J. Appl. Poult. Res.
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J APPL POULT RES 2008. 17:237-242. doi:10.3382/japr.2007-00067
© 2008 Poultry Science Association
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Research Reports

Recovery and Genetic Diversity of Escherichia coli Isolates from Deep Litter, Shallow Litter, and Surgical Shoe Covers

B. A. McCrea*,1, K. S. Macklin{dagger}, R. A. Norton{dagger}, J. B. Hess{dagger} and S. F. Bilgili{dagger}

* Department of Animal Science, University of California-Davis, Davis 95616; and {dagger} Department of Poultry Science, Auburn University, Auburn, AL 36849-5416

1 Corresponding author: bmccrea{at}desu.edu


   SUMMARY
TOP
 SUMMARY
DESCRIPTION OF PROBLEM
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS AND APPLICATIONS
 REFERENCES AND NOTES
 
Litter is a common source of infectious agents in poultry production environments. Typical sampling methods only examine bacteria on the surface; however, bacteria recovered with these methods may not be representative of the population in the litter. To test this hypothesis, both shallow (top 2 in.) and deep (bottom 2 in.) litter samples were taken and compared with a surface sampling method (i.e., surgical shoe covers). Escherichia coli isolates were recovered from all 3 sample types and examined using an automated ribotyping system to determine the genetic diversity of the isolates. Twenty-six unique E. coli strains were recovered from the 3 different sampling methods. There was no correlation among strains between visit age, house, flock, or farm and ribogroups. Based upon the patterns of E. coli recovery among the different sample types, our results suggest that surface sampling methods are equally capable of recovering common isolates from the litter. Surgical shoe covers were easy to use, provided the same core population of isolates, and were comparable to shallow litter in the number of strains recovered.

Key Words: Escherichia coli • litter • broiler • surgical shoe cover • automated ribotyping


   DESCRIPTION OF PROBLEM
TOP
 SUMMARY
 DESCRIPTION OF PROBLEM
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS AND APPLICATIONS
 REFERENCES AND NOTES
 
Samples of the broiler house environment, or the birds, may be used to determine the pathogen positive or negative status of a flock. This information can aid processing plant scheduling decisions. As an indicator of fecal contamination, Escherichia coli is scrutinized in the processing plant and is considered a commensal organism in poultry litter. Poultry litter E. coli levels as high as 108 cfu/mL have been reported [1]. Litter samples are easy to obtain. Grab samples can be replaced by drag swabs as an effective method by which to sample broiler flocks for salmonellae [2, 3]. Researchers have also investigated shoe covers as a simpler litter sampling method in broiler houses [4, 5] with better recovery of Salmonella isolates [6]. Both of these methods allow for greater surface areas to be sampled compared with grab samples that are limited by volume and location.

The objective of our study was to test the hypothesis that surface sampling may not allow a true bacterial assessment of the litter composition. We compared litter samples from different depths with surgical shoe cover samples obtained simultaneously. Isolate differences were assessed and compared through the use of automated ribotyping.


   MATERIALS AND METHODS
TOP
 SUMMARY
 DESCRIPTION OF PROBLEM
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 CONCLUSIONS AND APPLICATIONS
 REFERENCES AND NOTES
 
Experimental Design
Two totally cleaned and disinfected commercial broiler farms were utilized for samples in the study, each with 4 houses of comparable age, style (dark-out, drop ceiling, and tunnel-ventilated), and size (42 x 500 ft). Total cleanout involved the removal of all old litter, cleaning and disinfection of premises with a nonchlorinated (phosphoric acid and dodecylbenzenesul-fonic acid) cleaner, cleaning of water and feed lines, and application of new litter. Sampling took place during the third and fourth consecutive flocks after the annual total cleanout, between January and May 2004. Clean peanut hulls were used as the bedding material on both farms. Between each successive flock, caked litter was removed, and a top dressing of fresh litter was added in the brooding area. Each farm was sampled 3 times during 2 consecutive flocks. The first visit was performed on chick delivery day, before chick placement. The second visit was midway through growout (~3.5 wk), and the final visit was on the day of processing.

Sampling Procedure
Litter grab samples were collected from 2 random locations within each quadrant of the house and then pooled into a sterile plastic bag. Shallow litter samples consisted of grab samples from the top 2 in. of litter, whereas deep litter consisted of the bottom 2 in. of litter. To prevent cross-contamination, latex gloves were changed between deep and shallow litter samples. Samples were placed in an ice chest with ice packs during transport to the laboratory.

Surgical shoe covers [7] were placed aseptically over disposable plastic shoe covers and only worn inside the house. One pair of surgical shoe covers was worn for each house. After sampling, the surgical shoe covers were placed into a sterile Whirl-Pak containing 250 mL of sterile PBS. Samples were placed in an ice chest during transport to the laboratory.

Plating and Identification
Litter samples were mixed thoroughly and diluted 1:10, based upon a 20-g subsample in a sterile stomacher bag [8], using buffered peptone water. Samples were then massaged for 1 min. A sterile, cotton-tipped swab was placed in the buffered peptone water containing the sample and streaked onto a MacConkey Agar plate. After streaking for isolation, plates were incubated at 37°C for 18 to 24 h. Presumptive positive isolates were based on colony color (lactose-positive fermentation) and morphology [9]. Surgical shoe cover samples were stomached for 1 min, and all isolation procedures were identical to those of litter samples.

Cultures producing typical reactions for E. coli were restreaked onto brain-heart infusion agar and incubated at 37°C for 24 h in preparation for automated ribotyping. Cultures of E. coli grown on brain-heart infusion were examined to ensure purity. A small group of cells was selected from the plate for automated ribotyping [10]. The RiboPrinter Microbial Characterization System [11] was used to identify the bacterial genus, species, and subspecies through the analysis of genomic fragments of ribosomal RNA operons [12–14].


   RESULTS AND DISCUSSION
TOP
 SUMMARY
 DESCRIPTION OF PROBLEM
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
CONCLUSIONS AND APPLICATIONS
 REFERENCES AND NOTES
 
The majority, 95.8% (n = 46), of surgical shoe cover samples (n = 48) yielded E. coli suspects. Shallow and deep litter samples (n = 48 each) had a slightly lower recovery of E. coli suspects, yielding 83.3% (n = 40) and 85.4% (n = 41), respectively. Each visit to farms yielded E. coli for all 3 sampling methods, although not all houses on each visit produced suspect colonies. Of the 127 suspect colonies, 65.4% (n = 84) were confirmed as E. coli using the RiboPrinter. Surgical shoe covers, shallow litter, and deep litter samples yielded 34.5% (n = 29), 31.0% (n = 26), and 34.5% (n = 29) RiboPrinter-confirmed E. coli isolates, respectively. Additionally, 1.6% of the E. coli suspects were identified as other bacteria (n = 2). Unfortunately, 32.3% (n = 41) of the E. coli suspects were not identified using the RiboPrinter and were considered unknowns. The status of unknown was conferred upon an isolate if the riboprint pattern was not in the RiboPrinter database. Further testing of unknown isolates to determine E. coli status was not performed.

A total of 26 different strains of E. coli were revealed with 5 strains representing 62% of the confirmed isolates. Deep litter samples yielded the greatest diversity in the number of isolates recovered with 15 strains. Shallow litter and surgical shoe cover samples each recovered 13 strains. The majority of strains, 81% (n = 21), yielded less than 5 isolates. Five core isolates (19%) maintained a high incidence of recovery among all 3 methods, and strain number 14147 was found with the greatest frequency (n = 14). This same strain was found frequently (93%) in surgical shoe covers and deep litter samples, and only 1 isolate was located in shallow litter. Strain 14176 was fairly evenly distributed among the 3 sample types. In contrast, shallow litter samples yielded strains 14217 and 14194 more frequently than other samples, whereas deep litter samples yielded 50% of the isolates for strain 18663 (Table 1Go). Overall, among the 5 most frequently recovered isolates, surgical shoe covers, shallow litter, and deep litter samples yielded these isolates in 34.5, 31, and 34.5%, respectively, of their total number of isolates. All strains yielding multiple isolates, with the exception of 1 strain, were found on both farms.


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  		Table 1. Distribution of the 5 most frequently isolated strains of Escherichia coli from 2 broiler houses using 3 sampling methods
 
When all isolates were compared, grouping patterns within the dendrogram were randomly distributed between house, farm, age of visit, and flock. Embedded within the dendrogram were 2 tight clusters of strains. Strain 14176 was 90% homogeneous when compared with isolates from all samples (data not shown). The 5 most frequently isolated strains were 71% homogeneous. Based upon an 80% similarity level, cluster analysis of the surgical shoe cover, shallow litter, and deep litter samples yielded 7, 6, and 7 groups, respectively. Within the surgical shoe cover samples, there was 52.4% homogeneity between all isolates recovered (Figure 1Go). Within shallow litter isolates, there was 32.3% homogeneity between isolates (Figure 2Go), and deep litter isolates had the lowest homogeneity with 11.8% between isolates (Figure 3Go).


Figure 1
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  		Figure 1. Ribotypes of 29 Escherichia coli strains from surgical shoe cover samples. The dendrograms are created using a numerical analysis of 16S rRNA by EcoRI. Clusters show the patterns of all types obtained. VCA = RiboPrinter internal designation for an EcoR1 batch.

 

Figure 2
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  		Figure 2. Ribotypes of 26 Escherichia coli strains from shallow litter samples. The dendrograms are created using a numerical analysis of 16S rRNA by EcoRI. Clusters show the patterns of all types obtained. VCA = RiboPrinter internal designation for an EcoR1 batch.

 

Figure 3
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  		Figure 3. Ribotypes of 29 Escherichia coli strains from deep litter samples. The dendrograms are created using a numerical analysis of 16S rRNA by EcoRI. Clusters show the patterns of all types obtained. VCA = RiboPrinter internal designation for an EcoR1 batch.

 
Overall, when all 3 sample types were compared, the tightly clustered strain 14176 fell within its own ribogroup, indicating a clonal population. Interestingly, this strain was found in different houses, at different times on both farms, perhaps originating from a single source beyond the farm such as the breeder population, hatchery, or feed. Common sources of E. coli on the farm can include water, dust, vectors, and poorly maintained litter or ventilation systems [15]. Research by Singer et al. [16] reported that certain E. coli strains can become endemic to the farm environment and infect successive flocks. With the exception of strain 14176, all other strains were randomly distributed, indicating individual development rather than clonal spread. Based on these results, we suggest that automated ribotyping is capable of identifying patterns among samples potentially emanating from a common source [17].

Based upon the patterns of E. coli recovery among the different sample types, we suggest that any of the methods are capable of recovering the most common isolates. Multiple isolates were present for 2 consecutive flocks and were detectable at multiple sampling ages. Similar results in a study of Clostridium perfringens on broiler farms were found regarding multiple isolate ribotypes recovered from several farms and time periods [18]. Deep litter samples yielded 2 more strains than surgical shoe covers or shallow litter samples, but these strains were not among the most frequently isolated. Surgical shoe covers were easy to use, provided the same core population of isolates, and were comparable to shallow litter in the number of strains recovered.

Litter sampling remains an excellent method by which to monitor flocks [3], including the antimicrobial-resistant E. coli. Automated ribo-typing is capable of discriminating between fluoroquinolone-resistant E. coli [19]. Although automated ribotyping of litter isolates is valuable, there is very little similarity between pathogenic E. coli from lesions and E. coli from the litter [20]. The lack of variation among our isolates may be due to broilers sufficiently mixing the litter to allow for even distribution. Therefore, we suggest that surface sampling techniques accurately represent E. coli litter flora.

Accurate representations of the litter mi-croflora can be obtained by using surgical shoe covers. The increased genetic diversity of the deep litter isolates could be influenced by the harsher, possibly anaerobic, environment that may or may not contain the same amount of nutrients for the bacteria. The deviation between homogeneities in the surgical shoe cover and shallow litter isolates, given that they each recovered 13 strains, can be interpreted to mean that the genetic diversity of E. coli recovered from litter remains unpredictable.


   CONCLUSIONS AND APPLICATIONS
TOP
 SUMMARY
 DESCRIPTION OF PROBLEM
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS AND APPLICATIONS
REFERENCES AND NOTES
 

  1. The use of surgical shoe covers for litter microflora monitoring of broiler houses provides equivalent results to litter grab samples with regard to E. coli recovery.
  2. Surgical shoe covers are less labor intensive to prepare and are easy to apply in the field.


   REFERENCES AND NOTES
TOP
 SUMMARY
 DESCRIPTION OF PROBLEM
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS AND APPLICATIONS
 REFERENCES AND NOTES
 

  1. Macklin, K. S., J. B. Hess, S. F. Bilgili, and R. A. Norton. 2005. Bacterial levels of pine shavings and sand used as poultry litter. J. Appl. Poult. Res. 14:238–245.[Abstract/Free Full Text]
  2. Kingston, D. J. 1981. A comparison of culturing drag swabs and litter for identification of infections with Salmonella spp. in commercial chicken flocks. Avian Dis. 25:513–516.[CrossRef][Web of Science][Medline]
  3. Mallinson, E. T., C. R. Tate, R. G. Miller, B. Bennett, and E. Russek-Cohen. 1989. Monitoring poultry farms for Salmonella by drag-swab sampling and antigen-capture im-munoassay. Avian Dis. 33:684–690.[CrossRef][Web of Science][Medline]
  4. Caldwell, D. J., B. M. Hargis, D. E. Corrier, and J. R. DeLoach. 1998. Frequency of isolation of Salmonella from protective foot covers worn in broiler houses as compared to drag-swab sampling. Avian Dis. 42:381–384.[CrossRef][Web of Science][Medline]
  5. McCrea, B. A., S. F. Bilgili, R. A. Norton, K. S. Macklin, and J. B. Hess. 2004. Recovery of E. coli and Salmonella suspects from poultry house drag swabs and surgical shoe covers. Page 38 in Proceedings of the XXV Annual Southern Poultry Science Conference, Atlanta, GA.
  6. McCrea, B. A., R. A. Norton, K. S. Macklin, J. B. Hess, and S. F. Bilgili. 2005. Recovery and genetic similarity of Salmonella from broiler house drag swabs versus surgical shoe covers. J. Appl. Poult. Res. 14:694–699.[Abstract/Free Full Text]
  7. Product no. 3688571, KleenGuard Select Protective Apparel, Fisher Scientific, Waltham, MA.
  8. Product no. 01–002–54, Fisherbrand Bags for Stomacher Lab Blenders, Fisher Scientific, Waltham, MA.
  9. Up to 4 colonies from each sample were then subcultured to cryovials containing 0.75 mL of buffered peptone water and incubated overnight at 37°C. Glycerol (0.75 mL) was added to the cryovials, mixed, and then samples were stored at –85°C.
  10. The cells were suspended in 200 µL of sample buffer via vortexing. A 30-µL aliquot of this sample was transferred to a single well on an 8-well sample carrier. The sample carrier, buffer, and all further reagents were part of a kit designed for the RiboPrinter System. The restriction enzyme EcoRI was used to differentiate ribotypes.
  11. Qualicon Inc., Wilmington, DE.
  12. Bruce, J. 1996. Automated system rapidly identifies and characterizes microorganisms in food. Food Technol. 50:77–81.
  13. New England Biosystems Inc., Beverly, MA.
  14. Riboprint analysis. The resulting patterns were analyzed using BioNumerics software (Applied Maths, Austin, TX). This software created a dendrogram of the genetic similarity of a culture based on Pearson’s coefficient and clustered by an unweighted pair-group arithmetic averaging method.
  15. Barnes, H. J., J. Vaillancourt, and W. B. Gross. 2003. Colibacillosis. Pages 631–656 in Diseases of Poultry. 11th ed. Y. M. Saif, H. J. Barnes, J. R. Glisson, A. M. Fadly, L. R. McDougald, and D. E. Swayne, ed. Iowa State Press, Ames.
  16. Singer, R. S., J. S. Jeffrey, T. E. Carpenter, C. L. Cooke, E. R. Atwill, W. O. Johnson, and D. C. Hirsh. 2000. Persistence of cellulitis-associated Escherichia coli DNA fingerprints in successive broiler chicken flocks. Vet. Micro-biol. 75:59–71.[CrossRef][Web of Science][Medline]
  17. Norton, D. M., M. A. McCamey, K. L. Gall, J. M. Scarlett, K. J. Boor, and M. Wiemann. 2001. Molecular studies on the ecology of Listeria monocytogenes in the smoked fish processing industry. Appl. Environ. Microbiol. 67:198–205.[Abstract/Free Full Text]
  18. Craven, S. E., N. J. Stern, J. S. Bailey, N. A. Cox, and P. Fedorka-Cray. 2000. Ribotyping for the differentiation of Clostridium perfringens isolates from poultry production and processing. Page 75 in Proceedings of the 89th Annual Poultry Science Association Meeting, Montreal, Canada.
  19. Khan, A. A., M. S. Nawaz, C. Summage West, S. A. Khan, and J. Lin. 2005. Isolation and molecular characterization of fluoroquinolone-resistant Escherichia coli from poultry litter. Poult. Sci. 84:61–66.[Abstract/Free Full Text]
  20. Jeffrey, J. S., R. S. Singer, R. O’Connor, and E. R. Atwill. 2004. Prevalence of pathogenic Escherichia coli in the broiler house environment. Avian Dis. 48:189–195.[CrossRef][Web of Science][Medline]



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