James Gomez

Replication dynamics of Mycobacterium tuberculosis in chronically infected mice

ABSTRACT The dynamics of host-pathogen interactions have important implications for the design of new antimicrobial agents to treat chronic infections such as tuberculosis (TB), which is notoriously refractory to conventional drug therapy. In the mouse model of TB, an acute phase of exponential bacterial growth in the lungs is followed by a chronic phase characterized by relatively stable numbers of bacteria. This equilibrium could be static, with little ongoing replication, or dynamic, with continuous bacterial multiplication balanced by bacterial killing. A static model predicts a close correspondence between “viable counts” (live bacteria) and “total counts” (live plus dead bacteria) in the lungs over time. A dynamic model predicts the divergence of total counts and viable counts over time due to the accumulation of dead bacteria. Here, viable counts are defined as bacterial CFU enumerated by plating lung homogenates; total counts are defined as bacterial chromosome equivalents (CEQ) enumerated by using quantitative real-time PCR. We show that the viable and total bacterial counts in the lungs of chronically infected mice do not diverge over time. Rapid degradation of dead bacteria is unlikely to account for the stability of bacterial CEQ numbers in the lungs over time, because treatment of mice with isoniazid for 8 weeks led to a marked reduction in the number of CFU without reducing the number of CEQ. These observations support the hypothesis that the stable number of bacterial CFU in the lungs during chronic infection represents a static equilibrium between host and pathogen.

Sensitive, Specific Polymorphism Discovery in Bacteria Using Massively Parallel Sequencing

Our variant ascertainment algorithm, VAAL, uses massively parallel DNA sequence data to identify differences between bacterial genomes with high sensitivity and specificity. VAAL detected ∼98% of differences (including large insertion-deletions) between pairs of strains from three species while calling no false positives. VAAL also pinpointed a single mutation between Vibrio cholerae genomes, identifying an antibiotics site of action by identifying sequence differences between drug-sensitive strains and drug-resistant derivatives.

Color selection with a hygromycin-resistance-based Escherichia coli-mycobacterial shuttle vector ☆

Hygromycin-resistance (HyR)-based Escherichia coli-mycobacterial shuttle plasmids have high efficiencies of transformation and a broad mycobacterial host range. We have introduced a lacZ alpha (encoding the alpha-polypeptide fragment of beta-galactosidase (beta Gal))-multiple cloning site cassette into a HyR-based shuttle vector to generate a plasmid with nine unique cloning sites and the added feature of beta Gal color selection in appropriate E. coli host strains.

RNA signatures allow rapid identification of pathogens and antibiotic susceptibilities

With rising rates of drug-resistant infections, there is a need for diagnostic methods that rapidly can detect the presence of pathogens and reveal their susceptibility to antibiotics. Here we propose an approach to diagnosing the presence and drug-susceptibility of infectious diseases based on direct detection of RNA from clinical samples. We demonstrate that species-specific RNA signatures can be used to identify a broad spectrum of infectious agents, including bacteria, viruses, yeast, and parasites. Moreover, we show that the behavior of a small set of bacterial transcripts after a brief antibiotic pulse can rapidly differentiate drug-susceptible and -resistant organisms and that these measurements can be made directly from clinical materials. Thus, transcriptional signatures could form the basis of a uniform diagnostic platform applicable across a broad range of infectious agents.

Identification of Mycobacterium tuberculosis counterimmune (cim) mutants in immunodeficient mice by differential screening

ABSTRACT Tuberculosis (TB) is characterized by lifetime persistence of Mycobacterium tuberculosis. Despite the induction of a vigorous host immune response that curtails disease progression in the majority of cases, the organism is not eliminated. Subsequent immunosuppression can lead to reactivation after a prolonged period of clinical latency. Thus, while it is clear that protective immune mechanisms are engaged during M. tuberculosis infection, it also appears that the pathogen has evolved effective countermechanisms. Genetic studies with animal infection models and with patients have revealed a key role for the cytokine gamma interferon (IFN-γ) in resistance to TB. IFN-γ activates a large number of antimicrobial pathways. Three of these IFN-γ-dependent mechanisms have been implicated in defense against M. tuberculosis: inducible nitric oxide synthase (iNOS), phagosome oxidase (phox), and the phagosome-associated GTPase LRG-47. In order to identify bacterial genes that provide protection against specific host immune pathways, we have developed the strategy of differential signature-tagged transposon mutagenesis. Using this approach we have identified three M. tuberculosis genes that are essential for progressive M. tuberculosis growth and rapid lethality in iNOS-deficient mice but not in IFN-γ-deficient mice. We propose that these genes are involved in pathways that allow M. tuberculosis to counter IFN-γ-dependent immune mechanisms other than iNOS.

Genomic analysis of globally diverse Mycobacterium tuberculosis strains provides insights into the emergence and spread of multidrug resistance

Multidrug-resistant tuberculosis (MDR-TB), caused by drug-resistant strains of Mycobacterium tuberculosis, is an increasingly serious problem worldwide. Here we examined a data set of whole-genome sequences from 5,310 M. tuberculosis isolates from five continents. Despite the great diversity of these isolates with respect to geographical point of isolation, genetic background and drug resistance, the patterns for the emergence of drug resistance were conserved globally. We have identified harbinger mutations that often precede multidrug resistance. In particular, the katG mutation encoding p.Ser315Thr, which confers resistance to isoniazid, overwhelmingly arose before mutations that conferred rifampicin resistance across all of the lineages, geographical regions and time periods. Therefore, molecular diagnostics that include markers for rifampicin resistance alone will be insufficient to identify pre-MDR strains. Incorporating knowledge of polymorphisms that occur before the emergence of multidrug resistance, particularly katG p.Ser315Thr, into molecular diagnostics should enable targeted treatment of patients with pre-MDR-TB to prevent further development of MDR-TB.

Independent Large Scale Duplications in Multiple M. tuberculosis Lineages Overlapping the Same Genomic Region

Mycobacterium tuberculosis, the causative agent of most human tuberculosis, infects one third of the worlds population and kills an estimated 1.7 million people a year. With the world-wide emergence of drug resistance, and the finding of more functional genetic diversity than previously expected, there is a renewed interest in understanding the forces driving genome evolution of this important pathogen. Genetic diversity in M. tuberculosis is dominated by single nucleotide polymorphisms and small scale gene deletion, with little or no evidence for large scale genome rearrangements seen in other bacteria. Recently, a single report described a large scale genome duplication that was suggested to be specific to the Beijing lineage. We report here multiple independent large-scale duplications of the same genomic region of M. tuberculosis detected through whole-genome sequencing. The duplications occur in strains belonging to both M. tuberculosis lineage 2 and 4, and are thus not limited to Beijing strains. The duplications occur in both drug-resistant and drug susceptible strains. The duplicated regions also have substantially different boundaries in different strains, indicating different originating duplication events. We further identify a smaller segmental duplication of a different genomic region of a lab strain of H37Rv. The presence of multiple independent duplications of the same genomic region suggests either instability in this region, a selective advantage conferred by the duplication, or both. The identified duplications suggest that large-scale gene duplication may be more common in M. tuberculosis than previously considered.

Ribosomal mutations promote the evolution of antibiotic resistance in a multidrug environment

Antibiotic resistance arising via chromosomal mutations is typically specific to a particular antibiotic or class of antibiotics. We have identified mutations in genes encoding ribosomal components in Mycobacterium smegmatis that confer resistance to several structurally and mechanistically unrelated classes of antibiotics and enhance survival following heat shock and membrane stress. These mutations affect ribosome assembly and cause large-scale transcriptomic and proteomic changes, including the downregulation of the catalase KatG, an activating enzyme required for isoniazid sensitivity, and upregulation of WhiB7, a transcription factor involved in innate antibiotic resistance. Importantly, while these ribosomal mutations have a fitness cost in antibiotic-free medium, in a multidrug environment they promote the evolution of high-level, target-based resistance. Further, suppressor mutations can then be easily acquired to restore wild-type growth. Thus, ribosomal mutations can serve as stepping-stones in an evolutionary path leading to the emergence of high-level, multidrug resistance. DOI: http://dx.doi.org/10.7554/eLife.20420.001

Genomic Analysis of the Evolution of Fluoroquinolone Resistance in Mycobacterium tuberculosis Prior to Tuberculosis Diagnosis

ABSTRACT Fluoroquinolones (FQs) are effective second-line drugs for treating antibiotic-resistant tuberculosis (TB) and are being considered for use as first-line agents. Because FQs are used to treat a range of infections, in a setting of undiagnosed TB, there is potential to select for drug-resistant Mycobacterium tuberculosis mutants during FQ-based treatment of other infections, including pneumonia. Here we present a detailed characterization of ofloxacin-resistant M. tuberculosis samples isolated directly from patients in Taiwan, which demonstrates that selection for FQ resistance can occur within patients who have not received FQs for the treatment of TB. Several of these samples showed no mutations in gyrA or gyrB based on PCR-based molecular assays, but genome-wide next-generation sequencing (NGS) revealed minority populations of gyrA and/or gyrB mutants. In other samples with PCR-detectable gyrA mutations, NGS revealed subpopulations containing alternative resistance-associated genotypes. Isolation of individual clones from these apparently heterogeneous samples confirmed the presence of the minority drug-resistant variants suggested by the NGS data. Further NGS of these purified clones established evolutionary links between FQ-sensitive and -resistant clones derived from the same patient, suggesting de novo emergence of FQ-resistant TB. Importantly, most of these samples were isolated from patients without a history of FQ treatment for TB. Thus, selective pressure applied by FQ monotherapy in the setting of undiagnosed TB infection appears to be able to drive the full or partial emergence of FQ-resistant M. tuberculosis, which has the potential to confound diagnostic tests for antibiotic susceptibility and limit the effectiveness of FQs in TB treatment.

Loss of a Class A Penicillin-Binding Protein Alters β-Lactam Susceptibilities in Mycobacterium tuberculosis.

Recent studies have renewed interest in β-lactam antibiotics as a potential treatment for Mycobacterium tuberculosis infection. To explore the opportunities and limitations of this approach, we sought to better understand potential resistance mechanisms to β-lactam antibiotics in M. tuberculosis. We identified mutations in the penicillin-binding protein (PBP) ponA2 that were able to confer some degree of resistance to the cephalosporin subclass of β-lactams. Surprisingly, deletion of ponA2 also confers resistance, demonstrating that β-lactam resistance can spontaneously arise from PBP loss of function. We show that ponA2 mutants resistant to the cephalosporin subclass of β-lactams in fact show increased susceptibility to meropenem, a carbapenem that is known to target l,d-transpeptidases, thereby suggesting that in the absence of PonA2, an alternative mode of peptidoglycan synthesis likely becomes essential. Consistent with this hypothesis, a negative genetic selection identified the l,d-transpeptidase ldtMt2 as essential in the absence of ponA2. The mechanism of β-lactam resistance we outline is consistent with emerging models of β-lactam killing, while the investigation of ponA2 downstream and synthetic lethal genes sheds light on the mechanism of cell wall biosynthesis and the interaction between conventional PBPs and l,d-transpeptidases.