Matrix-assisted laser desorption ionization–time of flight mass spectrometry reveals Enterococcus and Enterobacter spp. in major insect species involved in food security with resistance to common antibiotics

  • Foteini F. Parlapani
  • Maria Kyritsi
  • Maria Sakka
  • Kleio Chatzinikolaou
  • Spyridon Donos
  • Ioannis S. Boziaris
  • Christos Hadjichristodoulou
  • Christos G. AthanassiouEmail author
Original Paper


Stored-product insects can transfer a wide range of serious pathogens involved in human health. The close contact of these insects with the food production chain makes these species extremely dangerous as carriers of severe infections. In addition, pathogenic bacteria, such as members of Enterococcus, are often resistant to antibiotics commonly used for human therapy. Herein we identified, using Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS), Enterococcus and coliform species isolated from 17 strains of different insect species associated with durable stored products. The antibiotic susceptibility of the isolated strains was also evaluated. MALDI-TOF MS revealed mainly the presence of Enterococcus (E. faecium, E. phoeniculicola and E. casseliflavus) and Enterobacter (Eb. cloacae and Eb. asburiae). E. casseliflavus was resistant to all antibiotics tested, while E. faecium and E. phoeniculicola were resistant to sulphonamides. Among E. faecium isolates, approx. 20% were found to be resistant to tetracycline, while Eb. cloacae and Eb. asburiae showed resistance to erythromycin. The current series of data clearly indicates that certain bacteria of the genera Enterococcus and Enterobacter are very common in stored-product insects, and, under certain circumstances, may seriously endanger public health, through potential introduction of antibiotic resistance.


Stored-product insects Amylaceous commodities Enterococcus Enterobacter MALDI-TOF-MS Antibiotic resistance 


Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with animals performed by any of the authors.

Supplementary material

10340_2019_1125_MOESM1_ESM.tif (36 kb)
Supplementary material 1 (TIFF 36 kb)


  1. Ahmad A, Ghosh A, Schal C, Zurek L (2011) Insects in confined swine operations carry a large antibiotic resistant and potentially virulent enterococcal community. BMC Microbiol 11:1–23. CrossRefGoogle Scholar
  2. Alanko K, Tuomi T, Vanhanen M et al (2000) Occupational IgE-mediated allergy to Tribolium confusum (confused flour beetle). Allergy 55:879–882CrossRefGoogle Scholar
  3. Asaniyan EK, Laseinde EAO, Agbede JO (2007) Prevalence of darkling beetles (Alphitobius diaperinus) and bacterial load in broiler litters. Intl J Poultry Sci 6:440–444CrossRefGoogle Scholar
  4. Böhme K, Fernández-No IC, Barros-Velazquez J, Gallardo JM, Cañas B, Calo-Mata P (2011a) Rapid species identification of seafood spoilage and pathogenic Gram-positive bacteria by MALDI-TOF mass fingerprinting. Electrophoresis 32:2951–2965CrossRefGoogle Scholar
  5. Böhme K, Fernández-No IC, Gallardo JM, Cañas B, Calo-Mata P (2011b) Safety assessment of fresh and processed seafood products by MALDI-TOF Mass fingerprinting. Food Bioprocess Technol 4:907–918CrossRefGoogle Scholar
  6. Böhme K, Fernandez-No IC, Pazos M et al (2013) Identification and classification of seafood-borne pathogenic and spoilage bacteria: 16S rRNA sequencing versus MALDI-TOF MS fingerprinting. Electrophoresis 34:877–887CrossRefGoogle Scholar
  7. Campbell A, Singh NB, Sinha RN (1976) Bioenergetics of the granary weevil, Sitophilus granarius (L) (Coleoptera: Curculionidae). Can J Zool 54:786–798. CrossRefGoogle Scholar
  8. Cetinkaya Y, Falk P, Mayhall CG (2000) Vancomycin-resistant enterococci. Clin Microbiol Rev 13:686–707CrossRefGoogle Scholar
  9. Channaiah LH, Subramanyam B, McKinney LJ, Zurek L (2010) Stored-product insects carry antibiotic-resistant and potentially virulent enterococci. FEMS Microbiol Ecol 74:464–471CrossRefGoogle Scholar
  10. Chong YP, Lee SO, Song EH et al (2010) Quinupristin–dalfopristin versus linezolid for the treatment of vancomycin-resistant Enterococcus faecium bacteraemia: efficacy and development of resistance. Scand J Infect Dis 42(6–7):491–499CrossRefGoogle Scholar
  11. Courvalin P (2006) Vancomycin resistance in Gram-positive cocci. Clin Infect Dis 42:S25–S34CrossRefGoogle Scholar
  12. Davin-Regli A, Pagès J-M (2015) Enterobacter aerogenes and Enterobacter cloacae; versatile bacterial pathogens confronting antibiotic treatment. Front Microbiol 6:392CrossRefGoogle Scholar
  13. De las Casas E, Harein PK, Pomeroy BS (1972) Bacteria and fungi within the lesser mealworm collected in poultry brooder houses. Environ Entomol 1:27–30. CrossRefGoogle Scholar
  14. Dordet-Frisoni E, Dorchies G, De Araujo C, Talon R, Leroy S (2007) Genomic diversity in Staphylococcus xylosus. J Appl Environ Microbiol 73(22):7199–7209CrossRefGoogle Scholar
  15. Dutkiewicz J, Mackiewicz B, Lemieszek MK, Golec M, Milanowski J (2016) Pantoea agglomerans: a mysterious bacterium of evil and good Part III Deleterious effects: infections of humans, animals and plants. Ann Agric Environ Med 23(2):197–205CrossRefGoogle Scholar
  16. Fernández-Álvarez C, Torres-Corral Y, Santos Y (2018) Use of ribosomal proteins as biomarkers for identification of Flavobacterium psychrophilum by MALDI-TOF mass spectrometry. J Proteomics 170:59–69CrossRefGoogle Scholar
  17. Fernandez-Baca V, Ballesteros F, Hervas JA et al (2001) Molecular epidemiological typing of Enterobacter cloacae isolates from a neonatal intensive care unit: three-year prospective study. J Hosp Infection 49:173–182. CrossRefGoogle Scholar
  18. Fernández-Caldas E (2013) On mite allergy in dogs and humans. Int Arch Allergy Immunol 160:329–330. CrossRefGoogle Scholar
  19. Fernández-No IC, Böhme K, Calo-Mata P, Cañas B, Gallardo JM, Barros-Velázquez J (2012) Isolation and characterization of Streptococcus parauberis from vacuum-packaging refrigerated seafood products. Food Microbiol 30:91–97CrossRefGoogle Scholar
  20. Fleurat-Lessard F (2002) Qualitative reasoning and integrated management of the quality of stored grain: a promising new approach. J Stored Prod Res 38:191–218. CrossRefGoogle Scholar
  21. Freitas AR, Sousa C, Novais C et al (2017) Rapid detection of high-risk Enterococcus faecium clones by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Diagn Microbiol Infect Dis 87:299–307CrossRefGoogle Scholar
  22. Gorham JR (1977) Training manual for analytical entomology in the food industry. FDA technical bulletin no, FDA US Department of Health, WashingtonGoogle Scholar
  23. Guzman Prieto AM, van Schaik W, Rogers MR et al (2016) Global emergence and dissemination of enterococci as nosocomial pathogens: attack of the clones? Front Microbiol 7:788CrossRefGoogle Scholar
  24. Hammerum AM (2012) Enterococci of animal origin and their significance for public health. Clin Microbiol Infect 18:619–625CrossRefGoogle Scholar
  25. Harein PK, de las Casas E, Larsen CT, Pomeroy BS (1972) Microbial relationship between the lesser mealworm and its associated environment in a turkey brooder house. Environ Entomol 1:189–194. CrossRefGoogle Scholar
  26. Hart PJ, Wey E, McHugh TD, Balakrishnan I, Belgacem O (2015) A method for the detection of antibiotic resistance markers in clinical strains of Escherichia coli using MALDI mass spectrometry. J Microbiol Methods 111:1–8CrossRefGoogle Scholar
  27. Healthcare Infection Control Practices Advisory Committee (HICPAC) (2016) Antibiotic stewardship statement for antibiotic guidelines—The Recommendations of the Healthcare Infection Control Practices Advisory CommitteeGoogle Scholar
  28. Hodges RJ, Buzby JC, Bennett B (2011) Post-harvest losses and waste in developed and less developed countries: opportunities to improve resource use. J Agric Sci 149:37–45. CrossRefGoogle Scholar
  29. Hubert J, Stejskal V, Athanassiou CG, Throne JE (2018) Health hazards associated with arthropod infestation of stored products. Annu Rev Entomol 63:553–573. CrossRefGoogle Scholar
  30. Ignasiak K, Maxwell A (2017) Antibiotic-resistant bacteria in the guts of insects feeding on plants: prospects for discovering plant-derived antibiotics. BMC Microbiol 17:223CrossRefGoogle Scholar
  31. Khan ID, Sahni AK, Bharadwaj R et al (2014) Emerging organisms in a tertiary healthcare set up. Med J Armed Forces 70:120–128CrossRefGoogle Scholar
  32. Kim E, Cho Y, Lee Y et al (2017) A proteomic approach for rapid identification of Weissella species isolated from Korean fermented foods on MALDI-TOF MS supplemented with an in-house database. Int J Food Microbiol 243:9–15CrossRefGoogle Scholar
  33. Larson Z, Subramanyam B, Zurek L, Herrman T (2008) Diversity and antibiotic resistance of enterococci associated with stored-product insects collected from feed mills. J Stored Prod Res 44:198–203. CrossRefGoogle Scholar
  34. Lasch P, Jacob D, Grunow R, Schwecke T, Doellinger J (2016) Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) for the identification of highly pathogenic bacteria Trends. Anal Chem 85:103–111. Google Scholar
  35. Law-Brown J, Meyers PR (2003) Enterococcus phoeniculicola sp nov, a novel member of the enterococci isolated from the uropygial gland of the Red-billed Woodhoopoe, Phoeniculus purpureus. Int J Syst Evol Microbiol 53:683–685CrossRefGoogle Scholar
  36. Lebreton F, Willems RJL, Gilmore MS (2014) Enterococcus diversity, origins in nature, and gut colonization. In: Gilmore MS, Clewell DB, Ike Y, Shankar N (eds) Enterococci: from commensals to leading causes of drug resistant infection. Massachusetts Eye and Ear Infirmary, BostonGoogle Scholar
  37. Lee CC, Lee NY, Yan JJ et al (2010) Bacteremia due to extended-spectrum-β-lactamase-producing Enterobacter cloacae: role of carbapenem therapy. Antimicrob Agents Chemother 54(9):3551–3556CrossRefGoogle Scholar
  38. Lopes ACS, Rodrigues JF, Cabral AB et al (2016) Occurrence and analysis of irp2 virulence gene in isolates of Klebsiella pneumoniae and Enterobacter spp from microbiota and hospital and community-acquired infections. Microb Pathog 96:15–19CrossRefGoogle Scholar
  39. McAllister JC, Steelman CD, Skeeles JK (1994) Reservoir competence of the lesser mealworm (Coleoptera: Tenebrionidae) for Salmonella typhimurium (Eubacteriales: Enterobacteriaceae). J Med Entomol 31:369–372CrossRefGoogle Scholar
  40. Moellering RC Jr (1992) Emergence of Enterococcus as a significant pathogen. Clin Infect Dis 14(6):1173–1176. CrossRefGoogle Scholar
  41. Morrison D, Woodford N, Cookson B (1997) Enterococci as emerging pathogens of humans. Soc Appl Bacteriol Symp Ser 83:89S–99SCrossRefGoogle Scholar
  42. Nacef M, Chevalier M, Chollet S, Drider D, Flahaut C (2017) MALDI-TOF mass spectrometry for the identification of lactic acid bacteria isolated from a French cheese: the Maroilles. Int J Food Microbiol 247:2–8CrossRefGoogle Scholar
  43. Noble WC, Virani Z, Cree RG (1992) Co-transfer of vancomycin and other resistance genes from Enterococcus faecalis NCTC 12201 to Staphylococcus aureus. FEMS Microbiol Lett 72:195–198CrossRefGoogle Scholar
  44. Omaye ST, Wirtz RA, Fruin JT (1981) Toxicity of substituted p-benzoquinones found in the secretion of tenebrionid flour beetles. Proc West Pharmacol Soc 24:169–171Google Scholar
  45. Perez-Mendoza J, Throne JE, Dowell FE, Baker JE (2003) Detection of insect fragments in wheat flour by near-infrared spectroscopy. J Stored Prod Res 39:305–312. CrossRefGoogle Scholar
  46. Perlatti Β, Luiz AL, Prieto EL et al (2017) MALDI-TOF MS identification of microbiota associated with pest insect Diabrotica speciosa. Agric For Entomol 19(4):408–417. CrossRefGoogle Scholar
  47. Regecová I, Pipová M, Jevinová P, Marušková K, Kmeť V, Popelka P (2014) Species identification and antimicrobial resistance of coagulase-negative staphylococci isolated from the meat of sea fish. J Food Sci 79(5):M898–M902CrossRefGoogle Scholar
  48. Reid KC, Cockerill FR III, Patel R (2001) Clinical and epidemiological features of Enterococcus casseliflavus/flavescens and Enterococcus gallinarum bacteremia: a report of 20 cases. Clin Infect Dis 32:1540–1546CrossRefGoogle Scholar
  49. Rice LB (2008) Federal funding for the study of antimicrobial resistance in nosocomial pathogens: no ESKAPE. J Infect Dis 197:1079–1081CrossRefGoogle Scholar
  50. Ruelle V, El Moualij B, Zorzi W, Ledent P, Pauw ED (2004) Rapid identification of environmental bacterial strains by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Rapid Commun Mass Spectrom 18(18):2013–2019CrossRefGoogle Scholar
  51. Santos T, Capelo JL, Santos HM et al (2015) Use of MALDI-TOF mass spectrometry fingerprinting to characterize Enterococcus spp and Escherichia coli isolates. J Proteomics 127:321–331CrossRefGoogle Scholar
  52. Sauget M, Valot B, Bertrand X, Hocquet D (2017) Can MALDI-TOF mass spectrometry reasonably type bacteria? Trends Microbiol 25(6):447–455CrossRefGoogle Scholar
  53. Shostak AW (2014) Hymenolepis diminuta infections in tenebrionid beetles as a model system for ecological interactions between helminth parasites and terrestrial intermediate hosts: a review and meta-analysis. J Parasitol 100:46–58CrossRefGoogle Scholar
  54. Siegel S, Lee N, Rohr A et al (1991) Evaluation of dermestid sensitivity in museum personnel. J Allergy Clin Immunol 87:190CrossRefGoogle Scholar
  55. Skov MN, Spencer AG, Hald B et al (2004) The role of litter beetles as potential reservoir for Salmonella enterica and thermophilic Campylobacter spp between broiler flocks. Avian Dis 48:9–18CrossRefGoogle Scholar
  56. Tanada Y, Kaya HK (1993) Insect Pathology. Academic Press, San DiegoGoogle Scholar
  57. Tandina F, Almeras L, Koné AK, Doumbo OK, Raoult D, Parola P (2016) Use of MALDI-TOF MS and culturomics to identify mosquitoes and their midgut microbiota. Parasit Vectors 9:495CrossRefGoogle Scholar
  58. Throne JE, Hallman GJ, Johnson JA, Follett PA (2003) Post-harvest entomology research in the United States Department of Agriculture-Agricultural Research Service. Pest Manag Sci 59:619–628CrossRefGoogle Scholar
  59. Top J, Willems R, Bonten M (2008) Emergence of CC17 Enterococcus faecium: from commensal to hospital-adapted pathogen. FEMS Immunol Med Microbiol 52:297–308CrossRefGoogle Scholar
  60. Wang A, Yao Z, Zheng W, Zhang H (2014) Bacterial communities in the gut and reproductive organs of Bactrocera minax (Diptera: Tephritidae) based on 454 pyrosequencing. PLoS ONE 9(9):e106988CrossRefGoogle Scholar
  61. Yezerski A, Cussatt G, Glick D, Evancho M (2005) The effects of the presence of stored product pests on the microfauna of a flour community. J Appl Microbiol 98:507–515CrossRefGoogle Scholar
  62. Zurek L, Gorhamm JR (2010) Insects as vectors of foodborne pathogens. In: Voeller JG (ed) Wiley handbook of science and technology for homeland security. Wiley, Hoboken, pp 1169–1683Google Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Foteini F. Parlapani
    • 1
  • Maria Kyritsi
    • 2
  • Maria Sakka
    • 3
  • Kleio Chatzinikolaou
    • 1
  • Spyridon Donos
    • 1
  • Ioannis S. Boziaris
    • 1
  • Christos Hadjichristodoulou
    • 2
  • Christos G. Athanassiou
    • 3
    Email author
  1. 1.Laboratory of Marketing and Technology of Aquatic Products and Foods, Department of Ichthyology and Aquatic Environment, School of Agricultural SciencesUniversity of ThessalyVolosGreece
  2. 2.Laboratory of Hygiene and Epidemiology, Faculty of Medicine, School of Health SciencesUniversity of ThessalyLarissaGreece
  3. 3.Laboratory of Entomology and Agricultural Zoology, Department of Agriculture, Crop Production and Rural EnvironmentUniversity of ThessalyVolosGreece

Personalised recommendations