About a month ago, a very depressing item was in the news: a Harris poll indicating that over two-thirds of Americans are unaware of the pressing issue of antibiotic resistance. This blog (and many news outlets) have highlighted the increasing prevalence of drug-resistant infections, but despite over half of American claiming to read about disease prevention weekly, the growing numbers of antibiotic-resistant infections in agriculture and the clinic have not resonated with the public.
New list of high-priority bacterial sources of infection in need of new antibiotics. Source.
Public awareness of this issue may have been helped, however, by the World Health Organization (WHO) publication of a priority list for antibiotic-resistant bacteria that require new drugs for treatment of infection. The list, divided into critical, high, and medium priorities (see figure), includes many of the usual suspects discussed on this blog and elsewhere: carbapenem-resistant Enterobacteriaceae, carbapenem-resistant Pseudomonas aeruginosa, and methicillin-resistant Staphylococcus aureus, among others. In publishing the list, the WHO hoped to bring attention to the dire need of research into new antimicrobial compounds for these bacterial groups.
While all of these bacterial species/classes are clinical problems that need to be addressed, the WHO list notably omitted one of the most prevalent drug-resistant infections: Mycobacterium tuberculosis. The notable omission of this species was justified by the WHO because of several ongoing drug-discovery programs specific for this organism, but that doesn’t negate the widespread incidence of multidrug-resistant (MDR) and extremely-drug-resistant (XDR) tuberculosis infections spreading around the world. Tuberculosis is one of the top 10 causes of death in the world, primarily in low-income countries with poor health system infrastructure, with over one-third of the worldwide population harboring latent (inactive) M. tuberculosis. In 2015, 10.4 million people developed active tuberculosis, with MDR-TB in 480,000 of these cases and XDR-TB in 45,600 cases.
These numbers highlight the need for research into new effective drugs or drug targets through a combination of clinical and basic research, as commented on in a recent mBio editorial from Christopher Kerantzas and Bill Jacobs. Several recent important reports have emphasized the issue of drug resistance in M. tuberculosis, from novel resistance mechanisms to faster diagnoses. In recognition of World TB Day, some of the reports and their ties to drug resistance are summarized below.
Novel drug resistance mechanism
A March 2017 Antimicrobial Agents and Chemotherapy study reports the emergence of carbapenem resistance in a lab-grown M. tuberculosis strain. Carbapenems are used to treat MDR- and XDR-TB without major resistance thus far, but the development of a carbapenem-resistant XDR-TB strain would be a huge clinical issue. Resistance discovery in a lab setting has important clinical applications, since clinicians and epidemiologists can then keep an eye out for lab-characterized genetic changes among clinical isolates. In this study, scientists working with senior researcher Gyanu Lamichhane sequenced the genome of 12 spontaneous mutants resistant to meropenem or biapenem, two carbapenem-class antibiotics used to treat TB patients.
A SNP in a previously uncharacterized M. tuberculosis gene confers drug resistance. Source.
The research team found that a single nucleotide polymorphism (SNP) in an uncharacterized gene was responsible for carbapenem resistance in these lab-generated mutants (see schematic, right). The gene, named crfA for carbapenem-resistance factor A, is highly expressed in all M. tuberculosis growth phases. The CrfA protein interacts with carbapenem-class compounds, and this interaction is blocked by the mutation, likely conferring resistance to the cell. The researchers posit that the SNP allows CrfA to maintain its normal cellular function while decreasing affinity for carbapenems like merobenem and biapenem. The mutation appears so far only to be found in the lab, since this SNP wasn’t recorded in the >100 GenBank-deposited M. tuberculosis genome sequences, but its discovery in all the lab-generated resistant strain genomes, combined with an increasing reliance on carbapenems for recalcitrant TB infections, means the mutation is one to watch out for in clinical isolates.
Improved tools for genetic studies
With the current state of genomic research, it may seem unusual that there are undiscovered genes with unknown functions in a commonly studied human pathogen like M. tuberculosis, but there are many genes yet uncharacterized in this organism. Gene essentiality is an important area of study to determine the genes that are required for M. tuberculosis growth. These studies are facilitated by mutagenesis studies that compile libraries of mutant bacteria, each missing a single gene. These libraries, generated by techniques like random transposon insertion, allow researchers to determine critical genes by measuring the ratio of organisms grown in different conditions. If a gene is necessary for growth in a certain environment – say, inside human cells – the mutant should grow without difficulty under lab conditions but be unable to grow in tissue culture. This is one way researchers can identify genes critical for infection that may be potential targets for drug development.
The transposon-insertion mutant libraries for M. tuberculosis studies have been non-saturating, meaning the transposon didn’t insert and interrupt every possible gene in the genome. These libraries interrupt 50-60% of the possible insertion sites, which for the commonly used Himar1 transposon is a TA dinucleotide sequence. An analysis of 14 independently generated libraries published in mBio in January 2017 provides more comprehensive genomic coverage and a tool to study gene essentiality. The combined information from these genomes creates a saturated model that was analyzed with a statistical framework to increase the ability of researchers to detect small essential open reading frames and other essential genome features like small RNAs, promoters, and terminator regions. This valuable tool from a research team led by Thomas Ioerger will help M. tuberculosis researchers assess gene essentiality, one of the first steps in discovering new drug targets.
Faster assessment of drug susceptibility
Assessing the genomes of M. tuberculosis of clinical isolates is another area of heavy research. While basic studies involving mutant libraries are essential [pun intended] to drug discovery, there remains the very real problem of drug-resistant infections already in the clinic. One of the hurdles that makes resistance a serious clinical issue is the length of time it takes to assess drug susceptibility in the slow-growing M. tuberculosis. Culture-based assessments remain the gold standard, but can take weeks to complete, due to the 24-h doubling time for this bacterium. A March 2017 Journal of Clinical Microbiology report demonstrates that whole-genome sequencing (WGS) directly from patient sputum can more quickly diagnose TB infection and, in many cases, assess drug susceptibility.
The report, from the lab of Zamin Iqbal, evaluates a relatively low-cost method to extract and sequence DNA using the Illumina MiSeq sequencer. Their method correctly identified M. tuberculosis in 39 patient isolates, and correctly predicted drug susceptibility in 24 isolates. The protocol took at most 48 hours to run from patient sample to completed analysis. The WHO endorses several molecular methods to test for susceptibility due to the long culture incubation time, but these test only a prescribed number of genes. Screening the entire genome for drug-resistant cassettes directly from a patient sputum sample (composed primarily of non-TB genomic material) represents progression toward point-of-care diagnostics and more accurate prescriptions for patients suffering with drug-resistant TB. Applying cutting-edge technologies like WGS and the discovery of lab-generated resistance mutations may even help identify new resistant mechanisms quickly and ensure that patients are given the most effective therapy possible.
Drug Resistance: A Crisis that Demands Global Solutions
M. tuberculosis is only one example of an infectious microbe in dire need of creative solutions to address the issue of drug resistance. The American Society for Microbiology recognizes the need for creative solutions. An ASM AMR Steering Committee was established and met in August 2016 to outline opportunities to combat the AMR crisis. “The Steering Committee is well positioned to advise the Society on interdisciplinary One Health approaches and opportunities to address critical data gaps and human resource needs to confront this multifaceted, urgent domestic and global challenge,” said Steering Committee co-chair James Tiedje, University Distinguished Professor and director of the NSF Center for Microbial Ecology at Michigan State University, a member of the Steering Committee. You can find a summary of the meeting here.
Inspired to engage further with the issues of tuberculosis or antibiotic-resistant infections?
Antibiotics: Challenges, Mechanisms, Opportunities. By Christopher Walsh and Timothy Wencewicz
Molecular Genetics of Mycobacteria, 2nd Ed. By Graham Hatfull and William Jacobs.
FAQ: The Threat of MRSA. 2015.