Novel Synergies and Isolate Specificities in the Drug Interaction Landscape of Mycobacterium abscessus.

Mycobacterium abscessus infections are difficult to treat and are often considered untreatable without tissue resection. Due to the intrinsic drug-resistant nature of the bacteria, combination therapy of three or more antibiotics is recommended. A major challenge in treating M. abscessus infections is the absence of a universal combination therapy with satisfying clinical success rates, leaving clinicians to treat infections using antibiotics lacking efficacy data. We systematically measured drug combinations in M. abscessus to establish a resource of drug interaction data and identify patterns of synergy to help design optimized combination therapies. We measured 191 pairwise drug combination effects among 22 antibacterials and identified 71 synergistic pairs, 54 antagonistic pairs, and 66 potentiator-antibiotic pairs. We found that commonly used drug combinations in the clinic, such as azithromycin and amikacin, are antagonistic in the lab reference strain ATCC 19977, whereas novel combinations, such as azithromycin and rifampicin, are synergistic. Another challenge in developing universally effective multidrug therapies for M. abscessus is the significant variation in drug response between isolates. We measured drug interactions in a focused set of 36 drug pairs across a small panel of clinical isolates with rough and smooth morphotypes. We observed strain-dependent drug interactions that cannot be predicted from single-drug susceptibility profiles or known drug mechanisms of action. Our study demonstrates the immense potential to identify synergistic drug combinations in the vast drug combination space and emphasizes the importance of strain-specific combination measurements for designing improved therapeutic interventions.

Morphological profiling of tubercle bacilli identifies drug pathways of action.

Morphological profiling is a method to classify target pathways of antibacterials based on how bacteria respond to treatment through changes to cellular shape and spatial organization. Here we utilized the cell-to-cell variation in morphological features of Mycobacterium tuberculosis bacilli to develop a rapid profiling platform called Morphological Evaluation and Understanding of Stress (MorphEUS). MorphEUS classified 94% of tested drugs correctly into broad categories according to modes of action previously identified in the literature. In the other 6%, MorphEUS pointed to key off-target activities. We observed cell wall damage induced by bedaquiline and moxifloxacin through secondary effects downstream from their main target pathways. We implemented MorphEUS to correctly classify three compounds in a blinded study and identified an off-target effect for one compound that was not readily apparent in previous studies. We anticipate that the ability of MorphEUS to rapidly identify pathways of drug action and the proximal cause of cellular damage in tubercle bacilli will make it applicable to other pathogens and cell types where morphological responses are subtle and heterogeneous.

Localization of EccA3 at the growing pole in Mycobacterium smegmatis.

BACKGROUND: Bacteria require specialized secretion systems for the export of molecules into the extracellular space to modify their environment and scavenge for nutrients. The ESX-3 secretion system is required by mycobacteria for iron homeostasis. The ESX-3 operon encodes for one cytoplasmic component (EccA3) and five membrane components (EccB3 - EccE3 and MycP3). In this study we sought to identify the sub-cellular location of EccA3 of the ESX-3 secretion system in mycobacteria. RESULTS: Fluorescently tagged EccA3 localized to a single pole in the majority of Mycobacterium smegmatis cells and time-lapse fluorescent microscopy identified this pole as the growing pole. Deletion of ESX-3 did not prevent polar localization of fluorescently tagged EccA3, suggesting that EccA3 unipolar localization is independent of other ESX-3 components. Affinity purification - mass spectrometry was used to identify EccA3 associated proteins which may contribute to the localization of EccA3 at the growing pole. EccA3 co-purified with fatty acid metabolism proteins (FAS, FadA3, KasA and KasB), mycolic acid synthesis proteins (UmaA, CmaA1), cell division proteins (FtsE and FtsZ), and cell shape and cell cycle proteins (MurS, CwsA and Wag31). Secretion system related proteins Ffh, SecA1, EccA1, and EspI were also identified. CONCLUSIONS: Time-lapse microscopy demonstrated that EccA3 is located at the growing pole in M. smegmatis. The co-purification of EccA3 with proteins known to be required for polar growth, mycolic acid synthesis, the Sec secretion system (SecA1), and the signal recognition particle pathway (Ffh) also suggests that EccA3 is located at the site of active cell growth.

Pharmacokinetics and Target Attainment of SQ109 in Plasma and Human-Like Tuberculosis Lesions in Rabbits.

SQ109 is a novel well-tolerated drug candidate in clinical development for the treatment of drug-resistant tuberculosis (TB). It is the only inhibitor of the MmpL3 mycolic acid transporter in clinical development. No SQ109-resistant mutant has been directly isolated thus far in vitro, in mice, or in patients, which is tentatively attributed to its multiple targets. It is considered a potential replacement for poorly tolerated components of multidrug-resistant TB regimens. To prioritize SQ109-containing combinations with the best potential for cure and treatment shortening, one must understand its contribution against different bacterial populations in pulmonary lesions. Here, we have characterized the pharmacokinetics of SQ109 in the rabbit model of active TB and its penetration at the sites of disease-lung tissue, cellular and necrotic lesions, and caseum. A two-compartment model with first-order absorption and elimination described the plasma pharmacokinetics. At the human-equivalent dose, parameter estimates fell within the ranges published for preclinical species. Tissue concentrations were modeled using an "effect" compartment, showing high accumulation in lung and cellular lesion areas with penetration coefficients in excess of 1,000 and lower passive diffusion in caseum after 7 daily doses. These results, together with the hydrophobic nature and high nonspecific caseum binding of SQ109, suggest that multiweek dosing would be required to reach steady state in caseum and poorly vascularized compartments, similar to bedaquiline. Linking lesion pharmacokinetics to SQ109 potency in assays against replicating, nonreplicating, and intracellular M. tuberculosis showed SQ109 concentrations markedly above pharmacokinetic-pharmacodynamic targets in lung and cellular lesions throughout the dosing interval.

Prediction of ultra-high-order antibiotic combinations based on pairwise interactions.

Drug combinations are a promising approach to achieve high efficacy at low doses and to overcome resistance. Drug combinations are especially useful when drugs cannot achieve effectiveness at tolerable doses, as occurs in cancer and tuberculosis (TB). However, discovery of effective drug combinations faces the challenge of combinatorial explosion, in which the number of possible combinations increases exponentially with the number of drugs and doses. A recent advance, called the dose model, uses a mathematical formula to overcome combinatorial explosion by reducing the problem to a feasible quadratic one: using data on drug pairs at a few doses, the dose model accurately predicts the effect of combinations of three and four drugs at all doses. The dose model has not yet been tested on higher-order combinations beyond four drugs. To address this, we measured the effect of combinations of up to ten antibiotics on E. coli growth, and of up to five tuberculosis (TB) drugs on the growth of M. tuberculosis. We find that the dose model accurately predicts the effect of these higher-order combinations, including cases of strong synergy and antagonism. This study supports the view that the interactions between drug pairs carries key information that largely determines higher-order interactions. Therefore, systematic study of pairwise drug interactions is a compelling strategy to prioritize drug regimens in high-dimensional spaces.

Rv0004 is a new essential member of the mycobacterial DNA replication machinery.

DNA replication is fundamental for life, yet a detailed understanding of bacterial DNA replication is limited outside the organisms Escherichia coli and Bacillus subtilis. Many bacteria, including mycobacteria, encode no identified homologs of helicase loaders or regulators of the initiator protein DnaA, despite these factors being essential for DNA replication in E. coli and B. subtilis. In this study we discover that a previously uncharacterized protein, Rv0004, from the human pathogen Mycobacterium tuberculosis is essential for bacterial viability and that depletion of Rv0004 leads to a block in cell cycle progression. Using a combination of genetic and biochemical approaches, we found that Rv0004 has a role in DNA replication, interacts with DNA and the replicative helicase DnaB, and affects DnaB-DnaA complex formation. We also identify a conserved domain in Rv0004 that is predicted to structurally resemble the N-terminal protein-protein interaction domain of DnaA. Mutation of a single conserved tryptophan within Rv0004's DnaA N-terminal-like domain leads to phenotypes similar to those observed upon Rv0004 depletion and can affect the association of Rv0004 with DnaB. In addition, using live cell imaging during depletion of Rv0004, we have uncovered a previously unappreciated role for DNA replication in coordinating mycobacterial cell division and cell size. Together, our data support that Rv0004 encodes a homolog of the recently identified DciA family of proteins found in most bacteria that lack the DnaC-DnaI helicase loaders in E. coli and B. subtilis. Therefore, the mechanisms of Rv0004 elucidated here likely apply to other DciA homologs and reveal insight into the diversity of bacterial strategies in even the most conserved biological processes.

Temporal and intrinsic factors of rifampicin tolerance in mycobacteria.

Mycobacteria grow and divide asymmetrically, creating variability in growth pole age, growth properties, and antibiotic susceptibilities. Here, we investigate the importance of growth pole age and other growth properties in determining the spectrum of responses of Mycobacterium smegmatis to challenge with rifampicin. We used a combination of live-cell microscopy and modeling to prospectively identify subpopulations with altered rifampicin susceptibility. We found two subpopulations that had increased susceptibility. At the initiation of treatment, susceptible cells were either small and at early stages of the cell cycle, or large and in later stages of their cell cycle. In contrast to this temporal window of susceptibility, tolerance was associated with factors inherited at division: long birth length and mature growth poles. Thus, rifampicin response is complex and due to a combination of differences established from both asymmetric division and the timing of treatment relative to cell birth.

Spatially distinct and metabolically active membrane domain in mycobacteria.

Protected from host immune attack and antibiotic penetration by their unique cell envelope, mycobacterial pathogens cause devastating human diseases such as tuberculosis. Seamless coordination of cell growth with cell envelope elongation at the pole maintains this barrier. Unraveling this spatiotemporal regulation is a potential strategy for controlling mycobacterial infections. Our biochemical analysis previously revealed two functionally distinct membrane fractions in Mycobacterium smegmatis cell lysates: plasma membrane tightly associated with the cell wall (PM-CW) and a distinct fraction of pure membrane free of cell wall components (PMf). To provide further insight into the functions of these membrane fractions, we took the approach of comparative proteomics and identified more than 300 proteins specifically associated with the PMf, including essential enzymes involved in cell envelope synthesis such as a mannosyltransferase, Ppm1, and a galactosyltransferase, GlfT2. Furthermore, comparative lipidomics revealed the distinct lipid composition of the PMf, with specific association of key cell envelope biosynthetic precursors. Live-imaging fluorescence microscopy visualized the PMf as patches of membrane spatially distinct from the PM-CW and notably enriched in the pole of the growing cells. Taken together, our study provides the basis for assigning the PMf as a spatiotemporally distinct and metabolically active membrane domain involved in cell envelope biogenesis.

Accelerating Early Antituberculosis Drug Discovery by Creating Mycobacterial Indicator Strains That Predict Mode of Action.

Due to the rise of drug-resistant forms of tuberculosis, there is an urgent need for novel antibiotics to effectively combat these cases and shorten treatment regimens. Recently, drug screens using whole-cell analyses have been shown to be successful. However, current high-throughput screens focus mostly on stricto sensu life/death screening that give little qualitative information. In doing so, promising compound scaffolds or nonoptimized compounds that fail to reach inhibitory concentrations are missed. To accelerate early tuberculosis (TB) drug discovery, we performed RNA sequencing on Mycobacterium tuberculosis and Mycobacterium marinum to map the stress responses that follow upon exposure to subinhibitory concentrations of antibiotics with known targets, ciprofloxacin, ethambutol, isoniazid, streptomycin, and rifampin. The resulting data set comprises the first overview of transcriptional stress responses of mycobacteria to different antibiotics. We show that antibiotics can be distinguished based on their specific transcriptional stress fingerprint. Notably, this fingerprint was more distinctive in M. marinum We decided to use this to our advantage and continue with this model organism. A selection of diverse antibiotic stress genes was used to construct stress reporters. In total, three functional reporters were constructed to respond to DNA damage, cell wall damage, and ribosomal inhibition. Subsequently, these reporter strains were used to screen a small anti-TB compound library to predict the mode of action. In doing so, we identified the putative modes of action for three novel compounds, which confirms the utility of our approach.

Exploitation of Mycobacterium tuberculosis reporter strains to probe the impact of vaccination at sites of infection.

Mycobacterium tuberculosis (Mtb) remains a major public health problem, with an effective vaccine continuing to prove elusive. Progress in vaccination strategies has been hampered by a lack of appreciation of the bacterium's response to dynamic changes in the host immune environment. Here, we utilize reporter Mtb strains that respond to specific host immune stresses such as hypoxia and nitric oxide (hspX'::GFP), and phagosomal maturation (rv2390c'::GFP), to investigate vaccine-induced alterations in the environmental niche during experimental murine infections. While vaccination undoubtedly decreased bacterial burden, we found that it also appeared to accelerate Mtb's adoption of a phenotype better equipped to survive in its host. We subsequently utilized a novel replication reporter strain of Mtb to demonstrate that, in addition to these alterations in host stress response, there is a decreased percentage of actively replicating Mtb in vaccinated hosts. This observation was supported by the differential sensitivity of recovered bacteria to the front-line drug isoniazid. Our study documents the natural history of the impact that vaccination has on Mtb's physiology and replication and highlights the value of reporter Mtb strains for probing heterogeneous Mtb populations in the context of a complex, whole animal model.

Quantitative analysis of pathways controlling extrinsic apoptosis in single cells.

Apoptosis in response to TRAIL or TNF requires the activation of initiator caspases, which then activate the effector caspases that dismantle cells and cause death. However, little is known about the dynamics and regulatory logic linking initiators and effectors. Using a combination of live-cell reporters, flow cytometry, and immunoblotting, we find that initiator caspases are active during the long and variable delay that precedes mitochondrial outer membrane permeabilization (MOMP) and effector caspase activation. When combined with a mathematical model of core apoptosis pathways, experimental perturbation of regulatory links between initiator and effector caspases reveals that XIAP and proteasome-dependent degradation of effector caspases are important in restraining activity during the pre-MOMP delay. We identify conditions in which restraint is impaired, creating a physiologically indeterminate state of partial cell death with the potential to generate genomic instability. Together, these findings provide a quantitative picture of caspase regulatory networks and their failure modes.

Protein complexes and proteolytic activation of the cell wall hydrolase RipA regulate septal resolution in mycobacteria.

Peptidoglycan hydrolases are a double-edged sword. They are required for normal cell division, but when dysregulated can become autolysins lethal to bacteria. How bacteria ensure that peptidoglycan hydrolases function only in the correct spatial and temporal context remains largely unknown. Here, we demonstrate that dysregulation converts the essential mycobacterial peptidoglycan hydrolase RipA to an autolysin that compromises cellular structural integrity. We find that mycobacteria control RipA activity through two interconnected levels of regulation in vivo-protein interactions coordinate PG hydrolysis, while proteolysis is necessary for RipA enzymatic activity. Dysregulation of RipA protein complexes by treatment with a peptidoglycan synthase inhibitor leads to excessive RipA activity and impairment of correct morphology. Furthermore, expression of a RipA dominant negative mutant or of differentially processed RipA homologues reveals that RipA is produced as a zymogen, requiring proteolytic processing for activity. The amount of RipA processing differs between fast-growing and slow-growing mycobacteria and correlates with the requirement for peptidoglycan hydrolase activity in these species. Together, the complex picture of RipA regulation is a part of a growing paradigm for careful control of cell wall hydrolysis by bacteria during growth, and may represent a novel target for chemotherapy development.

In vitro identification of underutilized ß-lactam combinations against methicillin-resistant Staphylococcus aureus bacteremia isolates.

Methicillin-resistant Staphylococcus aureus (MRSA) bacteremia is a serious clinical challenge with high mortality rates. Antibiotic combination therapy is currently used in cases of persistent infection; however, the limited development of new antibiotics will likely increase the need for combination therapy, and better methods are needed for identifying effective combinations for treating persistent bacteremia. To identify pairwise combinations with the most consistent potential for benefit compared to monotherapy with a primary anti-MRSA agent, we conducted a systematic study with an in vitro high-throughput methodology. We tested daptomycin and vancomycin each in combination with gentamicin, rifampicin, cefazolin, and oxacillin, and ceftaroline with daptomycin, gentamicin, and rifampicin. Combining cefazolin with daptomycin lowered the daptomycin concentration required to reach 95% growth inhibition (IC95) for all isolates tested and lowered daptomycin IC95 below the sensitivity breakpoint for five out of six isolates that had daptomycin minimum inhibitory concentrations at or above the sensitivity breakpoint. Similarly, vancomycin IC95s were decreased when vancomycin was combined with cefazolin for 86.7% of the isolates tested. This was a higher percentage than was achieved by adding any other secondary antibiotic to vancomycin. Adding rifampicin to daptomycin or vancomycin did not always reduce IC95s and failed to produce synergistic interaction in any of the isolates tested; the addition of rifampicin to ceftaroline was frequently synergistic and always lowered the amount of ceftaroline required to reach the IC95. These analyses rationalize further in vivo evaluation of three drug pairs for MRSA bacteremia: daptomycin+cefazolin, vancomycin+cefazolin, and ceftaroline+rifampicin.IMPORTANCEBloodstream infections caused by methicillin-resistant Staphylococcus aureus (MRSA) have a high mortality rate despite the availability of vancomycin, daptomycin, and newer antibiotics including ceftaroline. With the slow output of the antibiotic pipeline and the serious clinical challenge posed by persistent MRSA infections, better strategies for utilizing combination therapy are becoming increasingly necessary. We demonstrated the value of a systematic high-throughput approach, adapted from prior work testing antibiotic combinations against tuberculosis and other mycobacteria, by using this approach to test antibiotic pairs against a panel of MRSA isolates with diverse patterns of antibiotic susceptibility. We identified three antibiotic pairs-daptomycin+cefazolin, vancomycin+cefazolin, and ceftaroline+rifampicin-where the addition of the second antibiotic improved the potency of the first antibiotic across all or most isolates tested. Our results indicate that these pairs warrant further evaluation in the clinical setting.

Critical role of growth medium for detecting drug interactions in Gram-negative bacteria that model in vivo responses.

The rise in infections caused by multidrug-resistant (MDR) bacteria has necessitated a variety of clinical approaches, including the use of antibiotic combinations. Here, we tested the hypothesis that drug-drug interactions vary in different media, and determined which in vitro models best predict drug interactions in the lungs. We systematically studied pair-wise antibiotic interactions in three different media, CAMHB, (a rich lab medium standard for antibiotic susceptibility testing), a urine mimetic medium (UMM), and a minimal medium of M9 salts supplemented with glucose and iron (M9Glu) with three Gram-negative ESKAPE pathogens, Acinetobacter baumannii (Ab), Klebsiella pneumoniae (Kp), and Pseudomonas aeruginosa (Pa). There were pronounced differences in responses to antibiotic combinations between the three bacterial species grown in the same medium. However, within species, PaO1 responded to drug combinations similarly when grown in all three different media, whereas Ab17978 and other Ab clinical isolates responded similarly when grown in CAMHB and M9Glu medium. By contrast, drug interactions in Kp43816, and other Kp clinical isolates poorly correlated across different media. To assess whether any of these media were predictive of antibiotic interactions against Kp in the lungs of mice, we tested three antibiotic combination pairs. In vitro measurements in M9Glu, but not rich medium or UMM, predicted in vivo outcomes. This work demonstrates that antibiotic interactions are highly variable across three Gram-negative pathogens and highlights the importance of growth medium by showing a superior correlation between in vitro interactions in a minimal growth medium and in vivo outcomes. IMPORTANCE: Drug-resistant bacterial infections are a growing concern and have only continued to increase during the SARS-CoV-2 pandemic. Though not routinely used for Gram-negative bacteria, drug combinations are sometimes used for serious infections and may become more widely used as the prevalence of extremely drug-resistant organisms increases. To date, reliable methods are not available for identifying beneficial drug combinations for a particular infection. Our study shows variability across strains in how drug interactions are impacted by growth conditions. It also demonstrates that testing drug combinations in tissue-relevant growth conditions for some strains better models what happens during infection and may better inform combination therapy selection.

Massively parallel combination screen reveals small molecule sensitization of antibiotic-resistant Gram-negative ESKAPE pathogens.

Antibiotic resistance, especially in multidrug-resistant ESKAPE pathogens, remains a worldwide problem. Combination antimicrobial therapies may be an important strategy to overcome resistance and broaden the spectrum of existing antibiotics. However, this strategy is limited by the ability to efficiently screen large combinatorial chemical spaces. Here, we deployed a high-throughput combinatorial screening platform, DropArray, to evaluate the interactions of over 30,000 compounds with up to 22 antibiotics and 6 strains of Gram-negative ESKAPE pathogens, totaling to over 1.3 million unique strain-antibiotic-compound combinations. In this dataset, compounds more frequently exhibited synergy with known antibiotics than single-agent activity. We identified a compound, P2-56, and developed a more potent analog, P2-56-3, which potentiated rifampin (RIF) activity against Acinetobacter baumannii and Klebsiella pneumoniae. Using phenotypic assays, we showed P2-56-3 disrupts the outer membrane of A. baumannii. To identify pathways involved in the mechanism of synergy between P2-56-3 and RIF, we performed genetic screens in A. baumannii. CRISPRi-induced partial depletion of lipooligosaccharide transport genes (lptA-D, lptFG) resulted in hypersensitivity to P2-56-3/RIF treatment, demonstrating the genetic dependency of P2-56-3 activity and RIF sensitization on lpt genes in A. baumannii. Consistent with outer membrane homeostasis being an important determinant of P2-56-3/RIF tolerance, knockout of maintenance of lipid asymmetry complex genes and overexpression of certain resistance-nodulation-division efflux pumps - a phenotype associated with multidrug-resistance - resulted in hypersensitivity to P2-56-3. These findings demonstrate the immense scale of phenotypic antibiotic combination screens using DropArray and the potential for such approaches to discover new small molecule synergies against multidrug-resistant ESKAPE strains.

Polar assembly and scaffolding proteins of the virulence-associated ESX-1 secretory apparatus in mycobacteria.

The ESX-1 secretion system is required for pathogenicity of Mycobacterium tuberculosis (Mtb). Despite considerable research, little is known about the structural components of ESX-1, or how these proteins are assembled into the active secretion apparatus. Here, we exploit the functionally related ESX-1 apparatus of Mycobacterium smegmatis (Ms) to show that fluorescently tagged proteins required for ESX-1 activity consistently localize to the cell pole, identified by time-lapse fluoro-microscopy as the non-septal (old) pole. Deletions in Msesx1 prevented polar localization of tagged proteins, indicating the need for specific protein-protein interactions in polar trafficking. Remarkably, expression of the Mtbesx1 locus in Msesx1 mutants restored polar localization of tagged proteins, indicating establishment of the MtbESX-1 apparatus in M. smegmatis. This observation illustrates the cross-species conservation of protein interactions governing assembly of ESX-1, as well as polar localization. Importantly, we describe novel non-esx1-encoded proteins, which affect ESX-1 activity, which colocalize with ESX-1, and which are required for ESX-1 recruitment and assembly. This analysis provides new insights into the molecular assembly of this important determinant of Mtb virulence.

Physicochemical modelling of cell signalling pathways.

Physicochemical modelling of signal transduction links fundamental chemical and physical principles, prior knowledge about regulatory pathways, and experimental data of various types to create powerful tools for formalizing and extending traditional molecular and cellular biology.

Advances in the design of combination therapies for the treatment of tuberculosis.

INTRODUCTION: Tuberculosis requires lengthy multi-drug therapy. Mycobacterium tuberculosis occupies different tissue compartments during infection, making drug access and susceptibility patterns variable. Antibiotic combinations are needed to ensure each compartment of infection is reached with effective drug treatment. Despite drug combinations' role in treating tuberculosis, the design of such combinations has been tackled relatively late in the drug development process, limiting the number of drug combinations tested. In recent years, there has been significant progress using in vitro, in vivo, and computational methodologies to interrogate combination drug effects. AREAS COVERED: This review discusses the advances in these methodologies and how they may be used in conjunction with new successful clinical trials of novel drug combinations to design optimized combination therapies for tuberculosis. Literature searches for approaches and experimental models used to evaluate drug combination effects were undertaken. EXPERT OPINION: We are entering an era richer in combination drug effect and pharmacokinetic/pharmacodynamic data, genetic tools, and outcome measurement types. Application of computational modeling approaches that integrate these data and produce predictive models of clinical outcomes may enable the field to generate novel, effective multidrug therapies using existing and new drug combination backbones.

Mycobacterium tuberculosis grows linearly at the single-cell level with larger variability than model organisms.

The ability of bacterial pathogens to regulate growth is crucial to control homeostasis, virulence, and drug response. Yet, we do not understand the growth and cell cycle behaviors of Mycobacterium tuberculosis (Mtb), a slow-growing pathogen, at the single-cell level. Here, we use time-lapse imaging and mathematical modeling to characterize these fundamental properties of Mtb. Whereas most organisms grow exponentially at the single-cell level, we find that Mtb exhibits a unique linear growth mode. Mtb growth characteristics are highly variable from cell-to-cell, notably in their growth speeds, cell cycle timing, and cell sizes. Together, our study demonstrates that growth behavior of Mtb diverges from what we have learned from model bacteria. Instead, Mtb generates a heterogeneous population while growing slowly and linearly. Our study provides a new level of detail into how Mtb grows and creates heterogeneity, and motivates more studies of growth behaviors in bacterial pathogens.

Semi-automated colony-forming unit counting for biosafety level 3 laboratories.

Biosafety level 3 decontamination precautions motivate measuring microbial colonies using consumable photography instead of expensive automated plate counters or smartphones, and assaying drug treatments-with multiple concentrations per treatment, replicates, and controls-produces hundreds of images. Here, we present a protocol for semi-automated image analysis by hand-tuning three parameters. The parameters control for non-uniform colony growth and artifacts such as lid condensation, reflections, and plating streaks. We describe steps to prepare images, tune parameters, and plot dose-response relationships. For complete details on the use and execution of this protocol, please refer to Larkins-Ford et al.1.