A Rapid and Direct Plate Method for Enumerating Escherichia coli biotype I in Food

Direct enumeration of Escherichia coli biotype 1 in foods within 24 h has been achieved by a development of the method of Delaney, McCarthy & Grasso (1962) which is based on the production of indole at 44°. Indole positive E. coli growing at this temperature on a cellulose acetate membrane overlaying tryptone bile agar can be demonstrated. Characterization of 555 indole positive colonies from 843 samples of food showed that 95% were E. coli biotype 1 and a further 3·4% were 'faecal coliforms'. Anaerogenic and non-lactose fermenting E. coli are detected and the significance of these strains is discussed.     

A Membrane Filtration Technique for the Enumeration of Escherichia coli in Seawater

S ummary . Membrane filtration has become an accepted method for enumerating Escherichia coli in water, but little published evidence could be found to judge the specificity of the method to assess faecal contamination in either fresh or saline waters. The method is used in our laboratory to monitor the extent and degree of sewage pollution in coastal areas, but there is need for information on what proportion of lactose-fermenting colonies from seawater, developing at 44° on a 4% enriched Teepol medium, are E. coli type I. A total of 1352 colonies from seawater was tested for production of indole and for gas from lactose at 44°. In addition, 46% of the colonies were screened by the IMVEC series of tests. The proportion of colonies tested ranged from 10–100%, depending on the number of colonies on the membrane. Many of the colonies (81.9%) to which IMVEC tests were applied were E. coli type I; a further 10.9% were Irregular type I. The practical implications of these findings are discussed.     

Relationships between the density of different indicator organisms on sheep and beef carcasses and in frozen beef and sheep meat

AIM: To describe the relationship between the concentration of different indicator bacteria in red meat. METHODS AND RESULTS: Enumeration data for aerobic plate count (APC), Enterobacteriaceae, coliforms and Escherichia coli biotype I were analysed from an Australia-wide survey of beef carcasses, sheep carcasses, frozen beef and frozen sheep meat. In all commodities, there was only low-to-moderate rank correlation (0.16-0.47) between concentration of APC and concentration of each Gram-negative indicator. Rank correlations between counts of Gram-negative indicators were much higher (0.47-0.92) especially when nondetections were excluded from analysis (0.78-0.94). Receiver-operator characteristics analysis showed that detection of coliforms can predict the presence of E. coli biotype I with almost 100% sensitivity but fails to predict absence in 2.7-8.5% of samples not containing E. coli biotype I. CONCLUSIONS: Enumeration of coliforms is a useful adjunct to enumeration of E. coli biotype I or Enterobacteriaceae in red meat. The density of coliforms or Enterobacteriaceae can be used to predict the presence or absence of E. coli biotype I, although when the latter is at low prevalence errors in positive test prediction can be large. SIGNIFICANCE AND IMPACT OF THE STUDY: A quantitative basis is provided for comparing the concentration of different indicator bacteria measured in the production, regulation and trade of red meat.     

THE RAPID IDENTIFICATION OF ESCHERICHIA COLI I BY THE PRODUCTION AT 44° OF BOTH INDOLE AND GAS FROM LACTOSE

SUMMARY: The modification of the 44° test proposed by Mackenzie, Taylor & Gilbert (1948) is useful for the rapid identification of Escherichia coli I in water and foods. False positive tests caused by other coli-aerogenes bacteria, or by their association with other organisms, can be considered rare. Only a few E. coli I fail to produce indole or ferment lactose at 44°, and further confirmation for routine purposes is necessary only when the results at 44° are discordant, e.g. indole positive-lactose negative or indole negative-lactose positive.     

Rapid Methods for the Determination of Faecal Contamination in Oysters

S ummary . Two methods for the rapid detection and estimation of numbers of faecal coliforms and Escherichia coli type I in oysters have been developed. That for faecal coliforms involves incubation of tubes of MacConkey broth for 2 h at 37° and then for 22–24 h at 44°. The second method, a modification of MacKenzie, Taylor & Gilbert's (1948) specific method for E. coli type I, makes use of the same system of incubation, but requires the inoculation of tubes of peptone water as well as MacConkey broth, the former tubes being used for subsequent testing for indole formation. Both methods take only 24–26 h and are as sensitive and accurate as the Most Probable Number methods which are in common use and which take upwards of 72–96 h to complete.     

Selective enrichment with a resuscitation step for isolation of freeze-injured Escherichia coli O157:H7 from foods

We studied injury of Escherichia coli O157:H7 cells in 11 food items during freeze storage and methods of isolating freeze-injured E. coli O157:H7 cells from foods. Food samples inoculated with E. coli O157:H7 were stored for 16 weeks at -20 degrees C in a freezer. Noninjured and injured cells were counted by using tryptic soy agar and sorbitol MacConkey agar supplemented with cefixime and potassium tellurite. Large populations of E. coli O157:H7 cells were injured in salted cabbage, grated radish, seaweed, and tomato samples. In an experiment to detect E. coli O157:H7 in food samples artificially contaminated with freeze-injured E. coli O157:H7 cells, the organism was recovered most efficiently after the samples were incubated in modified E. coli broth without bile salts at 25 degrees C for 2 h and then selectively enriched at 42 degrees C for 18 h by adding bile salts and novobiocin. Our enrichment method was further evaluated by isolating E. coli O157:H7 from frozen foods inoculated with the organism prior to freezing. Two hours of resuscitation at 25 degrees C in nonselective broth improved recovery of E. coli O157:H7 from frozen grated radishes and strawberries, demonstrating that the resuscitation step is very effective for isolating E. coli O157:H7 from frozen foods contaminated with injured E. coli O157:H7 cells.     

Indole-inducible proteins in bacteria suggest membrane and oxidant toxicity

Oxidant toxicity of indole was demonstrated by the induction of alkylhydroperoxide reductase subunit C (AhpC) in Escherichia coli K12 and by the constitutive overproduction of AhpC in a variant of E. coli JM109 with enhanced resistance to indole. Oxidant toxicity was also indicated in an indole-adapted variant of Brevibacterium flavum by the indole-inducible overproduction of a novel 36-kDa protein with N-terminal sequence similarity to proteins involved in superoxide and singlet oxygen resistance. It is proposed that indole dissolved in membrane lipids, which caused membrane derangement and enabled direct interaction of redox-cycling isoprenoid quinones and dioxygen, resulting in the generation of superoxide. A direct indication of membrane derangement in E. coli may be the indole-inducible overproduction of spheroplast protein y (Spy).     

Repair and Enumeration of Injured Coliforms in Frozen Foods

Two strains of Escherichia coli manifested death and repairable injury after being frozen in water or sterile foods at -20 C. The injured survivors were inhibited from forming colonies on violet red bile agar (VRBA) or deoxycholate lactose agar; this inhibition was greater when enumeration was done by the pour plate method instead of the surface or surface-overlay method. Injured cells repaired rapidly in Trypticase soy broth (TSB), and the repair was about maximum after 1 h at 25 C. When the injured cells were added to different foods and incubated at 25 C, repair also occurred; however, recovery was better and more uniform when the samples were mixed with TSB and incubated 1 h at 25 C. Cell multiplication was not evident until after 90 to 120 min at 25 C. The enumeration of coliforms from commercially frozen foods was increased when the thawed samples were mixed with TSB and the cells were allowed to repair 1 h at 25 C. In some samples, the repair permitted at least a 20-fold increase in the coliform count. The associated flora in the commercially frozen foods gave no evidence of impairing the repair of coliforms, nor did they start multiplication prior to 90 min after being incubated in TSB at 25 C. Generally, the plating gave more reproducible recovery of coliforms than did the most probable number method. Also, a higher number of coliforms were obtained by the surface-overlay method of plating using VRBA.     

Discrepancies in the Enumeration of Escherichia coli

Stationary-phase cells of Escherichia coli were enumerated by the pour plate method on Trypticase soy agar containing 0.3% yeast extract (TSYA), violet red-bile agar, and desoxycholate-lactose agar, and by the most-probable-number method in Brilliant Green-bile broth and lauryl sulfate broth. Maximum counts were assumed to be those on TSYA. In general, numbers detected were lower with the selective solid media and higher with the selective liquid media. Inhibitory effects, especially on selective solid media varied with the strains of E. coli. The lower detection on selective solid media was partly due to the stress induced in some cells by the temperature of the melted media used in the pour plate method. These cells apparently failed to repair and form colonies in the selective media. Improved detection on the selective solid media was achieved by using 1% nonfat milk solids, 1% peptone, or 1% MgSO(4).7H(2)O in the dilution blanks. Higher detection on selective agar media was effected by surface plating or by surface-overlay plating of the cells. The surface-overlay method appeared to be superior for the direct enumeration of E. coli in foods.     

Using ribosomal RNA gene restriction patterns in distinguishing isolates of Pasteurella haemolytica from bighorn sheep (Ovis canadensis).

Pasteurella haemolytica isolates (n = 31) from two isolated captive herds of Rocky Mountain bighorn sheep (Ovis canadensis canadensis) were characterized and compared phenotypically (biotype, serotype, hemolytic activity) and by a genomic fingerprinting method known as ribotyping. Seven to nine distinct phenotypes were observed. Depending on the method used for serotyping, one to three phenotypes were common to both herds. Eighteen isolates, recovered from both herds, were non-hemolytic, biotype T, indirect hemagglutination assay serotype 4. Ribotyping, a method for highlighting genetically conserved deoxyribonucleic acid restriction site heterogeneity with a 32P-labelled Escherichia coli ribosomal ribonucleic acid probe, produced six to eight distinct ribotype pattern groups within the 31 P. haemolytica isolates, depending on the restriction enzyme used. In contrast to phenotypes, ribotypes appeared unique to each herd, and ribotyping helped to further differentiate some isolates of the same biotype and serotype. In addition, ribotyping provided an alternative means for evaluating relationships between isolates differing in hemolytic activity but which were otherwise phenotypically identical. We propose that ribotyping may be a useful adjunct to other bacterial characterization methods in studying the epizootiology of pasteurellosis in bighorn sheep.     

STUDIES OF THE DETERMINATION OF THE BACTERIUM COLI TYPE I CONTENT OF FARM WATER SUPPLIES

SUMMARY: A – comparison of the suitability of brilliant green bile broth and MacConkey's broth at 44° for the detection of Bacterium coli type I in farm water supplies, showed that 83.1% of the samples had no difference in the number of positive tubes at 44°, and only 5 samples (1.7%) had a significantly higher number of positive tubes in MacConkey's broth.
Of 707 strains of coli-aerogenes bacteria isolated from 44° positive tubes of both media, 94.5% were Bact. coli type I. Strains of Bact. coli type II and Bact. aerogenes type I which were 44° positive constituted 3.7% and 0.4% respectively, all of which were indole negative at 44°. In addition there were 10 strains (1.4%) of 44° positive Intermediate type II, 9 of which were indole positive at 44°.
An appreciable number (6.6%) of Bact. coli type I strains failed to give a positive indole reaction in 24 hr at 44°.     

A comparison of fluorochromes for direct viable counts by image analysis

Total direct and direct viable counts of fresh and injured cultures of Escherichia coli were determined by image analysis in preparations stained with acridine orange, ethidium bromide and 4',6-diamidino-2-phenyl indole (DAPI). Cells stained with DAPI were not detected by image analysis. Fresh cultures stained with acridine orange or ethidium bromide gave comparable counts. Injured E. coli stained with ethidium bromide gave higher counts that with acridine orange. Injured cultures stained with acridine orange contain high proportions of green cells which are less easily detected than red cells in image analysis. In certain cases it may be better to use ethidium bromide, which stains all cells red, for direct viable counts by image analysis.     

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 in cooked and raw meats by ion mobility spectrometry

I.D. OGDEN AND N.J.C. STRACHAN. 1993. A novel method for enumerating Escherichia coli in foods is described. It is based on the production of o -nitrophenol (ONP) from o -nitrophenyl-β-D-glucuronide as detected by an ion mobility spectrometer. The time taken for the detection of ONP in a food sample was inversely proportional to the logarithm of the initial E. coli population. Escherichia coli could be detected at a level of 10 g^-1 within 9 h.     

Rapid Method for Detection and Enumeration of Fecal Coliforms in Fresh Chicken

A rapid method for enumerating fecal coliforms in foods was developed employing an agar pour-plate medium. After 7 h of incubation at 41.5 +/- 0.05 C, this medium effectively allows the growth of fecal coliforms only. This rapid method was compared with the Association of Official Analytical Chemists multiple-tube dilution method for Escherichia coli, by using 21 samples of fresh, cut-up chicken and a surface rinsing procedure for sample preparation. Verification of picked colonies was carried out in EC broth using parallel incubation temperatures of 45.5 and 44.5 +/- 0.05 C. Verifications for these temperatures averaged 79 and 98%, respectively. All positively verified isolates were E. coli types I and II, as were the negatives. Geometric means for the verified 7-h plate count were within 12% of the standard means for both EC broth incubation temperatures.     

Coliform Behavior in Frozen Foods: I. Rapid Test for the Recovery of Escherichia coli from Frozen Foods

An assortment of 496 samples of frozen foods consisting of fish or marine products, variety types, and cream pie desserts were subjected to four parallel examinations for the recovery of Escherichia coli. The test procedures consisted of two low-temperature (35 C) and two high-temperature (44 C) presumptive tests, followed by an E C confirmatory test at 45.5 C. Of all the test methods examined, a single Lauryl Sulfate Tryptose (LST) presumptive test at 44 C gave best E. coli recovery (425). This recovery compared favorably with the lengthier Association of Official Analytical Chemists test with which only 420 E. coli cells were recovered. The LST (44 C) test saves much time, since it renders a follow-up 48-hr confirmatory test unnecessary. Moreover, since 96% of all the E. coli are recovered within 24 hr by LST (44 C), it is essentially a 24-hr test. The results of this study also confirmed earlier findings, in that it is possible to describe a specific coliform bacteriological test method by simple reproducible productivity ratios. E. coli recovery dilution data and coliform group behavior were also examined.     

Aerobic plate count and Escherichia coli determination on frozen ground-beef patties.

The aerobic plate count and Escherichia coli content of 604 samples of frozen ground-beef patties is reported. The results are discussed with regard to existing and proposed microbiological standards for raw meats.     

Domain organization and evolution of multifunctional autoprocessing repeats-in-toxin (MARTX) toxin in Vibrio vulnificus

The objective of this study was to analyze multifunctional autoprocessing repeats-in-toxin (MARTX) toxin domain organization within the aquatic species Vibrio vulnificus as well as to study the evolution of the rtxA1 gene. The species is subdivided into three biotypes that differ in host range and geographical distribution. We have found three different types (I, II, and III) of V. vulnificus MARTX (MARTX(Vv)) toxins with common domains (an autocatalytic cysteine protease domain [CPD], an α/β-hydrolase domain, and a domain resembling that of the LifA protein of Escherichia coli O127:H6 E2348/69 [Efa/LifA]) and specific domains (a Rho-GTPase inactivation domain [RID], a domain of unknown function [DUF], a domain resembling that of the rtxA protein of Photorhabdus asymbiotica [rtxA(PA)], and an actin cross-linking domain [ACD]). Biotype 1 isolates harbor MARTX(Vv) toxin types I and II, biotype 2 isolates carry MARTX(Vv) toxin type III, and biotype 3 isolates have MARTX(Vv) toxin type II. The analyzed biotype 2 isolates harbor two identical copies of rtxA1, one chromosomal and the other plasmidic. The evolutionary history of the gene demonstrates that MARTX(Vv) toxins are mosaics, comprising pieces with different evolutionary histories, some of which have been acquired by intra- or interspecific horizontal gene transfer. Finally, we have found evidence that the evolutionary history of the rtxA1 gene for biotype 2 differs totally from the gene history of biotypes 1 and 3.     

Comparison of commercially available kits with standard methods for the detection of coliforms and Escherichia coli in foods.

Three commercially available kits that were supplemented with substrates for enzyme reactions were evaluated to determine their abilities to detect coliforms and fecal coliforms in foods. Japanese and U.S. Food and Drug Administration standard methods, as well as two agar plate methods, were compared with the three commercial kits. A total of 50 food samples from various retailers were examined. The levels of detection of coliforms were high with the commercial kits (78 to 98%) compared with the levels of detection with the standard methods (80 to 83%) and the agar plate methods (56 to 83%). Among the kits tested, the Colilert kit had highest level of recovery of coliforms (98%), and the level of recovery of Escherichia coli as determined by beta-glucuronidase activity with the Colilert kit (83%) was comparable to the level of recovery obtained by the U.S. Food and Drug Administration method (87%). Isolation of E. coli on the basis of the beta-glucuronidase enzyme reaction was found to be good. Levine's eosine methylene blue agar, which has been widely used in various laboratories to isolate E. coli was compared with 4-methylumbelliferyl-beta-D-glucuronide (MUG)-supplemented agar for isolation of E. coli. Only 47% of the E. coli was detected when eosine methylene blue agar was used; however, when violet red bile (VRB)-MUG agar was used, the E. coli detection rate was twice as high. Of the 200 E. coli strains isolated, only 2 were found to be MUG negative, and the gene responsible for beta-glucuronidase activity (uidA gene) was detected by the PCR method in these 2 strains. Of the 90 false-positive strains isolated that exhibited various E. coli characteristic features, only 2 non-E.coli strains hydrolyzed MUG and produced fluorescent substrate in VRB-MUG agar. However, the PCR did not amplify uidA gene products in these VRB-MUG fluorescence-positive strains.