Published June 25, 2014

Journal of Environmental Quality

Reviews and Analyses

Primary Isolation of Shiga Toxigenic Escherichia coli from Environmental Sources Lisa M. Durso*

E

scherichia coli have been used for decades as indi-

Since the time of the first microbe hunters, primary culture and isolation of bacteria has been a foundation of microbiology. Like other microbial methods, bacterial culture and isolation methodologies continue to develop. Although fundamental concepts like selection and enrichment are as relevant today as they were over 100 yr ago, advances in chemistry, molecular biology and bacterial ecology mean that today’s culture and isolation techniques serve additional supporting roles. The primary isolation of Shiga toxigenic Escherichia coli (STEC) from environmental sources relies on enriching the target while excluding extensive background flora. Due to the complexity of environmental substrates, no single method can be recommended; however, common themes are discussed. Brilliant Green Bile Broth, with or without antibiotics, is one of many broths used successfully for selective STEC enrichment. Stressed cells may require a pre-enrichment recovery step in a nonselective broth such as buffered peptone water. After enrichment, immunomagnetic separation with serotype specific beads drastically increases the chances for recovery of STEC from environmental or insect sources. Some evidence suggests that acid treating the recovered beads can further enhance isolation. Although it is common in human clinical, food safety, and water quality applications to plate the recovered beads on Sorbitol MacConkey Agar, other chromogenic media, such as modified CHROMagar, have proven helpful in field and outbreak applications, allowing the target to be distinguished from the numerous background flora. Optimum conditions for each sample and target must be determined empirically, highlighting the need for a better understanding of STEC ecology.

cators of fecal pollution in environmental samples such as water, soil, and sediment. Escherichia coli thrive in the mammalian lower gastrointestinal tract (GIT) and are excreted into the environment via feces. Historically it was thought that E. coli do not survive long outside of the GIT, so the culture of E. coli from environmental sources was thought to reveal recent fecal contamination. Recently, this idea has been challenged by evidence that some E. coli survive and reproduce outside of the mammalian host (Bermudez and Hazen, 1988; Alm et al., 2003; Anderson et al., 2005). Nonetheless, the culture of generic E. coli from water, soil, and sediment is a classic technique that is broadly used in environmental microbiology. There are many different E. coli subtypes, and most are harmless or even beneficial gut inhabitants. Some E. coli, however, carry genes for a potent human toxin, called Shiga toxin. These E. coli are of special concern because a subset of Shiga toxigenic E. coli (STEC) cause serious disease in humans, including kidney failure and death. Shiga toxigenic E. coli is commonly isolated from animal manures (Keen et al., 2006; Arthur et al., 2010) and has been isolated from manure-impacted water (Tanaro et al., 2012; Cooley et al., 2013) and soil (Islam et al., 2004; Cooley et al., 2013), including soil used to grow fresh produce (Islam et al., 2004; Ma et al., 2012; Cooley et al., 2013). Like generic E. coli, human pathogenic STEC can survive for prolonged periods outside of the GIT (Durso et al., 2005b; Durso et al., 2010). The ability of STEC to survive in manure-impacted environments, including soil and water, and the use of manurebased fertilizers and irrigation waters in food production means that it is increasingly important to understand how STEC moves through agroecosystems. The ability to detect and isolate STEC from environmental sources is therefore important for protecting public safety. Detection involves determining if the bacteria are present in a sample, regardless of whether or not a pure culture of the bacteria is obtained. Detection-only methods are quicker and less expensive than traditional culture-based isolation techniques, but they come with their own set of limitations. Culture involves growing the bacteria and may or may not involve con-

Copyright © American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America. 5585 Guilford Rd., Madison, WI 53711 USA. All rights reserved. No part of this periodical may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher.

USDA–ARS, Agroecosystem Management Research Unit, 137 Keim Hall, Univ. of Nebraska-Lincoln East Campus, Lincoln, NE 68583. Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. Assigned to Associate Editor Abasiofiok Ibekwe.

J. Environ. Qual. 42:1295–1307 (2013) doi:10.2134/jeq2013.02.0035 Received 1 Feb. 2013. *Corresponding author ([email protected]).

Abbreviations: BGB, Brilliant Green Bile; GIT, gastro intestinal tract; IMS, immunomagnetic separation; MUG, 4-methylumbelliferyl b-D-glucuronide; PCR, polymerase chain reaction; RMAC, rhamnose-MacConkey; SMAC, Sorbitol MacConkey agar; STEC, Shiga toxigenic Escherichia coli; TSB, Trypticase soy broth.

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tinuing the procedure until a pure culture is obtained. The focus of this review is on methods that lead to a pure culture isolate of the target STEC bacteria.

Escherichia coli as a Laboratory Organism Escherichia coli is widely studied as a model laboratory bacterium. Originally discovered in healthy human feces by Theodor Escherich in 1885 and named “Bacterium coli,” this organism was the workhorse of microbiology laboratories in Europe and the United States during the golden age of microbiology. Descendants of these original laboratory strains remain important model bacteria and important tools in today’s microbiology, genetics, and molecular biology laboratories. The two most famous lab strains of E. coli are E. coli B and E. coli K-12, which originated from the collection of the Pasteur Institute in France in 1918 (Daegelen et al., 2009) and from human feces cultured at Stanford University in 1922 (Lederberg, 2004), respectively. Because the primary isolation of these laboratory strains of E. coli happened so long ago and because these strains have been cultured and subcultured for so many years, these E. coli have become adapted to their artificial laboratory environment and no longer closely resemble “wild” E. coli. For this reason, primary culture of E. coli from the environment requires a different strategy than the routine culture of laboratory E. coli strains. Also, laboratory E. coli strains are a poor choice for evaluating culture and isolation methods designed to target environmental E. coli.

Escherichia coli Nomenclature Serotyping

Escherichia coli subtypes are named based on the classic Kauffmann serotyping scheme developed in the 1940s (Kauffmann, 1947) and described by Ørskov et al. (1977) and Ørskov and Ørskov (1984). There are a number of surface proteins, called antigens, on the body (O), capsule (K), and flagella (H) of E. coli bacteria. The “O” antigens, also known as “somatic” antigens and designated with the letter O (not the number 0), were numbered 1 through 181, with a few (O31, O47, O67, O72, O94, O122) that have been retired because of cross reactions or duplications within the original scheme. Some additional O antigens have been categorized, but the majority of E. coli are classified using the original numbering system. Escherichia coli strains expressing the O antigen are considered “smooth” due to the smooth margins of the colonies when grown on agar. Escherichia coli strains that do not agglutinate with any of the known O antisera but which are smooth are considered “not typeable.” Escherichia coli strains that do not express the complete O antigen are considered “rough” due to uneven margins of their colonies. Rough strains cannot be typed using traditional phenotypic methods. However, molecular serotyping, which is based on the traditional serotyping scheme but looks at the O antigen genotype instead of the O antigen phenotype, can be used (Durso et al., 2005a; Durso et al., 2007). The O antigen genes have become popular targets for detection of pathogenic E. coli strains (DebRoy et al., 2011; Fratamico et al., 2011); however, the E. coli Reference Center at Penn State (the largest repository for E. coli strains in America) still uses an antisera-based assay to confirm O serotype status. 1296

The flagellar, or H antigen, serotypes of E. coli are numbered 1 through 56. The flagella must be expressed to determine the H antigen phenotype using traditional serotyping, but molecular methods can now be used to type nonmotile strains. In the early 2000s, various genotype-based methods were proposed as a way to gather serotyping information on the H antigen, including nonmotile strains (Reid et al., 1999; Machado et al., 2000), and in 2003, all 56 E. coli H flagellar types were sequenced by a group from the Reeves’ lab in Australia (Wang et al., 2003). The E. coli Reference Center now routinely serotypes the E. coli H antigen using DNA-based methods. There is a group of Shiga-toxigenic E. coli O157 strains from Germany that code for, but do not express, the H7 antigen (Karch et al., 2011). In these E. coli O157:H- strains, there is a 12 base-pair deletion that prevents transcriptional activation of the flagellum biosynthesis genes (Monday et al., 2004).

Shiga Toxigenic Escherichia coli Shiga toxigenic E. coli, by definition, carry one or both Shiga toxin genes (also sometimes referred to as Vero-toxin genes). The E. coli serotyping scheme refers exclusively to the antigenic properties of any particular isolate and does not of itself provide information on virulence or Shiga-toxin status. Some specific E. coli serotypes are associated with disease in humans and carriage of the Shiga toxins, but individual O serotypes are not a substitute for information on Shiga toxin status. The two are independent of one another. For example, there are many strains of E. coli with the O157 serotype but with no Shiga-toxin genes. In one study, up to 28% of cattle fecal samples contained plain E. coli O157 (no Shiga toxin genes) and STEC O157 (Durso and Keen, 2007). In regulatory situations especially, it is important to remember that E. coli O157 is not the same as STEC O157. Shiga toxigenic E. coli O157 is a subset of all E. coli O157. Shiga toxigenic E. coli O157 emerged as an important foodborne pathogen in the 1980s, and there are now regulations considering it an adulterant in meat products (Federal Register, 2002). There are six non-O157 STEC serotypes that are regulated in the United States: STEC O26, STEC O45, STEC O103, STEC O111, STEC O121, and STEC O145 (Federal Register, 2012). As with STEC O157, each E. coli serotype has many “wild” members that have the same O serotype but do not carry the Shiga-toxin or other virulence genes. High throughput DNA sequencing of many E. coli isolates from the same serotype is allowing researchers to find specific, single-nucleotide differences that are currently associated with just the subset of an individual serotype that carries the Shiga-toxin genes (Bono et al., 2007; Clawson et al., 2009, Norman et al., 2012). Finally, there are also many E. coli that carry the Shiga toxin genes that do not belong to one of the seven regulated STEC serotypes (Karmali, 1989). The recent STEC 104 outbreak in Germany is one example of a non-O157 STEC associated with human illness. Numerous studies document carriage of multiple STEC serotypes in ruminants (Leomil et al., 2003; Kijima-Tanaka et al., 2005). One review notes 155 different STEC serotypes found in beef, only 56 of which have a history of causing illness in humans (Hussein and Bollinger, 2005). A second study looking more broadly at cattle production environments reported 49 different STEC serotypes from 527 STEC cattle, water, and wildlife isolates. Of these, only 13.3% Journal of Environmental Quality

were positive for eaeA, another common virulence factor associated with human clinical STEC infections (Renter et al., 2005). In addition to cattle, water, and wildlife, a variety of other vectors and reservoirs have been screened for STEC. Insects have the potential to serve as mechanical vectors of zoonotic pathogens, and flies have been hypothesized to play a role in the ecology of STEC in food production environments, including animal (Alam and Zurek, 2004; Keen et al., 2006) and vegetable production systems (Talley et al., 2009). Although STEC O157 has been routinely cultured from insects such as flies (Alam and Zurek, 2004; Keen et al., 2006), data on the broader prevalence of fly-associated E. coli carrying the Shiga toxin genes are lacking. Isolation methods that target Shiga toxin production or Shiga toxin genes along with a specific serotype may result in isolation of nonpathogenic strains due to the absence of other important virulence factors.

Escherichia coli as a Naturally Occurring Organism Outside of the laboratory, E. coli occupies two ecological niches: (i) the warm, nutrient rich lower intestines of warmblooded animals (including humans, cattle, and other mammals) and (ii) the cool, nutrient-limiting environment external to the host (water, soil, and sediments) (Savageau, 1983). Most E. coli originate in the mammalian lower GIT via binary fission. Unlike many of the GIT microbes, which are obligate anaerobes, E. coli are facultative, meaning that they can grow aerobically or anaerobically. When fecal material is cultured in the lab, the majority of the gut bacteria are unable to grow in the presence of oxygen, so the E. coli have an advantage and can rapidly reproduce. In nature, E. coli leave the mammalian GIT via the feces. The water, soil, and sediment that receive the fecal matter and E. coli cells harbor a complex mixture of bacteria, many of which are aerobic or facultative. Also, although E. coli grow best in the warm (body temperature) conditions of the GIT, many environmental bacteria thrive at moderate and even cool temperatures. Thus, when culturing water, soil, and sediment, E. coli are at a disadvantage, and special methods must be used to enrich and select for the E. coli compared with the other background flora. Because of its association with the mammalian GIT, E. coli is widely used as a fecal indicator organism. As such, it is the target for a number of standardized tests and regulatory assays. Most of these assays are culture based. Recently, the USEPA has adopted molecular methods for detecting E. coli in recreational water samples, and the latest Bacteriological Analytical Manual from the USFDA also includes polymerase chain reaction (PCR)-based methods (USEPA, 2012; USFDA, 2012). The advantage of these methods is that they are rapid in comparison to the culture-based assays. The disadvantage is that they exclude phenotypic information and do not result in a living, bacterial isolate. Depending on the purpose of the assay, hybrid methods that combine molecular and culture-based strategies can be used.

Shiga toxigenic Escherichia coli as Naturally Occurring Organisms In many ways, any individual STEC cell is just another E. coli, and the techniques that select for total E. coli (pathogenic and nonpathogenic) from food, fecal, environmental, and insect www.agronomy.org • www.crops.org • www.soils.org

samples can also be used to select for STEC from those same samples. Generic and STEC E. coli frequently occupy the same ecological niche (Durso et al., 2004; Durso and Keen, 2007), and in natural environments different E. coli strains compete with each other for resources. Shiga toxigenic E. coli is routinely isolated from healthy humans and domestic animals (Beutin et al., 1993; Stephan et al., 2000; Alikhani et al., 2007; Fujihara et al., 2009) as well as cattle on grass-only and organic diets (Thran et al., 2001; Grauke et al., 2003; Hussein et al., 2003; Kuhnert et al., 2005; Jacob et al., 2008; Reinstein et al., 2009; Callaway et al., 2013). In the absence of in vivo fitness assays, there is no evidence to suggest that the carriage of the Shiga toxin genes, or other virulence genes, give STEC strains a competitive advantage over non-STEC for daily survival in the bovine GIT. Unlike in human clinical STEC infections, where STEC causes disease and is often shed in high numbers in the feces, STEC is only occasionally shed in high numbers from ruminants (Arthur et al., 2010). In animal GITs, the environment, and insects, E. coli (including STEC) is a minority component of a complex and diverse microbial community (Hungate, 1966; Yokoyama and Johnson, 1988). Thus, small numbers of pathogenic STEC strains can be masked by large numbers of commensal bacteria, including non-STEC E. coli. It is not clear what factors influence competitive fitness between STEC strains and non-STEC strains in complex environments, and no selective enrichment procedures exist that would target just the subset of E. coli that carry the Shiga toxin genes.

Shiga toxigenic Escherichia coli O157 Isolation The three basic steps involved in the primary isolation of STEC O157 from environmental sources, including insects, are enrichment, immunomagnetic separation (IMS), and plating on differential media (Table 1). The specifics of which media are best for the enrichment and plating stages depends on the target organism (STEC O157, non-O157 STEC, or any STEC) and the composition of the competing background flora in the sample. Immunomagnetic separation is a widely used standard step for isolation of STEC from environmental sources and can be instrumental in concentrating the target from the large number of background flora. Ideal conditions for the primary isolation of STEC from environmental samples must be determined empirically. When working with cattle fecal samples, it is occasionally possible to recover STEC O157 without an enrichment step, especially if the feces comes from a “super shedder” animal that is actively shedding >104 cfu g-1 (Arthur et al., 2010). When working with soil and insect samples, however, the target is rarely present in such high densities, and enrichment steps become necessary. The enrichment step is designed to increase the number of target bacteria by helping in the recovery of dormant or injured cells and/or by increasing the number of target bacteria in relationship to the background flora. Shiga toxigenic E. coli O157 enrichments can be single or multiple stages and range from those optimized to specifically enrich STEC O157 from a single source (i.e., dried feces, or environmental swabs, or flies) (Fig. 1) to those designed as the first step in high-throughput methods to enrich multiple pathogens from many sources. Enrichment times need to be determined empirically by sample type and media 1297

used and can have a great impact on recovery rates. Shiga toxigenic E. coli enrichment times generally range from 6 to 24 h. Immunomagnetic separation can be done manually with individual tubes or with an eight-channel magnet or can be done using an automated system. Beads that are recovered from the IMS step are immediately plated onto differential media or immediately added to a second enrichment. The Handbook of Microbiological Media (Atlas, 1997), Difco Manual (Zimbro et al., 2009), and the FDA Bacteriological Analytical Manual (BAM) (USFDA, 2012) are excellent references describing a wide variety of recovery, enrichment, selective, and differential media. Because STEC is a subset of E. coli, methods designed for generic E. coli are generally effective in the first stages of primary isolation of STEC as well. Environmental samples, especially soil, tend to be exceptionally diverse in physical and chemical properties compared with specific food matrices and feces. In addition, there is much greater microbial diversity in soil (Schloss and Handelsman, 2006) compared with foods such as ground beef (Ercolini et al., 2011). Environmental water samples, on the other hand, can be exceptionally nutrient limiting, with a very low cell density, providing a different set of challenges. Methods that have been optimized for food and feces can be a fine starting place for primary isolation from environmental samples, but for best results methods need to be optimized for the specific sample type. Insect enrichments, for example, are lipid-rich compared with soil and often require a modification of IMS methods to obtain optimum sensitivity. Because most environmental sources are heterogeneous and because the target bacteria is present in low numbers, the final sample size that is processed can also affect the recovery of the target organism (Ogden et al., 2000).

The Early Years Since its emergence as a foodborne pathogen in the early 1980s (Riley et al., 1983; Wells et al., 1983), the epidemiology of STEC O157:H7 and STEC O157:H- has been extensively studied, with most efforts focusing on carriage of STEC O157 by ruminant animals (Tuttle et al., 1999; Elder et al., 2000; LeJeune et al., 2004). Most of these studies were culture based. Two physiological traits distinguish most STEC O157 from generic E. coli: (i) slow or no sorbitol fermentation and (ii) the inability to ferment 4-methylumbelliferyl b-D-glucuronide (MUG). These traits were the foundation for early culture and isolation of STEC O157. Reports of the early STEC O157 outbreak isolates from Michigan and Oregon showed that they were sorbitol negative after 7 d of incubation (Wells et al., 1983). This is in contrast to the biochemical reaction results published by Edwards and Ewing (1972), showing that 93.5% (±1.1%) of generic E. coli ferment sorbitol within 48 h. This sorbitolnegative phenotype of STEC O157 served as the basis for a widely adopted modification of a standard E. coli MacConkey agar that substituted sorbitol for lactose (March and Ratnam, 1986). Most E. coli that can ferment sorbitol quickly turn pink on Sorbitol MacConkey agar (SMAC), whereas sorbitolnegative bacteria, such as STEC O157, remain a grey color (Fig. 2). One exception to the sorbitol fermentation phenotype is the large set of sorbitol positive STEC O157:H- strains common in Germany (Karch et al., 2011). There are also sorbitol-negative E. coli that are not STEC O157 (Edwards and Ewing, 1972; Hussein and Bollinger, 2008). In addition, some STEC O157 strains are slow fermenters of sorbitol. Other carbon-source utilization differences exist between STEC O157 and generic E. coli, including D-saccharic acid, D-serine, and glycolic acid

Table 1. Primary isolation of Shiga toxigenic Escherichia coli from environmental sources. Step Enrichment

Reagents

Notes Increases number of target cells

Alternative A: Selective media Brilliant Green Bile Broth, MacConkey, Gram-negative broth, modified E. coli Media, Rapid Check E. coli O157:H7, R&F broth, selective enrichment broth, Enteorhemmorrahgic E. coli enrichment broth, modified Trypticase soy broth. Commonly used antibiotics include cefixime, tellurite, and novobiocin (novobiocin for STEC O157 only) Alternative B: Nonselective media Buffered peptone water, Trypticase soy broth, brain-heart infusion medium, minimal lactose enrichment, universal pre-enrichment broth Optional: Postenrichment acid treatment Immunomagnetic separation

Inhibits background but can also inhibit stressed target cells Incubate at 37°C for 6–24 h. Static incubation favors facultative growth of E. coli compared with anaerobic competitors.

Incubations often performed at 42°C instead of 37°C for increased specificity. Incubate for 6–24 h. Concentrates target bacteria.

Manual or automated bead processing Plating For STEC† O157 CHROMagar O157 with reduced antibiotics, RAPID E. coli O157:H7 Agar, ChromID O157:H7 Agar, Sorbitol MacConkey agar. Commonly used antibiotics include cefixime and tellurite. For non-O157 STEC USMARC chromogenic medium, CHROMagarO157, CHROMagar STEC, Rainbow agar, Modified Rainbow agar, Enterohemolysin agar, Possé media (STEC O26, O103, O111, and O145), RMAC w/Cefixime, tellurite, X-GlcA, and MUG (for STECO26)

If beads “slide” down the side of the tube as wash water is removed, only remove half of the wash at a time and add additional wash steps. Phenotypically distinguishes target bacteria from background Sorbitol MacConkey agar phenotypes are not stable over time, and care should be taken to read plates promptly.

The diversity of serotypes in this group means that false-positive and false-negative colony morphologies are common regardless of the media used.

† Shiga toxigenic Escherichia coli. 1298

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Fig. 1. Enrichment of Shiga toxigenic Escherichia coli (STEC) O157 from environmental sources. Methods used to enrich for STEC O157 in the author’s laboratory. Due to the complexity of environmental substrates, no single method can be recommended. Optimum conditions for each sample and target must be determined empirically. BG, Brilliant Green Bile broth; GN, Gram-negative broth; IMS, immunomagnetic separation; VCC, vancomycin (8 mg L−1), cefixime (0.05 mg L−1), and cefsulodin (10 mg L−1). Chrom Agar with Tellurite = CHROMagar O157 containing 0.63 mg L−1 potassium tellurite (TCA). This is a lower concentration of tellurite than suggested in other sources. Arrows indicate STEC O157 suspects.

(Durso et al., 2004). Culture methods that make use of sorbitol fermentation to screen for and/or identify STEC O157 will miss sorbitol-positive STEC O157 strains. A second phenotypic characteristic that was instrumental in the early days of isolating STEC O157 is MUG. Most E. coli strains (94%) express the b-glucuronidase enzyme, which breaks down MUG, resulting in a fluorogenic product (Feng and Hartman, 1982). Shiga toxigenic E. coli O157 isolates are generally MUG negative due to a point mutation in the gene that codes for b-glucuronidase and therefore do not fluoresce. When MUG is added to solid media, a UV light can be used to distinguish MUG-positive generic E. coli (fluoresce) from MUG-negative STEC O157 (not fluorescing). DNA differences within the uidA (betaglucuonidase) gene have been widely used to identify STEC O157 (Feng, 1993; Feng and Lampel, 1994). As with the sorbitol-negative phenotype, this feature is neither unique to nor exclusive of STEC O157. Although these selective factors can increase the odds of culturing and identifying STEC O157, they can also result in false-negative and false-positive colonies. All isolates need to be confirmed by serotyping and assays for virulence factors and/or virulence genes.

Enrichment There are two basic selective enrichment strategies for the primary isolation of STEC O157 from environmental and insect sources, one using a selective enrichment broth and the other using a nonselective enrichment media. The use of a selective enrichment broth uses parameters such as specific carbon sources and pH to favor the replication of E. coli. These selective factors www.agronomy.org • www.crops.org • www.soils.org

can be paired with chemical measures that restrict the growth of competing microflora, including antibiotics. For example, Brilliant Green Bile Broth, with or without antibiotics, has been proven over many years to be an excellent enrichment for the isolation of STEC O157 from soil, environmental swabs, and pest fly pools (Durso et al., 2005b; Davies et al., 2005; Keen et al., 2006; Goode et al., 2009; Durso et al., 2010; Comstock et al., 2012). It contains lactose as a carbon source (lactose is a preferred carbon source for the enrichment of E. coli) and oxgall (bile), which is tolerated by bacteria like E. coli that naturally cycle through the GIT. Bile is inhibitory, however, to many environmental bacteria. Other selective enrichment broths that have been used to isolate STEC O157 include MacConkey broth (Tanaro et al., 2012), Gram-negative broth (Durso and Keen, 2007), modified E. coli media (Grant,2005; Baylis, 2008), RapidChek E. coli O157:H7 enrichment broth, R&F broth (Fratamico and Bagi, 2007), selective enrichment broth (Kim and Bhunia, 2008), enterohemorrhagic E. coli enrichment broth, and various modifications of Tryptic soy broth (Grant, 2005). A wide range of antibiotic concentrations have been used, and all have the potential to inhibit some STEC O157, especially stressed cells. The mechanisms of action of the commonly used antibiotics and their suitability for pairing with specific enrichment media are described in detail in Hussein and Bollinger (2008). When using selective enrichment media, the elements that restrict the growth of the nontarget bacteria can also impede the growth of stressed target cells. The addition of antibiotics, for instance, can greatly decrease the background flora, allowing the target organism to be selectively cultured. However, the 1299

et al., 2005b). Aliquots of the enrichment can then be subjected to further selection if needed. One report suggests that preconditioning of a selective enrichment media with growth of STEC, which is then filtered out, leaves behind a growth factor that results in improved recovery of small numbers of target cells (Ferenc et al., 2000). The final decision is based predominantly on the composition of the background flora, which varies with location even among the same type of samples. Ideally, one can screen the common background flora for sensitivity to commonly used selective agents to determine the best combination and concentrations to use. A second strategy is to use a rich nonselective broth or a specially designed recovery broth that is intended to improve isolation of stressed environmental cells. When working with samples that may contain starved cells from low-nutrient environments, such as water, the use of a nonselective recovery step can greatly increase the likelihood of success (Sata et al., 1999; Hussein and Bollinger, 2008). Buffered peptone water is often used. Nonselective broth enrichments are frequently paired with incubation at an increased temperature. By definition, coliform bacteria such as E. coli can ferment lactose at temperatures of up to 44.5°C. By incubating at 42 to 44.5°C instead of the more common 37°C, many environmental bacteria are inhibited. One study reported good growth of STEC O157 at 45°C in nonselective media but noted that a number of STEC O157 did not produce turbid growth or gas formation when selective media were used (Ferenc et al., 2000). Some media have been used specifically for the recovery of stressed E. coli cells. Buffered peptone water was recommended for recovery of STEC O157 from soils (USFDA, 1998). Other nonselective media that have been used include Trypticase soy broth (TSB) (Vimont et al., 2006), brain heart infusion (Hussein and Bollinger, Fig. 2. Colony morphologies of Shiga toxigenic Escherichia coli (STEC) and background flora on common media. Background flora isolates represent common phenotypes that 2008), minimal lactose enrichment (Shelton et al., 2004), and a universal pre-enrichment broth (Zhao are co-selected with the STEC targets on immunomagnetic beads. Even on differential media, the target and background morphologies can be similar. Although there is a fair and Doyle, 2001; Nam et al., 2004). A number of amount of consistency in the phenotypes of STEC O157 isolates across the different comparison studies have been performed evaluating media types, non-O157 isolates show a range of morphologies. The photos in this various selective and nonselective media; however, figure display only a single isolate from each non-O157 group. In primary isolation from insect and environmental sources, a range of colony morphologies can be no media have been determined to be superior for all observed for each O serotype. sample types. The inclusion of antibiotic and other selective factors helps to limit background flora addition of antibiotics can also inhibit some stressed STEC but has also been observed to inhibit stressed STEC cells. Even O157 cells from growing, potentially resulting in false-negative with nonselective media, not all positives are identified (Sata et results (Hussein and Bollinger, 2008). It is not uncommon for al., 1999; Hepburn et al., 2002; Foster et al., 2003; Lionberg et research laboratories that commonly rely on primary isolation to al., 2003; Vimont et al., 2006; Fratamico and Bagi, 2007; Baylis, use a reduced amount of the recommended antibiotics in their 2008; Grant, 2008; Hussein et al., 2008). Another enrichment enrichment or plating media (Dogan et al., 2003; Durso et al., technique that is commonly used is to incubate the enrichments 2005b; Vimont et al., 2006; Tillman et al., 2012). For the most statically (without shaking). Because E. coli are facultative, they thorough recovery of STEC from samples, multiple enrichments can continue to grow well even without aeration. Dilute or lowcan be run with and without antibiotics. If sample volumes are nutrient media and extended incubation times (i.e., 1:100 dilutions so low that only a single enrichment can be run and if high levels of TSB incubated for 24–72 h) can be helpful in the recovery of of background flora are expected (e.g., soil, flies, swabs, manureenvironmentally stressed E. coli (Bussmann et al., 2001). impacted runoff ), it is recommended that 1.5× Brilliant Green Escherichia coli are naturally acid resistant. In the life cycle of Bile (BGB) broth be used without additional antibiotics (Durso the organism, the cells cycle from the lower GIT, through the 1300

Journal of Environmental Quality

environment, and back into the mammalian host via the fecaloral route. These ingested cells must pass through the acid of the stomach before reaching the lower GIT, where conditions are favorable. As they go through this ecological life cycle, the cells are only exposed to acid after having been out in the cool, nutrient-limiting environment and right before they reach their preferred nutrient-rich habitat of the colon. Exposure to acid shock of the mammalian stomach is part of the natural life-cycle of E. coli (Savageau, 1983), providing one possible signal to cells in “survival mode” that they soon will be facing nutrient-rich conditions. Acid exposure has been manipulated to improve recovery of generic and Shiga toxigeinc environmental E. coli (Gauthier et al., 1989; Fukushima and Gomyoda, 1999a), food, and animal manure (Grant et al., 2009; Fukushima and Gomyoda, 1999a; Fukushima and Gomyoda, 1999b). A second mechanism by which acid treatment may improve primary isolation of STEC is by inhibiting the non-STEC background flora (Fukushima and Gomyoda, 1999a). Improved STEC enrichment has been reported in artificially inoculated laboratory experiments by exposing cells to a 1/8 mol L-1 hydrochloric acid treatment (Fukushima and Gomyoda, 1999a) and by pairing a prolonged exposure to pH 2 media with subsequent enrichment in a nonselective media (Grant, 2004). Acid treatment of magnetic beads before plating improved recovery of six regulated non-O157 STEC using Rainbow Agar (Tillman et al., 2012). For samples with high particulates, such as fly pools or soil, filter bags can be used. After enrichment, many labs use rapid immunological or PCR-based methods to screen samples for target STEC serotypes to decide which enrichments to move forward in the process. This strategy is the basis for the Food Safety and Inspection Service regulatory standard protocol for the detection and confirmation of STEC in food (Food Safety and Inspection Service, 1998). This is especially useful for high-throughput screening. However, it has been reported that culture-based methods are more sensitive than immunological or PCR-based methods for the detection of STEC O157 (Fratamico and Bagi, 2007), so some positive samples may be missed using this strategy.

The Introduction of Immunomagnetic Separation The availability of E. coli O157:H7 serotype-specific monoclonal antibodies (He et al., 1996; Westerman et al., 1997), combined with magnetic beads, greatly improved the sensitivity of primary culture methods to isolate STEC O157 from complex environmental matrices. The magnetic beads are coated with the serotype-specific monoclonal or polyclonal antibodies and are mixed with the sample directly or mixed with an enrichment. A magnet can then be used to pull out the magnetic bead–bacteria complex, thus separating the concentrated target bacteria from the rest of the sample and remaining bacteria. There are many types of magnetic beads, and differences in the bead size and specific antibody used can affect the efficacy of the IMS procedure (Stevens and Jaykus, 2004; Gee, 1998). Even when a monoclonal antibody is used, in practice, there is still cross-reaction on the beads, and the target bacteria, although concentrated, is not isolated (Fig. 1). Further steps must be taken to separate out the target serotype from other background flora on the beads. This can be done using selective and/or differential media. Sometimes, the IMS bacteria–bead complex is subjected www.agronomy.org • www.crops.org • www.soils.org

to a second selective enrichment. The necessity of including an IMS step for primary culture from complex manure-impacted environmental samples has been demonstrated repeatedly in outbreak situations, where use of an IMS step consistently resulted in isolation of the outbreak strain after standard human clinical methods, such as direct plating on SMAC media, had failed (Durso et al., 2005b; Davies et al., 2005; Goode et al., 2009; Durso et al., 2010; Comstock et al., 2012). Commercially produced IMS beads are available for E. coli O157 and the six regulated non-O157 serotypes, although the sensitivity and specificity varies among STEC serotypes. Some groups have reported success using a brief acid treatment on the IMS beads to reduce background microflora before plating (Tillman et al., 2012). It has been reported that use of a commercial wash buffer instead of PBS + Tween 20 for the IMS wash steps improved recovery of STEC O157 from fecal and beef samples (BarkocyGallagher et al., 2005). Immunomagnetic separation steps can be performed by hand, and multiple high-throughput automated methods also exist. The advantage of manual IMS processing is that bead recovery and quality can be monitored for each sample, and adjustments can be made to the procedure when necessary. The advantage of automated methods is that throughput can be increased. Some researchers suggest that STEC recovery can be improved when larger sample volumes are used (Ogden et al., 2000; Pearce et al., 2004; Echeverry et al., 2005). Most methods use 20 mL of beads and 1 mL of sample, but one common high volume systems use 250 mL of sample that is circulated over 50 mL of magnetic beads. In an evaluation of enrichment, IMS, and selective plating on recovery of STEC O157 from naturally contaminated bovine feces, the large volume system was more sensitive than the small-volume method but was also more expensive. A mix of STEC O157 and E. coli O157 without either of the Shiga toxin genes was isolated using both IMS procedures (Durso and Keen, 2007).

Differential Plating Direct plating of STEC O157 onto SMAC media was the foundation for early STEC detection, and SMAC is still very widely used, especially in clinical and regulatory settings and for the screening of ground and surface waters (LeJeune et al., 2001; Shelton et al., 2003; Sargeant et al., 2004; Shelton et al., 2004; Heijnen and Medema, 2006; Mull and Hill, 2009). Sorbitol MacConkey agar plates can be difficult to read: the target gray colonies are difficult to distinguish if there are many background colonies, and the STEC O157 sorbitol-negative phenotype is not very stable, requiring plates to be read and colonies to be picked in a fairly small window of time. The target phenotype can easily be obscured after storage of the plates, even for as little as 24 h. Finally, there can be many sorbitol-negative bacteria in soil and fly samples, and it is difficult to screen them all to find the target STEC O157. In outbreak investigations associated with agricultural fairs, CHROMagar O157 in conjunction with IMS has proven superior to SMAC for detection of STEC O157 from environmental sources (Durso et al., 2005b; Davies et al., 2005; Goode et al., 2009; Durso et al., 2010; Comstock et al., 2012). Researchers in food-based systems have reported success with Rainbow Agar O157 (Fratamico et al., 2011; Manafi and Kremsmaier, 2001; Grant, 2008). Many new selective and/or differential media are available to distinguish 1301

STEC O157 from other E. coli and other background flora. For example, CHROMagar O157 (BD Diagnostics), RAPID E. coli O157:H7 Agar (Bio-Rad), and ChromID O157:H7 Agar (BioMerieux Inc.) are widely used in the food industry and have received Performance Tested Methods certification from AOAC Research Institute. They have not been performance tested for environmental samples. As with enrichments, selective agents such as antibiotics can be added to the media to control background flora. Comparison studies, performed predominantly with ground beef and food samples, suggests that there is no single plating medium that is ideal for all STEC O157 strains (Grant, 2008), and in some instances IMS beads may be plated on multiple types of media. This strategy is recommended by some bead manufacturers to increase the likelihood of detecting positive colonies. In one commonly used procedure, the beads are suspended in 100 mL, and the entire volume of beads is plated onto a single or multiple plates. The volume of beads spread plated onto each plate can be varied. In environmental studies, 50 mL is routinely used. However, when background levels were high and sample volume was restricted, volumes as low as 10 mL per plate (spread onto each of 10 plates) were used.

Hybrid Methods Many modern detection methods rely partially or exclusively on molecular methods such as real-time PCR and quantitative real-time PCR The detection limits for these assays on environmental samples can be low compared with enrichment and culture, and there are concerns that the molecular methods allow for higher number of false positives due to detection of DNA from nonviable cells (Artz et al., 2006). Some methods pair an initial enrichment step with a real-time PCR step (Sen et al., 2011; Yoshitomi et al., 2012; Heijnen and Medema, 2006; Food Safety and Inspection Service, 1998) for rapid detection of the target cells, with the possibility of going back to the PCRpositive enrichments to obtain an isolate for confirmation. Enrichment cultures paired with PCR can also be used in a most probably number procedure to obtain quantitative information for specific samples (Heijnen and Medema, 2006). One factor that needs to be highlighted when using multiplex PCR assays to screen environmental samples is that it is common to have many kinds of E. coli within any sample. For example, a sample may contain a generic (non-STEC) O157 E. coli, an O153:H7 E. coli, and Shiga toxin–positive O82:H10 E. coli. A multiplex PCR that targets rfbO157, flicH7, and the stx genes will be positive for this sample even though the sample does not contain STEC O157.

Non-O157 Shiga toxigenic Escherichia coli There are many E. coli, in addition to STEC O157, that carry the Shiga toxin genes and have the potential to make people sick. These are referred to broadly as “STEC” or “non-O157 STEC.” Recently a subset of six specific non-O157 STEC serotypes was identified that are most frequently associated with human disease in the United States and that are targeted for regulation within the US food industry. The term “non-O157 STEC” is commonly used to refer to these “big six” serotypes: STEC O26, STEC O45, STEC O103, STEC O111, STEC O121, 1302

and STEC O145. Just as most STEC O157 are associated with a single H antigen (i.e., E. coli O157:H7), so too with nonO157 STEC: there are specific O:H serotypes that are most frequently associated with disease in humans (e.g., O26:H11 and O111:H8). Recently, another STEC serotype, STEC O104, was associated with a large foodborne outbreak (Scavia et al., 2011). The factor or combination of factors with which to predict the specific STEC serotypes that will cause disease has yet to be determined. Primary isolation of non-O157 STEC from environmental samples is much more challenging and time consuming than primary isolation of STEC O157. Isolation of this subset of E. coli bacteria from their natural habitats is a challenge that is complicated by the relatively low numbers of target organisms compared with the background flora and the similarity of STEC to other E. coli bacteria. There is a great diversity of serotypes included in the group and a lack of easily identifiable phenotypic characteristics that can be used to select for the non-O157 STEC while inhibiting the growth of other closely related E. coli. Modern field methods for tracking non-O157 in preharvest food and environmental samples combine enrichment, PCR, IMS, and selective plating on multiple media to detect the target organism (Cooley et al., 2013). Much of the early work defining the epidemiology of nonO157 STEC was done by screening large numbers of sorbitolfermenting E. coli colonies for production of the Shiga toxin protein via immunoblotting (Karch et al., 1986) or the presence of the Shiga toxin genes via colony hybridization (Karch and Meyer, 1989). Many of the currently used “big-six” STEC detection methods rely heavily on molecular assays. These methods are efficient for high-throughput and routine screening applications (Bosilevac and Koohmaraie, 2012). Recent DNA sequencing efforts have identified nucleotide polymorphisms within the O-antigen of the big-six STEC (Norman et al., 2012), and these differences can be exploited to improve the likelihood of successfully isolating the big-six STEC from environmental sources via screening of enrichment broths or screening of colonies from differential agar plates to identify samples that are likely positive for the target strain. Polymerase chain reaction– based screening of enrichments for the Shiga-toxin genes is also a common strategy in beef (Bosilevac and Koohmaraie, 2011) and water samples (Tanaro et al., 2012; Cooley et al., 2013). Food safety regulatory procedures for the detection of the big six non-O157 STEC serotypes from ground beef include an initial screening using PCR, followed by IMS and culture. It is not known if non-O157 STEC, as a group, are more likely to be detected by culture-based versus immunological or PCR-based methods. Culture is more sensitive for STEC O157 (Fratamico and Bagi, 2007), but STEC O157 culture methods are more refined than those available for non-O157 STEC. Although the sensitivity of PCR assays for detecting the specific big six O serotypes directly from environmental samples is unknown, these methods can be exceptionally helpful once an isolate is obtained. Screening can be done with ELISA (Brooks et al., 2005). New low-volume PCR formats can reduce reagent cost and have the scalability to work for modest and large-scale studies. As with STEC O157, a variety of selective agents can be used during the enrichment to help reduce the abundant background flora in environmental samples. However, non-O157 STEC Journal of Environmental Quality

may respond differently than STEC O157 to individual agents. For example, STEC O157 has been reported to be significantly more resistant to novobiocin (20 mg L-1) than non-O157 STEC (Vimont et al., 2007). Although STEC O157 represents a narrow lineage within E. coli, non-O157 STEC can represent many different serotypes and evolutionary lineages. It is more difficult, therefore, to find selective agents that specifically restrict generic E. coli but allow growth of the big six STEC serotypes.

Differential Media for Non-O157 Shiga Toxigenic Escherichia coli A small number of differential agars have been designed for the detection of the big six STEC. Some target all six serotypes; others focus on a single serotype. A new media has recently been developed by Kalchayanand et al. (2013) to distinguish all six serotypes. It has been extensively validated and field tested on postharvest samples such as beef and with feces. In addition to the media recipe, the authors provide extensive information on phenotypic screening of reference strains and background information on fundamental elements of differential media formulation (Kalchayanand et al., 2013). Another media, developed by Possé et al. (2008), is designed to be used for differentiation of four non-O157 STEC serotypes (O26, O103, O111, and O45) from human stools. CHROMagar O157, CHROMagar STEC, Rainbow agar, and modified Rainbow agar have also been used for detection and isolation of the big-six non-O157 STEC (Tillman et al., 2012; Wylie et al., 2012) (Fig. 2). A recent study by Cooley et al. (2013) screened over 13,000 water, soil, animal, and produce samples for non-O157 STEC using a combination of four different agars. Research studies performed at the U.S. Meat Animal Research Center since the early 2000s have resulted in the primary isolation of numerous STEC O26 and STEC O111 from environmental and pest fly samples, originating in multiple states. In published (Keen et al., 2006) and unpublished studies, BGB and Gramnegative broth were used as primary enrichment broths, followed by IMS with O26 or O111 serotype-specific magnetic beads and plating on rhamnose-MacConkey agar to detect STEC O26 and CHROMagar with reduced antibiotic concentrations to detect STEC O111. Environmental and fly isolates were screened using ELISA and confirmed as STEC using PCR for stx and eae genes (Keen et al., 2006). Isolates were serotyped using validated molecular serotyping methods (Durso et al., 2005a; Durso et al., 2007). Regardless of sample type, STEC O111 can occasionally be isolated from the STEC O157 magnetic beads. Shiga toxigenic E. coli O111 often displays a “dark center” morphology (pink colony with small blue center) on the CHROMagar O157 plates. Examination of frozen beef enrichments used PCR screening combined with colony hybridization to collect non-O157 STEC isolates (Arthur et al., 2002). These studies recovered 361 isolates belonging to 41 different O serotypes. Results indicate that nonO157 is common on beef carcasses (93% of lots surveyed had at least one STEC isolate) and that STEC O157 interventions were also effective at reducing other STEC on the product (only 8% of post-treatment carcasses had STEC-positive isolates). None of the postevisceration STEC belonged to the big six STEC serotypes (Arthur et al., 2002). www.agronomy.org • www.crops.org • www.soils.org

Shiga Toxigenic Eschrichia coli O26 Many STEC O26 strains have been reported to differentially ferment rhamnose, and rhamnose-MacConkey (RMAC) has been used by a number of laboratories for differential plating of this STEC serotype from mixed enrichment broths (Hiramatsu et al., 2002; Murinda et al., 2004). Shiga toxigenic E. coli O26 strains appear cream colored on RMAC, whereas nontarget E. coli strains are red. Fly homogenates, soil, and some environmental swabs can contain a significant amount of background flora that also appear cream colored on RMAC, so additional screening measures are needed when doing primary isolations. Cefixime and tellurite are sometimes added to RMAC in a manner similar to their addition in SMAC for detection of STEC O157. Evaluation of cefixime-tellurite RMAC for STEC O26 showed false-negative and false-positive results, and the authors conclude that cefixime-tellurite RMAC can be used for primary isolation, but some strains will be missed (Evans et al., 2008). Screening by Murinda et al. (2004) of STEC and non-STEC O26 on a variety of media and comparison with generic E. coli, STEC O157, and STEC O111 revealed that addition of 5-bromo-4-chloro3-indolyl-b-D-glucuronide (X-GlcA) to RMAC media changes the target color of rhamnose-negative strains (e.g., STEC O26) to a well-defined blue (Murinda et al., 2004). However, up to 40% of E. coli O26 without Shiga toxin genes were also rhamnose negative, so confirmation of Shiga toxin status was required even with the rhamnose-negative phenotype. The final recommendation is for RMAC supplemented with cefixime, tellurite, MUG, and X-GlcA, resulting in target STEC O26 colonies that are blue and that fluoresce under ultraviolet light (Murinda et al., 2004).

Shiga Toxigenic Escherichia coli O111 Two groups of E. coli O111 cause disease in humans (Nataro and Kaper, 1998): (i) enteropathogenic E. coli (EPEC) O111, which is associated with childhood diarrhea in developing countries (Trabulsi et al., 2002), and (ii) STEC O111, which differs from EPEC O111 in that it carries one or both Shiga toxin genes (Nataro and Kaper, 1998). Shiga toxigenic E. coli O111 has been associated with numerous outbreaks (Bettelheim and Goldwater, 2004; Brooks et al., 2004; Comstock et al., 2012). It was recently isolated from fecal and environmental sources associated with an outbreak at a correctional facility (Comstock et al., 2012). In that investigation, fecal and environmental samples were combined with 1.5× BGB, incubated at 37°C for 6 h, subjected to IMS using O111-specific beads, and spread plated (50 mL per plate) on CHROMagar O157, where the suspect STEC O111 isolates displayed a pink colony with a small dark blue center (this is different than the STEC O111 colonies on CHROMagar STEC, which are indistinguishable from other STEC serotypes). The standard IMS bead wash of PBS + 0.05% Tween 20 was used, and the wash steps were performed by hand. Some samples resulted in beads that did not adhere solidly to the magnetic device, as evidenced by their tendency to “slide” down the side of the tube as the wash water was removed. For these samples, only half of the wash solution was removed at a time, and additional wash steps were included.

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Screening for Shiga Toxin If the target for primary isolation is any E. coli that carries the Shiga toxin gene, then methods for generic E. coli can be used, with isolated E. coli colonies being screened for Shiga toxin genotype (via PCR) or phenotype. An early study reported a correlation between enterohemolysis, nonfermentation of L-rhamnose, and nonfermentation of D-sucrose associated with carriage of Shiga toxin genes, especially stx1 (Wieler et al., 1995). In other studies, washed blood agar was used to phenotypically screen for STEC with the idea that many E. coli that carry the Shiga toxin gene also carry a gene for entrohaemolysin (over 90% in one study) and will leave a zone of lysis around colonies growing on blood plates (Beutin et al., 1989; Bettelheim, 1995). The addition of mitomycin C appears to enhance the expression of the hemolysin in some strains and improves detection of STEC on blood plates (Sugiyama et al., 2001; Lin et al., 2012). Blood plates are often used directly in human clinical settings, but an enrichment step is recommended for environmental samples (Hussein and Bollinger, 2008). DNA colony hybridization has also been used to screen for Shiga toxin genes (García-Aljaro et al., 2005). For enrichments that will be screened for the production of Shiga toxin, animal-based media (i.e., brain-heart infusion medium) provides superior results (more Shiga toxin production) compared with enrichments using plant-based media (Hussein and Bollinger, 2008). A second strategy is to screen enrichments using immunological assays (Park et al., 2003; Klein et al., 2004; Karama et al., 2008; Willford et al., 2009) or cell culture assays (Blanco et al., 1996; Quiñones et al., 2009; Zheng et al., 2008).

Conclusions When STEC first emerged as a human pathogen, monitoring and control measures were focused primarily on meat. As we learn more about the ecology of STEC in agroecosystems and the ability of STEC to survive for prolonged periods outside of the mammalian GIT, the public health importance of STEC in environmental samples must be considered. Many environmental STEC detection and isolation methods exist, with STEC O157 methodology being more developed than that for nonO157 STEC. The use of quick, gene-based STEC detection methods can be complicated when the substrate is a mixed bacterial culture because it is possible for each of the targets to be carried by separate bacteria. For this reason, primary isolation of STEC from water, soil, and sediment samples remains an important tool for epidemiological and ecological studies as well as for monitoring and control. A three-step process, including enrichment, IMS, and selective plating, is effective at isolating STEC from even complex environmental samples.

Acknowledgments The author thanks Jennifer McGhee and Amy Mantz for technical assistance, Adam Shrek for sample collection, James Bono for sharing the non-O157 strains used for the photos, and Brian Wienhold for critical reading of the manuscript. This work was supported by the USDA–ARS, National Program 214.

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