Terminology

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Overview

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Chapter 4

Introduction

Escherichia coli, originally known as Bacterium coli commune, was identified in 1885 by the German pediatrician, Theodor Escherich (14, 29). E. coli is widely distributed in the intestine of humans and warm-blooded animals and is the predominant facultative anaerobe in the bowel and part of the essential intestinal flora that maintains the physiology of the healthy host (9, 29). E. coli is a member of the family Enterobacteriaceae (15), which includes many genera, including known pathogens such as Salmonella, Shigella, and Yersinia. Although most strains of E. coli are not regarded as pathogens, they can be opportunistic pathogens that cause infections in immunocompromised hosts. There are also pathogenic strains of E. coli that when ingested, causes gastrointestinal illness in healthy humans (see Chap. 4A).

In 1892, Shardinger proposed the use of E. coli as an indicator of fecal contamination. This was based on the premise that E. coli is abundant in human and animal feces and not usually found in other niches. Furthermore, since E. coli could be easily detected by its ability to ferment glucose (later changed to lactose), it was easier to isolate than known gastrointestinal pathogens. Hence, the presence of E. coli in food or water became accepted as indicative of recent fecal contamination and the possible presence of frank pathogens. Although the concept of using E. coli as an indirect indicator of health risk was sound, it was complicated in practice, due to the presence of other enteric bacteria like Citrobacter, Klebsiella and Enterobacter that can also ferment lactose and are similar to E. coli in phenotypic characteristics, so that they are not easily distinguished. As a result, the term "coliform" was coined to describe this group of enteric bacteria. Coliform is not a taxonomic classification but rather a working definition used to describe a group of Gram-negative, facultative anaerobic rod-shaped bacteria that ferments lactose to produce acid and gas within 48 h at 35°C. In 1914, the U.S. Public Health Service adopted the enumeration of coliforms as a more convenient standard of sanitary significance.

Although coliforms were easy to detect, their association with fecal contamination was questionable because some coliforms are found naturally in environmental samples (6). This led to the introduction of the fecal coliforms as an indicator of contamination. Fecal coliform, first defined based on the works of Eijkman (12) is a subset of total coliforms that grows and ferments lactose at elevated incubation temperature, hence also referred to as thermotolerant coliforms. Fecal coliform analyses are done at 45.5°C for food testing, except for water, shellfish and shellfish harvest water analyses, which use 44.5°C (1, 3, 30). The fecal coliform group consists mostly of E. coli but some other enterics such as Klebsiella can also ferment lactose at these temperatures and therefore, be considered as fecal coliforms. The inclusion of Klebsiella spp in the working definition of fecal coliforms diminished the correlation of this group with fecal contamination. As a result, E. coli has reemerged as an indicator, partly facilitated by the introduction of newer methods that can rapidly identify E. coli.

Currently, all 3 groups are used as indicators but in different applications. Detection of coliforms is used as an indicator of sanitary quality of water or as a general indicator of sanitary condition in the food-processing environment. Fecal coliforms remain the standard indicator of choice for shellfish and shellfish harvest waters; and E. coli is used to indicate recent fecal contamination or unsanitary processing. Almost all the methods used to detect E. coli, total coliforms or fecal coliforms are enumeration methods that are based on lactose fermentation (4). The Most Probable Number (MPN) method is a statistical, multi-step assay consisting of presumptive, confirmed and completed phases. In the assay, serial dilutions of a sample are inoculated into broth media. Analysts score the number of gas positive (fermentation of lactose) tubes, from which the other 2 phases of the assay are performed, and then uses the combinations of positive results to consult a statistical table (Appendix 2), to estimate the number of organisms present. Typically only the first 2 phases are performed in coliform and fecal coliform analysis, while all 3 phases are done for E. coli. The 3-tube MPN test is used for testing most foods. Analysis of seawater using a multiple dilution series should not use less than 3 tubes per dilution (5 tubes are recommended); in certain instances a single dilution series using no less than 12 tubes may also be acceptable. (For additional details, see: FDA. National Shellfish Sanitation Program, Manual of Operations. 2009 Revision. DHHS/PHS/FDA, Washington DC). Likewise, analysis of bivalve molluscan shellfish should be performed using a multiple dilution MPN series whereby no fewer than 5- tubes per dilution should be used, see section IV. There is also a 10-tube MPN method that is used to test bottled water or samples that are not expected to be highly contaminated (3). Analysis of citrus juice for E. coli is performed as an absence/presence method, see section V.

Also, there is a solid medium plating method for coliforms that uses Violet Red Bile Agar, which contains neutral red pH indicator, so that lactose fermentation results in formation of pink colonies. There are also membrane filtration tests for coliform and fecal coliform that measure aldehyde formation due to fermentation of lactose. This chapter also includes variations of above tests that use fluorogenic substrates to detect E. coli (18), special tests for shellfish analysis, a brief consideration of bottled water testing and a method for testing large volumes of citrus juices for presence of E. coli in conjunction with the Juice HACCP rule.

I. Conventional Method for coliforms, fecal coliforms and E. coli

C. MPN - Presumptive test for coliforms, fecal coliforms and E. coli

Weigh 50 g of food into sterile high-speed blender jar (see Chapter 1 and current FDA compliance programs for instructions on sample size and compositing) Frozen samples can be softened by storing for <18 h at 2-5°c, but do not thaw. Add 450 mL of Butterfield's phosphate buffered water and blend for 2 min. If <50 g of sample are available, weigh portion that is equivalent to half of the sample and add sufficient volume of sterile diluent to make a 1:10 dilution. The total volume in the blender jar should completely cover the blades.

Prepare decimal dilutions with sterile Butterfield's phosphate diluent or equivalent. Number of dilutions to be prepared depends on anticipated coliform density. Shake all suspensions 25 times in 30 cm arc or vortex mix for 7 s. Using at least 3 consecutive dilutions, inoculate 1 mL aliquots from each dilution into 3 LST tubes for a 3 tube MPN analysis (other analysis may require the use of 5 tubes for each dilution; See IV). Lactose Broth may also be used. For better accuracy, use a 1 mL or 5 mL pipet for inoculation. Do not use pipets to deliver<10% of their total volume; eg. a 10 mL pipet to deliver 0.5 mL. Hold pipet at angle so that its lower edge rests against the tube. Not more than 15 min should elapse from time the sample is blended until all dilutions are inoculated in appropriate media.

Incubate LST tubes at 35°C± 0.5°C . Examine tubes and record reactions at 24 ± 2 h for gas, i.e., displacement of medium in fermentation vial or effervescence when tubes are gently agitated. Re-incubate gas-negative tubes for an additional 24 h and examine and record reactions again at 48 ± 3 h. Perform confirmed test on all presumptive positive (gas) tubes.

D. MPN - Confirmed test for coliforms

From each gassing LST or lactose broth tube, transfer a loopful of suspension to a tube of BGLB broth, avoiding pellicle if present. (a sterile wooden applicator stick may also be used for these transfers). Incubate BGLB tubes at 35°C ± 0.5°C and examine for gas production at 48 ± 3 h. Calculate most probable number (MPN) (see Appendix 2) of coliforms based on proportion of confirmed gassing LST tubes for 3 consecutive dilutions.

E. MPN - Confirmed test for fecal coliforms and E. coli

From each gassing LST or Lactose broth tube from the Presumptive test, transfer a loopful of each suspension to a tube of EC broth (a sterile wooden applicator stick may also be used for these transfers). Incubate EC tubes 24 ± 2 h at 44.5°C and examine for gas production. If negative, reincubate and examine again at 48 ± 2 h. Use results of this test to calculate fecal coliform MPN. To continue with E. coli analysis, proceed to Section F below. The EC broth MPN method may be used for seawater and shellfish since it conforms to recommended procedures (1).

F. MPN - Completed test for E. coli.

To perform the completed test for E. coli, gently agitate each gassing EC tube, remove a loopful of broth and streak for isolation on a L-EMB agar plate and incubate for 18-24 h at 35°C ± 0.5°C . Examine plates for suspicious E. coli colonies, i.e., dark centered and flat, with or without metallic sheen. Transfer up to 5 suspicious colonies from each L-EMB plate to PCA slants, incubate them for 18-24 h at 35°C ± 0.5°C and use for further testing.

Identification of any 1 of the 5 colonies as E. coli is sufficient to regard that EC tube as positive; hence, not all 5 isolates may need to be tested.

Perform Gram stain. All cultures appearing as Gram-negative, short rods should be tested for the IMViC reactions below and also re-inoculated back into LST to confirm gas production.

Indole production. Inoculate tube of tryptone broth and incubate 24 ± 2 h at 35°C ± 0.5°C . Test for indole by adding 0.2-0.3 mL of Kovacs' reagent. Appearance of distinct red color in upper layer is positive test.

Voges-Proskauer (VP)-reactive compounds. Inoculate tube of MR-VP broth and incubate 48 ± 2 h at 35°C± 0.5°C . Transfer 1 mL to 13 × 100 mm tube. Add 0.6 mL α-naphthol solution and 0.2 mL 40% KOH, and shake. Add a few crystals of creatine. Shake and let stand 2 h. Test is positive if eosin pink color develops.

Methyl red-reactive compounds. After VP test, incubate MR-VP tube additional 48 ± 2 h at 35°C± 0.5°C . Add 5 drops of methyl red solution to each tube. Distinct red color is positive test. Yellow is negative reaction.

Citrate. Lightly inoculate tube of Koser's citrate broth; avoid detectable turbidity. Incubate for 96 h at 35°C ± 0.5°C . Development of distinct turbidity is positive reaction. Gas from lactose. Inoculate a tube of LST and incubate 48 ± 2 h at 35°C ± 0.5°C . Gas production (displacement of medium from inner vial) or effervescence after gentle agitation is positive reaction.

Interpretation: All cultures that (a) ferment lactose with gas production within 48 h at 35°C, (b) appear as Gram-negative nonsporeforming rods and (c) give IMViC patterns of ++-- (biotype 1) or -+-- (biotype 2) are considered to be E. coli. Calculate MPN (see Appendix 2) of E. coli based on proportion of EC tubes in 3 successive dilutions that contain E. coli.

Alternatively, instead of performing the IMViC test, use API20E or the automated VITEK biochemical assay to identify the organism as E. coli. Use growth from the PCA slants and perform these assays as described by the manufacturer.

G. Solid medium method - Coliforms

Prepare violet red bile agar (VRBA) according to manufacturer's instructions. Cool to 48°C before use. Prepare, homogenize, and decimally dilute sample as described in section I. C above so that isolated colonies will be obtained when plated. Transfer two 1 mL aliquots of each dilution to petri dishes, and use either of the following two pour plating methods, depending on whether injured or stressed cells are suspected to be present (1).

Pour 10 mL VRBA tempered to 48°C into plates, swirl plates to mix, and let solidify. To prevent surface growth and spreading of colonies, overlay with 5 mL VRBA, and let solidify. If resuscitation is necessary, pour a basal layer of 8-10 mL of tryptic soy agar tempered to 48°C. Swirl plates to mix, and incubate at room temperature for 2 ± 0.5 h. Then overlay with 8-10 mL of melted, cooled VRBA and let solidify.

Invert solidified plates and incubate 18-24 h at 35°C. Incubate dairy products at 32°C (2). Examine plates under magnifying lens and with illumination. Count purple-red colonies that are 0.5 mm or larger in diameter and surrounded by zone of precipitated bile acids. Plates should have 25-250 colonies. To confirm that the colonies are coliforms, pick at least 10 representative colonies and transfer each to a tube of BGLB broth. Incubate tubes at 35°C. Examine at 24 and 48 h for gas production.

If gas-positive BGLB tube shows a pellicle, perform Gram stain to ensure that gas production was not due to Gram-positive, lactose-fermenting bacilli.

Determine the number of coliforms per gram by multiplying the number of suspect colonies by percent confirmed in BGLB by dilution factor.

Alternatively, E. coli colonies can be distinguished among the coliform colonies on VRBA by adding 100 µg of 4-methyl-umbelliferyl-β-D-glucuronide (MUG) per mL in the VRBA overlay. After incubation, observe for bluish fluorescence around colonies under longwave UV light. (see LST-MUG section II for theory and applicability.)

H. Membrane Filtration (MF) Method - coliforms: see Section III. Bottled Water.

Food homogenates will easily clog filters, hence MF are most suitable for analysis of water samples; however, MF may be used in the analysis of liquid foods that do not contain high levels of particulate matter such as bottled water (see Section III for application of MF).

II. LST-MUG Method for Detecting E. coli in Chilled or Frozen Foods Exclusive of Bivalve Molluscan Shellfish

The LST-MUG assay is based on the enzymatic activity of β-glucuronidase (GUD), which cleaves the substrate 4-methylumbelliferyl β-D-glucuronide (MUG), to release 4 methylumbelliferone (MU). When exposed to longwave (365 nm) UV light, MU exhibits a bluish fluorescence that is easily visualized in the medium or around the colonies. Over 95% of E. coli produces GUD, including anaerogenic (non-gas-producing) strains. One exception is enterohemorrhagic E. coli (EHEC) of serotype O157:H7, which is consistently GUD negative (11, 17). The lack of GUD phenotype in O157:H7 is often used to differentiate this serotype from other E. coli, although GUD positive variants of O157:H7 do exist (24, 26). The production of GUD by other members of the family Enterobacteriaceae is rare, except for some shigellae (44 58%) and salmonellae (20-29%) (18, 27). However, the inadvertent detection of these pathogens by GUD-based assays is not considered a drawback from a public health perspective. Expression of GUD activity is affected by catabolite repression (8) so on occasion, some E. coli are GUD-negative, even though they carry the uidA gene (gusA) that encodes for the enzyme (19). In most analyses however, about 96% of E. coli isolates tested are GUD positive without the need for enzyme induction (27). MUG can be incorporated into almost any medium for use in detecting E. coli. But some media such as EMB, which contain fluorescent components, are not suitable, as they will mask the fluorescence of MU. When MUG is incorporated into LST medium, coliforms can be enumerated on the basis of gas production from lactose and E. coli are presumptively identified by fluorescence in the medium under longwave UV light, thus it is capable of providing a presumptive identification of E. coli within 24 h (18, 28). The LST-MUG method described below has been adopted as Official Final Action by the AOAC for testing for E. coli in chilled or frozen foods, exclusive of shellfish (28). See Sec. IV.4. D. for precautions in using MUG in testing shellfish.

To observe for fluorescence, examine inoculated LST-MUG tubes under longwave (365 nm) UV light in the dark. A 6-watt hand-held UV lamp is adequate and safe. When using a more powerful UV source, such as a 15-watt fluorescent lamp, wear protective glasses or goggles. Also, prior to use in MUG assays, examine all glass tubes for auto fluorescence. Cerium oxide, which is sometimes added to glass as a quality control measure, will fluoresce under UV light and interfere with the MUG test (25). The use of positive and negative control strains for MUG reaction is essential.

A. Equipment and material

see section I.A above and in addition,

1. New, disposable borosilicate glass tubes (100 × 16 mm)

2. New, disposable borosilicate glass Durham vials (50 × 9 mm) for gas collection

3. Longwave UV lamp, not to exceed 6-watt

B. Media and reagents

see section I.B above

C. Presumptive LST-MUG test for E. coli.

Prepare food samples and perform the MPN Presumptive test as described in section I.C. above, except use LST-MUG tubes instead of LST. Be sure to inoculate one tube of LST-MUG with a known GUD-positive E. coli isolate as positive control (ATCC 25922). In addition, inoculate another tube with a culture of Enterobacter aerogenes (ATCC 13048) culture of Enterobacter aerogenes (ATCC 13048) or a Klebsiella pneumoniae strain as negative control, to facilitate differentiation of sample tubes that show only growth from those showing both growth and fluorescence. Incubate tubes for 24 to 48 ± 2 h at 35°C. Examine each tube for growth (turbidity, gas) then examine tubes in the dark under longwave UV lamp (365 nm). A bluish fluorescence is a positive presumptive test for E. coli. Studies by Moberg et al. (28) show that a 24 h fluorescence reading is an accurate predictor of E. coli and can identify 83-95% of the E. coli-positive tubes. After 48 h of incubation, 96-100% of E. coli-positive tubes can be identified (28). Perform a confirmed test on all presumptive positive tubes by streaking a loopful of suspension from each fluorescing tube to L-EMB agar and incubate 24 ± 2 h at 35°C. Follow protocols outlined in I. F, above, for Completed test for E. coli. Calculate MPN of E. coli based on combination of confirmed fluorescing tubes in 3 successive dilutions.

III. Examination of Bottled Water

See Attached

IV. Examination of Shellfish and Shellfish Meats

See Attached

V. Analysis for E. coli in citrus juices

See Attached

VI. Other Methods for Enumerating Coliforms and E. coli

There are many other methods for enumerating coliforms and E. coli , including several that uses fluorogenic reagents like MUG or other chromogenic substrates for presumptive detection and identification of coliform and E. coli in foods. Many of these tests, such as the Petrifilm dry rehydratable film, the hydrophobic grid membrane filter/MUG (HGMF/MUG) method (13), ColiComplete disc (16), Colilert (AOAC 991.15), have been evaluated by collaborative studies and adopted as official first or final action by the AOAC. There are also many modifications of the membrane filtration assays that have been developed for testing for coliform, fecal coliform and E. coli and some of these may be useful in testing foods such as milk and beverages, but they are used mostly for water, environmental waters, and shellfish harvest waters analysis (5, 7, 20, 22, 23, 31).