16S-ARDRA and MALDI-TOF mass spectrometry as tools for identification of Lactobacillus bacteria isolated from poultry

Dec et al. BMC Microbiology (2016) 16:105 DOI 10.1186/s12866-016-0732-5 RESEARCH ARTICLE Open Access 16S-ARDRA and MALDI-TOF mass spectrometry as tools for identification of Lactobacillus bacteria isolated from poultry Marta Dec* , Andrzej Puchalski, Renata Urban-Chmiel and Andrzej Wernicki Abstract Background: The objective of our study is to evaluate the potential use of Amplified 16S Ribosomal DNA Restriction Analysis (16S-ARDRA) and MALDI-TOF mass spectrometry (MS) as methods for species identification of Lactobacillus strains in poultry. Results: A total of 80 Lactobacillus strains isolated from the cloaca of chicken, geese and turkeys were identified to the species level by MALDI-TOF MS (on-plate extraction method) and 16S-ARDRA. The two techniques produced comparable classification results, some of which were additionally confirmed by sequencing of 16S rDNA. MALDI-TOF MS enabled rapid species identification but produced more than one reliable identification result for 16. 25 % of examined strains (mainly of the species L. johnsonii). For 30 % of isolates intermediate log(scores) of 1.70–1. 99 were obtained, indicating correct genus identification but only presumptive species identification. The 16S-ARDRA protocol was based on digestion of 16S rDNA with the restriction enzymes MseI, HinfI, MboI and AluI. This technique was able to distinguish 17 of the 19 Lactobacillus reference species tested and enabled identification of all 80 wild isolates. L. salivarius dominated among the 15 recognized species, followed by L. johnsonii and L. ingluviei. Conclusions: The MALDI-TOF MS and 16S-ARDRA assays are valuable tools for the identification of avian lactobacilli to the species level. MALDI-TOF MS is a fast, simple and cost-effective technique, and despite generating a high percentage of results with a log(score) <2.00, the on-plate extraction method is characterized by high-performance. For samples for which Biotyper produces more than one reliable result, MALDI-TOF MS must be used in combination with genotypic techniques to achieve unambiguous results. 16S-ARDRA is simple, repetitive method with high power of discrimination, whose sole limitation is its inability to discriminate between species with very high 16S rDNA sequence homology, such as L. casei and L. zeae. The assays can be used for discrimination of Lactobacillus bacteria from different habitats. Keywords: Lactobacillus, Lactic acid bacteria, Identification, Poultry, MALDI-TOF MS, ARDRA, 16S rDNA Background Lactobacilli are Gram-positive, non-sporing, aerotolerant or anaerobic catalase-negative rods or coccobacilli. The genus Lactobacillus currently (December 2015) comproses 224 species [1] and is thus the most numerous group of lactic acid bacteria (LAB). The natural habitats of these bacteria are dairy products, healthy and rotting plants, and the mucous membranes of humans and * Correspondence: ; Sub-Department of Veterinary Prevention and Avian Diseases, Institute of Biological Bases of Animal Diseases, Faculty of Veterinary Medicine, University of Life Sciences in Lublin, Akademicka 12, 20-033 Lublin, Poland animals, including birds. They have been isolated from the GIT (gastrointestinal tract) of chickens [2], geese [3], ducks [4] and pigeons [5]. The most commonly identified species in these birds are L. salivarius, L. johnsonii, L. crispatus, L. reuteri and L. agilis [2–5]. Lactobacilli, as beneficial components of the gut microbiome, have a great impact on the health status of farm animals, including poultry. While maintaining the microbial balance of the mucous membranes, they provide protection against enteropathogenic infection [6, 7]. In addition, they improve digestion and nutrient assimilation, remove toxic substances, and enhance immunity © 2016 The Author(s). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Dec et al. BMC Microbiology (2016) 16:105 [8, 9]. Owing to their health-promoting properties Lactobacillus bacteria are used to produce probiotic preparations for humans and animals. Probiotics, through multi-pronged action, improve the health of animals and increase the efficiency of livestock production. Interest in the application of probiotics in poultry has grown since the introduction in the EU of a ban on antibiotic growth promoters in animals and the associated increase in the frequency of intestinal infections in birds, mainly induced by C. perfringens. The use of selected Lactobacillus strains as feed additives for poultry can produce similar effects to those of antibiotic growth promoters, manifested by increases in weight and better feed efficiency [10, 11], as well as resistance to pathogenic bacteria such as Salmonella sp. [12], C. perfringens [13, 14], E. coli [10] or Campylobacter sp. [14]. Moreover, supplementing the diet of broilers with Lactobacillus strains reduces fat deposition in the coelom [15] and increases the size, quality and production of eggs [16, 17]. Accurate taxonomic classification of lactobacilli to the species level is not an easy task. It is made difficult by the large and continually growing number of species belonging to this genus and their biochemical and genetic diversity. Identification by phenotypic methods is timeconsuming and has a low discriminatory level [18]. The commercial kit API CHL50 (Biomerieux) for lactic acid bacilli yields ambiguous results and even misidentifications [19]. Molecular methods have proven to be more reliable. The target most commonly used for bacterial identification is 16S rDNA. This ~1500 base-pair gene is characterized by slow rates of evolution and encodes 16S rRNA, a component of the 30S small subunit of prokaryotic ribosomes. In addition to highly conserved sites (used for binding of universal primers in PCR), 16S rRNA gene sequences contain hypervariable regions that can provide species-specific signature sequences useful in identifying bacteria and determining their phylogenetic position [20]. Despite its accuracy, the use of 16S rRNA gene sequence analysis is not widespread outside of reference laboratories because of technical and cost considerations. Sequencer purchase prices exceed the financial capacity of ordinary laboratories, and the costs of sequencing performed by outside labs offering this service is not cost-effective for identification of multiple strains. The high price (about €30 per sample) is dictated by the substantial length of 16S rDNA, which requires (...truncated)