|Year : 2014 | Volume
| Issue : 2 | Page : 51-57
Bacterial whole genome sequencing: The future of clinical bacteriology
Department of Medical Microbiology, Ahmadu Bello University Teaching Hospital, Shika, Zaria, Kaduna State, Nigeria
|Date of Web Publication||16-Mar-2015|
Department of Medical Microbiology, Ahmadu Bello University Teaching Hospital, Shika, Zaria, Kaduna State
Source of Support: None, Conflict of Interest: None
|How to cite this article:|
Aliyu S. Bacterial whole genome sequencing: The future of clinical bacteriology. Ann Nigerian Med 2014;8:51-7
The genomic revolution promises to transform the practice of medicine, including the specialty of clinical microbiology. Clinical microbiology is the branch of medicine that is concerned with the laboratory diagnosis, treatment, and prevention of infectious diseases. Clinical microbiology helps to direct the management of infections in individual patients (diagnostic microbiology), and helps to monitor and control the spread of infectious diseases (public health microbiology). Ongoing developments in sequencing technology will have a huge impact on the way clinical microbiologist identify and characterize all pathogens, namely bacteria, viruses, fungi, and parasites. However, because of the burden of infections caused by bacterial pathogens worldwide, this editorial focuses on the impact of whole genome sequencing (WGS) on clinical bacteriology.
This editorial begins with a brief history of the evolution of deoxyribonucleic acid (DNA) sequencing; and then gently introduces next generation sequencing (NGS), the technology that has made WGS possible. The ability of NGS technology to sequence large quantities of DNA in a very short period of time has made it an invaluable tool for WGS in the laboratory.
In 1977, Sanger et al. published two landmark papers on the methodology of rapid determination of DNA sequences. , This sequencing method known as "chain-terminator or dideoxy" Sanger sequencing, ushered in a new era of DNA sequencing that transformed the field of biotechnology. This "chain-terminator" sequencing method had been an improvement on the "plus and minus" Sanger sequencing method, which Fred Sanger and Alan R. Couldson had described 2 years earlier.  Sequencing technology continued to evolve over the next few decades. The next major development occurred in 1986, when the automated "dye-terminator" Sanger sequencing method was introduced.  This eventually culminated in the first full sequencing of two bacterial pathogens in 1995, Haemophilus influenzae and Mycoplasma genitalium. 
In 1995, a radically new and different approach to sequencing was described by Margulies et al. and Shendure et al. This new method which was called sequencing by synthesis, heralded the beginning of NGS. Basic descriptions of "dye-terminator" Sanger sequencing and NGS methods are outlined below.
| Dye-Terminator Sanger Sequencing Method|| |
The dye-terminator variant of Sanger sequencing involves the use of dideoxynucleotides to sequence DNA. The basic steps of dye-terminator Sanger sequencing are outlined below.  Dideoxynucleotides are similar to normal nucleotides, except that they contain a hydrogen group on the 3' carbon, instead of a hydroxyl group. When integrated into a sequence, dideoxynucleotides prevent the addition of further nucleotides; because a phosphodiester bond cannot form between the dideoxynucleotide and the next incoming nucleotide. This results in termination of DNA synthesis. For a particular DNA sample or template, four parallel sequencing reactions are set up. Each reaction involves a single-stranded template, a specific primer to start the reaction, DNA polymerase, the four standard deoxynucleotides (dATP, dGTP, dCTP, and dTTP), and one of the four dideoxynucleotides (ddATP, ddGTP, ddCTP, or ddTTP). The dideoxynucleotides are added to each reaction at a lower concentration than the standard deoxynucleotides. When the sequencing is started, a polymerase chain reaction (PCR) generates over 1 billion DNA molecules, with each of the four PCR reactions generating all of the possible terminating fragments for that particular base. This results in the generation of fragments of different sizes. These fragments are then separated by size, using gel or capillary tube electrophoresis. Each of the dideoxynucleotides is labeled with different color dyes that fluoresce at different wavelengths; allowing for detection of each dideoxynucleotides at the end of the newly synthesized fragments. This allows for the determination of each nucleotide type on the DNA template that was synthesized [Figure 1]. 
|Figure 1: Dye terminator Sanger sequencing — Dye terminator Sanger sequencing involves a short oligonucleotide which acts as a primer for the synthesis of new strands of deoxyribonucleic acid (DNA) complementary to a single-stranded template. Four ddNTPs (each labeled with a different colored fl uorescent tag) are present, and chain elongation will stop when a ddNTP inserted. A mixture of DNA chains with different lengths will be generated and followed by separating the DNA, by size using gel electrophoresis. The DNA sequence is decoded from the pattern of colors, which corresponds to the nucleotide sequence|
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| Next Generation Sequencing Method|| |
Next generation sequencing method, also known as high throughput sequencing or massively parallel sequencing, has now replaced Sanger sequencing as the method of choice for sequencing DNA. Millions or billions of DNA strands are sequenced in parallel, yielding substantially more throughput and minimizing the need for fragment-cloning, which was a feature of Sanger sequencing. These NGS platforms are broadly divided into two groups,  namely platforms that require production of libraries of clonally amplified DNA; and more recently, platforms that can sequence a single molecule of DNA, such as Oxford Nanopore's 4 inches portable sequencer called MinION™  [Figure 2].
Although most next generation platforms essentially utilize the sequencing by synthesis approach to DNA sequencing, they do differ slightly in the approach used to read the sequence. These differences are eloquently outlined in the review article by Loman et al. 
Sequencing by synthesis basically relies on detection of pyrophosphate release on nucleotide incorporation; rather than chain termination with dideoxynucleotides, which is utilized by Sanger sequencing. A slightly different approach used in some NGS platforms is synthesis by ligation,  where fluorescent probes undergo steps of hybridization and ligation to complementary positions in the template strand at the 5΄ end of the extending strand. This is followed by fluorescence detection of the ligated probe. The type of probe detected is used by the machine to determine the sequence of the template DNA.
Many companies produce NGS machines, with the most important players being © Illumina, © Pacific Biosciences, © Roche, and © Life Technologies. Illumina produces the most dominant sequencing technology today, with Illumina machines accounting for more than 90% of all DNA data produced as of 2014. 
Next-generation sequencing platforms exist as high-end instruments which are expensive, bulky, and deliver high throughput; and bench top instruments that have lower throughput and are more affordable.  The choice of platform largely depends on the intended use of the platform, and the funds available to purchase the sequencing platform, reagents, and other consumables.
| Microbial Whole Genome Sequencing|| |
Microbial WGS refers to the process of mapping genomes of novel microorganisms, completing genomes of known microorganisms, or comparing genomes of different microorganisms. The two methods employed in microbial WGS are De novo microbial genome sequencing and microbial whole-genome resequencing.  De novo whole-genome sequencing involves assembling a genome without the use of a genomic reference, and is often used to sequence novel microbial genomes; while microbial whole-genome resequencing involves sequencing the entire genome of a bacteria, virus, or other microbes, and comparing the sequence to that of a known reference.  The choice of method depends on the read length obtained from the NGS platform, the availability of a good reference sequence for comparison, and the intended biological application of the sequencing data obtained.  Stages of bacterial WGS using NGS platforms include template preparation, sequencing and imaging, and data analysis; as depicted in [Figure 3]. 
| Whole Genome Sequencing and Clinical Bacteriology|| |
Diagnostic bacteriology has remained a cornerstone of clinical microbiology. Culture-based bacteriological techniques have governed the way clinical bacteriology has been practiced for over a century. These techniques have been relatively cheap and easy to perform. However, one of the major challenges remains the characterization of nonculturable or difficult to grow organisms, where serology, antigen detection, and PCR have played a role. In the last 50 years, PCR has become a vital tool for the detection and characterization of bacteria, despite having some limitations.
Detection and identification of bacteria, drug susceptibility testing, identification of virulent determinants, and epidemiological typing are key components of clinical bacteriology. Each component requires utilization of specific routine microbiological techniques to complete; and in totality require a turnaround time of around 1-2 days for many samples, and longer for a few samples such as blood cultures. 
Recent advances in WGS have helped to lower the cost of sequencing a whole bacterial genome, to as low as £40 (about $62).  These advances have also eased the process of WGS using next generation sequencing techniques. For clinical microbiology, the choice of sequencing platform is also important. Three benchtop platforms, the 454 GS Junior™ (Roche), the Ion Torrent PGM™ (Life technologies), and the MiSeq™ (Illumina), are increasingly being used by clinical microbiologists  [Figure 4]. They are laser-printer sized and have modest set-up costs, when compared to the high-end sequencers. This makes them ideal for the clinical laboratory.
|Figure 4: MISeq™ bench top next generation sequencer (Adopted from www.illumina.com)|
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Four areas of clinical bacteriology that are likely to be impacted by WGS include detection and identification of bacteria, antimicrobial susceptibility testing (AST), outbreak detection and surveillance, and detection of virulence determinants.
| Detection and Identification of Bacteria|| |
Culture of bacteria and identification through routine methods still remain the cheapest and most reliable way of identifying and characterising bacteria. Despite this, bacterial WGS is increasingly being utilized in the clinical laboratory for this purpose. It provides a unique way of a characterising bacteria, which is independent of the physiological state of the microorganism or the composition of the growth medium.
Culture of bacteria prior to identification with WGS was standard practice, and this requires large number of bacterial colonies. However, in 2014, Köser et al. were able to demonstrate that it was possible to identify and fully characterize a clinical Salmonella isolate, using a single bacterial colony.  They were able to confirm that the test isolate was serovar Enteritidis, and simultaneously provided genome-level discrimination between this and other isolates. Rapid WGS has also been utilized to detect and characterize bacteria directly from clinical samples, to identify bacteria from polymicrobial urine samples, as well as detect pathogenic bacteria from culture-negative samples.  Also in a recent case report, NGS was used to diagnose a patient with a rare bacterial meningoencephalitis caused by leptospirosis, a condition that was undetectable using current clinical assays. 
Whole genome sequencing provides the highest resolution possible for identification and discrimination of bacteria; and may in fact revolutionise bacterial systematics and taxonomy.
| Antimicrobial Susceptibility Testing|| |
Resistance to antibacterial agents constitutes a serious threat to human health.  AST is important not only for the management of infectious diseases, but is also important for tracking the spread of antimicrobial resistance. The role of bacterial WGS in AST is likely to be limited for now. This is because phenotypic susceptibility testing is cheap and relatively easy to carry out, and more so, there is incomplete congruence between genotype and phenotype. However, phenotypic tests do have limitations, such as the duration it takes to complete phenotypic antimicrobial tests. This can be a few days for fast-growing organism and even longer for slower growing organisms. Furthermore, phenotypic testing can be unreliable in some situations. An example was the inability to detect the emergence of quinolone resistance in a Salmonella enterica subsp. enterica serovar typhi isolate by routine phenotypic testing.  This failure was found to have been caused by the emergence of a new resistance mechanism that was undetectable by current phenotypic methods. These and other limitations of phenotypic testing open a window of opportunity for bacterial WGS.
Whole genome has already been used to carry out drug susceptibility testing, with some encouraging results. In one such example, Gordon et al., identified a panel of resistance determinants by comparing routine DST results for 12 commonly used antibiotics of 501 isolates of Staphylococcus aureus, to WGS data. This panel was subsequently used to perform WGS DST for another 491 isolates, which yielded an overall sensitivity and specificity of 0.97 (95% confidence interval [95% CI], 0.95-0.98) and 0.99 (95% CI, 0.99-1), respectively.  The increasing speed of WGS means that resistance determinants can be identified quickly, thus informing patient management. A case in point was the use of WGS to rapidly detect the blaNDM-1 gene in a clinical isolate of Klebsiella pneumoniae that was isolated from a perinephric abscess. 
Whole genome sequencing is playing a vital role in our understanding of antibacterial resistance. An example is the discovery of mecC (a homolog of the methicillin-resistance gene mecA) in S. aureus, using WGS. This resulted in the development of new ways to detect methicillin resistance.  WGS also helped to discover that lineage 2 Mycobacterium tuberculosis complex (MTBC) isolates (East Asian lineage and Beijing sub lineage-the most frequent MTBC genotype globally) acquired rifampicin resistance in vitro at an elevated rate when compared with lineage 4 strains (Euro-American lineage).  This suggests that patients infected with drug-susceptible lineage 2 isolates have a 22-fold increased risk of having developed multidrug resistance (MDR-TB) by the time that they are first diagnosed. In another example, genome analysis of 33 isolates of Mycobacterium tuberculosis using WGS showed that the mutation rate during latent tuberculosis is similar to that during active infection.  The implications for treatment of latent tuberculosis cannot be overemphasized. These studies highlighted above give a glimpse of the potential of WGS in AST, as well as its use in tracking antimicrobial resistance.
| Outbreak Detection and Surveillance|| |
Outbreak detection and surveillance is one area where WGS has proven to be invaluable. It has been used to define transmission pathways of infectious pathogens and to support outbreak investigations.  Sequencing of the entire genome provides the ultimate resolution for epidemiological studies, as one is able to see every region of the genome; so as to identify subtle differences that are missed by earlier genotypic techniques. Several recent studies have highlighted the usefulness of WGS in outbreak investigations.
Whole genome sequencing provided strong evidence that Mycobacterium abscessus, which is intrinsically resistance to many antibiotics, is transmissible between cystic fibrosis (CF) patients in the hospital setting, and this was previously unknown.  On a more global scale, WGS showed that the epidemic Pseudomonas aeruginosa Liverpool strain spreading among patients in cystic fibrosis clinics in the UK had been the same strain spreading in North American cystic fibrosis clinics.  During an Escherichia coli O104:H4 outbreak in Germany and France in 2011, WGS was able to show that the outbreak isolates were more diverse than had been detected by standard tests.  Genome sequences and genome-wide restriction enzyme maps (optical maps) of the outbreak strains were also available in less than 3 days, demonstrating the power of WGS for the investigation of infectious outbreaks in real time.  Another good example of the usefulness of WGS in outbreak investigations was the epidemiological investigation carried in British Columbia, Canada, during an outbreak of tuberculosis.  Ultimately, whole-genome data revealed two genetically distinct lineages of M. tuberculosis with identical MIRU-VNTR genotypes, suggesting two concomitant outbreaks. Interestingly, genotyping and contact tracing alone failed to capture the true dynamics of the outbreak.
Whole genome sequencing has made it possible to detect direct transmission events, to determine the extent of local nosocomial or community-based outbreaks, and identify worldwide patterns of spread.  This will undoubtedly make WGS of bacterial pathogens an extremely valuable infection control tool for microbiologist, infectious disease specialist, and epidemiologists.
| Detection of Virulence Determinants|| |
Detection of genetic determinants of virulence phenotypes will improve our understanding of how pathogenic bacteria cause disease. Recently, analysis of whole-genome data from E. coli O104, showed that the strain was a novel E. coli O104:H4 variant that had acquired a prophage encoding Shiga toxin 2, and also had a distinct set of additional virulence determinants.  This ability to rapidly and simultaneously detect known and novel virulence determinants within a few days is unique to WGS.
| Future Prospects|| |
With the cost of NGS technology falling, and speed and reliability of DNA sequencing improving, the future holds great promise for bacterial WGS and its role in the clinical laboratory. The trend in the last 10 years has shown that sequencing capability has been doubling every 6-9 months, and a $1 bacterial genome sequence seems to be a possibility in the not so distant future. 
Functional genomics and metagenomics are two exciting fields of genomic research that will have a major impact on the practice of clinical bacteriology. Functional genomics, which involves the use of genomic data to study gene and protein expression and function on a genome-wide scale; focusing on gene transcription, translation, and protein-protein interactions; has now been made easier by high-throughput sequencing. This will improve our understanding of the complex relationship between genotype and phenotype, and will no doubt give microbiologist a better understanding of bacterial metabolism and bacterial pathogenesis. This will eventually lead to the discovery of new methods of treating and preventing bacterial infections.
Metagenomics which refers to the use of culture-independent methods to explore the genetic diversity, population structures, and interactions of microbial communities in their ecosystems;  has also been revolutionized by NGS and WGS. The ability to identify and characterize all the bacteria (both culturable and nonculturable) in a complex microbial community in the human host will have a major impact on the diagnosis of bacterial infectious diseases. Already, high-throughput DNA sequencing of the intestinal microbiota has been used to identify patients at high risk of developing bacterial sepsis, by showing that intestinal domination by Vancomycin Resistant Enterococci (VRE) preceded bloodstream infection in patients undergoing allogeneic hematopoietic stem cell transplantation.  In another example of the potential benefits of metagenomics, Rea et al. determined that the use of broad spectrum antibiotics (Vancomycin and Metronidazole) to treat pseudomembranous colitis, had a significant effect on the composition of the human gut microbiota.  Their study also demonstrated that a narrow spectrum bacteriocin such as thuricin CD, was effective at killing Clostridium difficile in the distal colon, but had no significant impact on the other members of the gut microbiota. This highlights the potential benefits of WGS in helping to assess the efficacy of new treatments and vaccines in the management of C. difficile infections; as outlined in the recent review by Mathur et al. 
Recently, WGS helped identify a novel antimicrobially active inhibitor of Vibrio cholera,  and also helped identify new drug targets in M. tuberculosis. All indications are that WGS is positioned to play a critical role in the fight against antibiotic resistance. 
New third generation single-molecule sequencing platforms such as the MiniION™ have been shown to be capable of sequencing entire bacterial genomes in a single run.  According to the Genomes OnLine Database (GOLD; http://www.genomesonline.org ), as of February 2015, there are about 44,430 bacterial WGS projects reported in the database.  This highlights the unprecedented WGS revolution occurring globally, and gives an idea of what is to come in the future.
| Conclusion|| |
Bacterial WGS is set to have a major impact on clinical bacteriology. This is because WGS has the potential to provide in one single step, nearly all the information required to detect and characterize bacteria, to carry out antimicrobial resistance testing, to identify virulence determinants, and ultimately inform public health measures. WGS protocols are being streamlined, so as to make them easier for routine clinical laboratory use. The reduction in the cost of new sequencing technologies has made it possible to generate bacterial genomes at timescales and price levels that a clinical relevant. Devices like the portable MiniION™ are likely to further decrease the cost of reagents and instrumentation.
Despite all this enthusiasm, several major challenges still exist. Bacterial WGS is still too expensive to be used routinely in a clinical laboratory, especially in developing countries. This is likely to remain so for many years to come. There is also the need for laboratory personnel to acquire the skills required to handle NGS technology. Furthermore, the interpretation of WGS data is still very cumbersome, and needs to be fully automated. This will help to make the information generated from WGS more readily accessible to the clinician. Many formats exist for presenting NGS data, and these need to be harmonized. Finally, there is a need to develop standardized methods and quality control guidelines for the use of WGS in clinical laboratories. WGS represents a paradigm shift in the field of microbiology and will no doubt define the practice of clinical microbiology in the next few decades.
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