Characterization and Survival of Environmental Escherichia coli O26 Isolates in Ground Beef and Environmental Samples Christine E. Palmer, Christy L. Bratcher, Manpreet Singh, and Luxin Wang
In addition to Escherichia coli O157:H7, shiga toxin-producing E. coli (STEC) O26 was added to the zerotolerance adulterant list together with other 5 non-O157 STEC serogroups in 2012. Four farm O26 isolates were used in this study; they were obtained from a on-farm survey study conducted in Alabama. The presence of 3 major pathogenic genes (stx1, stx2, and eaeA) was determined through multiplex polymerase chain reaction (PCR). Two major pathogenic gene profiles were observed: 3 of the farm isolates contain only the eaeA gene whereas 1 farm isolate has both the eaeA and the stx1 genes. No significant difference was seen among the 4 farm isolates in the antibiotic resistance tests. To test their survival in ground beef and environmental samples, 2 inoculums were prepared and inoculated at various concentrations into samples of ground beef, bovine feces, bedding materials, and trough water. One inoculum was made of 3 farm isolates containing only the eaeA gene and another inoculum contained the isolate with both the eaeA and stx1 genes. Inoculated beef samples were stored at 4 °C for 10 d and the inoculated environmental samples were stored at ambient temperature for 30 d. Results showed that virulence gene profiles do not have an impact on O26’s ability to survive in ground beef and in environment (P > 0.05). The inoculation levels, sample types as well as the storage times are the major factors that impact O26 survival (P < 0.05). Abstract:
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Keywords: pathogenic genes, Shiga toxin producing E. coli O26, survival
Escherichia coli O26 was the top one non-O157 shiga toxin-producing E. coli isolated in a cow/calf operation survey study conducted in Alabama. The capability of the 4 fecal O26 isolates being able to survive in food and environmental samples for an extended period of time indicated that good agriculture practices for small cow/calf operations are required and of great importance to prevent on-farm pathogenic O26 transfer.
Practical Application:
Introduction
serogroup has been frequently found in cattle with prevalence rates of 5.1% in Japan (Fukushima and Seki 2004) and 1.7% in Australia (Cobbold and Desmarchlier 2000). In France, a study showed that 15% of the dairy farms tested were positive for STEC O26 (Fremaux and others 2006). In a recent study conducted in the United States, 4 E. coli O26 strains were isolated from 3 of 7 (42.8%) cow/calf farms in Alabama (Palmer 2014). Several virulence factors are involved in the pathogenicity of non-O157 STEC. The presence of shiga toxin 1 (stx1), shiga toxin 2 (stx2), or a combination of these genes is the most important indicator of a bacterium’s virulence (Etcheverria and Padola 2013). Another important virulence factor of STEC is eae, the intimin gene, which is used by the bacteria to attach to epithelial cells. It works by inducing an attaching and effacing (A/E) lesion on the surface of the target cell (Etcheverria and Padola 2013). Even though the presence of virulence genes such as eae is a good indicator of pathogenicity, its absence does not necessarily mean it is not pathogenic (Beutin 2006). The presence of intimin can sometimes be associated with the more severe cases (Beutin and others 2004). Virulence profiles of STEC O26 have changed over the years. Zhang and others (2000) showed that all O26 isolates collected between 1965 and 1994 expressed only stx1 whereas most O26 isolates collected between 1995 and 1999 had either MS 20141539 Submitted 9/15/2014, Accepted 2/1/2015. Authors Palmer, Bratcher and Wang are with Dept. of Animal Sciences, Auburn Univ., Auburn, AL, stx2 alone or a combination of stx1 and stx2. A better understanding of the 4 O26 isolates is still needed. U.S.A. Authors Singh is with Dept. of Food Sciences, Purdue Univ., West Lafayette, IA, U.S.A. Direct inquiries to author Wang (E-mail: [email protected]). In addition to their pathogenic gene compositions and antibiotic resistance profiles, information on their survival capability in foods
Shiga toxin-producing Escherichia coli (STEC) strains are foodborne infectious agents that have caused a number of outbreaks. In past decades, studies on STEC have focused on E. coli O157:H7 due to its earlier discovery in human illness (Monaghan and others 2011) and its involvement in the 1982 Michigan and Oregon outbreaks (Lim and others 2010). In recent years, more reports about non-O157 STEC have attracted public attentions. Grant and others (2011) reported that 23 outbreaks of non-O157 STEC illnesses happened between 1990 and 2007 in the United States. Among non-O157 STEC serovars, E. coli O26, O111, O103, O121, O45, and O145 are now placed on the zero-tolerance adulterant list as of June 2012 (USDA 2011). Of the 6 non-O157 STEC strains, O26 has been more prominent and the focus of many studies due to its notoriety for the virulence factors that can make people sick (Mathusa and others 2010). It has been regarded as an important cause of STEC-associated diseases (Tarr and Neill 1996). It is estimated that O26 and O111 are responsible for 25% of all serious cases of infection with the risk of kidney failure and death (Sharma 2002). O26 has the history of being isolated from cattle farms. The O26
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doi: 10.1111/1750-3841.12827 Further reproduction without permission is prohibited
Survival, E. coli O26, beef, environment . . .
Materials and Methods Bacterial strains Four O26 farm isolates (LWP1, LWP2, LWP3, and LWP4) and 1 clinical strain (TWO8031) were used in this study. TWO8031 was obtained from the Thomas S. Whittam STEC Center at Michigan State Univ. and was originally isolated in 1999 from a human host living in the United States. The TWO8031 strain was used as the reference strain. Characterization of farm isolates Antibiotic susceptibility/sensitivity, finger printing (pulse field gel electrophoresis patterns), and the pathogenic profiles of 4 farm isolates were determined. The antibiotic susceptibility/sensitivity of these strains was determined using a disk diffusion method outlined by the Clinical and Laboratory Standards Inst. (CLSI 2012). The diameters of the inhibition zones were measured. Antibiotic disks were purchased from Becton Dickinson, U.S.A. The susceptibility of all isolates to a total of 11 antibiotics was examined. The pulse field gel electrophoresis (PFGE) protocol followed the CDC standard operating procedure for PulseNet PFGE of Escherichia coli Non-O157 STEC (CDC 2013). One restriction enzyme (XbaI; Promega, U.S.A.) was used. Salmonella ser. Braenderup H9812 (ATCC, BAA-664TM ) was used as the molecular weight standard. A multiplex PCR assay targeting 3 different virulence genes was applied to determine the virulence profiles of O26 isolates. Target virulence genes included the eaeA, stx1, and stx2. Primers were designed by Paton and Paton (1998) and were synthesized by Integrated DNA Technologies (IDT, Coralville, IA, R U.S.A.). DNA was extracted by using PrepMan ultra sample preparation reagent following the manufacturer’s manual (Life TechnologiesTM , Grand Island, NY, U.S.A.). The working solution for each primer was 10 μM. AccuStartTM II PCR Supermix (2×; Quanta BioSciences, San Jose, CA, U.S.A.) was used for the multiplex PCR assay. A 1:10 dilution of the DNA samples with ultrapure water was used as the PCR template. In each 0.1 mL PCR tube, the following reagents were mixed together: 5 μL of the template, 0.25 μL of each forward and reverse primer, 12.5 μL of AccuStart II PCR Supermix (2×), and 6 μL of nuclease-free water, giving a total volume of 25 μL per PCR tube. The PCR R R reaction was carried out in an Applied Biosystems Veriti 96-Well Fast Thermal Cycler (Life TechnologiesTM , Grand Island, NY, U.S.A.), with an initial 3 min 94 °C denaturation, and then 30 cycles of: 30 s 94 °C denaturation, 30 s 58 °C annealing, and 1 min 72 °C. PCR products were then held at 4 °C and examined by running the products in a 2% agarose gel for 40 min. Inoculum preparation In preparation of the inoculum, E. coli O26 strains were transformed with the pGLOTM plasmid containing the green fluorescence protein gene (GFP; Bio-Rad Laboratories, Hercules, CA, U.S.A.). The protocols of making competent cells and transferring plasmid followed the “Molecular cloning—a laboratory manual,” 3rd edition. The transformed strains were picked from the LB plates supplemented with 0.1 mg/mL ampicillin (Sigma-Aldrich, St. Louis, MO, U.S.A.) and 50 mg/mL arabinose (Alfa Aesar, Ward Hill, MA, U.S.A.) and their growth curves were proved to be
similar to the parent strains before use (P > 0.05). Two sets of culture inoculums were prepared: one was composed of 3 farm isolates with the pathogenic gene profile of eaeA gene and another one contained one farm isolate that have both the eaeA and the stx1 genes. Overnight bacterial cultures were prepared by transferring 100 μL of frozen cultures into 10 mL Luria-Bertani (LB) broth supplemented with ampicillin and arabinose and incubating at 37 °C. The concentrations of overnight cultures were determined by plating their dilutions onto LB plates supplemented with ampicillin and arabinose and counted the next day. To prepare the inoculum, cultures were washed by centrifugation (Eppendorf, Hauppauge, NY, U.S.A.) at 4000 × g for 5 min and resuspended in MilliQ water and were then mixed. For lower inoculation levels, stock inoculums were diluted in MilliQ water before being inoculated into beef and environmental samples.
Ground beef and environmental sample inoculation Ground beef was purchased from a local store (85% lean and 15% fat). Before inoculation, ground beef was confirmed to be STECfree by enriching and plating the samples on CHROMagarTM STEC plates (CHROMagar, Paris, France). Suspect colonies were further tested using multiplex PCR to determine if they contain stx or eae genes. Ground beef was used only if the results from both the CHROMagar STEC plating and the PCR results were negative. To prepare each inoculated beef sample, 25 g of ground R beef were weighed and put into filtered Whirl-pak bags (Nasco, Albuquerque, NM, U.S.A.) together with 1 mL of the chosen E. coli inoculum. Three final inoculation levels were reached, low (102 log CFU/g), medium (106 log CFU/g), and high (108 log CFU/g). Inoculated samples were stored in the refrigerator (4 °C) for 10 d. Composite fecal samples, bedding materials, and trough water were used in this study and were retrieved from E.V. Smith Research Center (Auburn Univ., Auburn, Ala., U.S.A.). Each composite sample was made with 3 individual subsamples taken from 3 different sites. To prepare each inoculated environmental sample, 25 g of fecal or bedding samples were measured into Whirl-pak bags (Nasco) and inoculated with 1 mL of the inoculum. To prepare each inoculated water sample, 100 mL of trough water was measured into a 125-mL sterile sample cup (VWR, Suwanee, GA, U.S.A.) and then inoculated with 1 mL of each inoculum. Two target inoculation levels were tested for environmental samples (106 and 104 log CFU/g or /mL). All inoculated environmental samples were kept at ambient temperature for 30 d. Enumeration of survived E. coli O26 Three replicate trials were conducted for every inoculation study. For each inoculated beef trial, 3 inoculated ground beef samples of every inoculation level were removed from the 4 °C refrigerator every day for 10 d. One hundred milliliter of 0.1% buffered peptone water was added to each bag. Samples were homogenized for 2 min using the SmasherTM lab blender (AES Chemunex, Marcy l’Etoile, U.S.A.). Bacteria were plated by spreading 100 μL of the smashed contents or dilutions on LB plates supplemented with 0.1 mg/mL ampicillin and 50 mg/mL arabinose. Plates were incubated at 37 °C and counted after 24 h. For each inoculated environmental sample trial, subsamples were taken every 5 d and the survived O26 cells were enumerated as described above. For samples in which the levels of O26 were below the limit of detection, sample suspensions were enriched by adding an additional 125 mL of 2× lactose broth and enriched for 24 h before streaking the enriched suspensions onto LB plates supplemented with Vol. 80, Nr. 4, 2015 r Journal of Food Science M783
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and environment will benefit the future development of efficient intervention and good agricultural practices. Thus, the objectives of this study were to characterize the 4 O26 isolates and evaluate the survival of STEC O26 in artificially inoculated ground beef and environmental samples.
Survival, E. coli O26, beef, environment . . . Table 1–Antibiotic resistance comparison between 4 farm STEC O26 isolates and one clinical isolate.
Antibiotics Amoxicillin-clavulanic acid Ampicillin Ceftriaxone Cephalothin Chloramphenicol Ciprofloxacin Cefoxitin Gentamicin Naladixic acid Sulfamethoxazole/trimethoprim Tetracylcine
Concentrations
Susceptible breakpoints (mma )
20 μg 10 μg 30 μg 30 μg 30 μg 5 μg 30 μg 10 μg 30 μg 23.75/1.25 μg 30 μg
18 17 23 18 18 21 18 15 19 16 15
Isolated from farm (mma ) LWP1 22.50 21.50 32.76 18.26 24.76 32.00 24.00 21.50 25.76 28.26 22.26
± ± ± ± ± ± ± ± ± ± ±
0.65 1.44 0.75 0.48 0.25 0.71 0.41 0.29 0.25 0.25 0.48
LWP2 21.26 20.00 32.26 17.50 23.00 32.26 24.76 21.50 25.00 29.00 22.00
± ± ± ± ± ± ± ± ± ± ±
0.85 0.58 0.25 0.29b 0.58 0.95 0.48 0.29 0.71 0.71 0.41
LWP3 20.76 20.50 30.00 18.76 22.00 33.76 25.50 21.00 24.50 27.52 21.00
± ± ± ± ± ± ± ± ± ± ±
0.75 1.19 0.58 0.25 0.58 1.03 1.44 0.00 0.29 0.87 0.41
LWP4 21.46 20.76 34.26 19.50 24.76 36.76 25.76 22.76 27.00 27.26 22.26
± ± ± ± ± ± ± ± ± ± ±
0.75 0.63 0.48 0.29 0.25 1.93 0.48 0.25 0.00 0.48 0.25
a Diameter b
of the inhibition zone. The intermediate zone diameter for Cephalothin is 15 to 17 mm.
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ampicillin and arabinose. All bacterial media were purchased from PCR reactions targeting stx1, stx2, and eaeA genes was conducted. As shown in Figure 1A, 3 of the 4 farm isolates (LWP1, LWP2, and Becton Dickinson unless otherwise stated. LWP4) contain only the eaeA gene and one farm isolate (LWP3) Statistical analysis had both the stx1 and eaeA genes. Strains were also compared Three replicates were done for the survival studies. The data using PFGE and enzyme XbaI was used. As shown in Figure 1B, were analyzed using a general linear model (GLM) procedure all PFGE patterns were different among all 4 isolates used. LWP1 in SAS 9.2 (SAS Inst. Inc., Cary, NC, U.S.A.) The effects of and 2 had the highest similarity whereas strain LWP3 and the 2 pathogenic gene profiles, different inoculation levels, materials clinical reference strain are more closely related compared to the (beef, bedding, feces, and water), and time (d) on O26 survival other 3. were evaluated. P values of less than 0.05 were considered to be significant.
Results and Discussion Characterization of O26 All 4 farm isolates showed susceptibility to the antibiotics tested (except for LWP2 on Cephalothin) and no antibiotic resistance was seen (Table 1). The breakpoint for cephalothin susceptibility is 18 mm. The diameter of the cephalothin inhibition zone on LWP2 was 17.5 ± 0.29, which is higher than the intermediate range of Cephalothin (15–17 mm) but a little lower than the susceptible breakpoint. DNA of all O26 strains was extracted and multiplex
Survival of E. coli O26 in Ground Beef E. coli O26 strains were transformed with GFP plasmids before use. As shown in Figure 2, transformed E. coli O26 cells showed green fluorescence under the UV light after 24 h incubation and could be easily differentiated from the background microflora and counted. The initial inoculation levels of ground beef were 7.54 ± 0.28 Log CFU/g, 5.53 ± 0.29 Log CFU/g, and 1.60 ± 0.24 Log CFU/g (high, medium, and low). As shown in Figure 3, E. coli O26 survived and persisted well in ground beef during the 10-day
Figure 1–(A) Multiplex PCR results of 4 farm O26 isolates and one clinical O26 strain; and (B) pulse field gel electrophoresis (PFGE) dendrogram of 4 farm isolates and one clinical strain.
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storage time, regardless of the inoculation levels. No significant farm water samples (Figure 4C), steep declines were seen over the 30-d trial period for all samples. After day 15, survived E. coli O26 difference was seen when comparing 2 inoculums (P > 0.05). (with the eaeA and stx1 genes) in water with an initial inoculation Survival of E. coli O26 in environmental samples level of 4.47 Log CFU/mL were below the limit of detection 0.6 The final inoculation levels for fecal samples were 5.05 ± 0.11 Log CFU/mL. With 24 h enrichment, O26 cells were able to and 3.25 ± 0.01 Log CFU/g. The survival of E. coli O26 in be confirmed by streaking the enriched broth on selective agar. fecal samples is shown in Figure 4A. An approximately 1 log Similar with the fecal and bedding material data, no significant increase was seen after 5 d from samples with the initial inoculation difference was seen between the 2 inoculums (P > 0.05). levels of 5.05 ± 0.11 Log CFU/g, regardless of the pathogenic As shown by statistical analysis, storage times, types of materigene profiles. A similar increase was seen for the fecal samples als, and the initial inoculation levels are the 3 major factors that inoculated with the inoculum containing only the eaeA gene at impacted the survival of E. coli O26 in this study (P < 0.05). The a level of an approximately 3.25 ± 0.01 Log CFU/g. Up to a 2 inoculums showed no significant difference in survival at each 2-Log reduction was seen for samples inoculated with 3.25 Log time point (P > 0.05); different pathogenic gene profiles had no CFU/g of E. coli O26, containing both the eaeA and stx genes impact on the survival capability of E. coli O26. after 10 d. One reason for the increases in O26 numbers may be The objectives of this study were to better characterize 4 farm due to the good nutrient availability in feces not available in the isolates obtained in a previous STEC prevalence study and to other environmental samples. Time played a significant role in E. find out the potential impact different pathogenic gene profiles coli O26’s survival (P < 0.05). However, no difference was seen can generate on cells’ survival in both meats and environmental between 2 inoculums at every time point (P > 0.05). samples. Among 4 farm isolates, only one of them contained both The initial inoculation levels for bedding materials were 5.19 ± eaeA and stx1 genes whereas the other 3 have only the eaeA gene, 0.04 and 3.29 ± 0.06 Log CFU/g. Results for bedding materials a gene used by the bacteria to attach to epithelial cells. As shown are shown in Figure 4B. Bacteria counts declined steadily over the by the PFGE results, the 3 farm isolates containing only the eaeA entire 30-d period. After 15 days, E. coli O26 counts with initial gene were grouped closely together whereas the farm isolate and inoculation levels of 3.29 ± 0.06 Log CFU/g were below the limit the clinical reference strain containing both eaeA and stx1 gene of detection (0.6 Log CFU/g). An approximately 2 Log reduction (LWP3 and TWO8031) were grouped together. None of the 4 was seen for samples inoculated with 5.19 ± 0.04 Log CFU/g. farm isolates exhibited any antibiotic resistance. No significant difference was seen between the 2 inoculums Fecal, bedding, and trough water sample can be vehicles by (P > 0.05). which zoonotic agents may enter and become disseminated The initial inoculation concentrations for trough water were throughout the agricultural environment. STEC serogroup O26 6.51 ± 0.08 Log CFU/mL and 4.47 ± 0.07 Log CFU/mL. For the has been regarded as an important cause of STEC-associated Figure 2–GFP transformed E. coli O26 isolates with background microflora plated on LB agar supplemented with ampicillin and arabinose. Colonies seen on LB plate under the regular light (left); GFP transformed colonies gave green fluorescence under the UV light (right).
Figure 3–Survival of O26 in ground beef stored at 4 °C. E. coli O26 with eaeA gene, high E. coli O26 with eaeA inoculation level; gene, medium inoculation level; E. coli O26 with eaeA gene, low inoculation level; ······ E. coli O26 with eaeA and stx1 genes, high inoculation level; —- E. coli O26 with eaeA and stx1 genes, medium inoculation level; –·–· E. coli O26 with eaeA and stx1 genes, low inoculation level.
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Survival, E. coli O26, beef, environment . . .
Survival, E. coli O26, beef, environment . . . diseases (Tarr and Neill 1996). E. coli O157:H7 can survive in bovine feces for long periods and can retain the potential to produce shiga toxin (Wang and others 1996). Unfortunately, the fate of O26, especially the impact of different pathogenic profiles, remains unclear. Unlike O157, STEC strains do not have common biochemical characteristics allowing the use of specific media for their isolation; in addition, the potential presence of pathogenic E. coli O26 in environmental samples could influence the enumeration of the survived inoculum. Thus, to better differentiate the inoculum and the background microflora, all O26 strains used this study were transformed with GFP plasmid. As discussed by Ehrmann and others, Vialette and others, and Fremaux and others (Ehrmann and others 2001; Vialette and others 2004; Fremaux
and others 2007b), the stability of GFP-labeled O26 has been confirmed. In addition, Fremaux and others (2007a) showed that no significant difference of behavior was observed between the transformed STEC O26 strains and the parental strains after their inoculation into cow slurry. In this study, the growth curves of GFP transformed O26 strains were compared with their parental strains and no significant difference was seen (data not shown). As shown in this study, regardless of inoculation levels, E. coli O26 survived in ground beef for 10 d without a significant decrease at the refrigerated temperature. These data indicate that intervention strategies applied during meat processing are of great importance to ensure the zero-tolerance regulation of non-O157 STEC in ground beef. When inoculating O26 into fecal and
Figure 4–Survival of E. coli O26 in fecal (A), bedding (B), and trough water (C) samples. ····· O26 with eaeA gene, high inoculation level; O26 with eaeA gene, low inoculation level; O26 with eaeA and stx1 genes, high inoculation level; - - - - O26 with eaeA and detected after 24-h enrichment.
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Survival, E. coli O26, beef, environment . . .
Conclusion The understanding of STEC serovars’ persistence in meats and environmental samples has practical implications for food safety; results of this study will provide fundamental information for designing and applying good practices at feedlots and transport trailers, as well as holding pens at slaughter facilities. In this study, no antibiotic resistance was seen from the 4 farm isolates. Although they have different pathogenic gene profiles, the presence of stx did not impact the isolate LWP3’s survival. Regardless of the isolates, O26 survived in beef and environmental samples for 10 and 30 d, respectively. When pathogenic E. coli survives in foods and the environment for an extended amount of time, it can not only become a contamination or cross-contamination source, it can also be a reservoir for keeping, transferring, or acquiring pathogenic genes and antibiotic resistance plasmids, posing higher public health risks. More research is needed to find the mechanisms by which the non-O157 STEC serovars can survive long term in the environment and also more accurate and rapid detection methods are urgently needed for non-O157 STEC serovars.
Author Contributions C. Palmer (M.S. student) conducted the experiments and worked on the manuscript. M. Singh and C. Bratcher helped with the experimental design and data interpretation. L. Wang designed the study, interpreted the data, and worked on the manuscript.
2007b. Growth and survival of non-O157:H7 shiga toxin producing Escherichia coli in cow manure. J Appl Microbiol 102:89–99. Fukushima H, Seki R. 2004. High numbers of Shiga toxin-producing Escherichia coli found in bovine faeces collected at slaughter in Japan. FEMS Microbiol Lett 238:189–97. Grant MA, Hedberg C, Johnson R, Harris J, Logue CM, Meng J, Sofos JN, Dickson JS. 2011. The significance of non-O157 shiga toxin producing Escherichia coli in food. Food Prot Trends 1:33–45. Lim JY, Jang YW, Hovde CJ. 2010. A brief overview of Escherichia coli O157:H7 and its plasmid O157. J Microbiol Biotechnol 20(1):5–14. Ma J, Ibekwe AM, Yi X, Wang H, Yamazaki A, Crowley DE, Yang C. 2011. Persistence of Escherichia coli O157:H7 and its mutants in soils. PLoSone 6(8):e23191. Mathusa EC, Chen Y, Enache E, Hontz L. 2010. Non-O157 Shiga toxin producing Escherichia coli in foods. J Food Prot 73(9):1721–36. Monaghan A, Byrne B, Fanning S, Sweeny T, McDowell D, Bolton DJ. 2011. Serotypes and Virulence Profiles of Non-O157 Shiga Toxin-Producing Escherichia coli Isolates from Bovine Farms. Appl Environ Biology 11(24): 8662-8668. Palmer C. 2014. Survival and prevalence of non-O157 shiga toxin producing Escherichia coli (STEC) in beef cattle preharvest and in the environment [Graduate thesis]. Auburn Univ., Auburn, Ala. Paton AW, Paton JC. 1998. Detection and characterization of Shiga toxigenic Escherichia coli by using multiplex PCR assays for stx1, stx2, eaeA, enterohemorrhagic E. coli hlyA, rfbO111 and rfbO157. J Clin Microbiol 36:598–602. Sharma V. 2002. Detection and quantitation of enterohemorrhagic Escherichia coli O157, O111, and O26 in beef and bovine feces by real-time polymerase chain reaction. J Food Prot 65(9):1371–80. Tarr PI, Neill MA. 1996. Perspective: the problem of non-O157:H7 shiga toxin (verocytotoxin)producing Escherichia coli. J Infect Dis 174:1136–39. USDA. 2011. USDA takes new steps to fight E. coli, protect the food supply. Designation extends zero tolerance policy for E. coli O157:H7 to six additional E. coli serogroups. U.S. Dept. of Agriculture. Available from: http://www.usda.gov/wps/portal/usda/ usdahome?contentidonly=true&contentid=2011/09/0400.xml. Accessed 2013 November 7. Vialette M, Jandos-Rudnik AM, Guyard C, Legeay O, Pinon A, Lange M. 2004. Validating the use of green fluorescent-marked Escherichia coli O157:H7 for assessing the organism behavior in foods. J Appl Microbiol 96:1097–104. Wang G, Zhao T, Doyle MP. 1996. Fate of enterohemorrhagic Escherichia coli O157:H7 in bovine feces. Appl Environ Microbiol 62:2567–70. Zhang W, Bielaszewska M, Liesgang A, Tschape H, Schmidt H, Bitzan M, Karch H. 2000. Molecular characteristics and epidemiology significance of Shiga toxin-producing Escherichia coli O26 strains. J Clin Microbiol 38:2134–40.
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bedding samples, E. coli O26 survived for up to 30 d, indicating References that fecal and bedding materials can be O26 reservoirs and con- Beutin L. 2006. Emerging enterohaemorrhagic Escherichia coli, causes and effects of the rise of a human pathogen. J Vet Med B53:299–305. taminate cattle that do not carry O26. Cells inoculated into the Beutin L, Krause MG, Zimmermann S, Kaulfuss S, Gleier K. 2004. Characterization of Shiga water samples died rapidly as there were not many nutrients availtoxin-producing Escherichia coli strains isolated from human patients in Germany over a three year period. J Clin Microbiol 42:1099–108. able. The finding from this study matches the previous literatures. Centers for Disease Control and Prevention [CDC]. 2013. Standard operating proIn the study conducted by Fremaux and others (2007a), when cedure for PulseNet PFGE of Escherichia coli O157:H7, Escherichia coli nonO157 (STEC), Salmonella serotypes, Shigella sonnei and Shigella flexneri. Available inoculating STEC O26 in cow fecal slurry at a level of 6.5 Log from: http://www.cdc.gov/pulsenet/PDF/ecoli-shigella-salmonella-pfge-protocol-508c.pdf. CFU/mL, O26 could survive in samples for 3 months. Accessed 2014 March 23. CLSI. 2012. Performance standards for antimicrobial susceptibility testing, 22 informational Not much research has been done to look at the impact of supplement. 32(3):44. pathogenic genes on STEC’s survival in food or environmental Cobbold R, Desmarchelier P. 2000. A longitudinal study of Shiga-toxigenic Escherichia coli (STEC) prevalence in three Australian dairy herds. Vet Microbiol 71:125–37. samples. Ma and others, 2011 showed that by knocking out the Ehrmann MA, Scheyhing CH, Vogel RF. 2001. In vitro stability and expression of green stx and/or eae genes from model strain E. coli O157:H7 EDL933, fluorescent protein under high pressure conditions. Lett Appl Microbiol 32:230–34. the survival of knockout strains E. coli EDL933- stx1, stx2, Etcheverria AI, Padola NL. 2013. Shiga toxin-producing Escherichia coli factors involved in virulence and cattle colonization. Virulence 4(5):366–72. stx1-2, and eae strains did not show any difference. Similar Fremaux B, Raynaud S, Beutin L, Vernozy-Rozand C. 2006. Dissemination and persistence of Shiga toxin-producing Escherichia coli (STEC) strains on French dairy farms. Vet Microbiol results were seen in this study. Although the strains used are not 117:180–91. isogenic in this study, the presence of different pathogenic profiles Fremaux B, Prigent-Combaret C, Delignette-Muller ML, Dothal M, Vernozy-Rozand C. 2007a. Persistence of Shiga toxin-producing Escherichia coli O26 in cow slurry. Lett Appl had no impact on the survival of 2 different E. coli O26 inoculums Microbiol 45:55–61. in beef, feces, bedding, and water. Fremaux B, Delignette-Muller ML, Prigent-Combaret C, Gleizzal A, Vernozy-Rozand C.
In addition to Escherichia coli O157:H7, shiga toxin-producing E. coli (STEC) O26 was added to the zerotolerance adulterant list together with other 5 non-O157 STEC serogroups in 2012. Four farm O26 isolates were used in this study; they were obtained from a on-farm survey study conducted in Alabama. The presence of 3 major pathogenic genes (stx1, stx2, and eaeA) was determined through multiplex polymerase chain reaction (PCR). Two major pathogenic gene profiles were observed: 3 of the farm isolates contain only the eaeA gene whereas 1 farm isolate has both the eaeA and the stx1 genes. No significant difference was seen among the 4 farm isolates in the antibiotic resistance tests. To test their survival in ground beef and environmental samples, 2 inoculums were prepared and inoculated at various concentrations into samples of ground beef, bovine feces, bedding materials, and trough water. One inoculum was made of 3 farm isolates containing only the eaeA gene and another inoculum contained the isolate with both the eaeA and stx1 genes. Inoculated beef samples were stored at 4 °C for 10 d and the inoculated environmental samples were stored at ambient temperature for 30 d. Results showed that virulence gene profiles do not have an impact on O26’s ability to survive in ground beef and in environment (P > 0.05). The inoculation levels, sample types as well as the storage times are the major factors that impact O26 survival (P < 0.05). Abstract:
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Keywords: pathogenic genes, Shiga toxin producing E. coli O26, survival
Escherichia coli O26 was the top one non-O157 shiga toxin-producing E. coli isolated in a cow/calf operation survey study conducted in Alabama. The capability of the 4 fecal O26 isolates being able to survive in food and environmental samples for an extended period of time indicated that good agriculture practices for small cow/calf operations are required and of great importance to prevent on-farm pathogenic O26 transfer.
Practical Application:
Introduction
serogroup has been frequently found in cattle with prevalence rates of 5.1% in Japan (Fukushima and Seki 2004) and 1.7% in Australia (Cobbold and Desmarchlier 2000). In France, a study showed that 15% of the dairy farms tested were positive for STEC O26 (Fremaux and others 2006). In a recent study conducted in the United States, 4 E. coli O26 strains were isolated from 3 of 7 (42.8%) cow/calf farms in Alabama (Palmer 2014). Several virulence factors are involved in the pathogenicity of non-O157 STEC. The presence of shiga toxin 1 (stx1), shiga toxin 2 (stx2), or a combination of these genes is the most important indicator of a bacterium’s virulence (Etcheverria and Padola 2013). Another important virulence factor of STEC is eae, the intimin gene, which is used by the bacteria to attach to epithelial cells. It works by inducing an attaching and effacing (A/E) lesion on the surface of the target cell (Etcheverria and Padola 2013). Even though the presence of virulence genes such as eae is a good indicator of pathogenicity, its absence does not necessarily mean it is not pathogenic (Beutin 2006). The presence of intimin can sometimes be associated with the more severe cases (Beutin and others 2004). Virulence profiles of STEC O26 have changed over the years. Zhang and others (2000) showed that all O26 isolates collected between 1965 and 1994 expressed only stx1 whereas most O26 isolates collected between 1995 and 1999 had either MS 20141539 Submitted 9/15/2014, Accepted 2/1/2015. Authors Palmer, Bratcher and Wang are with Dept. of Animal Sciences, Auburn Univ., Auburn, AL, stx2 alone or a combination of stx1 and stx2. A better understanding of the 4 O26 isolates is still needed. U.S.A. Authors Singh is with Dept. of Food Sciences, Purdue Univ., West Lafayette, IA, U.S.A. Direct inquiries to author Wang (E-mail: [email protected]). In addition to their pathogenic gene compositions and antibiotic resistance profiles, information on their survival capability in foods
Shiga toxin-producing Escherichia coli (STEC) strains are foodborne infectious agents that have caused a number of outbreaks. In past decades, studies on STEC have focused on E. coli O157:H7 due to its earlier discovery in human illness (Monaghan and others 2011) and its involvement in the 1982 Michigan and Oregon outbreaks (Lim and others 2010). In recent years, more reports about non-O157 STEC have attracted public attentions. Grant and others (2011) reported that 23 outbreaks of non-O157 STEC illnesses happened between 1990 and 2007 in the United States. Among non-O157 STEC serovars, E. coli O26, O111, O103, O121, O45, and O145 are now placed on the zero-tolerance adulterant list as of June 2012 (USDA 2011). Of the 6 non-O157 STEC strains, O26 has been more prominent and the focus of many studies due to its notoriety for the virulence factors that can make people sick (Mathusa and others 2010). It has been regarded as an important cause of STEC-associated diseases (Tarr and Neill 1996). It is estimated that O26 and O111 are responsible for 25% of all serious cases of infection with the risk of kidney failure and death (Sharma 2002). O26 has the history of being isolated from cattle farms. The O26
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Survival, E. coli O26, beef, environment . . .
Materials and Methods Bacterial strains Four O26 farm isolates (LWP1, LWP2, LWP3, and LWP4) and 1 clinical strain (TWO8031) were used in this study. TWO8031 was obtained from the Thomas S. Whittam STEC Center at Michigan State Univ. and was originally isolated in 1999 from a human host living in the United States. The TWO8031 strain was used as the reference strain. Characterization of farm isolates Antibiotic susceptibility/sensitivity, finger printing (pulse field gel electrophoresis patterns), and the pathogenic profiles of 4 farm isolates were determined. The antibiotic susceptibility/sensitivity of these strains was determined using a disk diffusion method outlined by the Clinical and Laboratory Standards Inst. (CLSI 2012). The diameters of the inhibition zones were measured. Antibiotic disks were purchased from Becton Dickinson, U.S.A. The susceptibility of all isolates to a total of 11 antibiotics was examined. The pulse field gel electrophoresis (PFGE) protocol followed the CDC standard operating procedure for PulseNet PFGE of Escherichia coli Non-O157 STEC (CDC 2013). One restriction enzyme (XbaI; Promega, U.S.A.) was used. Salmonella ser. Braenderup H9812 (ATCC, BAA-664TM ) was used as the molecular weight standard. A multiplex PCR assay targeting 3 different virulence genes was applied to determine the virulence profiles of O26 isolates. Target virulence genes included the eaeA, stx1, and stx2. Primers were designed by Paton and Paton (1998) and were synthesized by Integrated DNA Technologies (IDT, Coralville, IA, R U.S.A.). DNA was extracted by using PrepMan ultra sample preparation reagent following the manufacturer’s manual (Life TechnologiesTM , Grand Island, NY, U.S.A.). The working solution for each primer was 10 μM. AccuStartTM II PCR Supermix (2×; Quanta BioSciences, San Jose, CA, U.S.A.) was used for the multiplex PCR assay. A 1:10 dilution of the DNA samples with ultrapure water was used as the PCR template. In each 0.1 mL PCR tube, the following reagents were mixed together: 5 μL of the template, 0.25 μL of each forward and reverse primer, 12.5 μL of AccuStart II PCR Supermix (2×), and 6 μL of nuclease-free water, giving a total volume of 25 μL per PCR tube. The PCR R R reaction was carried out in an Applied Biosystems Veriti 96-Well Fast Thermal Cycler (Life TechnologiesTM , Grand Island, NY, U.S.A.), with an initial 3 min 94 °C denaturation, and then 30 cycles of: 30 s 94 °C denaturation, 30 s 58 °C annealing, and 1 min 72 °C. PCR products were then held at 4 °C and examined by running the products in a 2% agarose gel for 40 min. Inoculum preparation In preparation of the inoculum, E. coli O26 strains were transformed with the pGLOTM plasmid containing the green fluorescence protein gene (GFP; Bio-Rad Laboratories, Hercules, CA, U.S.A.). The protocols of making competent cells and transferring plasmid followed the “Molecular cloning—a laboratory manual,” 3rd edition. The transformed strains were picked from the LB plates supplemented with 0.1 mg/mL ampicillin (Sigma-Aldrich, St. Louis, MO, U.S.A.) and 50 mg/mL arabinose (Alfa Aesar, Ward Hill, MA, U.S.A.) and their growth curves were proved to be
similar to the parent strains before use (P > 0.05). Two sets of culture inoculums were prepared: one was composed of 3 farm isolates with the pathogenic gene profile of eaeA gene and another one contained one farm isolate that have both the eaeA and the stx1 genes. Overnight bacterial cultures were prepared by transferring 100 μL of frozen cultures into 10 mL Luria-Bertani (LB) broth supplemented with ampicillin and arabinose and incubating at 37 °C. The concentrations of overnight cultures were determined by plating their dilutions onto LB plates supplemented with ampicillin and arabinose and counted the next day. To prepare the inoculum, cultures were washed by centrifugation (Eppendorf, Hauppauge, NY, U.S.A.) at 4000 × g for 5 min and resuspended in MilliQ water and were then mixed. For lower inoculation levels, stock inoculums were diluted in MilliQ water before being inoculated into beef and environmental samples.
Ground beef and environmental sample inoculation Ground beef was purchased from a local store (85% lean and 15% fat). Before inoculation, ground beef was confirmed to be STECfree by enriching and plating the samples on CHROMagarTM STEC plates (CHROMagar, Paris, France). Suspect colonies were further tested using multiplex PCR to determine if they contain stx or eae genes. Ground beef was used only if the results from both the CHROMagar STEC plating and the PCR results were negative. To prepare each inoculated beef sample, 25 g of ground R beef were weighed and put into filtered Whirl-pak bags (Nasco, Albuquerque, NM, U.S.A.) together with 1 mL of the chosen E. coli inoculum. Three final inoculation levels were reached, low (102 log CFU/g), medium (106 log CFU/g), and high (108 log CFU/g). Inoculated samples were stored in the refrigerator (4 °C) for 10 d. Composite fecal samples, bedding materials, and trough water were used in this study and were retrieved from E.V. Smith Research Center (Auburn Univ., Auburn, Ala., U.S.A.). Each composite sample was made with 3 individual subsamples taken from 3 different sites. To prepare each inoculated environmental sample, 25 g of fecal or bedding samples were measured into Whirl-pak bags (Nasco) and inoculated with 1 mL of the inoculum. To prepare each inoculated water sample, 100 mL of trough water was measured into a 125-mL sterile sample cup (VWR, Suwanee, GA, U.S.A.) and then inoculated with 1 mL of each inoculum. Two target inoculation levels were tested for environmental samples (106 and 104 log CFU/g or /mL). All inoculated environmental samples were kept at ambient temperature for 30 d. Enumeration of survived E. coli O26 Three replicate trials were conducted for every inoculation study. For each inoculated beef trial, 3 inoculated ground beef samples of every inoculation level were removed from the 4 °C refrigerator every day for 10 d. One hundred milliliter of 0.1% buffered peptone water was added to each bag. Samples were homogenized for 2 min using the SmasherTM lab blender (AES Chemunex, Marcy l’Etoile, U.S.A.). Bacteria were plated by spreading 100 μL of the smashed contents or dilutions on LB plates supplemented with 0.1 mg/mL ampicillin and 50 mg/mL arabinose. Plates were incubated at 37 °C and counted after 24 h. For each inoculated environmental sample trial, subsamples were taken every 5 d and the survived O26 cells were enumerated as described above. For samples in which the levels of O26 were below the limit of detection, sample suspensions were enriched by adding an additional 125 mL of 2× lactose broth and enriched for 24 h before streaking the enriched suspensions onto LB plates supplemented with Vol. 80, Nr. 4, 2015 r Journal of Food Science M783
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and environment will benefit the future development of efficient intervention and good agricultural practices. Thus, the objectives of this study were to characterize the 4 O26 isolates and evaluate the survival of STEC O26 in artificially inoculated ground beef and environmental samples.
Survival, E. coli O26, beef, environment . . . Table 1–Antibiotic resistance comparison between 4 farm STEC O26 isolates and one clinical isolate.
Antibiotics Amoxicillin-clavulanic acid Ampicillin Ceftriaxone Cephalothin Chloramphenicol Ciprofloxacin Cefoxitin Gentamicin Naladixic acid Sulfamethoxazole/trimethoprim Tetracylcine
Concentrations
Susceptible breakpoints (mma )
20 μg 10 μg 30 μg 30 μg 30 μg 5 μg 30 μg 10 μg 30 μg 23.75/1.25 μg 30 μg
18 17 23 18 18 21 18 15 19 16 15
Isolated from farm (mma ) LWP1 22.50 21.50 32.76 18.26 24.76 32.00 24.00 21.50 25.76 28.26 22.26
± ± ± ± ± ± ± ± ± ± ±
0.65 1.44 0.75 0.48 0.25 0.71 0.41 0.29 0.25 0.25 0.48
LWP2 21.26 20.00 32.26 17.50 23.00 32.26 24.76 21.50 25.00 29.00 22.00
± ± ± ± ± ± ± ± ± ± ±
0.85 0.58 0.25 0.29b 0.58 0.95 0.48 0.29 0.71 0.71 0.41
LWP3 20.76 20.50 30.00 18.76 22.00 33.76 25.50 21.00 24.50 27.52 21.00
± ± ± ± ± ± ± ± ± ± ±
0.75 1.19 0.58 0.25 0.58 1.03 1.44 0.00 0.29 0.87 0.41
LWP4 21.46 20.76 34.26 19.50 24.76 36.76 25.76 22.76 27.00 27.26 22.26
± ± ± ± ± ± ± ± ± ± ±
0.75 0.63 0.48 0.29 0.25 1.93 0.48 0.25 0.00 0.48 0.25
a Diameter b
of the inhibition zone. The intermediate zone diameter for Cephalothin is 15 to 17 mm.
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ampicillin and arabinose. All bacterial media were purchased from PCR reactions targeting stx1, stx2, and eaeA genes was conducted. As shown in Figure 1A, 3 of the 4 farm isolates (LWP1, LWP2, and Becton Dickinson unless otherwise stated. LWP4) contain only the eaeA gene and one farm isolate (LWP3) Statistical analysis had both the stx1 and eaeA genes. Strains were also compared Three replicates were done for the survival studies. The data using PFGE and enzyme XbaI was used. As shown in Figure 1B, were analyzed using a general linear model (GLM) procedure all PFGE patterns were different among all 4 isolates used. LWP1 in SAS 9.2 (SAS Inst. Inc., Cary, NC, U.S.A.) The effects of and 2 had the highest similarity whereas strain LWP3 and the 2 pathogenic gene profiles, different inoculation levels, materials clinical reference strain are more closely related compared to the (beef, bedding, feces, and water), and time (d) on O26 survival other 3. were evaluated. P values of less than 0.05 were considered to be significant.
Results and Discussion Characterization of O26 All 4 farm isolates showed susceptibility to the antibiotics tested (except for LWP2 on Cephalothin) and no antibiotic resistance was seen (Table 1). The breakpoint for cephalothin susceptibility is 18 mm. The diameter of the cephalothin inhibition zone on LWP2 was 17.5 ± 0.29, which is higher than the intermediate range of Cephalothin (15–17 mm) but a little lower than the susceptible breakpoint. DNA of all O26 strains was extracted and multiplex
Survival of E. coli O26 in Ground Beef E. coli O26 strains were transformed with GFP plasmids before use. As shown in Figure 2, transformed E. coli O26 cells showed green fluorescence under the UV light after 24 h incubation and could be easily differentiated from the background microflora and counted. The initial inoculation levels of ground beef were 7.54 ± 0.28 Log CFU/g, 5.53 ± 0.29 Log CFU/g, and 1.60 ± 0.24 Log CFU/g (high, medium, and low). As shown in Figure 3, E. coli O26 survived and persisted well in ground beef during the 10-day
Figure 1–(A) Multiplex PCR results of 4 farm O26 isolates and one clinical O26 strain; and (B) pulse field gel electrophoresis (PFGE) dendrogram of 4 farm isolates and one clinical strain.
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storage time, regardless of the inoculation levels. No significant farm water samples (Figure 4C), steep declines were seen over the 30-d trial period for all samples. After day 15, survived E. coli O26 difference was seen when comparing 2 inoculums (P > 0.05). (with the eaeA and stx1 genes) in water with an initial inoculation Survival of E. coli O26 in environmental samples level of 4.47 Log CFU/mL were below the limit of detection 0.6 The final inoculation levels for fecal samples were 5.05 ± 0.11 Log CFU/mL. With 24 h enrichment, O26 cells were able to and 3.25 ± 0.01 Log CFU/g. The survival of E. coli O26 in be confirmed by streaking the enriched broth on selective agar. fecal samples is shown in Figure 4A. An approximately 1 log Similar with the fecal and bedding material data, no significant increase was seen after 5 d from samples with the initial inoculation difference was seen between the 2 inoculums (P > 0.05). levels of 5.05 ± 0.11 Log CFU/g, regardless of the pathogenic As shown by statistical analysis, storage times, types of materigene profiles. A similar increase was seen for the fecal samples als, and the initial inoculation levels are the 3 major factors that inoculated with the inoculum containing only the eaeA gene at impacted the survival of E. coli O26 in this study (P < 0.05). The a level of an approximately 3.25 ± 0.01 Log CFU/g. Up to a 2 inoculums showed no significant difference in survival at each 2-Log reduction was seen for samples inoculated with 3.25 Log time point (P > 0.05); different pathogenic gene profiles had no CFU/g of E. coli O26, containing both the eaeA and stx genes impact on the survival capability of E. coli O26. after 10 d. One reason for the increases in O26 numbers may be The objectives of this study were to better characterize 4 farm due to the good nutrient availability in feces not available in the isolates obtained in a previous STEC prevalence study and to other environmental samples. Time played a significant role in E. find out the potential impact different pathogenic gene profiles coli O26’s survival (P < 0.05). However, no difference was seen can generate on cells’ survival in both meats and environmental between 2 inoculums at every time point (P > 0.05). samples. Among 4 farm isolates, only one of them contained both The initial inoculation levels for bedding materials were 5.19 ± eaeA and stx1 genes whereas the other 3 have only the eaeA gene, 0.04 and 3.29 ± 0.06 Log CFU/g. Results for bedding materials a gene used by the bacteria to attach to epithelial cells. As shown are shown in Figure 4B. Bacteria counts declined steadily over the by the PFGE results, the 3 farm isolates containing only the eaeA entire 30-d period. After 15 days, E. coli O26 counts with initial gene were grouped closely together whereas the farm isolate and inoculation levels of 3.29 ± 0.06 Log CFU/g were below the limit the clinical reference strain containing both eaeA and stx1 gene of detection (0.6 Log CFU/g). An approximately 2 Log reduction (LWP3 and TWO8031) were grouped together. None of the 4 was seen for samples inoculated with 5.19 ± 0.04 Log CFU/g. farm isolates exhibited any antibiotic resistance. No significant difference was seen between the 2 inoculums Fecal, bedding, and trough water sample can be vehicles by (P > 0.05). which zoonotic agents may enter and become disseminated The initial inoculation concentrations for trough water were throughout the agricultural environment. STEC serogroup O26 6.51 ± 0.08 Log CFU/mL and 4.47 ± 0.07 Log CFU/mL. For the has been regarded as an important cause of STEC-associated Figure 2–GFP transformed E. coli O26 isolates with background microflora plated on LB agar supplemented with ampicillin and arabinose. Colonies seen on LB plate under the regular light (left); GFP transformed colonies gave green fluorescence under the UV light (right).
Figure 3–Survival of O26 in ground beef stored at 4 °C. E. coli O26 with eaeA gene, high E. coli O26 with eaeA inoculation level; gene, medium inoculation level; E. coli O26 with eaeA gene, low inoculation level; ······ E. coli O26 with eaeA and stx1 genes, high inoculation level; —- E. coli O26 with eaeA and stx1 genes, medium inoculation level; –·–· E. coli O26 with eaeA and stx1 genes, low inoculation level.
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Survival, E. coli O26, beef, environment . . .
Survival, E. coli O26, beef, environment . . . diseases (Tarr and Neill 1996). E. coli O157:H7 can survive in bovine feces for long periods and can retain the potential to produce shiga toxin (Wang and others 1996). Unfortunately, the fate of O26, especially the impact of different pathogenic profiles, remains unclear. Unlike O157, STEC strains do not have common biochemical characteristics allowing the use of specific media for their isolation; in addition, the potential presence of pathogenic E. coli O26 in environmental samples could influence the enumeration of the survived inoculum. Thus, to better differentiate the inoculum and the background microflora, all O26 strains used this study were transformed with GFP plasmid. As discussed by Ehrmann and others, Vialette and others, and Fremaux and others (Ehrmann and others 2001; Vialette and others 2004; Fremaux
and others 2007b), the stability of GFP-labeled O26 has been confirmed. In addition, Fremaux and others (2007a) showed that no significant difference of behavior was observed between the transformed STEC O26 strains and the parental strains after their inoculation into cow slurry. In this study, the growth curves of GFP transformed O26 strains were compared with their parental strains and no significant difference was seen (data not shown). As shown in this study, regardless of inoculation levels, E. coli O26 survived in ground beef for 10 d without a significant decrease at the refrigerated temperature. These data indicate that intervention strategies applied during meat processing are of great importance to ensure the zero-tolerance regulation of non-O157 STEC in ground beef. When inoculating O26 into fecal and
Figure 4–Survival of E. coli O26 in fecal (A), bedding (B), and trough water (C) samples. ····· O26 with eaeA gene, high inoculation level; O26 with eaeA gene, low inoculation level; O26 with eaeA and stx1 genes, high inoculation level; - - - - O26 with eaeA and detected after 24-h enrichment.
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Survival, E. coli O26, beef, environment . . .
Conclusion The understanding of STEC serovars’ persistence in meats and environmental samples has practical implications for food safety; results of this study will provide fundamental information for designing and applying good practices at feedlots and transport trailers, as well as holding pens at slaughter facilities. In this study, no antibiotic resistance was seen from the 4 farm isolates. Although they have different pathogenic gene profiles, the presence of stx did not impact the isolate LWP3’s survival. Regardless of the isolates, O26 survived in beef and environmental samples for 10 and 30 d, respectively. When pathogenic E. coli survives in foods and the environment for an extended amount of time, it can not only become a contamination or cross-contamination source, it can also be a reservoir for keeping, transferring, or acquiring pathogenic genes and antibiotic resistance plasmids, posing higher public health risks. More research is needed to find the mechanisms by which the non-O157 STEC serovars can survive long term in the environment and also more accurate and rapid detection methods are urgently needed for non-O157 STEC serovars.
Author Contributions C. Palmer (M.S. student) conducted the experiments and worked on the manuscript. M. Singh and C. Bratcher helped with the experimental design and data interpretation. L. Wang designed the study, interpreted the data, and worked on the manuscript.
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bedding samples, E. coli O26 survived for up to 30 d, indicating References that fecal and bedding materials can be O26 reservoirs and con- Beutin L. 2006. Emerging enterohaemorrhagic Escherichia coli, causes and effects of the rise of a human pathogen. J Vet Med B53:299–305. taminate cattle that do not carry O26. Cells inoculated into the Beutin L, Krause MG, Zimmermann S, Kaulfuss S, Gleier K. 2004. Characterization of Shiga water samples died rapidly as there were not many nutrients availtoxin-producing Escherichia coli strains isolated from human patients in Germany over a three year period. J Clin Microbiol 42:1099–108. able. The finding from this study matches the previous literatures. Centers for Disease Control and Prevention [CDC]. 2013. Standard operating proIn the study conducted by Fremaux and others (2007a), when cedure for PulseNet PFGE of Escherichia coli O157:H7, Escherichia coli nonO157 (STEC), Salmonella serotypes, Shigella sonnei and Shigella flexneri. Available inoculating STEC O26 in cow fecal slurry at a level of 6.5 Log from: http://www.cdc.gov/pulsenet/PDF/ecoli-shigella-salmonella-pfge-protocol-508c.pdf. CFU/mL, O26 could survive in samples for 3 months. Accessed 2014 March 23. CLSI. 2012. Performance standards for antimicrobial susceptibility testing, 22 informational Not much research has been done to look at the impact of supplement. 32(3):44. pathogenic genes on STEC’s survival in food or environmental Cobbold R, Desmarchelier P. 2000. A longitudinal study of Shiga-toxigenic Escherichia coli (STEC) prevalence in three Australian dairy herds. Vet Microbiol 71:125–37. samples. Ma and others, 2011 showed that by knocking out the Ehrmann MA, Scheyhing CH, Vogel RF. 2001. In vitro stability and expression of green stx and/or eae genes from model strain E. coli O157:H7 EDL933, fluorescent protein under high pressure conditions. Lett Appl Microbiol 32:230–34. the survival of knockout strains E. coli EDL933- stx1, stx2, Etcheverria AI, Padola NL. 2013. Shiga toxin-producing Escherichia coli factors involved in virulence and cattle colonization. Virulence 4(5):366–72. stx1-2, and eae strains did not show any difference. Similar Fremaux B, Raynaud S, Beutin L, Vernozy-Rozand C. 2006. Dissemination and persistence of Shiga toxin-producing Escherichia coli (STEC) strains on French dairy farms. Vet Microbiol results were seen in this study. Although the strains used are not 117:180–91. isogenic in this study, the presence of different pathogenic profiles Fremaux B, Prigent-Combaret C, Delignette-Muller ML, Dothal M, Vernozy-Rozand C. 2007a. Persistence of Shiga toxin-producing Escherichia coli O26 in cow slurry. Lett Appl had no impact on the survival of 2 different E. coli O26 inoculums Microbiol 45:55–61. in beef, feces, bedding, and water. Fremaux B, Delignette-Muller ML, Prigent-Combaret C, Gleizzal A, Vernozy-Rozand C.
