This website uses cookies in order to improve our services. If you proceed visiting this website you accept the usage of cookies. For more info please read our Data Privacy statement.


Next Generation Sequencing in Antibiotic Resistance Testing

Infectious diseases are the most common cause of death worldwide. A number of pathogens causing infectious diseases are on the advance. This includes not only human pathogens but also pathogens that can infect different species including farming animals. These pathogens are denoted as zoonotic pathogens.

Mycobacterium tuberculosis is the infectious agent of Tuberculosis, the estimated infection rate for 2004 worldwide was about 2 billion infections representing 30% of the total world population whereof 2-3 million people die each year (Kaneene et al. 2004). Other important pathogenic bacteria are for example members of the families or genera Clostridium, Neisseria, Salmonella, Bacillus, Staphylococcus, Streptococcus, Brucellosis, Pseudomonas, and Campylobacteraceae. For more information we recommend the World Health Organization (WHO) website. 

Clostridium difficile causes about half a million infections in the United States each year with an estimation of 15000 death directly associated with these infection. In the USA about two million people become infected with resistant bacteria whereof about 23.000 people die each year (CDC website).

Antibiotic Application and Resistance Development

As consequence of the development and application of antibiotics, a number of bacteria developed resistances against these chemicals. The extensive usage of antibiotics in the food industry and the less stringed control and application of antibiotics in parts of Asia and Africa are major causes for increase of resistant pathogens. Currently, we can find different resistance classes with pathogens that are resistance against certain antibiotics, multidrug resistant bacteria (MDR), extensively drug resistant bacteria (XDR), and most recently totally drug resistant bacteria (TDR).

Different bacterial resistance mechanisms are known, for more detailed information we refer to the Center for Disease Control and Prevention website. ( These resistance mechanisms are caused and regulated by genetic and epigenetic factors. The resistance can be caused by chromosomal mutations or can be plasmid mediated. A number of genes and plasmids are described that are related with certain drug resistances. For example in Salmonella typhi resistance to ampicillin, chloramphenicol, co-trimoxazole, and tetracycline is mediated by the IncHI1 plasmid. Quinolone resistance is caused by single point mutation in the topoisomerase gene gyrA, which encodes the DNA gyrase (Ugboko and De 2014).

Current Methods for Antibiotic Resistance Testing

Standard methods for antibiotic testing are based on bacterial growth in the presence of the corresponding test antibiotics. These methods are still time consuming and can take days depending on the bacteria of interest. The traditional bacterial growth on agar plates is more and more replaces by modern solutions that enable automated high throughput testing in reduced time. Different companies offer high throughput test systems for example the Vitek System (bioMerieux, France), Walk-Away System (Dade International, Sacramento, Calif.), Sensititre ARIS (Trek Diagnostic Systems, East Grinstead, UK), Avantage Test System (Abbott Laboratories, Irving, Texas), Micronaut (Merlin, Bornheim-Hesel, Germany), and the Phoenix system (BD Biosciences, Maryland).

Molecular Genetic Based Methods for Resistance Testing

Different molecular genetic methods are currently investigated for their application in antibiotic resistance testing. These are for example PCR based technics, Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), microarrays, and Next Generation Sequencing. PCR based technics include conventional PCR and real time PCR. These methods are relatively fast and can present results within a few hours, high throughput is possible, and PCR is cost effective. Different commercial PCR based test systems are available on the market for example the BD GeneOhm MRSA (Becton Dickinson, Heidelberg, Germany), GT MRSA Direct/GQ MRSA (Hain Lifescience, Nehren, Germany), and the Hyplex Staphylo Resist test (Amplex, Gießen, Germany). A major drawback of PCR is that multiplexing is limited. Microarrays enable high multiplexing and is very fast. Different research studies based on MALDI-TOF resistance testing are published. For more information we refer to the publication of Polido et al. 2013

Next Generation Sequencing for Bacterial Resistance Testing

Next Generation Sequencing enable the sequencing of billions short reads in one run and thus allows sequencing of large genome regions, whole genomes and multiple samples in one sequencing run. Barcoding of individual samples with specific sequence tags (barcodes) allows the sequencing of hundreds or even thousands of samples in one run. The capacity to multiplex is one major advantage of NGS over PCR based tests. NGS can be used for whole bacterial genome sequencing or for targeted sequencing of different genes of interests. Depends on the number of relevant genes the development of targeted gene panels, as available for heritability and cancer testing, is one option.

The below mentioned limitations for molecular based resistance testing methods apply likewise for NGS based tests. Additional drawbacks of this technology for antibiotic resistance testing is the relative long time to result, the complex workflow and analysis, and the high operation costs compared to conventional resistance testing methods. Currently, the time to result including sample preparation (DNA extraction), sequencing and data analysis will take even with optimized workflows 24 hours or more depending on the used sequencing technology. The so called third generation sequencing technologies propose sequencing in real time but DNA extraction, sample preparation, operation time for the technology and data analysis including result interpretation needs comparable amounts of time as second generation sequencing technologies (Illumina, Roche, Ion Torrent, Qiagen). High multiplexing of samples can partially reduce the operation cost of the NGS technology but the prerequisite is a high sample load that will normally not be available in most labs as delay of testing by collecting and storing of samples is not an option.

Another limitation is the still missing automated and reliable data analysis and interpretation especially for new and rare or complex mutations. The interaction of mutations and the impact of epigenetic factors and transcription levels is not evaluation for all individual mutations and resistances. The corresponding data need to be provided in order to enable reliable data analysis and result interpretation. Referring to the lately FDA published draft guidelines for NGS based tests in precision medicine there will be a number of challenges before NGS based tests will be accepted and approved for diagnostics in microbiology and resistance testing.

A number of scientific publications and research studies investigated different applications and developed different targeted gene panels for bacterial drug resistance testing.

Limitations of Molecular Based Resistance Testing Methods

One limitation of molecular based methods for resistance testing is that these methods do only detect the presence of genes or mutations that are associated with certain resistances. The presence of a resistance gene does not provides conclusive information about the resistance as the resistance phenotype might depend on interaction of different gene products, on gene expression rates, or on epigenetic factors as well. Nevertheless, the information about resistance genes, even though the bacteria is not resistance yet, can help to predict the likelihood of the bacteria to become resistant.

Another limitation is that only known genotypes that are associated with resistance phenotypes can be detected with these methods. New mutations cannot be assigned to specific resistance phenotypes and need to be evaluated and validated in bacterial growth tests.

Summery and Outlook for NGS Application in Bacterial Drug Resistance Testing

In summary, even though the research results are very promising, the implementation of NGS based tests in routine bacterial drug resistance testing is currently not practicable and depends on the future development of the technology in terms of costs, time to results and reliable data analysis and result interpretation.


Zoonosis Update Tuberculosis, Kaneene et al. JAVMA, Vol 224, No. 5, March 1, 2004
Mechanisms of Antibiotic resistance in Salmonella typhi, Ugboko and De, Int.J.Curr.Microbiol.App.Sci (2014) 3(12): 461-476
Progress on the development of rapid methods for antimicrobial susceptibility testing, Polido et al. J Antimicrob Chemother 2013; 68: 2710–2717