Skip to main content
SpringerLink
Log in

Microbiota Analysis of Eggshells in Different Areas and During Different Storage Time by Non-cultural Methods

  • Published:
Current Microbiology Aims and scope Submit manuscript

Abstract

This study is to investigate and characterize the microbiota composition on eggshells from 3 different areas of Shaanxi province (Yulin, Hanzhong and Xi’an). The eggs were stored at 25 °C for 56 days and bacterial samples were collected from eggshells on day 0, 14, 28, 42 and 56. Denaturing gradient gel electrophoresis and high-throughput sequencing of 16S rRNA hypervariable region V3–V4 were performed. Alpha diversity was applied for analyzing the diversity of samples through 6 indices, including Observed-species, Chao1, Shannon, Simpson, ACE and Good’s-coverage. Beta diversity was used to study the similarities or differences in the community composition of the samples. Totally, 36 phyla and 595 genera were classified by 16S rRNA gene sequencing. The composition of the microbial communities of different regions was quite different. Firmicutes (33–38% of total phyla) and Actinobacteria (36–61% of total phyla) were the most abundant phyla in all three regions. Proteobacteria were relatively more abundant (about 18% of total phyla) on eggs from Hanzhong. During storage time, the microbial communities mainly changed from Firmicutes to Actinobacteria on eggs from Yulin and Xi’an. Lactobacillus, Kocuria and Streptomyces were much higher at the genus level. Spoilage bacteria Staphylococcus, Streptococcus, Pseudomonas and Enterococcus were detected at the genus level. Campylobacter jejuni (< 1% of total bacteria), which might be related to human illness, was also detected. In conclusion, the structure, abundance, and composition of microbiota on eggshells differ among areas. The microbiota changed regularly during storage time. The current study may offer a new insight into bacterial species on eggshells.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

Data Availability

The data used to support the findings of this study is available upon request.

References

  1. Al-Bahry SN, Mahmoud IY, Al-Musharafi SK, Al-Ali MA (2012) Penetration of spoilage and food poisoning bacteria into fresh chicken egg: a public health concern. Glob J Biosci Biotechnol 1:33–33

    Google Scholar 

  2. Berrang ME, Frank JF, Buhr RJ, Bailey JS, Cox NA (1999) Eggshell membrane structure and penetration by Salmonella typhimurium. J Food Prot 62:73. https://doi.org/10.1111/j.1745-4530.1999.tb00500.x

    Article  CAS  PubMed  Google Scholar 

  3. Cason JA, Cox NA, Bailey JS (1994) Transmission of Salmonella typhimurium during hatching of broiler chicks. Avian Dis 38:583–588. https://doi.org/10.2307/1592082

    Article  CAS  PubMed  Google Scholar 

  4. Yang SE, Yu RC, Chou CC (2001) Influence of holding temperature on the growth and survival of Salmonella spp. and Staphylococcus aureus and the production of staphylococcal enterotoxin in egg products. Int J Food Microbiol 63:99–107. https://doi.org/10.1016/s0168-1605(00)00416-5

    Article  CAS  PubMed  Google Scholar 

  5. Peggy GB, Claudia W, Karsten F (2011) Experimental studies on the influence of washing of table eggs on their microbial quality. J Food Saf Food Qual 62:145–188. https://doi.org/10.2376/0003-925X-62-157

    Article  Google Scholar 

  6. Alloui N, Tlidjene M, Alloui-Lombarkia O, Zeghina D (2003) Effect of the hygienic status of poultry houses on performance and production. Br Poult Sci 44:771–772. https://doi.org/10.1080/00071668.2003.9610494

    Article  CAS  PubMed  Google Scholar 

  7. Grizard S, Dini-Andreote F, Tieleman BI, Salles JF (2014) Dynamics of bacterial and fungal communities associated with eggshells during incubation. Ecol Evol 4:1140–1157. https://doi.org/10.1002/ece3.1011

    Article  PubMed  PubMed Central  Google Scholar 

  8. Ercolini D (2013) High-throughput sequencing and metagenomics: moving forward in the culture-independent analysis of food microbial ecology. Appl Environ Microbiol 79:3148–3155. https://doi.org/10.1128/AEM.00256-13

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Rouger A, Tresse O, Zagorec M (2017) Bacterial contaminants of poultry meat: sources, species, and dynamics. Microorganisms. https://doi.org/10.3390/microorganisms5030050

    Article  PubMed  PubMed Central  Google Scholar 

  10. Wilson MR, Brown E, Keys C, Strain E, Luo Y, Muruvanda T, Grim C, Jean-Gilles Beaubrun J, Jarvis K, Ewing L, Gopinath G, Hanes D, Allard MW, Musser S (2016) Whole genome DNA sequence analysis of Salmonella subspecies enterica serotype Tennessee obtained from related peanut butter foodborne outbreaks. PLoS ONE 11:e0146929. https://doi.org/10.1371/journal.pone.0146929

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Blaser MJ, Dominguez-Bello MG, Contreras M, Magris M, Hidalgo G, Estrada I, Gao Z, Clemente JC, Costello EK, Knight R (2013) Distinct cutaneous bacterial assemblages in a sampling of South American Amerindians and US residents. ISME J 7:85–95. https://doi.org/10.1038/ismej.2012.81

    Article  CAS  PubMed  Google Scholar 

  12. Li W, Han L, Yu P, Ma C, Wu X, Moore JE, Xu J (2014) Molecular characterization of skin microbiota between cancer cachexia patients and healthy volunteers. Microb Ecol 67:679–689. https://doi.org/10.1007/s00248-013-0345-6

    Article  PubMed  Google Scholar 

  13. Muyzer G, de Waal EC, Uitterlinden AG (1993) Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA. Appl Environ Microbiol 59:695–700. https://doi.org/10.0000/PMID7683183

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Haas BJ, Gevers D, Earl AM, Feldgarden M, Ward DV, Giannoukos G, Ciulla D, Tabbaa D, Highlander SK, Sodergren E, Methe B, DeSantis TZ, Human Microbiome C, Petrosino JF, Knight R, Birren BW (2011) Chimeric 16S rRNA sequence formation and detection in Sanger and 454-pyrosequenced PCR amplicons. Genome Res 21:494–504. https://doi.org/10.1101/gr.112730.110

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Magoc T, Salzberg SL (2011) FLASH: fast length adjustment of short reads to improve genome assemblies. Bioinformatics 27:2957–2963. https://doi.org/10.1093/bioinformatics/btr507

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Bokulich NA, Subramanian S, Faith JJ, Gevers D, Gordon JI, Knight R, Mills DA, Caporaso JG (2013) Quality-filtering vastly improves diversity estimates from Illumina amplicon sequencing. Nat Methods 10:57–59. https://doi.org/10.1038/nmeth.2276

    Article  CAS  PubMed  Google Scholar 

  17. Caporaso JG, Kuczynski J, Stombaugh J, Bittinger K, Bushman FD, Costello EK, Fierer N, Pena AG, Goodrich JK, Gordon JI, Huttley GA, Kelley ST, Knights D, Koenig JE, Ley RE, Lozupone CA, McDonald D, Muegge BD, Pirrung M, Reeder J, Sevinsky JR, Turnbaugh PJ, Walters WA, Widmann J, Yatsunenko T, Zaneveld J, Knight R (2010) QIIME allows analysis of high-throughput community sequencing data. Nat Methods 7:335–336. https://doi.org/10.1038/nmeth.f.303

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Edgar RC, Haas BJ, Clemente JC, Quince C, Knight R (2011) UCHIME improves sensitivity and speed of chimera detection. Bioinformatics 27:2194–2200. https://doi.org/10.1093/bioinformatics/btr381

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Edgar RC (2013) UPARSE: highly accurate OTU sequences from microbial amplicon reads. Nat Methods 10:996–998. https://doi.org/10.1038/nmeth.2604

    Article  CAS  PubMed  Google Scholar 

  20. DeSantis TZ, Hugenholtz P, Larsen N, Rojas M, Brodie EL, Keller K, Huber T, Dalevi D, Hu P, Andersen GL (2006) Greengenes, a chimera-checked 16S rRNA gene database and workbench compatible with ARB. Appl Environ Microbiol 72:5069–5072. https://doi.org/10.1128/AEM.03006-05

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Wang Q, Garrity GM, Tiedje JM, Cole JR (2007) Naive Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy. Appl Environ Microbiol 73:5261–5267. https://doi.org/10.1128/AEM.00062-07

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Beissinger SR, Cook MI, Arendt WJ (2005) The shelf life of bird eggs: testing egg viability using a tropical climate gradient. Ecology 86:2164–2175. https://doi.org/10.2307/3450927

    Article  Google Scholar 

  23. Cook MI, Beissinger SR, Toranzos GA, Rodriguez RA, Arendt WJ (2003) Trans-shell infection by pathogenic micro-organisms reduces the shelf life of non-incubated bird's eggs: a constraint on the onset of incubation? Proc Biol Sci 270:2233–2240. https://doi.org/10.1098/rspb.2003.2508

    Article  PubMed  PubMed Central  Google Scholar 

  24. Cook MI, Beissinger SR, Toranzos GA, Rodriguez RA, Arendt WJ (2005) Microbial infection affects egg viability and incubation behavior in a tropical passerine. Behav Ecol 16:30–36. https://doi.org/10.1093/beheco/arh131

    Article  Google Scholar 

  25. Ruiz de Castañeda R, Vela AI, Lobato E, Briones V, Moreno J (2011) Bacterial loads on eggshells of the pied flycatcher: environmental and maternal factors. Condor 113:200–208. https://doi.org/10.1525/cond.2011.100035

    Article  Google Scholar 

  26. Cook MI, Beissinger SR, Toranzos GA, Arendt WJ (2005) Incubation reduces microbial growth on eggshells and the opportunity for trans-shell infection. Ecol Lett 8:532–537. https://doi.org/10.1111/j.1461-0248.2005.00748.x

    Article  PubMed  Google Scholar 

  27. Klomp JEM, Michael T, Smith SB, Mckay JE, Ferrera I, Reysenbach AL (2010) Cloacal microbial communities of female spotted towhees Pipilo maculatus: microgeographic variation and individual sources of variability. J Avian Biol 39:530–538. https://doi.org/10.1111/j.0908-8857.2008.04333.x

    Article  Google Scholar 

  28. Wei S, Morrison M, Yu Z (2013) Bacterial census of poultry intestinal microbiome. Poult Sci 92:671–683. https://doi.org/10.3382/ps.2012-02822

    Article  CAS  PubMed  Google Scholar 

  29. Vieira DAP, Cabral L, Noronha MF, Júnior GVL, Sant’Ana AS (2019) Microbiota of eggs revealed by 16S rRNA-based sequencing: from raw materials produced by different suppliers to chilled pasteurized liquid products. Food Control 96:194–204

    Article  CAS  Google Scholar 

  30. Neira C, Laca A, Laca A, Díaz M (2017) Microbial diversity on commercial eggs as affected by the production system. A first approach using PGM. Int J Food Microbiol 262:3–7. https://doi.org/10.1016/j.ijfoodmicro.2017.09.008

    Article  PubMed  Google Scholar 

  31. Long C, Wang J, Zhang HJ, Wu SG, Qi GH (2017) Effects of dietary rapeseed meal supplementation on cecal microbiota in laying hens with different flavin-containing monooxygenase 3 genotypes. Poult Sci 96:1748–1758. https://doi.org/10.3382/ps/pew449

    Article  CAS  PubMed  Google Scholar 

  32. Lee MD, Lu J, Harmon B, Hofacre CL, Maurer JJ (2006) Antimicrobial growth promoters: where do we go from here?. Molecular basis for AGP effects in poultry 978:149–163

  33. Lu JR, Hofacre CL, Lee M (2006) Emerging technologies in microbial ecology aid in understanding the effect of monensin in the diets of broilers in regard to the complex disease necrotic enteritis. J Appl Poult Res 15:145–153. https://doi.org/10.1093/japr/15.1.145

    Article  Google Scholar 

  34. Kohl KD (2012) Diversity and function of the avian gut microbiota. J Comp Physiol B 182:591–602. https://doi.org/10.1007/s00360-012-0645-z

    Article  PubMed  Google Scholar 

  35. Pan D, Yu Z (2014) Intestinal microbiome of poultry and its interaction with host and diet. Gut microbes 5:108–119. https://doi.org/10.4161/gmic.26945

    Article  PubMed  Google Scholar 

  36. Rehman HU, Vahjen W, Awad WA, Zentek J (2007) Indigenous bacteria and bacterial metabolic products in the gastrointestinal tract of broiler chickens. Arch AnimNutr 61:319–335. https://doi.org/10.1080/17450390701556817

    Article  CAS  Google Scholar 

  37. Stanley D, Hughes RJ, Moore RJ (2014) Microbiota of the chicken gastrointestinal tract: influence on health, productivity and disease. Appl Microbiol Biotechnol 98:4301–4310. https://doi.org/10.1007/s00253-014-5646-2

    Article  CAS  PubMed  Google Scholar 

  38. van der Wielen PW, Keuzenkamp DA, Lipman LJ, van Knapen F, Biesterveld S (2002) Spatial and temporal variation of the intestinal bacterial community in commercially raised broiler chickens during growth. Microb Ecol 44:286–293. https://doi.org/10.1007/s00248-002-2015-y

    Article  CAS  PubMed  Google Scholar 

  39. Waite DW, Taylor MW (2014) Characterizing the avian gut microbiota: membership, driving influences, and potential function. Front Microbiol 5:223. https://doi.org/10.3389/fmicb.2014.00223

    Article  PubMed  PubMed Central  Google Scholar 

  40. Pandey B, Ghimire P, Agrawal VP Climate, Health, ecology, management and conservation, Kathmandu. In: International conference on the great Himalayas, Canada, 2004. Kathmandu University and the Aquatic Ecosystem Health and Management Society

  41. Elbendary AA, Hessain AM, El-Hariri MD, Seida AA, Moussa IM, Mubarak AS, Kabli SA, Hemeg HA, El Jakee JK (2018) Isolation of antimicrobial producing Actinobacteria from soil samples. Saudi J Biol Sci 25:44–46. https://doi.org/10.1016/j.sjbs.2017.05.003

    Article  CAS  PubMed  Google Scholar 

  42. Grizard S, Versteegh MA, Ndithia HK, Salles JF, Tieleman BI (2015) Shifts in bacterial communities of eggshells and antimicrobial activities in eggs during incubation in a ground-nesting passerine. PLoS ONE 10:e0121716. https://doi.org/10.1371/journal.pone.0121716

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Holl L, Behr J, Vogel RF (2016) Identification and growth dynamics of meat spoilage microorganisms in modified atmosphere packaged poultry meat by MALDI-TOF MS. Food Microbiol 60:84–91. https://doi.org/10.1016/j.fm.2016.07.003

    Article  CAS  PubMed  Google Scholar 

  44. Zhang QQ, Han YQ, Cao JX, Xu XL, Zhou GH, Zhang WY (2012) The spoilage of air-packaged broiler meat during storage at normal and fluctuating storage temperatures. Poult Sci 91:208–214. https://doi.org/10.3382/ps.2011-01519

    Article  CAS  PubMed  Google Scholar 

  45. Suwannarach N, Kaewyana C, Yodmeeklin A, Kumla J, Matsui K, Lumyong S (2017) Evaluation of Muscodor cinnamomi as an egg biofumigant for the reduction of microorganisms on eggshell surfaces and its effect on egg quality. Int J Food Microbiol 244:52–61. https://doi.org/10.1016/j.ijfoodmicro.2016.12.021

    Article  CAS  PubMed  Google Scholar 

  46. Russell SM (2008) The effect of an acidic, copper sulfate-based commercial sanitizer on indicator, pathogenic, and spoilage bacteria associated with broiler chicken carcasses when applied at various intervention points during poultry processing. Poult Sci 87:1435. https://doi.org/10.3382/ps.2007-00339

    Article  CAS  PubMed  Google Scholar 

  47. Hinton A Jr, Cason JA, Ingram KD (2004) Tracking spoilage bacteria in commercial poultry processing and refrigerated storage of poultry carcasses. Int J Food Microbiol 91:155–165. https://doi.org/10.1016/S0168-1605(03)00377-5

    Article  PubMed  Google Scholar 

  48. Chaemsanit S, Akbar A, Anal AK (2015) Isolation of total aerobic and pathogenic bacteria from table eggs and its contents. Food Appl Biosci J 3:1–9

    Google Scholar 

  49. Kone AZ, Jan S, Le Marechal C, Grosset N, Gautier M, Puterflam J, Baron F (2013) Identifying risk factors for eggshell contamination by Bacillus cereus group bacteria in French laying farms. Br Poult Sci 54:298–305. https://doi.org/10.1080/00071668.2013.783900

    Article  CAS  PubMed  Google Scholar 

  50. Stern NJ, Clavero MR, Bailey JS, Cox NA, Robach MC (1995) Campylobacter spp. in broilers on the farm and after transport. Poult Sci 74:937–941. https://doi.org/10.3382/ps.0740937

    Article  CAS  PubMed  Google Scholar 

  51. Lee MD, Newell DG (2006) Campylobacter in poultry: filling an ecological niche. Avian Dis 50:1–9. https://doi.org/10.1637/7474-111605R.1

    Article  CAS  PubMed  Google Scholar 

  52. Wassenaar TM, Newell DG (2006) The genus campylobacter in the prokaryotes. In: Martin-Dworkin SF (ed) Proteobacteria: delta and epsilon subclasses. Deeply rooting bacteria, vol 7, 3rd edn. Springer, New York, pp 119–138

    Google Scholar 

  53. Gross WB (1990) Factors affecting the development of respiratory disease complex in chickens. Avian Dis 34:607–610. https://doi.org/10.2307/1591252

    Article  CAS  PubMed  Google Scholar 

  54. Smith HW, Cook JK, Parsell ZE (1985) The experimental infection of chickens with mixtures of infectious bronchitis virus and Escherichia coli. J Gen Virol 66(Pt 4):777–786. https://doi.org/10.1099/0022-1317-66-4-777

    Article  PubMed  Google Scholar 

  55. Praveen PK, Debnath C, Shekhar S, Dalai N, Ganguly S (2016) Incidence of Aeromonas spp. infection in fish and chicken meat and its related public health hazards: a review. Veterin World 9:6–11

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We would like to thank Xiaoqiang Wang and Zengguo Wang from Xi’an Center for Disease Control and Prevention; Hongli Liu and Yali Chen from Hanzhong Center for Disease Control and Prevention; Zhiping Wang and Yadong Jiao from Yulin Center for Disease Control and Prevention for the sample collection. We would also like to thank Prof. Chaofeng Ma from Xi’an Center for Disease Control and Prevention for the equipment and technical support.

Funding

This work was supported by [the project of Shaanxi Science and Technology Research and Development Plan] under Grant [2016SF-051]; [the Project of Open and Sharing Platform of Shaanxi Science and Technology Resources] under Grant [2016FWPT-12]; and [Shaanxi Provincial Health Research Fund] under Grant [2016D023].

Author information

Authors and Affiliations

  1. Department of Microbiology and Immunology, School of Medicine, Xi’an Jiaotong University, No. 76 Yanta West Road, Xi’an, 710061, Shaanxi, China

    Yi Shi, Lu Yuan, Huan Li, Siruo Zhang, Nosheen Mushtaq & Jiru Xu

  2. Shaanxi Center for Disease Control and Prevention, Xi’an, 710054, Shaanxi, China

    Yi Shi, Wenjuan Li, Dongli Liu, Guozhu Ma & Zheng Zhang

  3. Department of Public Health, Xi’an Medical University, Xi’an, 710021, Shaanxi, China

    Songwen Wu

Authors
  1. Yi Shi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Songwen Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Wenjuan Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Dongli Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Guozhu Ma
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Zheng Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Lu Yuan
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Huan Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Siruo Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Nosheen Mushtaq
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Jiru Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

JX designed the study, collected the funds and revised the manuscript. YS, SW, WL, DL, GM, ZZ, LY and HL carried out the experiments and analyzed the data. SZ and NM searched the literatures. YS drafted the manuscript. All authors contributed to this study and have approved the final manuscript.

Corresponding author

Correspondence to Jiru Xu.

Ethics declarations

Conflict of interests

The authors declare no conflict of interest.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Shi, Y., Wu, S., Li, W. et al. Microbiota Analysis of Eggshells in Different Areas and During Different Storage Time by Non-cultural Methods. Curr Microbiol 77, 3842–3850 (2020). https://doi.org/10.1007/s00284-020-02212-y

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00284-020-02212-y

Search

Navigation