Bacteriophage genotyping using BOXA repetitive-PCR

Damnjanovic et al. BMC Microbiology (2020) 20:154 https://doi.org/10.1186/s12866-020-01770-2 METHODOLOGY ARTICLE Open Access Bacteriophage genotyping using BOXA repetitive-PCR Dragica Damnjanovic, Xabier Vázquez-Campos, Daniel L. Winter, Melissa Harvey and Wallace J. Bridge* Abstract Background: Repetitive-PCR (rep-PCR) using BOXA1R and BOXA2R as single primers was investigated for its potential to genotype bacteriophage. Previously, this technique has been primarily used for the discrimination of bacterial strains. Reproducible DNA fingerprint patterns for various phage types were generated using either of the two primers. Results: The similarity index of replicates ranged from 89.4–100% for BOXA2R-PCR, and from 90 to 100% for BOXA1R-PCR. The method of DNA isolation (p = 0.08) and the phage propagation conditions at two different temperatures (p = 0.527) had no significant influence on generated patterns. Rep-PCR amplification products were generated from different templates including purified phage DNA, phage lysates and phage plaques. The use of this method enabled comparisons of phage genetic profiles to establish their similarity to related or unrelated phages and their bacterial hosts. Conclusion: The findings suggest that repetitive-PCR could be used as a rapid and inexpensive method to preliminary screen phage isolates prior to their selection for more comprehensive studies. The adoption of this rapid, simple and reproducible technique could facilitate preliminary characterisation of a large number of phage isolates and the investigation of genetic relationship between phage genotypes. Keywords: Bacteriophage, Phage genotyping, Repetitive-PCR Background Repetitive DNA sequences constitute a substantial component of both eukaryotic and prokaryotic genomes. In some higher plant species, they can account for up to 90% of the genomic DNA [1], while in humans DNA repeats comprise nearly half of the genome [2]. The presence or absence of certain types of repeats, diversity in their nucleotide sequences, their size, location and copy number per genome characterize various bacterial species, even those with the smallest genomes [3]. Interspersed repeats play a significant role in genomic rearrangements, such as inversions, deletions, duplications and translocations [3]. The proposed functional roles of repetitive sequences involve the regulation of coding * Correspondence: School of Biotechnology and Biomolecular Sciences, Faculty of Science, UNSW Sydney, Kensington, Australia sequence expression and the formatting necessary for genome packaging; DNA repair and restructuring; genome replication and transmission to progeny cells; formation of nucleoprotein complexes; and formation of a characteristic genome system organization that allows for evolutionary significant changes without altering coding sequences [4]. Specific families of interspersed DNA elements have been observed in many bacterial and archaeal genomes [3, 5], while bacteriophages are considered to carry few repetitive elements [5]. The BOX family of repetitive DNA elements, consisting of different combinations of three sequence sub-motifs, boxA, boxB, and boxC, was originally identified in Gram-positive Streptococcus pneumoniae [6]. Hybridization studies have shown that only boxA sequences are highly evolutionary conserved. The outwardly facing repetitive primers BOXA1, © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. 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 in a credit line to the data. Damnjanovic et al. BMC Microbiology (2020) 20:154 BOXA1R and BOXA2R that are complementary to the consensus sequences of boxA, when used as single primers in the repetitive-PCR, generated complex fingerprint patterns in various Gram-positive and Gramnegative bacterial species [7]. BOXA-based primers have since been used for genotyping diverse bacterial species in various ecological [8], epidemiological [9] and industrial application studies [10]. The optimal performance of dairy starter cultures is challenged by the risk of lytic bacteriophage (phage) infection [11, 12] and indeed, phage may pose a problem to any industry based on bacterial fermentation [13]. In recent years, there has been a renewed interest in phage from the perspective of their beneficial applications, such as phage therapy for treating pathogens [14, 15]. Of great utility for the dairy industry are multiplex PCR systems that can detect and classify the three main lactococcal phage species, 936, P335 and c2, as well as Streptococcus thermophilus and Lactobacillus delbrueckii phages [16, 17]. These rapid and sensitive tests are based on the generation of specific PCR amplification products for each phage species; however, they do not enable the identification of individual phage strains. Methods that can be applied for genetic characterization of phages involve restriction digestion of genomic DNA [18]; multilocus sequence typing (MLST) [19]; restriction fragment length polymorphism (RFLP) or a denaturing gradient gel electrophoresis (DGGE) of a particular gene [20]; random amplification of polymorphic DNA (RAPD)PCR [18, 21, 22] and genomic sequencing [23]. However, these methods are not necessarily the most suitable for routine use due to time or cost-associated constraints. MLST and DGGE require a priori genetic information [21]. Restriction digestion of genomic DNA and DNA/ DNA hybridizations are considered time-consuming and often require large quantities (μg) of pure DNA [21]. Additionally, the genomes of some phages can be resistant to restriction enzymes, which may be due to a scarcity of cleavage sites [24]; a base modification within the recognition sequence, genome methylation or other antirestriction mechanisms [18]. This imposes the need to use several restriction enzymes to ensure digestion [18]. While MLST can distinguish phages with the same RFLP pattern [19], it may not be universally applicable for fingerprinting all phage types. For example, it has proved suitable for phylogene (...truncated)