Skip to main content
SpringerLink
Log in

Identification of Clostridium spp. derived from a sheep and cattle slaughterhouse by matrix-assisted laser desorption and ionization-time of flight mass spectrometry (MALDI-TOF MS) and 16S rDNA sequencing

  • Original Article
  • Published:
Journal of Food Science and Technology Aims and scope Submit manuscript

Abstract

Clostridia are widespread and some of them are serious human pathogens. Identification of Clostridium spp. is important for managing microbiological risks in the food industry. Samples derived from sheep and cattle carcasses from a slaughterhouse in Iran were analyzed by MALDI-TOF MS using direct transfer and extended direct transfer sample preparation methods and 16S rDNA sequencing. MALDI-TOF MS could identify ten species in 224 out of 240 Clostridium isolates. In comparison to the 16S rDNA sequencing, correct identification rate of the Clostridium spp. at the species level by MALDI-TOF MS technique was 93.3%. 16 isolates were not identified by MALDI-TOF MS but 16s rDNA sequencing identified them as C. estertheticum, C. frigidicarnis, and C. gasigenes species. The most frequently identified Clostridium species were: C. sporogenes (13%), C. cadaveris (12.5%), C. cochlearium (12%) and C. perfringens (10%). Extended direct transfer method [2.26 ± 0.18 log (score)] in comparison to direct transfer method [2.15 ± 0.23 log (score)] improved Clostridium spp. identification. Using a cut-off score of 1.7 was sufficient for accurate identification of Clostridium species. MALDI-TOF MS identification scores for Clostridium spp. decreased with longer incubation time. Clostridium species predominantly were isolated from carcasses after skinning and evisceration steps in the slaughterhouse. MALDI-TOF MS could be an accurate way to identify Clostridium species. Moreover, continuous improvement of the database and MALDI-TOF MS instrument enhance its performance in food control laboratories.

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

Similar content being viewed by others

References

  • Adam KH, Flint SH, Brightwell G (2013) Reduction of spoilage of chilled vacuum-packed lamb by psychrotolerant clostridia. Meat Sci 93(2):310–315

    Article  PubMed  Google Scholar 

  • Agustini BC, Silva LP, Bloch C Jr, Bonfim TMB et al (2014) Evaluation of MALDI-TOF mass spectrometry for identification of environmental yeasts and development of supplementary database. Appl Microbiol Biotechnol 98(12):5645–5654

    Article  CAS  PubMed  Google Scholar 

  • Alispahic M, Christensen H, Hess C, Razzazi-Fazeli E et al (2011) Identification of Gallibacterium species by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry evaluated by multilocus sequence analysis. Int J Med Microbiol 301:513–522

    Article  CAS  PubMed  Google Scholar 

  • AlMogbel MS (2016) Matrix assisted laser desorption/ionization time of flight mass spectrometry for identification of Clostridium species isolated from Saudi Arabia. Braz J Microbiol 47(2):410–413

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Angeletti S (2017) Matrix assisted laser desorption time of flight mass spectrometry (MALDI-TOF MS) in clinical microbiology. J Microbiol Methods 138:20–29

    Article  CAS  PubMed  Google Scholar 

  • Aras Z, Hadimli HH (2015) Detection and molecular typing of Clostridium perfringens isolates from beef, chicken and turkey meats. Anaerobe 32:15–17

    Article  CAS  PubMed  Google Scholar 

  • Bakhtiary F, Sayevand HR, Remely M, Hippe B, Hosseini H, Haslberger AG (2016) Evaluation of bacterial contamination sources in meat production line. J Food Qual 39:750–756

    Article  CAS  Google Scholar 

  • Bano L, Drigo I, Tonon E, Pascoletti S et al (2017) Identification and characterization of Clostridium botulinum group III field strains by matrix-assisted laser desorption–ionization time-of-flight mass spectrometry (MALDI-TOF MS). Anaerobe 48:126–134

    Article  CAS  PubMed  Google Scholar 

  • Barba MJ, Fernandez A, Oviano M, Fernandez B et al (2014) Evaluation of MALDI-TOF mass spectrometry for identification of anaerobic bacteria. Anaerobe 30:126–128

    Article  CAS  PubMed  Google Scholar 

  • Bruggemann H, Brzuszkiewicz E, Chapeton-Montes D, Plourde L et al (2015) Genomics of Clostridium tetani. Res Microbiol 166(4):326–331

    Article  CAS  PubMed  Google Scholar 

  • Brunt J, Cross KL, Peck MW (2015) Apertures in the Clostridium sporogenes spore coat and exosporium align to facilitate emergence of the vegetative cell. Food Microbiol 51:45–50

    Article  PubMed  PubMed Central  Google Scholar 

  • Chean R, Kotsanas D, Francis MJ, Palombo EA et al (2014) Comparing the identification of Clostridium spp. by two matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry platforms to 16S rRNA PCR sequencing as a reference standard: a detailed analysis of age of culture and sample preparation. Anaerobe 30:85–89

    Article  CAS  PubMed  Google Scholar 

  • de Koster C, Brul S (2016) MALDI-TOF MS identification and tracking of food spoilers and food-borne pathogens. Curr Opin Food Sci 10:76–84

    Article  Google Scholar 

  • Doern CD (2013) Charting uncharted territory: a review of the verification and implementation process for matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for organism identification. Clin Microbiol Newsl 35(9):69–78

    Article  Google Scholar 

  • Doulgeraki AL, Ercolini D, Villani F, Nychas GJE (2012) Spoilage microbiota associated to the storage of raw meat in different conditions. Int J Food Microbiol 157(2):130–141

    Article  PubMed  Google Scholar 

  • Fournier R, Wallet F, Grandbastien B, Dubreuil L et al (2012) Chemical extraction versus direct smear for MALDI-TOF mass spectrometry identification of anaerobic bacteria. Anaerobe 18(3):294–297

    Article  CAS  PubMed  Google Scholar 

  • Grosse-Herrenthey A, Maier T, Gessler F, Schaumann R et al (2008) Challenging the problem of clostridial identification with matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS). Anaerobe 14(4):242–249

    Article  CAS  PubMed  Google Scholar 

  • Gutiérrez C, Gómez-Flechoso M, Belda I, Ruiz J et al (2017) Wine yeasts identification by MALDI-TOF MS: optimization of the preanalytical steps and development of an extensible open-source platform for processing and analysis of an in-house MS database. Int J Food Microbiol 254:1–10

    Article  CAS  PubMed  Google Scholar 

  • Höll 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

    Article  CAS  PubMed  Google Scholar 

  • Hsu YMS, Burnham CAD (2014) MALDI-TOF MS identification of anaerobic bacteria: assessment of pre-analytical variables and specimen preparation techniques. Diagn Microbiol Infect Dis 79(2):144–148

    Article  CAS  PubMed  Google Scholar 

  • Húngaro H, Caturla M, Horita C, Furtado M et al (2016) Blown pack spoilage in vacuum-packaged meat: a review on clostridia as causative agents, sources, detection methods, contributing factors and mitigation strategies. Trends Food Sci Technol 52:123–138

    Article  CAS  Google Scholar 

  • Kim J, Kim S, Park H, Park D et al (2016) MALDI-TOF MS is more accurate than VITEK II ANC card and API Rapid ID 32 A system for the identification of Clostridium species. Anaerobe 40:73–75

    Article  CAS  PubMed  Google Scholar 

  • Koohdar VA (2013) Study of beef carcass bacterial contamination in Karaj Slaughterhouse. J Food Hyg 3:43–51

    Google Scholar 

  • Kozma-Sipos Z, Szigeti J, Asvanyi B, Varga L (2010) Heat resistance of Clostridium sordellii spores. Anaerobe 16(3):226–228

    Article  PubMed  Google Scholar 

  • Lee J, Pascall MA (2012) Inactivation of Clostridium sporogenes spores on stainless-steel using heat and an organic acidic chemical agent. J Food Eng 110(3):493–496

    Article  CAS  Google Scholar 

  • Madden RH, Murray KA et al (2004) Determination of the principal points of product contamination during beef carcass dressing processes in Northern Ireland. J Food Prot 67(7):1494–1496

    Article  PubMed  Google Scholar 

  • Nacef M, Chevalier M, Chollet S, Drider D et al (2016) MALDI-TOF mass spectrometry for the identification of lactic acid bacteria isolated from a French cheese: the Maroilles. Int J Food Microbiol. https://doi.org/10.1016/j.ijfoodmicro.2016.07.005

    Article  PubMed  Google Scholar 

  • Naim F, Zareifard MR, Zhu S, Huizing RH et al (2008) Combined effects of heat, nisin, and acidification on the inactivation of Clostridium sporogenes spores in carrot-alginate particles: from kinetics to process validation. Food Microbiol 25(7):936–941

    Article  CAS  PubMed  Google Scholar 

  • Narvaez-Bravo C, Rodas Gonzalez A, Fuenmayor Y, Flores-Rondon C et al (2013) Salmonella on feces, hides, and carcasses in beef slaughter facilities in Venezuela. Int J Food Microbiol 166(2):226–230

    Article  PubMed  Google Scholar 

  • Prevost S, Cayol J, Zuber F, Tholozan J et al (2013) Characterization of clostridial species and sulfite-reducing anaerobes isolated from foie gras with respect to microbial quality and safety. Food Control 32(1):222–227

    Article  Google Scholar 

  • Randall LP, Lemma F, Koylass M, Rogers J et al (2015) Evaluation of MALDI-TOF as a method for the identification of bacteria in the veterinary diagnostic laboratory. Res Vet Sci 101:42–49

    Article  CAS  PubMed  Google Scholar 

  • Redondo-Solano M, Valenzuela-Martinez C, Cassada DA, Snow DD, Juneja VK, Burson DE et al (2013) Effect of meat ingredients (sodium nitrite and erythorbate) and processing (vacuum storage and packaging atmosphere) on germination and outgrowth of Clostridium perfringens spores in ham during abusive cooling. Food Microbiol 35(2):108–115

    Article  CAS  PubMed  Google Scholar 

  • Remely M, Dworzak S, Hippe B, Zwielehner J et al (2013) Abundance and diversity of microbiota in type 2 diabetes and obesity. J Diabetes Metab. https://doi.org/10.4172/2155-6156.1000253

    Article  Google Scholar 

  • Salmela SP, Fredriksson-Ahomaa M, Hatakka M, Nevas M (2013) Microbiological contamination of sheep carcasses in Finland by excision and swabbing sampling. Food Control 31(2):372–378

    Article  Google Scholar 

  • Salvador F, Porte L, Durin L, Marcotti A et al (2013) Breakthrough bacteremia due to Clostridium tertium in a patient with neutropenic fever, and identification by MALDI-TOF mass spectrometry. Int J Infect Dis 17(11):1062–1063

    Article  Google Scholar 

  • Seng P, Drancourt M, Gouriet F, La Scola B et al (2009) Ongoing revolution in bacteriology: routine identification of bacteria by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Clin Infect Dis 49(4):543–551

    Article  CAS  PubMed  Google Scholar 

  • Shafer D, Liu H, Dong J, Liu W et al (2017) Comparison of direct smear and chemical extraction methods for MALDI-TOF mass spectrometry identification of clinical relevant anaerobic bacteria. Front Lab Med 1(1):27–30

    Article  Google Scholar 

  • Shinha T (2015) Bacteremia Due to Clostridium cadaveris. Infect Dis Clin Pract 24(4):232–233

    Article  Google Scholar 

  • Silva AR, Tahara ACC, Chaves RD, Sant’ana AS et al (2012) Influence of different shrinking temperatures and vacuum conditions on the ability of psychrotrophic Clostridium to cause “blown pack” spoilage in chilled vacuum-packaged beef. Meat Sci 92(4):498–505

    Article  PubMed  Google Scholar 

  • TeKippe EME, Shuey S, Winkler DW, Butler MA et al (2013) Optimizing identification of clinically relevant Gram-positive organisms using the Bruker Biotyper MALDI-TOF MS system. J Clin Microbiol. https://doi.org/10.1128/JCM.02680-12

    Article  Google Scholar 

  • Veloo ACM, Erhard M, Welker M, Welling GW et al (2011a) Identification of Gram-positive anaerobic cocci by MALDI-TOF mass spectrometry. Syst Appl Microbiol 34(1):58–62

    Article  CAS  PubMed  Google Scholar 

  • Veloo ACM, Knoester M, Degener JE, Kuijper EJ et al (2011b) Comparison of two matrix-assisted laser desorption ionization-time of flight mass spectrometry methods for the identification of clinically relevant anaerobic bacteria. Clin Microbiol Infect 17(10):1501–1506

    Article  CAS  PubMed  Google Scholar 

  • Veloo ACM, Welling GW, Degener JE (2011c) The identification of anaerobic bacteria using MALDI-TOF MS. Anaerobe 17(4):211–212

    Article  CAS  PubMed  Google Scholar 

  • Veloo ACM, Elgersma PE, Friedrich AW, Nagy E et al (2014) The influence of incubation time, sample preparation and exposure to oxygen on the quality of the MALDI-TOF MS spectrum of anaerobic bacteria. Clin Microbiol Infect 20(12):1091–1097

    Article  CAS  Google Scholar 

  • Vidor C, Awad M, Lyras D (2015) Antibiotic resistance, virulence factors and genetics of Clostridium sordellii. Res Microbiol 166(4):368–374

    Article  CAS  PubMed  Google Scholar 

  • Wiegel J, Tanner R, Rainey FA (2006) an introduction to the family Clostridiaceae. Prokaryotes. https://doi.org/10.1007/0-387-30744-3_20

    Article  Google Scholar 

  • You MJ, Shin GW, Lee CS (2015) Clostridium tertium bacteremia in a patient with glyphosate ingestion. Am J Case Rep 16:4–7

    Article  PubMed  PubMed Central  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Nutritional Sciences, University of Vienna, Althanstrasse 14, UZAII;2D541, 1090, Vienna, Austria

    Farzaneh Bakhtiary, Hamid Reza Sayevand, Marlene Remely, Berit Hippe & Alexander G. Haslberger

  2. IMED AGES Vienna, Vienna, Austria

    Alexander Indra

  3. Faculty of Nutrition Sciences and Food Technology, National Nutrition and Food Technology Research Institute, Shahid Beheshti University of Medical Sciences, Tehran, Iran

    Hedayat Hosseini

Authors
  1. Farzaneh Bakhtiary
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Hamid Reza Sayevand
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Marlene Remely
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Berit Hippe
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Alexander Indra
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Hedayat Hosseini
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Alexander G. Haslberger
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Alexander G. Haslberger.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Bakhtiary, F., Sayevand, H.R., Remely, M. et al. Identification of Clostridium spp. derived from a sheep and cattle slaughterhouse by matrix-assisted laser desorption and ionization-time of flight mass spectrometry (MALDI-TOF MS) and 16S rDNA sequencing. J Food Sci Technol 55, 3232–3240 (2018). https://doi.org/10.1007/s13197-018-3255-2

Download citation

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s13197-018-3255-2

Keywords

Search

Navigation