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isolation of a new neisseria phage from the oral cavity of healthy humans

Research Article ºÚÁÏÍø
Ahmed N Aljarbou1,3 and Mohamad Aljofan1,2*
1Department of Pharmaceutics, College of Pharmacy, Qasim University, Buraidah, Saudi Arabia
2Department of Microbiology, School of Medicine and Health Sciences, Monash University, Australia
3Al-Ghad International College of Health Sciences, Al-Qassim, P.O. BOX 406. Buraidah 51411. Saudi Arabia
*Corresponding authors: Mohamad Aljofan
Department of Microbiology
School of Medicine and Health Sciences
Monash University, Australia 3600
Tel: +61399029356
Fax: +6139902950
E-mail: mo.aljofan@monash.edu
 
Received September 06, 2012; Published October 29, 2012
 
Citation: Aljarbou AN, Aljofan M(2012) Isolation of a New Neisseria Phage from the Oral Cavity of Healthy Humans. 1:416. doi:10.4172/scientificreports.416
 
Copyright: © 2012 Aljarbou AN, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
 
Abstract
 
The incidences of antibacterial drug resistant pathogens are rising, which necessitates the need for alternative therapies. As a result, research in the field of antibiotic drug development utilising bacteriophage is gaining momentum. The aim of this study is to provide proof of concept that phages can be isolated from healthy human individuals and that such phages may play a significant role in the maintenance of oral health. The results of sequence analyses using the available databases, confirmed the presence and identification of different phage proteins, which have been inserted into a bacterial host identified as Neisseria meningitidis. The results in this study warrant further investigation into the efficacy and suitability of the isolated phage as potential control for oral infections and possibly the development of a selective phage therapy treatment for Neisseria.
 
Keywords
 
Neisseria; Bacteriophage; DNA sequence
 
Introduction
 
There is no effective or definitive therapy to treat periodontal disease, which is a wide set of pathological alterations that occur in the human oral cavity to which different bacterial groups have been suggested to be the causative microorganism [1]. These bacterial groups are usually embedded in dental plaques and represent a complex microbial community, widely known to be the precursor for many infections including gingival [2]. While, gram negative anaerobic bacteria species have predominantly been isolated from infected individuals, they have also been isolated from healthy individuals [ 3]. The plaque biofilm is stabilised and protected by a bacteriallyproduced exopolysaccharide matrix [4]. Therefore, specific disruption of the bacterial polysaccharide may provide a new therapy to treat not only periodontal causing bacteria, but also to treat a wider group of antibiotic resistant bacterial strains.
 
Bacteriophages are bacteria-specific viruses that infect, and in the case of obligate lytic phages, destroy their host bacteria [5]. Since their discovery in the early nineteenth century, bacteriophages were clinically used as antibacterial agents until the discovery of penicillin, after which the use of bacteriophage as antibacterial agents was replaced by the new therapies [5]. Bacteriophages have the potential to regulate the oral microflora by i) lysing sensitive cells, ii) selecting mutants (which may have altered characteristics) and iii) by releasing bacterial components with pro-inflammatory activity [6]. Intriguingly, many examples of phage therapies at the clinical and commercial levels have been documented [7,8], including their use in dentistry where several bacteriophages that infect diverse oral bacteria have been isolated from saliva and dental plaques [9-11].
 
This study describes the isolation of a new phage from Neisseria meningitides strain (WUE2594), which were attained from 3 healthy human plaque samples. The study provides proof of concept that phages are present in dental plaques of healthy individuals and that they could potentially provide a selective antibacterial activity against their host species Neisseria.
 
2. Detecting, Purification and DNA Sequencing of a New Bacteriophage
 
LB and BHIB agar were used as a bottom agar, while 0.35% agarose LB and BHIB were used as the soft top agar, which were supplemented with horse blood at 2%, 3%, 5% v/v, to ensure growth for bacteria that grew better in the presence of blood. For infection, 100 μl of filtered sample of was added to 300 μl of the host cell culture that had been grown overnight. The viral particles were allowed to adsorb into the host cells for 15 min at room temperature, then the infected cells were added to 3 ml of the molten soft top agar in universal tubes and mixed well before being poured onto the bottom agar. This was left to set for a few minutes; the plates were then inverted and incubated at 37°C. After 24 to 48 h, they were checked for the appearance of plaques.
 
Completely lysed soft top agars were collected and added to 40 ml of SM buffer (Krackeler Scientific, US) and then incubated overnight at 10°C to allow the virus particles to diffuse from the soft top agar into the SM buffer. The sample was then centrifuged at 250× g for 25 min, and then the supernatant was filtered with a 0.45 μm and then a 0.22 μm Millipore filter to ensure the removal of agar and cell debris. The viral particles were mixed with 1/8 volume Polyethylene glycol (PEG) 6000 solution (2.5 M NaCl, 20% (w/v) PEG 6000) and incubated on ice for 30 min. Samples were then centrifuged at 16000× g for 10 min and the virus pellet was re-suspended in 0.5 ml of 10 mM Tris pH 7.5, 10 mM MgCl 2, 100 mM NaCl. Free nucleic acids were digested by adding 10 U of DNase and 10 μg/ml RNase A and incubating for 30 min at 37°C. Nucleic acids were then extracted using an equal volume of phenol: chloroform.
 
Extracted viral genomes were cut using restriction endonuclease (New England Biolabs). DNA fragments of 0.5 to 2 kb in size were ligated to the pGEM-T Easy® Promega vector, and introduced into competent E. coli JM109. The complete nucleotide sequence was analysed using Nucleotide Basic Local Alignment Search Tool (BLASTN) to search for highly similar sequence alignments within the nucleotide collection database. The complete contig was entered into MacVector 12.5 sequence analysis software and open reading frames (ORFs) 150 nucleotides or greater ≥ 50 amino acids) were identified. The ORFs were translated using MacVector 12.5 and the protein sequences were entered into Protein BLAST (BLASTP) and used to search for non-redundant protein sequence alignments.
 
While we have not attempted to characterise these viral proteins, our preliminary database search showed that the majority of these sequences have high homology to viral (phage) proteins (Table 1). These viral proteins were identified by setting up the selection search to the minimum size of the ORF’s to be more than 50 amino acids in length. Generally, the majority of the identified phage related proteins seemed to be associated with an insertion into the Neisseria genome. A significant number of the identified phage proteins have strong association with Neisseria meningitides, which might suggest that the identified virus might be a specific prophage for the bacteria Neisseria meningitidis. However, based on the fact that almost all of the rest of the identified proteins were associated with insertion into different Neisseria genome, we can safely speculate that the identified virus is a specific phage for the Neisseria genus.
 
Table 1: Gene 6- top 5 alignments
 
Furthermore, the preliminary sequence analyses have revealed a number of interesting observations including findings of putative bacterial promoter sequences, which were identified at -35 (TTGACA) and -10 (TATAAT). Intriguingly, these sequences were identified for the vast majority (79%, 19/24) of the identified phage related genes ( Figure 1). However, it is rather difficult to accurately identify the exact promoter sequence for the identified phage proteins without experimental validation. Hence, further analyses and characterisation of the identified phages are being undertaken at a high scale by our research group.
 
Figure 1: Identified putative bacterial phage related genes.
 
Discussion and Conclusion
 
There is an ever constant increase in the reported cases of multiple antibiotic-resistant pathogenic bacteria [12], which has prompted many researchers to revisit an older antibacterial therapy that utilises bacteriophages. Multi drug resistant bacterial pathogens pose a major threat to human health as well as to the long term efficacy of commonly used antibiotics [13]. The last few years have seen a significant increase in the number of new bacteriophage research programs, encompassing different delivery routes, the most popular being oral and parenteral [5,11]. Research that focuses on the oral route of ‘anti-bacterial’ therapy revolves around the scenario of the potential isolation of specific bacteriophages from the human oral cavity, and investigating the possibility of utilising these phages as potential antibacterial agents. Bacteriophages isolated from the human oral cavity will more likely be useful in the development of antibacterial therapies for antibiotics resistant oral pathogens. Also, most of the published reports on human oral lytic phage isolation have encountered and/or reported the formation of lysis zones [2].
 
Bachrach et al. (2003) [14] reported the isolation of a lytic bacteriophage which they speculate contributes to the ecosystem of the human oral cavity and also possibly to overall human health since the phage is ubiquitously associated with its bacterium host. Oral pathogens are therefore noted to be found both in healthy and diseased individuals [3]. Thus, the presence of phages in healthy individuals may, although does not necessarily prove, the theory that they, phages, somehow contribute to the overall health of the oral flora. While a number of studies who reported the isolation of bacteriophages from healthy human individuals indicated somewhat the likelihood of phages contributing on maintaining the oral flora [1,11,15], many others have argued otherwise [16,17].
 
This study provides proof of concept that bacteriophages may be isolated from healthy human individuals and that this fact (isolation from healthy individuals), prompted us to speculate that the isolated phage is likely to play a role in the maintenance of the oral flora. This warrants further investigations into the promising utility of bacteriophages therapy as an antibacterial modality. Furthermore, the identified bacteriophage could be utilised or further developed into making specific antibiotic treatments that could potentially target its host, Neisseria meningitidis, or the Neisseria species in general. Currently, we are further validating and characterising this isolated phage as well as determining the biological role that these phages might play in health and disease- which we believe can significantly improve the development of specific bacteriophage therapy.
 
Acknowledgment
 
The authors of the manuscripts would like to acknowledge Dr. Shaun Heaphy at the Department of Infection, Immunity and Inflammation, Leicester Medical School, University of Leicester for allowing us to avail research facilities to conduct this study, and Natalie Stevenson for her valuable help with the sequence alignment.
 
Conflict of Interest
 
Non declared.
 
 
References