 1990. Of the 116 bacterial isolates obtained from their body surfaces 6% were from parasites (mosquitoes), 59% from eusynanthropic arthropods (Tenebrionid beetles, flies, German cockroaches, wasps), 16% from hemisynanthropic arthropods (ants, spiders) and 19% from occasionally encountered insects (non-biting midges, moths, beetles). Most (88%) of the isolated bacteria were Gram-negative rods of the species E. coli, Enterobacter, Klebsiella, Citrobacter, Proteus, Serratia, Pseudomonas and Acinetobacter. Gram-positive cocci accounted for 13% of isolates and were primarily represented by coagulase-negative staphylococci. The highest isolation rates were from body surfaces of flies, German cockroaches, non-biting midges (Chironomids) and Tenebrionid beetles. About one third of all isolates were resistant to more than three antimicrobials using a standard disc diffusion assay. The presence of multiple resistance to antibiotics was observed in two thirds of Enterobacter isolates, namely those of Enterobacter cloacae from the body surface of Germany cockroaches, in 13% of Citrobacter spp and in 8% of Klebsiella spp as well as Acinetobacter calcoaceticus strains. Strains of Morganella and Hafnia species were very infrequent but all of them shared resistance to the antibiotics tested. In contrast, strains of Serratia spp were relatively antibiotic-sensitive. The group of isolated Gram-positive organisms was represented by two strains of Staphylococcus hominis and one strain of Enterococcus sp, all of them were multiply-resistant to antimicrobials.     

API 20E、VITEK-32在临床细菌鉴定中的应用

目的对API 20E、VITEK-32在临床细菌鉴定中的应用效果进行评价。方法利用API 20E、VITEK-32对临床收集的67株肠道杆菌进行了鉴定,并用常规生化和血清学方法对鉴定结果进行确认。结果 API 20E试条和VITEK-32对67株肠道杆菌的鉴定结果与常规鉴定法符合率为100%。结论 API 20E、VITEK-32鉴定系统快速、准确,可用于临床细菌的鉴定。     

Studies on microbial quality of filtered water in households of a university community in Nigeria

Water samples from home filters in nine residential areas of a Nigerian university community were studied. The membrane filter technique was used to determine the total coliform and faecal coliform counts/100 ml of water. Most of the 100 samples studied were grossly contaminated with total coliform counts/100 ml ranging from 0-442, faecal coliform counts/100 ml, 0-216 and the total aerobic plate count per millilitre ranged from 3.0 X 10(3) to 1.9 X 10(9) c.f.u. The source (dams) of water, fitness of filter candles, frequency of cleaning candles and pH of water did not significantly (P greater than 0.05; chi2) affect the microbial quality of either filtered boiled or unboiled tap water. Escherichia coli type I was isolated from 17.9% of the faecal coliforms tested but from only 2.3% of total coliforms. Enterobacter aerogenes was most predominant (38.5%) amongst faecal coliforms isolated while Enterobacter cloacae was the most frequent (37.2%) of the total coliform isolates. The gross contamination of filtered water from all households sampled calls for an enlightenment of residents on the proper use of home water filters.     

Studies on microbial quality of filtered water in households of a university community in Nigeria.

Water samples from home filters in nine residential areas of a Nigerian university community were studied. The membrane filter technique was used to determine the total coliform and faecal coliform counts/100 ml of water. Most of the 100 samples studied were grossly contaminated with total coliform counts/100 ml ranging from 0-442, faecal coliform counts/100 ml, 0-216 and the total aerobic plate count per millilitre ranged from 3.0 X 10(3) to 1.9 X 10(9) c.f.u. The source (dams) of water, fitness of filter candles, frequency of cleaning candles and pH of water did not significantly (P greater than 0.05; chi2) affect the microbial quality of either filtered boiled or unboiled tap water. Escherichia coli type I was isolated from 17.9% of the faecal coliforms tested but from only 2.3% of total coliforms. Enterobacter aerogenes was most predominant (38.5%) amongst faecal coliforms isolated while Enterobacter cloacae was the most frequent (37.2%) of the total coliform isolates. The gross contamination of filtered water from all households sampled calls for an enlightenment of residents on the proper use of home water filters.     

Vibrio cholerae in the environment: a simple method for reliable identification of the species

A simple screening and identification protocol was assessed for the efficient distinction of colonies of Vibrio cholerae species from others obtained on thiosulphate citrate bile salts sucrose agar after isolation from different environmental specimens. It was demonstrated here that the yellow colonies (sucrose-fermenting), which are able to grow on nutrient agar without added NaCl and which present a positive oxidase reaction, can be confidently considered as presumptive V. cholerae. Confirmation of the identification was carried out using the API 20E microtest and by species-specific ompW-based polymerase chain reaction: 809 of 925 isolates obtained by this screening procedure were identified as V. cholerae by API 20E and confirmed by PCR. The results showed that the direct use of the PCR-based method for the definite identification of the screened colonies gave better results than the API 20E method: of a selection of 100 isolates presumptively identified as V. cholerae according to the proposed screening procedure, all gave a positive result with PCR but only 94 were confirmed by API 20E. This protocol provides reliable identification of V. cholerae species and is adapted to the capabilities of routine clinical, food-testing and environmental microbiology laboratories.     

Effect of ionophore supplementation on the incidence of Escherichia coli O157:H7 and Salmonella and antimicrobial susceptibility of fecal coliforms in Stocker cattle

To examine the effect of ionophore supplementation on fecal shedding of Escherichia coli O157:H7 and Salmonella, crossbred beef calves (n=113; mean body weight [BW], 243 kg) were fed a mineral supplement with ionophore (1.76 g lasalocid/kg) for 61 days (d). Control calves received an identical mineral supplement without lasalocid. Calves were pastured on fescue/bermudagrass paddocks and supplemented with a corn/wheat midds/soybean meal supplement (1.5% of BW/d). Upon arrival, cattle were fed a commercial receiving ration containing 1 g chlorotetracycline/kg for 10 d. Sick calves were administered one or a combination of the following: Nuflor (florfenicol), Baytril (eurofloxacin), Micotil (tilmicosin), or LA 200 (oxytetracycline). Fecal samples were collected immediately prior to ionophore supplementation, approximately midway and at the end of the experimental period (60 d total ionophore feeding) for isolation of E. coli O157:H7 and Salmonella. Putative fecal coliforms were also isolated at these sampling times and examined for antimicrobial susceptibility. The study was replicated over a two year period (year 1, n=53 head; year 2, n=60 head). Ionophore supplementation had no effect (p>0.10) on the incidence of calves shedding E. coli O157:H7 or Salmonella. The percentage of calves shedding E. coli O157:H7 varied throughout the experimental period from 0 to 30%, while Salmonella was cultured from only three calves over the 2-year experimental period. Antimicrobial susceptibility profiles of putative fecal coliforms were consistent with antibiotic treatments administered during the study (observed resistance to chlortetracycline, florfenicol, oxytetracycline), while only one treatment effect was observed. Ionophore treatment resulted in a significantly higher number of coliform isolates resistant to ampicillin compared to controls in year 1, but not year 2. A number of fecal coliform isolates demonstrated resistance to multiple antibiotics, however, this was not affected (p>0.10) by ionophore supplementation. Mineral intakes, BW gain, and the number of sick calves were similar (p>0.10) among treatments. Ionophore supplementation had no affect on fecal shedding of E. coli O157:H7 or Salmonella and a negligible impact on antimicrobial susceptibility patterns of fecal coliforms in beef calves.     

API20E、PCR方法在沙门菌鉴定中的应用

为了评价API 20E试条、PCR方法在沙门菌鉴定中的应用，本研究利用API 20E、PcR方法对54株疑似沙门菌进行鉴定，利用常规生化和血清学方法对鉴定结果进行确认。结果发现API20E、PCR方法与常规鉴定方法的结果一致，51株为沙门菌，3株非沙门菌（1株为柠檬酸杆菌，2株为河生肠杆菌）。API20E、PCR方法具有简便、特异、敏感的特点，可适用于沙门菌鉴定。     

Molecular epidemiological survey of Citrobacter freundii misidentified as Cronobacter spp. (Enterobacter sakazakii) and Enterobacter hormaechei isolated from powdered infant milk formula

A total of 75 powdered infant milk formula (PIF) samples collected from pharmacies and drugstores in Western Sicily, Italy, and representative of 12 different brands were analyzed in this study to evaluate their microbiological quality. According to the U.S. Food and Drug Administration protocol, 32 samples out of 75 were contaminated by enterobacteria. Commercial biochemical API(r) 20E-system identification method indicated that six PIF samples were presumptively contaminated by Cronobacter spp., but further characterization by alpha-glucosidase based polymerase chain reaction (PCR) assay identification strongly suggested that these strains did not belong to the genus Cronobacter. Phylogenetic analysis of partial 16S rRNA (rrs) sequences combined with the results of biochemical tests allowed to identify the six strains as Citrobacter freundii. Similarly, rrs sequence analysis identified as Enterobacter hormaechei 23 strains originally ascribed to Enterobacter cloacae by the API 20E system. Characterization of C. freundii and E. hormaechei PIF isolates by the DiversiLab(r) repetitive sequence-based PCR (rep-PCR) typing method revealed a variety of amplification patterns, but the recovery of the same rep-PCR genotype in several products might indicate a special adaptation of genetic clones to this food or cross-contamination through common ingredients. Antibiotic-resistance profiles were also determined, but none of the strains tested was resistant to third-generation cephalosporins or fluoroquinolones and extended-spectrum beta-lactamase activity was not detected. Our results confirm that E. hormaechei contamination of PIF is widespread, thus making it a cause for concern. Similarly to what was demonstrated for E. hormaechei, we suggest that C. freundii also may be an under-reported cause of bacterial infection, especially in high-risk neonates, due to misidentification.     

Microbiological quality of fermented cassava flour 'kpor umilin'

A study was conducted on the microbiological quality of traditionally fermented cassava flour 'kpor umilin' and a laboratory modification of the method developed with a view to upgrading the traditional process. Microbial analysis of samples from both processing methods was evaluated using standard microbiological methods. The bacterial count in the traditionally processed flours ranged from 2.7 x 10(3) to 1.2 x 10(7) cfu/g, while the modified sample had a count of 3.5 x 10(2) cfu/g. The predominant flora were Leuconostoc spp., Lactobacillus spp., Staphylococcus spp., Micrococcus spp., Bacillus spp., Streptococcus spp. and coliforms such as Escherichia coli. Other isolates were Enterobacter spp. and proteus spp. The sample from the modified method had a negligible (4 MPN/100 ml) coliform count. The yeast and mould count ranged from 1.9 x 10(3) cfu/g to 3.9 x 10(5) cfu/g in the traditionally processed samples as compared with 1.5 x 10(3) cfu/g in the sample from the modified process. Aspergillus spp., Penicillium spp., Saccharomyces spp. and Geotrichum candidum were the predominant fungi. The poor microbiological quality of 'kpor umilin', processed traditionally, calls for urgency in upgrading the processing and preservation methods as developed in this study as well as proper education of the local processors on good manufacturing practices.     

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。     

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.